University of Southern California Dissertations and Theses

Investigating the role of O-GlcNAcylation in α-synuclein aggregation

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Investigating the role of O-GlcNAcylation in α-synuclein aggregation

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INVESTIGATING THE ROLE OF O-GlcNAcylation IN α-SYNUCLEIN
AGGREGATION

by

Ana Galesic

A Dissertation Presented to
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)

December 2020

Copyright 2020 Ana Galesic
ii

Acknowledgements

I would like to thank my parents, sister, aunt and uncle for their love, support, and caring. There
are no words to describe how much I am thankful for you instilling in me a love for science,
learning and encouraning me not to be afraid of new challanges. I would have not been able to
finish my PhD without you.

I would like to thank my thesis advisor, Matt Pratt, for his support, mentoring and for facilitating
my growth as a scientist and person. I am grateful to the members of my thesis advisory committee,
Peter Qin and Ralf Langen for their guidance, encourangment and advice during my PhD program.

I want to thank to my dear friend, Wei Yuan, for her support, friendship and much appreciated
advice when needed. You are a great mentor and amazing friend.

Finally, I would like to thank all my friends for your friendship, support and caring.

iii

Table of Contents

Acknowledgments ii

List of Figures v

Abstract ix

Chapter 1. Parkinson’s Disease and α-Synuclein
Introduction 1
Mechanism of α-Synuclein Aggregation and Propagation 3
Investigating the site specific effects of O-GlcNAc modifications in
PD using semisynthetic approach 13
Chapter One References 17

Chapter 2. α-Synuclein O-GlcNAcylation alters aggregation and toxicity, revealing
certain residues as potential inhibitors of Parkinson’s disease 34
Introduction 34
Results 39
Discussion 56
Materials and Methods 60
Supplemental Information 71
Chapter Two References 78

Chapter 3. Comparison of N-acetyl-glucosamine to other monosaccharides
reveals potentially special abilities of O-GlcNAc in amyloid inhibition 86
Introduction 86
Results and Discussion 89
Materials and Methods 95
Supplemental Information 106
Chapter Three References 108

Chapter 4. O-GlcNAc modification inhibits the calpain-mediated cleavage
of α-synuclein 112

Introduction 112
Results and Discussion 116
Conclusions 120
Materials and Methods 122
Chapter Four References 126

iv
Chapter 5. O-GlcNAcylation of α-Synuclein at Serine 87 Reduces Aggregation
without Affecting Membrane Binding 133

Introduction 133
Results and Discussion 136
Materials and Methods 146
Supplemental Information 153
Chapter Five References 160

References 165

v
List of Figures

Figure 1-1: Structural domains of α-synuclein. 2

Figure 1-2: The process of α-synuclein aggregation. 4

Figure 1-3: α-synuclein is a substrate for several post translational
modifications. 6

Figure 1-4: Harnessing inteins for expressed protein ligation (EPL). 15

Figure. 1-5: Synthesis of O-GlcNAcylated α-synuclein. 16

Figure 2-1: O-GlcNAc and α-synuclein. 36

Figure 2-2: Synthesis of site-specifically O-GlcNAcylated α-synuclein. 40

Figure 2-3: O-GlcNAcylation alters the nucleation step of α-synuclein
aggregation in a site-specific manner. 44

Figure 2-4: Most, but not all, O-GlcNAcylation sites inhibit the extension
step of α-synuclein aggregation. 49

Figure 2-5: O-GlcNAcylation site-specifically inhibits neuronal toxicity
in a membrane-based extension model. 52

Figure 2-6; O-GlcNAcylation can inhibit both steps in the aggregation of
an early-onset Parkinson’s disease mutant (A53T) of
α-synuclein. 55

Figure S 2-1: Characterization of unmodified and O-GlcNAcylated
α-synuclein. 71

Figure S 2-2: O-GlcNAc does not effect on the monomeric nature of
α-synuclein in solution. 72

Figure S 2-3: O-GlcNAc does not induce α-synuclein secondary structure. 72

Figure S 2-4: O-GlcNAc has very little effect on the micelle-bound
structure of α-synuclein. 73

Figure S 2-5: Representative TEM images from α-synuclein nucleation
reactions. 73

Figure S 2-6: α-Synuclein(gT72,75,81) inhibits the aggregation of
unmodified protein. 74
vi

Figure S 2-7: Representative TEM images from α-synuclein extension
reactions. 74

Figure S 2-8: Blow-up TEM images of α-synuclein(gT81) and (gS87)
extension reactions. 75

Figure S 2-9: PK digestion of the α-synuclein extension reactions is
consistent with site-selective inhibition by O-GlcNAc. 75

Figure S 2-10: O-GlcNAcylated α-synuclein monomers are not toxic to
primary neurons. 76

Figure S 2-11: Characterization of unmodified and O-GlcNAcylated
α-synuclein. 77

Figure S 2-12: O-GlcNAc does not affect the solution confirmation or
membrane binding of α-synuclein(A53T). 77

Figure S 2-13: α-Synuclein O-GlcNAcylation is not readily detected by
Western blotting with commonly used anti-O-GlcNAc
antibodies. 77

Figure 3-1: O-GlcNAc modification of α-synuclein. 87

Figure 3-2: Different monosaccharides have distinct effects
on α-synuclein aggregation. 88

Figure 3-3: Analysis of the different α-synuclein aggregates using
transmission electron microscopy (TEM). 93

Figure S 3-1: Synthesis and characterization of α-synuclein proteins. 106

Figure S 3-2: Different monosaccharides have distinct effects on
α-synuclein aggregation. 106

Figure S 3-3: Large format TEM images. 107

Figure 4-1: O-GlcNAc modification and calpain cleavage of α-synuclein. 114

Figure 4-2: Synthesis of α-synuclein bearing site-specific
O-GlcNAcylation. 115

Figure 4-3: O-GlcNAcylation blocks the cleavage of α-synuclein by
calpain. 118

vii
Figure 4-4: Identification of thea-synuclein-derived fragments after
calpain cleavage. 120

Figure 5-1: O-GlcNAcylation and α-synuclein. 134

Figure 5-2: Synthesis and characterization of unmodified and
O-GlcNAcylated α-synuclein (α-synuclein(gS87)). 137

Figure 5-3: O-GlcNAcylation of α-synuclein at S87 inhibits protein
aggregation. 138

Figure 5-4: O-GlcNAcylation at S87 inhibits α-synuclein aggregation
without affecting membrane binding. (A) O-GlcNAcylation
at S87 is more inhibitory toward aggregation at lower protein
concentrations. 141

Figure S 5-1: Characterization of α-synuclein protein fragments. 154

Figure S 5-2: Ligation of peptide 2 and protein 4 and the subsequent
deprotection to give protein fragment 5. 154

Figure S 5-3: Ligation of peptide 3 and protein 4 and the subsequent
deprotection to give protein fragment 6. 155

Figure S 5-4: Ligation of proteins 5 or 6 and protein-thioester 1 to give
proteins 7 or 8. 155

Figure S 5-5: Desulfurization of proteins 7 or 8. 156

Figure S 5-6: Structural characterization of α-synuclein(gS87) and α-
synuclein(S87E) using circular dichroism (CD) and
dynamic light scattering (DLS). 156

Figure S 5-7: Expression and characterization of α-synuclein(S87E). 157

Figure S 5-8: Structural characterization of synthetic and recombinant
unmodified α-synuclein using circular dichroism (CD) and
dynamic light scattering (DLS). 157

Figure S 5-9: Transmission electron microscopy (TEM) images of the
from the α-synuclein aggregation reactions. 158

Figure S 5-10: Characterization of the additional α-synuclein mutants S87A,
S87D, S87W, and S87K. 158

viii
Figure S 5-11: Analysis of mutant α-synuclein aggregation and membrane
binding. 159

ix
Abstract
Parkinson’s disease (PD) is the second most common neurodegenerative disease. The neuropathology
of PD is driven by the progressive degeneration of dopaminergic neurons which leads to a significant
reduction of stratal dopamine. The common hallmark of PD is the presence of insoluble inclusions
called Lewy bodies (LBs) or Lewy neurites (NTs). Current studies support the hypothesis that α-
synuclein plays a central role in Lewy pathology. Furthermore, abnormal α-synuclein expression,
impairment of proteostasis machinery, and various types of stresses are all implicated in pathogenesis
of α-synuclein. Additionally, various genetic mutations (SNCA, LRRK2, and others) are known to be
driving forces of α-synuclein pathology, and the presence of various post-translational modifications
(PTMs) of α-synuclein, such as ubiquitination, phosphorylation, O-GlcNAcylation, and SUMOylation
have been identified to play a role in the pathogenesis in vivo. These mutations and PTMs can either
exacerbate or diminish α-synuclein’s aggregation and toxicity, and, in the end, they add another layer
of complexity to the study of the pathology of α-synuclein. The molecular mechanisms by which
several of these cellular events promote neurodegenerative diseases are still not known. In the Pratt
lab, we seek to understand the roles played by various PTMs in α-synculein aggregation. In the past,
our lab extensively studied ubiquitinated and SUMOylated α-synuclein, whereas my main focus was
to understand the role of O-GlcNAcylation in the process of aggregation. Several proteomics studies
have identified nine O-GlcNAc-modified sites of α-synuclein in mouse and/or human tissue samples.
In addition, recent studies have shown that global O-GlcNAc levels decrease in neurodegeneration.
Initially, we wanted to delineate the modification’s site-dependent aggregative effects by synthesizing
five differently-modified O-GlcNAcylated proteins via solid phase peptide synthesis and native
chemical ligation. We hypothesized the O-GlcNAc moiety might alter the aggregation of α-synuclein
x
in site-specific manner. Once we obtained the variants, we used a variety of biochemical experiments
to show that O-GlcNAc inhibits protein aggregation, alters fibrillar structure, and abrogates aggregate
cytotoxicity in site-dependent manner. As the mechanism of how O-GlcNAcylation inhibits α-
synuclein aggregation is not yet known, we next asked if these effects are generalizable to other sources
of poly-hydroxylated steric bulk. Therefore, we synthesized and studied three α-synuclein variants
bearing different monosaccharides - glucose, N-acetyl-galactosamine (GalNAc) and mannose. Our data
has indicated the O-GlcNAc moiety as being especially inhibitory of α-synuclein aggregation when
compared to other monosaccharides. Next, we subjected two O-GlcNAc-modified proteins to protease
calpain, which has been found to co-localize in with α-synuclein in PD patient brains. The results show
that O-GlcNAcylation inhibits calpain cleavage, and thus indicates a protective role of O-
GlcNAcylation against calpain cleavage in the modulation of α-synuclein biology. Next, we
investigated the effects of O-GlcNAcylation at serine 87, which is also a phosphorylation site. While
we showed that this particular O-GlcNAc variant is also able to inhibit the process of aggregation, we
found that this modification does not affect the membrane-binding properties of α-synculein in the
same way as phosphorylation does. Overall, our data support the hypothesis that O-GlcNAcylation has
a protective role against protein aggregation, and highlight the promise of therapies that can elevate
the O-GlcNAcyation of α-synculei
1

Chapter 1. Parkinson’s Disease and α-Synuclein
Introduction
James Parkinson’s essay, “Shaking Palsy,” initially described a handful patients with tremors at
rest, akinesia, and bradykinesia over two centuries ago (Parkinson, 1817). These are manifestations
of a disease we now call Parkinson’s disease (PD), the second most common neurodegenerative
disease in the world after Alzheimer’s disease (Auluck et al., 2010; Tysnes and Storstein, 2017).
The main physiological characteristic of PD is the loss of dopaminergic neuronal cells in the
substantial nigra pars compact (SNpc), which is caused by Lewy bodies (LBs), amyloid-like
aggregates of α-synuclein found in the cytoplasm (Forno, 1996; Braak et al., 2003; Shulman et al.,
2011). Recently, the Lashuel lab showed the formation of Lewy bodies (LBs) involved various
cellular events such as internalization and truncation of PFFs, seeding of endogenous α-synuclein,
fibril elongation with incorporation of PTMs, lateral association of aggregates with organelles,
packing of the fibers into LB-lie inclusions and sequestration of proteins, lipids, membranes and
organelles. (Mahul-Mellier et al., 2020). α-Synuclein’s pathogenic role in PD and other LB-related
diseases is supported by various genetic data. For example, duplications of the gene encoding α-
synuclein (SNCA) or various point mutations in this gene (A53T, A30P and E46K) (Kruger et al.,
1998; Polymeropoulos et al., 1997; Zarranz et al., 2004; Simon-Sanchez et al., 2009; Golbe at al.,
1990), in lysosome-related genes (Robak et al., 2017), or in LRRK2 results in familial
parkinsonism (Funayama et al., 2002; Paisan-Ruiz et al., 2004; Zimprich et al., 2004)
α-Synuclein (Figure. 1-1) is a small protein of 140 amino acids (Ueda et al., 1993) that is present
at high concentrations ( ∼50 μM) at neuronal synapses (Wilhelm et al., 2014) This protein has three
structurally distinct regions: 1) the lysine-rich N-terminal region (1-60 residues) which contains
2
four repetitive segments of a hexametric motif (KTKEGV) that is responsible for α-synuclein’s
interactions with membranes (Ulmer et al., 2005), 2) the central fragment of α-synuclein, called
non-amyloid β component of AD amyloid plaques (NAC) (61-95 residues), that is a hydrophobic
region of the protein and is the main driving force for the process of aggregation (Giasson et al.,
2001), and 3) the C-terminal region of the protein (96-140 residues) that contains mostly acidic
and proline residues and interacts with N-terminal region, other proteins, and metals (Uverski and
Eliezer, 2009; Eliezer et al., 2001). While the individual roles/interacting partners of three
structural regions are somewhat known, the actual physiological role of α-synuclein and the
interplay of these various domains in the disease state needs to be elucidated. Thus far, it is known
that α-synuclein’s location is at presynaptic terminals where it associates with synaptic vesicles
and carries out its roles in vesicle trafficking and second-messenger release (Murphy et al., 2000;
Yavich et al., 2004). Nevertheless, after two centuries, more research needs to be done to
understand the fundamental nature of the biology of α-synuclein in order to develop therapeutics.

Figure 1-1. Structural domains of α-synuclein. α-Synuclein contains three main regions: 1) N
terminal region, amphipathic domain, 2) NAC, hydrophobic region, and 3) C terminal, acidic
domain.
3
Mechanism of α-Synculein Aggregation and Propagation
α-Synuclein exists as an unstructured monomer in solution and forms an extended α-helix in the
presence of cellular membranes or artificial lipids (Davidson et al., 1998; Eliezer et al., 2001),
which is important for its putative role in vesicle trafficking (Burre et al., 2015). While it is not
well understood whether α-synuclein monomers form tetramers in the cytosol (Dettmer et al.,
2015), a recent study from the Hiller lab indicates α-synuclein monomers interact with various
chaperones, such as HSP90. This might be a plausible way the cell prevents aggregation of α-
synuclein monomers. (Burmann et al., 2020) The interplay between α-synuclein’s cytosolic and
membrane-bound forms needs to be further investigated.
During the progression of PD and other synucleinopathies, there is a formation of β-sheet-rich
amyloid aggregates that are part of Lewy bodies (LBs) and are toxic to cells. The amyloid
aggregation occurs in the cytoplasm and in close proximity to membranes. (Lee and Lee, 2002)
This aggregation process is known to yield structurally different aggregates, but the overall process
consists of two concentration-dependent steps: nucleation and extension (Figure 1-2). The slow
initial step, nucleation, is initiated by the association of several α-synuclein monomers, and the
end product is the formation of oligomers and small fibers. The further extension or “seeding” of
these structures results in the formation of large fibers which are toxic to cells. This extension step
has very fast kinetics. (Wood et al., 1999) These fibers disturb various cellular machineries and
possibly disturb cellular membranes. (Tsigelny et al., 2012)

4

Figure 1-2. The process of α-synuclein aggregation. Monomers can facilitate the nucleation step
where oligomers and small fibers form with slow kinetics, while further addition of monomers
leads to the formation of large fibers. Eventually, the formation of Lewy bodies (LBs) leads to
impairment of cellular homeostasis.

The main feature of all neurodegenerative diseases is propagation and transmission of the toxic
species from one cell to another, thus spreading throughout different regions of the brain. (Luk et
al., 2009; Luk et al., 2013) The mechanism by which toxic extracellular α-synuclein species
transfer to other cells include endocytosis, direct penetration, trans-synaptic dissemination, and
membrane receptor binding (Lee et al., 2008; Kordower et al., 1998; Danzer et al., 2011; Tang et
al., 2012). These species serve as seeds which facilitate additional aggregation, leading to
impairment of cellular homeostasis and ultimately to exacerbation of the neurodegenerative
process. (Luk et al., 2009)
Although the two step aggregation mechanism is known, more research needs to be done to
understand its complexities. For example, the aggregation process yields various, structurally
heterogeneous aggregate species (Wood et al., 1999). It is not yet known what causes this
heterogeneity. Furthermore, it is not known how toxic species escape vesicles upon cellular uptake
to nucleate the aggregation process. Therefore, more research needs to be done to identify the
factors that influence the heterogeneity of α-synuclein aggregate species and their cellular toxicity
pathways.

5
SNCA gene gene multiplications, point mutations
α-Synuclein is encoded by the SNCA gene. Duplication or triplication of the SNCA gene leads to
sporadic Parkinsonism (Chartier-Harlin et al., 2004) by increasing intracellular α-synuclein levels
and thus inducing its accumulation. Where and how cellular system is impacted has yet to be
explored. In addition to gene multiplication, there are several familial missense mutations in SNCA
genes (A30P, E46K, A53T, H50Q, G51D) that are known to cause early-onset Parkinson’s disease
(Kruger et al., 1998; Zarranz et al., 2004; Polymeropoulos et al., 1997; Lesage et al., 2013; Golbe
et al., 1990; Khalaf et al., 2014). Its is important to note that all these mutations occur in the N-
terminal region of α-synuclein. For example, H50Q is known to induce greater levels of α-
synuclein aggregation than wild-type protein (Khalaf et al., 2014; Lashuel et al., 2013).

Impaired degradation systems
The proteostasis network consists of numerous biochemical pathways that are responsible for
maintaining proteins in their native state and minimizing non-reductive or harmful pathways which
are the hallmarks of neurodegenerative diseases. (Balch et al., 2008; Klaips et al., 2017) One of
most critical proteostasis components is the protein degradation system where toxic or unfolded
proteins are degraded. Failure to properly clear these proteins leads to ER stress, mitochondrial
dysfunction, oxidative stress, and, ultimately, impairment of cellular homeostasis (Klaips et al.,
2017). Two central components of the degradation system are the ubiquitin-proteasome system
and the autophagosomal-lysosomal system. (Ciecanover et al., 2017; Dikic et al., 2017;
Varskavsky et al., 2012) The ubiquitin-proteasome system mostly degrades damaged unfolded
proteins that are tagged by ubiquitin moieties by trafficking them to a proteasome for degradation.
Through this specific type of degradation, there is turnover of monomeric and soluble oligomeric
6
α-synuclein. In contrast, the autophagosomal-lysosomal system is responsible for the degradation
of oligomers via an encasing of these species by vesicles which can then fuse with lysosomes.
(McNaught and Jenner, 2001; McNaught et al., 2001) It is absolutely vital for the cell to have the
ability to degrade small or large oligomers and thus prevent lethal accumulation of aggregates. It
is important to note that the impaired function of lysosomal related genes is linked to Parkinson’s
disease. (Smolders and Broeckhover, 2020) For example, the mutation of GBA gene, which
encodes glucocerobrosidase (GCase), a lysosomal enzyme involved in the metabolism of
glucosylceramide, is more prevalent in people with PD when compared to control population.
(Verez-Paldo et al., 2012; Sindransky and Lopez, 2012) The biological pathway is yet to
discovered.

Post-translational modifications
Numerous studies have shown that α-synuclein is extensively post-translationally modified
(Schmid et al., 2013; Questali et al., 2010). For example, ubiqutination, SUMOylation,
phosphorylation, O-GlcNAcylation are just some of PTMs present. While we know they are
present, the effects of PTMs and the interplay between them are still subjects of current research.

Figure 1-3. α-synuclein is a substrate for several post translational modifications. Various
proteomics studies have identified α-synuclein to be modified by ubiqutination, phosphorylation,
SUMOylation and O-GlcNAcylation.
7
Ubiquitination
Ubiquitination is a wide-spread post-translational modification in eukaryotic cells where small, 76
amino-acid protein called ubiquitin is added to a target protein via covalent attachment. (Hershko
and Ciechanover, 1998; Oh et al., 2018) The most well known function of this PTM is protein
degradation though the 26S proteasome (Finley, 2009; Collins and Golberg, 2017; Yu and
Matousckek, 2017). It's also know to play a role in altering protein structure/function, changing
protein location and turning on/off various pathways (Doel et al., 2019). The specificity of
ubiquitination is determined by the type of ubiquitination linkage (Komander and Rape, 2012).
The target protein can be (multi-) monoubiquitinated and/or polyubiqutinated, and the addition of
ubiquitin molecule/s is catalyzed by three enzymes in a cascade: 1) E1 (ubiquitin-activation), 2)
E2 (ubiquitin-conjugation) and 3) E3 (ubiquitin-ligation). First, ubiquitin’s C-terminal Gly residue
is activated in ATP dependent manner to generate a thioester intermediate with the catalytic Cys
residue of E1 enzyme. Then ubiquitin molecule is transferred to E2 enzyme and then E3 ligase
catalyzes the isopeptide bond formation between Gly residue of ubiquitin and Lys residue of target
protein (Hershko et al., 2000) There are eight sites on ubiquitin that can be further elaborated into
various branch structures (M1, K6, K11, K27, K29, K33, K48, and K63) and thus the number of
possible chain types is staggering. Human genome encodes 2 E1 enzymes, ~40 E2 enzymes and
~600 E3 enzymes (Michelle et al, 2009; Yates et al., 2017; Li et al., 2008). Thus, E3 enzymes play
a pivotal role in ubiquitin signaling. There are three distinct classes of E3 enzymes: RING/U-box
(really interesting new gene), HECT (homologous to E6AP C-terminus) and RBR (RING-
between-RING)ligases (Doel et al., 2019). Additionally, deubiquitinases (DUBs) are enzymes that
are responsible for the hydrolysis of ubiquitin chains and there are roughly 100 enzymes in human
genome. (Clague et al., 2013, 2019; Mevissen and Komander, 2017) There are seven sub-
8
categories of DUB families: ubiquitin C-terminal hydrolases (UCHs) (Doel et al., 2019), ubiquitin
specific proteases (USPs), ovarian tumor proteases (OTUs), JAB1/MPN domain-associated
metalloisopeptidases (JAMM/MPN
+
) (Doel et al., 2019), the novel MIU-containing DUB family
MINDY (Abdul Rehman et al., 2016), and the zinc finger with UFM1- specific peptidase domain-
containing ZUFSP family. (Doel et al., 2019)
Recent studies had indicated the core of LBs is modified by ubiquitin. (Gomez-Tortosa et al., 2000)
Furthermore, the majority of α-synuclein species are either mono-, di-, and tri- modified, and thus
ubiquitination was hypothesized to play role in pathological properties of α-synuclein. (Hasegawa
et al, 2002; Sampathu et al., 2003) α-synuclein is known to interact with three ubiquitin E3 ligases:
seven in absentia homolog (SIAH), neuronal precursor cell-expressed, developmentally down-
regulated gene 4 (Nedd4) and C-terminal U-box domain of co-chaperone Hsp70-interacting
protein (CHIP). (Shin et al., 2005; Tofaris et al., 2011; Liani et al., 2004) Briefly, SIAH is known
to promote the formation of inclusion bodies in vivo (Rott et al., 2008). In brain samples, Nedd4
binds to endogenous α-synuclein and promotes the degradation by the lysosome pathway (Tofaris
et al., 2011). CHIP is co-localized with Lewy bodies where directs α-synuclein towards
proteosomal or lysosomal degradation. (Shin et al., 2005; Kalia et al., 2011)
The sites-specific effects of ubiquitination on aggregation were studies by Pratt lab using a semi-
synthetic strategy. Briefly, a ubiqutin-intein fusion construct was expressed, and the resulting
ubiquitin-intein thioester intermediate was treated with cysteamine, resulting in
transthioesterification and rearrangement to generate a ubiquitin product that contains C-terminal
thiol. After treatment with 2,2′-dithiobis(5-nitropyridine (DTNP) the final product, a ubiquitin
thiol-disulfide product is generated. Upon incubation of this complex with a protein bearing a
single free cysteine, site-directed ubiquitination occurs. This newly formed structure resembles
9
native protein-ubiquitin isopeptide bond. Briefly, ubiquitination of α-synuclein at position 10 and
23 causes the formation of the aggregates, while ubiquitination at position 6, 12 and 21 partially
inhibits the formation of fibers, and modification of the positions at 32, 34, 43 and 96 prevented
formation of fibers (Hejjaoui et al., 2011; Meier et al., 2012) Additionally, the incubation of these
proteins with a purified proteasome resulted in differential proteasomal degradation.
Monoubiquitination located at the N-terminal region of α-synuclein supports some proteasome
degradation. However, monoubiquitination of the central region of α-synuclein does not promote
degradation. Overall, these data indicate the importance of a specific location of
monoubiquitination on α-synuclein, as various sites have divergent effects on α-synuclein
degradation rates (Abeywardana et al., 2013). Furthermore, the Lashuel group has shown that N-
terminal ubiquitination prevents α-synuclein’s oligomerization and aggregation in vitro and
improves α-synuclein’s stability. (Hejjaoui et al., 2011) Additionally, the Brik group has
investigated the importance of the length of the polyubiquitinated chain, hypothesizing it might
play an important role in α-synuclein physiology, clearance, and pathology as well as mediating
the crosstalk between phosphorylation and ubiquitination. Briefly, their results indicated that
ubiquitination might occur after α-synuclein’s fibrillation and might be a cellular attempt to
degrade these toxic structures by the proteasome. (Hai-Yahya et al., 2013)

SUMOylation
SUMOylation is a ubiquitin-like post-translational modification that involves a covalent addition
of a Small Ubiquitin-like Modifier (SUMO) protein to a lysine residues on target consensus motif
of target proteins. (Matunis et al., 1996; Zhao et al., 2014) This type of modification is known to
regulate cellular events inside nucleus, such as cell division, transcription, nuclear transport and
10
DNA repair (Zhao 2018). Interestingly, a recent study indicated that cytoplasmic proteins that have
role in endocytosis or synaptic activity can also be a target by SUMO (Henley et al., 2018).
Furthermore, it is known that SUMO can change proteins structure, localization and stability
(Dorval and Fraser, 2006) SUMOylation is very similar to previously discussed ubiquitination,
where there are three enzymes (activating-conjugating-ligating) that are responsible for addition
of SUMO to protein of interest. (Muller et al., 2001)
α-Synuclein is know to be SUMO-lated at positions K96 and K102 by various in vitro studies and
immunoprecipitation studies from brain tissues. (Dorval and Fraser, 2006; Krumova et al., 2011;
Zhe et al., 2018) There are several SUMO-ligases, such as human Polycomb protein 2 (hPc2),
PIAS2 and TRIM28, that are known to be involved. (Krumova et al., 2011; Oh et al., 2011; Rott
et al., 2017; Rousseaux et al., 2018) The Pratt lab has investigated the role of SUMOylation by
disulfide approach discussed above for ubiquitin, and they found that SUMOlylation is responsible
for reduced ability of α-synuclein to form fibrils. Briefly, the Pratt lab has shown that α-synuclein’s
SUMOylation at position K102 inhibits the kinetics of aggregation more pronouncedly than does
modification at site K96. Interestingly enough, ubiquitination of K96, results in inhibition of the
formation of fibers. (Abeywardana and Pratt, 2015).

Phosphorylation
Phosphorylation is a dynamic post-translational modification of serine, threonine or tyrosine
residues of a target proteins. There are more then 500 kinases in human genome that perform ɣ-
phosphate addition. Also there are more than 150 phosphatases that can remove this type of
modification. (Okachi et al., 2000) Phosphorylation is one of the most widely studied PTMs on α-
synuclein. The main reason for the huge interest lies in the fact that Lewy Bodies 90% of deposited
11
α-synuclein is phosphorylated at Ser129. This modification pays an important role in α-synuclein’s
aggregation pathology in Parkinson’s disease. When compared to healthy sample brain tissues,
only 4% of total α-synuclein is phosphorylated. This interesting observation is not yet well
understood. Additionally, positions S87, Y125, Y133 and Y136 are also phosphorylated. (Ellis et
al., 2001; Nakamura et al., 2001; Takahaski et al. 2000)
Several studies have identified casein kinase I (CKI), casein kinase II (CKII), the G protein-
coupled receptor kinases (GRK), LRRK2 and polo-kinases (PLK) are involved in vitro
phosphorylation of α-synuclein at S129. (Okochi et al., 2000; Pronin et al., 2000; Inglis et al.,
2009) In summary, phosphorylation of α-synuclein at S129 is linked to toxicity where its known
to result in enhancing α-synuclein’s aggregation that ultimately leads to cellular toxicity. (Smith
et al., 2005) Briefly, the Lashuel and Li labs generated semi-synthetic phosphorylated α-synuclein
at position 129 (pS129). Upon incubation with lipids, the Li lab showed that pS129 formed weaker
α-helical structures, indicating that the phosphorylation might impair α-synuclein’s ability to bind
membranes. Furthermore, aggregation assays indicated that phosphorylation at pS129 promoted
α-synuclein aggregation when compared to the control protein. When control and pS129 fibrils
were assessed by transmission electron microscopy (TEM) and Proteinase K (PK), results
indicated pS129 results in the formation of a distinct stain conformation. (Ma et al., 2016; Fauvet
and Lashuel, 2016) Furthermore, the Lashuel and Eliezer labs prepared phosphorylated α-
synuclein at position Y39. While this protein did not promote α-synuclein’s aggregation, its
membrane binding ability was enhanced. (Dikiy et al., 2016) The Lashuel lab also synthetically
made phosphorylated α-synuclein at position Y125, and this protein bound to the membranes to
the same extent as the control protein. Also, there were no differences in the aggregation kinetics
between control and phosphorylated Y125 α-synuclein. (Hejjaoui et al., 2012)
12
O-GlcNAcylation
O-GlcNAcylation is a dynamic posttranslational modification that involves a single addition of O-
linked N-acetylglucosamine (O-GlcNAc) to serine and/or threonine residues of intracellular
proteins located in cytoplasm, nucleus and mitochondria. (Yuzwa et al., 2014; Wani et al., 2017)
This type of modification is a product of nutrient flux that is generated by hexosamine biosynthetic
pathway (HPB), as the end product of this pathway,uridine diphosphate GlcNAc (UDP-GlcNAc),
is a donor substrate for O-GlcNAcylation. (Vasconcelos-Santos et al., 2018) Unlike previously
discussed PTMs, this type of modification is dependent on nutrient availability. Additionally, this
type of modification is also very unique since O-GlcNAcylation addition is controlled only by the
enzyme O-GlcNac transferase (OGT) and removal is controlled by the enzyme O-GlcNAcase
(OGA). (Yuzma et al., 2014; Wani et al., 2017) One of great outstanding question in the field is
how does a single pair of enzymes control very diverse sets of protein substrates.
Changes in O-GlcNAc levels have been documented in various human diseases such as cancer,
neurodegenerative diseases and diabetes. More specifically, in neurodegenerative diseases O-
GlcNAcylation is believed to play a protective role. For example, the aggregation kinetics of
enzymatically O-GlcNAc modified tau is significantly decreased when compared to its control
protein. (Yuzma et al., 2014; Wani et al., 2017) Interestingly enough, several labs has shown
recently lower levels of O-GlcNAcylation in disease brain samples when compared to control.
(Liu et al., 2004; Liu et al., 2009; Aguilar et al., 2017; Pinho et al., 2020)
The responsible mechanism for this observation is yet to be determined. Several proteomics studies
have identified nine different O-GlcNAc modified sites (T33, T43, T54, T59, T64, T72, T75, T81,
S87). (Wang et al., 2009; Wang et al., 2010; Alfraro et al., 2012; Morris et al., 2015) Perviously,
the Pratt lab prepared a semi-synthetic α-synuclein and in vitro studies have indicated that O-
13
GlcNAc has a protective role against α-synuclein aggregation and toxicity. (Marotta et al., 2015;
Lewis et al., 2017) Furthermore, we continued to investigate the effects of O-GlcNAcylation on
α-synuclein aggregation and toxicity. Briefly, we prepared semi-synthetic variants of α-synuclein
(T75, T81, S87 and T727581). In vitro studies had indicated that these proteins affect the
aggregation kinetics in site specific manner, some are less and some more inhibitory. In addition,
these variants are also inhibitory to neuronal toxicity in site specific effect. Overall, current data
indicated the protective role of O-GlcNAcylation in neurodegenerative diseases. However, there
are several outstanding questions that needs to be answered first. 1) For example, what is the
interplay between O-GlcNAcylation and phosphorylation? 2) What is the mechanism of down-
regulation of O-GlcNAcylation in neurodegenerative diseases? More research needs to be done to
better understand the O-GlcNAc biology so therapeutics could be developed.

Investigating the site specific effects of O-GlcNAc modifications in PD using semisynthetic
approach
The enzymatic approach for O-GlcNAc installation to recombinant proteins has been done in
several cases in the past. For example, enzymatically modified tau exhibits reduced aggregation in
vitro, and increasing the amounts of O-GlcNAcylation slows neurodegeneration in a mouse model
of Alzheimer’s disease. (Yuzwa et al., 2012; Borghgreaef et al., 2013; Graham et al., 2014;
Hastings et al., 2017) While this approach was somewhat informative, it leads to heterogeneous
mixtures of differently O-GlcNAc-modified proteins that cannot be readily separated and thus do
not provide any useful information about the importance of site-specific O-GlcNAcylation. Thus,
we decided to utilize the application of protein ligation techniques to prepare site-specifically O-
GlcNAcylated α-synuclein.
14
More specifically, we took advantage of expressed protein ligation (EPL), an extension of native
chemical ligation (NCL). NCL takes advantage of a reaction between a peptide thioester and
peptide or protein bearing an N-terminal cysteine to create a native amide bond. (Dowson et al.,
1994) More specifically, the first step of an NCL reaction is a reversible transthioesterification
between a C-terminal thioester of one peptide and an N-terminal cysteine residue of the other.
Once this intermediate forms, an S-N acyl shift results in the formation of a native amide bond.
While this technology can be used successfully for the generation of site-specifically modified
proteins, the major drawback is the size limitation of solid phase peptide synthesis (SPPS). The
solution to this problem, termed expressed protein ligation (EPL) (Muir et al., 1998), involves the
generation of recombinant protein-thioesters, which occur naturally as intermediates during
protein splicing. Protein splicing is a posttranslational process wherein an intein fragment is
removed from the primary sequence, which results in an amide bond formation between the two
flanking segments or exteins. (Muir and Vila-Perello, 2010) While the biological role of inteins is
not clear, the splicing mechanism of inteins can be used to generate protein thioesters. Proteins of
interest are recombinantly expressed as simple N-terminal fusion to an intein engineered to be
catalytically incompetent, which can be cleaved in the presence of exogenous thiols, yielding a
recombinant C-terminal protein thioester. (Figure 1-4 A) This protein thioester can then participate
in an NCL reaction. (Figure 1-4 B) EPL allows the incorporation of synthetic peptides that contain
PTMs to be incorporated into proteins. Our route involves a protein thioester, a synthetic O-
GlcNAc-modified peptide and a protein fragment generated from E. coli.
15

Figure 1-4. Harnessing inteins for expressed protein ligation (EPL). (A) Generation of
protein thioester. (B) Thioester in native chemical ligation (NCL).

We used EPL to synthesize α-synuclein with a single or multiple O-GlcNAc modifications at
various threonine or serine residues. Here, we will present the synthesis for α-synuclein(gT72) as
an example protein case (Figure 1-4 or whatever). Specifically, O-GlcNAc modified threonine (De
Leon et al., 2018) is first incorporated into a synthetic peptide thioester (A) using SPPS on the
Dawson thioester resin. Next, peptide A undergoes a ligation reaction with recombinant fragment
B, obtained by heterologous expression in E. coli. The N-terminal thiazolidine of the resulting
product is then removed to give intermediate D, which readily undergoes a second ligation reaction
with protein thioester C to yield full-length O-GlcNAcylated α-synuclein. α-Synuclein contains no
native cysteines residues. Therefore, the cysteines required for our synthesis can be transformed
into the native alanine residues by a final desulfurization reaction. Importantly, we have applied
this same general strategy to O-GlcNAc modifications at threonines 72, 75, and 81 or serine 87 by
using the different ligation sites highlighted in Fig. 2b. (discussed in chapter 2). This is
representative of the synthesis of site-specific O-GlcNAcylated α-synuclein, but can be applicable
toward the synthesis of other synthetic proteins bearing site-specific PTMs.

16

Figure 1-5. Synthesis of O-GlcNAcylated α-synuclein. (a) Synthetic route of α-synuclein(gT72).
(b) The primary sequence of human α-synuclein with the different O-GlcNAcylation sites (blue)
that we have prepared and the ligation sites (red) that we have utilized.

