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Uncovering the protective role of protein glycosylation in Parkinson's disease utilizing protein semi-synthesis

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Uncovering the protective role of protein glycosylation in Parkinson's disease utilizing protein semi-synthesis

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Content Uncovering the protective role of protein glycosylation in
Parkinson’s Disease utilizing protein semi-synthesis
by
Nicholas P. Marotta

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
December 2015
Copyright 2015 Nicholas P. Marotta

I

Acknowledgements
This work would not have been possible without a great deal of support from
my friends, co-workers, and family. I would like to thank Matt Pratt for being a great
mentor, as well as the members of my committee Ralf Langen and Peter Qin. Thank
you to all of the Pratt Lab members past and present, especially my contemporaries
Yu Hsuan Lin and Tharindumala Abeywardana. Thank you to my friends and
roommates for putting up with me all these years. Thank you to my family: my
siblings Katie, Molly, Adam, and Gracie, and especially to my parents Margaret and
Paul. Finally, thank you to Christina for keeping me sane through all of this.

II

Abstract
Parkinson’s Disease (PD) is the second most prevalent neurodegenerative
disease that is characterized by resting muscle tremors and slowing or stiffness of
movement. These symptoms are the result of the progressive loss of dopamine
producing neurons in a region of the brain called the Substantia nigra pars compacta
(SNpc). The SNpc supplies the neurotransmitter dopamine (DA) to the striatum, and
loss of DA is the cause of these motor deficits. Progressive neuron loss in PD
patients’ brains is also accompanied by the accumulation of intracellular deposits
called Lewy bodies (LB). These deposits are a pathological hallmark of the disease
and are strongly linked to neuron death. LBs are proteinaceous, and consist mainly of
aggregated protein fibers composed of the protein α-synuclein (αSyn). In addition to
being the most abundant protein in LBs, αSyn is also genetically linked to PD, with
the familial forms of the disease being linked to several mutations to the αSyn gene.
Additional copies of the αSyn gene also leads to early onset of PD. While the driving
force behind onset of sporadic PD (i.e. non-inherited), which comprises ~95% of
cases, is still a mystery, the link between αSyn, it’s aggregated form in LBs, and
progressive neuron loss in PD is common factor in all cases.
αSyn is an unusual protein in that it does not fold into a regular structure in
solution. It instead exists as a random coil with only weak and transient globular
structure. αSyn has a high affinity for cellular lipid membranes and it does adopt a
regular, extended, α-helical structure when it associates with membranes or vesicles.
This helical structure is strongly linked to the physiological role αSyn plays in
neurons where it is involved in neurotransmitter release and maintenance of synaptic

III

vesicles. In the disease-associated state, αSyn monomers aggregate together to form a
variety of higher molecular weight structures, including soluble oligomers and long
protein fibers. In these aggregates αSyn adopts as fold that is very high in β-sheet
content and is very stable to unfolding and degradation. These aggregates have also
been shown to be cytotoxic to neuronal cells in a variety of contexts, further
strengthening the link between αSyn and PD pathology.
Currently, treatments for PD are limited, with the major treatment being DA
replacement therapy to supplement the loss of naturally produced DA. Transplant of
new DA producing neurons has also shown some promise, however both of these
therapies can only treat the symptoms of the disease, not the underlying problem of
αSyn aggregation. The aggregation of proteins such as αSyn is very difficult to target
using traditional drug design paradigms, which usually target the function of enzymes
or other specific cellular components. Protein aggregates are driven by protein-
protein interactions between monomers, and the driving forces for these interactions
are the same that drive the correct folding of all other cellular proteins. Thus
selectively targeting the protein-protein interactions responsible for disease-
associated aggregation is not yet a viable approach. One important aspect of αSyn
biology that may make it a better therapeutic target is it’s high level of post-
translational modification. Post-translational modifications (PTMs) are chemical
modifications that occur on a protein after it has been synthesized, and they take a
variety of forms. The key is that most PTMs are installed by enzymes. If a PTM could
be identified that effected the aggregation behavior of αsyn, the relevant enzymes for
controlling that modification could be targets of study for new therapies.

IV

One PTM of αSyn that has, to date, received little attention from the field is
the modification by the O-linked monosaccharide N-acetyl-Glucosamine (O-
GlcNAc). This modification has been identified on several sites of αSyn but it’s
cellular consequences are still a complete mystery. This particular modification is of
interest as it is tightly linked to cell metabolism, which is greatly altered in PD brains,
and it has generally shown to help solubilize target substrates and is protective against
a variety of cell stressors such as heat shock and oxidative damage. Thus, the focus of
the research presented herein was to uncover the biochemical consequence of αSyn
O-GlcNAc modification, especially on it’s aggregation behavior. This aim was
accomplished through a combination of biochemistry, cell biology, and synthetic
protein chemistry. As the results will indicate, modification of αSyn by O-GlcNAc
was shown to strongly inhibit both aggregate formation and toxicity in neurons. This
result shows great promise for future investigation and suggests O-GlcNAc
modification, and the enzymes that regulate it, are possible targets in the search for
better PD therapeutics.

V

Table of Contents
Acknowledgements ....................................................................................................... I
Abstract ........................................................................................................................ II
Table of Figures ......................................................................................................... IX
Chapter 1. Parkinson's Disease and the Pathological protein
αSynuclein…………..……………….…… ......................................................... .…. 1
1.1 Introduction to Parkinson's Disease .............................................................. 1
1.2 Pysiology of α-Synuclein .............................................................................. 3
1.3 α-Synuclein Aggregation and Toxicity ......................................................... 6
1.4 Mechanism of Toxicity ................................................................................. 9
1.5 Propagation of aggregates between Cells .................................................... 13
1.6 Conclusion ................................................................................................... 14
1.7 Reference ..................................................................................................... 15
Chapter 2. Post-Translational Modifications of α-Synuclein add further
complexity .................................................................................................................. 20

2.1 An Overview of Post-Translational Modification on αSyn ........................ 20
2.2 Non-enzymatic modifications ..................................................................... 20
2.3 Phosphorylation ........................................................................................... 22
2.4 Nα-Acetylation ............................................................................................ 23
2.5 Ubiquitin and Ubiquitin-like modifiers ....................................................... 24
2.6 O-GlcNAc modificatoin .............................................................................. 25
2.7 Conclusion ................................................................................................... 28
2.8 References ................................................................................................... 29

VI

Chapter 3. O-GlcNAc modification prevents peptide-dependent acceleration of
α-Synuclein Aggregation ........................................................................................... 34

3.1 Introduction ................................................................................................. 34
3.2 Results ......................................................................................................... 38
3.3 Discussion ................................................................................................... 54
3.4 Materials and Methods ................................................................................ 55
3.4.1 General ............................................................................................ 55
3.4.2 Plasmid Construction ...................................................................... 56
3.4.3 Co-expression of αSyn with OGT ................................................... 56
3.4.4 Western Blotting .............................................................................. 57
3.4.5 Expression of wild-type αSyn ......................................................... 57
3.4.6 Synthesis of O-GlcNAc Threonine Cassette ................................... 59
3.4.7 Solid-phase Peptide Synthesis ......................................................... 63
3.4.8 Aggregation Assay .......................................................................... 65
3.5 Reference ..................................................................................................... 68
Chapter 4. Native Chemical Ligation: a powerful tool for protein biochemistry ...... 76

4.1 Introduction ................................................................................................. 76
4.2 Expansion of scope through new chemistry ................................................ 78
4.3 Expressed Protein Ligation ......................................................................... 81
4.4 Conclusion ................................................................................................... 84
4.5 References ................................................................................................... 85

VII

Chapter 5. O-GlcNAc modification blocks the aggregation and toxicity of the
Parkinson's Disease associated protein α-Synuclein .................................................. 87
5.1 Abstract ....................................................................................................... 87
5.2 Introduction ................................................................................................. 88
5.3 Results ......................................................................................................... 93
5.3.1 An α-Synuclein loss-of-function O-GlcNAcylation mutant has
compromised aggregation ........................................................................... 93
5.3.2 Synthesis of O-GlcNAcylated α-Synuclein ..................................... 95
5.3.3 O-GlcNAcylation blocks αsyn aggregation but has no effect on
membrane binding ..................................................................................... 104
5.3.4 O-GlcNacylation affects subsequent α-synuclein
phosphorylation ......................................................................................... 110
5.3.5 O-GlcNAcylation inhibits α-Synuclein toxicity ............................ 112
5.3.6 O-GlcNAcylation largely prevents incorporation of α-Synuclein
monomers into aggregates ......................................................................... 115
5.4 Discussion ................................................................................................. 121
5.5 Materials and Methods .............................................................................. 124
5.5.1 General .......................................................................................... 124
5.5.2 Plasmid construction ..................................................................... 125
5.5.3 Expression of recombinant α-synuclein ........................................ 125
5.5.4 Expressoin of αSyn C-terminal fragment ...................................... 126
5.5.5 Expression of αSyn N-terminal fragment ...................................... 127
5.5.6 Solid-phase synthesis of thioester peptides 1 and 6 ...................... 128
5.5.7 Unmodified α-Synuclein synthesis ................................................ 129
5.5.8 α-Synuclein(gT72) synthesis ......................................................... 130
5.5.9 Aggregation reactions .................................................................... 132
5.5.10 Circular Dichroism ........................................................................ 132

VIII

5.5.11 Dynamic Light Scattering ............................................................. 133
5.5.12 Thioflavin T fluorescence ............................................................. 133
5.5.13 Transmission electron microscopy ................................................ 133
5.5.14 SEC-MALS ................................................................................... 134
5.5.15 Circular Dichroism of αSyn in the presence of lipids ................... 134
5.5.16 Transmission electron microscopy of membrane tubulation ........ 135
5.5.17 Cellular Toxicity assay .................................................................. 135
5.5.18 In Vitro phosphorylation reactions and Western Blotting ............. 137
5.5.19 Analysis of aggregates by SDS-PAGE and/or Western Blot ........ 138
5.5.20 Aggregation reactions with pre-formed fibers .............................. 139
5.6 References ................................................................................................. 140
Bibliography ............................................................................................................ 148

IX

Table of Figures
Figure 1.1. α-Synuclein primary structure and native monomer behavior .................. 5
Figure 1.2. α-Synuclein forms β-sheet rich aggregates ............................................... 8

Figure 2.1. αSyn is heavily decorated with PTMs ..................................................... 22
Figure 2.2. Synthesis and Incorporation of O-GlcNAc modification ........................ 26
Figure 3.1. Co-expression of α-Syn with OGT .......................................................... 37
Figure 3.2. Aggregation of αSyn with peptide 2 ........................................................ 39

Figure 3.3. Characterization of wild-type αSyn ......................................................... 40

Figure 3.4. Characterization of peptide 1 and aggregation assay for αSyn+1 ........... 41
Figure 3.5. Dynamic Light Scattering ........................................................................ 43
Figure 3.6. Circular Dichroism spectra of αSyn ........................................................ 44
Figure 3.7. Full ThT fluorescence data ...................................................................... 45
Figure 3.8. Synthesis of GlcNAc Threonine .............................................................. 47
Figure 3.9. Aggregation of α-synuclein with peptides 3 and 4 .................................. 50
Figure 3.10. Transmission electron micrographs of mature fibers ............................ 52
Figure 3.11. Comparison of αSyn aggregation with peptides .................................... 53
Figure 4.1. Mechanism of Native Chemical Ligation ................................................ 78

Figure 4.2. Mechanism of radical desulfurization ..................................................... 80
Figure 4.3. Recombinant thioester from an intein fusion .......................................... 83

Figure 5.1. O-GlcNAc modification and α-synuclein ................................................ 92
Figure 5.2. Characterization of full-length, recombinant αSyn and
αSyn(T72A) mutant ................................................................................................... 94

Figure 5.3. α-Synuclein(T72A) displays reduced aggregation compared to
wild-type α-synuclein ................................................................................................ 94

X

Figure 5.4. Semisynthesis of α-synuclein .................................................................. 96

Figure 5.5. Characterization of thioester peptide 1 .................................................... 97
Figure 5.6. Characterization of protein fragment 2 .................................................... 98
Figure 5.7. Ligation of peptide 1 and protein 2 ......................................................... 98
Figure 5.8. N-terminal deprotection of the thiazolidine of protein 4 to yield
protein fragment 5 ...................................................................................................... 99

Figure 5.9. Expression and characterization of protein thioester fragment 3 ............ 99
Figure 5.10. Ligation of protein thioester 3 and protein 5 to give full-length
α-synuclein ............................................................................................................... 100

Figure 5.11. Desulfurization of α-synuclein 6 to give synthetic α-synuclein .......... 100

Figure 5.12. Characterization of O-GlcNAcylated thioester peptide 7 ................... 101
Figure 5.13. Ligation of protein 2 and glycopeptide 7 to give O-GlcNAcylated
protein fragment 8 .................................................................................................... 102

Figure 5.14. N-terminal deprotection of thiazolidin of protein 8 to yield
glycoprotein fragment 9 ........................................................................................... 103

Figure 5.15. Ligation of protein thioester 3 and gylcoprotein 9 to give
full-length O-GlcNAcylated α-Synuclein ................................................................ 103

Figure 5.16. Desulfurization of α-synuclein 10 to give O-GlcNAcylated
α-synuclein [α-synuclein(gT72)] ............................................................................. 104

Figure 5.17. Structural characterization of synthetic and recombinant
synucleins using circular dichroism and dynamic light scattering .......................... 105

Figure 5.18. O-GlcNAcylation blocks α-synuclein aggregation ............................. 106
Figure 5.19. TEM images of protein aggregation reactions for modified and un-
modified α-synuclein ............................................................................................... 108

Figure 5.20. O-GlcNAcylation has no effect on α-synuclein membrane binding or
bending ..................................................................................................................... 110

Figure 5.21. O-GlcNAcylation affects subsequent phosphorylation of αSyn ......... 112

XI

Figure 5.22. Analysis of cellular toxicity samples by TEM .................................... 113
Figure 5.23. O-GlcNAcylation blocks α-synuclein toxicity .................................... 115
Figure 5.24. O-GlcNAcylated αSyn is largely excluded from aggregates .............. 117

Figure 5.25. O-GlcNAcylation inhibits aggregation by preventing incorporation
of α-synuclein into aggregates ................................................................................. 119

Figure 5.26. O-GlcNAcylation does not strongly inhibit aggregation in the
presence of pre-formed fibers .................................................................................. 120

Figure 5.27. Model of α-synuclein aggregation ....................................................... 122
Chapter 1. Parkinson’s Disease and the Pathological protein α-Synuclein
1.1 Introduction to Parkinson’s Disease
Parkinson’s Disease (PD), is a progressive, neurodegenerative disease
characterized by symptoms of stiffness and slowness of movement (bradykinesia),
postural instability, and resting muscle tremors. Dementia is also common at late stages
of the disease. PD is the second most common neurodegenerative disease, after
Alzheimer’s Disease (AD), afflicting 1% of the population over the age of 65 and 5% of
the population over 80(Farrer, 2006). PD results from the loss of neurons that produce the
neurotransmitter dopamine (DA) in a region of the brain called the Substantia Nigra pars
compacta (SNpc). This small region supplies DA to the striatum, which is responsible for
initiation and control motor function, and progressive neuron loss leads to development
of PD symptoms. Symptoms do not usually manifest until about 50% of DA neurons in
the SNpc and 70-80% of striatal DA levels are lost(Marsden, 1990). The pathological
hallmarks of PD observed in patient autopsies are intracellular inclusions called Lewy
bodies (LB) and Lewy neurites (LN). These inclusions are proteinaceous and observed in
the cell bodies or processes, respectively, of neurons of the SNpc and other degenerative
regions of patient brains.
The majority of PD cases are idiopathic, and the disease was long thought to have
no underlying genetic cause. However, a familial form of the disease that also exhibited
early age of onset was discovered, and attributed to a mutation in the SNCA gene that
1
codes for the protein α-Synuclein (αSyn)(Athanassiadou et al., 1999). This mutation,
A53T, was followed by the discovery of additional missense mutations A30P(Krüger et
al., 1998) and E46K(Zarranz et al., 2004) in the same protein that also cause heritable
forms of the disease. This protein, αSyn, had previously been identified from patients
with AD as a 140 amino acid, natively-unfolded protein(Weinreb et al., 1996) but after its
link to familiar PD was discovered, it was also shown to be the major protein component
of LB and LN deposits(Spillantini et al., 1998). Taken together, these findings suggest a
critical role for αSyn in the progression of PD.
2
1.2 Physiology of α-Synuclein
α-Syn is a small cytosolic protein of only 140 amino acids that is heavily
expressed in neurons and localized to the presynaptic nerve terminal. In solution it lacks
any persistent secondary structure, but it does form an extended α-helical structure when
it interacts with lipid membranes(Clayton and George, 1999). Its sequence is divided into
three regions (Fig. 1.1) with distinct properties: the N-terminal region (aa 1-60) is lysine-
rich and contains four of the protein’s six imperfect hexameric repeats of the sequence
KTKEGV . These repeats help drive the formation of an amphipathic helix when αSyn
interacts with membranes. The N-terminal region is also home to the three familial point
mutations listed above. Amino acids 61-90 are referred to as the Non-Amyloid
Component (NAC) and contains an abundance of hydrophobic amino acids. This region
has been shown to be required for the disease-related, aggregated form of the protein
described below. Finally, the C-terminal region is a highly soluble domain containing
many acidic residues as well as several prolines. This region is also implicated in a
variety of interaction between αSyn and metal cations(Breydo et al., 2012), small
molecules(Mazzulli et al., 2006), and other proteins(Jin et al., 2007).
The exact physiological role of αSyn in neurons is not fully understood and is still
an area of intense investigation. However, many overlapping lines of evidence are
beginning to illuminate this problem. SNCA-knockout mice are non-lethal, suggesting
the role of αSyn is nonessential, however these mice do have impaired synaptic activity
and response over prolonged periods of stimulation. These findings suggest these mice
3
were deficient in replenishing synaptic vesicles from the reserve pools and trafficking
them to the site of release. Alternatively, mice over-expressing human αSyn exhibited
impaired rates of vesicle exocytosis and had decreased amount of neurotransmitter
release(Lashuel et al., 2013). This effect on vesicle trafficking and cycling is likely
exerted through the natural propensity for αSyn to interact with synaptic vesicles in its α-
helical conformation. In addition to these observations, αSyn has also been shown to act
as a chaperone for SNARE proteins, thus expanding it’s role in regulation of vesicle
fusion and neurotransmitter release(Burré et al., 2010). Cell culture experiments have
also demonstrated that αSyn plays a role in protecting neurons from oxidative stress,
particularly related to the toxicity of oxidized DA species. However, these results were
highly sensitive to levels αSyn expression, as higher expression led to increased
toxicity(Bellucci et al., 2012).

4
Figure 1.1 α-Synuclein primary structure and native monomer behavior. The sequence of αSyn is
broken up into three regions based on functional behavior. In solution the monomer adopts a
random coil conformation but it forms an extended α-helical structure when it interacts with lipid
membranes
5
1.3 α-Synuclein aggregation and toxicity
While αSyn is natively a natively unfolded monomer in solution, in the disease
state it forms aggregates that are rich in β-sheet secondary structure. In it’s aggregated
form, the NAC region of αSyn forms a core consisting of five parallel β-sheet. Monomers
stack on top of one another with these β-sheets in register(Emanuele and Chieregatti,
2015). Aggregates can extend in this manner to form long, unbranched fibrils hundreds of
microns long. This is the form in which αSyn is deposited in LB, and fibrils grown in
vitro are nearly identical to those observed in patient samples and from animal
models(Goedert, 2001). Aggregation of αSyn follows a nucleation dependent kinetic
model with an initial lag phase, followed by an explosive growth in the amount of fibrils
before finally reaching a plateau (Fig. 1.2). From a thermodynamic point of view, the lag
phase is due to an initial organization of the unfolded monomer(s) requiring a high degree
of order before creating an aggregation-capable nucleus that can be extended(Guo and
Lee, 2014). In practical terms this means two or more αSyn monomers must first come
together in an oligomeric species of some kind before undergoing a structural
rearrangement to the core β-sheet structure required for eventual fibril growth. This
mechanism has been a topic of much debate as oligomeric species of αSyn have proven
to be both diverse and difficult to characterize cleanly. Some oligomers have been
observed that are protofibrillar, with a large degree of β-sheet character, some with more
α-helical structure, or with little regular structure at all. Some oligomers also form ring or
pore-like structures that interact with and may permeabilize membranes(Breydo et al.,
6
2012). Not all forms of oligomers are interconvertible and not all oligomers are on the
path from monomers to fibrils.
Some of the most illustrative data on the oligomer species involved in fibril
formation comes from a single-molecule fluorescence microscopy study that used αSyn
monomers with two fluorophores to identify oligomeric species in solution by
FRET(Cremades et al., 2012). Briefly, FRET, or Forster Resonance Energy Transfer,
allows for the transfer of energy between fluorophores with overlapping emission and
excitation spectra, but only across short distances (r to the power of -12). FRET was only
observed from oligomers and the FRET efficiencies and size of the oligomers was used to
identify two classes of oligomers. The first proceeds directly from monomers, forms
quickly, and is not stable to proteinase K digestion. The second oligomeric species
proceeds from the first and is more densely packed and resistant to degradation. The
second oligomeric species also readily elongates to form mature fibrils. The transition
between the two is very slow and it’s half life is on the same order of time as the typically
observed lag phase for aggregation kinetics. These results, along with others suggest that
αSyn monomers likely populate many less stable forms of soluble oligomers, but once
one undergoes the slow rearrangement to an ordered β-sheet structure, it proceeds
downhill towards fibril elongation more readily than reverting back.
7
Figure 1.2 α-Synuclein forms β-sheet rich aggregates. a) Illustration of the general pathway from
monomers to mature fibrils shows the rate limiting transition from unstable, transient oligomers
to stable, aggregation-competent oligomers. b) Representation of the kinetics of the nucleation
dependent aggregation pathway. The slow rate of accumulation of stable oligomers leads to a lag
phase that is followed by a rapid growth phase as pre-fibrillar oligomers extend to form fibrils.
8
1.4 Mechanism of toxicity
A crucial consideration for understanding the pathological role of αSyn is: what is
the mechanism, or mechanisms, by which it is cytotoxic? Evidence from PD patients
suggests that αSyn deposits and neuron loss are interdependent, but the exact causative
relationship between αSyn, its aggregation, and the fate of DA neurons is still unclear.
Animal models have been helpful in elucidating this relationship, but most animal models
do a poor job or replicating all of the key factors of PD, namely progressive DA neuron
loss, LB type inclusion, and symptomatic motor deficiency. Similarly, in vitro and cell
culture studies are invaluable but can not replicate every aspect of the disease
system(Blesa et al., 2012). Thus, discerning the cytotoxic mechanism at work in PD has
proven challenging, with a good deal of seemingly contradictory data across many types
of experiments. Cytotoxicity due to αSyn aggregation must come from either a toxic loss
of function due to a lack of functional αSyn monomer, a toxic gain of function due to
some action of the aggregated species, or some combination of the two.
The point mutations linked to familial PD mentioned above offer an important
line of evidence for this problem. The A53T mutant aggregates far more readily than WT
αSyn, which results in increase fibril formation and greater toxicity in both animal and
cell culture models. At first this suggests that pushing αSyn into fibrils drives
cytotoxicity. However, the A30P mutant forms fibrils more slowly than WT, but is still
significantly more toxic. It has also been shown that A30P has decreased affinity for lipid
membranes and does not form α-helical structures as readily. This would lead to higher
9
effective levels of A30P in solution and also provide the monomers with fewer possible
pathways for oligomer formation. However, with slower rates of fibril formation, A30P
would presumably build up higher levels of oligomeric species before forming
fibrils(Farrer, 2006). In fact, mutants that are biased towards oligomer production and
away from mature fibril formation are often more toxic in cell based assays than WT.
This implies that the most cytotoxic species must be an intermediate
oligomer(Taschenberger et al., 2011). Indeed (Cremades et al., 2012) showed that the
more stable, protofibril oligomer generates significantly more reactive oxygen species
(ROS) when used to treat cultured neurons, when compared to the less stable oligomer
and the mature fibrils. This and other such observations have lead to the toxic oligomer
hypothesis, which states that the most damaging state for αSyn to occupy is an oligomer
between monomer and fibril, with both extreme ends of the spectrum being less toxic. An
interesting corollary to this theory is that fibril formation, and by extension LBs, are
actually cytoprotective because they sequester toxic oligomers from the cytosol.
However, this theory is far from complete. For instance, the same
paper(Cremades et al., 2012)showed that, in the absence of monomer, fibrils will shed
toxic oligomers, thus acting as a reversible store of toxic species. Additionally, mutants
that will form oligomers but can not eventually form fibrils are more toxic when
overexpressed in rats, but only over a short period of time, and furthermore do not lead to
sustained and progressive degeneration of neurons. This suggests that while oligomeric
10
species may do the most harm when present, the ability to form fibrils is likely necessary
for prolonged toxicity(Taschenberger et al., 2011).
As mentioned above, the mode of action by which αSyn exerts its neurotoxic
effect is not fully understood, but it is likely a combination of gain of function on behalf
of the aggregates and loss of function from the monomer. The monomer, as described
above, is heavily involved in synaptic vesicle homeostasis, through the action of its α-
helical, membrane bound form(Murphy et al., 2000; Yavich, 2004). Loss of functional
monomers would lead to a delay in vesicle trafficking and neurotransmitter release, as
well as re-uptake and recycling. This would serve to put stress on the cell by disrupting
cellular transit machinery and axonal transport(Lundblad et al., 2012). Failure to release
synaptic vesicles would also see a build up of DA within the cell, which had been shown
to cause oxidative stress via it’s oxidized quinone byproducts. This disruption of neuronal
function would also be compounded by the presence of aggregated αSyn deposits that
would likely interfere with the movement of vesicles and other cargo(Choi et al., 2013).
Oligomeric αSyn has been shown to inhibit chaperone mediated autophagy, which
not only reduces clearance of aggregates but also places the cell under additional
stress(Tanik et al., 2013). As stated above, protofibrillar oligomers that have a stable β-
sheet structure can induce high levels of ROS inside cells. This is, in part, because they
have a large amount of exposed hydrophobic surface that can interact with and disrupt the
stability of folded proteins. This would add additional unfolded protein load the cell’s
protein turnover machinery, exacerbating the problem of turning over αSyn aggregates.
11
Finally, annular oligomers that interact with membranes to form pores can disrupt
a variety of cellular functions in an afflicted neuron(Tosatto et al., 2012). Mitochondrial
dysfunction is a major symptom of αSyn pathology, due in part to disruption of
mitochondrial membranes and loss of membrane potential. This will hinder the cells
ability to generate ATP as well as release additional ROS equivalents and potentially
trigger apoptosis(Zaltieri et al., 2015). More significantly, permeabilization of the outer
plasma membrane can lead to aberrant calcium flux into the cell, causing further
oxidative stress and ROS generation. Given that oxidation of αSyn has been shown to
promote the transition from monomer to aggregates, this would also result in a feed
forward loop driving more cytotoxicity.
12
1.5 Propagation of aggregates between cells
Once αSyn aggregates begin to disrupt cellular processes within an afflicted cell,
they can then promote their release out of the cell and into the extracellular space(Guo
and Lee, 2014). This can be accomplished via passive release through disruptions or
pores in the cell membrane, or upon cell death. However there are several mechanisms of
active export that lead to the spread of aggregates. Continued impairment of the
lysosomal/autophagic turnover machinery can lead to secretion of aggregates in
exosomes(Alvarez-Erviti et al., 2011). Continued cell stress will also lead to aggregates
being released by non-canonical exocytosis or released by direct synaptic transfer(Jang et
al., 2010). Once outside of the cell, less stable oligomers will likely break up and
disperse, but more stable oligomeric and fibrillar species will persist and can then be
taken up by surrounding cells. Endocytosis and degradation of protein aggregates may
actually be a natural response to these species, but this system can become overwhelmed,
resulting in release of the aggregates inside the cell(Lee et al., 2008). Oligomers and
fibrils are also thought to enter the cell through both active and passive transmembrane
transporters, however the identity of such proteins remains unknown. Regardless of their
mode of entry, once inside the aggregates will seed the growth of more aggregates and
propagate the disease phenotype even if the cell had previously lacked αSyn
pathology(V olpicelli-Daley et al., 2011). This mechanism explains why and how αSyn
aggregates are observed to move throughout the brain of patients progressively with the
disease through regions that share neuronal connections. This same phenomenon can be
13
recapitulated in mice using human A53T(Luk et al., 2012). Local injection of a small
amount of αSyn aggregates initiates a progressive and predictable spread of aggregate
pathology and neuron loss. Finally, extracellular aggregates can also be taken up by non-
neuronal cells such as microglial. Activation of glial cells by exogenous aggregates leads
to neuronal inflammation, which will lead to further damage and loss of surrounding
neurons(Sacino et al., 2014).
1.6 Conclusion
αSyn is a small protein with an unusual lack of defined structure or definite function.
However it is a crucial component in the not yet fully understood mechanism of PD
development and progression. Its uniquely variable secondary structure allows it to both
perform a variety of useful cellular functions, and to adopt a wide range of high
molecular weight aggregates, many of which are strongly associated with toxicity and
neuron loss. It appears that αSyn can modulate many factors that contribute to PD, for
better or for worse. In order to improve the current available options for PD treatment, it
will be necessary to understand how αSyn can be influenced in order to favor its native
physiological functions over its disease associated behavior. While research in this field is
constantly uncovering more about the biological function of αSyn, a good therapeutic
target by which to control or modulate it’s behavior remains to be found.
14
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and Cooper, J.M. (2011). Neurobiology of Disease. Neurobiology of Disease 42, 360–
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Athanassiadou, A., V outsinas, G., Psiouri, L., Leroy, E., Polymeropoulos, M.H., Ilias, A.,
Maniatis, G.M., and Papapetropoulos, T. (1999). Genetic analysis of families with
Parkinson disease that carry the Ala53Thr mutation in the gene encoding alpha-synuclein.
Am. J. Hum. Genet. 65, 555–558.
Bellucci, A., Zaltieri, M., Navarria, L., Grigoletto, J., Missale, C., and Spano, P. (2012).
From α-synuclein to synaptic dysfunctions: New insights into the pathophysiology of
Parkinson's disease. Brain Res 1476, 183–202.
Blesa, J., Phani, S., Jackson-Lewis, V ., and Przedborski, S. (2012). Classic and New
Animal Models of Parkinson's Disease. Journal of Biomedicine and Biotechnology 2012,
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Breydo, L., Wu, J.W., and Uversky, V .N. (2012). Biochimica et Biophysica Acta -
Molecular Basis of Disease 1822, 261–285.
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Choi, B.-K., Choi, M.-G., Kim, J.-Y ., Yang, Y ., Lai, Y ., Kweon, D.-H., Lee, N.K., and
Shin, Y .-K. (2013). Large α-synuclein oligomers inhibit neuronal SNARE-mediated
vesicle docking. Proc Natl Acad Sci USA 110, 4087–4092.
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Clayton, D.F., and George, J.M. (1999). Synucleins in synaptic plasticity and
neurodegenerative disorders. J Neurosci Res 58, 120–129.
Cremades, N., Cohen, S.I.A., Deas, E., Abramov, A.Y ., Chen, A.Y ., Orte, A., Sandal, M.,
Clarke, R.W., Dunne, P., Aprile, F.A., et al. (2012). Direct Observation of the
Interconversion of Normal and Toxic Forms of α-Synuclein. Cell 149, 1048–1059.
Emanuele, M., and Chieregatti, E. (2015). Mechanisms of Alpha-Synuclein Action on
Neurotransmission: Cell-Autonomous and Non-Cell Autonomous Role. Biomolecules 5,
865–892.
Farrer, M.J. (2006). Genetics of Parkinson disease: paradigm shifts and future prospects.
Nat Rev Genet 7, 306–318.
Goedert, M. (2001). Alpha-synuclein and neurodegenerative diseases. Nat Rev Neurosci
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Guo, J.L., and Lee, V .M.Y . (2014). Cell-to-cell transmission of pathogenic proteins in
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Jang, A., Lee, H.-J., Suk, J.-E., Jung, J.-W., Kim, K.P., and Lee, S.-J. (2010). Non-
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Jin, J., Li, G.J., Davis, J., Zhu, D., Wang, Y ., Pan, C., and Zhang, J. (2007). Identification
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Krüger, R.R., Kuhn, W.W., Müller, T.T., Woitalla, D.D., Graeber, M.M., Kösel, S.S.,
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Lee, H.-J., Suk, J.-E., Bae, E.-J., Lee, J.-H., Paik, S.R., and Lee, S.-J. (2008). Assembly-
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Luk, K.C., Kehm, V .M., Zhang, B., O'Brien, P., Trojanowski, J.Q., and Lee, V .M.Y .
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Neuron 72, 57–71.
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18
Yavich, L. (2004). Role of -Synuclein in Presynaptic Dopamine Recruitment. Journal of
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19
Chapter 2. Post-Translational modifications of α-Synuclein add further complexity
2.1 An overview of the many Post-Translation Modification on αSyn
For being such a small protein, αSyn boasts a great number and variety of post-
translational modifications (PTMs) (Fig. 2.1). These modifications can be both
enzymatically installed or result from chemical modification by the cellular environment,
they can be cycle on and off or persist for the life of the protein, and they can either have
functional consequences or be largely passive in their effect. Unfortunately, many of the
modifications have been shown to be at play before, during, or after αSyn aggregation,
thus necessitating an attempt to understand their consequences if one is to fully
comprehend the behavior of αSyn in both healthy and diseased cells. This chapter will
present a brief overview of the many PTMs of αSyn and place emphasis on those
modifications that have the most bearing on PD pathology or that offer promising targets
for disease intervention. This chapter will also address one class of modification in
particular, O-GlcNAc modification, which is little understood but offers significant
interest for investigation, as well as unique challenges.
2.2 Non-enzymatic modifications
These modifications are the result of direct chemical reactions of side chains as a
result of an oxidizing environment within the cell. Both of these modifications can alter
20
αSyn aggregation, and represent a direct connection between the diseased state of the
protein and the diseased cellular state.
Methionine oxidation results from the direct oxidation of methionine side chain
thioether to a sulfoxide and then to a sulfone. αSyn has four methionines (M1, M5,
M116, M127) that are readily oxidized(Glaser et al., 2005). Methionine oxidation adds
increased hydrophilicity and in the case of αSyn helps to inhibit the formation of
oligomers or aggregates. The degree of inhibition is proportional to the number of
possible methionines that are fully oxidized. Methionine oxidation thus favors either the
unfolded monomer or unstructured oligomers that have been shown to be non-
toxic(Rekas et al., 2010). This may present a natural mechanism by which cells can
counteract the drive of cellular ROS towards aggregation. There is also some evidence
that this reducing capacity of αSyn may be a natural function to help buffer the redox
homeostasis of DA neurons(Zaltieri et al., 2015).
Tyrosine nitration is the result of peroxynitrite reacting with tyrosine side chains
to produce a 3-Nitro-tyrosine adduct. This modification makes the tyrosine more bulky,
more polar, and more acidic(Ischiropoulos, 2003). All four of the tyrosines in αSyn (Y39,
125, 133, and 136) have been shown to be susceptible to nitration, but the predominant
modification in patients is found on Y39(Danielson et al., 2009). On its own this
modification disfavors its own aggregation, but the similarity to methionine oxidation
ends there. 3-Nitro Y39 accelerates the aggregation of WT αSyn and is highly cytotoxic
on its own(Hodara et al., 2004; Yu et al., 2010). This is likely because the nitrated adduct
21
can form a stable oligomeric species that is toxic and can be further stabilized by covalent
cross-linking between oxidized tyrosines.
Figure 2.1 α-Synuclein is heavily decorated with PTMs. This is a simplistic depiction of the
various sites and types of modification. While these PTMs all occur at different rates, there is still
a high degree of complexity.
2.3 Phosphorylation
Phosphorylation is the addition of a phosphate group to the hydroxyl group of
serine and threonine side chains, or to the phenolic oxygen of tyrosines. This
modification is installed by a class of enzymes called kinases, using ATP as a phosphate
source, and can subsequently be removed by enzymes called phosphatases. Kinases and
phosphatases are abundant generally have high substrate or sequence specificity. The
tightly controlled cycling on and off of phosphorylation events is responsible for a great
deal of cell signaling and control. αSyn has three significant sites of phosphorylation:
S87, S129, and Y125(Inglis et al., 2008; Nakamura et al., 2002). Modification at Y125
was shown to have little effect on the native structure of the protein or its membrane
affinity. This site of modification also showed little to no influence on the protein’s ability
22
to form aggregates, when compared to the WT(Hejjaoui et al., 2012). Both sites of serine
phosphorylation (87 and 129) were discovered in model cell culture experiments. Serine
129 phosphorylation is probably the most disease-relevant of the modifications and by far
the most studied. This modification was shown to occur on aggregate αSyn in LB and
tissues of patients(Anderson, 2006; Fujiwara et al., 2002). This association was
recapitulated in animal models, however there arouse some degree of conflicting data
about wether pS129 does or does not promote aggregation on it’s own(Paleologou et al.,
2008). It was only recently demonstrated recently that αSyn aggregates can be
phosphorylated at S129 after forming and this modification can actually lead to
degradation of the protein(Oueslati et al., 2013). Phosphorylation at S87 is generally
found to be disruptive of any aggregates, likely due to it’s location in the NAC region.
This site can also contribute to protein turnover, though not to the extent of
pS129(Paleologou et al., 2010).
2.4 Nα-Acetylation
Acetylation of the N-terminal amine has been found to occur on the majority of
αSyn material isolated from aggregates or in solution(Anderson, 2006). This modification
is not currently known to have a significant effect on natural or disease related αSyn
behavior, but is believed to increase the α-helical propensity of the free monomer.
Synthetic preparation of N-acetylated αSyn showed no significant difference in stability
or aggregation when compared to WT, and while it was shown to have a similar affinity
23
for lipid membranes, it is unknown if any of its cellular functions regarding membrane
binding would be similarly unaffected(Fauvet et al., 2012).
2.5 Ubiquitin and ubiquitin-like modifiers
Ubiquitin (Ub) and other homologous modifiers are small, globular protein that
are activated by a series of enzymes at their C-terminus before being transferred to the
target lysine side chain. The protein can subsequently be removed by the action of
proteases called deUbiquitylases. Additionally, Ub can be polymerized through addition
of additional Ub monomers to one of seven lysines and/or the N-terminal amine. This
imparts a great deal of complexity to the Ub signaling system(Welchman et al., 2005).
Ubiquitination is an abundant modification on αSyn, with nine lysine residues, most of
them in the N-terminal region, capable of being modified with a single Ub. LBs are
highly immunoreactive to Ub and, like pS129, Ub was thought to be closely related to
aggregate formation(Hasegawa et al., 2002; Manetto et al., 1988). Initial results from
studying Ub from cells or using recombinant Ub ligase led to some conflicting results
about whether it increased or decreased aggregation. Further complicating the picture was
the fact that Ub modification can also target αSyn for degradation(Tofaris et al., 2011). In
order to approach this complex system, several groups synthesized various Ub-αSyn
conjugates, utilizing a variety of protein synthesis techniques to create the necessary
isopeptide bond(Hejjaoui et al., 2010; Meier et al., 2012). This synthetic approach has
yielded great insight, reveal that Ub can have various effects on αSyn aggregation
24
behavior in site specific manner. The previously contradictory results likely can be
explained in the context of heterogeneous mixtures of sites modified to give divergent
phenotypes. Mono-Ub was generally found to be protective against aggregation, with
effectiveness varying by site. Synthetic preparation of mono-Ub modified αSyn was also
successful in demonstrating the site specific sensitivity of αSyn to Ub as a signal for
proteasomal degradation(Abeywardana et al., 2013). Finally, the small, ubiquitin-like
modifiers (SUMOs) which is installed and behaves analogously to Ub, can be installed on
Lys96 and Lys102. These protein adducts were similarly prepared and studied, to reveal
that they too have a protective effect against aggregation(Abeywardana and Pratt, 2015).
2.6 O-GlcNAc modification
O-GlcNAc modification, or O-GlcNAcylation, is the transfer of the single
monosaccharide N-acetyl glucosamine to the hydroxyl group of serine and threonine side
chains. It is installed by the enzyme O-GlcNAc Transferase (OGT), and unlike most
glycosylations, can be cycled off by the enzyme O-GlcNAcase (OGA) in a time less than
the lifetime of the protein. The O-GlcNAc moiety is derived from the high energy donor
uridine diphosphatidyl GlcNAc (UDP-GlcNAc) (Fig 2.2). This compound is closely tied