17
Chapter One References

Abeywardana, T. and Pratt, M.R., 2015. Extent of inhibition of α-synuclein aggregation in vitro
by SUMOylation is conjugation site-and SUMO isoform-selective. Biochemistry, 54(4), pp.959-
961.
Abeywardana, T., Lin, Y.H., Rott, R., Engelender, S. and Pratt, M.R., 2013. Site-specific
differences in proteasome-dependent degradation of monoubiquitinated α-synuclein. Chemistry &
Biology, 20(10), pp.1207-1213.
Aguilar, A.L., Hou, X., Wen, L., Wang, P.G. and Wu, P., 2017. A chemoenzymatic histology
method for O-GlcNAc detection. Chembiochem: a European journal of chemical biology, 18(24),
p.2416.
Alfaro, J.F., Gong, C.X., Monroe, M.E., Aldrich, J.T., Clauss, T.R., Purvine, S.O., Wang, Z.,
Camp, D.G., Shabanowitz, J., Stanley, P. and Hart, G.W., 2012. Tandem mass spectrometry
identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc
transferase targets. Proceedings of the National Academy of Sciences, 109(19), pp.7280-7285.
Auluck, P. K., Caraveo, G., and Lindquist, S., 2010. alpha-Synuclein: membrane interactions and
toxicity in Parkinson’s disease. Annu. Rev. Cell Dev. Biol. 26, 211–233.
Balch, W. E., Morimoto, R. I., Dillin, A. & Kelly, J. W., 2008. Adapting proteostasis for disease
intervention. Science 319, 916–919.
Borghgraef, P., Menuet, C., Theunis, C., Louis, J.V., Devijver, H., Maurin, H., Smet-Nocca, C.,
Lippens, G., Hilaire, G., Gijsen, H. and Moechars, D., 2013. Increasing brain protein O-GlcNAc-
ylation mitigates breathing defects and mortality of Tau. P301L mice. PloS one, 8(12), p.e84442.
18
Braak, H., Del Tredici, K., Rub, U., de Vos, R. A., Jansen Steur, E. N., and Braak, E., 2003. Staging
of brain pathology related to sporadic Parkinson’s disease. Neurobiol. Aging 24, 197–211.
Burre, J., 2015. The synaptic function of alpha-synuclein. J. Parkinsons Dis. 5, 699–713.
Burmann, B.M., Gerez, J.A., Matečko-Burmann, I., Campioni, S., Kumari, P., Ghosh, D., Mazur,
A., Aspholm, E.E., Šulskis, D., Wawrzyniuk, M. and Bock, T., 2020. Regulation of α-synuclein
by chaperones in mammalian cells. Nature, 577(7788), pp.127-132.
Chartier-Harlin, M.C., Kachergus, J., Roumier, C., Mouroux, V., Douay, X., Lincoln, S.,
Levecque, C., Larvor, L., Andrieux, J., Hulihan, M. and Waucquier, N., 2004. α-Synuclein locus
duplication as a cause of familial Parkinson's disease. The Lancet, 364(9440), pp.1167-1169.
Ciechanover, A., 2017. Intracellular protein degradation: from a vague idea thru the lysosome and
the ubiquitin-proteasome system and onto human diseases and drug targeting. Best Pract. Res.
Clin. Haematol. 30, 341–355.
Clague, M. J., Barsukov, I., Coulson, J. M., Liu, H., Rigden, D. J., and Urbe, S., 2013.
Deubiquitylases from genes to organism. Physiol. Rev. 93, 1289–1315.
Clague, M. J., Heride, C., and Urbé, S., 2019. The demographics of the ubiquitin system. Trends
Cell Biol. 25, 417–426.
Collins, G.A. and Goldberg, A.L., 2017. The logic of the 26S proteasome. Cell, 169(5), pp.792-
806.
Danzer, K.M., Ruf, W.P., Putcha, P., Joyner, D., Hashimoto, T., Glabe, C., Hyman, B.T. and
19
McLean, P.J., 2011. Heat ‐shock protein 70 modulates toxic extracellular α ‐synuclein oligomers
and rescues trans ‐synaptic toxicity. The FASEB journal, 25(1), pp.326-336.
Davidson, W. S., Jonas, A., Clayton, D. F., and George, J. M., 1998. Stabilization of alpha-
synuclein secondary structure upon binding to synthetic membranes. J. Biol. Chem. 273, 9443–
9449.
Dawson, P.E., Muir, T.W., Clark-Lewis, I. and Kent, S.B., 1994. Synthesis of proteins by native
chemical ligation. Science, 266(5186), pp.776-779.
De Leon, C.A., Lang, G., Saavedra, M.I. and Pratt, M.R., 2018. Simple and efficient preparation
of O-and S-GlcNAcylated amino acids through InBr3-catalyzed synthesis of β-N-acetylglycosides
from commercially available reagents. Organic letters, 20(16), pp.5032-5035.
Dettmer U, Newman AJ, von Saucken VE, Bartels T, Selkoe D., 2015. KTKEGV repeat motifs
are key mediators of normal α-synuclein tetramerization: Their mutation causes excess monomers
and neurotoxicity. Proc Natl Acad Sci USA 112:9596–9601.
Dikic, I., 2017. Proteasomal and autophagic degradation systems. Annu. Rev. Biochem. 86, 193–
224.
Dikiy, I., Fauvet, B., Jovicic, A., Mahul-Mellier, A. L., Desobry, C., El-Turk, F., Gitler, A. D.,
Deol, K.K., Lorenz, S. and Strieter, E.R., 2019. Enzymatic logic of ubiquitin chain
assembly. Frontiers in physiology, 10, p.835.
Dorval, V. and Fraser, P.E., 2006. Small ubiquitin-like modifier (SUMO) modification of natively
unfolded proteins tau and α-synuclein. Journal of Biological Chemistry, 281(15), pp.9919-9924.
20
Eliezer, D., Kutluay, E., Bussell, R. Jr., and Browne, G., 2001. Conformational properties of alpha-
synuclein in its free and lipid-associated states. J. Mol. Biol. 307, 1061–1073.
Fauvet, B., and Lashuel, H. A., 2016. Semisynthesis and enzymatic preparation of post-
translationally modified alpha-synuclein. Methods Mol. Biol. 1345, 3−20.
Forno, L. S., 1996. Neuropathology of Parkinson’s disease. J. Neuropathol. Exp. Neurol. 55, 259–
272.
Finley, D., 2009. Recognition and processing of ubiquitin-protein conjugates by the
proteasome. Annual review of biochemistry, 78, pp.477-513.
Funayama, M., Hasegawa, K., Kowa, H., Saito, M., Tsuji, S. and Obata, F., 2002. A new locus for
Parkinson's disease (PARK8) maps to chromosome 12p11. 2–q13. 1. Annals of Neurology:
Official Journal of the American Neurological Association and the Child Neurology
Society, 51(3), pp.296-301.
Giasson, B.I., Murray, I.V., Trojanowski, J.Q. and Lee, V.M.Y., 2001. A hydrophobic stretch of
12 amino acid residues in the middle of α-synuclein is essential for filament assembly. Journal of
Biological Chemistry, 276(4), pp.2380-2386.
Golbe, L. I., Di Iorio, G., Bonavita, V., Miller, D. C., and Duvoisin, R. C., 1990. A large kindred
with autosomal dominant Parkinson’s disease. Ann. Neurol. 27, 276–282.
Gómez-Tortosa, E., Newell, K., Irizarry, M.C., Sanders, J.L. and Hyman, B.T., 2000. α-Synuclein
immunoreactivity in dementia with Lewy bodies: morphological staging and comparison with
ubiquitin immunostaining. Acta neuropathologica, 99(4), pp.352-357.
21
Graham, D.L., Gray, A.J., Joyce, J.A., Yu, D., O'Moore, J., Carlson, G.A., Shearman, M.S.,
Dellovade, T.L. and Hering, H., 2014. Increased O-GlcNAcylation reduces pathological tau
without affecting its normal phosphorylation in a mouse model of
tauopathy. Neuropharmacology, 79, pp.307-313.
Hastings, N.B., Wang, X., Song, L., Butts, B.D., Grotz, D., Hargreaves, R., Hess, J.F., Hong,
K.L.K., Huang, C.R.R., Hyde, L. and Laverty, M., 2017. Inhibition of O-GlcNAcase leads to
elevation of O-GlcNAc tau and reduction of tauopathy and cerebrospinal fluid tau in rTg4510
mice. Molecular neurodegeneration, 12(1), p.39.
Haj-Yahya, M., Fauvet, B., Herman-Bachinsky, Y., Hejjaoui, M., Bavikar, S.N., Karthikeyan,
S.V., Ciechanover, A., Lashuel, H.A. and Brik, A., 2013. Synthetic polyubiquitinated α-Synuclein
reveals important insights into the roles of the ubiquitin chain in regulating its
pathophysiology. Proceedings of the National Academy of Sciences, 110(44), pp.17726-17731.
Henley, J.M., Carmichael, R.E. and Wilkinson, K.A., 2018. Extranuclear SUMOylation in
neurons. Trends in neurosciences, 41(4), pp.198-210.
Hejjaoui, M., Haj ‐Yahya, M., Kumar, K.A., Brik, A. and Lashuel, H.A., 2011. Towards
Elucidation of the Role of Ubiquitination in the Pathogenesis of Parkinson’s Disease with
Semisynthetic Ubiquitinated α ‐Synuclein. Angewandte Chemie International Edition, 50(2),
pp.405-409.
Hershko, A., 2000. Basic medical research award. The ubiquitin system. Nat Med, 6, pp.1073-
1081.
Hershko, A. and Ciechanover, A., 1998. The ubiquitin system. Annual review of
22
biochemistry, 67(1), pp.425-479.
Inglis, K.J., Chereau, D., Brigham, E.F., Chiou, S.S., Schöbel, S., Frigon, N.L., Yu, M.,
Caccavello, R.J., Nelson, S., Motter, R. and Wright, S., 2009. Polo-like kinase 2 (PLK2)
phosphorylates α-synuclein at serine 129 in central nervous system. Journal of Biological
Chemistry, 284(5), pp.2598-2602.
Kalia, L. V., Kalia, S. K., Chau, H., Lozano, A. M., Hyman, B. T., and McLean, P. J., 2011.
Ubiquitinylation of alpha-synuclein by carboxyl terminus Hsp70-interacting protein (CHIP) is
regulated by Bcl-2-associated athanogene 5 (BAG5). PLoS One 6:e14695.
Khalaf, O., Fauvet, B., Oueslati, A., Dikiy, I., Mahul-Mellier, A.L., Ruggeri, F.S., Mbefo, M.K.,
Vercruysse, F., Dietler, G., Lee, S.J. and Eliezer, D., 2014. The H50Q mutation enhances α-
synuclein aggregation, secretion, and toxicity. Journal of Biological Chemistry, 289(32),
pp.21856-21876.
Klaips, C. L., Jayaraj, G. G. & Hartl, F. U., 2017. Pathways of cellular proteostasis in aging and
disease. J. Cell Biol. 217, 51–63.
Kordower, J. H., Freeman, T. B. & Olanow, C. W., 1998. Neuropathology of fetal nigral grafts in
patients with Parkinson’s disease. Mov. Disord. 13, 88–95.
Komander, D. and Rape, M., 2012. The ubiquitin code. Annual review of biochemistry, 81, pp.203-
229.
Krüger, R., Kuhn, W., Müller, T., Woitalla, D., Graeber, M., Kösel, S., Przuntek, H., Epplen, J.T.,
Schols, L. and Riess, O., 1998. AlaSOPro mutation in the gene encoding α-synuclein in Parkinson's
23
disease. Nature genetics, 18(2), pp.106-108.
Krumova, P., Meulmeester, E., Garrido, M., Tirard, M., Hsiao, H.H., Bossis, G., Urlaub, H.,
Zweckstetter, M., Kügler, S., Melchior, F. and Bähr, M., 2011. Sumoylation inhibits α-synuclein
aggregation and toxicity. Journal of Cell Biology, 194(1), pp.49-60.
Lashuel, H.A., Overk, C.R., Oueslati, A. and Masliah, E., 2013. The many faces of α-synuclein:
from structure and toxicity to therapeutic target. Nature Reviews Neuroscience, 14(1), pp.38-48.
Lashuel, H. A., and Eliezer, D., 2016. Semisynthetic and in vitro phosphorylation of alpha-
synuclein at Y39 promotes functional partly helical membrane-bound states resembling those
induced by PD mutations. ACS Chem. Biol. 11, 2428−2437.
Lee, H.J., Suk, J.E., Bae, E.J., Lee, J.H., Paik, S.R. and Lee, S.J., 2008. Assembly-dependent
endocytosis and clearance of extracellular a-synuclein. The international journal of biochemistry
& cell biology, 40(9), pp.1835-1849.
Lee, H.J. and Lee, S.J., 2002. Characterization of cytoplasmic α-synuclein aggregates fibril
formation is tightly linked to the inclusion-forming process in cells. Journal of Biological
Chemistry, 277(50), pp.48976-48983.
Lesage, S., Anheim, M., Letournel, F., Bousset, L., Honoré, A., Rozas, N., Pieri, L., Madiona, K.,
Dürr, A., Melki, R. and Verny, C., 2013. G51D α ‐synuclein mutation causes a novel Parkinsonian–
pyramidal syndrome. Annals of neurology, 73(4), pp.459-471.
Lewis, Y.E., Galesic, A., Levine, P.M., De Leon, C.A., Lamiri, N., Brennan, C.K. and Pratt, M.R.,
2017. O-GlcNAcylation of α-synuclein at serine 87 reduces aggregation without affecting
24
membrane binding. ACS chemical biology, 12(4), pp.1020-1027
Li, W., Bengtson, M. H., Ulbrich, A., Matsuda, A., Reddy, V. A., Orth, A., et al. (2008). Genome-
wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial
E3 that regulates the organelle’s dynamics and signaling. PLoS One 3:e1487.
Liani, E., Eyal, A., Avraham, E., Shemer, R., Szargel, R., Berg, D., Bornemann, A., Riess, O.,
Ross, C.A., Rott, R. and Engelender, S., 2004. Ubiquitylation of synphilin-1 and α-synuclein by
SIAH and its presence in cellular inclusions and Lewy bodies imply a role in Parkinson's
disease. Proceedings of the National Academy of Sciences, 101(15), pp.5500-5505.
Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G.W. and Gong, C.X., 2004. O-GlcNAcylation regulates
phosphorylation of tau: a mechanism involved in Alzheimer's disease. Proceedings of the National
Academy of Sciences, 101(29), pp.10804-10809.
Liu, F., Shi, J., Tanimukai, H., Gu, J., Gu, J., Grundke-Iqbal, I., Iqbal, K. and Gong, C.X., 2009.
Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer's
disease. Brain, 132(7), pp.1820-1832.
Luk, K.C., Song, C., O'Brien, P., Stieber, A., Branch, J.R., Brunden, K.R., Trojanowski, J.Q. and
Lee, V.M.Y., 2009. Exogenous α-synuclein fibrils seed the formation of Lewy body-like
intracellular inclusions in cultured cells. Proceedings of the National Academy of
Sciences, 106(47), pp.20051-20056.
Luk, K.C., Kehm, V.M., Zhang, B., O’Brien, P., Trojanowski, J.Q. and Lee, V.M., 2012.
Intracerebral inoculation of pathological α-synuclein initiates a rapidly progressive
neurodegenerative α-synucleinopathy in mice. Journal of Experimental Medicine, 209(5), pp.975-
25
986.
Ma, M. R., Hu, Z. W., Zhao, Y. F., Chen, Y. X., and Li, Y. M., 2016. Phosphorylation induces
distinct alpha-synuclein strain formation. Sci. Rep. 6, 37130.
Mahul-Mellier, A.L., Burtscher, J., Maharjan, N., Weerens, L., Croisier, M., Kuttler, F., Leleu, M.,
Knott, G.W. and Lashuel, H.A., 2020. The process of Lewy body formation, rather than simply α-
synuclein fibrillization, is one of the major drivers of neurodegeneration. Proceedings of the
National Academy of Sciences, 117(9), pp.4971-4982.
Marotta, N.P., Lin, Y.H., Lewis, Y.E., Ambroso, M.R., Zaro, B.W., Roth, M.T., Arnold, D.B.,
Langen, R. and Pratt, M.R., 2015. O-GlcNAc modification blocks the aggregation and toxicity of
the protein α-synuclein associated with Parkinson's disease. Nature chemistry, 7(11), pp.913-920.
Matunis, M.J., Coutavas, E. and Blobel, G., 1996. A novel ubiquitin-like modification modulates
the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the
nuclear pore complex. The Journal of cell biology, 135(6), pp.1457-1470.
McNaught, K. S., Belizaire, R., Isacson, O., Jenner, P., and Olanow, C. W. (2003). Altered
proteasomal function in sporadic Parkinson’s disease. Exp. Neurol. 179, 38–46.
McNaught, K. S., Belizaire, R., Jenner, P., Olanow, C. W., and Isacson, O., 2002. Selective loss
of 20S proteasome alpha-subunits in the substantia nigra pars compacta in Parkinson’s
disease. Neurosci. Lett. 326, 155–158.
McNaught, K. S., and Jenner, P., 2001. Proteasomal function is impaired in substantia nigra in
Parkinson’s disease. Neurosci. Lett. 297, 191–194.
26
McNaught, K. S., Olanow, C. W., Halliwell, B., Isacson, O., and Jenner, P., 2001. Failure of the
ubiquitin-proteasome system in Parkinson’s disease. Nat. Rev. Neurosci. 2, 589–594.
Meier, F., Abeywardana, T., Dhall, A., Marotta, N.P., Varkey, J., Langen, R., Chatterjee, C. and
Pratt, M.R., 2012. Semisynthetic, site-specific ubiquitin modification of α-synuclein reveals
differential effects on aggregation. Journal of the American Chemical Society, 134(12), pp.5468-
5471.
Mevissen, T. E. T., and Komander, D., 2017. Mechanisms of deubiquitinase specificity and
regulation. Annu. Rev. Biochem. 86, 159–192.
Michelle, C., Vourc’H, P., Mignon, L., and Andres, C. R., 2009. What was the set of ubiquitin and
ubiquitin-like conjugating enzymes in the eukaryote common ancestor? J. Mol. Evol. 68, 616–628.
Morris, M., Knudsen, G.M., Maeda, S., Trinidad, J.C., Ioanoviciu, A., Burlingame, A.L. and
Mucke, L., 2015. Tau post-translational modifications in wild-type and human amyloid precursor
protein transgenic mice. Nature neuroscience, 18(8), pp.1183-1189.
Murphy, D. D., Rueter, S. M., Trojanowski, J. Q., & Lee, V. M. Y., 2000. Synucleins are
developmentally expressed, and α-synuclein regulates the size of the presynaptic vesicular pool in
primary hippocampal neurons. Journal of Neuroscience, 20(9), 3214-3220.
Müller, S., Hoege, C., Pyrowolakis, G. and Jentsch, S., 2001. SUMO, ubiquitin's mysterious
cousin. Nature reviews Molecular cell biology, 2(3), pp.202-210.
Muir, T.W., Sondhi, D. and Cole, P.A., 1998. Expressed protein ligation: a general method for
protein engineering. Proceedings of the National Academy of Sciences, 95(12), pp.6705-6710.
27
Oh, E., Akopian, D. and Rape, M., 2018. Principles of ubiquitin-dependent signaling. Annual
Review of Cell and Developmental Biology, 34, pp.137-162.
Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P.J.
and Haass, C., 2000. Constitutive phosphorylation of the Parkinson's disease associated α-
synuclein. Journal of Biological Chemistry, 275(1), pp.390-397.
Oueslati A, Fournier M, Lashuel HA., 2010. Role of post-translational modifications in
modulating the structure, function and toxicity of alpha-synuclein: Implications for Parkinson’s
disease pathogenesis and therapies. Prog Brain Res 183:115–145.
Paisán-Ruı
́ z, C., Jain, S., Evans, E.W., Gilks, W.P., Simón, J., Van Der Brug, M., De Munain,
A.L., Aparicio, S., Gil, A.M., Khan, N. and Johnson, J., 2004. Cloning of the gene containing
mutations that cause PARK8-linked Parkinson's disease. Neuron, 44(4), pp.595-600
Parkinson, J., 1817. An essay on the shaking palsy. J. Neuropsychiatry Clin. Neurosci. 14, 223–
236.
Pinho, T.S., Correia, S.C., Perry, G., Ambrósio, A.F. and Moreira, P.I., 2019. Diminished O-
GlcNAcylation in Alzheimer's disease is strongly correlated with mitochondrial
anomalies. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1865(8), pp.2048-
2059.
Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide, S.E., Dehejia, A., Dutra, A., Pike, B., Root,
H., Rubenstein, J., Boyer, R. and Stenroos, E.S., 1997. Mutation in the α-synuclein gene identified
in families with Parkinson's disease. science, 276(5321), pp.2045-2047.
28
Pronin, A. N., Morris, A. J., Surguchov, A., and Benovic, J. L., 2000. Synucleins are a novel class
of substrates for G protein-coupled receptor kinases. J. Biol. Chem. 275, 26515–26522.
Robak, L.A., Jansen, I.E., Van Rooij, J., Uitterlinden, A.G., Kraaij, R., Jankovic, J., Heutink, P.
and Shulman, J.M., 2017. Excessive burden of lysosomal storage disorder gene variants in
Parkinson’s disease. Brain, 140(12), pp.3191-3203.
Rott, R., Szargel, R., Shani, V., Hamza, H., Savyon, M., Abd Elghani, F., Bandopadhyay, R. and
Engelender, S., 2017. SUMOylation and ubiquitination reciprocally regulate α-synuclein
degradation and pathological aggregation. Proceedings of the National Academy of
Sciences, 114(50), pp.13176-131811.
Rousseaux, M.W., Revelli, J.P., Vázquez-Vélez, G.E., Kim, J.Y., Craigen, E., Gonzales, K.,
Beckinghausen, J. and Zoghbi, H.Y., 2018. Depleting Trim28 in adult mice is well tolerated and
reduces levels of α-synuclein and tau. Elife, 7, p.e36768.
Sampathu, D.M., Giasson, B.I., Pawlyk, A.C., Trojanowski, J.Q. and Lee, V.M.Y., 2003.
Ubiquitination of α-synuclein is not required for formation of pathological inclusions in α-
synucleinopathies. The American journal of pathology, 163(1), pp.91-100.
Schmid AW, Fauvet B, Moniatte M, Lashuel HA., 2013. Alpha-synuclein post-translational
modifications as potential biomarkers for Parkinson disease and other synucleinopathies. Mol Cell
Proteomics 12:3543–3558.
Shin, Y., Klucken, J., Patterson, C., Hyman, B. T., and McLean, P. J., 2005. The co-chaperone
carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation
decisions between proteasomal and lysosomal pathways. J. Biol. Chem. 280, 23727–23734.
29
Shulman, J. M., De Jager, P. L., and Feany, M. B., 2011. Parkinson’s disease: genetics and
pathogenesis. Annu. Rev. Pathol. 6, 193–222.
Sidransky, E. and Lopez, G., 2012. The link between the GBA gene and parkinsonism. The Lancet
Neurology, 11(11), pp.986-998.
Simon-Sanchez, J., Schulte, C., Bras, J.M., Sharma, M., Gibbs, J.R., Berg, D., Paisan-Ruiz, C.,
Lichtner, P., Scholz, S.W., Hernandez, D.G. and Krüger, R., 2009. Genome-wide association study
reveals genetic risk underlying Parkinson's disease. Nature genetics, 41(12), p.1308.
Smith, W.W., Margolis, R.L., Li, X., Troncoso, J.C., Lee, M.K., Dawson, V.L., Dawson, T.M.,
Iwatsubo, T. and Ross, C.A., 2005. α-Synuclein phosphorylation enhances eosinophilic
cytoplasmic inclusion formation in SH-SY5Y cells. Journal of Neuroscience, 25(23), pp.5544-
5552.
Smolders, S. and Van Broeckhoven, C., 2020. Genetic perspective on the synergistic connection
between vesicular transport, lysosomal and mitochondrial pathways associated with Parkinson’s
disease pathogenesis. Acta Neuropathologica Communications, 8, pp.1-28
Tang, B., Becanovic, K., Desplats, P.A., Spencer, B., Hill, A.M., Connolly, C., Masliah, E.,
Leavitt, B.R. and Thomas, E.A., 2012. Forkhead box protein p1 is a transcriptional repressor of
immune signaling in the CNS: implications for transcriptional dysregulation in Huntington
disease. Human molecular genetics, 21(14), pp.3097-3111.
Tofaris, G. K., Kim, H. T., Hourez, R., Jung, J. W., Kim, K. P., and Goldberg, A. L., 2011.
Ubiquitin ligase NEDD4 promotes alpha-synuclein degradation by the endosomal-lysosomal
pathway. Proc. Natl. Acad. Sci. U.S.A. 108, 17004–17009.
30
Tofaris, G. K., Razzaq, A., Ghetti, B., Lilley, K. S., and Spillantini, M. G., 2003. Ubiquitination
of alpha-synuclein in Lewy bodies is a pathological event not associated with impairment of
proteasome function. J. Biol. Chem. 278, 44405–44411.
Tsigelny, I.F., Sharikov, Y., Wrasidlo, W., Gonzalez, T., Desplats, P.A., Crews, L., Spencer, B.
and Masliah, E., 2012. Role of α ‐synuclein penetration into the membrane in the mechanisms of
oligomer pore formation. The FEBS journal, 279(6), pp.1000-1013.
Tysnes, O. B., and Storstein, A., 2017. Epidemiology of Parkinson’s disease. J. Neural
Transm. 124, 901–905.
Uéda, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D.A., Kondo, J.,
Ihara, Y. and Saitoh, T., 1993. Molecular cloning of cDNA encoding an unrecognized component
of amyloid in Alzheimer disease. Proceedings of the National Academy of Sciences, 90(23),
pp.11282-11286.
Ulmer, T.S., Bax, A., Cole, N.B. and Nussbaum, R.L., 2005. Structure and dynamics of micelle-
bound human α-synuclein. Journal of Biological Chemistry, 280(10), pp.9595-9603.
Uversky, V. N., and Eliezer, D., 2009. Biophysics of Parkinson’s disease: structure and
aggregation of alpha-synuclein. Curr. Protein Pept. Sci. 10, 483–499.
Varshavsky, A., 2012. The ubiquitin system, an immense realm. Annu. Rev. Biochem. 81, 167–
176.
Vasconcelos-Dos-Santos A, de Queiroz RM, da Costa Rodrigues B, Todeschini AR, Dias WB,
2015. Hyperglycemia and aberrant O-GlcNAcylation: contributions to tumor progression. J
31
Bioenerg Biomembr. 50:175–87.
Vila-Perelló, M. and Muir, T.W., 2010. Biological applications of protein splicing. Cell, 143(2),
pp.191-200.
Velez-Pardo, C., Lorenzo-Betancor, O., Jimenez-Del-Rio, M., Moreno, S., Lopera, F., Cornejo-
Olivas, M., Torres, L., Inca-Martinez, M., Mazzetti, P., Cosentino, C. and Yearout, D., 2019. The
distribution and risk effect of GBA variants in a large cohort of PD patients from Colombia and
Peru. Parkinsonism & related disorders, 63, pp.204-208.
Wang, Z., Park, K., Comer, F., Hsieh-Wilson, L.C., Saudek, C.D. and Hart, G.W., 2009. Site-
specific GlcNAcylation of human erythrocyte proteins: potential biomarker (s) for
diabetes. diabetes, 58(2), pp.309-317.
Wang, Z., Udeshi, N.D., O'Malley, M., Shabanowitz, J., Hunt, D.F. and Hart, G.W., 2010.
Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of
chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass
spectrometry. Molecular & Cellular Proteomics, 9(1), pp.153-160.
Wani WY, Chatham JC, Darley-Usmar V, McMahon LL, Zhang J., 2017. O-GlcNAcylation and
neurodegeneration. Brain Res Bull 133:80–87.
Wilhelm, B.G., Mandad, S., Truckenbrodt, S., Kröhnert, K., Schäfer, C., Rammner, B., Koo, S.J.,
Claßen, G.A., Krauss, M., Haucke, V. and Urlaub, H., 2014. Composition of isolated synaptic
boutons reveals the amounts of vesicle trafficking proteins. Science, 344(6187), pp.1023-1028.
32
Wood, S.J., Wypych, J., Steavenson, S., Louis, J.C., Citron, M. and Biere, A.L., 1999. α-Synuclein
fibrillogenesis Is nucleation-dependent Implications for the pathogenesis of Parkinson′ s
disease. Journal of Biological Chemistry, 274(28), pp.19509-19512.
Yates, B., Braschi, B., Gray, K.A., Seal, R.L., Tweedie, S. and Bruford, E.A., 2016. Genenames.
org: the HGNC and VGNC resources in 2017. Nucleic acids research, p.gkw1033.
Yavich, L., Tanila, H., Vepsäläinen, S., & Jäkälä, P., 2004. Role of α-synuclein in presynaptic
dopamine recruitment. Journal of Neuroscience, 24(49), 11165-11170.
Yu, H. and Matouschek, A., 2017. Recognition of client proteins by the proteasome. Annual review
of biophysics, 46, pp.149-173.
Yuzwa SA, Vocadlo DJ., 2014. O-GlcNAc and neurodegeneration: Biochemical mechanisms and
potential roles in Alzheimer’s disease and beyond. Chem Soc Rev 43:6839–6858.
Yuzwa, S.A., Shan, X., Macauley, M.S., Clark, T., Skorobogatko, Y., Vosseller, K. and Vocadlo,
D.J., 2012. Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against
aggregation. Nature chemical biology, 8(4), pp.393-399.
Zarranz, J.J., Alegre, J., Gómez ‐Esteban, J.C., Lezcano, E., Ros, R., Ampuero, I., Vidal, L.,
Hoenicka, J., Rodriguez, O., Atarés, B. and Llorens, V., 2004. The new mutation, E46K, of α ‐
synuclein causes parkinson and Lewy body dementia. Annals of Neurology: Official Journal of
the American Neurological Association and the Child Neurology Society, 55(2), pp.164-173.
Zhao, Q., Xie, Y., Zheng, Y., Jiang, S., Liu, W., Mu, W., Liu, Z., Zhao, Y., Xue, Y. and Ren, J.,
2014. GPS-SUMO: a tool for the prediction of sumoylation sites and SUMO-interaction
33
motifs. Nucleic acids research, 42(W1), pp.W325-W330.
Zhao, X., 2018. SUMO-mediated regulation of nuclear functions and signaling
processes. Molecular cell, 71(3), pp.409-418.
Zimprich, A., Biskup, S., Leitner, P., Lichtner, P., Farrer, M., Lincoln, S., Kachergus, J., Hulihan,
M., Uitti, R.J., Calne, D.B. and Stoessl, A.J., 2004. Mutations in LRRK2 cause autosomal-
dominant parkinsonism with pleomorphic pathology. Neuron, 44(4), pp.601-607.

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Chapter 2. α-Synuclein O-GlcNAcylation alters aggregation and toxicity, revealing certain
residues as potential inhibitors of Parkinson’s disease
Introduction
O-GlcNAcylation (Figure 2-1A) is a widespread intracellular posttranslational modification of
serine and threonine residues. In contrast to most other forms of protein glycosylation, O-
GlcNAcylation involves the sole addition of the monosaccharide N-acetylglucosamine and can be
a dynamic modification through addition by O-GlcNAc transferase (OGT) and subsequent
removal by the enzyme O-GlcNAcase (OGA). While the physiological roles of O-GlcNAcylation
are diverse, a variety of in vivo and biochemical experiments support an important role for this
modification in neurodegenerative diseases. (Yuzma and Vocadlo, 2014; Wani et al., 2017) For
example, tissue-specific knockout of OGT in neurons or even specifically in the forebrain of mice
results in neurodegeneration and neuron death. (O’Donnell et al., 2004; Wang et al., 2016)
Measurement of O-GlcNAc levels in human brains has shown decreased modification in
Alzheimer’s disease compared with healthy controls. (Lie et al., 2004; Aguilar et al., 2017) In
mice, increasing the amounts of O-GlcNAcylation with a small-molecule inhibitor of OGA
(Yuzma et al., 2008) slows neurodegeneration and the formation of tau aggregates in a model of
Alzheimer’s disease. (Yuzma et al., 2012) Additionally, enzymatic O-GlcNAcylation of
recombinant tau by OGT inhibits the aggregation of this protein in vitro. (Yuzma et al., 2014;
Yuzma et al., 2012) Finally, we have previously contributed to this area by using a semisynthetic
strategy to site-specifically O-GlcNAcylate the Parkinson’s disease causing protein α-synuclein at
residues 72 or 87 and demonstrated that these modifications display site-specific differences in
their ability to inhibit protein aggregation. (Marotta et al., 2015; Lewis et al., 2017) Together, these
35
results support a hypothesis where proper O-GlcNAcylation of certain proteins prevents their
aggregation, and loss of this modification is a contributing factor in the development of
neurodegenerative diseases. This has encouraged the application of OGA inhibitors that raise the
brain O-GlcNAcylation levels as potential treatments for Alzheimer’s and Parkinson’s diseases.
(Yuzwa et al., 2008) However, significant fundamental questions remain. For those proteins that
contain multiple O-GlcNAcylation sites, like tau and α-synuclein, which of those sites is most
inhibitory to aggregation and/or toxicity and should receive the largest attention? Does O-GlcNAc
simply stabilize the monomeric state of proteins or can it change the structure of the aggregate,
potentially in ways that are less toxic? What are the levels of O-GlcNAcylation of these proteins
and do they change during the progression of disease? And finally, how can OGA inhibitors best
be tested to explore their potential for the treatment of neurodegenerative diseases? Here, we
present results that make progress toward answering all of these questions in the context of α-
synuclein and Parkinson’s disease.

______________
Paul Levine, Aaron Balana, Mariana Navarro, Cesar De Leon (University of Southern California),
and Anne-Laure Mahul-Mellier and Hilal Lashuel (EPLF) contributed to the work presented in
this chapter.

36

Figure 2-1. O-GlcNAc and α-synuclein. (A) O-GlcNAcylation is the dynamic addition of the
monosaccharide N-acetyl-glucosamine (GlcNAc) to serine and threonine side chains of
intracellular proteins. (B) α-Synuclein is a small protein consisting of three domains: an N-terminal
repeat domain that mediates its interactions with membranes, the central nonamyloid component
(NAC) domain that is responsible for protein aggregation, and a C-terminal acidic domain. Nine
different serines and threonines in α-synuclein have been found to be O-GlcNAcylated, including
the four modifications in bold studied here. (C) α-Synuclein can aggregate to form amyloid fibers
in a two-step process. Monomer can nucleate into oligomers and small fibers that will then stack
additional monomers in an extension step to form lager fiber structures.

α-Synuclein (Figure 2-1B) is a short, 140-amino-acid protein (Lashuel et al., 2013) that exists at
relatively high concentrations ( ∼50 μM) at neuronal synapses. (Wilhelm et al., 2014) This protein
exists as an unstructured monomer in solution but will form an extended α-helix in the presence
of cellular membranes or artificial lipids (Jao et al., 2008; Varkey et al., 2010; Mizuno et al., 2012),
where it can carry out its roles in vesicle trafficking and second-messenger release (Emanuele and
Chieregatti, 2015). During neurodegeneration in Parkinson’s disease and other synucleinopathies,
the protein will form β-sheet rich amyloid aggregates that are toxic to cells (Fink, 2006). This
aggregation process is likely somewhat heterogeneous but broadly consists of two concentration-
37
dependent steps: nucleation and extension (Figure 2-1C) (Wood et al., 1999). Nucleation is the
initiation of aggregate formation from α-synuclein monomers to oligomeric structures and small
fibers while extension involves the rapid “seeding” of further monomer aggregation by these initial
small fibers. Notably, we have shown that seeding capacity plays a key role in cellular toxicity
(Jan et al., 2011; Mahul-Mellier et al., 2015), and this process is increasingly accepted as an
important component of the progressive spread of aggregates throughout the brain in vivo
(Brettschneider et al., 2015; Recasens et al., 2014).
α-Synuclein can be modified by a variety of posttranslational modifications, including O-GlcNAc,
that have the potential to significantly impact its stability, aggregation propensity, and toxicity in
vivo (Queslati et al, 2010; Schmid et al., 2013). A variety of in vivo proteomics studies from mice
and humans have identified up to nine different sites of O-GlcNAc modification on α-synuclein,
including several located within the region of the protein required for aggregation that spans
approximately residues 61–95 (Figure 2-1B). (Wang et al., 2009; Wang et al., 2010; Alfaro et al.,
2012; Morris et al., 2015; Wang et al.. 2017) Unlike some other posttranslational modifications
(e.g., phosphorylation and acetylation), the only way to directly investigate the site-specific effects
of O-GlcNAcylation on protein biophysics and biochemistry is the application of synthetic protein
chemistry to build the protein from the “ground up.” Previously, we have applied a powerful
version of synthetic protein chemistry, expressed protein ligation (EPL) (Muir et al., 1998), which
enables the use of recombinant and synthetic protein fragments, to investigate the consequences
of α-synuclein O-GlcNAcylation at residues 72 or 87. (Marotta et al., 2015; Lewis et al., 2017)
We found that modification of threonine 72 (T72) is more inhibitory than O-GlcNAcylation of
serine 87 (S87) but also that neither modification completely prevents the aggregation of α-
synuclein.
38
Here, we dramatically expand the characterization of α-synuclein O-GlcNAcylation. First, we
synthesized five different modified protein variants bearing O-GlcNAc at threonines T72, T75, or
T81 or serine S87, as well as a triply O-GlcNAcylated protein at T72, T75, and T81. The choice
of these sites was motivated by a variety of proteomic, structural, and biological studies. For
example, the recent solid-state NMR and cryo-EM structures of the α-synuclein fiber place all four
of these modifications in the core of the aggregate. (Tuddle et al., 2016; Guerrrero-Ferreira et al.,
2018; Li et al., 2018) O-GlcNAc modification of T72 and T81 has been found in at least two
independent proteomics experiments (Wang et al., 2010; Alfraro et al., 2012; Wang et al., 2017),
including one from human brain tissue, and T75 and S87 O-GlcNAcylation has been found in mice
and human red blood cells, respectively (Wang et al., 2009; Morris et al., 2015). We have also
shown that S87 is subjected to reciprocal phosphorylation (Paleologou et al., 2010), and this
position is asparagine in rodent α-synuclein, a mutation that has been shown to alter protein
aggregation (Fares M-B et al., 2016: Luk et al., 2016). Using a variety of biophysical and
biochemical techniques we show that individual modification sites uniquely affect the two steps
of α-synuclein aggregation, while having very little effect on membrane binding, with O-
GlcNAcylation of T75 or T81 having the greatest overall inhibitory potential in vitro. Additionally,
multiple O-GlcNAc modifications on α-synuclein endowed it with the ability to inhibit the
aggregation of unmodified protein. We also use our previously established fibril-growth assay in
neuronal cell culture (Mahul-Mellier et al., 2015), which we demonstrated plays a key role in
neuron apoptosis, to demonstrate that modification at T75 could be the most important O-
GlcNAcylation site for slowing the progression of Parkinson’s disease. These results support the
application of OGA inhibitors, although the specific sites of dynamic O-GlcNAcylation still need
to be characterized in vivo. However, robust animal models often rely on the expression of an
39
aggressive aggregation mutant (A53T) of α-synuclein that causes a familial early-onset version of
Parkinson’s disease. Therefore, we also synthesized the triply O-GlcNAcylated analog of this
mutant and found that these modifications also blocked both steps of aggregation of this protein,
indicating that OGA inhibitors could be tested in a range of preclinical models. Finally, we
demonstrate that the most widely used pan-antibodies for O-GlcNAc detection can at best only
visualize modification at T72 but at none of the other positions and that additional modification
prevents even this limited recognition of T72 O-GlcNAcylation. This result could explain why
these modifications have been difficult to detect in the past. Unfortunately, it also demonstrates
that novel approaches will be needed to determine the levels and dynamics of α-synuclein O-
GlcNAcylation not only in cells, but also in healthy and Parkinson’s disease patients. However,
our results further support an important role for O-GlcNAcylation in preventing protein
aggregation in neurodegenerative diseases.

Results
To investigate the effect of site-specific O-GlcNAcylation on α-synuclein, we used EPL to prepare
these proteins semisynthetically from synthetic and recombinant fragments. An EPL-based
strategy requires cysteines residues at any ligation sites between these fragments, but α-synuclein
contains no native cysteine residues. This apparent limitation is actually a feature, however, as it
allows us to introduce cysteine residues in place of any alanine in α-synuclein and then chemically
desulfurize these mutations back to the native sequence at the end of our synthetic route. With
these considerations in mind, we deconstructed α-synuclein into three fragments (Figure 2-2): an
N-terminal protein-thioester synthon that can be obtained from recombinant expression as an in-
frame fusion to an intein from Anabaena variabilis, a synthetic peptide containing a chemically
40
placed O-GlcNAc modification, and a recombinant C-terminal fragment from Escherichia coli.
Using this general strategy, we generated five differentially O- GlcNAcylated α-synuclein proteins
with glycosylation at either threonine 72 (gT72), threonine 75 (gT75), threonine 81 (gT81), serine
87 (gS87), or a triply modified protein at all three threonine residues (gT72,75,81) (Figure 2-2).
At each step of the synthesis, the peptides/proteins were purified by RP-HPLC and their identity
was confirmed by electrospray ionization mass spectrometry, including the final semisynthetic
products (Supplemental Information, Figure 2-S1). Critically, we previously demonstrated that
semisynthetic α-synuclein with no modifications prepared by the same route behaves identically
to recombinant full-length protein prepared in E. coli. (Marotta et al., 2015: Lewis et al., 2017)

Figure 2-2. Synthesis of site-specifically O-GlcNAcylated α-synuclein. Five differentially O-
GlcNAcylated versions of α-synuclein were retrosynthetically deconstructed into a single
glycopeptide prepared by solid-phase peptide synthesis and two recombinant proteins. The C-
terminal fragments were obtained by recombinant expression in E. coli, while the N-terminal
protein thioesters were prepared by recombinant expression as an intein fusion followed by
thiolysis. Notably, the sites of ligation that require a cysteine residue are natively alanine in α-
synuclein, allowing for radical-based desulfurization of the proteins to afford α-synuclein with no
primary sequence mutations.

With these semisynthetic proteins in hand, we first set out to determine if any of the O-GlcNAc
modification events changed the native monomeric and unfolded state of α-synuclein in solution.
Comparison of the five O-GlcNAcylated proteins with recombinant, unmodified α-synuclein using
41
dynamic light scattering gave very similar Stokes radii for all of the proteins and confirmed their
monomeric states (Supplemental Information, Figure S 2-2). Furthermore, circular dichroism
revealed that none of the O-GlcNAc modifications resulted in the formation of any significant
secondary structure (Supplemental Information, Figure S 2-3), indicating that these modifications
would have no effect on soluble α-synuclein in the cytosol. As mentioned above, the ability of α-
synuclein to bind and bend membranes is likely a major physiological function of this protein in
neurons by helping to regulate second messenger release. Accordingly, we tested whether O-
GlcNAc affected the interaction of α-synuclein with the negatively charged vesicles by incubating
the different proteins with a large excess (1:100) of the negatively charged lipid 1-palmitoyl-2-
oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) for 20 min. Subsequent analysis by circular
dichroism readily shows the formation of the expected α-helix (minima around 208 and 222 nm)
by unmodified α-synuclein in each of the experiments (Supplemental Information, Figure S 2-4).
Individual O-GlcNAc modifications at all four sites had essentially no effect on α-helix formation.
The triply modified protein [α-synuclein(gT72,75,81)] displayed a slight decrease in secondary
structure formation, which may not be surprising as it would represent a cumulative effect of the
modifications. This indicates that O-GlcNAcylation may not have a large effect on the normal
biology of α-synuclein in contrast to phosphorylation that has been shown to more strongly alter
the micelle-bound structure (Paleologou et al., 2010). However, these in vitro systems are artificial
and need to be corroborated with cellular assays before any definitive conclusions can be drawn.
We next moved to test if O-GlcNAcylation can block the initiation of α-synuclein aggregation by
inhibiting the nucleation of protein monomers. To perform this analysis, we simultaneously
subjected either unmodified α-synuclein or an O-GlcNAcylated variant to aggregation conditions
(50 μM protein concentration, agitation at 1,000 rpm, and 37 °C) for 7 d. Notably, this
42
concentration is within the measured physiological range of α-synuclein concentrations in vivo
(Wihelm et al., 2014). Reaction aliquots were removed after different lengths of time (0, 48, 96,
and 168 h) and then added to a solution of the dye thioflavin T (ThT), which displays increased
fluorescence upon binding of amyloid aggregates. In all of the experiments, unmodified α-
synuclein showed robust aggregation with a 20- to 30-fold increase in ThT fluorescent signal
(Figure. 2-3A). However, the O-GlcNAcylated proteins showed interesting differences in their
inhibitory capacity (Figure. 2-3A). Consistent with our previous results, α-synuclein(gT72) was a
very poor aggregator while α-synuclein(gS87) did form amyloid fibers but with slightly slower
kinetics. α-Synuclein(gT75) displayed a similar inhibitor capacity compared with α-
synuclein(gT72), while α-synuclein(gT81) was the most inhibitory of all of the individual O-
GlcNAc modifications. Notably, α-synuclein(gT72,75,81) was completely refractory to any
aggregation. To examine the structure of any aggregates that formed in these reactions, we
analyzed the 168-h timepoint using transmission electron microscopy (TEM) and visualized
structures that were highly consistent with the ThT data (Figure. 2-3B and Supplemental
Information, Figure. S 2-5). Unmodified α-synuclein formed long regular fibers, as expected,
while α-synuclein(gS87) formed fibers that were shorter and of a grossly different shape. This was
followed by α-synuclein(gT72) that formed very small and broken fiber segments and α-
synuclein(gT75) and (gT81) that formed rare and highly irregular aggregates. Finally, we did not
observe any aggregates from α-synuclein(gT72,75,81) despite scanning significant areas of the
TEM grid. Importantly, these results are largely consistent with our previously published analysis
of α-synuclein(gT72) and (gS87) but highlight the potentially greater importance of O-
GlcNAcylation at T75 or T81.
We next moved to test if O-GlcNAcylation can block the initiation of α-synuclein aggregation by
43
inhibiting the nucleation of protein monomers. To perform this analysis, we simultaneously
subjected either unmodified α-synuclein or an O-GlcNAcylated variant to aggregation conditions
(50 μM protein concentration, agitation at 1,000 rpm, and 37 °C) for 7 d. Notably, this
concentration is within the measured physiological range of α-synuclein concentrations in vivo
(Wilhelm et al., 2014). Reaction aliquots were removed after different lengths of time (0, 48, 96,
and 168 h) and then added to a solution of the dye thioflavin T (ThT), which displays increased
fluorescence upon binding of amyloid aggregates. In all of the experiments, unmodified α-
synuclein showed robust aggregation with a 20- to 30-fold increase in ThT fluorescent signal
(Figure. 2-3A). However, the O-GlcNAcylated proteins showed interesting differences in their
inhibitory capacity (Figure. 2-3A). Consistent with our previous results, α-synuclein(gT72) was a
very poor aggregator while α-synuclein(gS87) did form amyloid fibers but with slightly slower
kinetics. α-Synuclein(gT75) displayed a similar inhibitor capacity compared with α-
synuclein(gT72), while α-synuclein(gT81) was the most inhibitory of all of the individual O-
GlcNAc modifications. Notably, α-synuclein(gT72,75,81) was completely refractory to any
aggregation. To examine the structure of any aggregates that formed in these reactions, we
analyzed the 168-h timepoint using transmission electron microscopy (TEM) and visualized
structures that were highly consistent with the ThT data (Figure. 2-3B and Supplemental
Information, Figure S 2-5). Unmodified α-synuclein formed long regular fibers, as expected, while
α-synuclein(gS87) formed fibers that were shorter and of a grossly different shape. This was
followed by α-synuclein(gT72) that formed very small and broken fiber segments and α-
synuclein(gT75) and (gT81) that formed rare and highly irregular aggregates. Finally, we did not
observe any aggregates from α-synuclein(gT72,75,81) despite scanning significant areas of the
TEM grid. Importantly, these results are largely consistent with our previously published analysis
44
of α-synuclein(gT72) and (gS87) but highlight the potentially greater importance of O-
GlcNAcylation at T75 or T81.