to the metabolic state of the cell, as it requires building blocks from several major
metabolic pathways, and increased levels of UDP-GlcNAc can actually alter the effective
Km of OGT for substrate proteins(Bond and Hanover, 2015; Hardivillé and Hart, 2014).
Thus both the quantity and variety of modifications depends on cell metabolism. O-
25
GlcNAcylation of αSyn has been identified in both rodent and human cells at T53 (mouse
only), T64, T72, and S87, with T72 being the most commonly identified.
Figure 2.2 Synthesis and incorporation of O-GlcNAc modifications. Glucose is shunted into the
Hexosamine biosynthetic pathway from as fructose 6-phosphate and is converted to UDP-
GlcNAc. The modification is cycled on and off by OGT and OGA respectively.
Next to nothing is known about the cellular consequences of these modifications,
likely for a variety of challenges involved in studying O-GlcNAcylation. O-GlcNAc
proteins are often more difficult to isolate and identify compared to other modification
such as phosphorylation and Ubiquitination. In particular O-GlcNAc modified peptides
are not well detected by traditional proteomics techniques. Additionally OGA and OGT
are the only enzymes that manipulate this modification, making selective modulation
26
with small molecules less powerful. OGT sometimes requires accessory proteins to bind
and modify substrates, meaning in vitro or recombinant techniques may give false
negatives.
Despite the challenges listed above and the dearth of information about the
consequence of this modification, it still presents some compelling reasons for
investigating further. O-GlcNAcylation is generally known to increase solubility or
stabilize protein that it modifies, which, combined with the fact the at the known sites of
modification are all in or near the NAC region required for aggregation, suggests that this
modification is positioned to act against αSyn aggregation. Additionally, glucose
metabolism is known to be reduced in cortical and subcortical regions of patients’ brains
in PD and other forms of neurodegeneration(Eberling et al., 1994; Siebner et al., 2003).
Furthermore, genome wide analysis has revealed that several gene sets involved in
glucose uptake and utilization are under-expressed in PD patients even at early stages of
the disease.(Ciron et al., 2015) Together, this suggests that alter glucose metabolism may
contribute to altered O-GlcNAc modification in PD versus healthy brains, which could
have an effect on αSyn in diseased cells. Finally, there is evidence from other
neurodegenerative diseases, namely AD, that an aggregation prone protein responsible for
forming proteinaceous deposits (Tau) in diseased neurons that is protected from
aggregating by a single O-GlcNAc modification(Yuzwa et al., 2012).
27
2.7 Conclusion
Despite its short length of only 140 amino acids, αSyn is heavily decorated with a
wide variety of post-translational modifications. These PTMs add to the already
complicated view of αSyn function in the cell. The study of αSyn PTMs is a growing
field, as more and more evidence demonstrates the strong effect PTMs can have on the
biological function and fate of αSyn. It is now crucial to discern which of these
modifications can exert some effect on αSyn that favors its natural function or disfavors
its disease related behavior. With this information in hand, attention can be turned to
targeting those specific modifications for new therapies. Based on evidence from other
aggregating proteins such as Tau, given the placement of modification sites in the core of
the NAC region, and given the strong line of evidence linking glucose metabolism to PD,
O-GlcNAc modification of αSyn appears to be a PTM that is poised to exert an effect on
αSyn function in the context of PD.
28
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Yu, Z., Xu, X., Xiang, Z., Zhou, J., Zhang, Z., Hu, C., and He, C. (2010). Nitrated α-
Synuclein Induces the Loss of Dopaminergic Neurons in the Substantia Nigra of Rats.
PLoS ONE 5, e9956.
32
Yuzwa, S.A., Shan, X., Macauley, M.S., Clark, T., Skorobogatko, Y ., V osseller, K., and
V ocadlo, D.J. (2012). Increasing O-GlcNAc slows neurodegeneration and stabilizes tau
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Who’s on First? Parkinson’S Disease 2015, 1–10.
33
Chapter 3. O-GlcNAc modification prevents peptide-dependent acceleration of α-
synuclein aggregation
3.1 Introduction
Aberrant protein folding and aggregation are implicated in numerous diseases and
represent a key challenge to biological and chemical investigations aimed at eventual
therapies. One class is neurodegenerative diseases associated with the aggregation of the
protein α-synuclein (α-syn), including dementia with Lewy bodies, Lewy body variant
Alzheimer’s disease, and Parkinson’s disease (PD)(Bellucci et al., 2012). A pathological
hallmark of all of these “synucleinopathies” is the presence of proteinaceous, intracellular
deposits called Lewy bodies (LBs), of which the major component is α-syn. PD is the
most prevalent of the synucleinopathies, affecting 1% of the population over 65 and 4 to
5% of those over 85(Farrer, 2006). It is a progressive, neurodegenerative disease resulting
from the loss of dopaminergic neurons in the Substantia nigra, accompanied by
significant accumulation of aggregated α-syn(Spillantini et al., 1998). α-Syn is a 140
amino-acid protein that is prevalent in pre-synaptic neurons of the central nervous
system. Its sequence is comprised of three domains: an N-terminal repeat domain, the
central non-amyloid component (NAC) required for aggregation, and the C-terminal
acidic domain. Native α-syn populates at least three conformations. In the cytosol, the
protein is most probably a combination of a helically folded tetramer and unfolded
monomer,(Bartels et al., 2011; Weinreb et al., 1996) while adopting an extended α-helical
34
conformation when associated with cellular membranes(Ferreon et al., 2009). In contrast,
in diseased cells, α-syn consists of β-sheet rich, high molecular-weight aggregates(Fink,
2006). This disease state closely resembles the oligomers and fibrils that are readily
formed by α-syn in vitro. Importantly, a range of oligomers, fibrils, and aggregates are
highly toxic to cells and model organisms, biochemically supporting their role in
disease(Cremades et al., 2012). Additionally, a variety of point mutations, duplications,
and triplications in the α-syn gene result in familial and early-onset forms of PD(Chartier-
Harlin et al., 2004; Kruger et al., 1998; Polymeropoulos et al., 1997; Singleton, AB et al.,
2003; Zarranz et al., 2004).
α-Syn is also a substrate for a variety of posttranslational modifications (PTMs)
(Oueslati et al., 2010) including proteolysis(Li et al., 2005), ubiquitination(Gómez-
Tortosa et al., 2000), phosphorylation(Chen et al., 2009; Fujiwara et al., 2002; Hejjaoui et
al., 2012; Paleologou et al., 2010), acetylation(Öhrfelt et al., 2011), and
glycosylation(Shimura et al., 2001); thus adding further complexity to its structure and
aggregation potential. A variety of in vitro and cellular experiments have shown that
ubiquitination can dramatically alter α-syn aggregation and toxicity, and that individual
ubiquitin modifications sites have different biochemical consequences(Hejjaoui et al.,
2010; Meier et al., 2012). In contrast, one PTM that has received very little attention is
the modification of serine and threonine residues by the monosaccharide N-acetyl-D-
glucosamine (O-GlcNAc), which has been identified as an endogenous modification of α-
syn at residues 53, 64, 72, and 87(Alfaro et al., 2012; Wang et al., 2010); however, the
35
biochemical consequences of these modifications are currently unknown. The
overwhelming majority of O-GlcNAc modification (O-GlcNAcylation) occurs on
cytosolic and nuclear proteins and is absolutely required for development in
mammals(Shafi et al., 2000) and Drosophila(Sinclair et al., 2009). O-GlcNAcylation is
dynamic through addition by the enzyme uridine diphosphate-N-acetyl-D-
glucosamine:polypeptidyl transferase (OGT) and subsequent removal by the enzyme O-
GlcNAcase (OGA)(Hart et al., 2007). O-GlcNAc modification can alter protein function
through changes in protein stability, localization, and molecular interactions. The levels
of O-GlcNAcylation are in part controlled by the concentration of the OGT substrate
uridine 5’-diphospho-N-acetylglucosamine (UDP-GlcNAc), which in turn is generated
from glucose through the hexosamine biosynthetic pathway(Hanover et al., 2010).
Therefore, the amount of O-GlcNAc modification is intimately linked to glucose
metabolism. Additionally, the global levels of O-GlcNAcylation are dramatically
increased by a variety of cellular stresses and this increase is protective against cell
death(Ngoh and Jones, 2008; Zachara et al., 2004). Glucose metabolism in the brain is
decreased in Alzheimer’s disease(Siebner et al., 2003) as well as in PD(Borghammer et
al., 2010; Eberling et al., 1994), and several genes associated with mitochondrial function
and glucose metabolism have been shown to be underexpressed in PD patients(Zheng et
al., 2010). In addition, protein aggregation is associated with cellular stress resulting from
impaired protein folding and degradation(Ong and Kelly, 2011), implicating a potential
role for O-GlcNAc modification in disease pathology. Indeed, in vitro analysis of the
36
Alzheimer’s disease associated protein tau revealed that O-GlcNAc modification directly
inhibits protein aggregation(Yuzwa et al., 2012). Furthermore, increasing O-
GlcNAcylation levels using a small-molecule inhibitor of OGA in a transgenic mouse
model of Alzheimer’s raises the amount of tau O-GlcNAc modification and reduces the
number of neurofibrillary tangles and neuronal cell loss(Yuzwa et al., 2012; 2008).
Figure 3.1 Co-expression of α-syn with OGT. Western blotting against OGlcNAcylation with
RL2 antibody for α-syn expressed with or without O-GlcNAc transferase (OGT) and TAB-1
expressed with or without OGT. Coomaisie stained gels presented to show even loading.
37
3.2 Results
Given this information, we set out to determine if O-GlcNAcylation of α-syn can
also inhibit protein aggregation and potentially play a protective role in
synucleinopathies. In support of this hypothesis, the identified sites of α-syn O-GlcNAc
modification are located within the central NAC region that is required for formation of
aggregates. Of particular interest is the most common O-GlcNAc modification site at
threonine 72 (T72) as mutation of this residue to either glutamic acid (T72E) or proline
(T72P) has been shown to inhibit aggregate formation in vitro(Koo et al., 2009). Thus, we
wanted to explore the potential effect of O-GlcNAc modification at T72 on the
aggregation of α-syn. Similar studies on tau and the TAK-1 binding protein used a
coexpression technology where OGT is expressed with a substrate protein of choice in
Escherichia coli, resulting in substrate O-GlcNAcylation(Yuzwa et al., 2008). However,
α-syn was not modified under these conditions (Fig. 3.1). To overcome this roadblock,
we took advantage of the observation that α-syn fiber formation can be accelerated by the
addition of short peptides corresponding to the NAC region, which participate in
aggregation(Du et al., 2006; Kim et al., 2009). Herein, we describe the use of peptide-
accelerated aggregation to demonstrate that NAC peptides bearing an O-GlcNAc
modification at T72 do not participate in α-syn aggregation, suggesting an inhibitory role
for O-GlcNAcylation on full-length α-syn fibrillization and potentially a protective role
in synucleinopathies.
38
Figure 3.2 Aggregation of α-synuclein with peptide 2. A) Primary sequences of peptides 1 and 2
with threonine 72 (T72) underlined. B) CD spectra of purified α-synuclein (α-Syn) with our
without peptide 2. C) Purified α-synuclein (α-Syn) at a concentration of 50 µM was incubated at
37 °C with or without the indicated molar ratios of peptide 2 before analysis by ThT fluorescence
(450 nm Ex/482 nm Em) at the indicated time points. The experiments were performed in
triplicate and error bars represent standard deviation.
39
Absorbance (mAU)
1800
1500
1200
900
600
300
60
0
Retention time (min)
Intensity (arbitrary)
2.5E
6
2500 500
Mass (m/z)
1000 1500 2000 3000
1.2E
7
Mass (m/z)
2.0E
4
1.0E
4
Intensity
6+
7+
8+
9+
10+
11+
12+
13+
14+
15+
16+
14464±2
10 20 30 40 50
Figure 3.3 Characterization of wild-type α-synuclein. RP-HPLC trace over C4 analytical column
(Higgins Analytical), 0-70% B gradient over 60 min, tR = 43.96 min. Mass spectrum acquired
with electrospray ionization, +ve detection mode, 800-3000 amu.
It has previously been demonstrated that incubating α-syn in the presence of a
short peptide spanning residues in the NAC region results in an acceleration of
aggregation(Du et al., 2006; Kim et al., 2009). To take advantage of this observation, we
synthesized a peptide corresponding to residues 68-77 (1, Fig. 3.2A, Fig. 3.4A, B) of α-
syn using standard Fmoc solid-phase peptide synthesis (SPPS) with HBTU/DIEA. We
then confirmed its ability to accelerate α-syn aggregation by incubation recombinant α-
syn (50 µM) with or without 1 (50 µM) under constant agitation at 37 °C for three days.
Upon initiation of the aggregation reaction, the samples were immediately analyzed by
circular dichroism (CD) and dynamic light scattering (DLS) (Fig. 3.4C, D). CD spectra
for α-syn and α-syn+1 were similar and consistent with the expected random coil
structure. DLS showed the absence of any preformed oligomers with a Stokes radius of
10 to 50 nm.
40
Figure 3.4 Characterization of peptide 1 and aggregation assay for α-syn, α-syn+1. A) RP-HPLC
trace of 1 over C4 analytical column (Higgins Analytical), 0-50% B gradient over 60 min . B)
Mass spectrum acquired with electrospray ionization, +ve detection mode, 200-1800 amu. C) CD
spectra of α-syn, α-syn+1. D) DLS for α-syn, α-syn+1. E) ThT fluorescence data for α-syn, α-syn
+1. Error bars represent standard deviation for triplicate reactions.
41
Aliquots were removed from each reaction at regular intervals and analyzed by ThT
fluorescence (Fig. 3.4E) Addition of 1 resulted in a significant increase in ThT
fluorescence.
Unfortunately, due to the highly hydrophobic nature of this peptide sequence, it
proved too insoluble to test at higher concentrations. Therefore, we shifted the peptide
sequence to incorporate more hydrophilic amino acids and synthesized peptide 2
spanning residues 71-82 (Fig. 3.2A)(Kim et al., 2009). Although we expected this peptide
to have less accelerating potential than 1, recombinant α-syn (50 µM) was incubated with
or without 2 (50 µM) under constant agitation at 37 °C for three days. As before, the
samples were immediately analyzed by CD and DLS. The CD spectra of α-syn and α-syn
with 2 were very similar and consistent with an unfolded protein conformation (Fig.
3.2B), demonstrating that addition of 2 does not alter the starting α-syn conformation.
Again, DLS showed that α-syn in both reactions had Stokes radii of 2-3 nm,
corresponding to monomeric protein, and no evidence of preformed oligomeric structures
at 10 to 50 nm (Fig. 3.5).
42
Figure 3.5 DLS for α-syn, α-syn+2, α-syn+3, and α-syn+4 at t0 for all molar ratios.
43
Figure 3.6 CD spectra of α-syn, α-syn+2, α-syn+3, and α-syn+4 at t0 for all molar ratios.
44
Figure 3.7 Full ThT fluorescence data for α-syn, α-syn+2, α-syn+3, and α-syn+4 for all molar
ratios. Error bars represent standard deviation for triplicate reactions.
45
To determine the acceleration potential of 2, aggregation reaction aliquots were
removed at 6 and 12 hours on the first day and every 12 hours thereafter, and added to a
solution of ThT before analysis using fluorescence (Fig. 3.2C and Fig. 3.7). The ThT
fluorescence for α-syn shows a lag phase of 12 hours before increasing rapidly and then
leveling off by 48 hours. The addition of 2 increased the fluorescence intensity, most
significantly in the first 48 hours. α-Syn+2 shows a lag phase of only 6 hours and reaches
the steady-state maximum in only 36 hours. We next asked if the observed acceleration of
aggregation would be more dramatic at higher molar ratios (3:1 and 5:1) of 2 to α-syn.
Accordingly, α-syn (50 µM) was incubated with 2 (150 µM or 250 µM) as above for five
days, and aliquots were analyzed by ThT fluorescence (Figure 3.2C). As expected,
increasing the concentration of 2 further decreased the lag phase and accelerated the
progression of aggregation. At 5 fold excess of 2, the lag phase lasted less than 6 hours
and the ThT intensity was nearly at the maximum value in only 12 hours. For both
reactions conditions (3:1 and 5:1) we again analyzed samples by CD and DLS
immediately after preparation of the reactions to confirm that the increase in aggregation
at high concentrations of 2 was not due to a change in the initial state of α-syn (Fig. 3.5
and Fig. 3.6). Again, the CD spectra of α-syn+2 at 3:1 and 5:1 were consistent with a
random coil conformation. DLS showed the Stokes radius of α-syn was unchanged and
did not detect the presence of any oligomeric species.
46
Figure 3.8. Synthesis of GlcNAc Threonine a) 1. TrocCl, 1M NaOH, H2O, 0 °C to rt, 68%; 2.
Ac2O, pyridine, 73%; b) thiophenol or ethanethiol, BF3•Et2O, CH2Cl2, 0 °C to rt, 86 or 83%; c)
1. benyzlcholorformate, DIEA, DMAP, CH2Cl2, 0 °C, 85%; 2. TFA, 80%; d) NIS, TfOH, CH2Cl2,
-20 °C, 87% for thiophenol or 58% for thioethyl; e) 1. Zn, HCl, Ac2O, 75%; 2. H2, Pd/C, 2%
AcOH in MeOH, 72%; f) pentafluorophenyl trifluoroacetate, pyridine, DMF, 92%.
47
Given that peptide 2 was able to dramatically accelerate the aggregation of α-syn,
we next set out to synthesize peptides spanning residues 68-77 bearing either an O-
GlcNAc modification at T72 (3) or a threonine 72 to proline mutation, T72P (4), which
has been previously shown to not accelerate α-syn aggregation(Kim et al., 2009). To
generate 3, we used a selectively-protected glycosylated threonine cassette (5) strategy in
combination with Fmoc-SPPS (Fig. 3.8). Specifically, commercially available
glucosamine hydrochloride salt (6) was selectively protected over two steps to yield
7(Yan et al., 2003), which was then converted to the corresponding thiophenyl glycoside
8(Zhang and Magnusson, 1996). N
α
-Fmoc-L-threonine benzyl ester (10)(Mitchell et al.,
2001) was synthesized from commercially available N
α
-Fmoc-O-tert-butyl-L-threonine
(9) in two steps and then glycosylated at the γ oxygen with 8 to yield the fully protected
gylcosyl amino acid 11. Synthesis of 11 using 8 showed increased yield (87%) over the
use of the thioethyl-glycoside analog 12 (58%)(Simanek et al., 1998). Selective de-
protection of 11 over two steps gave 5, which was then activated for Fmoc-SPPS as the
pentafluorphenyl ester 13. Upon completion of 3 on resin, the O-acetyl groups on the
GlcNAc moiety were then cleaved using hydrazine, and the peptide was cleaved and
deprotected using trifluoroacetic acid, followed by purification by RP-HPLC. With
peptide 3 (Fig. 3.9A) in hand, we then probed its effect on the aggregation of α-syn using
our ThT assay. We incubated α-syn (50 µM) with or without peptide 3 (50 µM) under the
same conditions used with peptide 2 and aliquots were analyzed at the same time points
using ThT fluorescence (Fig. 3.9C). Preservation of the initial, unfolded state was
48
confirmed by CD for α-syn and α-syn+3, with both reactions showing similar spectra
consistent with a random coil conformation (Fig. 3.9B). DLS was again used to confirm
that no oligomeric species with a Stokes radius of 10 to 50 nm were present at the
initiation of the assay (Fig. 3.5). At a 1:1 ratio, α-syn+3 did not show any significant
increase in aggregation over three days or any appreciable change in the length of the lag
phase, compared to α-syn alone. Encouraged by this result, we repeated the assay at
higher concentrations of 3 (150 µM and 250 µM) for five days. No acceleration of α-syn
aggregation or shortening of the lag phase was observed at higher molar excess of 3, in
contrast to what we observed with α-syn+2 (Fig. 3.9C). As an additional control, we
incubated α-syn (50 µM) in the presence and absence of peptide 4 (Fig. 3.9A), which is
known to not accelerate aggregation(Kim et al., 2009). We conducted our aggregation
assay at three different concentrations of 4 (50, 150, and 250 µM) and analyzed samples
over three or five days by ThT fluorescence, as with 2 and 3. α-Syn+4 did not show any
acceleration of aggregation compared to α-syn alone, with the lag phase and degree of
aggregation unchanged (Fig. 3.9C). This result mirrors what was observed for α-syn+3
for the same conditions. As with the other peptides, we used CD to analyze the
conformation of α-syn at the initiation of the assay with and without 4, and the CD
spectra were again consistent with α-syn being a random coil (Fig. 3.9B). We also
confirmed the absence of any oligomeric species by DLS (Fig. 3.5).
49
Figure 3.9. Aggregation of α-synuclein with peptides 3 and 4. A) Primary sequences of peptides
3 and 4 with threonine 72 (T72) or the corresponding proline mutant (T72P) underlined. B) CD
spectra of purified α-synuclein (α-Syn) with our without peptides 3 or 4. C) Purified α-synuclein
(α-Syn) at a concentration of 50 µM was incubated at 37 °C with or without the indicated molar
ratios of peptides 3 or 4 before analysis by ThT fluorescence (450 nm Ex/482 nm Em) at the
indicated time points. The experiments were performed in triplicate and error bars represent
standard deviation.
50
Finally, we analyzed α-syn alone and with each of our peptides (2, 3, or 4) at the
three molar ratios probed before (1:1, 3:1, and 5:1) for five days under the same
conditions described above and analyzed the resulting aggregates by transmission
electron microscopy (TEM). We did not observe any gross changes in fiber morphology
between α-syn and α-syn with any of our peptides for all molar ratios tested (Fig. 3.10).
We were pleased to observe that our glycopeptide 3 behaved similarly to the T72P mutant
peptide (4). In contrast to what we observed with wild-type peptide 2, which significantly
increased α-syn aggregation, peptides 3 and 4 did not accelerate aggregation of full-
length α-syn, even at five-fold molar excess (Fig. 3.11). Circular dichroism confirmed
that the native, random coil structure of α-syn was not altered at the initiation of our
assays by any of the three peptides and dynamic light scattering measurements confirmed
that the reactions were not seeded by oligomeric species. Thus we attribute the increase in
aggregation observed with peptide 2 to direct participation of 2 with α-syn at one or more
stages in the aggregation pathway. This is corroborated by both the shortening of the lag
phase and the increase in the amount of fibrils at earlier time points for α-syn+2, as
measured by ThT fluorescence. The lack of any observable increase in aggregation or
change in the lag phase for either α-syn+3 or α-syn+4 leads us to concluded that neither 3
nor 4 is participating in aggregation with α-syn under our assay conditions.
51
Figure 3.10 A) Transmission electron
micrographs of mature fibers, 1:1 molar
ratio.
B) Transmission electron micrographs of
mature fibers, 3:1 molar ratio
C) Transmission electron micrographs of
mature fibers, 5:1 molar ratio.
52
Figure 3.11 Comparison of α-synuclein aggregation. Purified α-synuclein (α-Syn) at a
concentration of 50 µM was incubated at 37 °C with or without the indicated molar ratios of
peptides 2, 3, or 4 before analysis by ThT fluorescence (450 nm Ex/482 nm Em) at the indicated
time points. The experiments were performed in triplicate and error bars represent standard
deviation.
53
3.3 Discussion
In summary, we have used a peptide-based aggregation acceleration assay to
investigate the consequences of O-GlcNAc modification on threonine 72 of α-syn. This
method allowed us to determine that the corresponding O-GlcNAc modification renders a
peptide compromised of α-syn residues 68-77, which would otherwise dramatically
accelerate the aggregation of α-syn, completely benign. Furthermore, this O-
GlcNAcylated peptide behaves almost identically to the same sequence mutated to
proline at residue 72 (T72P). As stated above, full-length α-syn bearing only the same
T72P mutation is completely resistant to aggregation. Taken together with the data
presented herein, this strongly suggests that full-length α-syn modified by O-GlcNAc at
T72 will not form fibers and aggregates. Furthermore, increasing the levels of O-
GlcNAcylation, which occurs at T72 in vivo, may be a viable strategy for the treatment of
Parkinson’s Disease. Encouraged by these results, we are currently exploring the effect of
O-GlcNAcylation of full-length α-syn using semi-synthetically prepared protein.
54
3.4 Materials and Methods
3.4.1 General
All solvents and reagents were purchased from commercial sources (Sigma-
Aldrich, Fluka, EMD, Novagen) and used without any further purification. All aqueous
solutions were prepared using ultrapure laboratory grade water (deionized, filtered,
sterilized) obtained from an in-house MilliQ
®
purification system and filter sterilized
with 0.4µm syringe filters (Genesee Scientific) before use. Growth media (LB broth,
Miller, Novagen and S.O.C. broth, Sigma) were prepared, sterilized, stored, and used
according to the manufacturer and/or published protocol.