Figure 2-3. O-GlcNAcylation alters the nucleation step of α-synuclein aggregation in a site-
specific manner. (A) O-GlcNAc largely inhibits the aggregation of α-synuclein monomers into
fibers. Unmodified α-synuclein or the indicated O-GlcNAcylated proteins (50 μM) were subjected
to aggregation conditions (agitation at 37 °C). After different lengths of time, aliquots were
removed and analyzed by ThT fluorescence (λex = 450 nm, λem = 482 nm). The y axis shows fold
change in fluorescence compared with the same conditions at t = 0 h. Results are mean ± SEM of
three experimental replicates. (B) The same reactions were analyzed by TEM after 168 h. (C) PK
digestion reveals site-specific differences imposed by O-GlcNAc on fiber stability and structure.
After 168 h, aggregation reactions were subjected to the indicated concentrations of PK for 30 min
before separation by SDS/PAGE and visualization by Coomassie staining. The persistence of full-
length protein correlates with the stability of the aggregate while the banding pattern indicates
differences in the fiber structure. These results are indicative of at least three different experiments.

To evaluate qualitative structural differences between different α-synuclein aggregates proteinase-
K (PK) digestion has regularly been employed. PK displays broad selectivity in the α-synuclein
primary sequence and will essentially completely degrade the unfolded protein. However, when
aggregates (either fibers or oligomers) are formed, they will inhibit the accessibility of the
aggregated region to PK, resulting in stabilized fragments that can be visualized by SDS/PAGE
and staining. This type of analysis, although operating at a very low resolution level, can provide
key pieces of information. The PK digestion is performed on the entire aggregation reaction that
45
can contain a mixture of fibers, oligomers, and monomers, depending on the α-synuclein variant
used. Therefore, the degradation of full-length α-synuclein with increasing amounts of PK gives
qualitative insight into the amounts of aggregates formed and corresponding stability of those
aggregates. The banding pattern of the partially digested fragments of α-synuclein also allows for
a relative comparison of any differences in the core of the aggregate between different α-synuclein
variants. Accordingly, we aggregated unmodified α-synuclein and the five different O-
GlcNAcylated proteins for 168 h, followed by treatment of the entire reaction with increasing
amounts of PK for 30 min. The protein products were then separated by SDS/PAGE and visualized
by Coomassie staining (Figure. 2-3C). The stability of full-length α-synuclein in this experiment
nicely matched our ThT and TEM data. Not surprisingly, unmodified protein showed the greatest
stability, while triply O-GlcNAcylated protein was completely lost even at the lowest amounts of
PK. Importantly, the complete degradation of α-synuclein(gT72,75,81) indicates that O-
GlcNAcylation itself does not inhibit the PK digestion. Of the singly O-GlcNAc modified proteins,
full-length α-synuclein(gS87) was as stable, if not more, than unmodified protein. This was
followed by α-synuclein(gT72), and finally α-synuclein(gT75) and (gT81). Notably, the banding
pattern of all of the O-GlcNAcylated proteins differed from unmodified α-synuclein, which
showed the expected pattern of five major bands based on previous publications (Luk et al., 2016).
In contrast, all of the individually O-GlcNAc modified proteins had at most three bands of differing
stability and molecular weights. α-Synuclein(gT72), (gT75), and (gS87) all display a band of ∼12
kDa that likely corresponds closely to the prominent degradation product of unmodified protein.
An additional prominent band at a slightly higher molecular weight is clearly visible from α-
synuclein(gS87) aggregates. PK digestion of α-synuclein(gT81) results in a different pattern of
bands with a notable band at a uniquely low molecular weight. Finally, triply O-GlcNAcylated
46
protein formed only one band that was essentially totally destroyed upon increased PK treatment,
further demonstrating that this protein does not form any aggregates. Again, these PK results are
very consistent with our ThT and TEM analysis.
Together, our ThT, TEM, and PK results demonstrate that α-synuclein O-GlcNAcylation has a
generally inhibitory effect on the aggregation of α-synuclein monomers in solution, the nucleation
step of the process. Additionally, they demonstrate that when aggregates do form, the site-specific
nature of the O-GlcNAc modification can “force” the protein into aggregate structures that differ
from unmodified protein.
Next, we tested the possibility that O-GlcNAcylated α-synuclein would inhibit the aggregation of
unmodified protein when they are both present in the same reaction. We previously found that this
is not the case with the single modification of α-synuclein(gT72) (Marotta et al., 2015). Given this,
combined with the fact that the triply modified protein completely prevents any fiber formation,
we chose to focus on α-synuclein(gT72,75,81). Specifically, we subjected unmodified α-synuclein
at either 25 or 50 μM concentrations or a one-to-one mixture of α-synuclein and α-
synuclein(gT72,75,81) at a total concentration of 50 μM to aggregation conditions. As expected,
analysis by ThT fluorescence and TEM shows that unmodified α-synuclein at 25 μM forms fibers
more slowly than the corresponding 50 μM reaction conditions (Supplemental Information, Figure
S 2-6). Intriguingly, the one-to-one mixture of modified and unmodified α-synuclein yielded the
least aggregation, indicating that higher levels of O-GlcNAcylation on individual α-synuclein
proteins could act substoichiometrically to slow fiber formation.
We then examined whether O-GlcNAcylation can inhibit the extension step of protein aggregation
that appears to play an important role in the spread of Parkinson’s disease pathology and
neurodegeneration in the brain. First, we generated unmodified α-synuclein fibers by subjecting
47
the corresponding monomeric protein (50 μM concentration) to aggregation conditions for 7 d.
After this time, the mature aggregates were separated from any remaining monomer by
centrifugation and then subjected to tip sonication to generate a high concentration of seeds, or
preformed fibers (PFFs), that have the potential to be extended by additional monomeric α-
synuclein. These seeds were then added to either buffer, additional unmodified protein, or the
different O-GlcNAcylated α-synuclein variants (25 μM monomeric protein concentration, 20%
PFFs by weight based on monomeric protein) and the resulting mixtures agitated for 48 h. After
different lengths of time, aliquots were removed and analyzed by ThT fluorescence (Figure. 2-
4A). As expected, the PFFs added to buffer showed no increase in ThT signal, while their addition
to unmodified α-synuclein resulted in a 30- to 50-fold increase in fluorescence by the termination
of the assay, resulting from the extension of the seeds. When the PFFs were aggregated with the
triply modified protein, α-synuclein(gT72,75,81), the extension into larger aggregates was
completely blocked. Each of the individually O-GlcNAcylated proteins also inhibited the
extension reaction to different extents. Notably, however, the inhibitory capacity differed from the
trend seen in the nucleation reactions above. Consistent with our previous results (Marotta et al.,
2015), α-synuclein(gT72) increases the lag phase of aggregation over 48 h, but not the plateau of
ThT fluorescence. The next least inhibitory modification at S87 more dramatically increased the
lag phase of aggregation and resulted in a plateau ∼50% of the ThT fluorescence compared with
unmodified monomer at the conclusion of the assay. Finally, α-synuclein(gT75) and (gT81) were
more potent and very similar in both the increase in aggregation lag time and lower plateau levels.
Gratifyingly, visualization of the aggregates formed after 48 h by TEM confirmed our ThT results
(Figure. 2-4B and Supplemental Information, Figure. S 2-7). As expected, long fibers were easily
visible from PFFs extended with either unmodified protein or α-synuclein(gT72), while the fibers
48
formed after α-synuclein(gS87) treatment are a combination of long fibers and short “plate-like”
oligomers (Supplemental Information, Figure S 2-8). α-Synuclein(gT81) formed a combination of
irregular fiber-like structures as well as the structures similar to the oligomers formed by α-
synuclein(gS87) (Supplemental Information, Figure S 2-8). Finally, the structures formed from α-
synuclein(gT75) or (gT72,75,81) appear to be at best PFFs surrounded by amorphous protein
aggregates (gT75) or no material at all (gT72,75,81). These findings suggest that this triple-
modification pattern alters the structures that monomeric α-synuclein can adopt and makes it
incompatible or incapable of adopting a confirmation that is aligned with that of α-synuclein at the
ends of the growing fibrils. To evaluate the structure and stability of these aggregates, we again
employed PK digestion (Supplemental Information, Figure S 2-9). Interestingly, all of the proteins
showed a similar digestion pattern, indicating that unmodified PFFs template and control the
structure of the fibers in the extension reaction. However, the different O-GlcNAcylation sites
again showed different amounts of stable fibers, highly consistent with our other data. These
results show that O-GlcNAcylation at certain sites on α-synuclein is capable of inhibiting the
extension of unmodified PFFs. Notably, the PK digestion suggests that any fibers formed during
this extension reaction are determined by the confirmation of the unmodified PFFs. For certain O-
GlcNAcylation sites, gT81 and gS87, this inhibition potentially promotes the formation of
oligomers, and unmodified oligomers have previously been shown to be toxic (Buell et al., 2014).

49

Figure 2-4. Most, but not all, O-GlcNAcylation sites inhibit the extension step of α-synuclein
aggregation. (A) PFFs were formed by aggregation unmodified α-synuclein (50 μM) for 168 h,
followed by sonication. PFFs were then added to either buffer, additional unmodified α-synuclein,
or the indicated O-GlcNAc modified proteins (25 μM monomeric protein concentration, 20%
PFFs). These mixtures were subjected to aggregation conditions (agitation at 37 °C) and aliquots
were removed at the indicated times for analysis by ThT fluorescence (λex = 450 nm, λem = 482
nm). The y axis shows fold change in fluorescence compared with PFFs only at t = 0 h. Results
are mean ± SEM of three experimental replicates. Aggregation of α-synuclein(gT72) and (gS87)
were performed simultaneously and compared with the same unmodified protein control.
Likewise, α-synuclein(gT75) and (gT871) aggregation reactions were performed simultaneously.
(B) The same reactions were analyzed by TEM after 168 h.

Next, we explored whether O-GlcNAc modifications reduce the toxicity of exogenous α-synuclein
administered to neurons in culture. While the initial steps of α-synuclein aggregation are thought
to be largely intracellular, a causative role for extracellular α-synuclein in the progression of
Parkinson’s disease has been established in cell culture and in vivo (Luk et al., 2012; Recasens et
al., 2014). For example, intracerebral injection of PFFs from recombinant α-synuclein causes
progressive neurodegeneration and seeds additional aggregation in mice (Luk et al., 2012).
Critically, these results also allow the toxicity of different recombinant and synthetic variants to
be tested in a physiologically relevant fashion. Recently, we established an assay in neuronal
culture that uses a combination of PFFs and α-synuclein monomer to monitor the toxicity
associated with overall amyloid formation (Mahul-Mellier et al, 2015). Importantly, we showed
that this process at the neuronal membranes results in increased apoptosis of neurons compared
with treatment with PFFs alone. We chose to employ this assay here, as it would more likely reflect
50
the endogenous biology where combinations of unmodified and O-GlcNAcylated α-synuclein are
likely to coexist. Additionally, it tests the potential for O-GlcNAcylated protein to slow the spread
of aggregates and the progression of disease. Accordingly, hippocampal neurons were isolated
from the brains of P0 mouse pups and cultured for 14 d. At this time, the neurons were treated with
monomeric, unmodified α-synuclein or the different glycosylated variants for six additional days.
The integrity of the cellular membranes was then determined using both lactate dehydrogenase
(LDH) release and the SYTOX Green fluorescence, and neuron apoptosis was measured by
visualizing caspase-3 activity (Supplemental Information, Figure S 2-10). As expected from our
previous experiments, the unmodified monomer induced no detectable toxicity, which was
replicated by all of the O-GlcNAcylated variants.
Simultaneously, we also treated neurons with either unmodified PFFs alone or in combination with
the different monomeric proteins (11% PFFs and 89% monomer by weight based on monomeric
protein) and assayed cellular toxicity using the same assays (Figure. 2-5A). As expected, PFFs
exhibited some toxicity that was increased upon the cotreatment of PFFs and unmodified
monomer. Again, we observed interesting site-specific differences for the different O-
GlcNAcylated proteins. In agreement with our in vitro aggregation reactions (Figure. 2-4),
cotreatment of PFFs with a-synuclein(gT75) or (gT72,75,81) resulted in significantly less toxicity
compared with PFFs plus unmodified monomer and similar to PFFs alone. Additionally, α-
synuclein(gT81) and α-synuclein(gS87), which both formed oligomers (Figure. 2-
4 and Supplemental Information, Figure S 2-8) showed increased toxicity compared with PFFs
alone (P < 0.05–0.01, one-way ANOVA test followed by Dunnett test). These results are consistent
with these oligomers being toxic to neurons. However, it is also possible that α-synuclein
aggregation in the presence of membranes is different from the same reaction in solution, as it
51
requires the PFFs and α-synuclein monomers to be accommodated on the membrane in an
orientation that enables PFF extension (Ysselstein et al., 2015). Finally, we observed no increase
in toxicity upon cotreatment of PFFs and α-synuclein(gT72), despite its ability to form mature
fibers in our in vitro extension assay (Figure. 2–4). Again, this observation could reflect
membrane-associated differences in α-synuclein aggregation.

52

Figure 2-5. O-GlcNAcylation site-specifically inhibits neuronal toxicity in a membrane-based
extension model. (A) Hippocampal neurons were plated in 96-well plates and treated with either
Tris buffer, unmodified α-synuclein PFFs, or these PFFs plus the indicated monomeric proteins.
After 6 d, compromised cellular membranes were measured using SYTOX Green or LDH release,
and cell death was measured using caspase-3 activation. In the case of STYOX Green and LDH
release, cell fixation [paraformaldehyde (PFA)] and cell lysate were used as positive controls. Data
shown represent the mean ± SEM of three independent biological experiments, each performed in
triplicate. Statistical significance was determined using a one-way ANOVA test followed by
Dunnett test (PFFs plus unmodified α-synuclein versus PFFs plus O-GlcNAcylated proteins). N.S.,
not significant. (B) Hippocampal primary neurons were plated on coverslips and treated with PFFs
in the absence or in the presence of the indicated monomeric proteins. At the indicated time, the
neurons were washed three times before fixation and stained using a total α-synuclein antibody
(epitope 1–20). Neurons were counterstained by MAP2, a specific neuronal marker (green), and
the nucleus were stained by DAPI. White arrows show the deposition of the PFFs at the outer
plasma membrane. Asterisks indicate a growing aggregate at the cell plasma membrane. The data
are consistent between two biological replicates.

To better understand the toxicity of α-synuclein(gT72) and (gT81), we next investigated the
aggregation of these proteins on the neurons by immunocytochemistry (ICC). Primary neurons
were again treated with PFFs alone or in combination with unmodified α-synuclein, α-
53
synuclein(gT72), or (gT81) for up to 6 d (Figure. 2-5B). As previously shown (Jan et al., 2011),
the addition of PFFs alone resulted in the formation of a thick layer of α-synuclein at the cell
plasma membrane that is visible in as little as 6 h and relatively consistent over 6 d (Figure. 2-5B,
indicated by the white arrows). Also in line with our published results, the addition of a mixture
of PFFs and unmodified α-synuclein monomers resulted in the formation of an entangled network
of α-synuclein aggregates that grew over time (Figure. 2-5B, indicated by the white asterisks). In
this model, α-synuclein PFFs that bind to the extracellular face of the plasma membrane serve as
seeds for the aggregation of the additional monomeric protein, leading to the formation of formed
toxic fibrils that could in turn serve as secondary nucleation sites for the formation of highly toxic
oligomeric and fibrillar species (Mahul-Mellier et al., 2015; Buell et al., 2014; Cohen et al., 2013;
Jeong et al., 2013). Treatment with PFFs and α-synuclein(gT72) monomers also led to the
deposition of fibrils at the plasma membrane and promoted the growth of large aggregates over
time (Figure. 2-5B, white asterisks). These data are largely in line with our in vitro seeding assay
showing that O-GlcNAcylation at T72 does not prevent the extension of unmodified PFFs (Figure.
2-4). However, these aggregates are different in shape and appear less “compact” and uniform
compared with the analogous treatment with unmodified monomers, potentially explaining their
reduced toxicity. Finally, incubation with PFFs and α-synuclein(gT81) was largely
indistinguishable from treatment with PFFs alone (Figure. 2-5B, white arrows). This result
corroborates our in vitro finding that gT81 inhibits the extension of PFFs, resulting in the formation
of irregular fibril-like structures and oligomers (Figure 2-4 and Supplemental Information, Figure
S 2-8). Based on all of our data, we believe that the mixture of PFFs and α-synuclein(gT81)
monomers promotes the formation of toxic oligomers instead of fibers, which could be driving the
toxicity in neurons and is an important topic of future study.
54
All of the above data suggest that increasing the levels of α-synuclein O-GlcNAcylation in the
brain with an OGA inhibitor might slow the progression of Parkinson’s disease. Unfortunately,
the vast majority of Parkinson’s cases are sporadic and undetected until the presentation of
symptoms that are thought to arise from an already significant burden of protein aggregates.
Therefore, it is not necessarily surprising that any treatment strategy aimed at inhibiting further α-
synuclein aggregation would be minimally efficacious. Additionally, genetic mouse models of
Parkinson’s disease built around wild-type α-synuclein have yielded variable results (Visanji et
al., 2016). Importantly, there are several mutations in α-synuclein that similarly cause early-onset
Parkinson’s disease. We chose to focus on the most well-characterized of these mutations, alanine
53 to threonine (A53T) (Polymeropoulos et al., 1997). α-Synuclein(A53T) forms aggregates more
rapidly than wild-type protein in vitro and causes Parkinson’s symptoms in patients in their 40s,
and has been used to create mouse models that give some clinical phenotypes. Accordingly, we
first prepared the triply glycosylated version of this mutant protein, α-synuclein(A53T,
gT72,75,81), using the same synthetic scheme that we employed for the wild-type proteins
(Supplemental Information, Figure S 2-11). Consistent with our results on α-
synuclein(gT72,75,81), O-GlcNAcylation of α-synuclein(A53T) did not affect the unstructured,
monomeric state of the protein and only had a small effect on its micelle-bound structure
(Supplemental Information, Figure S 2-12). In contrast, these three O-GlcNAc modifications were
again able to completely block both the nucleation and extension steps of α-synuclein(A53T)
aggregation, as determined by ThT fluorescence, TEM, and PK cleavage (Figure. 2-6). These
results support the application of OGA inhibitors in α-synuclein(A53T) expressing mice to begin
to examine the potential for O-GlcNAcylation to slow the progression of Parkinson’s disease
symptoms in these models.
55

Figure 2-6. O-GlcNAcylation can inhibit both steps in the aggregation of an early-onset
Parkinson’s disease mutant (A53T) of α-synuclein. (A) Unmodified α-synuclein(A53T) or the
triply O-GlcNAcylated protein, α-synuclein(A53T,gT72,75,81) (50 μM) were subjected to
aggregation conditions (agitation at 37 °C). After different lengths of time, aliquots were removed
and analyzed by ThT fluorescence (λex = 450 nm, λem = 482 nm). The y axis shows fold change
in fluorescence compared with the same conditions at t = 0 h. Results are mean ± SEM of three
experimental replicates. (B) The same reactions were analyzed by TEM after 168 h. (C) After 168
h, aggregation reactions were subjected to the indicated concentrations of PK for 30 min before
separation by SDS/PAGE and visualization by Coomassie staining. Unmodified, wild-type α-
synuclein is added for comparison. (D) PFFs were formed by aggregation unmodified α-
synuclein(A53T) (50 μM) for 168 h, followed by sonication. PFFs were then added to either buffer,
additional unmodified α-synuclein(A53T), or α-synuclein(A53T,gT72,75,81) (25 μM monomeric
protein concentration, 20% PFFs). These mixtures were subjected to aggregation conditions
(agitation at 37 °C) and aliquots were removed at the indicated times for analysis by ThT
fluorescence (λex = 450 nm, λem = 482 nm). The y axis shows fold change in fluorescence
compared with PFFs only at t = 0 h. Results are mean ± SEM of three experimental replicates. (E)
The same reactions were analyzed by TEM after 168 h.

56
Discussion
Here, we used synthetic protein chemistry to prepare a small panel of differentially O-
GlcNAcylated α-synuclein proteins, which are otherwise inaccessible via standard protein
expression systems, and determined how they affect protein aggregation and toxicity. More
specifically, we found that modification of T72, T75, or T81 strongly inhibits the nucleation step
of aggregation, but O-GlcNAc at S87 only slightly slows the kinetics of this process (Figure. 2-3).
Additionally, all four modifications, including at S87, significantly alter the structure of the
aggregates that do form (Figure. 2-3C). Interestingly, O-GlcNAcylation of T75, T81, or S87
inhibits the extension of PFFs formed from unmodified α-synuclein, but modification at T72 does
not (Figure. 2-4). However, O-GlcNAcylation of T81 or S87 promotes the formation of plate-like
oligomers (Supplemental Information, Figure S 2-8), while modification at T75 does not. These
data on T72 and S87 O-GlcNAcylation are largely consistent with our previous studies (Marotta
et al., 2015, Lewis et al., 2017). Our prior analysis of T72 O-GlcNAcylation showed stronger
inhibition of aggregation by this modification. However, our current aggregation reaction setup is
better temperature controlled, which we believe explains these differences by more efficiently
promoting aggregation. The triply glycosylated material was completely refractory to both steps
of aggregation in vitro (Figures. 2-3 and 2-4). Additionally, this higher level of modification was
able to inhibit the aggregation of unmodified protein in a coaggregation experiment (Supplemental
Information, Figure S 2-6).
Data from the structural models of the α-synuclein fiber can be used to explain many of our
observations (Tuttle et al., 2016; Guerrero-Ferreira et al., 2018; Li et al., 2018). While all of the
modification sites that we analyzed here are in the core of the aggregate, T72 faces toward a portion
of the fiber (residues 51–67) that was more flexible in the NMR, providing one explanation as to
57
why it can be more easily accommodated into a growing fiber in the extension step of aggregation.
S87 lies toward the end of the aggregate core, approximately residues 61–95 as determined by
NMR and EPR spectroscopy and cryo-EM. (Tuttle et al., 2016; Guerrero-Ferreira et al., 2018; Li
et al., 2018; Chen et al., 2007; Vilar et al., 2008) This potentially explains how O-GlcNAcylation
at this site alters the structure of the aggregate core as visualized by PK digestion. Based again on
the structural models, we hypothesize that modification of S87 “shrinks” the core of the fiber by
preventing the participation of the last 10–15 amino acids, although other scenarios are certainly
possible. In either case, it is consistent with experiments from the Lee laboratory showing that
mutation of S87 to asparagine, the native residue of mouse α-synuclein, also results in changes to
the aggregate structure and toxicity (Luk et al., 2016); however, the changes induced by O-GlcNAc
are different from those imparted by this S87N mutation. Interestingly, this is one of only a few
examples that we are aware of where O-GlcNAc directly alters the conformation of a protein, in
this case an amyloid, instead of simply changing protein–protein interactions. The inhibition of
unmodified α-synuclein by α-synuclein(gT72,75,81) is very interesting and generates the
possibility that substoichiometric O-GlcNAcylation may have a significant effect on fiber
formation. We do not know the mechanism of this inhibition, but it could arise from the “capping”
of small fibers by α-synuclein(gT72,75,81) that prevents further extension. Alternatively,
unmodified protein and α-synuclein(gT72,75,81) could participate in the formation of small
complexes that are aggregation incompetent. Elucidating this exact mechanism with fluorescently
labeled semisynthetic proteins is something that we are keen to explore in the future.
When we next tested the consequences of O-GlcNAcylation on neuronal toxicity in an extension
model of aggregation, we obtained results that are largely consistent with our in vitro aggregation
and structural studies (Figures. 2-4 and 2-5). Both α-synuclein(gT75) and α-
58
synuclein(gT72,75,81) were essentially harmless in the toxicity assays (Figure. 2-5A). In contrast,
α-synuclein(gT81) and α-synuclein(gS87) that form oligomeric species when added to PFFs
(Figure. 2-4 and Supplemental Information, Figure S 2-8) were toxic to cells (Figure. 2-5A). This
result is not necessarily surprising, as oligomers are known to be highly toxic. In support of this
model, we used ICC to directly observe this extension reaction on neurons in culture, and we did
not find any extension of the PFFs by α-synuclein(gT81) into large extracellular aggregates
(Figure. 2-5B). As noted above, our results with α-synuclein(gT72) are somewhat divergent, as
this protein does not inhibit the extension of PFFs in vitro (Figure. 2-4) but does not induce toxicity
in cultured neurons (Figure. 2-5A). Analysis by ICC potentially explains this difference. The
addition of PFFs and α-synuclein(gT72) results in the formation of large aggregates at the cell
membrane, but they appear different in their shape and compactness compared with the
unmodified monomer control (Figure. 2-5B). This might be explained by the fundamental
differences in α-synuclein aggregation in the absence or presence of cellular membranes
(Ysselstein et al., 2015). Notably, T72 has been shown to point directly away from the lipid bilayer
when α-synuclein forms its extended α-helix (Jao et al., 2008). This alignment potentially explains
how O-GlcNAcylation at T72 could alter the interaction of α-synuclein monomers with PFFs and
therefore extension and toxicity at membranes. However, there are other potential toxicity
mechanisms at play. For example, it has been consistently observed that upon internalization,
extracellular PFFs can serve as seeds and promote the formation and accumulation of toxic,
intracellular α-synuclein inclusions over time. (Luk et al., 2009; Luk et al., 2012; Volpicelli-Daley
et al., 2011) Therefore, it is possible that the different O-GlcNAc modifications also affect this
process. We believe that it is actually likely that multiple aggregation pathways exist that could be
exerting their toxic effects through multiple cellular mechanisms, and we plan to test the effects of
59
O-GlcNAc on the internalization and seeding of PFFs in neurons in the future.
Together, these results indicate that O-GlcNAcylation of T75 and potentially T72/T81 are the most
important modifications from the perspective of α-synuclein aggregation and potentially
neurodegeneration, as the relative importance of the different aggregation steps and conditions
(i.e., on-membrane versus solution) has yet to be determined. An obvious next step will be the
exploration of these modifications in animal models of Parkinson’s disease, and OGA inhibitors
of the function in the brain are available (Yuzwa et al., 2008; Yuzwa et al., 2012). However, many
Parkinson’s disease animal models that recapitulate α-synuclein aggregation and
neurodegeneration rely on familial mutants of α-synuclein. (Visanji et al., 2016) We show here
that O-GlcNAcylation is also capable of inhibiting the aggregation of one such mutant, α-
synuclein(A53T). We hope that this encourages the targeted testing of OGA inhibitors in these
animal models. Finally, it is still unknown how much α-synuclein is O-GlcNAcylated in healthy
neurons, whether this modification is lower in Parkinson’s disease patients, or even if the amounts
of this modification can be increased in cells or in vivo. To determine if current Western blotting
reagents could be used to explore this question, we attempted to visualize the O-GlcNAc
modifications on our synthetic proteins using the most widely used global O-GlcNAc antibodies
CTD110.6 (Comer et al., 2001) and RL2 (Snow et al., 1987) (Supplemental Information, Figure
S2-13). We found that CTD110.6 did not recognize any of the O-GlcNAc modifications, while
RL2 only weakly binds to modification at T72 and does not recognize the remainder of the singly
O-GlcNAcylated sties. Furthermore, RL2 identification of the T72 modification is blocked by
additional O-GlcNAcylation at T75 and T81. While this result can help explain why α-synuclein
O-GlcNAcylation remained undiscovered until relatively recently with the advent of modern
proteomic ionization techniques, it also means that alternative approaches (i.e., chemoenzymatic
60
detection) will need to be utilized to determine the in vivo relevance of α-synuclein O-
GlcNAcylation in Parkinson’s disease patients and to evaluate animal models, a problem we are
currently exploring.
In summary, our data presented here further support a model where O-GlcNAcylation may inhibit
aggregation and play a protective role in Parkinson’s disease and other protein aggregation
disorders. The process of synuclein aggregation and toxicity is highly complex in Parkinson’s
disease. There are multiple types of aggregation reactions that are all thought to contribute to the
formation of toxic species, including nucleation of synuclein intracellularly, extension of PFFs
both intracellularly and on membranes, secondary nucleation on PFFs, and uptake and seeding of
PFFs into previously healthy cells. Here, we show that several O-GlcNAcylation sites inhibit the
nucleation of aggregation in vitro and that modification of T75 or multiple O-GlcNAc sites can
also block the extension of PFFs in solution. Additionally, these same sites and α-synuclein(gT72)
can block toxicity during PFF extension on cellular membranes. It remains to be seen whether O-
GlcNAc can also affect the uptake and intracellular seeding of monomeric α-synuclein, a question
we are currently exploring.

Material and Methods
General.
All solvents and reagents were purchased from commercial sources (EMD, Sigma-Aldrich, Fluka,
Novagen, etc.) and used without any further purification. Growth media (Luria Bertani-Miller,
Terrific broth - EMD) were prepared, sterilized, stored, and used according to the manufacturer.
Antibiotics were prepared as stock solutions at a working concentration of 1,000x (ampicillin
sodium salt, EMD 100 mg mL-1, kanamycin sulfate, EMD, 50 mg mL-1) and stored at -20 °C.
Reverse phase high performance liquid chromatography (RP-HPLC) was performed using an
61
Agilent Technologies 1200 Series HPLC with Diode Array Detector. Unless otherwise stated the
HPLC buffers used were buffer A: 0.1% TFA in H2O, buffer B: 0.1% TFA, 90% ACN in H2O.
Mass spectra were acquired on an API 150EX (Applied Biosystems/MDS SCIEX).
Plasmid Construction.
A pRK172 construct was generated containing wild-type human α-synuclein inserted into Nde I
and Hind III restriction sites using standard molecular cloning techniques, as has been described
previously1 (citation). The C-terminal fragment of α-synuclein (aa 91-140) was introduced into a
pET42b vector using Nde I and Spe I restriction sites and standard molecular cloning techniques.
The N-terminal fragment of α-synuclein (1-68 or 1-75) was introduced into a modified pTXB1
construct containing the Ava-DnaE N137A intein using NdeI and Bpu10I restriction sites and
standard molecular cloning techniques. The A53T mutant was introduced using QuikChange
(Agilent) site-directed mutagenesis. All plasmids were confirmed by sequencing and detailed
plasmid maps and cloning strategies are available upon request.
Expression of recombinant α-synuclein and α-synuclein(A53T).
BL21(DE3) E. coli (EMD Milipore) were transformed with the pRK172 construct containing wild-
type human α-synuclein of A53T α-synuclein using standard molecular biology techniques. Single
colonies were selected and used to inoculate starter cultures, which were grown at 37 °C with
shaking at 250 rpm for 16 h. Each starter culture was used to inoculate a 300 mL TB-amp culture.
These cultures were grown to an OD600 of 0.6-0.7 at 37 °C with shaking at 250 rpm, and then
expression was induced with IPTG (final concentration: 0.5 mM) at 25 °C with shaking at 250 rpm
for 18 h. Bacteria were harvested by centrifugation (8,000 x g, 30 min, 4 °C), and the cell pellets
were lysed by three freeze thaw cycles, using liquid N2 and a 37 °C water bath. Cell lysates were
resuspended, on ice, in 10 mL (per 300 mL of culture) of lysis buffer (500 mM NaCl, 100 mM
62
Tris, 10 mM β-mercaptoethanol (βME), 1 mM EDTA, pH 8.0). Cell lysates were boiled at 80 °C
for 10 min, allowed to cool down to RT, before the addition of protease inhibitor cocktail (mini
complete EDTA free, Roche). The resulting solution was incubated on ice for 30 min and then
cleared by centrifugation (7,000 x g, 30 min, 4 °C). The pH of the supernatant was adjusted to pH
3.5 with 1 M HCl and the supernatant was incubated on ice for 30 min before centrifuging again
(7,000 x g, 30 min, 4 °C). The resulting supernatant was dialyzed against 3 x 1 L of 1% acetic acid
in water (degassed with N2). The dialyzed protein solution was then characterized and purified by
ESI-MS and RP-HPLC over a C4 semi-preparative column (Higgins Analytical). The yield was
determined by Pierce BCA assay (Thermo Scientific). Purified material was flash frozen in liquid
N2 and lyophilized.
Expression of α-synuclein C-terminal fragment.
BL21(DE3) chemically competent E. coli (EMD Milipore) were transformed with the appropriate
pET42b construct by heat shock and plated on selective LB agar plates containing 50 µg mL-1
kanamycin (LB-kan). Expression and purification was carried out as described above for
recombinant α-synuclein. Expression of α-synuclein N-terminal thioester: BL21(DE3) chemically
competent E. coli (VWR) were transformed with the modified pTXB1 construct by heat shock,
plated on selective LB agar plates containing 100 µg mL-1 ampicillin (LB-amp), and incubated at
37 °C for 16 h. Bacteria were cultured and induced as described above for full-length protein. After
harvesting bacteria by centrifugation (8,000 x g, 30 min, 4 °C), the cell pellet was resuspended on
ice in 10 mL (per 300 mL of TB culture) of cold lysis buffer (50 mM NaH2PO4, 300 mM NaCl,
5 mM imidazole, 2 mM TCEP HCl, pH 8.0) plus protease inhibitor cocktail and lysed by tip
sonication (35% amplitude, 30 sec pulse duration, 30 sec rest for 12 min) while on ice. The crude
cell lysate was cleared by centrifugation (42,000 x g, 30 min, 4 °C) and the supernatant was
63
incubated with a Ni-ATA Agarose resin (Qiagen), which was previously washed with the wash
buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 2 mM TCEP HCl, pH 7.4), and the
mixture was incubated for 1 h with shaking at 4 °C. The matrix was then washed with wash buffer
(~5 column volumes), and the protein was eluted with elution buffer (50 mM NaH2PO4, 300 mM
NaCl, 250 mM imidazole, 2 mM TCEP HCl, pH=7.8). Elution S8 fractions were concentrated by
using spin-column concentrators (Amicon Ultra 3 kDa MW cut-off, Millipore) to a volume of 1
mL and then buffer exchanged into thiolysis buffer (100 mM NaH2PO4, 150 mM NaCl, 1 mM
EDTA, 1 mM TCEP HCl, pH=7.4). Sodium mercaptoethane sulfonate (MESNa) was added to a
final concentration of 250 mM, and the thiolysis reaction was incubated at room temperature to
generate the protein thioester. Upon completion, the thiolysis reaction was purified over a C4 semi-
prep column (Higgins Analytical) and stored as a lyophilized solid. Pure protein thioesters were
characterized by analytical RP-HPLC and ESI-MS.
Solid phase synthesis of thioester peptides.
All solid-phase peptide syntheses were conducted manually using Dawson Dbz AM resin
(Novabiochem) on a 0.1 mmol scale. Commercially available N-Fmoc and side chain protected
amino acids (5 eq, Novabiochem) were activated for 5 min with HBTU (5 eq, Novabiochem) and
DIEA (10 eq, Sigma) and then coupled to the resin for 45 min. Following coupling, the terminal
Fmoc group was removed with 20% v/v piperidine in DMF for 15 min. For the incorporation of
O-GlcNAcylated threonine, pentafluorophenyl (PFP) activated O-GlcNAc Fmoc-threonine was
synthesized and purified as described previously (1). Two equivalents of this amino acid was
incubated with the peptide resin overnight, followed by standard coupling cycles for the remaining
amino acids. When peptide synthesis was completed, O-GlcNAc amino acids containing peptides
were subjected to hydrazine hydrate (80% v/v in MeOH) twice for 45 min to remove the O-acetate
64
groups on the sugar. The Dawson linker was then activated with treatment of paranitrophenyl
chloroformate (5 equiv in CH2Cl2) for 1 h, followed by incubation with excess DIEA (5 equiv in
DMF) for 30 min. Peptides were cleaved from the resin by incubating in cleavage cocktail
(95:2.5:2.5 TFA/H2O/Triisopropylsilane) for 3.5 h at room temperature. The crude peptides were
then diluted ~1/10 in cold diethyl ether and precipitated over night at -80 °C. The resulting
suspension was centrifuged (5,000 x g, 30 min, 4 °C) and the pellet was then resuspended in H2O,
flash frozen, and lyophilized. This crude lyophized material was resuspended in thiolysis buffer
(150 mM NaH2PO4, 150 mM MESNa, pH 7.4) and incubated at room temperature for 2 h before
being purified on C18 semi-preparative column (Higgins Analytical). Purified peptides were
characterized by ESI-MS.
α-Synuclein synthesis.
Purified O-GlcNAc modified peptide thioester (4 mM, 1 equiv) and α-synuclein C-terminal
fragment (2 equiv) were resuspended in ligation buffer (3 M guanidine-HCl, 300 mM phosphate,
30 mM TCEP, 30 mM 4-mercaptophenylacetic acid “MPAA”, pH 7.5), and the resulting solution
was rocked for 8 h at 25 °C. The reaction was monitored by RP-HPLC (0−70 % B over 60 min).
Once complete, methoxyamine HCl salt (150 mM final concentration) was added, and the resulting
solution was incubated for 12 h at rt. The deprotection of thioproline was confirmed by ESI-MS,
resulting in pure α-synuclein fragments (69-140 or 75-140). This product was purified using a C18
semi-prep column (Higgins Analytical) and lyophilized. Subsequently, the purified and
lyophilized product (1 equiv, 2 mM) and N-terminal thioester (2 equiv) were resuspended in the
same ligation buffer as above. The reaction was rocked at 25 °C and monitored by RP-HPLC (0-
70 % B over 60 min). Once the reaction was completed, the product was purified by C4 semi-prep
column (Higgins Analytical) and lyophilized. Radical catalyzed desulfurization was performed in
65
degassed buffer (6 M guanidine-HCl, 300 mM phosphate, 300 mM TCEP, 2.5% v/v ethanethiol,
10% v/v tertbutylthiol, pH 7.0) and the addition of a radical initiator, VA-061 (200 mM in MeOH,
2 mM final concentration). The reaction was incubated at 37 °C with constant agitation for 16 h.
The product was purified and characterized with C4 analytical column (Higgins Analytical) and
ESI-MS.
Circular Dichroism.
All circular dichroism (CD) spectra were taken with a Jasco-J-815 spectrometer at RT. Sample
aliquots were diluted to 7.5 µM with phosphate buffer (10 mM phosphate, pH 7.4) in a 1 mm path
length quartz cuvette and far UV spectra (195 nm-250 nm) were obtained by averaging three scans
with a 50 nm min−1 scanning speed, 1 nm bandwidth, 0.1 nm step size, and data integral speed of
4 sec. The blank buffer readings were subtracted for all samples, and the data were converted into
mean residue ellipticity.
Dynamic light scattering.
Dynamic light scattering data were obtained with Wyatt Technologies Dynastar. All samples were
at t = 0 h of aggregation reactions (50 µM). For all data, an average of 10 scans at 25 °C was
obtained with laser power adjusted to intensity of 2.6E6 counts s−1. To calculate radii, Raleigh
sphere approximation was used.
Circular Dichroism of α-synuclein in the presence of lipids.
S9 All circular dichroism (CD) spectra were collected with Jasco-J-815 spectrometer at RT.
Samples were prepared by mixing 1:100 ratio of protein and lipid mixture and incubated at RT for
20 min. Lipid vesicles were prepared with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-RAC-(1-
glycerol)] (POPG). Dried lipid films were solubilized in 10 mM phosphate buffer at pH 7.4 by
vortexing. All spectra (190−250 nm) were collected with a scan rate of 50 nm min−1, bandwidth
66
of 1 nm, data integration time of 8 s, and 0.1 nm step resolution. Appropriate buffer spectra were
subtracted from the final spectra.
Aggregation reactions.
Recombinant protein or synthetic proteins (gT72, gT75, gT81, gS87, gT72,75,81, A53T, or
A53T,gT7,27,581) were dissolved by bath sonication in reaction buffer (10 mM phosphate, 0.05%
sodium azide, pH 7.4). The solution was centrifuged at 15,000 rpm for 15 min at 4 °C to remove
any debris, and the supernatant was aliquoted into triplicate reactions. The samples were incubated
at 37 °C with constant agitation (1,000 rpm) in a Thermomixer F1.5 (Eppendorf) for 7 days.
Aliquots were taken after different lengths of time for analysis by ThT or TEM.
Thioflavin T fluorescence.
The degree of α-synuclein aggregation was quantified by Thioflavin T fluorescence. Samples were
prepared by diluting samples from aggregation reactions (final α-synuclein concentration = 1.25
µM) in 20 µM Thioflavin T in aggregation reaction buffer (above). Samples were diluted, vortexed
briefly, and then incubated 2 min before analyzing. Spectra were collected using a NanoLog
Spectro-fluorometer (Horiba), λex = 450 nm, ex slit = 4 nm, λem = 482, em slit = 3 nm, 10 mm
path length, integration time = 0.1 sec, 3 averaged accumulations. Data was measured in triplicate
for all aggregation reaction conditions.
Transmission electron microscopy.
A 10 µL droplet from each sample was deposited on formvar coated copper grid (150 mesh,
Electron Microscopy Sciences) and allowed to sit for 5 min. The excess liquid was removed with
filter paper. Grids were then negatively stained for 2 min with 1% uranyl acetate, washed three
times with 1% uranyl acetate, each time removing excess liquid with filter paper. The grids were
desiccated for 24 h in a vacuum desiccator and then imaged using a JOEL JEM-2100F transmission
67
electron microscope operated at 200 kV, 60,000x magnification, and an Orius Pre-GIF CCD.
Proteinase K Digestion.
Ten micrograms of protein from aggregation reactions or five micrograms from extension
reactions were incubated with Proteinase K (Sigma Aldrich P2308) at the indicated concentrations
for 30 min at 37 °C. Reactions were quenched by the addition of sample loading buffer (2% final
SDS concentration) and boiling at 95 °C for 10 min. Digestion products were separated by SDS-
PAGE using precast 12% Bis-Tris gels (Bio-Rad, Criterion XT) with MES running buffer (Bio-
Rad). Bands were visualized with either Coommassie Brilliant Blue (Bio-Rad) for aggregation
reactions or silver stain (Thermo Scientific Pierce Silver Stain Kit) for extension reactions.
Aggregation reactions with pre-formed fibers.
Lyophilized wild-type α-synuclein was resuspended in reaction buffer (10 mM phosphate, 0.05%
sodium azide, pH 7.4) to a concentration of 50 µM. After 10 min of bath sonication, debris was
removed with centrifugation, and the protein was incubated at 37°C under continuous shaking
(1,000 rpm) in an Eppendorf thermomixer for 7 days. This aggregate reaction was then bath
sonicated for 20 min, and subsequently tip-sonicated (8 X 1 sec pulses separated by 1 sec, 20 %
amplitude). Sonicated aggregates were aliquoted into three sets of triplicates. To each set, the equal
volume of either the reaction buffer, unmodified α-synuclein monomer, or O-GlcNAcylated α-
synuclein monomer were added to give a final 1:5 ratio of fibers to monomer. The resulting
mixtures were incubated under continuous shaking (1,000 rpm) at 37 °C in the Eppendorf
thermomixer for indicated times. Aliquots were taken after different lengths of time for analysis
by ThT or TEM.
Preparation and characterization of α-synuclein fibrils for cellular toxicity.
As previously described (2), monomeric α-synuclein resuspended in Tris aggregation solution (50
68
mM Tris, 150 mM NaCl, pH 7.5) was filtered (100 kDa filter) (Millipore, Switzerland) and then
incubated under constant orbital agitation (1000 rpm) (Peqlab, Thriller, Germany) at 37°C. After
five days, α-synuclein Pre-Formed fibrils (PFFs) were sonicated on ice with a fine tip for 5 sec at
amplitude of 40% (Sonics Vibra cell). α-synuclein PFFs were then fully characterized by ThT
assay, SDS-PAGE gel and Coomassie staining and Transmission Electron Microscopy (TEM) (3).
S10
Primary culture of hippocampal neurons.
Pregnant female C57BL/6J Rcc Hsd were purchased from Harlan Laboratories (France) and were
housed at EPFL according to the Swiss legislation and the European Community Council directive
(86/609/EEC). As previously described, primary Hippocampal cultures were prepared from mice
brains from P0 pups (4). The hippocampi were first isolated stereoscopically and neurons were
dissociated by trituration in medium containing papain (20U/mL, Sigma-Aldrich, Switzerland).
The neurons were plated on black 96 well plates (Costar, Switzerland) coated with poly-L-lysine
0.1% w/v in water (Brunschwig, Switzerland). Neurons were cultured for two weeks in Neurobasal
medium containing B27 supplement (Life Technologies, Switzerland), L-glutamine and
penicillin/streptomycin (100U/mL, Life Technologies, Switzerland).
Neuronal toxicity assay.
After 14 days in vitro, the hippocampal neurons were treated as previously described (3) with Tris
buffer (as negative control) or extracellular α-synuclein monomers (at a final concentration of 20
µM) or α-synuclein pre-formed fibrils (PFFs) (at a final concentration of 2µM) or a mixture of
monomeric and fibrillar α-synuclein (respectively 18 µM and 2 µM as final concentration). After
six days of treatment, cell death was quantified using three distinct methods. Following the
manufacturer’s instructions, we first measured the release of lactate dehydrogenase (LDH) from
69
damage cells into the extracellular media using the colorimetric Cytox-Tox 96 Non-radioactive
Assay (Promega). We next measured the plasma membrane breakdown using the vital dye
exclusion method. Briefly, the impermeant Sytox Green dye (SG, Life technology) was added to
the neurons at a final concentration of 330nM. After 15 mins, neurons were washed twice in PBS
and fluorescence (Excitation/Emission : 487 nm/519 nm) was measured using the Tecan infinite
M200 Pro plate reader (Tecan, Switzerland). Finally, apoptotic cell death was confirmed by the
quantification of active Caspase 3 using the SRFLICA® Caspase-3/7 Assay Kit
(ImmunoChemistry technologies, USA) following the kit instructions.
Immunocytochemistry (ICC).
Hippocampal primary neurons were seeded onto coverslips coated with poly-L-lysine (Life
Technologies). After 14 days of differentiation, neurons were treated, for up to 6 days, with
extracellular α-syn fibrils alone or prepared as a mixture with α-synuclein monomers (unmodified
or gT72 or gT81). At the indicated time-point, cells were washed 3 times with PBS and then fixed
in 4% PFA (paraformaldehyde) for 15 min at rt. After blocking with 3% BSA (Bovine Serum
Albumin) in 0.1% Triton X-100 PBS (Phosphate Buffer Saline) (PBS-T) for 30 minutes at RT,
hippocampal primary neurons were incubated, for 2 hours at rt, with primary antibodies: rabbit
anti-α-synuclein (epitope: 1-20). Neurons were counterstained with a chicken anti-MAP2 antibody
(abcam). The cells were rinsed five times in PBS-T and subsequently incubated for one hour with
the secondary anti-rabbit Alexa568 and anti-chicken Alexa488 at a dilution of 1/800 in PBST. The
cells were washed five times in PBST and incubated 30 minutes at rt in DAPI at 2 µg/mL (Sigma-
Aldrich), before mounting in polyvinyl alcohol mounting medium with DABCO (Sigma-Aldrich).
The cells were then examined with confocal laser-scanning microscope (LSM 700, Carl Zeiss
Microscopy, Germany) with a 40X objective and analyzed using Zen software.
70
Western Blotting.
Ten micrograms of protein from aggregation reactions were boiled with sample loading buffer
(2% final SDS concentration) at 95°C for 10 minutes. These were loaded onto precast 4-20% Tris-
Glycine gel (BioRad Criterion TGX) with Tris-Glycine running buffer. The proteins were
transferred to 0.2 um pore size nitrocellulose membrane with Towbin buffer. The transferred
proteins were fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature.
Membranes were then washed 3 times in PBS for 5 minutes each before blocking for 1 hour at
room temperature with OneBlock Western-CL buffer (Genessee Scientific) for RL2 antibody
(Thermo Fisher), 5% BSA in TBST (Cell Signaling Technologies) for CTD110.6 (Cell Signaling
Technologies) or 5% milk in TBST (Cell Signaling Technologies) for syn211 antibody
(Invitrogen). Primary antibodies were added to the blocking buffer at 1:5000 dilution and the
membranes were incubated for 16 hours at 4 °C. The membranes were washed three times in TBST
for 10 minutes each, after which incubated with anti-mouse-HRP secondary antibodies (1:10,000
in blocking buffer) for one hour at room temperature. Membranes were washed again with TBST
three times for 10 minutes each before developing in western blotting substrate (Bio-Rad Clarity
Western ECL).
1. Marotta NP, Cherwien CA, Abeywardana T, Pratt MR (2012) O-GlcNAc Modification
Prevents Peptide-Dependent Acceleration of α-Synuclein Aggregation. ChemBioChem
13(18):2665–2670.
2. Fauvet B, et al. (2012) α-Synuclein in central nervous system and from erythrocytes,
mammalian cells, and Escherichia coli exists predominantly as disordered monomer. J Biol
Chem 287(19): 15345–15364.
3. Mahul-Mellier AL, et al. (2015) Fibril growth and seeding capacity play key roles in α-
71
synucleinmediated apoptotic cell death. Cell Death Differ 22(12):2107–2122.
4. Steiner P, et al. (2002) Modulation of receptor cycling by neuron-enriched endosomal protein
of 21 kD. J Cell Biol 157(7):1197–1209.

Supplemental Information

Figure S 2-1. Characterization of unmodified and O-GlcNAcylated α-synuclein. Analytical
RP-HPLC traces and ESI-MS of the indicated recombinant (unmodified) and synthetic proteins.

72

Figure S 2-2. O-GlcNAc does not effect on the monomeric nature of α-synuclein in solution.
The indicated proteins were analyzed using dynamic light scattering (DLS) at 50 µM
concentration. All preparations showed a single peak with a Stoke’s Radius less than the 10-100
nm range, consistent with monomeric protein.

Figure S 2-3. O-GlcNAc does not induce α-synuclein secondary structure. Circular dichroism
(CD) spectra were collected for freshly dissolved samples of the indicated proteins at 7.5 µM
concentration. The single minimum at approximately 200 nm indicates natively unfolded protein
in all cases.

73

Figure S 2-4. O-GlcNAc has very little effect on the micelle-bound structure of α-synuclein.
The indicated proteins were incubated with an 100-fold excess of POPG vesicles for 20 min and
then analyzed using circular dichroism (CD). Minima at 208 and 222 nm are consistent with α-
helix formation.

Figure S 2-5. Representative TEM images from α-synuclein nucleation reactions. Additional
TEM images collected from the aggregation reactions in Figure 3.

74

Figure S 2-6. α-Synuclein(gT72,75,81) inhibits the aggregation of unmodified protein. a,
Unmodified αsynuclein at either 25 or 50 µM concentration or a 1:1 mixture of unmodified α-
synuclein and α-synuclein(gT72,75,81) (50 µM total concentration) were subjected to aggregation
conditions (agitation at 37 °C). After different lengths of time, aliquots were removed and analyzed
by ThT fluorescence (λex = 450 nm, λem = 482 nm). The y-axis shows fold change in fluorescence
compared with the same conditions at t = 0 h. Results are mean ± s.e.m. of four experimental
replicates. b, The same reactions were analyzed by TEM after 168 h.

Figure S 2-7. Representative TEM images from α-synuclein extension reactions. Additional
TEM images collected from the aggregation reactions in Figure 4. Remaining PFFs are shown
with red arrows.

75

Figure S 2-8. Blow-up TEM images of α-synuclein(gT81) and (gS87) extension reactions.
Plate-like αsynuclein oligomers can be seen in these close-up TEM images.

Figure S 2-9. PK digestion of the α-synuclein extension reactions is consistent with site-
selective inhibition by O-GlcNAc. The extension reactions in Figure 4 were incubated with the
indicated concentrations of PK for 30 min before separation by SDS-PAGE and visualization by
silver staining. The persistence of full-length protein correlates with the stability of the aggregate
while the banding pattern indicates differences in the fiber structure. These results are indicative
of at least 3 different experiments.

76

Figure S 2-10. O-GlcNAcylated α-synuclein monomers are not toxic to primary neurons.
Hippocampal neurons were plated in 96-well plates and treated with either Tris buffer or the
indicated monomeric proteins. After 6 days, compromised cellular membranes were measured
using SYTOX Green or LDH release and cell death was measured using caspase-3 activation. In
the case of STYOX Green and LDH release, cell fixation (PFA) and cell lysate were used as
positive controls. Data shown represent the mean ±s.e.m. of three independent biological
experiments, each performed in triplicate.

77

Figure S 2-11. Characterization of unmodified and O-GlcNAcylated α-synuclein. Analytical
RP-HPLC traces and ESI-MS of the indicated recombinant (unmodified) and synthetic proteins.

Figure S 2-12. O-GlcNAc does not affect the solution confirmation or membrane binding of
α-synuclein(A53T). a, O-GlcNAc does not effect on the monomeric nature of α-synuclein(A53T)
in solution. The indicated proteins were analyzed using dynamic light scattering (DLS) at 50 µM
concentration. Both proteins showed a single peak with a Stoke’s Radius less than the 10-100 nm
range, consistent with monomeric protein. b, O-GlcNAc does not induce α-synuclein(A53T)
secondary structure. Circular dichroism (CD) spectra were collected for freshly dissolved samples
of the indicated proteins at 7.5 µM concentration. The single minimum at approximately 200 nm
indicates natively unfolded protein in all cases. c, O-GlcNAc has very little effect on α-synuclein
membrane-binding. The indicated proteins were incubated with an 100-fold excess of POPG
vesicles for 20 min and then analyzed using circular dichroism (CD). Minima at 208 and 222 nm
are consistent with α-helix formation.

Figure S 2-13. α-Synuclein O-GlcNAcylation is not readily detected by Western blotting with
commonly used anti-O-GlcNAc antibodies. The indicated proteins were separated by SDS-
PAGE, subjected to transfer to a PVDF membrane and Western blotting with either a pan-selective
anti-O-GlcNAc antibody (CDT110.6 or RL2) or an anti-α-synuclein antibody.

78
Chapter Two References

Aguilar, A.L., Hou, X., Wen, L., Wang, P.G. and Wu, P., 2017. A chemoenzymatic histology
method for O-GlcNAc detection. Chembiochem: a European journal of chemical biology, 18(24),
p.2416.
Alfaro, J.F., Gong, C.X., Monroe, M.E., Aldrich, J.T., Clauss, T.R., Purvine, S.O., Wang, Z.,
Camp, D.G., Shabanowitz, J., Stanley, P. and Hart, G.W., 2012. Tandem mass spectrometry
identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc
transferase targets. Proceedings of the National Academy of Sciences, 109(19), pp.7280-7285.
Brettschneider, J., Del Tredici, K., Lee, V.M.Y. and Trojanowski, J.Q., 2015. Spreading of
pathology in neurodegenerative diseases: a focus on human studies. Nature Reviews
Neuroscience, 16(2), pp.109-120.
Buell, A.K., Galvagnion, C., Gaspar, R., Sparr, E., Vendruscolo, M., Knowles, T.P., Linse, S. and
Dobson, C.M., 2014. Solution conditions determine the relative importance of nucleation and
growth processes in α-synuclein aggregation. Proceedings of the National Academy of
Sciences, 111(21), pp.7671-7676.
Chen, M., Margittai, M., Chen, J. and Langen, R., 2007. Investigation of α-synuclein fibril
structure by site-directed spin labeling. Journal of Biological Chemistry, 282(34), pp.24970-
24979.
Cohen, S.I., Linse, S., Luheshi, L.M., Hellstrand, E., White, D.A., Rajah, L., Otzen, D.E.,
Vendruscolo, M., Dobson, C.M. and Knowles, T.P., 2013. Proliferation of amyloid-β42 aggregates
79
occurs through a secondary nucleation mechanism. Proceedings of the National Academy of
Sciences, 110(24), pp.9758-9763.
Comer, F.I., Vosseller, K., Wells, L., Accavitti, M.A. and Hart, G.W., 2001. Characterization of a
mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Analytical
biochemistry, 293(2), pp.169-177.
Emanuele, M. and Chieregatti, E., 2015. Mechanisms of alpha-synuclein action on
neurotransmission: cell-autonomous and non-cell autonomous role. Biomolecules, 5(2), pp.865-
892.
Fares, M.B., Maco, B., Oueslati, A., Rockenstein, E., Ninkina, N., Buchman, V.L., Masliah, E.
and Lashuel, H.A., 2016. Induction of de novo α-synuclein fibrillization in a neuronal model for
Parkinson’s disease. Proceedings of the National Academy of Sciences, 113(7), pp.E912-E921.
Fink, A.L., 2006. The aggregation and fibrillation of α-synuclein. Accounts of chemical
research, 39(9), pp.628-634.
Guerrero-Ferreira, R., Taylor, N.M., Mona, D., Ringler, P., Lauer, M.E., Riek, R., Britschgi, M.
and Stahlberg, H., 2018. Cryo-EM structure of alpha-synuclein fibrils. Elife, 7, p.e36402.
Jan, A., Adolfsson, O., Allaman, I., Buccarello, A.L., Magistretti, P.J., Pfeifer, A., Muhs, A. and
Lashuel, H.A., 2011. Aβ42 neurotoxicity is mediated by ongoing nucleated polymerization process
rather than by discrete Aβ42 species. Journal of Biological Chemistry, 286(10), pp.8585-8596.
80
Jao, C.C., Hegde, B.G., Chen, J., Haworth, I.S. and Langen, R., 2008. Structure of membrane-
bound α-synuclein from site-directed spin labeling and computational refinement. Proceedings of
the National Academy of Sciences, 105(50), pp.19666-19671.
Jeong, J.S., Ansaloni, A., Mezzenga, R., Lashuel, H.A. and Dietler, G., 2013. Novel mechanistic
insight into the molecular basis of amyloid polymorphism and secondary nucleation during
amyloid formation. Journal of molecular biology, 425(10), pp.1765-1781.
Lashuel, H.A., Overk, C.R., Oueslati, A. and Masliah, E., 2013. The many faces of α-synuclein:
from structure and toxicity to therapeutic target. Nature Reviews Neuroscience, 14(1), pp.38-48.
Lewis, Y.E., Galesic, A., Levine, P.M., De Leon, C.A., Lamiri, N., Brennan, C.K. and Pratt, M.R.,
2017. O-GlcNAcylation of α-synuclein at serine 87 reduces aggregation without affecting
membrane binding. ACS chemical biology, 12(4), pp.1020-1027.
Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G.W. and Gong, C.X., 2004. O-GlcNAcylation regulates
phosphorylation of tau: a mechanism involved in Alzheimer's disease. Proceedings of the National
Academy of Sciences, 101(29), pp.10804-10809.
Luk, K.C., Covell, D.J., Kehm, V.M., Zhang, B., Song, I.Y., Byrne, M.D., Pitkin, R.M., Decker,
S.C., Trojanowski, J.Q. and Lee, V.M.Y., 2016. Molecular and biological compatibility with host
alpha-synuclein influences fibril pathogenicity. Cell reports, 16(12), pp.3373-3387.
Luk, K.C., Song, C., O'Brien, P., Stieber, A., Branch, J.R., Brunden, K.R., Trojanowski, J.Q. and
Lee, V.M.Y., 2009. Exogenous α-synuclein fibrils seed the formation of Lewy body-like
intracellular inclusions in cultured cells. Proceedings of the National Academy of
Sciences, 106(47), pp.20051-20056.
81
Luk, K.C., Kehm, V., Carroll, J., Zhang, B., O’Brien, P., Trojanowski, J.Q. and Lee, V.M.Y.,
2012. Pathological α-synuclein transmission initiates Parkinson-like neurodegeneration in
nontransgenic mice. Science, 338(6109), pp.949-953.
Li, Y., Zhao, C., Luo, F., Liu, Z., Gui, X., Luo, Z., Zhang, X., Li, D., Liu, C. and Li, X., 2018.
Amyloid fibril structure of α-synuclein determined by cryo-electron microscopy. Cell
research, 28(9), pp.897-903.
Marotta, N.P., Lin, Y.H., Lewis, Y.E., Ambroso, M.R., Zaro, B.W., Roth, M.T., Arnold, D.B.,
Langen, R. and Pratt, M.R., 2015. O-GlcNAc modification blocks the aggregation and toxicity of
the protein α-synuclein associated with Parkinson's disease. Nature chemistry, 7(11), pp.913-920.
Mahul-Mellier, A.L., Vercruysse, F., Maco, B., Ait-Bouziad, N., De Roo, M., Muller, D. and
Lashuel, H.A., 2015. Fibril growth and seeding capacity play key roles in α-synuclein-mediated
apoptotic cell death. Cell Death & Differentiation, 22(12), pp.2107-2122.
Mizuno, N., Varkey, J., Kegulian, N.C., Hegde, B.G., Cheng, N., Langen, R. and Steven, A.C.,
2012. Remodeling of lipid vesicles into cylindrical micelles by α-synuclein in an extended α-
helical conformation. Journal of Biological Chemistry, 287(35), pp.29301-29311.
Morris, M., Knudsen, G.M., Maeda, S., Trinidad, J.C., Ioanoviciu, A., Burlingame, A.L. and
Mucke, L., 2015. Tau post-translational modifications in wild-type and human amyloid precursor
protein transgenic mice. Nature neuroscience, 18(8), pp.1183-1189.
Muir, T.W., Sondhi, D. and Cole, P.A., 1998. Expressed protein ligation: a general method for
protein engineering. Proceedings of the National Academy of Sciences, 95(12), pp.6705-6710.
82
O'Donnell, N., Zachara, N.E., Hart, G.W. and Marth, J.D., 2004. Ogt-dependent X-chromosome-
linked protein glycosylation is a requisite modification in somatic cell function and embryo
viability. Molecular and cellular biology, 24(4), pp.1680-1690.
Oueslati, A., Fournier, M. and Lashuel, H.A., 2010. Role of post-translational modifications in
modulating the structure, function and toxicity of α-synuclein: implications for Parkinson’s disease
pathogenesis and therapies. In Progress in brain research (Vol. 183, pp. 115-145). Elsevier.
Paleologou, K.E., Oueslati, A., Shakked, G., Rospigliosi, C.C., Kim, H.Y., Lamberto, G.R.,
Fernandez, C.O., Schmid, A., Chegini, F., Gai, W.P. and Chiappe, D., 2010. Phosphorylation at
S87 is enhanced in synucleinopathies, inhibits α-synuclein oligomerization, and influences
synuclein-membrane interactions. Journal of Neuroscience, 30(9), pp.3184-3198.
Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide, S.E., Dehejia, A., Dutra, A., Pike, B., Root,
H., Rubenstein, J., Boyer, R. and Stenroos, E.S., 1997. Mutation in the α-synuclein gene identified
in families with Parkinson's disease. science, 276(5321), pp.2045-2047.
Recasens, A. and Dehay, B., 2014. Alpha-synuclein spreading in Parkinson’s disease. Frontiers in
neuroanatomy, 8, p.159.
Recasens, A., Dehay, B., Bové, J., Carballo ‐Carbajal, I., Dovero, S., Pérez ‐Villalba, A., Fernagut,
P.O., Blesa, J., Parent, A., Perier, C. and Fariñas, I., 2014. Lewy body extracts from Parkinson
disease brains trigger α ‐synuclein pathology and neurodegeneration in mice and monkeys. Annals
of neurology, 75(3), pp.351-362.
83
Schmid, A.W., Fauvet, B., Moniatte, M. and Lashuel, H.A., 2013. Alpha-synuclein post-
translational modifications as potential biomarkers for Parkinson disease and other
synucleinopathies. Molecular & Cellular Proteomics, 12(12), pp.3543-3558.
Snow, C.M., Senior, A. and Gerace, L., 1987. Monoclonal antibodies identify a group of nuclear
pore complex glycoproteins. The Journal of cell biology, 104(5), pp.1143-1156.
Tuttle, M.D., Comellas, G., Nieuwkoop, A.J., Covell, D.J., Berthold, D.A., Kloepper, K.D.,
Courtney, J.M., Kim, J.K., Barclay, A.M., Kendall, A. and Wan, W., 2016. Solid-state NMR
structure of a pathogenic fibril of full-length human α-synuclein. Nature structural & molecular
biology, 23(5), pp.409-415.
Varkey, J., Isas, J.M., Mizuno, N., Jensen, M.B., Bhatia, V.K., Jao, C.C., Petrlova, J., Voss, J.C.,
Stamou, D.G., Steven, A.C. and Langen, R., 2010. Membrane curvature induction and tubulation
are common features of synucleins and apolipoproteins. Journal of Biological Chemistry, 285(42),
pp.32486-32493.
Visanji, N.P., Brotchie, J.M., Kalia, L.V., Koprich, J.B., Tandon, A., Watts, J.C. and Lang, A.E.,
2016. α-Synuclein-based animal models of Parkinson's disease: challenges and opportunities in a
new era. Trends in neurosciences, 39(11), pp.750-762.
Vilar, M., Chou, H.T., Lührs, T., Maji, S.K., Riek-Loher, D., Verel, R., Manning, G., Stahlberg,
H. and Riek, R., 2008. The fold of α-synuclein fibrils. Proceedings of the National Academy of
Sciences, 105(25), pp.8637-8642.
84
Volpicelli-Daley, L.A., Luk, K.C., Patel, T.P., Tanik, S.A., Riddle, D.M., Stieber, A., Meaney,
D.F., Trojanowski, J.Q. and Lee, V.M.Y., 2011. Exogenous α-synuclein fibrils induce Lewy body
pathology leading to synaptic dysfunction and neuron death. Neuron, 72(1), pp.57-71.
Wang, Z., Park, K., Comer, F., Hsieh-Wilson, L.C., Saudek, C.D. and Hart, G.W., 2009. Site-
specific GlcNAcylation of human erythrocyte proteins: potential biomarker (s) for
diabetes. diabetes, 58(2), pp.309-317.
Wang, Z., Udeshi, N.D., O'Malley, M., Shabanowitz, J., Hunt, D.F. and Hart, G.W., 2010.
Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of
chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass
spectrometry. Molecular & Cellular Proteomics, 9(1), pp.153-160.
Wang, S., Yang, F., Petyuk, V.A., Shukla, A.K., Monroe, M.E., Gritsenko, M.A., Rodland, K.D.,
Smith, R.D., Qian, W.J., Gong, C.X. and Liu, T., 2017. Quantitative proteomics identifies altered
O ‐GlcNAcylation of structural, synaptic and memory ‐associated proteins in Alzheimer's
disease. The Journal of pathology, 243(1), pp.78-88.
Wang, A.C., Jensen, E.H., Rexach, J.E., Vinters, H.V. and Hsieh-Wilson, L.C., 2016. Loss of O-
GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration. Proceedings of
the National Academy of Sciences, 113(52), pp.15120-15125.
Wilhelm, B.G., Mandad, S., Truckenbrodt, S., Kröhnert, K., Schäfer, C., Rammner, B., Koo, S.J.,
Claßen, G.A., Krauss, M., Haucke, V. and Urlaub, H., 2014. Composition of isolated synaptic
boutons reveals the amounts of vesicle trafficking proteins. Science, 344(6187), pp.1023-1028.
85
Wood, S.J., Wypych, J., Steavenson, S., Louis, J.C., Citron, M. and Biere, A.L., 1999. α-Synuclein
fibrillogenesis Is nucleation-dependent Implications for the pathogenesis of Parkinson′ s
disease. Journal of Biological Chemistry, 274(28), pp.19509-19512.
Yuzwa, S.A. and Vocadlo, D.J., 2014. O-GlcNAc and neurodegeneration: biochemical
mechanisms and potential roles in Alzheimer's disease and beyond. Chemical Society
Reviews, 43(19), pp.6839-6858.
Wani, W.Y., Chatham, J.C., Darley-Usmar, V., McMahon, L.L. and Zhang, J., 2017. O-
GlcNAcylation and neurodegeneration. Brain research bulletin, 133, pp.80-87.
Ysselstein, D., Joshi, M., Mishra, V., Griggs, A.M., Asiago, J.M., McCabe, G.P., Stanciu, L.A.,
Post, C.B. and Rochet, J.C., 2015. Effects of impaired membrane interactions on α-synuclein
aggregation and neurotoxicity. Neurobiology of disease, 79, pp.150-163.
Yuzwa, S.A., Macauley, M.S., Heinonen, J.E., Shan, X., Dennis, R.J., He, Y., Whitworth, G.E.,
Stubbs, K.A., McEachern, E.J., Davies, G.J. and Vocadlo, D.J., 2008. A potent mechanism-
inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo. Nature chemical
biology, 4(8), pp.483-490.
Yuzwa, S.A., Shan, X., Macauley, M.S., Clark, T., Skorobogatko, Y., Vosseller, K. and Vocadlo,
D.J., 2012. Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against
aggregation. Nature chemical biology, 8(4), pp.393-399.
Yuzwa, S.A., Cheung, A.H., Okon, M., McIntosh, L.P. and Vocadlo, D.J., 2014. O-GlcNAc
modification of tau directly inhibits its aggregation without perturbing the conformational
properties of tau monomers. Journal of molecular biology, 426(8), pp.1736-175.
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Chapter 3. Comparison of N-acetyl-glucosamine to other monosaccharides reveals
potentially special abilities of O-GlcNAc in amyloid inhibition
Introduction
O-GlcNAc modification is the dynamic addition of the N-acetylglucosamine to serine and
threonine residues of intracellular proteins (Figure 3-1A) and appears to play multiple important
roles in the inhibition of neurodegenerative diseases.

(Yang et al., 2017) For example, direct O-
GlcNAc modification of the amyloid-forming proteins tau and α-synuclein inhibits their
aggregation in vitro. Additionally, a small molecule inhibitor of the enzyme that removes O-
GlcNAc, O-GlcNAcase (OGA), increases overall modification levels, slows neurodegeneration in
mouse models of Alzheimer’s disease, increases autophagy, (Zhu et al., 2018) and inhibits the
uptake and toxicity of α-synuclein amyloid fibers in primary neurons. (Tavassoly et al., 2020)
Conversely, conditional knockout of the enzyme that adds O-GlcNAc, O-GlcNAc (Borghgraef et
al., 2013) transferase (OGT), in mouse forebrains leads to neurodegeneration. Finally, multiple
studies have found that O-GlcNAc is lower in Alzheimer’s disease patients compared to age-
matched controls. (Liu et al., 2004; Liu et al., 2009; Pinho et al., 2019; Wang et al., 2016)

______________
Ananya Rakshit, Giuliano Cutolo and Aaron Balana (University of Southern California)
contributed to the work presented in this chapter.

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Figure 3-1. O-GlcNAc modification of α-synuclein. a) O-GlcNAc is the dynamic addition of N-
acetyl-glucosamine to intracellular proteins. b) α-Synuclein is modified at nine different positions
with O-GlcNAc and the effects of these modifications can be studied in a site-specific fashion
using protein semisynthesis. c) Here, we test the importance of O-GlcNAc for α-synuclein amyloid
formation by comparing it to other monosaccharides.

Our own work in this area has focused on the major amyloidogenic protein in Parkinson’s disease,
α-synuclein. α-Synuclein (Uniprot: P37840) is a short, 140 amino acid protein found at relatively
high concentrations (~50 μM) at pre-synaptic termini. This protein is natively unstructured in
solution but can form an α-helix on the surface of lipid vesicles where it participates in membrane
remodeling and vesicle trafficking. Unfortunately, α-synuclein can also form toxic amyloid
oligomers and fibers. These fibers can then spread from cell to cell resulting in the progressive loss
of dopaminergic neurons and the onset of Parkinson’s disease symptoms. (Lashuel et al., 2013;
Wilhelm et al., 2014) Proteomic analysis from human and mouse tissue has found α-synuclein to
be O-GlcNAc modified at nine different positions (Figure 3-1B). (Wang et al., 2010; Morris et al.,
2015) OGT does not display a strong primary sequence dependance but does prefer unstructured
regions of proteins. Therefore, it is not necessarily surprising that a natively unfolded protein like
α-synuclein could be this broadly modified. To understand the consequences of O-GlcNAc on α-
synuclein aggregation, we employ synthetic protein chemistry to prepare site-specifically and
homogeneously modified protein for biological studies. Specifically, we take advantage of
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expressed protein ligation (EPL) to combine synthetic and recombinant protein fragments (Figure
3-1B). (Muir et al., 1998) α-Synuclein contains no native cysteines require for the ligation
reactions, but we can simply introduce cysteine in the place of any of the numerous alanine
residues and subject them to desulfurization at the end of the synthesis. (Wan et al., 2007) Using
this synthetic strategy, we previously described the preparation and characterization of six different
O-GlcNAc modified variants of α-synuclein. We found that all of the O-GlcNAc modifications
inhibit at least the kinetics of α-synuclein aggregation and several appear to affect the structure of
any amyloid fibers that do form, suggesting that increasing O-GlcNAc might be a therapeutic
strategy to slow the progression of Parkinson’s disease. (Marotta et al., 2015; Lewis et al., 2017;
Levine et al., 2017; Levine et al., 2019)

Figure 3-2. Different monosaccharides have distinct effects on α-synuclein aggregation. a)
Analysis of amyloid formation using thioflavin T (ThT) fluorescence (λex = 450 nm, λem = 482
nm). The indicated α-synuclein proteins (50 μM) were subjected to aggregation conditions and
analyzed by fluorescence at the indicated timepoints. b) The same reactions (168 h timepoint) were
analyzed by dot-blotting using an α-synuclein amyloid-selective antibody (A17183A). c) The
aggregation reaction mixtures (168 h timepoint) were subjected to the indicted concentrations of
proteinase K (PK) before analysis by SDS-PAGE and staining with Coomassie blue. The
persistence of bands correlates with the amount and gross structure of amyloids formed.

During the course of describing these results, we have been often asked if any monosaccharide
would provide similar aggregation, as the simplest hypothesis to explain O-GlcNAc’s ability to
89
inhibit amyloid formation is that it is acting as a relatively hydrophilic, steric block. If this is true,
one would expect that many similarly structured monosaccharides would behave in the same
fashion. Here, we test this possibility through the synthesis of four different α-synuclein proteins
bearing O-GlcNAc, β-O-GalNAc, β-O-glucose, or α-O-mannose at threonine 72 (Figure 3-1C).
We then used a variety of biochemical techniques to determine the effects of the different
monosaccharides on amyloid aggregation. Consistent with our previous publications, O-GlcNAc
at threonine 72 strongly inhibited α-synuclein aggregation. Interestingly, we found that despite
only small differences in their structures, the other monosaccharides were all less inhibitory. This
suggests that there is something potentially special about O-GlcNAc as an amyloid inhibitor with
interesting implications for the evolution of this type of glycosylation.

Results and Discussion
We chose the β-O-GalNAc and β-O-glucose monosaccharides as they provide a limited structural
activity relationship profile of the O-GlcNAc, and we picked α-O-mannose because it has been
shown to potentially be a replacement for O-GlcNAc in Sacchromyces cerevisiae. (Halim et al.,
2015) We performed a stepwise assembly of three protein fragments to yield the final glycosylated
proteins (Supporting Information, Figure S 3-1A). We first synthesized the glycosylated threonine
building-blocks using our InBr
19
3 catalyzed glycosylation. This was followed by standard (Leon
et al., 2018) Fmoc-based solid phase peptide synthesis on hydrazine resin resulting C-terminal
hydrazide peptides (1-4). We then reacted these peptides with a recombinant thioester protein 5,
which was previously expressed as an intein fusion. The C-terminal hydrazides were then
transformed to the corresponding thioesters and reacted the N-terminal cysteine protein 6, (Flood
et al., 2018) which we expressed recombinantly in E. coli by taking advantage of its endogenous
90
methionine aminopeptidase. Finally, we took advantage of desulfurization chemistry to transform
the cysteines required for the ligation reactions back to their native alanines in the α-synuclein
primary sequence. The final protein products, α-synuclein(GlcNAc), (Glc), (GalNAc), and (Man),
were characterized by RP-HPLC and mass spectrometry (Supporting Information, Figure S 3-1B).
With these proteins in hand, we next moved to examine how they affect the amounts of α-synuclein
aggregation using three methods: Thioflavin T (ThT) fluorescence, dot-blotting, and proteinase K
(PK) digestion. We first subjected all four modified proteins, as well as unmodified recombinant
α-synuclein, to aggregation conditions (50 μM protein concentration, agitation at 1,000 rpm, and
37 °C) in phosphate buffer (pH 7.4) for 7 days. Aliquots of the reaction mixture were removed
after 48, 96, and 168 h, mixed the ThT, and analyzed by plate reader (Figure 2a). ThT is a member
of environmentally sensitive dyes that display increased fluorescence in the presence of amyloid
fibers due to interpolation of the dye into hydrophobic grooves that form along the side of the
aggregates. As expected from our previous results, unmodified α-synuclein rapidly aggregated
over the course of the assay, while α-synuclein(GlcNAc) displays slower aggregation kinetics and
lower overall ThT signal at the end of the assay. (Levine et al., 2019) All three of the other
monosaccharides also displayed lower ThT signals, indicating reduced formation of α-synuclein
aggregates. Notably, however, α-synuclein(Man) appeared to be more inhibitory than α-
synuclein(Glc) or α-synuclein(GalNAc). We next analyzed the aggregation reactions at the end of
the assay (168 h) by dot-blotting with an antibody (A17183A) that broadly recognizes α-synuclein
amyloid aggregates, including oligomers and fibers (Figure 3-2B). The results were consistent with
the ThT measurements, with all four glycosylated proteins showing fewer amyloids and α-
synuclein(GlcNAc)/α-synuclein(Man) having the least. Next, we subjected the 168 h reaction
mixtures to digestion by PK (Figure 3-2C). PK is a highly promiscuous protease that will
91
completely digest monomeric α-synuclein. However, certain amyloid structures restrict the access
of PK to the internal segments of α-synuclein, resulting in stable bands that can be visualized by
SDS-PAGE. This banding pattern gives a low resolution picture of qualitative differences in the
amyloid structure. Because the digestion is performed on the entire aggregation reaction, a mixture
of monomers, oligomers, and fibers, it can also be used as a proxy for aggregate stability. (Luk et
al., 2016) Specifically, the proteolysis of full-length α-synuclein corresponds to the amount of
aggregation and the stability of those amyloids. For unmodified α-synuclein, we observed stability
of the full-length band and five overall bands in the pattern observed for “typical” stable amyloid-
fibers. Similar to our published results, we found much less full-length protein upon digestion of
α-synuclein(GlcNAc) and a three-band pattern. Despite the relatively high signal from ThT
fluorescence and dot-blotting, we observed essentially complete degradation of α-
synuclein(GalNAc) by PK. Finally, we found that digestion of both α-synuclein(Glc) and α-
synuclein(Man) resulted in three bands that are similar to α-synuclein(GlcNAc). However, those
bands had different stabilities depending on the monosaccharide.
Next, we set out to confirm our results by repeating the aggregation reactions. However, this time
we extended the timeframe of the aggregation to potentially accentuate any differences between
the different monosaccharides. Overall, we observed results that were very consistent with the
previous set of experiments. ThT fluorescence showed that all of the different sugars inhibited the
kinetics of α-synuclein aggregation, and that α-synuclein(GlcNAc)/α-synuclein(Man) were more
inhibitory (Supporting Information, Figure S 3-2a). Notably, at the extended timepoint of this
assay (192 h), we found that α-synuclein(GalNAc) displayed more ThT signal than α-
synuclein(Glc) (Supporting Information, Figure S 3-2a). Dot-blotting once again largely
confirmed these results (Supporting Information, Figure S3-2b). We also analyzed the 192 h
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reaction mixture by PK digestion (Supporting Information, Figure S3-2c). Once again, digestion
of α-synuclein(GlcNAc) and α-synuclein(GalNAc) resulted in almost no detectable stable bands.
As expected from the longer aggregation, and therefore more amyloid formation, we in general
observed more stable bands for both α-synuclein(Glc) and α-synuclein(Man).
Finally, we analyzed the 192 h timepoint from this second aggregation reaction by transmission
electron microscopy to characterize the structure of the any amyloids that formed (Figure 3-3 &
Supporting Information, Figure S3-3). As expected from our prior work, unmodified α-synuclein
formed long fibers consistent with typical amyloids, while α-synuclein(GlcNAc) only yielded
short and broken fiber structures. α-Synuclein(
18
Glc) and α-synuclein(GalNAc) both formed long
amyloid fibers more similar to unmodified α-synuclein than α-synuclein(GlcNAc). Finally, we
found a mixture of both long fibers and short, broken structures formed by α-synuclein(Man).

93

Figure 3-3. Analysis of the different α-synuclein aggregates using transmission electron
microscopy (TEM). The 192 h timepoint of the aggregation reactions in Figure S2 were
visualized using TEM.