Antibiotics (ampicillin Na salt,
EMD) were prepared as stock solutions at a working concentration of 1000x (100 mg
mL
-1
) and stored at -20°C. All media and cultures were handled under sterile conditions
under open flame. All silica gel column chromatography was performed using 60 Å silica
gel (EMD) and all thin-layer chromatography performed using 60 Å, F254 silica gel plates
(EMD) with detection by ceric ammonium molybdate (CAM) or UV light. Reverse phase
high performance liquid chromatography (RP-HPLC) was performed using an Agilent
Technologies 1200 Series HPLC with Diode Array Detector. Mass spectra were acquired
on an API 3000 LC/MS-MS System (Applied Biosystems/MDS SCIEX).
1
H NMR
spectra were acquired on either a Varian Mercury 400 MHz or Varian VNMRS 500 MHz
magnetic resonance spectrometer housed at the USC Dept. of Chemistry Instrument
Facility.
55
3.4.2 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 previously(Der-Sarkissian et al., 2003). Wild-type
human α-synuclein was introduced into a pET28a vector using Nde I and BamH I
restriction sites and standard molecular cloning techniques.
3.4.3 Co-expression of α-syn with OGT
The pET28a vector containing hexahistidine-tagged, wild-type human α-synuclein
was co-transformed with wild-type OGT in a pMal-c2X vector(Yuzwa et al., 2010)
(provided by Dr. David J. V ocadlo) in E. coli Turner cells (Stratagene). For expression,
the co-transformed cells were induced with IPTG (final concentratino: 0.5 mM) for 18 h
at 25°C. Cells were harvested by centrifugation (8,000 rcf, 30 min, 4°C), the pellet was
resuspended in 30 mL column binding buffer (20 mM Na2PO4, 500 mM NaCl, 5 mM
imidazole, pH 7.4), and the cells lysed by addition of 2 mg mL-1 lysozyme (EMD) in the
presence of protease inhibitor cocktail (mini complete EDTA free, Roche). The cell
suspension was sonicated at ~30% power for six cycles (20 s on, 40 s rest) using a Vibra-
cell VC505 (Sonics & Materials) and then cleared by centrifugation (42,000 rcf, 30 min,
4°C). The supernatant was loaded onto a HisTrap FF Ni-NTA column (GE scientific),
washed with of column washing buffer (90 mL, 20 mM Na2PO4, 500 mM NaCl, 60 mM
imidazole, pH 7.4), and then eluted with column elution buffer (20 mM Na2PO4, 500 mM
56
NaCl, 250 mM imidazole, pH 7.4). Elution fractions were dialyzed against PBS for
western blotting. As a positive control, TAK1-binding protein 1 (TAB-1)(Pathak et al.,
2012) in pET28a vector (provided by Dr. David J. V ocadlo) was transformed into Turner
cells and expressed as above. Crude lysates were dialyzed as above and analyzed by
western blot without HisTrap FF Ni-NTA purification.
3.4.4 Western Blotting
Proteins were separated by SDS-PAGE and subsequently transferred to PVDF
membrane (Bio-Rad). Blots were blocked in TBST (0.1% Tween-20, 150mM NaCl,
10mM Tris, pH8.0) containing 5% BSA, Fraction V (Calbiochem) for 1 h at room
temperature. Then the blots were incubated with RL2 antibody (Thermo Scientific) at
1:1,000 dilutions in blocking buffer for 1 h at room temperature. Following that the blots
were washed three times with TBST and incubated with HRP-conjugated anti-mouse
antibody (Jackson ImmunoResearch) at 1:10,000 in blocking buffer for 1 h at room
temperature. Blots were washed with TBST three times and developed using ECL
reagents (Bio-Rad) and the ChemiDoc XRS+ molecular imager (Bio-Rad).
3.4.5 Expression of wild-type α-syn
BL21(DE3) chemically competent E. coli (GeneChoice) were transformed with
the pRK172 construct containing wild-type human α-synuclein by heat shock, plated on
selective LB agar plates containing 100 µg mL
-1
ampicillin (LB-amp), and incubated at
57
37°C for 16 h. Single colonies were selected and used to inoculate two LB-amp liquid
cultures (5 mL), which were grown at 37°C with shaking at 250 rpm for 16 h. Each 5 mL
culture was used to inoculate a 1 L LB-amp culture. These cultures were grown to an
OD600 of 0.6-0.7 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. Bacteria were
harvested by centrifugation (8,000 rcf, 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 1 L of culture) lysis buffer (500 mM NaCl, 100 mM
Tris, 10 mM β-mercaptoethanol, 1 mM EDTA, pH 8.0). Cell lysates were boiled at 80°C
for 10 min, allowed to cool to rt and then placed on ice. Protease inhibitor cocktail (mini
complete EDTA free, Roche) was added and lysates were incubated on ice for 20 min and
then cleared by centrifugation (42,000 rcf, 30 min, 4°C). The resulting supernatant was
acidified, on ice, to pH 3.5 with HCl and then incubated on ice an additional 20 min
before centrifuging again (42,000 rcf, 30 min, 4°C). The resulting supernatant was
dialyzed against 3 x 1 L of 1% acetic acid in water (degassed with N2, 1 h per L). The
dialyzed protein solution was then purified by RP-HPLC over a C4 semi-preparative
column (Higgins Analytical) using a 40-50% B linear gradient over 30 min, tR = 25.21
min (buffer A: 0.1% TFA in H2O, buffer B: 0.1% TFA, 90% ACN in H2O). Purified
material was flash frozen in liquid N2 and lyophilized. Pure α-synuclein was
characterized by C4 analytical RP-HPLC and ESI-MS (M + H
+
) and yield was
58
determined by Pierce
®
BCA assay (Thermo). Yield = 42.5 mg L
-1
culture, observed =
14,464.7 ± 2.0 Da, expected = 14,460.1 Da.
3.4.6 Synthesis of O-GlcNAc Threonine cassette
1,3,4,6-tetra-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbonyl)-amino-D-
glucopyranose (7)(Yan et al., 2003). Glucosamine HCl (6, 1.0 eq) was dissolved in 1 M
NaOH (2.0 eq) and cooled to 0°C. 2,2,2-trichloroethyl chloroformate (1.5 eq) was added
dropwise over 30 min with stirring. Reaction was allowed to warm to rt overnight,
yielding a white precipitate that was collected by filtration, washed with cold H2O, and
dried by suction (yield = 68%). This white solid (1.0 eq) was dissolved in pyridine (~20
eq) and acetic anhydride (10 eq) was added. Reaction was stirred at rt overnight and then
concentrated and azeotroped 3x with Toluene. Residual solution was dissolved in EtOAc,
washed 3x with 1 M HCl, 3x H2O, 1x brine, dried over Na2SO4, and concentrated.
Product was a white foam (yield = 73%).
1
H-NMR (500 MHz, CDCl3) δ 6.23 (d, 1, J =
3.6), 5.57 (d, 1, J = 9.4), 5.30 (t, 1, J = 9.8), 5.19 (t, 1, J = 9.9), 4.82 (d, 1, J = 12.1), 4.63
(d, 1, 12.1), 4.24 (m, 2), 4.05 (m, 2), 2.19 (s, 3), 2.08 (s, 3), 2.04 (s, 3).
Phenyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbonyl)-amino-1-thio-β-
D-glucopyranoside (8)(Zhang and Magnusson, 1996). 7 (1.0 eq) was dissolved in dry
CH2Cl2 under argon and cooled to 0°C. Thiophenol (5.0 eq) was added, followed by
boron trifluoride diethyl etherate (10 eq) and the reaction was stirred and allowed to
59
warm to rt overnight. Reaction was washed 3x each with concentrated NaHCO3, H2O,
and brine, dried over Na2SO4, and concentrated. Crude was a viscous, colorless oil that
was recrystallized from EtOAc by addition of hexanes and cooling to -20°C to give white
needles (yield = 86%).
1
H-NMR (500 MHz, CDCl3) δ 7.48 (m, 2), 7.25 (m, 2), 5.84 (d, 1,
J = 9.3), 5.28 (t, 1, J = 9.8), 4.97 (t, 1, J = 9.7), 4.85 (d, 1, J = 10.4), 4.73 (dd, 2, J = 12.1,
36.1), 4.16 (m, 2), 3.74 (m, 2), 2.03 (s, 3), 1.97 (s, 3), 1.92 (s, 3).
Ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbonyl)-amino-1-thio-β-D-
glucopyranoside (12)(Simanek et al., 1998). 7 (1.0 eq) was dissolved in dry CH2Cl2 under
argon and cooled to 0°C. Ethane thiol (5.0 eq) was added, followed by boron trifluoride
diethyl etherate (10 eq) and the reaction was stirred and allowed to warm to rt overnight.
Reaction was washed 3x each with concentrated NaHCO3, H2O, and brine, dried over
Na2SO4, and concentrated. Crude was a viscous, colorless oil that was recrystallized from
EtOAc by addition of hexanes and cooling to -20°C to give white needles (yield = 83%).