Taken together, our results yield some interesting and somewhat expected conclusions. O-GlcNAc
is the only monosaccharide tested that inhibits the amyloid aggregation of α-synuclein using all
four different characterization techniques: ThT fluorescence, dot-blotting, PK digestion, TEM
imaging. On the other end of the spectrum, we found that glucose modification was the least
inhibitory, with relatively high ThT fluorescence and dot-blot signal attributable to amyloid
oligomers and/or fibers, as well as several bands that were stable to PK digestion and long fibers
visualized by TEM. We found intermediate results for both α-synuclein(GalNAc) and α-
94
synuclein(Man). In the case of α-synuclein(GalNAc), the ThT fluorescence, dot-blotting, and TEM
all showed the formation of notable amounts of long amyloid fibers. However, these amyloids
were not stable to PK digestion. Interestingly, these results are consistent with a recent report on
α-synuclein “needles” that are a structurally different polyform of the α-synuclein amyloid.
(Peduzzo et al., 2020) We believe that GalNAc at T72 may be inducing the formation of needles,
or something similar, instead of the more traditional fiber amyloid structure. There is precedent
for PTMs causing different α-synuclein polymorphs, including our own work on O-GlcNAc at
serine 87 (Lewis et al., 2017) and phosphorylation at tyrosine (Zhao et al., 2020) Finally, α-
synuclein(Man) gave ThT and dot-blot signals that were similar to α-synuclein(GlcNAc), α-
synuclein(Man) also yield a mixture of broken and long amyloids visualized by TEM that were at
least partially stable to PK digestion. It is possible that mannose is also forcing the formation of a
polyform, but we are not aware of any previously characterized amyloid that share these
characteristics. α-Synuclein(Man) may display a more bifurcated aggregation process that results
in a mixture of small, broken structures similar to α-synuclein(GlcNAc) and longer amyloids.
Currently there is no physiological relevance to modification of α-synuclein by glucose, GalNAc,
or mannose, as these modifications have never been observed. However, these data demonstrate
that O-GlcNAc may particularly effective at inhibiting amyloid aggregation, at least in the case of
α-synuclein. At the outset of these experiments, we assumed that any monosaccharide would
function similarly by acting as a hydrophilic, steric impediment to the formation of the largely
hydrophobic core of the amyloid aggregate. That is clearly not the case, and even small changes
to the structure of the monosaccharide can have fairly large consequences. In summary, our results
suggest that O-GlcNAc is a potentially special inhibitor of protein aggregation with interesting
implications for the evolution of this PTM over other potential monosaccharides.
95
Materials and Methods
General.
Unless otherwise mentioned, all solvents and reagents were purchased from Sigma-Aldrich, EMD,
Fluka, Novagen, VWR etc. and used without any further purification. Luria Bertani-Miller and
Terrific broth medium were procured from EMD and solutions were prepared, autoclaved, stored
and used following protocols from the manufacturer. Aqueous solutions were prepared using
deionized, distilled and filtered water (18 MΩ at 25 °C). E. coli BL21 (DE3) chemically
competent cells were obtained from VWR. Precasted 12% Bis-Tris gels, MES running buffer and
Coomassie Brilliant Blue were purchased from Bio-Rad. N-Fmoc and side chain protected amino
acids, HBTU, as well as resin beads for peptide synthesis were obtained from NovaBiochem.
Fmoc-Thr(ß-O-Ac4Glc)-OH was purchased from Polt laboratory, University of Arizona. Stock
solutions of ampicillin sodium salt and kanamycin sulfate were prepared at concentration of 100
mg mL
-1
and 50 mg mL
-1
respectively and stored at -20 °C. All silica gel column chromatography
was performed using 60 Å silica gel (EMD) and all thin-layer chromatography (TLC) was
performed using 60 Å, F254 silica gel plates (EMD) with detection by ultraviolet light and staining
with either ceric ammonium molybdate (CAM) or ninhydrin.
1
H and
13
C NMR spectra were
collected in either CDCl3 or CD3OD as a solvent (Cambridge Isotope Laboratories, Cambridge,
MA) at 25 °C on a Varian 600 MHz spectrometer. All chemical shifts are reported in the standard
notation of parts per million (ppm) using the peaks of proton and carbon signals of residual solvents
for calibration (CDCl3:
1
H - 7.26 ppm and
13
C - 77.16 ppm and CD3OD: 1H - 4.78, 3.31 ppm and
13
C - 49.15 ppm) and coupling constants (J) given in Hertz. The abbreviations used for the proton
spectra multiplicities are: s, singlet; b, broad; d, doublet; t, triplet; q, quartet; m, multiplet. A
Biotage Isolera Spektra FLASH system (solvent A, 0.1% TFA in water; solvent B, 0.1 % TFA in
96
acetonitrile) or an Agilent 1200 Series HPLC (solvent A: 0.1 % TFA in water; solvent B: 0.1 %
TFA and 90 % acetonitrile in water) system was used for reverse-phase high performance liquid
chromatography (RP-HPLC). Mass spectra was recorded either on an API-150EX (Applied
Biosystems/MDS SCIEX) or on an Agilent HPLC/Q TOF MS/MS Spectrometer.
Unmodified α-synuclein expression and purification.
E. coli BL21 (DE3) cells were transformed with α-synuclein-coding pRK172
construct{Meier:2012hq} via a 30 s heat shock at 42 °C. The cells were grown on an LB agar
ampicillin plate (100 μg mL
-1
) at 37 °C for 12 h and then stored at 4 °C. A started culture was used
to inoculate 300 mL of TB culture (100 μg mL
-1
ampicillin) which was shaken at 250 rpm at 37
°C till an OD600nm of around 0.6-0.7. To induce α-synuclein expression, IPTG (0.5 M) was added
to the cultures with shaking at same conditions for 16 h. Bacteria were harvested by centrifugation
at 6,000 × g for 15 min at 4 °C. Cell pellets were re-suspended in lysis buffer (100 mM Tris, 500
mM NaCl, 10 mM 2-mercaptoethanol, 1 mM EDTA, pH 8) after performing freeze thaw cycles
thrice using liquid nitrogen and a 37 °C incubator respectively. The cells were then boiled at 80
°C for 10 mins while agitating the resuspension every minute. The lysed cells were then kept at
room temperature for 30 mins. PMSF solution in isopropanol (100 mM stock) was added to the
cell lysates to give a 2 mM final concentration, and cells were incubated on ice for another 30 min.
After that, the cell lysate was centrifuged (6000 × g, 4 °C, 20 min) and the supernatant was
collected and acidified to pH 3.5 using 1M HCl. It was again incubated on ice for 30 mins before
another centrifugation (6000 × g, 4 °C, 20 min). After centrifugation, the supernatant was dialyzed
into a 2 L degassed, 1% acetic acid solution using SnakeSkin dialysis tubing (3.5K MWCO). After
12 h, the dialyzed protein solution was centrifuged and the clear solution was purified by a C4
semi-preparative column (Higgins Analytical) using an Agilent 1200 Series HPLC system (20-70
97
% solvent B in solvent A gradient for 40 mins, 2.5 ml/min flow rate) and then characterized by
ESI-MS. Pure protein was lyophilized and stored at -20 °C. The amount of pure protein was
calculated by Pierce BCA assay (Thermo Scientific).
Expression of α-synuclein N-terminal (1-68) thioester.
E. coli BL21 (DE3) cells were transformed with a modified pTXB1 plasmid encoding α-
synuclein(1-68) fused to the Ava-DnaE N137A intein{Levine:2019ea} by heat shock at 42 °C for
30 s, plated on a LB agar plate containing ampicillin (100 µg mL
-1
) and incubated overnight at 37
°C. N-terminal (1-68) fragment was expressed in E. coli cells as wild type α-synuclein protein.
After centrifuging (6,000 x g, 15 min, 4 °C), the cell pellet was resuspended in 40 mL (per 2L of
TB culture) of cold lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 5 mM imidazole, 2 mM PMSF,
2 mM TCEP.HCl, pH 7.4) and subjected to tip sonication (45% amplitude, 30 sec pulse ON, 30
sec rest, 8 min, on ice). The cell lysate was centrifuged again (6,000 x g, 45 min, 4 °C) to remove
cell debris and supernatant was incubated with Co-NTA Agarose resin beads, previously
equilibrated with the wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 2 mM
TCEP.HCl, pH 7.4), for 1 h with continuous agitation at 4 °C. The resin beads were thoroughly
washed with wash buffer before treating with elution buffer (50 mM NaH2PO4, 300 mM NaCl,
250 mM imidazole, 2 mM TCEP.HCl, pH 7.4). The eluted protein was dialyzed into a 0.5X DPBS
(2 L) solution using SnakeSkin dialysis tubing (3.5K MWCO) overnight at 4 °C followed by
addition of sodium mercaptoethane sulfonate (MESNa) (250 mM) to induced intein cleavage and
thioester generation. The thiolysis reaction was carried out for 12 h at room temperature. After
completion, the reaction mixture was purified from the intein fusion by C4 column using a Biotage
Isolera Spektra FLASH system and stored as lyophilized powder at -20 °C. The purity of α-
synuclein N-terminal (1-68) thioester fragment was confirmed by HPLC and ESI-MS.
98
Expression of α-synuclein C-terminal fragment (76-140).
E. coli BL21 (DE3) cells were transformed with the specific pET42b construct1 for α-
synuclein(A76C-140) via a 30 s heat shock at 42 °C and plated on a kanamycin (50 µg mL
-1
)
containing LB agar plate. Protein was expressed and purified following protocol as described
above for full length, unmodified α-synuclein.
Solid phase synthesis of 69-75 peptides.
Peptides were synthesized manually by using solid phase peptide synthesis strategy on 2-Cl-(Trt)-
Cl resin (loading 0.68 mmol/g resin) on a 0.1 mmol scale. These resin beads were activated to 2-
Cl-(Trt)-NHNH2 with hydrazine hydrate solution following a previously reported
procedure.{Zheng:2013gn} Before coupling, commercially available N-Fmoc and side chain
protected amino acids (5 eq) were activated for 5 min with HBTU (5 eq) and DIEA (10 eq) and
then coupled to the resin for 1 hour. After coupling, the terminal Fmoc group was deprotected with
20% v/v piperidine in DMF for 7 min twice. For the first coupling, N-Fmoc protected amino acid
was coupled twice for 45 mins. After that, the resin beads were capped with a capping solution
(156 µl Ac2O, 40 µl pyridine in 2 mL DMF) for 30 mins. Deprotection of Fmoc group was then
carried out. For the incorporation of the glycosylated threonine amino acids, pentafluorophenyl
(PFP) activated O-modified Fmoc-threonines were synthesized and purified as described in the
synthesis section. Two equivalents of this amino acid were incubated with the peptide resin
overnight, followed by standard coupling cycles for the remaining amino acids. After the final
Fmoc-deprotection, peptides were cleaved from resin by treatment with 3 mL of cleavage cocktail
(TFA:H2O:Triisopropylsilane; 95:2.5:2.5) for 4 h at room temperature with constant rocking. The
crude peptide was filtered and diluted in cold diethyl ether (35 mL) and precipitated overnight at
-80 °C. The resulting suspension was then centrifuged (6,000 x g, 30 min, 4 °C). The pellet was
99
dried under nitrogen flow to remove traces of diethylether and then resuspended in 20%
acetonitrile in water solution before HPLC purification using a C18 semi-preparative column.
Purified peptides were then characterized by HPLC and ESI-MS.
α-Synuclein semi-synthesis.
A common ligation procedure was followed to make all four glycosylated proteins. N-terminal (1-
68) thioester fragment (2 mM, 1 eq) and an individual peptide (4 mM, 2 eq) were dissolved in
ligation buffer (3 M guanidine-HCl, 300 mM phosphate, pH 7.0). To this solution were added
TCEP (300 mM stock in ligation buffer) and 4-mercaptophenylacetic acid “MPAA” (250 mM
stock in ligation buffer) to a final concentration of 30 mM each. The mixture was rocked for 12 h
at room temperature after adjusting the pH of the reaction to 7.0 with 3M NaOH solution. After
12 h, ligation product (α-synuclein(1-75)-NHNH2 fragment) was purified using a C18 semi-prep
column (Higgins Analytical) and lyophilized.
The α-synuclein(1-75)-NHNH2 fragment was first activated using the Dawson Knorr-Pyrazole
method.{Flood:2018fe}. Specifically, the fragment was dissolved in activation buffer (6 M
guanidine-HCl, 200 mM MPAA, pH 3.5) and acetylacetone (1:20 v/v) in activation buffer at pH
3.5 and stirred at room temperature until the MPAA activation is complete (~2 h). The thioester
formation was monitored by RP-HPLC (0−70 % of solvent B over 60 min) and activation was
confirmed by ESI-MS. After completion, the α-synuclein(A76C-140) fragment was added to the
reaction mixture and equal volume of ligation buffer (6 M guanidine-HCl, 200 NaH2PO4, pH 8.3)
was added followed by TCEP.HCl (50 mM final concentration). The pH of the resulting solution
was adjusted to 7.0 with addition of 3M NaOH solution. The reaction mixture was stirred for 12 h
at room temperature. This product was purified using a C4 semi-prep column (Higgins Analytical)
and lyophilized. Deacetylation of the monosaccharide on the lyophilized full length protein was
100
done in presence of 5 % hydrazine monohydrate aqueous solution at room temperature for 1 h and
then quenched with 5% acetic acid solution. The de-acetylated full length α-synuclein was then
purified using a C4 semi-prep column (Higgins Analytical) and lyophilized. Desulfurization on
the full length protein was carried out in degassed buffer (6 M guanidine-HCl, 300 mM phosphate,
300 mM TCEP, 2.5% v/v ethanethiol, 10% v/v tertbutylthiol, pH 7.0) in the presence of a radical
initiator VA-061 (200 mM in MeOH, 2 mM final concentration) under inert atmosphere. The
reaction was stirred at 37 °C for 16 h. The product was purified and characterized by C4 analytical
column (Higgins Analytical) and ESI-MS respectively.
α-Synuclein aggregation.
Purified unmodified α-synuclein and the glycosylated derivatives were individually dissolved in
aggregation buffer (10 mM phosphate, 0.05% NaN3, pH 7.4, filtered) and sonicated for 10 mins.
The solutions were then centrifuged at 20,000 x g for 20 min at 4 °C to remove any pre-formed
seeds. The supernatant was transferred into a fresh tube and BCA assay was performed to exactly
determine the concentration of proteins in solution. The volume of solution was adjusted with
aggregation buffer to get a final concentration of 50 µM of each protein which was then partitioned
into triplicates with a final volume of 150 µl in each tube. These triplicates solutions were then left
at 37 °C incubator with constant agitation (1,000 x g) in a Thermomixer F1.5 (Eppendorf) for the
indicated lengths of time, and aliquots were collected at different time points and stored at -80 °C
for analysis by Thioflavin T.
Thioflavin T fluorescence.
The progression of α-synuclein aggregation was quantified by Thioflavin T fluorescence. Samples
from the aggregation assay reaction mixture were diluted in a 96-well plate to a concentration of
1.25 μM with reaction buffer (10 mM PBS, pH 7.4 and 0.05% NaN3) containing 20 μM Thioflavin
101
T (dissolved from a 2000X stock prepared in DMSO). Fluorescence was measured using a Synergy
H4 hybrid reader (BioTek). The plate was shaken for 3 min, and data was collected (λex = 450
nm, 9 nm band path, λem = 482 nm, 9 nm band path). Triplicate measurements were performed
for all aggregation reaction conditions.
Dot blotting.
Ten nanograms (10 ng) of protein from aggregation reactions were spotted onto nitrocellulose
membrane and air dried for 30 min. The proteins were fixed with 4% paraformaldehyde in PBS
for 30 minutes at room temperature. Membrane was washed 3 times in PBS for 5 minutes each
before blocking for 1 hour at room temperature with OneBlock Western-CL buffer (Genessee
Scientific). Primary antibody (A17183A, BioLegend) was added to the blocking buffer at 1:5000
dilution and the membrane was incubated for 16 hours at 4 °C. The membrane was washed three
times in TBST for 10 minutes each, after which it was incubated with anti-rat-HRP secondary
antibody (1:10,000 in blocking buffer) for one hour at room temperature. The membrane was
washed again with TBST three times for 10 minutes each before developing in Western blotting
substrate (Bio-Rad Clarity Western ECL).
Transmission electron microscopy.
At the end of the aggregation process, each protein solution was diluted to 15 μM by adding the
aggregation reaction buffer, and the diluted solution (10 μL) was incubated with a Formvar coated
copper grid (150 mesh, Electron Microscopy Science) for 5 min. Subsequently, the grid was
incubated with 1% uranyl acetate three times for 2 min. Each time, excess liquid was removed
with filter paper. The grid was dried for 24 h. Grids were visualized with a JEOL JEM-2100F
transmission electron microscope operated at 200 kV and 600,000× magnification and an Orius
Pre-GIF CCD.
102
Proteinase K Digestion.
Ten micrograms of aggregation protein sample were incubated with Proteinase K (Sigma Aldrich
P2308) at the indicated concentrations for 30 min at 37 °C. Reactions were quenched by the
addition of sample loading buffer (2% final SDS concentration) and boiling samples at 95 °C for
10 min. Digestion products were separated by SDS- PAGE using precast 12% Bis-Tris gels with
MES running buffer. Bands were visualized with Coommassie Brilliant Blue.
β-O-Ac3GlcNAc-threonine pentafluorophenyl ester was synthesized as previously described.
(DeLeon et al., 2018)
General glycosylation reaction for the preparation of glycosyl-threonines. (DeLeon et al.,
2018)
A solution of the per-O-acetylated donor sugar (3 equiv.), InBr3 (0.6 equiv., 20 mol % with respect
to donor), and the Fmoc-threonine acceptor (1 equiv.) in 1,2-dichloroethane (200 mM
concentration of the acceptor) was refluxed for 16 h at 85 °C under nitrogen atmosphere. The
reaction was monitored by TLC (7:2:1: EtOAc: MeOH: H2O). The reaction was allowed to cool
to room temperature and concentrated under vacuum. The crude was then resuspended in
dichloromethane (DCM), loaded onto a flash column chromatography and purified with a mixture
of 5% MeOH in DCM (0.1% AcOH). The amounts of individual reagents can be found in the
specific methods below.
General preparation of the pentafluorophenyl amino-acid esters.
Pentafluorophenyl trifluoroacetate (3 equiv.) was added to a stirring solution of the free acid (1
equiv.), anhydrous pyridine (4 equiv.) in anhydrous dimethylformamide (100 mM concentration
of the amino acid) under N2. The reaction was allowed to stir at room temperature overnight. The
reaction progress was monitored by TLC (4:6 EtOAc: Hexane). After the reaction went to
103
completion, the mixture was concentrated in vacuum and the residue was then suspended in DCM
and purified by flash chromatography (Hexane/ Acetone, 7/3). The amounts of individual reagents
can be found in the specific methods below.
O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-
threonine pentafluorophenyl ester (β-O-Ac4Glc-threonine).
Following the general pentafluorophenyl ester synthesis using commercially available β-O-
Ac4Glc-threonine (250 mg), β-O-Ac4Glc-threonine pentafluorophenyl ester was obtained as a
white or light brown foam (310 mg, 97%).
1
H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz, 2H),
7.67 – 7.59 (m, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 5.72 (d, J = 8.9 Hz, 1H),
5.23 (t, J = 9.6 Hz, 1H), 5.08 (t, J = 9.7 Hz, 1H), 4.97 (dd, J = 9.8,
7.9 Hz, 1H), 4.72 (dd, J = 8.9, 2.8 Hz, 1H), 4.59 – 4.54 (m, 2H),
4.50 – 4.39 (m, 2H), 4.26 (t, J = 7.1 Hz, 1H), 4.20 (dd, J = 12.3,
4.8 Hz, 1H), 4.05 (dd, J = 12.3, 2.5 Hz, 1H), 3.69 (ddd, J = 9.9,
4.8, 2.5 Hz, 1H), 2.05 (s, 3H), 2.03 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H), 1.31 (d, J = 6.3 Hz, 3H).
13
C
NMR (101 MHz, CDCl3) δ 170.56, 170.23, 169.32, 169.28, 166.30, 166.28, 156.45, 143.78,
143.52, 141.31, 127.77, 127.75, 127.09, 127.08, 125.10, 119.99, 119.98, 98.30, 73.87, 72.40,
71.87, 71.29, 68.23, 67.44, 61.67, 58.48, 47.11, 20.63, 20.60, 20.56, 20.42, 16.74.
O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl)-N-(9-
fluorenylmethyloxycarbonyl)-L-threonine (β-O-Ac3GalNAc-threonine).
Following the general glycosylation reaction: β-Ac4GalNAc (3.14 g, 8.07 mmol), Fmoc-Thr-OH
(800 mg, 2.69 mmol), and InBr3 (572 mg, 1.61 mmol). Following purification, β-O-Ac3GalNAc
threonine was obtained as a white or light brown foam (550 mg, 61% yield).
1
H NMR (400 MHz,
CD3OD) δ 7.77 (d, J = 7.5 Hz, 1H), 7.68 (t, J = 7.2 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.30 (t, J =
104
6.8 Hz, 2H), 5.33 (d, J = 3.3 Hz, 1H), 5.08 (dd, J = 11.3, 3.4 Hz, 2H), 4.59 (d, J = 8.5 Hz, 1H),
4.45 – 4.3 9 (m, 1H), 4.35 (dd, J = 7.1, 4.2 Hz, 2H), 4.25 – 4.21 (m, 2H), 4.18 – 4.02 (m, 3H), 3.97
(t, J = 6.8 Hz, 1H), 2.09 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.94 (s,
3H).
13
C NMR (100 MHz, CD3OD) δ 173.95, 173.33, 172.13,
172.04, 171.69, 158.98, 145.29, 145.05, 142.51, 128.78, 128.20,
128.19, 126.34, 126.30, 120.93, 101.57, 76.72, 71.79, 71.57,
68.19, 67.81, 62.19, 59.93, 51.62, 48.33, 22.95, 20.59, 20.55, 17.99.
O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl)-N-(9-
fluorenylmethyloxycarbonyl)-L-threonine pentafluorophenyl ester (β-O-Ac3GalNAc
threonine pentafluorophenyl ester).
Following the general pentafluorophenyl ester synthesis using β-
O-Ac3GalNAc Threonine (330 mg, 8.07 mmol), β-O-
Ac3GalNAc threonine pentafluorophenyl ester was obtained as
a white or light brown foam after purification (365 mg, 87%
yield).
1
H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 7.5 Hz, 2H), 7.66 – 7.59 (m, 2H), 7.34 (t, J = 7.4
Hz, 2H), 7.26 (t, J = 7.2 Hz, 2H), 6.07 (dd, J = 8.8, 3.9 Hz, 2H), 5.30 (d, J = 3.5 Hz, 1H), 5.20 (dd,
J = 11.4, 3.4 Hz, 1H), 4.70 – 4.63 (m, 2H), 4.56 – 4.51 (m, 1H), 4.47 – 4.44 (m, 3H), 4.22 (t, J =
7.2 Hz, 1H), 4.02 – 3.92 (m, 3H), 3.85 (t, J = 6.6 Hz, 1H), 2.10 (s, 3H), 1.96 (s, 3H), 1.95 (s, 3H),
1.92 (s, 3H), 1.25 (d, J = 6.3 Hz, 3H).
13
C NMR (100 MHz, CDCl3) δ 207.16, 170.71, 170.55,
170.31, 170.26, 166.51, 156.65, 143.84, 143.56, 141.24, 127.70, 127.67, 127.06, 127.02, 125.19,
125.13, 119.91, 98.53, 73.18, 70.46, 69.65, 67.30, 66.41, 61.15, 58.84, 51.41, 47.08, 30.85, 23.37,
20.61, 20.47, 20.42, 16.36.

105
O-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)-N-(9-fluorenylmethyloxycarbonyl)-L-
threonine pentafluorophenyl ester (α-O-Ac4Man-threonine pentafluorophenyl ester).
Following the general glycosylation reaction: O-Ac5Man (3.90 g, 10.09 mmol), Fmoc-Thr-OH
(1.00 g, 3.36 mmol), and InBr3 (715 mg, 2.02 mmol); α-O-Ac4Man-threonine was first purified
by column chromatography using 5% MeOH in DCM (0.1% AcOH). The product was obtained
as a mixture of compounds and used for the next step without further purification. Following the
general pentafluorophenyl ester synthesis using α-O-
Ac4Man-threonine (600 mg), α-O-Ac4Man-threonine
pentafluorophenyl ester was obtained as a white or light
brown foam (650 mg, 24% yield over two steps).
1
H NMR
(400 MHz, CDCl3) δ 7.77 (d, J = 6.6 Hz, 2H), 7.69 – 7.64
(m, 1H), 7.46 – 7.22 (m, 5H), 5.72 (d, J = 8.3 Hz, 1H), 5.34 – 5.21 (m, 2H), 5.15 (s, 1H), 5.00 (s,
1H), 4.83 (d, J = 9.5 Hz, 1H), 4.62 – 4.42 (m, 3H), 4.34 – 4.22 (m, 2H), 4.19 – 4.02 (m, 2H), 2.14
(s, 3H), 2.09 (s, 3H), 2.08 – 2.06 (m, 3H), 1.99 (s, 3H), 1.48 – 1.39 (m, 3H).
13
C NMR (101 MHz,
CDCl3) δ 170.50, 169.84, 169.66, 169.62, 166.47, 156.41, 143.68, 143.56, 141.31, 127.79, 127.13,
125.13, 120.03, 120.00, 99.16, 69.34, 69.20, 68.66, 67.73, 66.26, 62.56, 58.53, 47.10, 29.68, 20.59,
18.07.

106
Supporting Information

Figure S 3-1. Synthesis and characterization of α-synuclein proteins. a) Differentially
glycosylated versions of α-synuclein were retrosynthetically deconstructed into a recombinant
protein thioester, synthetic peptide thioesters prepared by solid phase peptide synthesis, and a
recombinant N-terminal cysteine protein. b) Analytical RP-HPLC traces and ESI-MS of the
indicated synthetic proteins.

Figure S 3-2. Different monosaccharides have distinct effects on α-synuclein aggregation. a)
Analysis of amyloid formation using thioflavin T (ThT) fluorescence (λex = 450 nm, λem = 482
nm). The indicated α-synuclein proteins (50 μM) were subjected to aggregation conditions and
analyzed by fluorescence at the indicated timepoints. b) The same reactions (192 h timepoint) were
analyzed by dot-blotting using an α-synuclein amyloid-selective antibody (A17183A). c) The
aggregation reaction mixtures (192 h timepoint) were subjected to the indicted concentrations of
proteinase K (PK) before analysis by SDS-PAGE and staining with Coomassie blue. The
persistence of bands correlates with the amount and gross structure of amyloids formed.
107

Figure S 3-3. Large format TEM images. Representative TEM images from Figure 3.

108
Chapter Three References

Borghgraef, P., Menuet, C., Theunis, C., Louis, J.V., Devijver, H., Maurin, H., Smet-Nocca, C.,
Lippens, G., Hilaire, G., Gijsen, H. and Moechars, D., 2013. Increasing brain protein O-GlcNAc-
ylation mitigates breathing defects and mortality of Tau. P301L mice. PloS one, 8(12), p.e84442.
Flood, D.T., Hintzen, J.C., Bird, M.J., Cistrone, P.A., Chen, J.S. and Dawson, P.E., 2018.
Leveraging the Knorr pyrazole synthesis for the facile generation of thioester surrogates for use in
native chemical ligation. Angewandte Chemie, 130(36), pp.11808-11813.
Halim, A., Larsen, I.S.B., Neubert, P., Joshi, H.J., Petersen, B.L., Vakhrushev, S.Y., Strahl, S. and
Clausen, H., 2015. Discovery of a nucleocytoplasmic O-mannose glycoproteome in
yeast. Proceedings of the National Academy of Sciences, 112(51), pp.15648-15653.
Lashuel, H.A., Overk, C.R., Oueslati, A. and Masliah, E., 2013. The many faces of α-synuclein:
from structure and toxicity to therapeutic target. Nature Reviews Neuroscience, 14(1), pp.38-48.
Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G.W. and Gong, C.X., 2004. O-GlcNAcylation regulates
phosphorylation of tau: a mechanism involved in Alzheimer's disease. Proceedings of the National
Academy of Sciences, 101(29), pp.10804-10809.
Liu, F., Shi, J., Tanimukai, H., Gu, J., Gu, J., Grundke-Iqbal, I., Iqbal, K. and Gong, C.X., 2009.
Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer's
disease. Brain, 132(7), pp.1820-1832.
Lewis, Y.E., Galesic, A., Levine, P.M., De Leon, C.A., Lamiri, N., Brennan, C.K. and Pratt, M.R.,
2017. O-GlcNAcylation of α-synuclein at serine 87 reduces aggregation without affecting
membrane binding. ACS chemical biology, 12(4), pp.1020-1027.
109
Levine, P.M., Galesic, A., Balana, A.T., Mahul-Mellier, A.L., Navarro, M.X., De Leon, C.A.,
Lashuel, H.A. and Pratt, M.R., 2019. α-Synuclein O-GlcNAcylation alters aggregation and
toxicity, revealing certain residues as potential inhibitors of Parkinson’s disease. Proceedings of
the National Academy of Sciences, 116(5), pp.1511-1519.
De Leon, C.A., Lang, G., Saavedra, M.I. and Pratt, M.R., 2018. Simple and efficient preparation
of O-and S-GlcNAcylated amino acids through InBr3-catalyzed synthesis of β-N-acetylglycosides
from commercially available reagents. Organic letters, 20(16), pp.5032-5035.
Luk, K.C., Covell, D.J., Kehm, V.M., Zhang, B., Song, I.Y., Byrne, M.D., Pitkin, R.M., Decker,
S.C., Trojanowski, J.Q. and Lee, V.M.Y., 2016. Molecular and biological compatibility with host
alpha-synuclein influences fibril pathogenicity. Cell reports, 16(12), pp.3373-3387.
Marotta, N.P., Lin, Y.H., Lewis, Y.E., Ambroso, M.R., Zaro, B.W., Roth, M.T., Arnold, D.B.,
Langen, R. and Pratt, M.R., 2015. O-GlcNAc modification blocks the aggregation and toxicity of
the protein α-synuclein associated with Parkinson's disease. Nature chemistry, 7(11), pp.913-920.
Morris, M., Knudsen, G.M., Maeda, S., Trinidad, J.C., Ioanoviciu, A., Burlingame, A.L. and
Mucke, L., 2015. Tau post-translational modifications in wild-type and human amyloid precursor
protein transgenic mice. Nature neuroscience, 18(8), pp.1183-1189.
Muir, T.W., Sondhi, D. and Cole, P.A., 1998. Expressed protein ligation: a general method for
protein engineering. Proceedings of the National Academy of Sciences, 95(12), pp.6705-6710.
Peduzzo, A., Linse, S. and Buell, A.K., 2020. The properties of α-synuclein secondary nuclei are
dominated by the solution conditions rather than the seed fibril strain. Acs Chemical
Neuroscience, 11(6), pp.909-918.
110
Pinho, T.S., Correia, S.C., Perry, G., Ambrósio, A.F. and Moreira, P.I., 2019. Diminished O-
GlcNAcylation in Alzheimer's disease is strongly correlated with mitochondrial
anomalies. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1865(8), pp.2048-
2059.
Tavassoly, O., Yue, J. and Vocadlo, D., 2020. Pharmacological inhibition and knockdown of O ‐
GlcNAcase reduces cellular internalization of α ‐synuclein pre ‐formed ‐fibrils. The FEBS Journal.
Wang, A.C., Jensen, E.H., Rexach, J.E., Vinters, H.V. and Hsieh-Wilson, L.C., 2016. Loss of O-
GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration. Proceedings of
the National Academy of Sciences, 113(52), pp.15120-15125.
Wang, Z., Udeshi, N.D., O'Malley, M., Shabanowitz, J., Hunt, D.F. and Hart, G.W., 2010.
Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of
chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass
spectrometry. Molecular & Cellular Proteomics, 9(1), pp.153-160.
Wan, Q. and Danishefsky, S.J., 2007. Free ‐radical ‐based, specific desulfurization of cysteine: a
powerful advance in the synthesis of polypeptides and glycopolypeptides. Angewandte
Chemie, 119(48), pp.9408-9412.
Wilhelm, B.G., Mandad, S., Truckenbrodt, S., Kröhnert, K., Schäfer, C., Rammner, B., Koo, S.J.,
Claßen, G.A., Krauss, M., Haucke, V. and Urlaub, H., 2014. Composition of isolated synaptic
boutons reveals the amounts of vesicle trafficking proteins. Science, 344(6187), pp.1023-1028.
Yang, X. and Qian, K., 2017. Protein O-GlcNAcylation: emerging mechanisms and
functions. Nature Reviews Molecular Cell Biology, 18(7), p.452.
111
Yuzwa, S.A., Shan, X., Jones, B.A., Zhao, G., Woodward, M.L., Li, X., Zhu, Y., McEachern, E.J.,
Silverman, M.A., Watson, N.V. and Gong, C.X., 2014. Pharmacological inhibition of O-
GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP
mutant mice. Molecular Neurodegeneration, 9(1), pp.1-14.
Zhao, K., Lim, Y.J., Liu, Z., Long, H., Sun, Y., Hu, J.J., Zhao, C., Tao, Y., Zhang, X., Li, D. and
Li, Y.M., 2020. Parkinson's disease-related phosphorylation at Tyr39 rearranges α-synuclein
amyloid fibril structure revealed by cryo-EM. bioRxiv.
Zhu, Y., Shan, X., Safarpour, F., Erro Go, N., Li, N., Shan, A., Huang, M.C., Deen, M., Holicek,
V., Ashmus, R. and Madden, Z., 2018. Pharmacological inhibition of O-GlcNAcase enhances
autophagy in brain through an mTOR-independent pathway. ACS chemical neuroscience, 9(6),
pp.1366-1379.

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Chapter 4. O-GlcNAc modification inhibits the calpain-mediated cleavage of α-synuclein
Introduction
O-GlcNAc modification (O-GlcNAcylation) refers to the modification of serine or threonine side
chains by the monosaccharide N-acetyl-glucosamine (Figure 4-1 A). (Zachara and Hart, 2002;
Bond and Hanover, 2015) This posttranslational modification occurs on intracellular proteins and
has been found in the nucleus, cytosol, and mitochondria. This modification is added by the
enzyme O-GlcNAc transferase (OGT) and is removed by the enzyme O-GlcNAcase (OGA),
(Vocadlo, 2012) neither which display a strong preference for any particular amino acid sequence.
Many different classes of proteins are known to be modified by O-GlcNAc, including several of
the proteins that are known to form toxic aggregates in neurodegenerative diseases, (Yuzwa and
Vocadlo, 2014) suggesting that O-GlcNAc may affect the normal and/or pathological functions of
these proteins. Notably, Vocadlo and co-workers demonstrated that enzymatic O-GlcNAcylation
of the microtubule stabilizing protein tau inhibited its aggregation in vitro. (Yuzwa et al.,
2012) Furthermore, they developed a potent small-molecule inhibitor of OGA, Thiamet-G, that
can raise O-GlcNAcylation levels in both cells and in vivo. Treatment of an Alzheimer's
disease mouse model with this inhibitor resulted in lower levels of tau hyperphosphorylation, a
hallmark of tau aggregation, and fewer protein aggregates in the mouse brains. (Yuzwa et al., 2012;
Yuzwa et al., 2008) Together these results support a potential therapeutic strategy where increasing
O-GlcNAcylation levels will slow protein aggregation and the progression of disease.
______________
Paul M. Levine, Yuka E. Lewis, Cesar A. De Leon, Aaron Balana and Nicholas Marotta
(University of Southern California) contributed to the work presented in this chapter.

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We have focused on the roles of posttranslational modifications, including O-GlcNAcylation, on
the aggregation and degradation of α-synuclein, the major aggregating protein in Parkinson’s
disease (PD) and related Lewy body dementias. (Lashuel et al., 2013) α-Synuclein is 140 amino
acids in length and is enriched at pre-synaptic termini, where it appears to play a role in vesicle
trafficking and membrane remodeling. (Emanuele et al., 2015) α-Synuclein exists as a natively
unstructured protein in the cytosol but will form an extended α-helix when it comes into contact
with cellular membranes,( Jao et al., 2008; Varkey et al., 2010; Mizuno et al., 2012) and this
equilibrium is likely important for its normal functions. During the progression
of neurodegeneration, however, α-synuclein forms β-sheet rich aggregates that
contain amyloid fibers. (Fink, 2006) Several point mutants of α-synuclein that increase its
aggregation have been identified that cause early-onset PD, demonstrating a causative role for this
process in human disease. (Polymeropoulos et al., 1997; Kruger et al., 1998; Zarrnz et al., 2004;
Chartier-Harlin et al., 2004; Singleton et al., 2003) Notably, a growing set of data shows that these
fibers can spread along neuronal connections where they can seed the aggregation of additional
protein. (Brettschneider et al., 2015; Recasens and Dehay, 2014) α-Synuclein is subjected to a
range of posttranslational modifications that can play important roles in PD. (Oueslati et al.,
2010) While enzymatic modification of α-synuclein has enabled the analysis of certain
modifications, their site-specific installation using synthetic protein chemistry has played an
instrumental role in the evaluation of modified α-synuclein biochemistry. (Pratt et al., 2015) For
example, Brik, Lashuel, and we used protein chemistry to demonstrate that ubiquitination of α-
synuclein can inhibit its aggregation and promote its degradation by the proteasome. (Hejjaoui et
al., 2010; Haj-Yahya et al., 2013; Meier et al., 2012; Abeywardana et al., 2013)

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Figure 4-1. O-GlcNAc modification and calpain cleavage of α-synuclein. (A) O-GlcNAc
modification is the dynamic addition of N-acetyl-glucosamine to serine and threonine residues of
intracellular proteins. (B) α-Synuclein, the major aggregating protein in Parkinson’s disease, is O-
GlcNAcylated at several residues in vivo. (C) Monomeric α-synuclein has been shown to be
proteolytically cleaved by the enzyme calpain after several residues.
We also used a similar chemical strategy to show that these effects are dependent on the site of
modification and that the ubiquitin-like modifier SUMO will also inhibit α-synuclein aggregation.
(Abeywardana et al., 2015) In the case of O-GlcNAcylation, a variety of in
vivo proteomics experiments have identified many different O-GlcNAcylation sites on α-
synuclein (Figure 4-1B). ( Wang et al., 2010; Alfaro et al., 2012; Wang et al., 2009; Morris et al.,
2015) We have previously used synthetic protein chemistry to prepare α-synuclein with O-
GlcNAcylation at two of these sites, threonine 72 (T72) and serine 87 (S87). (Marotta et al., 2015;
Lewis et al., 2017) Using a variety of biochemical experiments, we found that neither of these
modifications inhibit the interaction of α-synuclein with membranes; however, both modifications
inhibit protein aggregation, with O-GlcNAcylation at T72 having a stronger inhibitory effect.

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Figure 4-2. Synthesis of α-synuclein bearing site-specific O-GlcNAcylation. (A) α-Synuclein
bearing an O-GlcNAc modification at either threonine 72 (T72) or serine 82 (S87) was
retrosynthetically deconstructed into two recombinant proteins and a synthetic peptide that were
assembled by native chemical ligation. (B) Both protein products were characterized by RP-HPLC
and ESI-MS.

Here, we use synthetic protein chemistry to explore the potential cross-talk between these
modifications and another critical posttranslational modification of α-synuclein, proteolytic
cleavage. While the majority of aggregated α-synuclein in the brains of PD patients is full-length,
several truncations have been observed. (Li et al., 2005; Liu et al., 2005) Many of these cleavages
remove the C-terminal region of α-synuclein and increase protein aggregation in vitro and in over
expression experiments. (Anderson et al., 2006) Although it is not exactly clear
which proteases are responsible for the cleavage of α-synuclein in vivo, several different enzymes
have been implicated. One of those enzymes is the calcium-dependent protease calpain. Calpain
has been shown to cleave soluble α-synuclein to generate several different fragments including C-
terminal truncated forms, 1–57, 1–73, 1–75, and 1–83 (Figure 4-1C). (Mishizen-Eberz et al.,
2003) Notably, these cleavage sites lie within the region of α-synuclein that is necessary for
aggregation and therefore inhibit aggregation in vitro. (Mishizen-Eberz et al., 2005 Additionally,
α-synuclein fibers can be cleaved near the C-terminus after residues 114 and 122, which promotes
the seeded aggregation of additional soluble protein. (Mishizen-Eberz et al., 2005) The same α-
116
synuclein fragments are found in PD brains where they co-localize with calpain, (Duftly et al.,
2007) and the levels of calpain activity are correlated with disease progression in PD mouse
models. (Crocker et al., 2003; Diepenbroek et al., 2014) Given, the close proximity of the some
calpain cleavage sites to O-GlcNAc modification at T72 and S87, we chose to directly test if these
O-GlcNAcylation events would affect α-synuclein proteolysis. To accomplish this goal, we first
used an expressed protein ligation strategy to prepare site-specifically O-GlcNAcylated α-
synuclein, which we then incubated with calpain. As expected, we found that this protease can
rapidly cleave unmodified α-synuclein to generate the previously identified protein fragments. In
contrast, both sites of O-GlcNAcylation strongly inhibited proteolysis, and the minor cleavage
products that were formed differed between the two modified α-synuclein proteins. These results
suggest that in addition to its direct inhibitory effect on protein aggregation, α-synuclein O-
GlcNAcylation has the potential to inhibit α-synuclein proteolysis.

Results and Discussion
In order to prepare α-synuclein with O-GlcNAcylation at either T72 or S87, we followed our
previously published synthetic routes. α-Synuclein O-GlcNAcylated at T72, α-synuclein(gT72),
was retrosynthetically deconstructed (Figure 4-2A) into a recombinant
protein thioester 1 consisting of residues 1–68, a synthetic O-GlcNAcylated peptide 2 (residues
69–75), and a recombinant protein 3 (residues 76–140) with an N-terminal cysteine residue.
Likewise, α-synuclein(gS87) can be constructed (Figure 4-2A) from a recombinant protein
thioester 4 (residues 1–75), a glycopeptide thioester 5 (residues 76–90), and a recombinant
protein 6 corresponding to residues 91–140. For both proteins, the recombinant thioesters were
prepared using intein-fusions. More specifically, the synuclein fragments were cloned as in-frame
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genetic fusions to an engineered DnaE intein from Anabaena variabilis and a histidine-
tag for purification developed by the Muir lab. (Shah et al., 2012) These proteins were
heterologously expressed in E. coli, followed by purification by nickel chromatography,
and thiolysis of the intein. The corresponding protein thioesters (1 and 4) where then isolated by
RP-HPLC and concentrated by lyophilization. The glycopeptides 2 and 5 were prepared by solid
phase peptide synthesis on commercially available Dawson Dbz resin, which enables the
generation of peptide thioesters. Notably, these peptides contained a N-
terminal thioproline residue to prevent auto-ligation. The O-
GlcNAcylated serine and threonine building blocks were prepared from the corresponding Fmoc-
protected amino acids and glucosamine using a thioglycoside approach. (Marotta et al., 2012)
Recombinant proteins 3 and 6 were expressed in E. coli followed by RP-HPLC purification, and
conveniently the initiator methionine residues were removed during expression. With these
proteins in hand, iterative ligation reactions were carried out by first reacting
fragments 2 and 3 or 5 and 6, followed by removal of the N-terminal protecting group. After
purification by RP-HPLC these proteins were then incubated with the appropriate protein thioester
(either 1 or 3) to give full-length α-synuclein. Finally, radical-based desulfurization was use to
transform the cysteine residues required for the ligation reactions to the native alanine residues,
resulting in α-synuclein(gT72) or α-synuclein(gS87) with no primary sequence mutations. These
synthetic proteins were purified by RP-HPLC and the identities were confirmed by ESI-MS as
previously described (Figure 4-2B).

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Figure 4-3. O-GlcNAcylation blocks the cleavage of α-synuclein by calpain. Unmodified α-
synuclein or O-GlcNAcylated protein, α-synuclein(gT72) or α-synuclein(gS87), were incubated
with calpain for the indicated lengths of time before termination of the enzymatic reaction by
addition of SDS-loading buffer. The proteins were then separated by SDS-PAGE and visualized
by colloidal silver staining.

To determine if O-GlcNAcylation at T72 or S87 affected α-synuclein cleavage by calpain, we used
both SDS-PAGE and RP-HPLC. Recombinant calpain (2 units mL−1) was incubated with 50 μM
concentrations of either unmodified α-synuclein (from recombinant expression), α-
synuclein(gT72), or α-synuclein(gS87) in HEPES buffer containing 5 mM DTT (pH = 7.2) at
37 °C for up to 30 min. After different lengths of time (0, 5, 10, 15, and 30 min), the enzymatic
reactions were quenched by addition of SDS-containing loading buffer and heating to 100 °C for
10 min. The proteins were then separated by SDS-PAGE and then detected by colloidal silver
staining and imaged on a ChemiDoc XRS (Bio-Rad) (Figure 4-3). As expected, wild-type α-
synuclein was rapidly degraded while, α-synuclein(gT72) and α-synuclein(gS87) were very
resistant to cleavage. Interestingly, the pattern of cleavage bands that appear at lower molecular
weights are different for the two O-GlcNAcylated proteins, indicating that the site of the
modification can play a role in the inhibition. In an effort to identify the calpain-dependent
119
cleavage products that were formed, we also analyzed the enzymatic reaction by RP-HPLC and
ESI-MS. The same reactions were initiated and allowed to proceed for 30 min. At this time, the
reactions were quenched by heating at 100 °C for 10 min. Upon cooling, 100 μL of reaction was
directly injected into the HPLC and fractions were collected for mass analysis (Figure 4-4).
Consistent with SDS-PAGE, no full-length, unmodified α-synuclein was detected after 30 min of
incubation with calpain. The major peaks detected in the unmodified cleavage reaction were
analyzed by ESI-MS and corresponded to known cleavage sites of calpain after residues 57, 73,
75, and 83. Consistent with our SDS-PAGE analysis, full-length α-synuclein(gT72) and α-
synuclein(gS87) were identified as the main peak in their respective chromatograms. The majority
of the small cleavage products that were analyzed by ESI-MS did not produce strong signals due
to their low abundance. However, we were able to identify cleavage of α-synuclein(gT72) after
residue 33 and α-synuclein(gS87) after residue 57. Cleavage after residue 33 is notable, as it has
not been observed from the proteolysis of unmodified α-synuclein.

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Figure 4-4.Identification of thea-synuclein-derived fragments after calpain cleavage. (A)
Unmodifieda-synuclein was incubated with calpain for 30 min and the enzymaticreaction was then
quenched by heating to 100°C. Protein fragments were then separated by RP-HPLC and identified
using ESI-MS. (B and C)a-Synuclein(gT72) ora-synuclein(gS87) were subjected to calpain
cleavage and analysis as in A.

Conclusion
121
Here, we have described the further application of synthetic protein chemistry to investigate the
roles of posttranslational modifications on proteins. Specifically, we demonstrate that α-
synuclein that has been site-specifically O-GlcNAcylated is notably resistant to cleavage by the
enzyme calpain, which has been shown to proteolyze α-synuclein in vitro and potentially in PD.
The exact consequences of calpain proteolysis are unknown in PD. α-Synuclein fragments that can
result from calpain cleavage have been found in aggregates from diseased brains, (Dufty et al.,
2007) but cleavage of α-synuclein at many of the sites in the middle of the protein are known to
generate fragments that do not aggregate in vitro. (Mishizen-Eberz et al., 2003) In vivo models of
PD show that calpain activity correlates with severity of the disease phenotype, but this could be
due to reasons other than α-synuclein cleavage. (Crocker et al., 2003; Diepenbroek et al.,
2014) Therefore, the exact physiological consequences of O-GlcNAc blockage of α-synuclein
proteolysis is not clear. However, since O-GlcNAc directly inhibits protein aggregation, we
believe that it should still be viewed as an overall protective modification. Additionally, we predict
that O-GlcNAc could similarly inhibit the cleavage of α-synuclein by the as of yet
unidentified proteases that generate aggregation-prone protein fragments of α-synuclein in PD.
Notably, O-GlcNAcylation is able to inhibit α-synuclein cleavage at sites that are fairly distant in
the primary protein sequence. For example, while α-synuclein(gS87) is only cleaved at low levels
after residue 57. This suggests that O-GlcNAc is not simply acting as steric bulk in the active site
of calpain. We believe that this is due to the natively unfolded nature of α-synuclein, which we
predict can cause non-specific interactions with a variety of proteins. These interactions that
mediate some of the binding to calpain could be distant from the protease active site, enabling
other areas of α-synuclein to be cleaved. Our previous experiments with O-GlcNAcylation and
protein aggregation indicate that this modification promotes the solubility of α-synuclein, which
122
could explain our results here by preventing non-specific interactions with calpain. In summary,
we have used synthetic protein chemistry to demonstrate that O-GlcNAcylation of α-synuclein can
have site-specific inhibitory effects on its cleavage, with potentially important implications for the
generation of aggregation-prone fragments in PD.
Material and Methods
Peptide synthesis.
All peptides were synthesized using standard Fmoc solid-phase chemistry on Dawson Dbz AM®
(Novabiochem, 0.49 mmol g−1) resin using HBTU (5 eq, Novabiochem) and DIEA (10 eq,
Sigma) in DMF for 1 hr. For activated glycosylated amino acids, 2 eq of monomer in 3 mL DMF
was coupled overnight. When peptides were completed, acetyl groups were deprotected
with hydrazine monohydrate (80% v/v in MeOH) twice for 30 min with mixing. Following
deprotection, Dawson linker peptides were activated with p-nitrophenyl-chloroformate (5 eq in
DCM, 1.5 h, mixing) followed by treatment with excess DIEA (0.5 M in DMF) for 30 min to
cyclize the Dbz linker. All peptides were then cleaved (95:2.5:2.5 TFA/H2O/Triisopropylsilane)
for 3.5 h at room temperature, precipitated out of cold ether and lyophilized. Following
lyophilization, crude peptides were resuspended in thiolysis buffer (150 mM NaH2PO4, 150 mM
MESNa, pH 7.0) and incubated at room temperature for 2 h prior to purification. All peptides were
purified by reverse-phase HPLC using preparative chromatography, characterized by mass
analysis using ESI-MS, and sequence purity was assessed by analytical HPLC.
Protein expression and purification.
General expression and purification methods were similar to those previously reported. Briefly,
BL21(DE3) chemically competent E. coli (VWR) were transformed with plasmid DNA by heat
shock and plated on selective LB agar plates containing 50 μg mL−1 kanamycin (C-terminus) or
123
100 μg mL−1 ampicillin (N-terminus). Single colonies were then inoculated, grown to OD600 of
0.6 at 37 °C shaking at 250 rpm, and then expression was induced with IPTG (final concentration:
0.5 mM) at 25 °C shaking at 250 rpm for 18 h. Pellets were harvested by centrifugation (8000g,
30 min, 4 °C) and lysed. C-terminus was acidified (pH 3.5 with HCl), centrifuged, dialyzed against
3 × 1 L of 1% acetic acid in water (degassed with N2, 1 h per L), and purified by HPLC. N-
terminus was loaded onto a Ni-NTA purification column (HisTrap FF Crude, GE Healthcare) and
eluted fractions were dialyzed against 3 × 1 L (100 mM NaH2PO4, 150 mM NaCl, 1 mM EDTA,
1 mM TCEP HCl, pH 7.2) and concentrated. Sodium mercaptoethane sulfonate (MESNa) was
added to a final concentration of 200 mM along with fresh TCEP (2 mM final concentration)
overnight to generate the protein thioester, and then purified by HPLC. Pure protein fragments
were characterized by analytical RP-HPLC and ESI-MS.
Expressed protein ligation.
General ligation and purification methods were similar to those previously reported. Briefly, C-
terminal fragment (2 mM) and peptide fragment (4 mM) were dissolved in ligation buffer
(300 mM NaH2PO4, 6 M guanidine HCl, 100 mM MESNa, 1 mM TCEP, pH 7.8) and allowed to
react at room temperature. Following completion, as monitored by HPLC, the pH was adjusted to
4 with HCl and methoxylamine was added (final concentration 100 mM) to deprotect the N-
terminal thiazolidine group. Following HPLC purification, N-terminal fragment (8 mM) and
ligation product fragment (2 mM) were dissolved in ligation buffer, and following completion was
purified by HPLC. Finally, radical desulfurization was carried out using desulfurization buffer
(200 mM NaH2PO4, 6 M guanidine HCl, 300 mM TCEP, pH 7.0) containing 2% v/v ethanethiol,
10% v/v tertbutyl-thiol, and the radical initiator VA-061 (as a 0.2 M stock in MeOH). The reaction
was heated to 37 °C for 15 h and then purified by RP-HPLC to yield synthetic α-synuclein.
124
Proteolysis reaction.
Cleavage of purified α-synuclein proteins (50 μM, 150 μL reaction volume) was carried out
using calpain (EMD Millpore, 2 units mL−1) in buffer containing 40 mM HEPES, pH 7.5, and
5 mM dithiothreitol at 37 °C. Calpain cleavage was initiated by the addition of calcium (1 mM
final concentration). Aliquots were removed from the reaction mixture and quenched with SDS-
containing loading buffer at various time points, heated at 100 °C for 10 min, and stored at –20 °C
until SDS-PAGE was performed.
Silver staining.
SDS-PAGE was performed using 16.5% Criterion Tris-Tricine polyacrylamide precast
gels. Silver staining of the gels was performed in accordance with the instructions provided in the
Pierce Silver Stain Plus. Briefly, the resolved proteins were fixed by incubating the gel in 200 ml
of fixative enhancer solution (30% ethanol, 10% acetic acid, 60% deionized distilled water) for
30 min at 25 °C. Following that, the gel was rinsed twice with 10% ethanol for 10 min and twice
with 10% deionized distilled water for 10 min. Gel was then subjected to sensitizer solution
(100 μL sensitizer in 50 mL deionized distilled water) for 1 min followed by washing with
deionized distilled water for 2 min. Next, gel was stained (1 mL enhancer in 50 mL stain solution)
for 30 min followed by washing with deionized distilled water for 1 min. Lastly, gel was developed
(1 mL enhancer in 50 mL developer) for 2 min followed by a 5% acetic acid wash for 10 min.
Stained gel was then imaged using ChemiDoc XRS (Bio-Rad).
HPLC/ESI-MS analysis.
Reverse phase high performance liquid chromatography (RP-HPLC) analysis was performed on
digested proteins following a 30 min incubation with calpain. Briefly, the reactions were quenched
by heating to 100 °C for 30 min followed by direct injection onto the HPLC (0–70% buffer B over
125
60 min). The chromatogram was monitored at 210 nm and fractions were collected for ESI-MS.
Each individual fraction was then acquired on an API 3000 LC/MS-MS system to identify the
corresponding cleaved peptide products. Buffer A: 0.1% TFA in H2O, buffer B: 0.1% TFA, 90%
ACN in H2O.

126

Chapter Four References
Abeywardana, T., Lin, Y. H., Rott, R., Engelender, S., & Pratt, M. R., 2013. Site-specific
differences in proteasome-dependent degradation of monoubiquitinated α-synuclein. Chemistry &
Biology, 20(10), 1207-1213.
Abeywardana, T., & Pratt, M. R., 2015. Extent of inhibition of α-synuclein aggregation in vitro by
SUMOylation is conjugation site-and SUMO isoform-selective. Biochemistry, 54(4), 959-961.
Alfaro, J.F., Gong, C.X., Monroe, M.E., Aldrich, J.T., Clauss, T.R., Purvine, S.O., Wang, Z.,
Camp, D.G., Shabanowitz, J., Stanley, P. and Hart, G.W., 2012. Tandem mass spectrometry
identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc
transferase targets. Proceedings of the National Academy of Sciences, 109(19), pp.7280-7285.
Anderson, J.P., Walker, D.E., Goldstein, J.M., De Laat, R., Banducci, K., Caccavello, R.J.,
Barbour, R., Huang, J., Kling, K., Lee, M. and Diep, L., 2006. Phosphorylation of Ser-129 is the
dominant pathological modification of α-synuclein in familial and sporadic Lewy body
disease. Journal of Biological Chemistry, 281(40), pp.29739-29752.
Bond, M. R., & Hanover, J. A., 2015. A little sugar goes a long way: the cell biology of O-
GlcNAc. Journal of Cell Biology, 208(7), 869-880.
Brettschneider, J., Del Tredici, K., Lee, V.M.Y. and Trojanowski, J.Q., 2015. Spreading of
pathology in neurodegenerative diseases: a focus on human studies. Nature Reviews
Neuroscience, 16(2), pp.109-120.
127
Chartier-Harlin, M.C., Kachergus, J., Roumier, C., Mouroux, V., Douay, X., Lincoln, S.,
Levecque, C., Larvor, L., Andrieux, J., Hulihan, M. and Waucquier, N., 2004. α-synuclein locus
duplication as a cause of familial Parkinson's disease. The Lancet, 364(9440), pp.1167-1169.
Crocker, S.J., Smith, P.D., Jackson-Lewis, V., Lamba, W.R., Hayley, S.P., Grimm, E., Callaghan,
S.M., Slack, R.S., Melloni, E., Przedborski, S. and Robertson, G.S., 2003. Inhibition of calpains
prevents neuronal and behavioral deficits in an MPTP mouse model of Parkinson's
disease. Journal of Neuroscience, 23(10), pp.4081-4091.
Diepenbroek, M., Casadei, N., Esmer, H., Saido, T.C., Takano, J., Kahle, P.J., Nixon, R.A., Rao,
M.V., Melki, R., Pieri, L. and Helling, S., 2014. Overexpression of the calpain-specific inhibitor
calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in
[A30P] αSyn transgenic mice. Human molecular genetics, 23(15), pp.3975-3989.
Dufty, B.M., Warner, L.R., Hou, S.T., Jiang, S.X., Gomez-Isla, T., Leenhouts, K.M., Oxford, J.T.,
Feany, M.B., Masliah, E. and Rohn, T.T., 2007. Calpain-cleavage of α-synuclein: connecting
proteolytic processing to disease-linked aggregation. The American journal of pathology, 170(5),
pp.1725-1738.
Emanuele, M., & Chieregatti, E., 2015. Mechanisms of alpha-synuclein action on
neurotransmission: cell-autonomous and non-cell autonomous role. Biomolecules, 5(2), 865-892.
Fink Acc Chem Res, 39 (2006), The Aggregation and Fibrillation of α-Synuclein, pp. 628-634
Haj-Yahya, M., Fauvet, B., Herman-Bachinsky, Y., Hejjaoui, M., Bavikar, S.N., Karthikeyan,
S.V., Ciechanover, A., Lashuel, H.A. and Brik, A., 2013. Synthetic polyubiquitinated α-Synuclein
reveals important insights into the roles of the ubiquitin chain in regulating its
128
pathophysiology. Proceedings of the National Academy of Sciences, 110(44), pp.17726-17731.
Hejjaoui, M., Haj ‐Yahya, M., Kumar, K. A., Brik, A., & Lashuel, H. A., 2011. Towards
Elucidation of the Role of Ubiquitination in the Pathogenesis of Parkinson’s Disease with
Semisynthetic Ubiquitinated α ‐Synuclein. Angewandte Chemie International Edition, 50(2), 405-
409.
Jao, C. C., Hegde, B. G., Chen, J., Haworth, I. S., & Langen, R., 2008. Structure of membrane-
bound α-synuclein from site-directed spin labeling and computational refinement. Proceedings of
the National Academy of Sciences, 105(50), 19666-19671.
Krüger R, Kuhn W, Müller T, Woitalla D, Graeber M, Kösel S, Przuntek H, Epplen JT, Schols L,
Riess O., 1998. AlaSOPro mutation in the gene encoding α-synuclein in Parkinson's disease.
Nature genetics. 1;18(2):106-8.
Lashuel, H. A., Overk, C. R., Oueslati, A., & Masliah, E., 2013. The many faces of α-synuclein:
from structure and toxicity to therapeutic target. Nature Reviews Neuroscience, 14(1), 38-48.
Levine, P. M., De Leon, C. A., Galesic, A., Balana, A., Marotta, N. P., Lewis, Y. E., & Pratt, M.
R., 2017. O-GlcNAc modification inhibits the calpain-mediated cleavage of α-
synuclein. Bioorganic & medicinal chemistry, 25(18), 4977-4982.
Li, W., West, N., Colla, E., Pletnikova, O., Troncoso, J.C., Marsh, L., Dawson, T.M., Jäkälä, P.,
Hartmann, T., Price, D.L. and Lee, M.K., 2005. Aggregation promoting C-terminal truncation of
α-synuclein is a normal cellular process and is enhanced by the familial Parkinson's disease-linked
mutations. Proceedings of the National Academy of Sciences, 102(6), pp.2162-2167.
129
Liu, C. W., Giasson, B. I., Lewis, K. A., Lee, V. M., DeMartino, G. N., & Thomas, P. J., 2005. A
Precipitating Role for Truncated α-Synuclein and the Proteasome in α-Synuclein Aggregation
IMPLICATIONS FOR PATHOGENESIS OF PARKINSON DISEASE. Journal of Biological
Chemistry, 280(24), 22670-22678.
Marotta, N. P., Cherwien, C. A., Abeywardana, T., & Pratt, M. R., 2012. O ‐GlcNAc Modification
Prevents Peptide ‐Dependent Acceleration of α ‐Synuclein Aggregation. ChemBioChem, 13(18),
2665-2670.
Meier, F., Abeywardana, T., Dhall, A., Marotta, N.P., Varkey, J., Langen, R., Chatterjee, C. and
Pratt, M.R., 2012. Semisynthetic, site-specific ubiquitin modification of α-synuclein reveals
differential effects on aggregation. Journal of the American Chemical Society, 134(12), pp.5468-
5471.
Morris, M., Knudsen, G. M., Maeda, S., Trinidad, J. C., Ioanoviciu, A., Burlingame, A. L., &
Mucke, L., 2015. Tau post-translational modifications in wild-type and human amyloid precursor
protein transgenic mice. Nature neuroscience, 18(8), 1183-1189.
Morris, M., Knudsen, G. M., Maeda, S., Trinidad, J. C., Ioanoviciu, A., Burlingame, A. L., &
Mucke, L., 2015. Tau post-translational modifications in wild-type and human amyloid precursor
protein transgenic mice. Nature neuroscience, 18(8), 1183-1189.
Mishizen ‐Eberz, A.J., Guttmann, R.P., Giasson, B.I., Day III, G.A., Hodara, R., Ischiropoulos, H.,
Lee, V.M.Y., Trojanowski, J.Q. and Lynch, D.R., 2003. Distinct cleavage patterns of normal and
pathologic forms of α ‐synuclein by calpain I in vitro. Journal of neurochemistry, 86(4), pp.836-
847.
130
Mishizen-Eberz, A.J., Norris, E.H., Giasson, B.I., Hodara, R., Ischiropoulos, H., Lee, V.M.Y.,
Trojanowski, J.Q. and Lynch, D.R., 2005. Cleavage of α-synuclein by calpain: potential role in
degradation of fibrillized and nitrated species of α-synuclein. Biochemistry, 44(21), pp.7818-7829.
Mizuno, N., Varkey, J., Kegulian, N. C., Hegde, B. G., Cheng, N., Langen, R., & Steven, A. C.,
2012. Remodeling of lipid vesicles into cylindrical micelles by α-synuclein in an extended α-
helical conformation. Journal of Biological Chemistry, 287(35), 29301-29311.
Oueslati, A., Fournier, M., & Lashuel, H. A., 2010. Role of post-translational modifications in
modulating the structure, function and toxicity of α-synuclein: implications for Parkinson’s disease
pathogenesis and therapies. In Progress in brain research (Vol. 183, pp. 115-145). Elsevier.
Pratt, M. R., Abeywardana, T., & Marotta, N. P., 2015. Synthetic Proteins and Peptides for the
Direct Interrogation of α-Synuclein Posttranslational Modifications. Biomolecules, 5(3), 1210-
1227.
Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide, S.E., Dehejia, A., Dutra, A., Pike, B., Root,
H., Rubenstein, J., Boyer, R. and Stenroos, E.S., 1997. Mutation in the α-synuclein gene identified
in families with Parkinson's disease. science, 276(5321), pp.2045-2047.
Recasens, A. and Dehay, B., 2014. Alpha-synuclein spreading in Parkinson’s disease. Frontiers in
neuroanatomy, 8, p.159.
Shah, N. H., Dann, G. P., Vila-Perelló, M., Liu, Z., & Muir, T. W., 2012. Ultrafast protein splicing
is common among cyanobacterial split inteins: implications for protein engineering. Journal of the
American Chemical Society, 134(28), 11338-11341.
131
Singleton, A.B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J., Hulihan, M.,
Peuralinna, T., Dutra, A.N., Lincoln, S. and Crawley, A., 2003. [alpha]-synuclein locus triplication
causes Parkinson’s disease. Science, 302(5646), pp.841-842.
Varkey, J., Isas, J. M., Mizuno, N., Jensen, M. B., Bhatia, V. K., Jao, C. C., & Langen, R., 2010.
Membrane curvature induction and tubulation are common features of synucleins and
apolipoproteins. Journal of Biological Chemistry, 285(42), 32486-32493.
Vocadlo, D. J., 2012. O-GlcNAc processing enzymes: catalytic mechanisms, substrate specificity,
and enzyme regulation. Current opinion in chemical biology, 16(5-6), 488-497.
Wang, Z., Udeshi, N. D., O'Malley, M., Shabanowitz, J., Hunt, D. F., & Hart, G. W., 2010.
Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of
chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass
spectrometry. Molecular & Cellular Proteomics, 9(1), 153-160.
Wang, Z., Park, K., Comer, F., Hsieh-Wilson, L.C., Saudek, C.D. and Hart, G.W., 2009. Site-
specific GlcNAcylation of human erythrocyte proteins: potential biomarker (s) for
diabetes. diabetes, 58(2), pp.309-317.
Yuzwa, S. A., & Vocadlo, D. J., 2014. O-GlcNAc and neurodegeneration: biochemical
mechanisms and potential roles in Alzheimer's disease and beyond. Chemical Society
Reviews, 43(19), 6839-6858.
Yuzwa, S. A., Shan, X., Macauley, M. S., Clark, T., Skorobogatko, Y., Vosseller, K., & Vocadlo,
D. J., 2012. Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against
aggregation. Nature chemical biology, 8(4), 393-399.
132
Yuzwa, S. A., Macauley, M. S., Heinonen, J. E., Shan, X., Dennis, R. J., He, Y., & Vocadlo, D.
J., 2008. A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau
in vivo. Nature chemical biology, 4(8), 483-490.
Zachara, N. E., and Hart. G.W., 2002. The emerging significance of O-GlcNAc in cellular
regulation Chem Rev, 102, pp. 431-438
Zarranz, J.J., Alegre, J., Gómez ‐Esteban, J.C., Lezcano, E., Ros, R., Ampuero, I., Vidal, L.,
Hoenicka, J., Rodriguez, O., Atarés, B. and Llorens, V., 2004. The new mutation, E46K, of α ‐
synuclein causes parkinson and Lewy body dementia. Annals of Neurology: Official Journal of
the American Neurological Association and the Child Neurology Society, 55(2), pp.164-173.

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Chapter 5. O-GlcNAcylation of α-Synuclein at Serine 87 Reduces Aggregation without
Affecting Membrane Binding
Introduction
The addition of the monosaccharide N-acetyl-glucosamine, or O-GlcNAc modification (Figure 5-
1A), occurs in plants and animals and has been found on hundreds of proteins in the cytosol,
nucleus, and mitochondria. (Hardiville and Hart, 2014) The sugar is added to the side chain
hydroxyl groups of serine and threonine residues of substrate proteins by the enzyme O-GlcNAc
transferase (OGT) and can be rendered dynamic through subsequent removal by O-GlcNAcase
(OGA). There are several lines of evidence that implicate the misregulation of O-GlcNAcylation
in neurodegenerative diseases. (Yuzma et al., 2014; Zhu at al., 2014) Mice with neuron-specific
deletion of OGT have hyperphosphorylated tau and suffer from locomotor defects before dying
within 10 days of birth. (O’Donnell et al., 2004) Decreased global levels of O-GlcNAcylation has
also been observed in the brains of patients who succumbed to Alzheimer’s disease (AD). (Liu et
al., 2004) Furthermore, increasing the levels of O-GlcNAcylation in a mouse model of AD with a
small-molecule inhibitor of OGA slowed the pace of neurodegeneration, (Yuzwa et al., 2012) and
the same small-molecule reduced the amount of hyperphosphorylated tau in healthy rats. (Yuzwa
et al., 2008) Notably, several of the proteins that form the toxic aggregates that are hallmarks of
neurodegenerative diseases are directly modified by O-GlcNAcylation, which can result in
inhibition of protein aggregation. For example, Vocadlo and co-workers enzymatically O-
GlcNAcylated the AD-associated protein tau in a heterogeneous fashion and showed that this
modified protein had a decreased propensity for aggregation. (Yuzwa et al., 2012)
______________
Yuka E. Lewis, Paul M. Levine, Cesar A. De Leon, Natalie Lamiri and Caroline K. Brennan
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(University of Southern California) contributed to the work presented in this chapter.

Figure 5-1. O-GlcNAcylation and α-synuclein. (A) O-GlcNAcylation is the addition of N-
acetyl-glucosamine to serine and threonine residues of intracellular proteins. The modification is
added by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). (B) Various
proteomics experiments on rodent brain samples and human erythrocytes have identified nine
different O-GlcNAcylation sites on αsynuclein.

We previously used a synthetic strategies to build site-specifically O-GlcNAcylated α-synuclein
peptides and the full-length protein, the major aggregating protein in Parkinson’s disease (PD),
and found that the modification completely blocks its aggregation. (Marotta et al., 2012; Marotta
et al., 2015)
α-Synuclein is a small (140 amino acids) protein that is highly enriched in presynaptic neurons of
the central nervous system, (Lashuel et al., 2013) where it appears to be involved in vesicle
remodeling and trafficking. (Emanuele et al., 2015) When in contact with membranes, the protein
forms an extended α-helix that can induce membrane bending, (Dao et al., 2008; Varkey et al.,
2010; Mizuno et al., 2012) while it exists as predominantly an unstructured monomer in solution
and the cytosol. In PD and other synucleinopathies, however, α-synuclein is found in aggregates
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that have the features of the β-sheet rich fibers that are common to all amyloid proteins. (Fink et
al., 2006) Solid-state NMR and EPR experiments have defined the region of α-synuclein that forms
the core of these aggregates to be approximately residues 61–95. (Chen et al., 2007; Vilar et al.,
2008; Tuttle et al., 2016) Several proteomics studies from both neurons and erythrocytes, which
also have high concentrations of α-synuclein, have identified nine different in vivo sites of O-
GlcNAcylation (Figure 5-1B). (Wang et al., 2010; Alfaro et al., 2012; Wang et al., 2009; Morris
et al., 2015) This modification being present at many sites is not necessarily surprising as OGT
prefers to modify unstructured regions of proteins. As mentioned above, we previously used a
synthetic protein chemistry strategy to investigate the consequences of O-GlcNAcylation at α-
synuclein T72. (Marotta et al., 2015) We found that modification at T72 completely blocks in
vitro the first step of the aggregation process, termed nucleation, and had a slight inhibitory effect
on the second elongation step that generates larger fiber structures. Here, we continue to explore
the effects of O-GlcNAcylation on the biophysical properties of α-synuclein. More specifically,
we chose to focus on modification at residue S87. Notably, this serine is mutated to glutamine in
rodent α-synucleins, and mouse synuclein was recently found to inhibit the aggregation of the
human protein. (Fares et al., 2010) Furthermore, S87 is also a site of phosphorylation, and both
the enzymatically phosphorylated protein and the phosphomimetic mutation S87E inhibit α-
synuclein aggregation, although phosphorylation at this site also inhibits membrane binding.
(Paleologou et al., 2010) In a similar strategy to our previous effort, we used chemical ligation to
prepare α-synuclein with O-GlcNAcylation at S87 and found that this modification also inhibits
protein aggregation, albeit to a lesser extent than O-GlcNAcylation at T72 or the S87E mutation.
However, in contrast to phosphorylation, O-GlcNAcylation had no observable effect on the
membrane binding properties of α-synuclein. These results add additional support for the targeting
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of OGA to raise O-GlcNAcylation levels as a treatment strategy for PD. Furthermore, they
demonstrate biophysical differences between O-GlcNAcylation and phosphorylation at the same
site, encouraging the further study of the effects of sterics versus charge in the modulation of
protein aggregation.

Results and Discussion
In order to directly test the effect of O-GlcNAcylation at S87 on α-synuclein aggregation and
membrane binding, we set out to first prepare the modified protein using expressed protein ligation
(EPL). Traditional EPL takes advantage of cysteine residues at the ligation sites through a
transthioesterification reaction followed by an S to N acyl shift. α-Synuclein contains no native
cysteines, so we decided to introduce these residues at positions 76 and 91 in the primary sequence,
which are alanine residues in the native protein. These cysteine residues then enable us to
retrosynthetically deconstruct α-synuclein (Figure 5-2A) into a recombinant protein thioester (1,
residues 1–75), a synthetic peptide (2 or 3, residues 76–90), and a recombinant C-terminal
fragment (4, residues 91–140).

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Figure 5-2. Synthesis and characterization of unmodified and OGlcNAcylated α-synuclein
(α-synuclein(gS87)). (A) α-Synuclein can be retrosynthetically deconstructed into an N-terminal
protein thioester (1), a synthetic peptide (2 or 3), and a C-terminal recombinant protein (4). (B)
Characterization of both synthetic proteins by RP-HPLC and ESI-MS: unmodified α-synuclein
expected mass is 14 460 Da, and the observed mass was 14 461 ± 2 Da; αsynuclein(gS87) expected
mass is 14 663 Da, and the observed mass was 14 666 ± 2 Da.

The recombinant protein thioester 1 was prepared by expression of the corresponding intein fusion
in E. coli followed by thiolysis. The peptides 2 (unmodified) or 3 (O-GlcNAcylated at S87) were
synthesized as the corresponding thioesters by Fmoc-based solid phase peptide synthesis using the
Dawson aminobenzyol resin. (Blanco-Canosa et al., 2008) Finally, fragment 4 was prepared by
expression in E. coli where the critical N-terminal cysteine residue was generated by the action of
an endogenous methionine aminopeptidase. All of these peptide and protein fragments were then
purified by RP-HPLC and characterized by ESI-MS (Supporting Information Figure 5-1).
Incubation of either peptide 2 or 3 and fragment 4 resulted in formation of the ligation product
(Supporting Information Figures 5-2 and 5-3). At this time, the pH of the buffer was reduced, and
the N-terminal cysteine protecting-group was removed using methoxylamine. The resulting
proteins 5 and 6 were purified by RP-HPLC and characterized by ESI-MS (Supporting
Information Figures 5-2 and 5-3). These proteins were then incubated separately with the
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recombinant protein thioester 1 to yield the corresponding unmodified or O-GlcNAcylated
proteins 7 and 8 (Supporting Information Figure 5-4). In order to generate the α-synuclein proteins
with no mutations, the cysteine residues at the ligation sites were then transformed to the native
alanine residues using radical-mediated desulfurization (Supporting Information Figure 5-5). The
final products 9 and 10 were then purified by RP-HPLC and characterized by ESI-MS (Figure 5-
2B).

Figure 5-3. O-GlcNAcylation of α-synuclein at S87 inhibits protein aggregation. (A)
Synthetic, unmodified α-synuclein aggregates in the same fashion as recombinant protein.
Recombinant or unmodified, synthetic α-synuclein was incubated under aggregation conditions
(50 μM concentration and agitation at 37 °C) for the indicated lengths of time before analysis by
ThT fluorescence (λex = 450 nm, λem = 482 nm). The y-axis shows the foldincrease of
fluorescence compared with the corresponding protein at t = 0. Error bars represent ± SEM from
the mean of biological replicates (n = 3). (B) The structures formed in the same aggregation
reactions were visualized by TEM after 168 h. Scale bars, 500 nm. (C) O-GlcNAcylation and a
phosphomimetic mutation at S87 inhibit aggregation. Recombinant α-synuclein, α-
synuclein(gS87), or α-synuclein(S87E) were subjected to aggregation conditions (50 μM
concentration and agitation at 37 °C) for indicated lengths of time before analysis by ThT
fluorescence (λex = 450 nm, λem = 482 nm). The y-axis shows the fold-increase of fluorescence
compared with the corresponding protein at t = 0. Error bars represent ± SEM from the mean of
biological replicates (n = 3), and statistical significance was calculated using a two-tailed Student’s
t test. (D) The same reactions were analyzed by TEM after 168 h. (E) Aliquots from the same
reactions were collected and the soluble fractions collected by centrifugation. These soluble
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proteins were then separated by SDS-PAGE and visualized by Coomassie blue staining. The data
are representative of two biological experiments.
With the synthetic proteins in hand, we first compared the unmodified, synthetic α-synuclein to
fully recombinant protein. Analysis by circular dichroism (CD) spectroscopy showed that both
proteins were unstructured in solution (Supporting Information Figure 5-6A) and dynamic light
scattering (DLS) analysis demonstrated that both proteins were monomeric in nature (Supporting
Information Figure 5-6B). These two proteins were then subjected in triplicate to aggregation
conditions at a concentration of 50 μM for 7 days. Reaction aliquots were removed after 72, 120,
and 168 h for visualization of any fiber formation using thioflavin T (ThT) fluorescence (Figure
5-3A), which demonstrated that synthetic and recombinant α-synuclein aggregated with essentially
the same kinetics. To visualize the structure of the fibers that formed, aliquots from the end of the
aggregation assay were collected, and transmission electron microscopy (TEM) was performed.
Both the recombinant and synthetic proteins formed α-synuclein fibers that are consistent with
amyloid structures (Figure 5-3B). These data demonstrate that our synthetic preparation of α-
synuclein did not affect its biophysical properties. Next, we set out to determine the consequences
of O-GlcNAcylation at S87 using our synthetic protein, termed α-synuclein(gS87). As mentioned
above, S87 is also a known site of phosphorylation, so we also expressed the S87E mutant of α-
synuclein (α-synuclein(S87E), Supporting Information Figure 5-7), as this glutamic acid mutant
has been shown to recapitulate some of the effects of phosphorylation at the same site. CD
spectroscopy showed that neither O-GlcNAcylation nor the S87E mutation induced any secondary
structure in α-synuclein (Supporting Information Figure 5-8A), and both proteins were monomeric
in nature as determined by DLS (Supporting Information Figure 5-8B). We next simultaneously
examined the aggregation of unmodified α-synuclein, α-synuclein(gS87), and α-synuclein(S87E)
using a combination of ThT fluorescence, SDS-PAGE analysis, and TEM. First, aggregation
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reactions at a concentration of 50 μM were again initiated in triplicate. After 48, 72, 120, and 168
h, aliquots were removed and analyzed by ThT fluorescence (Figure 5-3C). These data showed
that α-synuclein(gS87) still aggregated but with slower kinetics than the unmodified protein, and
that α-synuclein(S87E) was essentially totally resistant to aggregation, consistent with previously
published results. Next, the structure of any aggregates that formed after 168 h was visualized
using TEM (Figure 5-3D and Supporting Information Figure 5-9). As expected, the unmodified α-
synuclein formed large, regular fibers. In contrast, α-synuclein(S87E) formed only amorphous
structures that are consistent with protein precipitation during the preparation of the TEM grids
and what could be small, irregular structures. α-Synuclein(gS87) formed both shorter fibers and
small structures that are similar to those formed by protein precipitation during TEM preparation.
As a complementary assay, identical aliquots were first subjected to centrifugation to separate the
aggregates from soluble material. The soluble fraction was then concentrated by lyophilization and
then subjected to SDS-PAGE and visualized by Coomassie staining (Figure 5-3E). Consistent with
the ThT fluorescence, the unmodified protein was lost from the soluble fraction to a much higher
extent than either α-synuclein(gS87) or α-synuclein(S87E), further demonstrating that both O-
GlcNAcylation and the phosphomimetic mutation inhibit aggregation. These data support a model
where both O-GlcNAcylation and phosphorylation at S87 have a similar effect on the formation
of fibers, namely the inhibition of the formation of fibers and the formation of small, irregular
structures. However, the phosphomimetic is more strongly inhibitory compared to O-
GlcNAcylation at the same site. The aggregation of α-synuclein is known to be concentration
dependent. Therefore, we next asked if the inhibitory capacity of O-GlcNAcylation at S87 is
dependent on the concentration of the protein, and aggregation reactions were performed at a
concentration of 25 μM. As expected, analysis of protein aggregation by ThT fluorescence (Figure
141
5-4A) showed that aggregation of unmodified α-synuclein over the same time frame was reduced
by ∼5-fold. Again, aggregation mixtures with α-synuclein(S87E) did not give any ThT signal over
the course of the reaction. Interestingly, the consequences of O-GlcNAcylation were more
significant, suggesting that it could have a reasonable inhibitory effect in neurons where the
concentration of α-synuclein has been estimated to be between 10 and 100 μM.

Figure 5-4. O-GlcNAcylation at S87 inhibits α-synuclein aggregation without affecting
membrane binding. (A) O-GlcNAcylation at S87 is more inhibitory toward aggregation at
lower protein concentrations. Recombinant α-synuclein, α-synuclein(gS87), or α-
synuclein(S87E) were subjected to aggregation conditions (25 μM concentration and agitation at
37 °C) for the indicated lengths of time before analysis by ThT fluorescence (λex = 450 nm, λem
= 482 nm). The y-axis shows the fold-increase of fluorescence compared with the corresponding
protein at t = 0. Error bars represent ± SEM from the mean of biological replicates (n = 3), and
statistical significance was calculated using a two-tailed Student’s t test. (B) O-GlcNAcylation at
S87 has no effect on α-synuclein membrane binding. Recombinant α-synuclein, αsynuclein(gS87),
or α-synuclein(S87E) were incubated with a 100-fold excess of the indicated, preformed vesicles
and analyzed using circular dichroism (CD). In the presence of negatively charged vesicles (POPG
or POPS), all of the proteins gave essentially indistinguishable CD spectra consistent with the
formation of an extended α-helix. The introduction of a zwitterionic lipid (POPC) reduced the α-
helix formation equally for both proteins. POPG = 1-palmitoyl-2-oleoyl-snglycero-3-[phospho-
RAC-(1-glycerol)]; POPS = 1-palmitoyl-2-oleoylsn-glycero-3-phospho-L-serine; POPC = 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

We next set out to determine the effects of O-GlcNAcylation on the endogenous function of α-
142
synuclein. As mentioned above, α-synuclein forms an extended α-helix when it comes into contact
with negatively charged membranes, and α-synuclein can remodel membranes in isolation and can
collaborate with other proteins to alter vesicle trafficking. Notably, α-synuclein that has been
enzymatically phosphorylated at S87 has reduced affinity for membranes and therefore may inhibit
these functions; however, the pseudophosphate mutation S87E had no observable effect.
(Paleologou et al., 2010) To determine if O-GlcNAcylation at S87 has consequences on membrane
binding, unmodified α-synuclein, α-synuclein(gS87), or α-synuclein(S87E) was incubated with an
excess of vesicles for 20 min before measuring the induction of any secondary structure by CD
spectroscopy (Figure 5-4B). As expected from previous results, unmodified protein and α-
synuclein(S87E) formed α-helical structures in the presence of the negatively charged lipid
vesicles made up of POPG or POPS, and this binding to POPS vesicles can be reduced by in
introduction of the zwitterionic lipid POPC. Importantly, these same vesicles have been used
extensively for the analysis of α-synuclein membrane interactions. (Auluck et al, 2010; Pfefferkorn
et al., 2012) We also observed no major difference upon O-GlcNAcylation of α-synuclein at S87,
indicating that this modification does not have a notable effect on the affinity or binding mode of
α-synuclein to membranes.

Finally, we set out to explore the effect of different substitutions at residue 87 in more detail. More
specifically, we chose a further set of mutations (S87A, S87D, S87W, and S87K) and expressed
and purified the corresponding α-synuclein proteins (Supporting Information Figure 5-10). We
chose S87A as it represents the loss-of-function mutant that cannot be modified and has been used
in the past to understand the effect of phosphorylation at this position. (Pfefferkorn et al., 2012;
Oueslati et al., 2012) α-Synuclein(S87D) was chosen as it also contains a negative charge, the
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exact position of that charge with respect to the protein backbone is altered compared to S87E.
The S87W mutation increases the steric bulk of this position similarly to O-GlcNAcylation;
however, unlike the carbohydrate, tryptophan is largely hydrophobic in nature. Finally, we chose
S87K to reverse the charge at this position compared to S87E. Wild-type α-synuclein and each of
these mutants were then subjected to aggregation conditions at 25 μM concentration before
analysis by ThT fluorescence (Supporting Information Figure 5-11A). In the case of S87A, we
found that this mutation was slightly inhibitory to aggregation, while S87D was also inhibitory to
a larger extent. In contrast, the S87W and S87K mutations increased the aggregation, with S87K
having a larger effect. Together these data indicate that inhibition of aggregation by O-
GlcNAcylation and phosphorylation (or the pseudophosphate S87E) at this position is not driven
by sterics alone but rather by hydrophilicity or negative charge, respectively. Next, we also
examined the effect(s) of these mutations on α-synuclein membrane binding by incubating the
proteins in excess of POPG vesicles for 20 min before analysis by CD spectroscopy (Supporting
Information Figure 5-11B). Interestingly, all of the mutations besides S87D had very little effect
on the induction of the α-synuclein α-helix, similar to O-GlcNAcylation and S87E. In contrast, α-
synuclein(S87D) showed less membrane binding that is quite similar in magnitude to
phosphorylation at this residue, (Pfefferkorn et al., 2012) suggesting that α-synuclein is very
sensitive to the exact position of the negative charge at serine 87.
Here, we have reported the synthesis of α-synuclein with site-specific O-GlcNAcylation at residue
87 and show that this modification inhibits protein aggregation. However, this modification is less
inhibitory when compared to O-GlcNAcylation at T72, which we previously prepared and
investigated. More specifically, α-synuclein(gT72) is completely resistant to aggregation at 50 μM
concentration, while α-synuclein(gS87) shows significant aggregation at this concentration and
144
even some aggregation at 25 μM. We speculate that this observation can be explained by the effects
of O-GlcNAcylation on the equilibrium between monomeric α-synuclein in solution and protofiber
structures that will seed a rapid extension reaction that forms mature fibers. In the case of O-
GlcNAcylation at T72, the equilibrium strongly favors monomeric protein, while modification at
S87 has a smaller effect that can be overcome at higher monomer concentrations. The site-specific
differences are also consistent with a recent NMR structure of the α-synuclein fiber, (Tuttle et at.,
2016) as well as previous structural models that have been developed using EPR and NMR
spectroscopy, (Chen et al., 2007; Vilar et al., 2008) as well as cryo-electron microscopy.
(Rodriguez et al., 2015) More specifically, T72 lies in the middle of a β-strand in the core structure
that makes up the individual monomers in the fiber, while S87 is toward the edge of this core in a
potentially more flexible turn between two β-strands that may more readily accommodate the steric
bulk of the O-GlcNAcylation. Interestingly, we also find that O-GlcNAcylation is less inhibitory
than the pseudophosphate S87E mutation. Importantly, our data showing that α-synuclein(S87E)
completely blocks aggregation at these concentrations is totally consistent with previous reports.
(Paleologou et al., 2010) Other mutations at S87 ranged from partial inhibition, like S87D, to
potentiation (S87W and S87K) of protein aggregation. Again, these observations can be
rationalized by the NMR structure. (Tuttle et at., 2016) The residues that surround S87 in the fiber
structure all mostly hydrophobic in nature except for a single glutamic acid at residue 82.
Therefore, the negative charge(s) on S87E, S87D, and phosphorylated S87 would strongly promote
interactions in this area with bulk water, while the uncharged carbohydrate could be more easily
desolvated to allow for the formation of the hydrophobic core of the fiber and suggest that the
increased steric bulk of O-GlcNAc at S87 is not sufficient to completely block aggregation.
Furthermore, the relatively hydrophobic bulk of tryptophan in S87W is well tolerated in the
145
aggregate-formation process. Interestingly, the S87K mutation shows the most aggregation, which
might be explained through a favorable interaction with the glutamic acid at residue 82 in α-
synuclein. This result means that we cannot rule out a charge repulsion to explain the strong
inhibition of the longer S87E compared to S87D, as it may be in closer physical proximity to E82.
In contrast to the effects on protein aggregation, we find that O-GlcNAcylation at S87 has
essentially no effect on the ability of α-synuclein to interact with lipid vesicles. This stands in
contrast to the observations that have been made with α-synuclein that has been enzymatically
phosphorylated at S87. (Paleologou et al., 2010) Again, these data fit with previous the structural
characterization of the α-synuclein extended helix using EPR spectroscopy. (Jao et al.,
2008) According to the model developed from these data, S87 lies toward the end of the α-helical
structure (residue 90) and close to the lipid-solvent interface near the phosphate headgroups.
Therefore, it is not surprising that the doubly charged phosphorylated S87 would experience more
electrostatic repulsion compared to either S87E or O-GlcNAcylation at this position. Notably,
however, α-synuclein(S87D) also shows less membrane binding, demonstrating that the exact
position of the negative charge may be equally important. This suggests that O-GlcNAcylation
may have a smaller effect on the healthy functions of α-synuclein when compared to
phosphorylation at the same site. In total, these data add further support for increasing O-
GlcNAcylation as a potential therapeutic strategy in PD and highlight the utility of synthetic
approaches for testing the consequences of protein post-translational modifications.