1
H-NMR (500 MHz, CDCl3) δ 5.40 (m, 1), 5.22 (t, 1, J = 9.7), 5.05 (t, 1, 9.3), 4.77 (d, 1,
12.0), 4.64 (m, 2), 4.21 (dd, 1, J = 4.7, 5.1), 4.11 (dd, 1, 1.8, 11.4), 3.77 (q, 1, J = 9.9),
3.70 (m, 1), 2.71 (m, 3), 2.06 (s, 3), 2.01 (s, 3), 1.99 (s, 3), 1.25 (t, 3, J = 7.3).
N
α
-Fmoc-L-threonine benzyl ester (10)(Mitchell et al., 2001). N
α
-Fmoc-O-tertbutyl-L-
threonine (9, 1.0 eq) was dissolved in dry CH2Cl2 under argon and cooled to 0°C. N,N-
diisopropylethylamine (1.1 eq) and 4-dimethylamino-pyridine (0.1 eq) were added,
60
followed by addition of benzyl chloroformate (1.0 eq) dropwise, over 30 min. The
reaction stirred for 2 h and then concentrated and azeotroped 3x with toluene. The crude
product was purified by silica gel column in 4:1 hexanes/EtOAc, yielding a colorless oil
(yield = 85%). This product was then dissolved in a minimum amount of trifluoracetic
acid and stirred under argon for 2 h at rt. When complete by TLC (1:4, EtOAc/hexanes)
the reaction was diluted with an equal amount of CH2Cl2 and concentrated. This was
repeated with 3x 100 mL CH2Cl2, followed by azeotroping 3x with toluene. The resulting
white solid was recrystallized from EtOAc by addition of hexanes and cooling to -20°C
(yield = 80%).
1
H-NMR (400 MHz, CDCl3) δ 7.77 (d, 2, J = 7.5), 7.61 (d, 2, J = 7.3), 7.35
(m, 9), 5.67 (d, 1, J = 8.7), 5.22 (m, 2), 4.41 (m, 4), 4.23 (t, 1, J = 7.0), 2.05 (br s, 1), 1.25
(d, 3, J = 6.0).
N
α
-Fmoc-O-(3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbonyl)-amino-β-D-
glucopyranosyl)-L-threonine benzyl ester (11)(Simanek et al., 1998). 8 (or 12) (1.2 eq)
and 10 (1.0 eq) were combined and azeotroped 3x with toluene and then placed under
high vacuum for 24 h. This mixture was dissolved in dry CH2Cl2 under argon and
cannulated into a flask containing freshly activated 4 Å molecular sieves. After stirring
with sieves for 30 min at rt, the resulting slurry was cooled to -20°C. N-iodosuccinimide
(5.0 eq) was added, followed by dropwise addition of triflic acid (1.0 eq). Reaction was
stirred at -20°C for 40 min, until complete by TLC (3:7, EtOAc/hexanes). The reaction
was filtered through celite, which was then thoroughly flushed with EtOAc. The filtrate
61
was was washed 2x each with concentrated NaHCO3 and concentrated Na2S2O3, 1x each
with H2O and brine, then dried over MgSO4. This crude product was concentrated and
then column purified in 3:7 EtOAc/hexanes yielding a pale yellow oil (yield = 87% for 8,
58% for 12).
1
H-NMR (500 MHz, CDCl3) δ 7.75 (d, 2, J = 7.5), 7.65 (t, 2, J = 7.3), 7.34
(m, 9), 5.91 (d, 1, J = 9.1), 5.86 (d, 1, J = 8.2), 5.22 (m, 3), 5.02 (t, 1, J = 9.6), 4.71 (dd, 2,
J = 12.2, 36.4), 4.58 (d, 1, J = 7.7), 4.46 (m, 3), 4.25 (m, 3), 4.03 (d, 1, J = 11.3), 3.62 (q,
1, J = 9.2) 3.51 (d, 1, J = 9.4), 2.02 (s, 3), 2.00 (s, 3), 1.99 (s, 3), 1.23 (d, 3, J = 6.0).
N
α
-Fmoc-O-(3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-β-D-glucopyranosyl)-L-threonine
(5)(Simanek et al., 1998). 11 (1 eq) was dissolved in acetic anhydride and stirred under
argon. Zinc powder (equal amount w/w, ~15 eq) was activated with HCl and added to the
reaction and stirred over night at rt. The reaction was then filtered through celite,
concentrated, azeotroped 3x with toluene, and then purified by silica gel column in 1:4
hexanes/EtOAc to give a yellow crystalline solid (yield = 75%). This solid (1.0 eq) was
then dissolved 2% AcOH in MeOH under argon. Palladium charcoal (5% Pd, w/w) was
added in equal amount, by mass, to the starting material (0.35 eq Pd) and the atmosphere
was purged and replaced with H2. The reaction was stirred vigorously under an H2
atmosphere for 3 h. The reaction was then filtered through celite, concentrated, and
azeotroped with toluene to yield a white solid (yield = 72%).
1
H-NMR (500 MHz,
CDCl3) δ 7.67 (d, 2, J = 7.4), 7.55 (t, 2, J = 6.2), 7.26 (dt, 4, J = 7.2, 42.1), 6.03 (br s, 1),
62
5.82 (br s, 1), 5.20 (t, 1, J = 9.6), 4.98 (t, 1, J = 9.5), 4.69 (d, 1, J = 6.8), 4.22 (m, 7), 3.74
(q, 1, J = 7.5), 3.60 (d, 1, J = 8.6), 1.98 (s, 3), 1.93 (s, 3), 1.84 (s, 3), 1.17 (d, 3, J = 5.6).
N
α
-Fmoc-O-(3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-β-D-glucopyranosyl)-L-threonine
pentafluorophenyl ester (13). 5 (1.0 eq) was dissolved in DMF and stirred under argon at
rt. pyridine (1.1 eq) was added, followed by dropwise addition of pentafluorophenyl
trifluoroacetate (1.2 eq). The reaction was monitored by TLC (9:1, CH2Cl2/MeOH) and
was complete after 2 h. The reaction was then concentrated and purified by silica gel
column in 94:5:1, CH2Cl2:MeOH:AcOH. Fractions containing product were pooled and
concentrated to yield an off-white solid (yield = 92%), which was dissolved in DMF and
used directly for coupling to incomplete peptide 3 on resin.
3.4.7 Solid phase peptide synthesis
All solid-phase peptide syntheses were conducted manually on a using
unprotected Rink amide ChemMatrix
®
resin, (PCAS BioMatrix) with an estimated
loading of 0.6 mmol g
-1
. Commercially available N-Fmoc and side chain protected amino
acids (10 eq, Novabiochem) were activated for 20 min with HBTU (10 eq,
Novabiochem) and DIEA (20 eq, Sigma) and then coupled to the resin for 1 h, bubbling
with N2 to mix. Reaction completion was checked using the Kaiser test. Briefly, a small
amount of resin was incubated with equal volumes of 5% w/v ninhydrin in EtOH, 80%
w/v phenol in EtOH, and 20µM KCN in pyridine and heated to 99°C for 5 min in a
63
sealed tube. If necessary, a second coupling was conducted with 10 eq amino acid, 10 eq
HOBt (Novabiochem) and 12 eq DCC (Sigma) for 2 h, with N2 mixing. After successful
coupling, the terminal Fmoc group was removed with 20% v/v piperidine in DMF for 5
min with N2 mixing, and then for an addition 15 min with fresh 20% piperidine in DMF.
When peptides were completed, the final Fmoc group was removed as described above
and the N-terminal amine was acetylated with 5 eq each of pyridine and acetic anhydride
in DMF. At this point, peptide 3 was deprotected with hydrazine hydrate (80% v/v in
MeOH) three times for 30, 30, and 60 min respectively, with N2 mixing. Peptides were
then 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 peptide was then diluted ~1/10 in
cold diethyl ether and precipitated over night (-80°C). The resulting suspension was
centrifuged (5,000 rcf, 30 min, 4°C) and the pellet was resuspended in fresh Et2O and
centrifuged again (5,000 rcf, 30 min, 4°C). The pellet was then resuspended in H2O, flash
frozen, and lyophillized. This crude lyophillized material was purified by RP-HPLC
(0-50% B gradient over 60 min) over a C18 semi-preparative column (Vydac). Purified
peptides were characterized by RP-HPLC (0-50% B gradient over 60 min) over an
analytical C18 column (Vydac) and ESI-MS. Yields were determined by A214 using
calculated molar extinction coefficients(Kuipers and Gruppen, 2007). 1 tR = 27.90,
observed = 914.9 amu, expected = 914.5 (M + H
+
) 2 tR = 29.45 min, yield = 7.44 µmol,
observed = 1187.0 amu, expected = 1187.7 (M + H
+
). 3 tR = 27.40 min, yield = 1.68
64
µmol, observed = 1140.1 amu, expected = 1139.6 (M + Na
+
). 4 tR = 30.95 min, yield =
25.89 µmol, observed = 932.6 amu, expected = 932.5 (M + Na
+
).
3.4.8 Aggregation assay
Each peptide and recombinant, wild-type α-syn was aliquoted and lyophillized.
Lyophillized peptides were resuspended in reaction buffer (10 mM PO4, 0.05% NaN3, pH
7.4) to the appropriate concentration (50, 150, or 250 µM). To lyophilized α-syn aliquots
was then added either the corresponding peptide solution or buffer only, in the case of α-
syn alone, and all material was dissolved using a bath sonicator. Reactions were
centrifuged at rcf to remove any debris or aggregated material. The supernatant was then
split into triplicate reaction in 2 mL screw cap vials (Genesee). To each reaction was
added a 2x2 mm teflon coated stir bar. All reactions were then arranged symmetrically
and placed on a magnetic stir plate in a 37°C incubator. Sample aliquots for analysis at t0
were removed prior to heating to 37°C.
Circular Dichroism: Circular dichroism spectra were collected on a Jasco J-815
CD Spectrometer. Sample aliquots were diluted to 7.5 µM α-syn with reaction buffer
containing no NaN3. Spectra were collected from 250-195 nm with a 0.1 nm data pitch,
50 nm min
-1
scanning speed, data integration time of 4 s, 1 nm bandwidth, 1 mm path
length with 3 accumulations, at 25°C. Raw CD data was corrected to account for the
additional peptide bonds present with the addition of peptide to α-syn. All elipticity
values were converted to molar elipticity values by multiplying by the mean residue
65
weight divided by 10 times the net concentration times the pathlength. [θ]=MRW*θ/
(10c*d)
Dynamic light scattering: Light scattering data was collected with a Dynapro
Titan temperature controlled microsampler (Wyatt). Samples taken directly from
aggregation reactions (50 µM α-syn ± peptide) were analyzed with ten 10 s acquisition, at
25°C, with laser power adjusted to give an intensity of 2.0E
6
counts s
-1
. Radii were
calculated using a Raleigh sphere approximation.
Thioflavin T fluorescence: The degree of α-syn aggregation was quantified by
Thioflavin T fluorescence. Samples were prepared by diluting samples from aggregation
reaction (final [α-syn] = 1.25 µM) in 20 µM Thioflavin T in reaction buffer (above).
Samples were diluted, vortexed briefly, and then incubated 3 min before analyzing.
Spectra were collected using a NanoLog Spectrofluorometer (Horiba), λex = 450 nm, ex
slit = 4 nm, λem = 482, em slit = 3 nm, 10 mm path length, integration time = 0.1 s, 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 grids (150 mesh, Electron Microscopy Sciences) and
allowed to sit for 5 min and then excess liquid was removed with filter paper. Grids were
then negatively stained 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
66
48 h in a vacuum desiccator. Grids were imaged using a JOEL JEM 2100 LaB6
transmission electron microscope operated at 200 kV , 10,000x magnification.
67
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and Siaylated Lactosamine Oligosaccharide Donors. J. Org. Chem. 68, 2426–2431.
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inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo. Nature
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Yuzwa, S.A., Shan, X., Macauley, M.S., Clark, T., Skorobogatko, Y ., V osseller, K., and
V ocadlo, D.J. (2012). Increasing O-GlcNAc slows neurodegeneration and stabilizes tau
against aggregation. Nature Chemical Biology 8, 393–399.
Yuzwa, S.A., Yadav, A.K., Skorobogatko, Y ., Clark, T., V osseller, K., and V ocadlo, D.J.
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(2004). Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to
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Zarranz, J.J., Alegre, J., Gómez Esteban, J.C., Lezcano, E., Ros, R., Ampuero, I., Vidal,
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Chapter 4. Native Chemical Ligation: a powerful tool for protein biochemistry
4.1 Introduction
The study of protein structure and function was once solely the purview of
biological study, while their combination of numerous reactive functional groups and
repetitive structure offered significant challenges to preparation by synthetic chemists.
This changed, however, with the development of solid-phase peptide synthesis (SPPS) by
Merrifield(Merrifield, 1963), which offered the first efficient strategy for preparing
synthetic polypeptides. With this approach came a new dimension in control over the
possible peptide structures available for study. Chemical modifications including both
modified and non-natural amino acid side chains as well as non-peptide backbone
linkages could now be introduced in a straight forward and site-specific manner.
However, despite its many advantages, SPPS has an inherent length limit. Even with
highly efficient reaction conditions, the length of synthetic peptides is usually limited to
~50 amino acids (Dawson and Kent, 2000). Thus the preparation of most full-length
proteins is beyond the scope of direct synthesis on solid-phase.
This challenge led to burgeoning investigation in chemical methods to couple
multiple peptides together to create longer and longer synthetic polypeptides. Many
synthetic methodologies were employed using a variety of orthogonally reactive
functional groups that allowed the selective ligation of two polypeptides. These included
reactions resulting in non-amide linkages such as hydrazones, oximes, and
76
thiazolidines(Thapa et al., 2014)as well as those that result in amide bond linkages such
as the alkylation thioacids or the Staudinger ligation between peptidyl azides and aryl
phosphines-containing thioesters(Tam et al., 2001). All of these approaches offer
advantages but most require some degree of side chain protection or the installation of
unstable reactive functionalities. By far the most robust synthetic solution for the ligation
of polypeptides was developed by Kent and coworkers, termed Native Chemical Ligation
(NCL)(Dawson et al., 1994). This reaction, which occurs between an N-terminal cysteine
thiol and a polypeptide bearing a C-terminal thioester, results in the generation of a native
amide bond linkage (Fig. 4.1). The first step is attack by the cysteine thiol on the
thioester, resulting in a n intermolecular transthioesterification. This new thioester species
can then undergo an intramolecular S to N acyl shift, resulting in amide bond formation
and regenerating the cysteine side chain. The reaction can be performed in aqueous buffer
at physiological pH and temperature, without the need for any side chain protection, and
because the first step is reversible, even internal free cysteines are tolerated.
77
Figure 4.1 Mechanism of Native Chemical Ligation. The N-terminal thiol of peptide 2 attacks the
thioester of peptide 1 to give a thioester conjugate. Following the S to N acyl shift, the two
peptides are linked by an amide bond.
4.2 Expansion of scope through new chemistry
The development of NCL greatly expanded the number of protein targets
accessible by synthetic preparation, with targets as large as ~250 amino acids well within
reach(Chandrudu et al., 2013). However, even though this reaction was a large leap
forward for protein synthesis, it is not without drawbacks. The first being that the
required cysteine residue is not a very abundant amino acid, making selection of
appropriate ligation sites difficult. The second drawback is the limited number of
available methods for introducing the thioester component. Thankfully, the utility of this
technique has inspired a great deal of effort and success in addressing these challenges.
78
One solution to the lack of suitable cysteine residues, or any cysteines at all, in the
target sequence has been addressed by moving the the reactive thiol group on the N-
terminus from a side chain to an auxiliary molecule appended to the terminal free
amine(Tam et al., 2001). This thiol bearing auxiliary can perform the job of reacting with
the C-terminal thioester and presenting it in proximity to the free N-terminal amine for
the acyl shift. Following amide bond formation, this auxiliary is then selectively cleaved
from the backbone amide in order to yield a fully native peptide structure. Another
solution to the lack of an N-terminal cysteine is to replace another side chain with a thiol
bearing analog and then chemically removing the thiol group after the ligation to restore
the native sequence. The simplest form would be substituting an alanine, which are much
more common residue, with cysteine. Following a normal NCL reaction, the free cysteine
side chain is chemically converted to alanine by removing the thiol group in a process
called desulfurization. This approach is not a limited to alanine, and has been
demonstrated with thiol bearing analogs of phenylalanine, valine, and lysine(Rohde and
Seitz, 2010). Removal of the thiol group can be achieved by direct reduction using Raney
nickel or palladium (Pd/Al2O3, Pd/charcoal, Pd/BaSO4)(Yan and Dawson, 2001).
This method does have the significant drawback of sometimes non-selectively
hydrogenating phenylalanine or tryptophan side chains and of protein products
sometimes adsorbing to the catalyst metal surface. An alternate solution to desulfurization
developed by the Danishefsky lab is a radical based chemistry that selectively removes
free thiol groups(Wan and Danishefsky, 2007). This reaction utilizes a unique reaction
79
between and alkylthiyl radical and an akylphosphine which results in an intermediate
phosphoranyl radical, which decomposes to give a free alkyl radical, which then abstracts
a proton (Fig. 4.2) This reaction is quite mild and tolerates a wide variety of sulfur
containing functional groups. This important, since any native cysteines that need to be
protected through the desulfurization can be protected as an acetamidomethyl (Acm) and
N-terminal cysteines can be protected as a thioazolidine (Thz) moiety, both of which are
tolerated by the radical chemistry.
Figure 4.2 Mechanism of radical desulfurization. Facile generation of a thiol center radical leads
to selective reaction with the phosphine TCEP, followed by bond homolysis and generation of the
alkyl radical. This radical abstracts a proton to yield the desulfurized side chain.
When it comes to the challenge of generating peptide species with C-terminal
thioesters, one approach is to use specialized linker molecules in the SPPS preparation
that act as masked thioesters. Such a linker can survive the reactivity of peptide synthesis
and then, upon peptide completion, be activated and converted to a thioester. Such linkers
are readily available for Boc-SPPS chemistry(Camarero et al., 2000), but these linkers are
labile under the basic conditions of Fmoc synthesis that may be required for modified
peptides due to harsh conditions required for Boc synthesis and cleavage. This goal can
80
be achieved the with so-called safety catch resin(Kenner et al., 1971), which features an
acylsulfonamide which is very resistant to basic hydrolysis. However N-methylation
makes it readily cleavable by nucleophiles, including thiols. Another alternative,
developed by the Dawson lab, makes use of a diaminobenzoic acid linker, that can be
cyclized following peptide completion to generate an N-acyl-benzimidazolinone leaving
group that can be cleaved by thiols to generate a thioester(Blanco-Canosa and Dawson,
2008).
4.3 Expressed protein ligation
The utility of NCL was further expanded when a technique for the recombinant
production of proteins bearing C-terminal thioesters was developed. This approach relies
on the activity of a class of polypeptides called inteins. Inteins are protein sequences that
post-translationally catalyze their own excision from a protein, while simultaneously
ligating the flanking sequences, called exteins, together with a native amide bond(Paulus,
2000). The intein acts with cysteine protease-like activity to cleave it’s N-terminal end as
a thioester. This thioester is then intercepted by a C-terminal cysteine residue to create a
branched intermediate. Finally the intein is cyclized at a conserved asparagine. The
thioester linkage is cleaved by an S to N acyl shift that ligates the two flanking exteins in
a process analogous to the NCL reaction (Fig. 4.3). This process is referred to as protein
splicing, as it is analogous to the splicing of exons in mRNA. By mutating crucial
residues in this process, it is possible to generate inteins that will undergo the first step of
81
the process and generated a thioester linkage without proceeding to full protein
splicing(Xu and Perler, 1996). If a protein of interest is expressed as an N-terminal fusion
to one of these engineered inteins, the resulting intramolecular thioester species can be
cleaved by the addition of exogenous thiol to generate a C-terminal thioester analog of
the protein of interest and a free intein. A protein thioester produced in this way could
then be ligated to a synthetic or recombinant polypeptide bearing a cysteine, or cysteine
surrogate, at its N-terminus in a process called Expressed Protein Ligation (EPL)(Muir,