146

Material and Methods
General.
All chemicals and solvents were purchased from commercial venders (Fluka, EMD, Novagen,
Sigmα-Aldrich, etc.) and used without any additional purification. Growth media (Luria–Bertani,
Miller) were prepared and sterilized according to the manufacture protocol. Antibiotics
(Kanamycin sulfate, EMD, and Ampicillin sodium salt, EMD) were prepared as a stock solution
(50 mg mL–1 and 100 mg mL–1 respectively) and stored at −20 °C. Analytical thin-layer
chromatography was performed on 60 Å F254 silica plates with detection by UV light and/or ceric
ammonium molybdate (CAM). An Agilent Technologies 1200 Series HPLC with Diode Array
Detector was used for reverse phase high performance liquid chromatography (RP-HPLC). Unless
otherwise noted, the HPLC buffers were buffer A, water with 0.1% TFA; buffer B, 90%
acetonitrile, 10% water with 0.1% TFA. Mass spectra were obtained using API 150EX (Applied
Biosystems/MDS SCIEX).

Expression of Recombinant Wild Type α-Synuclein and Mutant α-Synuclein Proteins.
BL21(DE3) E. coli transformed with pRK172 construct containing human wild-type α-synuclein
or α-synuclein(S87E) was grown until its OD600 was above 0.6. The culture was induced by the
addition of 0.5 mM IPTG and incubation for 20 h at 25 °C. The culture was pelleted by
centrifugation at 6000 rpm. The pellet was lysed by three freeze and thaw cycles. The resulting
lysate was resuspended in lysis buffer (500 mM NaCl, 100 mM Tris, 10 mM beta-mercaptoethanol,
1 mM EDTA, pH 8.0) and heated at 80 °C for 10 min. The lysate was allowed to cool down to RT
before the addition of protease inhibitor cocktail (mini complete EDTA free, Roche). The resulting
147
solution was incubated in ice for 30 min, and the cell debris was pelleted by centrifugation at
15 000 rpm for 30 min at 4 °C. The pH of the supernatant was adjusted to 3.5 with 1 M HCl, and
the resulting solution was incubated on ice for an additional 30 min. The lysate was cleared by
centrifugation (15 000 rpm, 30 min, 4 °C) and then dialyzed against 3 × 1 L of a degassed 1%
acetic acid solution. The dialyzed solution was cleared by centrifugation (6000 rpm, 15 min, 4 °C).
α-Synuclein was purified on RP-HPLC (40–60% B over 60 min) and lyophilized. The purified
protein was characterized by RP-HPLC and ESI-MS. The expected mass of wild type α-synuclein
is 14 460 Da, and the observed mass was 14 460 ± 3 Da. The expected mass of α-synuclein(S87E)
is 14 502 Da, and the observed mass was 14 504 ± 1 Da. The expected mass of α-synuclein(S87A)
is 14 435 Da, and The observed mass was 14 435 ± 1 Da. The expected mass of α-synuclein(S87D)
is 14 488 Da, and the observed mass was 14 489 ± 1 Da. The expected mass of α-synuclein(S87W)
is 14 560 Da, and the observed mass was 14 562 ± 2.5 Da. The expected mass of α-
synuclein(S87K) is 14 501 Da, and the observed mass was 14 503 ± 1 Da.
Expression and Purification of α-Synuclein C-Terminal Fragment.
Transformed BL21(DE3) E. coli with pET42b plasmid containing α-synuclein(C91–140) was
expressed and semipurified by HCl treatment and centrifugation as described above for full-length
α-synuclein. α-Synuclein(C91–140) was then further purified on RP-HPLC (10–45% B linear
gradient over 60 min). The purified fragment was characterized by RP-HPLC and ESI-MS. The
expected mass is 5593 Da, and the observed mass was 5595 ± 2 Da.
Expression and Purification of α-Synuclein N-Terminal Thioester.
Transformed BL21(DE3) E. coli with pTXB1 plasmid containing α-synuclein(1–75)-AvaDnaE-
Histag was grown to OD600 above 0.6. The induction of culture was conducted by the addition of
148
0.5 mM IPTG and incubation for 17 h at 25 °C. The culture was centrifuged (6000 rpm, 30 min, 4
°C) and resuspended with lysis buffer (50 mM phosphate, 5 mM imidazole, 300 mM NaCl, pH
8.0). The cells were lysed by tip sonication (30 s/30 s on/off cycle, 6 min total, 4 °C). The lysate
was centrifuged (15 000 rpm, 30 min, 4 °C), and the supernatant was loaded on HisTrap column
(GE healthcare). The protein was bound to the column by washing with five column volumes
(CVs) of buffer A (50 mM phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0) and eluted with
five CVs of buffer B (50 mM phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0). Elution
fractions were pooled and dialyzed against 3× 1 L of degassed buffer C (100 mM phosphate, 150
mM NaCl, 1 mM TCEP, 1 mM EDTA at pH 7.5). The dialyzed solution was incubated with fresh
TCEP (2 mM final concentration) and mercaptoethanesulfonate (MesNa, 200 mM final
concentration) for 3 days to generate C-terminal thioester group. The resulting solution was
purified on C4 semiprep RP-HPLC (35–55% B over 60 min). The expected mass is 7686 Da, and
the observed mass was 7686 ± 1 Da.
Solid Phase Synthesis of Peptide Thioesters.
All peptide synthesis was performed manually using protected Dawson linker resin (Millipore).
Commercially available side chain and N-Fmoc protected amino acids (10 equiv) were activated
by the incubation with DIEA (20 equiv) and HBTU (10 equiv) for 15 min and coupled for 1 h 30
min with nitrogen gas agitation. The completion of reaction was confirmed using Kaiser test. If
needed, a second coupling was performed by incubating 10 equiv of amino acids, 10 equiv of
HOBt, and 12 equiv of DCC for 2 h with nitrogen gas agitation. For incorporation of O-
GlcNAcylated serine, pentafluorophenyl (PFP) activated O-GlcNAc Fmoc-Serine was synthesized
and purified as described previously. (Marotta et al., 2012) Two equivalents of this amino acid
were incubated with the peptide resin overnight. Terminal N-Fmoc protecting groups were
149
removed by 20% piperidine (in DMF) incubation for 5 min, followed by 20 min incubation with
flesh 20% piperidine. For O-GlcNAc modified peptide, the O-acetyl groups were removed after
the completion of the peptide synthesis by treatment of hydrazine hydrate (80% in MeOH) for 30
min twice on resin. Before the cleavage of peptides, the Dawson linker was activated with
treatment of para-nitrophenyl chloroformate (5 equiv in CH2Cl2) for 1 h, followed by incubation
with excess DIEA (5 equiv in DMF) for 30 min. The peptides were cleaved from resin by
incubation in cleavage cocktail (95% TFA, 2.5% water, 2.5% TIS) for 4 h and precipitated in ice-
cold diethyl ether overnight. The pellet was then collected by centrifugation (6000g, 30 min, 4 °C)
and resuspended with water/acetonitrile mixture. The solution was lyophilized, resolubilized, and
incubated in thiolysis buffer (6 M guanidine-HCl, 200 mM phosphate, 100 mM MesNa, pH 7.5)
for 4 h. The desired peptide-thioester was purified on C18 semiprep RP-HPLC (0–30% B over 60
min). Purified peptides were characterized by ESI-MS. The expected mass for unmodified peptide
is 1540 Da, and the observed mass was 1541 Da. The expected mass for O-GlcNAc modified
peptide is 1743 Da, and the observed mass was 1742 Da.
Unmodified α-Synuclein Synthesis.
Lyophilized peptide thioester (4 mg, 1 equiv, 4 mM) and C-terminal fragment (26 mg, 2 equiv)
were solubilized in ligation buffer (6 M guanidine-HCl, 300 mM phosphate, 30 mM TCEP, 30
mM MPAA, pH 7.5) and rocked at RT. The reaction was monitored by RP-HPLC (10–45% B over
60 min). Once the completion of the ligation reaction was confirmed by RP-HPLC and ESI-MS,
the reaction mixture was diluted to 2 mM and acidified to pH 4 with HCl. Methoxyamine (100
mM final concentration) was added, and the resulting solution was incubated at RT for an
additional 4 h. The deprotection of thioproline was confirmed by ESI-MS. The product was
purified on C18 semiprep RP-HPLC and lyophilized. Subsequently, the purified and lyophilized
150
product (1 equiv, 2 mM) and N-terminal thioester (2 equiv) were resuspended in the same ligation
buffer as above. The reaction was rocked at 25 °C and monitored by RP-HPLC (25–60% B over
60 min). Once the reaction was completed, the product was purified by C4 semiprep RP-HPLC
and lyophilized. Radical catalyzed desulfurization was performed by solubilizing the full length
protein at the concentration of 0.75 mg mL–1 in buffer (6 M guanidine-HCl, 300 mM phosphate,
300 mM TCEP, 2.5% v/v ethanethiol, 10% v/v tertbutylthiol, pH 7.0) and the addition of a radical
initiator, VA-061 (200 mM in MeOH, 2 mM final concentration). The reaction was incubated at
37 °C with constant agitation for 16 h. The product was purified with C4 analytical RP-HPLC (25–
60% B over 60 min). The purified unmodified α-synuclein was characterized by RP-HPLC (0–
70% B over 60 min) and ESI-MS. The expected mass is 14 460 Da, and the observed mass was
14 461 ± 2 Da.
Synthesis of O-GlcNAc Modified α-Synuclein.
Purified O-GlcNAc modified peptide thioester (4 mM, 1 equiv) and α-synuclein C-terminal
fragment (2 equiv) were resuspended in the ligation buffer (6 M guanidine-HCl, 300 mM
phosphate, 30 mM TCEP, 30 mM MPAA, pH 7.5), and the resulting solution was rocked for 15 h
at 25 °C. The reaction was monitored by RP-HPLC (10–45% B over 60 min). Once the completion
of the ligation reaction was confirmed by RP-HPLC and ESI-MS, the reaction mixture was diluted
to 2 mM and acidified to pH 4 by the addition of HCl. Methoxyamine (100 mM final concentration)
was added to the solution and incubated for an additional 4 h. Deprotection of thioproline was
observed by ESI-MS. The product was purified by RP-HPLC (10–45% B over 60 min) and
lyophilized. Subsequently, the lyophilized product (1 equiv, 2 mM) was dissolved in the ligation
buffer with N-terminal thioester (2 equiv), and rocked at RT for 30 h. The reaction was monitored
by RP-HPLC. Once the reaction was completed, the product was isolated on C4 analytical RP-
151
HPLC (25–60% B over 60 min). The product fractions were pooled and lyophilized. Finally,
radical-catalyzed desulfurization was performed by resuspending full length O-GlcNAc modified
α-synuclein with cysteines in desulfurization buffer (6 M guanidine-HCl, 200 mM phosphate, 300
mM TCEP, 2.5% ethanethiol, 10% tertbutylthiol, pH 7.0). The reaction was initiated with the
addition of a radical initiator, VA-061 (200 mM in MeOH, 2 mM final concentration), and the
reaction solution was incubated at 37 °C with constant agitation in inert gas for 12 h. Desulfurized
product was purified on C4 analytical RP-HPLC (25–60% B over 60 min). The purified O-GlcNAc
modified α-synuclein was characterized by RP-HPLC and ESI-MS. The expected mass is 14 663
Da, and the observed mass was 14 666 ± 2 Da.
Aggregation Reaction.
Synthetic or recombinant protein was dissolved with bath sonication in a reaction buffer (10 mM
phosphate, 0.05% sodium azide, pH 7.4) to make its final concentration at either 50 μM or 25 μM.
The solution was centrifuged at 15 000 rpm for 15 min at 4 °C to remove any debris, and the
supernatant was aliquoted into triplicate reactions. The samples were incubated at 37 °C with
constant agitation (1000 rpm) in a Thermomixer F1.5 (Eppendorf) for 7 days. At each time point,
solution was aliquoted for ThT analysis.
Circular Dichroism (CD) Spectroscopy.
All circular dichroism (CD) spectra were taken with a Jasco-J-815 spectrometer at RT. Sample
aliquots were diluted to 7.5 μM with the aggregation reaction buffer without sodium azide in a 1
mm path length quartz cuvette at 25 °C. The far UV spectra (195 nm-250 nm) were obtained by
averaging three scans with a 50 nm min–1 scanning speed, 1 nm bandwidth, 0.1 nm step size, and
data integral speed of 4. The buffer readings were subtracted for all samples, and the data were
152
converted into mean residue ellipticity.
Dynamic Light Scattering (DLS).
Dynamic light scattering data were obtained with Wyatt Technologies Dynastar. All samples were
at t = 0 h of aggregation reaction (50 μM). For all data, an average of 10 scans at 25 °C was
obtained with laser power adjusted to intensity of 2.6E6 counts s–1. To calculate radii, Raleigh
sphere approximation was used.
Thioflavin T (ThT) Fluorescence.
α-Synuclein aggregation progression was quantified by ThT fluorescence. Samples from the
aggregation reaction were diluted to 1.25 μM protein concentration with 20 μM ThT dye in the
reaction buffer, followed by brief vortexing and incubation for 2 min. Samples in a 10 mm path
length quartz cuvette were analyzed using a NanoLog spectrofluorometer (Horiba), λex at 450 nm
with a 4 nm slit, λem at 482 nm with a 4 nm slit, and data integration time of 0.1 s, averaging 3
scans. Data were measured in triplicate for all aggregation reaction conditions.
Transmission Electron Microscopy (TEM).
At the end of the aggregation reactions, protein solution was diluted to 15 μM by adding the
reaction buffer, and the diluted solution (10 μL) was incubated with a Formvar coated copper grid
(150 mesh, Electron Microscopy Science) for 5 min. Subsequently, the grid was negatively stained
with 1% uranyl acetate for 2 min and washed three times with 1% uranyl acetate. Each time, excess
liquid was removed with filter paper. The grid was dried for 48 h. Grids were visualized with a
JEOL JEM-2100F transmission electron microscope operated at 200 kV and 600 000×
magnification and an Orius Pre-GIF CCD.
153
SDS-PAGE Analysis.
At each time point, 10 uL of aggregation reaction sample was aliquoted and centrifuged at
20 000g for 1 h at 25 °C. The supernatant was transferred into a new tube and lyophilized to
dryness. The lyophilized sample was solubilized in fresh 8 M urea and 20 mM HEPES buffer (pH
8.0) with subsequent bath sonication for 20 min. The sample was boiled for 10 min with 4× SDS
loading buffer and loaded on 4–20% Criterion precast gel (BioRad) and separated by SDS-PAGE
at 195 V. The gel was stained with Coomassie brilliant blue for 30 min and destained with 1:4:5
acetic acid/water/methanol solution overnight.
Circular Dichroism (CD) of α-Synuclein with Lipids.
All circular dichroism (CD) spectra were collected with Jasco-J-815 spectrometer at RT. Samples
were prepared by mixing 1:100 ratio of a protein and desired lipid mixture and incubated at RT
for 20 min. Lipid vesicles were prepared with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-RAC-
(1-glycerol)] (POPG) or by mixing a different ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-
phospho-l-serine (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Dried
lipid films were solubilized in 10 mM phosphate buffer at pH 7.4 by vortexing. All spectra (190–
250 nm) were collected with a scan rate of 50 nm min–1, bandwidth of 1 nm, data integration time
of 8 s, and 0.1 nm step resolution. Appropriate buffer spectra were subtracted from the final
spectra.
Supplemental Information
154

Figure S 5-1. Characterization of α-synuclein protein fragments. Recombinant protein-
thioester (1) and the recombinant C-terminus (4) were characterized by RP-HPLC and ESI-MS.
1 expected mass is 7,686 Da, and the observed mass was 7,686 ± 1 Da; 4 expected mass is 5,593
Da, and the observed mass was 5,595 ± 2 Da.

Figure S 5-2. Ligation of peptide 2 and protein 4 and the subsequent deprotection to give
protein fragment 5. The ligation reaction was followed by RP-HPLC and the identity of
intermediate product C was confirmed by ESI-MS. This was followed by deprotection in the same
pot to give the product D, which again was confirmed by ESI-MS. C expected mass is 6,992 Da,
and the observed mass was 6,993 ± 1 Da; D expected mass is 6,980 Da, and the observed mass
was 6,979 ± 1 Da.
155

Figure S 5-3. Ligation of peptide 3 and protein 4 and the subsequent deprotection to give
protein fragment 6. The ligation reaction was followed by RP-HPLC and the identity of
intermediate product C was confirmed by ESI-MS. This was followed by deprotection in the same
pot to give the product D, which again was confirmed by ESI-MS. C expected mass is 7,195 Da,
and the observed mass was 7,195 ± 1 Da; D expected mass is 7,184 Da, and the observed mass
was 7,184 ± 1 Da.

Figure S 5-4. Ligation of proteins 5 or 6 and protein-thioester 1 to give proteins 7 or 8. The
ligation reactions were followed by RP-HPLC and the identities of the products C were confirmed
by ESI-MS. Unmodified C expected mass is 15,524 Da, and the observed mass was 15,525 ± 2
Da; O-GlcNAcylated C expected mass is 14,728 ± 2 Da, and the observed mass was 14,728 ± 2
Da.
156

Figure S 5-5. Desulfurization of proteins 7 or 8. The desulfurization reactions were followed by
RP-HPLC and the identities of the products B were confirmed by ESI-MS. Unmodified B expected
mass is 14,460 Da, and the observed mass was 14,461 ± 1 Da; O-GlcNAcylated B expected mass
is 14,663 Da, and the observed mass was 14,666 ± 2 Da.

Figure S 5-6. Structural characterization of α-synuclein(gS87) and α-synuclein(S87E) using
circular dichroism (CD) and dynamic light scattering (DLS). (A) α-Synuclein(gS87) and α-
synuclein(S87E) are unstructured in solution. CD spectra were collected for freshly dissolved
proteins at 7.5 μM concentration. (B) Both proteins were monomeric in nature with Stoke’s Radii
of approximately 4 nm, and no significant, larger peaks. The indicated proteins were analyzed
using DLS at 50 μM concentration.

157

Figure S 5-7. Expression and characterization of α-synuclein(S87E). Recombinant α-
synuclein(S87E) was characterized by RP-HPLC and ESI-MS. Expected mass is 14,502 Da and
the observed mass was 14,504 ± 1 Da.

Figure S 5-8. Structural characterization of synthetic and recombinant unmodified α-
synuclein using circular dichroism (CD) and dynamic light scattering (DLS). (A) Both
recombinant and synthetic αsynuclein are unstructured in solution. CD spectra were collected for
freshly dissolved proteins at 7.5 μM concentration. (B) Both proteins were monomeric in nature
with Stoke’s Radii of approximately 4 nm, and no significant, larger peaks. The indicated proteins
were analyzed using DLS at 50 μM concentration.

158

Figure S 5-9. Transmission electron microscopy (TEM) images of the from the α-synuclein
aggregation reactions. Unmodified α-synuclein, α-synuclein(gS87), or α-synuclein(S87E)
were subjected to aggregation conditions (50 μM concentration and agitation at 37 °C) for
168 h before visualization of any aggregate structures by TEM. Scale bars, 500 nm.

Figure S 5-10. Characterization of the additional α-synuclein mutants S87A, S87D, S87W,
and S87K. The indicated recombinant α-synuclein mutants were characterized by RP-HPLC and
ESI-MS.Expected mass of α-synuclein(S87A) is 14,435 Da, and observed mass was 14,435 ± 1
Da. Expected mass of αsynuclein(S87D) is 14,488 Da, and observed mass was 14,489 ± 1 Da.
Expected mass of αsynuclein(S87W) is 14,560 Da, and observed mass was 14,562 ± 2.5 Da.
Expected mass of αsynuclein(S87K) is 14,501 Da, and observed mass was 14,503 ± 1 Da.

159

Figure S 5-11. Analysis of mutant α-synuclein aggregation and membrane binding. (A)
Mutations of αsynuclein at S87 have different effects on protein aggregation. Recombinant α-
synuclein or the indicated α-synuclein mutants were individually subjected to aggregation
conditions (25 μM concentration and agitation at 37 °C) for the indicated lengths of time before
analysis by ThT fluorescence (λex = 450 nm, λem = 482 nm). The y-axis shows the fold-increase
of fluorescence compared with the corresponding protein at t = 0. Error bars represent ±s.e.m from
the mean of biological replicates (n = 3), and statistical significance was calculated using a two-
tailed Student’s t-test. (B) Mutation of S87 to D inhibits αsynuclein membrane binding.
Recombinant α-synuclein or the indicated α-synuclein mutants were incubated with an 100-fold
excess of the indicated, preformed vesicles and analyzed using circular dichroism (CD). In the
presence of negatively charged vesicles (POPG), most of the proteins gave essentially
indistinguishable CD spectra consistent with the formation of an extended α-helix. However, α-
synuclein(S87D) showed diminished signal corresponding to less membrane binding and α-helix
formation. POPG = 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-RAC-(1-glycerol)].

160
Chapter Five References
Alfaro, J. F., Gong, C.-X., Monroe, M. E., Aldrich, J. T., Clauss, T. R. W., Purvine, S. O., Wang,
Z., Camp, D. G., Shabanowitz, J., Stanley, P., Hart, G. W., Hunt, D. F., Yang, F., and Smith, R.
D., 2012. Tandem mass spectrometry identifies many mouse brain OGlcNAcylated proteins
including EGF domain-specific O-GlcNAc transferase targets. Proc. Natl. Acad. Sci. U. S. A. 109,
7280−7285.
Auluck, P. K., Caraveo, G., and Lindquist, S., 2010. αSynuclein: membrane interactions and
toxicity in Parkinson’s disease. Annu. Rev. Cell Dev. Biol. 26, 211−233.
Blanco-Canosa, J. B., and Dawson, P. E., 2008. An efficient Fmoc-SPPS approach for the
generation of thioester peptide precursors for use in native chemical ligation. Angew. Chem., Int.
Ed. 47, 6851−6855.
Bond, M. R., and Hanover, J. A., 2015. A little sugar goes a long way: the cell biology of O-
GlcNAc. J. Cell Biol. 208, 869−880.
Chen, M., Margittai, M., Chen, J., and Langen, R., 2007. Investigation of alpha-synuclein fibril
structure by site-directed spin labeling. J. Biol. Chem. 282, 24970−24979.
Emanuele, M., and Chieregatti, E., 2015. Mechanisms of AlphaSynuclein Action on
Neurotransmission: Cell-Autonomous and NonCell Autonomous Role. Biomolecules 5, 865−892.
Fares, M.-B., Maco, B., Oueslati, A., Rockenstein, E., Ninkina, N., Buchman, V. L., Masliah, E.,
and Lashuel, H. A., 2016. Induction of de novo α-synuclein fibrillization in a neuronal model for
Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A. 113, E912−21.
161
Fink, A. L., 2006. The Aggregation and Fibrillation of αSynuclein. Acc. Chem. Res. 39, 628−634.
Hardiville, S., and Hart, G. W., 2014. Nutrient Regulation of ́ Signaling, Transcription, and Cell
Physiology by O-GlcNAcylation. Cell Metab. 20, 208−213.
Jao, C. C., Hegde, B. G., Chen, J., Haworth, I. S., and Langen, R., 2008. Structure of membrane-
bound alpha-synuclein from sitedirected spin labeling and computational refinement. Proc. Natl.
Acad. Sci. U. S. A. 105, 19666−19671.
Lashuel, H. A., Overk, C. R., Oueslati, A., and Masliah, E., 2013. The many faces of α-synuclein:
from structure and toxicity to therapeutic target. Nat. Rev. Neurosci. 14, 38−48.
Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G. W., and Gong, C.-X., 2004. O-GlcNAcylation
regulates phosphorylation of tau: a mechanism involved in Alzheimer’s disease. Proc. Natl. Acad.
Sci. U. S. A. 101, 10804−10809.
Marotta, N. P., Cherwien, C. A., Abeywardana, T., and Pratt, M. R., 2012. O-GlcNAc
Modification Prevents Peptide-Dependent Acceleration of α-Synuclein Aggregation.
ChemBioChem 13, 2665− 2670.
Marotta, N. P., Lin, Y. H., Lewis, Y. E., Ambroso, M. R., Zaro, B. W., Roth, M. T., Arnold, D.
B., Langen, R., and Pratt, M. R., 2015. O-GlcNAc modification blocks the aggregation and toxicity
of the protein α-synuclein associated with Parkinson’s disease. Nat. Chem. 7, 913− 920.
Mizuno, N., Varkey, J., Kegulian, N. C., Hegde, B. G., Cheng, N., Langen, R., and Steven, A. C.,
2012. Remodeling of lipid vesicles into cylindrical micelles by α-synuclein in an extended α-
helical conformation. J. Biol. Chem. 287, 29301−29311.
162
Morris, M., Knudsen, G. M., Maeda, S., Trinidad, J. C., Ioanoviciu, A., Burlingame, A. L., and
Mucke, L., 2015. Tau posttranslational modifications in wild-type and human amyloid precursor
protein transgenic mice. Nat. Neurosci. 18, 1183−1189.
O’Donnell, N., Zachara, N. E., Hart, G. W., and Marth, J. D., 2004. Ogt-dependent X-
chromosome-linked protein glycosylation is a requisite modification in somatic cell function and
embryo viability. Mol. Cell. Biol. 24, 1680−1690.
Oueslati, A., Paleologou, K. E., Schneider, B. L., Aebischer, P., and Lashuel, H. A., 2012.
Mimicking Phosphorylation at Serine 87 Inhibits the Aggregation of Human -Synuclein and
Protects against Its Toxicity in a Rat Model of Parkinson’s Disease. J. Neurosci. 32, 1536− 1544.
Paleologou, K. E., Oueslati, A., Shakked, G., Rospigliosi, C. C., Kim, H.-Y., Lamberto, G. R.,
Fernandez, C. O., Schmid, A., Chegini, F., Gai, W. P., Chiappe, D., Moniatte, M., Schneider, B.
L., Aebischer, P., Eliezer, D., Zweckstetter, M., Masliah, E., and Lashuel, H. A., 2010.
Phosphorylation at S87 is enhanced in synucleinopathies, inhibits alpha-synuclein
oligomerization, and influences synuclein-membrane interactions. J. Neurosci. 30, 3184−3198.
Pfefferkorn, C. M., Jiang, Z., and Lee, J. C., 2012. Biophysics of α-synuclein membrane
interactions. Biochim. Biophys. Acta, Biomembr. 1818, 162−171.
Rodriguez, J. A., Ivanova, M. I., Sawaya, M. R., Cascio, D., Reyes, F. E., Shi, D., Sangwan, S.,
Guenther, E. L., Johnson, L. M., Zhang, M., Jiang, L., Arbing, M. A., Nannenga, B. L., Hattne, J.,
Whitelegge, J.,Brewster, A. S., Messerschmidt, M., Boutet, S., Sauter, N. K., Gonen, T., and
Eisenberg, D. S., 2015. Structure of the toxic core of αsynuclein from invisible crystals. Nature
525, 486−490.
163
Tuttle, M. D., Comellas, G., Nieuwkoop, A. J., Covell, D. J., Berthold, D. A., Kloepper, K. D.,
Courtney, J. M., Kim, J. K., Barclay, A. M., Kendall, A., Wan, W., Stubbs, G., Schwieters, C. D.,
Lee, V. M.- Y., George, J. M., and Rienstra, C. M., 2016. Solid-state NMR structure of a
pathogenic fibril of full-length human α-synuclein. Nat. Struct. Mol. Biol. 23, 409−415.
Varkey, J., Isas, J. M., Mizuno, N., Jensen, M. B., Bhatia, V. K., Jao, C. C., Petrlova, J., Voss, J.
C., Stamou, D. G., Steven, A. C., and Langen, R., 2010. Membrane curvature induction and
tubulation are common features of synucleins and apolipoproteins. J. Biol. Chem. 285,
32486−32493.
Vilar, M., Chou, H.-T., Lü hrs, T., Maji, S. K., Riek-Loher, D., Verel, R., Manning, G., Stahlberg,
H., and Riek, R., 2008. The fold of alpha-synuclein fibrils. Proc. Natl. Acad. Sci. U. S. A. 105,
8637−8642.
Wang, Z., Udeshi, N. D., O’Malley, M., Shabanowitz, J., Hunt, D. F., and Hart, G. W., 2010.
Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/
Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass
Spectrometry. Mol. Cell. Proteomics 9, 153−160.
Wang, Z., Park, K., Comer, F., Hsieh-Wilson, L. C., Saudek, C. D., and Hart, G. W., 2009. Site-
Specific GlcNAcylation of Human Erythrocyte Proteins Potential Biomarker(s) for Diabetes.
Diabetes 58, 309−317.
Yuzwa, S. A., and Vocadlo, D. J., 2014. O-GlcNAc and neurodegeneration: biochemical
mechanisms and potential roles in Alzheimer’s disease and beyond. Chem. Soc. Rev. 43,
6839−6858.
164
Yuzwa, S. A., Shan, X., Macauley, M. S., Clark, T., Skorobogatko, Y., Vosseller, K., and Vocadlo,
D. J., 2012. Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation.
Nat. Chem. Biol. 8, 393−399.
Yuzwa, S. A., Macauley, M. S., Heinonen, J. E., Shan, X., Dennis, R. J., He, Y., Whitworth, G.
E., Stubbs, K. A., McEachern, E. J., Davies, G. J., and Vocadlo, D. J., 2008. A potent mechanism-
inspired OGlcNAcase inhibitor that blocks phosphorylation of tau in vivo. Nat. Chem. Biol. 4,
483−490.
Zhu, Y., Shan, X., Yuzwa, S. A., and Vocadlo, D. J., 2014. The emerging link between O-GlcNAc
and Alzheimer disease. J. Biol. Chem. 289, 34472−34481.

165
References
Abeywardana, T. and Pratt, M.R., 2015. Extent of inhibition of α-synuclein aggregation in vitro
by SUMOylation is conjugation site-and SUMO isoform-selective. Biochemistry, 54(4), pp.959-
961.
Abeywardana, T., Lin, Y.H., Rott, R., Engelender, S. and Pratt, M.R., 2013. Site-specific
differences in proteasome-dependent degradation of monoubiquitinated α-synuclein. Chemistry &
Biology, 20(10), pp.1207-1213.
Aguilar, A.L., Hou, X., Wen, L., Wang, P.G. and Wu, P., 2017. A chemoenzymatic histology
method for O-GlcNAc detection. Chembiochem: a European journal of chemical biology, 18(24),
p.2416.
Alfaro, J.F., Gong, C.X., Monroe, M.E., Aldrich, J.T., Clauss, T.R., Purvine, S.O., Wang, Z.,
Camp, D.G., Shabanowitz, J., Stanley, P. and Hart, G.W., 2012. Tandem mass spectrometry
identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc
transferase targets. Proceedings of the National Academy of Sciences, 109(19), pp.7280-7285.
Anderson, J.P., Walker, D.E., Goldstein, J.M., De Laat, R., Banducci, K., Caccavello, R.J.,
Barbour, R., Huang, J., Kling, K., Lee, M. and Diep, L., 2006. Phosphorylation of Ser-129 is the
dominant pathological modification of α-synuclein in familial and sporadic Lewy body
disease. Journal of Biological Chemistry, 281(40), pp.29739-29752.
Auluck, P. K., Caraveo, G., and Lindquist, S., 2010. alpha-Synuclein: membrane interactions and
toxicity in Parkinson’s disease. Annu. Rev. Cell Dev. Biol. 26, 211–233.
Balch, W. E., Morimoto, R. I., Dillin, A. & Kelly, J. W., 2008. Adapting proteostasis for disease
166
intervention. Science 319, 916–919.
Blanco-Canosa, J. B., and Dawson, P. E., 2008. An efficient Fmoc-SPPS approach for the
generation of thioester peptide precursors for use in native chemical ligation. Angew. Chem., Int.
Ed. 47, 6851−6855.
Borghgraef, P., Menuet, C., Theunis, C., Louis, J.V., Devijver, H., Maurin, H., Smet-Nocca, C.,
Lippens, G., Hilaire, G., Gijsen, H. and Moechars, D., 2013. Increasing brain protein O-GlcNAc-
ylation mitigates breathing defects and mortality of Tau. P301L mice. PloS one, 8(12), p.e84442.
Bond, M. R., & Hanover, J. A., 2015. A little sugar goes a long way: the cell biology of O-
GlcNAc. Journal of Cell Biology, 208(7), 869-880.
Braak, H., Del Tredici, K., Rub, U., de Vos, R. A., Jansen Steur, E. N., and Braak, E., 2003. Staging
of brain pathology related to sporadic Parkinson’s disease. Neurobiol. Aging 24, 197–211.
Brettschneider, J., Del Tredici, K., Lee, V.M.Y. and Trojanowski, J.Q., 2015. Spreading of
pathology in neurodegenerative diseases: a focus on human studies. Nature Reviews
Neuroscience, 16(2), pp.109-120.
Buell, A.K., Galvagnion, C., Gaspar, R., Sparr, E., Vendruscolo, M., Knowles, T.P., Linse, S. and
Dobson, C.M., 2014. Solution conditions determine the relative importance of nucleation and
growth processes in α-synuclein aggregation. Proceedings of the National Academy of
Sciences, 111(21), pp.7671-7676.
Burre, J., 2015. The synaptic function of alpha-synuclein. J. Parkinsons Dis. 5, 699–713.
167
Burmann, B.M., Gerez, J.A., Matečko-Burmann, I., Campioni, S., Kumari, P., Ghosh, D., Mazur,
A., Aspholm, E.E., Šulskis, D., Wawrzyniuk, M. and Bock, T., 2020. Regulation of α-synuclein
by chaperones in mammalian cells. Nature, 577(7788), pp.127-132.
Chartier-Harlin, M.C., Kachergus, J., Roumier, C., Mouroux, V., Douay, X., Lincoln, S.,
Levecque, C., Larvor, L., Andrieux, J., Hulihan, M. and Waucquier, N., 2004. α-Synuclein locus
duplication as a cause of familial Parkinson's disease. The Lancet, 364(9440), pp.1167-1169.
Chen, M., Margittai, M., Chen, J., and Langen, R., 2007. Investigation of alpha-synuclein fibril
structure by site-directed spin labeling. J. Biol. Chem. 282, 24970−24979.
Ciechanover, A., 2017. Intracellular protein degradation: from a vague idea thru the lysosome and
the ubiquitin-proteasome system and onto human diseases and drug targeting. Best Pract. Res.
Clin. Haematol. 30, 341–355.
Clague, M. J., Barsukov, I., Coulson, J. M., Liu, H., Rigden, D. J., and Urbe, S., 2013.
Deubiquitylases from genes to organism. Physiol. Rev. 93, 1289–1315.
Clague, M. J., Heride, C., and Urbé, S., 2019. The demographics of the ubiquitin system. Trends
Cell Biol. 25, 417–426.
Cohen, S.I., Linse, S., Luheshi, L.M., Hellstrand, E., White, D.A., Rajah, L., Otzen, D.E.,
Vendruscolo, M., Dobson, C.M. and Knowles, T.P., 2013. Proliferation of amyloid-β42 aggregates
occurs through a secondary nucleation mechanism. Proceedings of the National Academy of
Sciences, 110(24), pp.9758-9763.
Collins, G.A. and Goldberg, A.L., 2017. The logic of the 26S proteasome. Cell, 169(5), pp.792-
168
806.
Comer, F.I., Vosseller, K., Wells, L., Accavitti, M.A. and Hart, G.W., 2001. Characterization of a
mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Analytical
biochemistry, 293(2), pp.169-177.
Crocker, S.J., Smith, P.D., Jackson-Lewis, V., Lamba, W.R., Hayley, S.P., Grimm, E., Callaghan,
S.M., Slack, R.S., Melloni, E., Przedborski, S. and Robertson, G.S., 2003. Inhibition of calpains
prevents neuronal and behavioral deficits in an MPTP mouse model of Parkinson's
disease. Journal of Neuroscience, 23(10), pp.4081-4091.
Danzer, K.M., Ruf, W.P., Putcha, P., Joyner, D., Hashimoto, T., Glabe, C., Hyman, B.T. and
McLean, P.J., 2011. Heat ‐shock protein 70 modulates toxic extracellular α ‐synuclein oligomers
and rescues trans ‐synaptic toxicity. The FASEB journal, 25(1), pp.326-336.
Davidson, W. S., Jonas, A., Clayton, D. F., and George, J. M., 1998. Stabilization of alpha-
synuclein secondary structure upon binding to synthetic membranes. J. Biol. Chem. 273, 9443–
9449.
Dawson, P.E., Muir, T.W., Clark-Lewis, I. and Kent, S.B., 1994. Synthesis of proteins by native
chemical ligation. Science, 266(5186), pp.776-779.
Deol, K.K., Lorenz, S. and Strieter, E.R., 2019. Enzymatic logic of ubiquitin chain
assembly. Frontiers in physiology, 10, p.835.
De Leon, C.A., Lang, G., Saavedra, M.I. and Pratt, M.R., 2018. Simple and efficient preparation
of O-and S-GlcNAcylated amino acids through InBr3-catalyzed synthesis of β-N-acetylglycosides
169
from commercially available reagents. Organic letters, 20(16), pp.5032-5035.
Dettmer U, Newman AJ, von Saucken VE, Bartels T, Selkoe D., 2015. KTKEGV repeat motifs
are key mediators of normal α-synuclein tetramerization: Their mutation causes excess monomers
and neurotoxicity. Proc Natl Acad Sci USA 112:9596–9601.
Dikic, I., 2017. Proteasomal and autophagic degradation systems. Annu. Rev. Biochem. 86, 193–
224.
Diepenbroek, M., Casadei, N., Esmer, H., Saido, T.C., Takano, J., Kahle, P.J., Nixon, R.A., Rao,
M.V., Melki, R., Pieri, L. and Helling, S., 2014. Overexpression of the calpain-specific inhibitor
calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in
[A30P] αSyn transgenic mice. Human molecular genetics, 23(15), pp.3975-3989.
Dikiy, I., Fauvet, B., Jovicic, A., Mahul-Mellier, A. L., Desobry, C., El-Turk, F., Gitler, A. D.,
Dorval, V. and Fraser, P.E., 2006. Small ubiquitin-like modifier (SUMO) modification of natively
unfolded proteins tau and α-synuclein. Journal of Biological Chemistry, 281(15), pp.9919-9924.
Dufty, B.M., Warner, L.R., Hou, S.T., Jiang, S.X., Gomez-Isla, T., Leenhouts, K.M., Oxford, J.T.,
Feany, M.B., Masliah, E. and Rohn, T.T., 2007. Calpain-cleavage of α-synuclein: connecting
proteolytic processing to disease-linked aggregation. The American journal of pathology, 170(5),
pp.1725-1738.
Eliezer, D., Kutluay, E., Bussell, R. Jr., and Browne, G., 2001. Conformational properties of alpha-
synuclein in its free and lipid-associated states. J. Mol. Biol. 307, 1061–1073.
170
Emanuele, M. and Chieregatti, E., 2015. Mechanisms of alpha-synuclein action on
neurotransmission: cell-autonomous and non-cell autonomous role. Biomolecules, 5(2), pp.865-
892.
Fauvet, B., and Lashuel, H. A., 2016. Semisynthesis and enzymatic preparation of post-
translationally modified alpha-synuclein. Methods Mol. Biol. 1345, 3−20.
Fares, M.B., Maco, B., Oueslati, A., Rockenstein, E., Ninkina, N., Buchman, V.L., Masliah, E.
and Lashuel, H.A., 2016. Induction of de novo α-synuclein fibrillization in a neuronal model for
Parkinson’s disease. Proceedings of the National Academy of Sciences, 113(7), pp.E912-E921.
Finley, D., 2009. Recognition and processing of ubiquitin-protein conjugates by the
proteasome. Annual review of biochemistry, 78, pp.477-513.
Fink, A.L., 2006. The aggregation and fibrillation of α-synuclein. Accounts of chemical
research, 39(9), pp.628-634.
Flood, D.T., Hintzen, J.C., Bird, M.J., Cistrone, P.A., Chen, J.S. and Dawson, P.E., 2018.
Leveraging the Knorr pyrazole synthesis for the facile generation of thioester surrogates for use in
native chemical ligation. Angewandte Chemie, 130(36), pp.11808-11813.
Forno, L. S., 1996. Neuropathology of Parkinson’s disease. J. Neuropathol. Exp. Neurol. 55, 259–
272.
Funayama, M., Hasegawa, K., Kowa, H., Saito, M., Tsuji, S. and Obata, F., 2002. A new locus for
Parkinson's disease (PARK8) maps to chromosome 12p11. 2–q13. 1. Annals of Neurology:
Official Journal of the American Neurological Association and the Child Neurology
171
Society, 51(3), pp.296-301.
Giasson, B.I., Murray, I.V., Trojanowski, J.Q. and Lee, V.M.Y., 2001. A hydrophobic stretch of
12 amino acid residues in the middle of α-synuclein is essential for filament assembly. Journal of
Biological Chemistry, 276(4), pp.2380-2386.
Golbe, L. I., Di Iorio, G., Bonavita, V., Miller, D. C., and Duvoisin, R. C., 1990. A large kindred
with autosomal dominant Parkinson’s disease. Ann. Neurol. 27, 276–282.
Gómez-Tortosa, E., Newell, K., Irizarry, M.C., Sanders, J.L. and Hyman, B.T., 2000. α-Synuclein
immunoreactivity in dementia with Lewy bodies: morphological staging and comparison with
ubiquitin immunostaining. Acta neuropathologica, 99(4), pp.352-357.
Graham, D.L., Gray, A.J., Joyce, J.A., Yu, D., O'Moore, J., Carlson, G.A., Shearman, M.S.,
Dellovade, T.L. and Hering, H., 2014. Increased O-GlcNAcylation reduces pathological tau
without affecting its normal phosphorylation in a mouse model of
tauopathy. Neuropharmacology, 79, pp.307-313.
Guerrero-Ferreira, R., Taylor, N.M., Mona, D., Ringler, P., Lauer, M.E., Riek, R., Britschgi, M.
and Stahlberg, H., 2018. Cryo-EM structure of alpha-synuclein fibrils. Elife, 7, p.e36402.
Hastings, N.B., Wang, X., Song, L., Butts, B.D., Grotz, D., Hargreaves, R., Hess, J.F., Hong,
K.L.K., Huang, C.R.R., Hyde, L. and Laverty, M., 2017. Inhibition of O-GlcNAcase leads to
elevation of O-GlcNAc tau and reduction of tauopathy and cerebrospinal fluid tau in rTg4510
mice. Molecular neurodegeneration, 12(1), p.39.
Haj-Yahya, M., Fauvet, B., Herman-Bachinsky, Y., Hejjaoui, M., Bavikar, S.N., Karthikeyan,
172
S.V., Ciechanover, A., Lashuel, H.A. and Brik, A., 2013. Synthetic polyubiquitinated α-Synuclein
reveals important insights into the roles of the ubiquitin chain in regulating its
pathophysiology. Proceedings of the National Academy of Sciences, 110(44), pp.17726-17731.
Halim, A., Larsen, I.S.B., Neubert, P., Joshi, H.J., Petersen, B.L., Vakhrushev, S.Y., Strahl, S. and
Clausen, H., 2015. Discovery of a nucleocytoplasmic O-mannose glycoproteome in
yeast. Proceedings of the National Academy of Sciences, 112(51), pp.15648-15653.
Hardiville, S., and Hart, G. W., 2014. Nutrient Regulation of ́ Signaling, Transcription, and Cell
Physiology by O-GlcNAcylation. Cell Metab. 20, 208−213.
Hejjaoui, M., Haj ‐Yahya, M., Kumar, K.A., Brik, A. and Lashuel, H.A., 2011. Towards
Elucidation of the Role of Ubiquitination in the Pathogenesis of Parkinson’s Disease with
Semisynthetic Ubiquitinated α ‐Synuclein. Angewandte Chemie International Edition, 50(2),
pp.405-409.
Henley, J.M., Carmichael, R.E. and Wilkinson, K.A., 2018. Extranuclear SUMOylation in
neurons. Trends in neurosciences, 41(4), pp.198-210.
Hershko, A., 2000. Basic medical research award. The ubiquitin system. Nat Med, 6, pp.1073-
1081.
Hershko, A. and Ciechanover, A., 1998. The ubiquitin system. Annual review of
biochemistry, 67(1), pp.425-479.
Inglis, K.J., Chereau, D., Brigham, E.F., Chiou, S.S., Schöbel, S., Frigon, N.L., Yu, M.,
Caccavello, R.J., Nelson, S., Motter, R. and Wright, S., 2009. Polo-like kinase 2 (PLK2)
173
phosphorylates α-synuclein at serine 129 in central nervous system. Journal of Biological
Chemistry, 284(5), pp.2598-2602.
Jan, A., Adolfsson, O., Allaman, I., Buccarello, A.L., Magistretti, P.J., Pfeifer, A., Muhs, A. and
Lashuel, H.A., 2011. Aβ42 neurotoxicity is mediated by ongoing nucleated polymerization process
rather than by discrete Aβ42 species. Journal of Biological Chemistry, 286(10), pp.8585-8596.
Jao, C.C., Hegde, B.G., Chen, J., Haworth, I.S. and Langen, R., 2008. Structure of membrane-
bound α-synuclein from site-directed spin labeling and computational refinement. Proceedings of
the National Academy of Sciences, 105(50), pp.19666-19671.
Jeong, J.S., Ansaloni, A., Mezzenga, R., Lashuel, H.A. and Dietler, G., 2013. Novel mechanistic
insight into the molecular basis of amyloid polymorphism and secondary nucleation during
amyloid formation. Journal of molecular biology, 425(10), pp.1765-1781.
Kalia, L. V., Kalia, S. K., Chau, H., Lozano, A. M., Hyman, B. T., and McLean, P. J., 2011.
Ubiquitinylation of alpha-synuclein by carboxyl terminus Hsp70-interacting protein (CHIP) is
regulated by Bcl-2-associated athanogene 5 (BAG5). PLoS One 6:e14695.
Khalaf, O., Fauvet, B., Oueslati, A., Dikiy, I., Mahul-Mellier, A.L., Ruggeri, F.S., Mbefo, M.K.,
Vercruysse, F., Dietler, G., Lee, S.J. and Eliezer, D., 2014. The H50Q mutation enhances α-
synuclein aggregation, secretion, and toxicity. Journal of Biological Chemistry, 289(32),
pp.21856-21876.
Klaips, C. L., Jayaraj, G. G. & Hartl, F. U., 2017. Pathways of cellular proteostasis in aging and
disease. J. Cell Biol. 217, 51–63.
174
Kordower, J. H., Freeman, T. B. & Olanow, C. W., 1998. Neuropathology of fetal nigral grafts in
patients with Parkinson’s disease. Mov. Disord. 13, 88–95.
Komander, D. and Rape, M., 2012. The ubiquitin code. Annual review of biochemistry, 81, pp.203-
229.
Krüger, R., Kuhn, W., Müller, T., Woitalla, D., Graeber, M., Kösel, S., Przuntek, H., Epplen, J.T.,
Schols, L. and Riess, O., 1998. AlaSOPro mutation in the gene encoding α-synuclein in Parkinson's
disease. Nature genetics, 18(2), pp.106-108.
Krumova, P., Meulmeester, E., Garrido, M., Tirard, M., Hsiao, H.H., Bossis, G., Urlaub, H.,
Zweckstetter, M., Kügler, S., Melchior, F. and Bähr, M., 2011. Sumoylation inhibits α-synuclein
aggregation and toxicity. Journal of Cell Biology, 194(1), pp.49-60.
L
Lashuel, H.A., Overk, C.R., Oueslati, A. and Masliah, E., 2013. The many faces of α-synuclein:
from structure and toxicity to therapeutic target. Nature Reviews Neuroscience, 14(1), pp.38-48.
Lashuel, H. A., and Eliezer, D., 2016. Semisynthetic and in vitro phosphorylation of alpha-
synuclein at Y39 promotes functional partly helical membrane-bound states resembling those
induced by PD mutations. ACS Chem. Biol. 11, 2428−2437.
Lee, H.J., Suk, J.E., Bae, E.J., Lee, J.H., Paik, S.R. and Lee, S.J., 2008. Assembly-dependent
endocytosis and clearance of extracellular a-synuclein. The international journal of biochemistry
& cell biology, 40(9), pp.1835-1849.
Lee, H.J. and Lee, S.J., 2002. Characterization of cytoplasmic α-synuclein aggregates fibril
175
formation is tightly linked to the inclusion-forming process in cells. Journal of Biological
Chemistry, 277(50), pp.48976-48983.
Lesage, S., Anheim, M., Letournel, F., Bousset, L., Honoré, A., Rozas, N., Pieri, L., Madiona, K.,
Dürr, A., Melki, R. and Verny, C., 2013. G51D α ‐synuclein mutation causes a novel Parkinsonian–
pyramidal syndrome. Annals of neurology, 73(4), pp.459-471.
Lewis, Y.E., Galesic, A., Levine, P.M., De Leon, C.A., Lamiri, N., Brennan, C.K. and Pratt, M.R.,
2017. O-GlcNAcylation of α-synuclein at serine 87 reduces aggregation without affecting
membrane binding. ACS chemical biology, 12(4), pp.1020-1027.
Lewis, Y.E., Galesic, A., Levine, P.M., De Leon, C.A., Lamiri, N., Brennan, C.K. and Pratt, M.R.,
2017. O-GlcNAcylation of α-synuclein at serine 87 reduces aggregation without affecting
membrane binding. ACS chemical biology, 12(4), pp.1020-1027.
Levine, P.M., Galesic, A., Balana, A.T., Mahul-Mellier, A.L., Navarro, M.X., De Leon, C.A.,
Lashuel, H.A. and Pratt, M.R., 2019. α-Synuclein O-GlcNAcylation alters aggregation and
toxicity, revealing certain residues as potential inhibitors of Parkinson’s disease. Proceedings of
the National Academy of Sciences, 116(5), pp.1511-1519.
De Leon, C.A., Lang, G., Saavedra, M.I. and Pratt, M.R., 2018. Simple and efficient preparation
of O-and S-GlcNAcylated amino acids through InBr3-catalyzed synthesis of β-N-acetylglycosides
from commercially available reagents. Organic letters, 20(16), pp.5032-5035.
Li, W., Bengtson, M. H., Ulbrich, A., Matsuda, A., Reddy, V. A., Orth, A., et al. (2008). Genome-
wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial
E3 that regulates the organelle’s dynamics and signaling. PLoS One 3:e1487.
176
Liani, E., Eyal, A., Avraham, E., Shemer, R., Szargel, R., Berg, D., Bornemann, A., Riess, O.,
Ross, C.A., Rott, R. and Engelender, S., 2004. Ubiquitylation of synphilin-1 and α-synuclein by
SIAH and its presence in cellular inclusions and Lewy bodies imply a role in Parkinson's
disease. Proceedings of the National Academy of Sciences, 101(15), pp.5500-5505.
Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G.W. and Gong, C.X., 2004. O-GlcNAcylation regulates
phosphorylation of tau: a mechanism involved in Alzheimer's disease. Proceedings of the National
Academy of Sciences, 101(29), pp.10804-10809.
Liu, F., Shi, J., Tanimukai, H., Gu, J., Gu, J., Grundke-Iqbal, I., Iqbal, K. and Gong, C.X., 2009.
Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer's
disease. Brain, 132(7), pp.1820-1832.
Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G.W. and Gong, C.X., 2004. O-GlcNAcylation regulates
phosphorylation of tau: a mechanism involved in Alzheimer's disease. Proceedings of the National
Academy of Sciences, 101(29), pp.10804-10809.
Li, Y., Zhao, C., Luo, F., Liu, Z., Gui, X., Luo, Z., Zhang, X., Li, D., Liu, C. and Li, X., 2018.
Amyloid fibril structure of α-synuclein determined by cryo-electron microscopy. Cell
research, 28(9), pp.897-903.
Li, W., West, N., Colla, E., Pletnikova, O., Troncoso, J.C., Marsh, L., Dawson, T.M., Jäkälä, P.,
Hartmann, T., Price, D.L. and Lee, M.K., 2005. Aggregation promoting C-terminal truncation of
α-synuclein is a normal cellular process and is enhanced by the familial Parkinson's disease-linked
mutations. Proceedings of the National Academy of Sciences, 102(6), pp.2162-2167.
177
Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G.W. and Gong, C.X., 2004. O-GlcNAcylation regulates
phosphorylation of tau: a mechanism involved in Alzheimer's disease. Proceedings of the National
Academy of Sciences, 101(29), pp.10804-10809.
Liu, F., Shi, J., Tanimukai, H., Gu, J., Gu, J., Grundke-Iqbal, I., Iqbal, K. and Gong, C.X., 2009.
Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer's
disease. Brain, 132(7), pp.1820-1832.
Liu, C. W., Giasson, B. I., Lewis, K. A., Lee, V. M., DeMartino, G. N., & Thomas, P. J., 2005. A
Precipitating Role for Truncated α-Synuclein and the Proteasome in α-Synuclein Aggregation
IMPLICATIONS FOR PATHOGENESIS OF PARKINSON DISEASE. Journal of Biological
Chemistry, 280(24), 22670-22678.
Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G. W., and Gong, C.-X., 2004. O-GlcNAcylation
regulates phosphorylation of tau: a mechanism involved in Alzheimer’s disease. Proc. Natl. Acad.
Sci. U. S. A. 101, 10804−10809.
Luk, K.C., Song, C., O'Brien, P., Stieber, A., Branch, J.R., Brunden, K.R., Trojanowski, J.Q. and
Lee, V.M.Y., 2009. Exogenous α-synuclein fibrils seed the formation of Lewy body-like
intracellular inclusions in cultured cells. Proceedings of the National Academy of
Sciences, 106(47), pp.20051-20056.
Luk, K.C., Kehm, V.M., Zhang, B., O’Brien, P., Trojanowski, J.Q. and Lee, V.M., 2012.
Intracerebral inoculation of pathological α-synuclein initiates a rapidly progressive
neurodegenerative α-synucleinopathy in mice. Journal of Experimental Medicine, 209(5), pp.975-
986.
178
Luk, K.C., Covell, D.J., Kehm, V.M., Zhang, B., Song, I.Y., Byrne, M.D., Pitkin, R.M., Decker,
S.C., Trojanowski, J.Q. and Lee, V.M.Y., 2016. Molecular and biological compatibility with host
alpha-synuclein influences fibril pathogenicity. Cell reports, 16(12), pp.3373-3387.
Ma, M. R., Hu, Z. W., Zhao, Y. F., Chen, Y. X., and Li, Y. M., 2016. Phosphorylation induces
distinct alpha-synuclein strain formation. Sci. Rep. 6, 37130.
Mahul-Mellier, A.L., Burtscher, J., Maharjan, N., Weerens, L., Croisier, M., Kuttler, F., Leleu, M.,
Knott, G.W. and Lashuel, H.A., 2020. The process of Lewy body formation, rather than simply α-
synuclein fibrillization, is one of the major drivers of neurodegeneration. Proceedings of the
National Academy of Sciences, 117(9), pp.4971-4982.
Marotta, N.P., Lin, Y.H., Lewis, Y.E., Ambroso, M.R., Zaro, B.W., Roth, M.T., Arnold, D.B.,
Langen, R. and Pratt, M.R., 2015. O-GlcNAc modification blocks the aggregation and toxicity of
the protein α-synuclein associated with Parkinson's disease. Nature chemistry, 7(11), pp.913-920.
Marotta, N. P., Cherwien, C. A., Abeywardana, T., & Pratt, M. R., 2012. O ‐GlcNAc Modification
Prevents Peptide ‐Dependent Acceleration of α ‐Synuclein Aggregation. ChemBioChem, 13(18),
2665-2670.
Matunis, M.J., Coutavas, E. and Blobel, G., 1996. A novel ubiquitin-like modification modulates
the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the
nuclear pore complex. The Journal of cell biology, 135(6), pp.1457-1470.
McNaught, K. S., Belizaire, R., Isacson, O., Jenner, P., and Olanow, C. W. (2003). Altered
proteasomal function in sporadic Parkinson’s disease. Exp. Neurol. 179, 38–46.
179
McNaught, K. S., Belizaire, R., Jenner, P., Olanow, C. W., and Isacson, O., 2002. Selective loss
of 20S proteasome alpha-subunits in the substantia nigra pars compacta in Parkinson’s
disease. Neurosci. Lett. 326, 155–158.
McNaught, K. S., and Jenner, P., 2001. Proteasomal function is impaired in substantia nigra in
Parkinson’s disease. Neurosci. Lett. 297, 191–194.
McNaught, K. S., Olanow, C. W., Halliwell, B., Isacson, O., and Jenner, P., 2001. Failure of the
ubiquitin-proteasome system in Parkinson’s disease. Nat. Rev. Neurosci. 2, 589–594.
Meier, F., Abeywardana, T., Dhall, A., Marotta, N.P., Varkey, J., Langen, R., Chatterjee, C. and
Pratt, M.R., 2012. Semisynthetic, site-specific ubiquitin modification of α-synuclein reveals
differential effects on aggregation. Journal of the American Chemical Society, 134(12), pp.5468-
5471.
Mevissen, T. E. T., and Komander, D., 2017. Mechanisms of deubiquitinase specificity and
regulation. Annu. Rev. Biochem. 86, 159–192.
Michelle, C., Vourc’H, P., Mignon, L., and Andres, C. R., 2009. What was the set of ubiquitin and
ubiquitin-like conjugating enzymes in the eukaryote common ancestor? J. Mol. Evol. 68, 616–628.
Mizuno, N., Varkey, J., Kegulian, N.C., Hegde, B.G., Cheng, N., Langen, R. and Steven, A.C.,
2012. Remodeling of lipid vesicles into cylindrical micelles by α-synuclein in an extended α-
helical conformation. Journal of Biological Chemistry, 287(35), pp.29301-29311.
Morris, M., Knudsen, G.M., Maeda, S., Trinidad, J.C., Ioanoviciu, A., Burlingame, A.L. and
Mucke, L., 2015. Tau post-translational modifications in wild-type and human amyloid precursor
180
protein transgenic mice. Nature neuroscience, 18(8), pp.1183-1189.
Morris, M., Knudsen, G. M., Maeda, S., Trinidad, J. C., Ioanoviciu, A., Burlingame, A. L., &
Mucke, L., 2015. Tau post-translational modifications in wild-type and human amyloid precursor
protein transgenic mice. Nature neuroscience, 18(8), 1183-1189.
Murphy, D. D., Rueter, S. M., Trojanowski, J. Q., & Lee, V. M. Y., 2000. Synucleins are
developmentally expressed, and α-synuclein regulates the size of the presynaptic vesicular pool in
primary hippocampal neurons. Journal of Neuroscience, 20(9), 3214-3220.
Müller, S., Hoege, C., Pyrowolakis, G. and Jentsch, S., 2001. SUMO, ubiquitin's mysterious
cousin. Nature reviews Molecular cell biology, 2(3), pp.202-210.
Muir, T.W., Sondhi, D. and Cole, P.A., 1998. Expressed protein ligation: a general method for
protein engineering. Proceedings of the National Academy of Sciences, 95(12), pp.6705-6710.
Mishizen ‐Eberz, A.J., Guttmann, R.P., Giasson, B.I., Day III, G.A., Hodara, R., Ischiropoulos, H.,
Lee, V.M.Y., Trojanowski, J.Q. and Lynch, D.R., 2003. Distinct cleavage patterns of normal and
pathologic forms of α ‐synuclein by calpain I in vitro. Journal of neurochemistry, 86(4), pp.836-
847.
Mishizen-Eberz, A.J., Norris, E.H., Giasson, B.I., Hodara, R., Ischiropoulos, H., Lee, V.M.Y.,
Trojanowski, J.Q. and Lynch, D.R., 2005. Cleavage of α-synuclein by calpain: potential role in
degradation of fibrillized and nitrated species of α-synuclein. Biochemistry, 44(21), pp.7818-7829.
Mizuno, N., Varkey, J., Kegulian, N. C., Hegde, B. G., Cheng, N., Langen, R., & Steven, A. C.,
2012. Remodeling of lipid vesicles into cylindrical micelles by α-synuclein in an extended α-
181
helical conformation. Journal of Biological Chemistry, 287(35), 29301-29311.
O'Donnell, N., Zachara, N.E., Hart, G.W. and Marth, J.D., 2004. Ogt-dependent X-chromosome-
linked protein glycosylation is a requisite modification in somatic cell function and embryo
viability. Molecular and cellular biology, 24(4), pp.1680-1690.
O’Donnell, N., Zachara, N. E., Hart, G. W., and Marth, J. D., 2004. Ogt-dependent X-
chromosome-linked protein glycosylation is a requisite modification in somatic cell function and
embryo viability. Mol. Cell. Biol. 24, 1680−1690.
Oh, E., Akopian, D. and Rape, M., 2018. Principles of ubiquitin-dependent signaling. Annual
Review of Cell and Developmental Biology, 34, pp.137-162.
Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P.J.
and Haass, C., 2000. Constitutive phosphorylation of the Parkinson's disease associated α-
synuclein. Journal of Biological Chemistry, 275(1), pp.390-397.
Oueslati A, Fournier M, Lashuel HA., 2010. Role of post-translational modifications in
modulating the structure, function and toxicity of alpha-synuclein: Implications for Parkinson’s
disease pathogenesis and therapies. Prog Brain Res 183:115–145.
Oueslati, A., Paleologou, K. E., Schneider, B. L., Aebischer, P., and Lashuel, H. A., 2012.
Mimicking Phosphorylation at Serine 87 Inhibits the Aggregation of Human -Synuclein and
Protects against Its Toxicity in a Rat Model of Parkinson’s Disease. J. Neurosci. 32, 1536− 1544.

182
Paisán-Ruı
́ z, C., Jain, S., Evans, E.W., Gilks, W.P., Simón, J., Van Der Brug, M., De Munain,
A.L., Aparicio, S., Gil, A.M., Khan, N. and Johnson, J., 2004. Cloning of the gene containing
mutations that cause PARK8-linked Parkinson's disease. Neuron, 44(4), pp.595-600.
Paleologou, K.E., Oueslati, A., Shakked, G., Rospigliosi, C.C., Kim, H.Y., Lamberto, G.R.,
Fernandez, C.O., Schmid, A., Chegini, F., Gai, W.P. and Chiappe, D., 2010. Phosphorylation at
S87 is enhanced in synucleinopathies, inhibits α-synuclein oligomerization, and influences
synuclein-membrane interactions. Journal of Neuroscience, 30(9), pp.3184-3198.
Parkinson, J., 1817. An essay on the shaking palsy. J. Neuropsychiatry Clin. Neurosci. 14, 223–
236.
Pfefferkorn, C. M., Jiang, Z., and Lee, J. C., 2012. Biophysics of α-synuclein membrane
interactions. Biochim. Biophys. Acta, Biomembr. 1818, 162−171.
Pinho, T.S., Correia, S.C., Perry, G., Ambrósio, A.F. and Moreira, P.I., 2019. Diminished O-
GlcNAcylation in Alzheimer's disease is strongly correlated with mitochondrial
anomalies. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1865(8), pp.2048-
2059.
Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide, S.E., Dehejia, A., Dutra, A., Pike, B., Root,
H., Rubenstein, J., Boyer, R. and Stenroos, E.S., 1997. Mutation in the α-synuclein gene identified
in families with Parkinson's disease. science, 276(5321), pp.2045-2047.
Pratt, M. R., Abeywardana, T., & Marotta, N. P., 2015. Synthetic Proteins and Peptides for the
Direct Interrogation of α-Synuclein Posttranslational Modifications. Biomolecules, 5(3), 1210-
1227.
183
Pronin, A. N., Morris, A. J., Surguchov, A., and Benovic, J. L., 2000. Synucleins are a novel class
of substrates for G protein-coupled receptor kinases. J. Biol. Chem. 275, 26515–26522.
Recasens, A. and Dehay, B., 2014. Alpha-synuclein spreading in Parkinson’s disease. Frontiers in
neuroanatomy, 8, p.159.
Recasens, A., Dehay, B., Bové, J., Carballo ‐Carbajal, I., Dovero, S., Pérez ‐Villalba, A., Fernagut,
P.O., Blesa, J., Parent, A., Perier, C. and Fariñas, I., 2014. Lewy body extracts from Parkinson
disease brains trigger α ‐synuclein pathology and neurodegeneration in mice and monkeys. Annals
of neurology, 75(3), pp.351-362.
Robak, L.A., Jansen, I.E., Van Rooij, J., Uitterlinden, A.G., Kraaij, R., Jankovic, J., Heutink, P.
and Shulman, J.M., 2017. Excessive burden of lysosomal storage disorder gene variants in
Parkinson’s disease. Brain, 140(12), pp.3191-3203.
Rodriguez, J. A., Ivanova, M. I., Sawaya, M. R., Cascio, D., Reyes, F. E., Shi, D., Sangwan, S.,
Guenther, E. L., Johnson, L. M., Zhang, M., Jiang, L., Arbing, M. A., Nannenga, B. L., Hattne, J.,
Whitelegge, J.,Brewster, A. S., Messerschmidt, M., Boutet, S., Sauter, N. K., Gonen, T., and
Eisenberg, D. S., 2015. Structure of the toxic core of αsynuclein from invisible crystals. Nature
525, 486−490.
Rousseaux, M.W., Revelli, J.P., Vázquez-Vélez, G.E., Kim, J.Y., Craigen, E., Gonzales, K.,
Beckinghausen, J. and Zoghbi, H.Y., 2018. Depleting Trim28 in adult mice is well tolerated and
reduces levels of α-synuclein and tau. Elife, 7, p.e36768
Rott, R., Szargel, R., Shani, V., Hamza, H., Savyon, M., Abd Elghani, F., Bandopadhyay, R. and
Engelender, S., 2017. SUMOylation and ubiquitination reciprocally regulate α-synuclein
184
degradation and pathological aggregation. Proceedings of the National Academy of
Sciences, 114(50), pp.13176-131811.
Sampathu, D.M., Giasson, B.I., Pawlyk, A.C., Trojanowski, J.Q. and Lee, V.M.Y., 2003.
Ubiquitination of α-synuclein is not required for formation of pathological inclusions in α-
synucleinopathies. The American journal of pathology, 163(1), pp.91-100.
Schmid AW, Fauvet B, Moniatte M, Lashuel HA., 2013. Alpha-synuclein post-translational
modifications as potential biomarkers for Parkinson disease and other synucleinopathies. Mol Cell
Proteomics 12:3543–3558.
Shah, N. H., Dann, G. P., Vila-Perelló, M., Liu, Z., & Muir, T. W., 2012. Ultrafast protein splicing
is common among cyanobacterial split inteins: implications for protein engineering. Journal of the
American Chemical Society, 134(28), 11338-11341.
Shin, Y., Klucken, J., Patterson, C., Hyman, B. T., and McLean, P. J., 2005. The co-chaperone
carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation
decisions between proteasomal and lysosomal pathways. J. Biol. Chem. 280, 23727–23734.
Shulman, J. M., De Jager, P. L., and Feany, M. B., 2011. Parkinson’s disease: genetics and
pathogenesis. Annu. Rev. Pathol. 6, 193–222.
Sidransky, E. and Lopez, G., 2012. The link between the GBA gene and parkinsonism. The Lancet
Neurology, 11(11), pp.986-998.
Simon-Sanchez, J., Schulte, C., Bras, J.M., Sharma, M., Gibbs, J.R., Berg, D., Paisan-Ruiz, C.,
Lichtner, P., Scholz, S.W., Hernandez, D.G. and Krüger, R., 2009. Genome-wide association study
185
reveals genetic risk underlying Parkinson's disease. Nature genetics, 41(12), p.1308.
Singleton, A.B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J., Hulihan, M.,
Peuralinna, T., Dutra, A.N., Lincoln, S. and Crawley, A., 2003. [alpha]-synuclein locus triplication
causes Parkinson’s disease. Science, 302(5646), pp.841-842.
Smith, W.W., Margolis, R.L., Li, X., Troncoso, J.C., Lee, M.K., Dawson, V.L., Dawson, T.M.,
Iwatsubo, T. and Ross, C.A., 2005. α-Synuclein phosphorylation enhances eosinophilic
cytoplasmic inclusion formation in SH-SY5Y cells. Journal of Neuroscience, 25(23), pp.5544-
5552.
Smolders, S. and Van Broeckhoven, C., 2020. Genetic perspective on the synergistic connection
between vesicular transport, lysosomal and mitochondrial pathways associated with Parkinson’s
disease pathogenesis. Acta Neuropathologica Communications, 8, pp.1-28.
Snow, C.M., Senior, A. and Gerace, L., 1987. Monoclonal antibodies identify a group of nuclear
pore complex glycoproteins. The Journal of cell biology, 104(5), pp.1143-1156.
T
Tang, B., Becanovic, K., Desplats, P.A., Spencer, B., Hill, A.M., Connolly, C., Masliah, E.,
Leavitt, B.R. and Thomas, E.A., 2012. Forkhead box protein p1 is a transcriptional repressor of
immune signaling in the CNS: implications for transcriptional dysregulation in Huntington
disease. Human molecular genetics, 21(14), pp.3097-3111.
Tavassoly, O., Yue, J. and Vocadlo, D., 2020. Pharmacological inhibition and knockdown of O ‐
GlcNAcase reduces cellular internalization of α ‐synuclein pre ‐formed ‐fibrils. The FEBS Journal.
186
Tofaris, G. K., Kim, H. T., Hourez, R., Jung, J. W., Kim, K. P., and Goldberg, A. L., 2011.
Ubiquitin ligase NEDD4 promotes alpha-synuclein degradation by the endosomal-lysosomal
pathway. Proc. Natl. Acad. Sci. U.S.A. 108, 17004–17009.
Tofaris, G. K., Razzaq, A., Ghetti, B., Lilley, K. S., and Spillantini, M. G., 2003. Ubiquitination
of alpha-synuclein in Lewy bodies is a pathological event not associated with impairment of
proteasome function. J. Biol. Chem. 278, 44405–44411.
Tsigelny, I.F., Sharikov, Y., Wrasidlo, W., Gonzalez, T., Desplats, P.A., Crews, L., Spencer, B.
and Masliah, E., 2012. Role of α ‐synuclein penetration into the membrane in the mechanisms of
oligomer pore formation. The FEBS journal, 279(6), pp.1000-1013.
Tuttle, M.D., Comellas, G., Nieuwkoop, A.J., Covell, D.J., Berthold, D.A., Kloepper, K.D.,
Courtney, J.M., Kim, J.K., Barclay, A.M., Kendall, A. and Wan, W., 2016. Solid-state NMR
structure of a pathogenic fibril of full-length human α-synuclein. Nature structural & molecular
biology, 23(5), pp.409-415.
Tysnes, O. B., and Storstein, A., 2017. Epidemiology of Parkinson’s disease. J. Neural
Transm. 124, 901–905.
Uéda, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D.A., Kondo, J.,
Ihara, Y. and Saitoh, T., 1993. Molecular cloning of cDNA encoding an unrecognized component
of amyloid in Alzheimer disease. Proceedings of the National Academy of Sciences, 90(23),
pp.11282-11286.
Ulmer, T.S., Bax, A., Cole, N.B. and Nussbaum, R.L., 2005. Structure and dynamics of micelle-
bound human α-synuclein. Journal of Biological Chemistry, 280(10), pp.9595-9603.
187
Uversky, V. N., and Eliezer, D., 2009. Biophysics of Parkinson’s disease: structure and
aggregation of alpha-synuclein. Curr. Protein Pept. Sci. 10, 483–499.
Vasconcelos-Dos-Santos A, de Queiroz RM, da Costa Rodrigues B, Todeschini AR, Dias WB,
2015. Hyperglycemia and aberrant O-GlcNAcylation: contributions to tumor progression. J
Bioenerg Biomembr. 50:175–87.
Varkey, J., Isas, J.M., Mizuno, N., Jensen, M.B., Bhatia, V.K., Jao, C.C., Petrlova, J., Voss, J.C.,
Stamou, D.G., Steven, A.C. and Langen, R., 2010. Membrane curvature induction and tubulation
are common features of synucleins and apolipoproteins. Journal of Biological Chemistry, 285(42),
pp.32486-32493.
Varshavsky, A., 2012. The ubiquitin system, an immense realm. Annu. Rev. Biochem. 81, 167–
176.
Vila-Perelló, M. and Muir, T.W., 2010. Biological applications of protein splicing. Cell, 143(2),
pp.191-200.
Vilar, M., Chou, H.T., Lührs, T., Maji, S.K., Riek-Loher, D., Verel, R., Manning, G., Stahlberg,
H. and Riek, R., 2008. The fold of α-synuclein fibrils. Proceedings of the National Academy of
Sciences, 105(25), pp.8637-8642.
Visanji, N.P., Brotchie, J.M., Kalia, L.V., Koprich, J.B., Tandon, A., Watts, J.C. and Lang, A.E.,
2016. α-Synuclein-based animal models of Parkinson's disease: challenges and opportunities in a
new era. Trends in neurosciences, 39(11), pp.750-762.
Velez-Pardo, C., Lorenzo-Betancor, O., Jimenez-Del-Rio, M., Moreno, S., Lopera, F., Cornejo-
188
Olivas, M., Torres, L., Inca-Martinez, M., Mazzetti, P., Cosentino, C. and Yearout, D., 2019. The
distribution and risk effect of GBA variants in a large cohort of PD patients from Colombia and
Peru. Parkinsonism & related disorders, 63, pp.204-208.
Volpicelli-Daley, L.A., Luk, K.C., Patel, T.P., Tanik, S.A., Riddle, D.M., Stieber, A., Meaney,
D.F., Trojanowski, J.Q. and Lee, V.M.Y., 2011. Exogenous α-synuclein fibrils induce Lewy body
pathology leading to synaptic dysfunction and neuron death. Neuron, 72(1), pp.57-71.
Vocadlo, D. J., 2012. O-GlcNAc processing enzymes: catalytic mechanisms, substrate specificity,
and enzyme regulation. Current opinion in chemical biology, 16(5-6), 488-497.
Wan, Q. and Danishefsky, S.J., 2007. Free ‐radical ‐based, specific desulfurization of cysteine: a
powerful advance in the synthesis of polypeptides and glycopolypeptides. Angewandte
Chemie, 119(48), pp.9408-9412.
Wang, Z., Udeshi, N. D., O'Malley, M., Shabanowitz, J., Hunt, D. F., & Hart, G. W., 2010.
Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of
chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass
spectrometry. Molecular & Cellular Proteomics, 9(1), 153-160.
Wang, Z., Park, K., Comer, F., Hsieh-Wilson, L.C., Saudek, C.D. and Hart, G.W., 2009. Site-
specific GlcNAcylation of human erythrocyte proteins: potential biomarker (s) for
diabetes. diabetes, 58(2), pp.309-317.
Wang, A.C., Jensen, E.H., Rexach, J.E., Vinters, H.V. and Hsieh-Wilson, L.C., 2016. Loss of O-
GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration. Proceedings of
the National Academy of Sciences, 113(52), pp.15120-15125.
189
Wang, S., Yang, F., Petyuk, V.A., Shukla, A.K., Monroe, M.E., Gritsenko, M.A., Rodland, K.D.,
Smith, R.D., Qian, W.J., Gong, C.X. and Liu, T., 2017. Quantitative proteomics identifies altered
O ‐GlcNAcylation of structural, synaptic and memory ‐associated proteins in Alzheimer's
disease. The Journal of pathology, 243(1), pp.78-88.
Wani WY, Chatham JC, Darley-Usmar V, McMahon LL, Zhang J., 2017. O-GlcNAcylation and
neurodegeneration. Brain Res Bull 133:80–87.
Wilhelm, B.G., Mandad, S., Truckenbrodt, S., Kröhnert, K., Schäfer, C., Rammner, B., Koo, S.J.,
Claßen, G.A., Krauss, M., Haucke, V. and Urlaub, H., 2014. Composition of isolated synaptic
boutons reveals the amounts of vesicle trafficking proteins. Science, 344(6187), pp.1023-1028.
Wood, S.J., Wypych, J., Steavenson, S., Louis, J.C., Citron, M. and Biere, A.L., 1999. α-Synuclein
fibrillogenesis Is nucleation-dependent Implications for the pathogenesis of Parkinson′ s
disease. Journal of Biological Chemistry, 274(28), pp.19509-19512.
Yang, X. and Qian, K., 2017. Protein O-GlcNAcylation: emerging mechanisms and
functions. Nature Reviews Molecular Cell Biology, 18(7), p.452.
Yavich, L., Tanila, H., Vepsäläinen, S., & Jäkälä, P., 2004. Role of α-synuclein in presynaptic
dopamine recruitment. Journal of Neuroscience, 24(49), 11165-11170.
Yates, B., Braschi, B., Gray, K.A., Seal, R.L., Tweedie, S. and Bruford, E.A., 2016. Genenames.
org: the HGNC and VGNC resources in 2017. Nucleic acids research, p.gkw1033.

190
Ysselstein, D., Joshi, M., Mishra, V., Griggs, A.M., Asiago, J.M., McCabe, G.P., Stanciu, L.A.,
Post, C.B. and Rochet, J.C., 2015. Effects of impaired membrane interactions on α-synuclein
aggregation and neurotoxicity. Neurobiology of disease, 79, pp.150-163.
Yu, H. and Matouschek, A., 2017. Recognition of client proteins by the proteasome. Annual review
of biophysics, 46, pp.149-173.
Yuzwa, S.A. and Vocadlo, D.J., 2014. O-GlcNAc and neurodegeneration: biochemical
mechanisms and potential roles in Alzheimer's disease and beyond. Chemical Society
Reviews, 43(19), pp.6839-6858.
Yuzwa, S.A., Macauley, M.S., Heinonen, J.E., Shan, X., Dennis, R.J., He, Y., Whitworth, G.E.,
Stubbs, K.A., McEachern, E.J., Davies, G.J. and Vocadlo, D.J., 2008. A potent mechanism-
inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo. Nature chemical
biology, 4(8), pp.483-490.
Yuzwa, S.A., Shan, X., Macauley, M.S., Clark, T., Skorobogatko, Y., Vosseller, K. and Vocadlo,
D.J., 2012. Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against
aggregation. Nature chemical biology, 8(4), pp.393-399.
Yuzwa, S.A., Shan, X., Jones, B.A., Zhao, G., Woodward, M.L., Li, X., Zhu, Y., McEachern, E.J.,
Silverman, M.A., Watson, N.V. and Gong, C.X., 2014. Pharmacological inhibition of O-
GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP
mutant mice. Molecular Neurodegeneration, 9(1), pp.1-14.
Zachara, N. E., and Hart. G.W., 2002. The emerging significance of O-GlcNAc in cellular
regulation Chem Rev, 102, pp. 431-438.
191
Zarranz, J.J., Alegre, J., Gómez ‐Esteban, J.C., Lezcano, E., Ros, R., Ampuero, I., Vidal, L.,
Hoenicka, J., Rodriguez, O., Atarés, B. and Llorens, V., 2004. The new mutation, E46K, of α ‐
synuclein causes parkinson and Lewy body dementia. Annals of Neurology: Official Journal of
the American Neurological Association and the Child Neurology Society, 55(2), pp.164-173.
Zhao, Q., Xie, Y., Zheng, Y., Jiang, S., Liu, W., Mu, W., Liu, Z., Zhao, Y., Xue, Y. and Ren, J.,
2014. GPS-SUMO: a tool for the prediction of sumoylation sites and SUMO-interaction
motifs. Nucleic acids research, 42(W1), pp.W325-W330.
Zhao, X., 2018. SUMO-mediated regulation of nuclear functions and signaling
processes. Molecular cell, 71(3), pp.409-418.
Zhao, K., Lim, Y.J., Liu, Z., Long, H., Sun, Y., Hu, J.J., Zhao, C., Tao, Y., Zhang, X., Li, D. and
Li, Y.M., 2020. Parkinson's disease-related phosphorylation at Tyr39 rearranges α-synuclein
amyloid fibril structure revealed by cryo-EM. bioRxiv.
Zhu, Y., Shan, X., Safarpour, F., Erro Go, N., Li, N., Shan, A., Huang, M.C., Deen, M., Holicek,
V., Ashmus, R. and Madden, Z., 2018. Pharmacological inhibition of O-GlcNAcase enhances
autophagy in brain through an mTOR-independent pathway. ACS chemical neuroscience, 9(6),
pp.1366-1379.
Zhu, Y., Shan, X., Yuzwa, S. A., and Vocadlo, D. J., 2014. The emerging link between O-GlcNAc
and Alzheimer disease. J. Biol. Chem. 289, 34472−34481.
Zimprich, A., Biskup, S., Leitner, P., Lichtner, P., Farrer, M., Lincoln, S., Kachergus, J., Hulihan,
M., Uitti, R.J., Calne, D.B. and Stoessl, A.J., 2004. Mutations in LRRK2 cause autosomal-
dominant parkinsonism with pleomorphic pathology. Neuron, 44(4), pp.601-607.

Abstract (if available)

Abstract Parkinson’s disease (PD) is the second most common neurodegenerative disease. The neuropathology of PD is driven by the progressive degeneration of dopaminergic neurons which leads to a significant reduction of stratal dopamine. The common hallmark of PD is the presence of insoluble inclusions called Lewy bodies (LBs) or Lewy neurites (NTs). Current studies support the hypothesis that α-synuclein plays a central role in Lewy pathology. Furthermore, abnormal α-synuclein expression, impairment of proteostasis machinery, and various types of stresses are all implicated in pathogenesis of α-synuclein. Additionally, various genetic mutations (SNCA, LRRK2, and others) are known to be driving forces of α-synuclein pathology, and the presence of various post-translational modifications (PTMs) of α-synuclein, such as ubiquitination, phosphorylation, O-GlcNAcylation, and SUMOylation have been identified to play a role in the pathogenesis in vivo. These mutations and PTMs can either exacerbate or diminish α-synuclein’s aggregation and toxicity, and, in the end, they add another layer of complexity to the study of the pathology of α-synuclein. The molecular mechanisms by which several of these cellular events promote neurodegenerative diseases are still not known. In the Pratt lab, we seek to understand the roles played by various PTMs in α-synculein aggregation. In the past, our lab extensively studied ubiquitinated and SUMOylated α-synuclein, whereas my main focus was to understand the role of O-GlcNAcylation in the process of aggregation. Several proteomics studies have identified nine O-GlcNAc-modified sites of α-synuclein in mouse and/or human tissue samples. In addition, recent studies have shown that global O-GlcNAc levels decrease in neurodegeneration. Initially, we wanted to delineate the modification’s site-dependent aggregative effects by synthesizing five differently-modified O-GlcNAcylated proteins via solid phase peptide synthesis and native chemical ligation. We hypothesized the O-GlcNAc moiety might alter the aggregation of α-synuclein in site-specific manner. Once we obtained the variants, we used a variety of biochemical experiments to show that O-GlcNAc inhibits protein aggregation, alters fibrillar structure, and abrogates aggregate cytotoxicity in site-dependent manner. As the mechanism of how O-GlcNAcylation inhibits α-synuclein aggregation is not yet known, we next asked if these effects are generalizable to other sources of poly-hydroxylated steric bulk. Therefore, we synthesized and studied three α-synuclein variants bearing different monosaccharidesㅡglucose, N-acetyl-galactosamine (GalNAc) and mannose. Our data has indicated the O-GlcNAc moiety as being especially inhibitory of α-synuclein aggregation when compared to other monosaccharides. Next, we subjected two O-GlcNAc-modified proteins to protease calpain, which has been found to co-localize in with α-synuclein in PD patient brains. The results show that O-GlcNAcylation inhibits calpain cleavage, and thus indicates a protective role of O-GlcNAcylation against calpain cleavage in the modulation of α-synuclein biology. Next, we investigated the effects of O-GlcNAcylation at serine 87, which is also a phosphorylation site. While we showed that this particular O-GlcNAc variant is also able to inhibit the process of aggregation, we found that this modification does not affect the membrane-binding properties of α-synculein in the same way as phosphorylation does. Overall, our data support the hypothesis that O-GlcNAcylation has a protective role against protein aggregation, and highlight the promise of therapies that can elevate the O-GlcNAcyation of α-synculein.

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Asset Metadata

Creator Galesic, Ana (author)

Core Title Investigating the role of O-GlcNAcylation in α-synuclein aggregation

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 12/11/2020

Defense Date 10/28/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag aggregation,amyloid,OAI-PMH Harvest,O-GlcNAc,Parkinson’s disease,α-synuclein

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Pratt, Matthew R. (committee chair), Langen, Ralf (committee member), Qin, Peter (committee member)

Creator Email anagalesic1@gmail.com,galesic@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-405031

Unique identifier UC11667194

Identifier etd-GalesicAna-9192.pdf (filename),usctheses-c89-405031 (legacy record id)

Legacy Identifier etd-GalesicAna-9192.pdf

Dmrecord 405031

Document Type Dissertation

Rights Galesic, Ana

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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