2003; Muir et al., 1998). This technique allows the ligation of any combination of
synthetic and recombinant protein fragments bearing the necessary thioester and N-
terminal thiol. It also removes the length restrictions associated with relying on SPPS to
generate thioester-bearing polypeptides. Thus EPL takes advantage of both synthetic
peptide chemistry’s ability to generate modified or unnatural peptides as well as
recombinant expression’s ability to produce protein sequences of virtually any length
with ease.
82
Figure 4.3 Recombinant thioester from an intein fusion. By expressing the protein of interest
(POI) as a fusion with the modified intein, the thioester species can be intercepted by a thiol, for
example MESNa, to generate the ligation ready POI-thioester.
83
4.4 Conclusion
Synthetic preparation of proteins offers a level of structural control that is not
possible by biological means, allowing for unnatural amino acids, non-native backbone
linkages, and side chain modifications and all with site-specific resolution. Unfortunately
the main route for synthetic preparation of polypeptides, solid-phase peptide synthesis, is
inherently limited by the number of successive residues that can be coupled. This lead to
the development of protein semi-synthesis, whereby a large polypeptide or even a full-
length protein could be synthesized by ligating several smaller polypeptides together. The
major road blocks to such synthetic methods involve the need for highly selective or even
orthogonal reaction by which to couple the fragments. This is done to avoid excessive
side chain protection and deprotection for each coupling. Reaction would ideally also
work well in aqueous media at pH and salt concentrations amendable to protein folding
and stability. One methodology that successfully navigated these roadblocks Native
Chemical Ligation. NCL proceeds rapidly in aqueous buffer and requires no additional
protection steps. The only drawback is the need for both an N-terminal cysteine and a
reactive thioester, but a great deal of synthetic progress has been made to make both
components easily accessible. Finally, the development of a recombinant system for
generating C-terminal protein thioesters from intein fusions, dubbed Expressed Protein
Ligation, overcame the still significant length limit on thioester-bearing fragments
produced by SPPS. Taken together, these techniques allow for the straightforward
synthesis of large proteins with all the synthetic control of SPPS.
84
4.5 References
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.
Camarero, J.A., Adeva, A., and Muir, T.W. (2000). 3-Thiopropionic acid as a highly
versatile multidetachable thioester resin linker. Letters in Peptide Science 7, 17-21.
Chandrudu, S., Simerska, P., and Toth, I. (2013). Chemical Methods for Peptide and
Protein Production. Molecules 18, 4373–4388.
Dawson, P.E., and Kent, S.B. (2000). Synthesis of native proteins by chemical ligation.
Annu. Rev. Biochem. 69, 923–960.
Dawson, P.E., Muir, T.W., Clark-Lewis, I., and Kent, S.B. (1994). Synthesis of proteins
by native chemical ligation. Science 266, 776–779.
Kenner, G.W., McDermott, J.R., and Sheppard, R.C. (1971). The safety catch principle in
solid phase peptide synthesis. J. Chem. Soc. D 12, 636–637.
Merrifield, R.B. (1963). Solid phase peptide synthesis. I. The synthesis of a tetrapeptide.
J. Am. Chem. Soc. 85, 2149–2154
Muir, T.W. (2003). Semisynthesis of Proteins by expressed protein ligation. Annu. Rev.
Biochem. 72, 249–289.
85
Muir, T., Sondhi, D., and Cole, P. (1998). Expressed protein ligation: A general method
for protein engineering. P Natl Acad Sci Usa 95, 6705–6710.
Paulus, H. (2000). Protein splicing and related forms of protein autoprocessing. Annu.
Rev. Biochem. 69, 447–496.
Rohde, H., and Seitz, O. (2010). Invited reviewligation-Desulfurization: A powerful
combination in the synthesis of peptides and glycopeptides. Biopolymers 94, 551–559.
Tam, J.P., Xu, J., and Eom, K.D. (2001). Methods and strategies of peptide ligation.
Biopolymers 60, 194–205.
Thapa, P., Zhang, R.-Y ., Menon, V ., and Bingham, J.-P. (2014). Native Chemical
Ligation: A Boon to Peptide Chemistry. Molecules 19, 14461–14483.
Wan, Q., and Danishefsky, S.J. (2007). Free-Radical-Based, Specific Desulfurization of
Cysteine: A Powerful Advance in the Synthesis of Polypeptides and Glycopolypeptides.
Angew. Chem. Int. Ed. 46, 9248–9252.
Xu, M., and Perler, F. (1996). The mechanism of protein splicing and its modulation by
mutation. Embo J 15, 5146–5153.
Yan, L.Z., and Dawson, P.E. (2001). Synthesis of Peptides and Proteins without Cysteine
Residues by Native Chemical Ligation Combined with Desulfurization. J. Am. Chem.
Soc. 123, 526–533.
86
Chapter 5. O-GlcNAc modification blocks the aggregation and toxicity of the Park-
inson’s Disease associated protein α-synuclein
5.1 Abstract
Several aggregation-prone proteins associated with neurodegenerative diseases
can be modified by O-linked N-acetyl- glucosamine (O-GlcNAc) in vivo. One of these
proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies,
including Parkinson’s disease. However, the effect of O-GlcNAcylation on α-synuclein is
not clear. Here, we use synthetic protein chemistry to generate both unmodified α-
synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physio-
logically relevant threonine residue 72. We show that this single modification has a nota-
ble and substoichiometric inhibitory effect on α-synuclein aggregation, while not affect-
ing the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also
shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-
synuclein that was exogenously added to cells in culture. These results suggest that in-
creasing O-GlcNAcylation may slow the progression of synucleinopathies and further
support a general function for O-GlcNAc in preventing protein aggregation.
87
5.2 Introduction
O-GlcNAc modification (Fig. 5.1a) is the enzymatic addition of the single mono-
saccharide N-acetyl-glucosamine to proteins(Bond and Hanover, 2015; Hardivillé and
Hart, 2014) in the cytosol, nucleus and mitochondria . This modification, which occurs
on serine and threonine side chains, is a uniquely dynamic form of glycosylation through
addition by the enzyme O-GlcNAc transferase (OGT) and removal by the enzyme O-
GlcNAcase (OGA). Several lines of evidence link O-GlcNAcylation to neurodegenera-
tive diseases(Yuzwa and V ocadlo, 2014; Zhu et al., 2014). Neuron-specific knockout of
OGT in mice results in locomotor defects, increased hyperphosphorylated tau and death
within 10 days of birth(O'Donnell et al., 2004). Several aggregation-prone proteins that
contribute directly to neurodegeneration are themselves modified by O-GlcNAc, includ-
ing tau(Lefebvre et al., 2003; Liu et al., 2004) and α-synuclein(Alfaro et al., 2012; Wang
et al., 2010). Increasing O-GlcNAcylation in vivo using small-molecule inhibitors of
OGA has been shown to reduce hyperphosphorylation of tau in healthy rats(Yuzwa et al.,
2008) and slow neurodegeneration in a mouse model of Alzheimer’s disease(Yuzwa et
al., 2012). Additionally, experiments where recombinant tau was enzymatically O-
GlcNAcylated have demonstrated that this modification can directly block that aggrega-
tion of tau in vitro(Yuzwa et al., 2012). Therefore, the maintenance of ‘normal’ O-
GlcNAcylation levels may be a protective mechanism that inhibits protein aggregation
and is potentially lost in neurodegenerative diseases.
88
We are interested in examining the consequences of O-GlcNAcylation on α-
synuclein, the aggregating protein in Parkinson’s disease and other synucleinopathi-
es(Martí et al., 2003). α-Synuclein is a 140-amino-acid protein that is prevalent in pre-
synaptic neurons of the central nervous system(Lashuel et al., 2013). In the cytosol, the
protein is an unfolded monomer, but it adopts an extended α-helical conformation when
associated with cellular membranes, where it performs its likely physiological roles in
vesicle trafficking(Emanuele and Chieregatti, 2015). In diseased cells, however, α-
synuclein consists of β-sheet-rich aggregates that are amyloid in structure (Fig.
5.1b)(Fink, 2006). A range of aggregate species that form in vitro closely resemble the
aggregates in Parkinson’s disease brain samples, biochemically supporting their impor-
tance in the disease. Furthermore, recent experiments in human patients, animal models
and cell culture have shown that extracellular α-synuclein aggregates are toxic and that
the protein is probably transferred from neuron to neuron, where it can propagate disease
pathology(Brettschneider et al., 2015; George et al., 2013; Recasens and Dehay, 2014).
O-GlcNAc has been identified as an in vivo, endogenous modification of α-synuclein at
threonine 64 and 72 in mice and serine 87 in humans, residues that are conserved be-
tween the species (Alfaro et al., 2012; Wang et al., 2009; 2010), raising the possibility
that O-GlcNAcylation may affect α-synuclein aggregation (Fig. 5.1c). In support of this
hypothesis, we have previously demonstrated that the addition of synthetic peptides cor-
responding to fragments of the aggregation-prone region of α-synuclein (residues 61–95)
will accelerate the in vitro aggregation of full-length protein(Marotta et al., 2012). How-
89
ever, the O-GlcNAcylated peptides at threonine 72 (T72) do not have any effect on the
kinetics of aggregation. Unfortunately, in the same study we also found that although re-
combinant tau is O-GlcNAcylated by OGT(Yuzwa et al., 2012; 2010), α-synuclein is
not(Marotta et al., 2012). This is not necessarily surprising, as OGT appears to require
accessory proteins to modify some of its substrates(Cheung et al., 2008), but it does pre-
vent the use of this method to study α-synuclein O-GlcNAcylation.
Here, we investigate the consequences of T72 O-GlcNAcylation on full-length α-
synuclein. We selected T72 as our first target for O-GlcNAc modification because it has
been identified in multiple proteomics experiments(Alfaro et al., 2012; Wang et al., 2010)
and because it lies within the region of α-synuclein that is required for aggregation in vi-
tro (residues 71–82). First, we demonstrate that mutation of α-synuclein residue 72 to al-
anine (T72A), a step that would prevent O-GlcNAcylation at this position, dramatically
reduces the aggregation of the protein. This would make any loss-of-function studies in
cell culture or animals extremely difficult to interpret. To overcome this roadblock, we
used synthetic protein chemistry to generate site-specifically O-GlcNAcylated α-
synuclein at T72 for subsequent gain-of-function experiments. Comparing this protein to
either synthetic, unmodified material or completely recombinant material, a well-
accepted standard in the field of α-synuclein biochemistry, we show that the single O-
GlcNAc modification at T72 completely blocks the formation of both fiber and oligomer
aggregates but has no effect on membrane binding or bending. We then demonstrate that
O-GlcNAcylation inhibits the toxicity of α-synuclein when it is exogenously added to
90
neurons in culture. Because O-GlcNAcylation can affect subsequent phosphorylation, we
also show that O-GlcNAcylation at T72 alters physiologically relevant phosphorylation
events on α-synuclein by three different kinases. We further demonstrate that O-
GlcNAcylation can act in a substoichiometric fashion to slow α-synuclein aggregation.
Finally, to explore the mechanism behind these observations, we find that O-
GlcNAcylation primarily blocks aggregation by preventing the incorporation of α-
synuclein into aggregates, thereby lowering the effective concentration of aggregation-
prone material. These studies support an important role for O-GlcNAcylation in poten-
tially inhibiting the progression of not only Alzheimer’s disease but also Parkinson’s dis-
ease.
91
Figure 5.1 O-GlcNAc modification and α-synuclein. a) O-GlcNAc modification (O-
GlcNAcylation) is a dynamic modification of intracellular proteins by the monosaccharide N-
acetyl-glucosamine (GlcNAc). b) The protein α-synuclein forms toxic amyloid aggregates that
contribute to the progression of neurodegenerative diseases, including Parkinson’s disease. c) α-
Synuclein is O-GlcNAcylated at three different sites but the effects on protein aggregation and
toxicity were unknown. Here, we determine the consequences of O-GlcNAcylation at threonine
residue 72.
92
5.3 Results
5.3.1 An α-synuclein loss-of-function O-GlcNAcylation mutant has compromised aggre-
gation
One common method to investigate the effects of post-translational modifications
in living cells or animal models is the expression of a loss-of-function point mutant of the
protein of interest that cannot be endogenously modified. For example, the consequences
of phosphorylation of α-synuclein serine 129 have been studied using a serine to alanine
(S129A) mutation(Oueslati et al., 2010). One possible avenue to understanding the role
of O-GlcNAcylation would therefore be the overexpression of either wild-type or a T72A
mutant protein and a comparison of their effects in living cells. However, several studies
have found that mutations in the region of α-synuclein that is responsible for aggregation,
including T72 to proline or glutamic acid, can themselves have dramatic effects on the
aggregation of the protein(Koo et al., 2009). We therefore recombinantly expressed both
wild-type α-synuclein and the mutant protein α-synuclein(T72A) (Fig. 5.2) and subjected
them to aggregation conditions (50 µM protein concentration at 37 °C with constant agi-
tation) for 4 days.
93
a
b
Figure 5.2 Characterization of full-length, recombinant α-synuclein and α-synuclein(T72A).
a)Analytical RP-HPLC trace and ESI-MS of purified, recombinant WT α-synuclein. b) Analytical
RP-HPLC trace and ESI-MS of purified, recombinant α-synuclein(T72A).
Figure 5.3 α-Synuclein(T72A) displays reduced aggregation compared to wild-type α-synuclein.
a) α-Synuclein or α-synuclein(T72A) (50 µM) were subjected to aggregation conditions (agitation
at 37 °C) before analysis by ThT fluorescence (λex = 450 nm, λex = 482 nm) at the indicated time
points. y-Axis is fold-change in fluorescence compared to unmodified α-synuclein at t = 0 h. b)
The same reactions were analyzed by TEM after 7 days; scale bar: 500 nm.
94
To determine the extent of aggregation, a combination of thioflavin T (ThT) fluo-
rescence and transmission electron microscopy (TEM) were used. Notably, we chose to
use an end-point-type assay, as the presence of ThT in continuous assays has recently
been shown to accelerate aggregation(Coelho-Cerqueira et al., 2014). Analysis by fluo-
rescence showed that wild-type protein aggregated with the expected kinetics beginning
around 24 h, while α-synuclein (T72A) only displayed a small amount of ThT signal at
72 and 96h (Fig. 5.3a). Analysis by TEM showed that both proteins form fiber structures
of the expected diameter of ∼10 nm (Fig. 5.3b). These data demonstrate that mutation of
α-synuclein at T72 to prevent O-GlcNAcylation has a direct inhibitory effect on the ag-
gregation of the protein, rendering any loss-of-function experiments by expression living
cells impossible to interpret accurately.
5.3.2. Synthesis of O-GlcNAcylated α-synuclein.
Because the loss-of-function mutation (T72A) in α-synuclein itself inhibits aggre-
gation, we chose to test directly the effects of O-GlcNAcylation at this residue by prepar-
ing the protein semi-synthetically by expressed protein ligation (EPL). The only absolute
requirement for the semi-synthesis of proteins using traditional EPL is a cysteine residue
at any ligation sites. α-Synuclein contains no native cysteine residues, so cysteines were
introduced at residues 69 and 76, which are normally alanine residues in the native pri-
mary sequence. Introduction of these cysteine residues enables the retrosynthesis of α-
synuclein (Fig. 5.4a) into a synthetic peptide (1, residues 69–75), a recombinant protein
95
with an N-terminal cysteine residue (2, residues 76–140) and a recombinant protein
thioester (3, residues 1–68). We reasoned that the introduced cysteines could be chemi-
cally desulfurized to alanine residues, yielding semisynthetic α-synuclein with no amino-
acid mutations.
Figure 5.4 Semisynthesis of α-synuclein. a) α-Synuclein was retrosynthetically deconstructed
into a synthetic thioester-peptide (1), a recombinant protein (2) and a recombinant protein thioes-
ter (3) obtained using intein chemistry. b) Synthetic scheme outlining the preparation of α-
synuclein. Synthetic thioester–peptide 1 was first incubated with recombinant protein 2, resulting
in the ligation reaction that yielded protein 4. The N-terminal thioproline of 4 was then trans-
formed to the corresponding cysteine (5) by treatment with methoxylamine. Protein 5 was then
ligated to the protein thioester 3 to give full-length α-synuclein. The cysteines required for the
ligation reactions were then desulfurized to the native alanine residues to give synthetic α-
synuclein with no amino acid mutations. c) Characterization of synthetic α-synuclein using RP-
HPLC and electrospray ionization mass spectrometry (ESI-MS). Analysis by RP-HPLC showed
that synthetic α-synuclein was pure, as evidenced by the appearance of only one, sharp peak.
Characterization by ESI-MS gave a range of charge states that could be deconvoluted to a mo-
lecular mass (14,460 ± 3 Da) in excellent agreement with the predicted weight of 14,460 Da.
As shown schematically in Fig. 5.4b, peptide 1 was prepared using standard
Fmoc-based solid-phase peptide synthesis on the Dawson aminobenzyol resin that en-
ables the generation of C-terminal peptide thioesters (Fig. 5.5)(Blanco-Canosa and Daw-
96
son, 2008). Importantly, the N-terminal cysteine residue remained protected as a thiopro-
line to prevent autoligation. Protein 2 was heterologously expressed in Escherichia coli
(Fig. 5.6). Notably, we found that the initiating methionine residue was conveniently re-
moved during expression by an endogenous methionine aminopeptidase. Incubation of
peptide 1 and protein 2 resulted in formation of ligation product 4 in high yield (Fig 5.7).
The new N-terminal cysteine residue of 4 was then deprotected by treatment with
methoxylamine to give protein 5 (Fig. 5.8). To prepare protein thioester 3, the appropriate
α-synuclein fragment (residues 1–68) was recombinantly expressed as an N-terminal fu-
sion with an engineered DnaE intein from Anabaena variabilis (Fig 5.9). Subsequent in-
cubation of thioester 3 with protein 5 gave full-length, unmodified α-synuclein (Fig.
5.10). Finally, radical-based desulfurization was used to convert the two cysteine residues
required for the ligations to the native alanine residues (synthetic α-synuclein, Fig. 5.4c
and Fig. 5.11).
Figure 5.5 Characterization of thioester peptide 1. Analytical RP-HPLC trace and ESI-MS of
purified thioester peptide 1 (* = guanidine HCl used for solubilization).
97
Figure 5.6 Characterization of protein fragment 2. Analytical RP-HPLC and ESI-MS trace of
purified C-terminal protein fragment 2.
Figure 5.7 Ligation of peptide 1 and protein 2 to give protein fragment 4. a) Monitoring of the
ligation reaction of 1 and 2 by RP-HPLC. b) Analytical RP-HPLC trace and ESI-MS of purified
product 4.
98
Figure 5.8 N-terminal deprotection of the thiazolidine of protein 4 to yield protein fragment 5. a)
Monitoring the thiazolidine (NThz) deprotection reaction of 4 by RP-HPLC. b) Analytical RP-
HPLC trace and ESI-MS of purified product 5.
Figure 5.9 Expression and characterization of protein-thioester fragment 3. a) Monitoring the
intein-fusion thiolysis to generate protein thioester 3 by RP-HPLC. b) Analytical RP-HPLC trace
and ESI-MS of purified product 3.
99
Figure 5.10 Ligation of protein-thioester 3 and protein 5 to give full-length α-synuclein 6. a)
Monitoring the ligation of fragments 3 and 5 by RP-HPLC. b) Analytical RP-HPLC trace and
ESI-MS of purified product 6.
Figure 5.11 Desulfurization of α-synuclein 6 to give synthetic α-synuclein. a) Monitoring the
desulfurization reaction of 6. b) Analytical RP-HPLC trace and ESI-MS of purified full-length,
synthetic α-synuclein.
100
We next embarked on the synthesis of α-synuclein bearing an O-GlcNAc modifi-
cation at T72, which we termed α-synuclein(gT72). The synthesis followed the same
route as that for unmodified α-synuclein, with peptide 1 replaced by glycopeptide 7 (Fig.
5.18a). The selectively protected O-GlcNAcylated threonine residue was first synthesized
using high-yielding thioglycoside chemistry(Marotta et al., 2012), followed by Fmoc-
based solid-phase peptide synthesis. On-resin deprotection of the O-GlcNAc moiety was
accomplished using hydrazine before cleavage and purification of the glycopeptide by
RP-HPLC (Fig. 5.12). Notably, hydrazine treatment did not result in any deprotection of
the N-terminal cysteine residue. Following the same reaction scheme as in Fig. 5.4b, ∼10
mg of α-synuclein(gT72) was readily prepared (Fig. 5.12-5.16).
Figure 5.12. Characterization of O-GlcNAcylated thioester-peptide 7. Analytical RP-HPLC trace
and ESI-MS of purified thioester peptide 7.
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Figure 5.13. Ligation of protein 2 and glycopeptide 7 to give O-GlcNAcylated protein fragment
8. a) Monitoring the ligation reaction of 2 and 7. The reaction proceeded rapidly at first but was
slow to reach completion. b) Analytical RP-HPLC trace and ESI-MS of purified product 8. MS
signal strength was decreased by ionization of the GlcNAc moiety and cleavage of the scissile
glycosidic bond.
102
Figure 5.14. N-terminal deprotection of the thiazolidine of protein 8 to yield glycoprotein frag-
ment 9. a) Monitoring of the thiazolidine (NThz) deprotection reaction of 8. b) Analytical RP-
HPLC trace and ESI-MS of purified product 9. MS signal strength was decreased as with 8.
Figure 5.15. Ligation of protein-thioester 3 and glycoprotein 9 to give full-length O-
GlcNAcylated α-synuclein 10. a) Monitoring the ligation reaction of 3 and 9. b) Analytical trace
from t96 of the same reaction. b, Analytical RP-HPLC trace and ESI-MS of purified product 10.
103
Figure 5.16. Desulfurization of α-synuclein 10 to give O-GlcNAcylated α-synuclein [α-
synuclein(gT72)]. a) Monitoring the desulfurization reaction of 10. b) Analytical RP-HPLC trace
and ESI-MS of purified full-length, synthetic α-synuclein(gT72).
5.3.3 O-GlcNAcylation blocks α-synuclein aggregation but has no effect on membrane
binding.
Comparison of unmodified α-synuclein to α-synuclein(gT72) using circular di-
chroism revealed that O-GlcNAcylation does not result in the formation of any signifi-
cant secondary structure (Fig. 5.17a), and dynamic light scattering showed no formation
of oligomeric or aggregate structures during synthesis or purification (Fig. 5.17b). Re-
combinant α-synuclein, synthetic α-synuclein or α-synuclein(gT72) at a concentration of
50 µM were then simultaneously subjected to aggregation conditions (agitation at 37 °C)
for 7 days. After 72, 120 and 168 h, reaction aliquots were removed and added to a solu-
104
tion of ThT. Analysis by fluorescence (λex = 450 nm, λex = 482 nm) showed that recom-
binant and synthetic α-synuclein formed fibril aggregates, but α-synuclein(gT72) resulted
in no ThT fluorescence over the entire assay (Fig. 5.18b).
Figure 5.17 Structural characterization of synthetic and recombinant proteins using circular di-
chroism (CD) and dynamic light scattering (DLS). a) CD spectra were collected for freshly dis-
solved samples of recombinant α-synuclein, synthetic α-synuclein, or α-synuclein(gT72) at 7.5
µM concentration. All samples show similar spectra that are consistent with a random-coil secon-
dary structure. b) The indicated proteins were analyzed using DLS at 50 µM concentration. All
three preparations showed a single peak with a Stoke’s Radius of approximately 4 nm, and no
significant peaks in the 10-100 nm range, consistent with monomeric protein.
105
Figure 5.18 O-GlcNAcylation blocks α-synuclein aggregation. a) α-Synuclein bearing an O-
GlcNAc modification at threonine 72 [α-synuclein(gT72)] was prepared using the same semisyn-
thetic route outlined in Fig. 5.4b, replacing peptide 1 with glycopeptide 7. Analysis of purified α-
synuclein(gT72) by RP-HPLC showed only one, sharp peak. Characterization by ESI-MS gave a
range of charge states that could be deconvoluted to a molecular mass (14,667 ± 2 Da) in good
agreement with the predicted weight of 14,663 Da. b) Recombinant or synthetic α-synuclein or α-
synuclein(gT72) (50 µM) were subjected to aggregation conditions before analysis by ThT fluo-
rescence at the indicated time points. The y-axis shows the fold change in fluorescence compared
with recombinant α-synuclein at t = 0 h. Results are the mean ± s.e.m. of three separate experi-
ments. Both recombinant and synthetic α-synuclein give strong ThT fluorescence signals over the
course of the aggregation assay, while α-synuclein(gT72) results in no detectable increase in the
signal. c) The same reactions were analyzed by TEM after 7 days. Scale bars, 500 nm. Mature,
rigid fibers with an approximate diameter of 10 nm, consistent with amyloid structures, were
readily visualized in the recombinant and synthetic α-synuclein aggregation reactions, but were
completely lacking in the α-synuclein(gT72) reaction. d) The majority of α-synuclein(gT72) re-
mains soluble during aggregation. After 7 days, aggregation reactions were also separated by cen-
trifugation, resuspended in 8 M urea to disassociate any aggregates and analyzed by SDS–PAGE
and Coomassie staining. Essentially all of the recombinant α-synuclein formed insoluble aggre-
gates, while the majority of α-synuclein(gT72) was found in the soluble fraction. e) O-
GlcNAcylation blocks α-synuclein oligomer formation. Aggregation reactions were analyzed by
SEC-MALS after 7 days. Faster eluting peaks corresponding to molecular weights of ∼1,000 and
100 kDa were detectable in the recombinant α-synuclein aggregation reaction, but were essen-
tially absent in the α-synuclein(gT72) reaction.
106
To visualize the structure of any aggregates that formed, TEM was performed af-
ter 7 days. Both unmodified α-synuclein preparations formed protein fibers characteristic
of amyloidogenic proteins (Fig. 5.18c and Fig. 5.19a). In stark contrast, α-
synuclein(gT72) did not form any fibrous aggregates (Fig. 5.18c and Fig 5.19a), and the
only protein aggregates visualized by TEM were rare and amorphous in nature (Fig.
5.19b). To determine the relative amounts of protein that remained in solution after 7
days of aggregation, the insoluble and soluble fractions were separated by centrifugation.
The soluble fraction was then concentrated by lyophilization, and both fractions were re-
suspended in 8 M urea to break up any aggregates. Analysis by SDS–polyacrylamide gel
electrophoresis (SDS–PAGE) showed that essentially all of the unmodified α-synuclein
was incorporated into insoluble structures, while a large majority of α-synuclein(gT72)
remained in solution (Fig. 5.18d). We next tested whether O-GlcNAcylation inhibits the
formation of oligomeric structures that are also toxic. Protein samples that had been sub-
jected to 7 days of aggregation were analyzed using SEC–MALS (size exclusion chroma-
tography with inline multi-angle light scattering), which enables the size determination of
protein complexes (Fig. 5.18e). As expected, recombinant α-synuclein formed oligomers
that are consistent in size with those found in previous SEC-MALS experiments(Loren-
zen et al., 2014; Paslawski et al., 2014). In contrast, α-synuclein(gT72) formed essentially
no oligomeric species. Taken together, these data demonstrate that O-GlcNAcylation
blocks the formation of both fibers and oligomers and promotes the solubility of α-
synuclein under aggregation conditions.
107
a
b
Figure 5.19 a) Large-scale representation of the transmission electron microscopy (TEM) images
of the protein aggregates aggregation reactions from in Fig. 5.18c. Both recombinant and syn-
thetic α-synuclein, as well as α-synuclein(gT72) (all at 50 µM) were subjected to aggregation
conditions (agitation at 37 °C) for 7 days, after which samples were deposited on formvar coated
copper grids and stained with 1% uranyl acetate. b) TEM images of the amorphous protein depos-
ited from the α-synuclein(gT72) aggregation reactions (50 µM). These samples were largely de-
void of protein aggregates, consistent with the sample being predominantly monomeric. When
these grids were scanned for the presence of any larger protein structures, only amorphous depos-
its, and not fibrils or oligomers, were observed.
α-Synuclein binds strongly to cellular membranes and negatively charged vesi-
cles, where it forms an extended α-helix(Jao et al., 2008), and we have previously shown
that α-synuclein can independently induce membrane curvature and convert vesicles into
tubular structures in vitro (Mizuno et al., 2012; Varkey et al., 2010). Notably, the forma-
tion of small vesicles and tubes by α-synuclein has also been reported in vivo and in neu-
108
ron culture (Boassa et al., 2013) and recent studies on the role of α-synuclein in synaptic
vesicle endocytosis demonstrated that α-synuclein acts at the earliest stages of this proc-
ess in neurons(Vargas et al., 2014). Together, these data establish membrane binding and
bending as a relevant readout on the endogenous function of α-synuclein. To test whether
O-GlcNAcylation had any effect on these properties, recombinant α-synuclein or α-
synuclein(gT72) were incubated with a large excess (1:100) of membrane vesicles for 20
min, and the extent of α-helix formation was measured with circular dichroism (Fig.
5.20a). As expected, recombinant α-synuclein formed an α-helix in the presence of nega-
tively charged lipid vesicles (POPG or POPS; see caption to Fig. 5.20) that could be
competed by the addition of the zwitterionic lipid (POPC). Notably, we observed essen-
tially no differences for α-synuclein (gT72), which demonstrates that there are no signifi-
cant changes in the affinity and mode of binding upon O-GlcNAcylation. Next, we incu-
bated either recombinant α-synuclein or α-synuclein(gT72) with POPG (1:20 ratio) for 20
min and found that both proteins induced the formation of tubular structures with a di-
ameter of ∼30nm, as visualized by TEM (Fig. 5.20b). Together, these data indicate that
O-GlcNAcylation will probably have little to no effect on α-synuclein’s ability to bind or
remodel membranes during its endogenous functions.
109
Figure 5.20 O-GlcNAcylation has no effect on α-synuclein membrane binding or bending. a) O-
GlcNAcylation has no effect on α-synuclein α-helix formation upon membrane binding. Recom-
binant α-synuclein or α-synuclein (gT72) were incubated with a 100-fold excess of the indicated
preformed vesicles and analyzed by circular dichroism. In the presence of negatively charged
vesicles (POPG or POPS), both recombinant α-synuclein and α-synuclein(gT72) gave indistin-
guishable circular dichroism spectra consistent with the formation of an extended α-helix. The
introduction of a zwitterionic lipid (POPC) reduced α-helix formation equally for both proteins.
b) Unmodified α-synuclein and α-synuclein(gT72) bend membranes into tubules equally. Recom-
binant α-synuclein or α-synuclein(gT72) were incubated with a 20-fold excess of POPG vesicles
and analyzed using TEM. Both proteins remodelled the vesicles into tube structures with diame-
ters of ∼30 nm. POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-RAC-(1-glycerol)]; POPS,1-
palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine.
5.3.4 O-GlcNAcylation affects subsequent α-synuclein phosphorylation.
In addition to its direct effects, O-GlcNAcylation has been demonstrated to affect
the phosphorylation of several substrate proteins. α-Synuclein is phosphorylated at multi-
ple residues, including serine 87 (S87) and 129 (S129)(Oueslati et al., 2010). Both of
110
these phosphorylation sites have been shown to be closely associated with protein aggre-
gates in vivo, indicating that they may have detrimental effects in Parkinson’s disease.
However, another model has emerged from a series of in vitro, cellular and in vivo ex-
periments that suggests a protective role for these phosphorylation events(Mbefo et al.,
2010; Oueslati et al., 2013; Paleologou et al., 2010; 2008). Briefly, these experiments
showed that simultaneous phosphorylation at S87 and S129 by casein kinase 1 (CK1) can
inhibit aggregation, and they demonstrated that polo-like kinases (PLKs) and G protein-
coupled receptor kinase 5 (GRK5) can phosphorylate α-synuclein aggregates and pro-
mote their degradation. Although the exact consequences of α-synuclein phosphorylation
still need to be completely elucidated, we chose to test if O-GlcNAcylation affects these
phosphorylation events. Recombinant α-synuclein or α-synuclein(gT72) were incubated
with CK1, polo-like kinase 3 (PLK3) or GRK5. The proteins were then separated by
SDS–PAGE and analyzed by western blotting (Fig. 5.21). O-GlcNAcylation may have a
small effect of increasing phosphorylation at S87 by CK1, while inhibiting the modifica-
tion at S129 by all three kinases. It is therefore possible that O-GlcNAcylation of α-
synuclein at T72 may not only directly block aggregation but also effect phosphorylation
events that may play protective or detrimental roles in Parkinson’s disease.
111
Figure 5.21 O-GlcNAcylation affects the subsequent phosphorylation of α-synuclein. Unmodi-
fied or O-GlcNAcylated α-synuclein were incubated with the indicated kinases for 16 h. Phos-
phorylation status was then visualized by SDS-PAGE followed by Western blotting. Results are
representative of two biological replicates.
5.3.5 O-GlcNAcylation inhibits α-synuclein toxicity.
Although O-GlcNAcylation blocks the formation of α-synuclein fibers and
oligomers, it is possible that it does not inhibit the genesis of other insoluble or soluble
species that are still toxic. α-Synuclein aggregates are found intracellularly in Parkinson’s
disease. However, many α-synuclein overexpression studies in neurons or cell lines did
not observe toxicity without additional cellular stress(Khalaf et al., 2014; Ko et al., 2008;
Lee et al., 2001; Tabrizi et al., 2000). Additionally, we have shown here that α-synuclein
(T72A) has an intrinsic aggregation deficiency (Fig. 5.3) that prevents the direct effects
of O-GlcNAcylation at this site from being meaningfully tested in an overexpression
model. Notably, a growing body of evidence supports the extracellular, cell-to-cell trans-
mission of α-synuclein aggregates to previously healthy neurons(George et al., 2013;
112
Olanow and Brundin, 2013). In particular, the exogenous addition of toxic α-synuclein
species to culture media results in the death of neurons and neuronal culture models, ena-
bling us to directly test the effects of O-GlcNAcylation with our synthetic material. Ac-
cordingly, we initiated aggregation reactions containing recombinant α-synuclein or α-
synuclein(gT72). After 7 days, aggregates were first collected by centrifugation and then
resuspended in culture media using sonication. To control for the presence of any species
that remained soluble, the supernatant was concentrated by lyophilization and also resus-
pended in culture media. These samples (25 µM concentration based on monomer) were
analyzed by TEM (Fig. 5.22) and then added to either primary rat cortical neurons or SH-
SY5Y cells, a neuroblastoma cell line that expresses endogenous α-synuclein. After 60 h,
cell toxicity was measured using the small molecule ethidium homodimer. Ethidium ho-
modimer is an environmentally sensitive fluorescent dye that is excluded from healthy
cells but gains access through the disrupted membranes of dying cells, where it interca-
lates into DNA.
Figure 5.22 Analysis of cellular toxicity samples by transmission electron microscopy (TEM).
Unmodified or O-GlcNAcylated α-synuclein were subjected to aggregation conditions for 7 days.
At this time, the aggregated and soluble material were separated by centrifugation and resus-
pended by sonication in water followed by analysis by TEM.
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Recombinant α-synuclein aggregates caused a substantial increase in ethidium
homodimer signal in both cell types (Fig. 5.23). In contrast, treatment with α-
synuclein(gT72) resulted in no significant change in toxicity in neurons and less death in
SH-SY5Y cells compared with unmodified protein (Fig. 5.23). Notably, treatment of cells
with either of the supernatants resulted in no significant changes in toxicity (Fig. 5.23).
Because SH-SY5Y cells divide in culture, we also measured the effect of α-synuclein on
their proliferation (Fig. 5.23). Consistent with the ethidium homodimer signal, unmodi-
fied α-synuclein aggregates inhibited proliferation, while α-synuclein(gT72) had no ef-
fect. Again, the soluble material from both proteins had no significant effect on prolifera-
tion (Fig. 5.23). Together, these data indicate that α-synuclein O-GlcNAcylation prevents
the formation of toxic species and that it might slow the cell-to-cell spread of toxicity in
the brain.
114
Figure 5.23 O-GlcNAcylation blocks α-synuclein toxicity. Primary rat cortical neurons in culture
or SH-SY5Y cells were treated for 60 h with vehicle or insoluble material or remaining soluble
material (25 µM based on monomer concentration) collected from aggregation reactions initiated
with either α-synuclein(gT72) or recombinant α-synuclein. In both cell types, toxicity was meas-
ured with ethidium homodimer fluorescence (λex = 528 nm, λex = 617 nm). Treatment with re-
combinant α-synuclein resulted in significantly more toxicity than α-synuclein(gT72) in both cell
types tested. Cellular proliferation was measured in SH-SY5Y cells by cell counting. Again,
treatment with recombinant α-synuclein slowed cellular proliferation to a significantly larger ex-
tent than α-synuclein(gT72). Results are the mean ± s.e.m. of three separate experiments. Statisti-
cal significance (two-tailed, t-test): *P < 0.05, **P = < 0.01. NS, not significant.
5.3.6 O-GlcNAcylation largely prevents incorporation of α-synuclein monomers into ag-
gregates.
To investigate how O-GlcNAcylation inhibits α-synuclein aggregation, we first
asked whether O-GlcNAcylation has a substoichiometric effect on fiber formation. Ag-
gregation reactions were prepared that contained either recombinant or synthetic α-
synuclein, α-synuclein(gT72) or different ratios of α-synuclein(gT72) and unmodified α-
synuclein. Analysis by ThT fluorescence (Fig. 5.24a) demonstrated that as little as 10%
O-GlcNAcylated α-synuclein resulted in delayed aggregation kinetics. Larger percent-
ages (25 and 50%) increased this delay and yielded overall lower levels of ThT fluores-
115
cence. Examination by TEM showed that increasing percentages did not have a notice-
able effect on the structure of the fibers that were formed (Fig. 5.24b). The aggregation of
α-synuclein is concentration-dependent. Because O-GlcNAcylation does not change the
structure of the fibers formed, its substoichiometric effect could be explained by the ex-
clusion of α-synuclein(gT72) from the fibers, which would in effect simply lower the
concentration of aggregation-competent protein. To test this possibility, aggregation reac-
tions were initiated containing either unmodified α-synuclein at 50 µM, the same protein
at 25 µM, or a 1:1 mixture of unmodified α-synuclein (25 µM) and α-synuclein(gT72)
(25 µM). Analysis of the aggregation kinetics by ThT fluorescence showed that aggrega-
tion was less efficient at 25 µM and in the 1:1 mixture compared to 50 µM α-synuclein,
with the 1:1 mixture showing slightly more aggregation at later time points (Fig. 5.24c).
116
Figure 5.24 O-GlcNAcylated α-synuclein is largely excluded from the protein aggregates. a) Re-
combinant α-synuclein or synthetic α-synuclein (50 µM) or the indicated mixtures of α-
synuclein(gT72) and recombinant α-synuclein (50 µM total concentration) were subjected to ag-
gregation conditions (agitation at 37 °C) before analysis by ThT fluorescence (λex = 450 nm, λex
= 482 nm) at the indicated time points. The y-axis shows the fold change in fluorescence com-
pared with recombinant α-synuclein at t = 0 h. Increasing percentages of α-synuclein(gT72) com-
pared with recombinant α-synuclein resulted in slower aggregation kinetics and lower overall
ThT fluorescence levels. b) The same reactions were analyzed by TEM after 7 days. Scale bars,
500 nm. Increasing percentages of α-synuclein(gT72) compared with recombinant α-synuclein
resulted in the formation of fewer overall fibre structures but did not change the gross structures
of the fibers formed. c) Recombinant α-synuclein at 50 or 25 µM or a 1:1 mixture of recombinant
α-synuclein (25 µM) and α-synuclein (gT72) (25 µM) were subjected to aggregation conditions
(agitation at 37 °C) before analysis by ThT fluorescence (λex = 450 nm, λex = 482 nm) at the in-
dicated time points. The y axis shows the fold change in fluorescence compared with recombinant
α-synuclein at t = 0 h. The 1:1 mixture of recombinant α-synuclein and α-synuclein(gT72) (50
µM concentration) gave less aggregation than α-synuclein alone, but more than recombinant α-
synuclein at a concentration of 25 µM. d) The same reactions were analyzed by TEM after 7 days.
Scale bars, 500 nm. All three aggregation reactions formed fibers that were similar in structure
and size. All results are the mean ± s.e.m. of at least three separate experiments. Statistical sig-
nificance (two-tailed, t-test): *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, not sig-
nificant.
117
Visualization of the aggregates by TEM again showed no major changes to the
fiber structure (Fig. 5.24d). At the 168 h time point, aggregates from the 25 µM and 1:1
reactions were isolated by centrifugation and the supernatant was concentrated by lyophi-
lization. Both samples were dissolved in an 8 M urea solution to break up any aggregates
and then separated by SDS–PAGE. Western blotting showed that the vast majority of α-
synuclein(gT72) remained in solution during the aggregation reaction (Fig. 5.25), sug-
gesting that a small fraction of α-synuclein(gT72) is incorporated into aggregates but the
majority remains in solution. Finally, we tested whether O-GlcNAcylation inhibits the
incorporation of α-synuclein monomers into a large amount of preformed fibers. Specifi-
cally, fibers were formed by subjecting unmodified α-synuclein to aggregation conditions
for 7 days. At this time the fibers were sonicated to break the fibers and increase the
available ‘ends’ for elongation. They were then added to either buffer or unmodified α-
synuclein or α-synuclein(gT72) to give a mixture of 25 µM fibers and 25 µM monomeric
protein. Aggregation conditions were then reinitiated and the extent of fiber formation
was measured using ThT fluorescence (Fig. 5.26). Fibers that were added to buffer gave a
relatively stable ThT fluorescence signal over the course of the experiment. In contrast,
fibers mixed with unmodified α-synuclein yielded an immediate increase in ThT signal
that plateaued within 24 h. Fibers mixed with α-synuclein(gT72) showed a similar in-
crease in ThT signal with a small but statistically significant decrease in aggregation ki-
netics. Analysis of the aggregates that formed in these reactions by TEM (Fig. 5.26)
showed that addition to unmodified α-synuclein led to the formation of long, straight fi-
118
bers (∼10 nm in diameter), while those formed by α-synuclein(gT72) were somewhat
more irregular in shape, suggesting that O-GlcNAcylation may have an effect on the
structure of the fibers when it is incorporated into them. Together, these three experiments
indicate that O-GlcNAcylated α-synuclein remains more soluble during the aggregation
reaction and is incorporated at a reduced efficiency compared with unmodified α-
synuclein. However, this equilibrium between soluble and aggregated α-synuclein(gT72)
can be driven by the presence of a large amount of preformed, unmodified fibers (Fig.
5.27).
Figure 5.25 O-GlcNAcylation inhibits aggregation by preventing incorporation of α-synuclein
into aggregates. Recombinant α-synuclein (25 µM) alone or in the presence of α-synuclein(gT72)
(25 µM) underwent aggregation for 7 days, followed by centrifugation and analysis by SDS-
PAGE and Western blotting of the aggregate and soluble fractions.
119
Figure 5.26 O-GlcNAcylation does not strongly inhibit aggregation in the presence of pre-
formed fibers. a) Unmodified α-synuclein (50 µM) was subjected to aggregation for 7 days. At
this time, these pre-formed fibers were sonicated and mixed with either buffer or unmodified α-
synuclein monomers or O-GlcNAcylated α-synuclein monomers to give a mixture of fibers (25
µM) and monomers (25 µM). These mixtures were were subjected to aggregation conditions (agi-
tation at 37 °C) before analysis by ThT fluorescence (λex = 450 nm, λex = 482 nm) at the indicated
time points. y-Axis is fold-change in fluorescence compared to only pre-formed fibers at t = 0 h.
b) The same reactions were analyzed by TEM after 60 h; scale bar: 500 nm. All results are the
mean ±s.e.m. of three separate experiments. Statistical significance (two-tailed, t-test): *P < 0.05,
**P < 0.01.
120
5.4 Discussion
Here, we have used synthetic protein chemistry to show that site-specific O-
GlcNAcylation of α-synuclein at T72 inhibits protein aggregation, most probably by
shifting the equilibrium of the aggregation reaction towards soluble material. Although
there is currently no atomic-scale structural information about α-synuclein aggregates,
our data are consistent with experiments using electronic spin resonance(Chen et al.,
2007) and solid-state NMR(Vilar et al., 2008) spectroscopies, which demonstrate that
T72 lies in the core of a fibre. O-GlcNAcylation also inhibits the toxicity of α-synuclein
in a well-established extracellular assay when added to primary neurons or a neuronal
cell line. We believe that this difference probably results from both a reduction in the di-
rect toxicity of aggregates in culture and reduced uptake of the protein, as aggregates
have been shown to be endocytosed more efficiently than monomeric protein(George et
al., 2013). In contrast to the inhibition of aggregation by O-GlcNAcylation, the modifica-
tion does not interfere with membrane binding or bending by α-synuclein, indicating that
it would not disrupt the normal biological functions of the protein. This has important
implications for neurodegenerative diseases. OGA inhibitors that raise O-GlcNAcylation
levels should be pursued as possible therapeutics in both Alzheimer’s disease and synu-
cleinopathies, particularly in cases where genetic screening can inform early treatment
decisions to potentially slow the onset of protein aggregation.
121
Figure 5.27 Model of α-synuclein aggregation. a) In the α-synuclein fiber are protein monomers
composed of 5 β-strands (residues ~35-90). These monomers stack to give parallel, in-register
structure that is conserved in other amyloid-forming proteins. O-GlcNAcylation is located at the
core of the fiber structure in a region (residues 71-82) that are critical to aggregate formation. b)
Aggregation of α-synuclein into fibers is a concentration dependent process where individual
monomers are added to the growing fiber structure. c) Our model for inhibition of aggregation by
O-GlcNAcylation at T72.
Coupled with previous data on the aggregation-prone proteins tau and TAB111, as
well as recent evidence that O-GlcNAc prevents the aggregation of the Polycomb protein
Polyhomeotic (Ph) in Drosophila(Gambetta and Müller, 2014), these data add further
122
support to a model where O-GlcNAcylation prevents indiscriminate protein aggregation
in general. Finally, we also found that a T72A mutant of α-synuclein that would prevent
O-GlcNAcylation has a large effect on the aggregation of α-synuclein, preventing the
straightforward interpretation of any loss-of-function experiments (for example, overex-
pression of wild-type α-synuclein and α-synuclein(T72A) in cell culture or animal mod-
els). This highlights the unique utility of synthetic protein chemistry to investigate the
direct effects of post-translational modifications on proteins, including modification of α-
synuclein by O-GlcNAcylation (this study), ubiquitination(Haj-Yahya et al., 2013;
Hejjaoui et al., 2010), phosphorylation(Hejjaoui et al., 2012), acetylation(Fauvet et al.,
2012) and nitration(Burai et al., 2015).
123
5.5 Materials and Methods
5.5.1 General
All solvents and reagents were purchased from commercial sources (Sigma-
Aldrich, Fluka, EMD, Novagen, etc.) and used without any further purification. All aque-
ous solutions were prepared using ultrapure laboratory grade water (deionized, filtered,
sterilized) obtained from an in-house ELGA water purification system and filter sterilized
with 0.45 µm syringe filters (VWR) before use. Growth media (LB broth, Miller, No-
vagen and S.O.C. broth, Sigma) were prepared, sterilized, stored, and used according to
the manufacturer. Antibiotics were prepared as stock solutions at a working concentration
of 1000x (ampicillin sodium salt, EMD 100 mg mL
-1
, kanamycin sulfate, EMD, 50 mg
mL
-1
) and stored at -20 °C. All bacterial growth media and cultures were handled under
sterile conditions under open flame. All silica gel column chromatography was performed
using 60 Å silica gel (EMD) and all thin-layer chromatography performed using 60 Å,
F254 silica gel plates (EMD) with detection by ceric ammonium molybdate (CAM) and/
or UV light. Reverse phase high performance liquid chromatography (RP-HPLC) was
performed using an 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 3000 LC/MS-
MS System (Applied Biosystems/MDS SCIEX).
1
H NMR spectra were acquired on ei-
ther a Varian Mercury 400 MHz or Varian VNMRS 500 MHz magnetic resonance spec-
trometer.
124
5.5.2 Plasmid Construction
A pRK172 construct was generated containing wild-type human α-synuclein in-
serted into Nde I and Hind III restriction sites using standard molecular cloning tech-
niques, as has been described previously(Meier et al., 2012). The C-terminal fragment of
α-synuclein 2 (aa 76-140, A76C) was introduced into a pET42b vector using Nde I and
Spe I restriction sites and standard molecular cloning techniques. The N-terminal frag-
ment of α-synuclein 3 (aa 1-68) was introduced into a modified pTXB1 construct con-
taining the Ava-DnaE N137A intein(Shah et al., 2012) using Nde I and Bpu10I restriction
sites and standard molecular cloning techniques.
5.5.3 Expression of recombinant α-synuclein
BL21(DE3) chemically competent E. coli (VWR) were transformed with the
pRK172 construct containing wild-type human α-synuclein or α-synuclein(T72A) 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. Single colonies were selected and used to inoculate two
5 mL LB-amp liquid cultures, which were grown at 37 °C with shaking at 250 rpm for 16
h. Each 5 mL culture was used to inoculate a 1 L LB-amp culture. These cultures were
grown to an OD600 of 0.6-0.7 at 37 °C shaking at 250 rpm, and then expression was in-
duced with IPTG (final concentration: 0.5 mM) at 25 °C shaking at 250 rpm for 18 h.
Bacteria were harvested by centrifugation (8,000 x g, 30 min, 4 °C), and the cell pellets
125
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 1 L of culture) of lysis buffer (500 mM
NaCl, 100 mM 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 to room temp, and then placed
on ice. Protease inhibitor cocktail (mini complete EDTA free, Roche) was added and
lysates were incubated on ice for 20 min and then cleared by centrifugation (42,000 x g,
30 min, 4 °C). The resulting supernatant was acidified, on ice, to pH 3.5 with HCl and
then incubated on ice an additional 20 min before centrifuging again (42,000 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, 1 h per L). The dialyzed protein solution was then purified by RP-
HPLC over a C4 semi-preparative column (Vydac). Purified material was flash frozen in
liquid N2 and lyophilized. Pure α-synuclein was characterized by C4 analytical RP-HPLC
column (Vydac) and ESI-MS (M+H
+
) and yield was determined by Pierce BCA assay
(Thermo Scientific).
5.5.4 Expression of α-synuclein C-terminal fragment (2)
BL21(DE3) chemically competent E. coli (VWR) were transformed with the
pET42b construct containing 2 by heat shock and plated on selective LB agar plates con-
taining 50 µg mL
-1
kanamycin (LB-kan). Expression and purification of 2 was carried out
as described above for recombinant α-synuclein.
126
5.5.5 Expression of α-synuclein N-terminal thioester (3)
BL21(DE3) chemically competent E. coli (VWR) were transformed with the
modified pTXB1 construct containing 3 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. After harvesting bacteria by centrifugation
(8,000 x g, 30 min, 4 °C), the cell pellet was resuspended on ice in 10 mL (per 1 L of cul-
ture) 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 loaded onto
a Ni-NTA purification column (HisTrap FF Crude, GE Healthcare). The column was
washed with 5 column volumes (CV) of lysis buffer, 5 CV of wash buffer 1 (lysis buffer,
20 mM imidazole), 3 CV of wash buffer 2 (lysis buffer, 50 mM imidazole), and then
eluted in 4 x 1 CV of elution buffer (lysis buffer, 250 mM imidazole). Elution fractions
were dialyzed against 3 x 1 L (100 mM NaH2PO4, 150 mM NaCl, 1 mM EDTA, 1mM
TCEP HCl, pH 7.2) and then concentrated approximately 5-fold in spin-column concen-
trators (Amicon Ultra 3 kDa MW cut-off, Millipore). Sodium mercaptoethane sulfonate
(MESNa) was added to a final concentration of 200 mM along with fresh TCEP (2 mM
final concentration), and the thiolysis reaction was incubated at room temperature to gen-
erate the protein thioester. Reaction progression was monitored by analytical RP-HPLC.
Upon completion, the thiolysis reaction was purified over a C4 semi-prep column and
127
stored as a lyophilized solid. Pure thioester 3 was characterized by analytical RP-HPLC
and ESI-MS.
5.5.6 Solid phase synthesis of thioester peptides 1 and 6
All solid-phase peptide syntheses were conducted manually using unprotected
Rink amide ChemMatrix
®
resin, (PCAS BioMatrix) with an estimated loading of 0.45
mmol g
-1
using a 4-amino benzoic acid linker(Blanco-Canosa and Dawson, 2008). Com-
mercially available N-Fmoc and side chain protected amino acids (10 eq, Novabiochem)
were activated for 20 min with HBTU (10 eq, Novabiochem) and DIEA (20 eq, Sigma)
and then coupled to the resin for 1 h, bubbling with N2 to mix. Reaction completion was
checked using the Kaiser test. Briefly, a small amount of resin was incubated with equal
volumes of 5% w/v ninhydrin in EtOH, 80% w/v phenol in EtOH, and 20µM KCN in
pyridine and heated to 99 °C for 5 min in a sealed tube. If necessary, a second coupling
was conducted with 10 eq amino acid, 10 eq HOBt (Novabiochem) and 12 eq DCC
(Sigma) for 2 h, with N2 mixing. After successful coupling, the terminal Fmoc group was
removed with 20% v/v piperidine in DMF for 5 min with N2 mixing, and then for an ad-
dition 15 min with fresh 20% piperidine in DMF. When peptides were completed, peptide
7 was deprotected with hydrazine hydrate (80% v/v in MeOH) twice for 30 min, with N2
mixing. Both peptides were then acetylated at the free amine of the Dbz linker with p-
Nitrophenyl-chloroformate (5 eq in DCM, N2 mixing) followed by treatment with excess
DIEA (0.5 M in DMF) for 15 min to cyclize the Dbz linker. Peptides were then cleaved
128
from the resin by incubating in cleavage cocktail (95:2.5:2.5 TFA/H2O/
Triisopropylsilane) for 3.5 h at room temperature. The peptide was then diluted ~1/10 in
cold diethyl ether and precipitated over night (-80 °C). The resulting suspension was cen-
trifuged (5,000 x g, 30 min, 4 °C) and the pellet was resuspended in fresh Et2O and cen-
trifuged again (5,000 x g, 30 min, 4 °C). The pellet was then resuspended in H2O, flash
frozen, and lyophilized. This crude lyophilized material was resuspended in thiolysis
buffer (150 mM NaH2PO4, 150 mM MESNa, pH 7.0) and incubated at room temperature
for 24 h before being purified by RP-HPLC (0-50% buffer B over 60 min) over a C18
semi-preparative column (Vydac). Purified peptides were characterized by RP-HPLC (0-
70% B gradient over 60 min) over an analytical C18 column (Vydac) and ESI-MS.
5.5.7 Unmodified α-synuclein synthesis
Lyophilized WT thioester peptide 1 (4 mM) and α-synuclein C-terminal fragment
2 (2 mM) were dissolved in ligation buffer (300 mM NaH2PO4, 5.5 M guanidine HCl,
100 mM MESNa, 1 mM TCEP, pH 7.8) and allowed to react at room temperature. The
reaction pH was readjusted with concentrated NaOH as needed after dissolving both
components. Reaction progress was monitored by RP-HPLC over an analytical C18 col-
umn with a gradient of 0-70% B over 60 min. The reaction was complete after 48 h and
purified by HPLC to yield pure α-synuclein fragment (69-140) 4. Product was confirmed
by ESI-MS. The N-terminal thiazolidine (NThz) protecting group of 4 was then removed
with methoxylamine to yield the free N-terminal cysteine fragment 5. Specifically, ly-
129
ophilized 4 (2 mM) was dissolved in deprotection buffer (100 mM NaAcO, 5.5 M gua-
nidine HCl, 100 mM NaCl, 250 mM MeONH2 HCl, pH 5.0) and heated to 37 °C for 48 h.
Upon completion, βME was added to reduce protein disulfides and the reaction was puri-
fied by RP-HPLC and lyophilized. Product was confirmed by ESI-MS. Lyophilized 5 (2
mM) was then dissolved in freshly prepared ligation buffer. This solution was then added
to lyophilized α-synuclein (1-68) thioester 3 (8 mM) and the pH of the solution was ad-
justed back to 7.8 with conc. NaOH. The reaction was complete after 72 h at room tem-
perature and fresh TCEP was added to reduce any MES disulfides. The reaction was puri-
fied by RP-HPLC to yield full-length α-synuclein 6. Finally, radical desulfurization was
used to convert the two cysteines in 6 to native alanines using the radical initiator V A-061
(Wako). Briefly, protein 6 was dissolved in buffer (200 mM NaH2PO4, 6 M guanidine
HCl, 300 mM TCEP, pH 7.0) to which was added 2% v/v ethanethiol, and 10% v/v tert-
butylthiol. V A-061 (as a 0.2 M stock in MeOH) was then added to a final concentration
of 2 mM (final protein concentration was 0.75 mg mL
-1
). The reaction was heated to 37
°C for 15 h and then purified by RP-HPLC to yield synthetic, full-length WT α-synuclein.
5.5.8 α-Synuclein(gT72) synthesis
Lyophilized O-GlcNAc thioester peptide 7 (2 mM) and α-synuclein C-terminal
fragment 2 (1.6 mM) were dissolved in ligation buffer (300 mM NaH2PO4, 5.5 M gua-
nidine HCl, 100 mM MESNa, 1 mM TCEP, pH 7.8) and allowed to react at room tem-
perature. The reaction pH was readjusted with concentrated NaOH as needed after dis-
130
solving both components. Reaction progress was monitored by RP-HPLC. The reaction
proceeded very slowly and was allowed to continue for 192 h, after which it was purified
by HPLC to yield pure O-GlcNAcylated α-synuclein fragment (69-140) 8. Product was
confirmed by ESI-MS. The N-terminal thiazolidine (NThz) protecting group of 8 was
removed with methoxylamine to yield the free N-terminal cysteine fragment 9. Specifi-
cally, lyophilized 8 (2 mM) was dissolved in deprotection buffer (100 mM NaAcO, 5.5 M
guanidine HCl, 100 mM NaCl, 250 mM MeONH2 HCl, pH 5.0) and heated to 37 °C for
72 h. Upon completion the reaction was purified by RP-HPLC and lyophilized. Product
was confirmed by ESI-MS. Lyophilized 9 (2 mM) was dissolved in freshly prepared liga-
tion buffer. This solution was then added to lyophilized α-synuclein (1-68) thioester 3 (8
mM) and the pH of the solution was adjusted back to 7.8 with concentrated NaOH. The
reaction was complete after 96 h at room temperature and fresh TCEP was added to re-
duce any MES disulfides. The reaction was purified by RP-HPLC to yield full-length α-
synuclein 10. Radical desulfurization was used to convert the two cysteines in 10 to na-
tive alanines using V A-061. Protein 10 was dissolved in buffer (200 mM NaH2PO4, 6 M
guanidine HCl, 300 mM TCEP, pH 7.0) to which was added 2% v/v ethanethiol, and 10%
v/v tertbutylthiol. V A-061 (as a 0.2 M stock in MeOH) was added to a final concentration
of 2 mM (final protein concentration was 0.75 mg mL
-1
). The reaction was heated to 37
°C for 16 h and then purified by RP-HPLC to yield synthetic, full-length O-GlcNAc α-
synuclein [α-synuclein (gT72)].
131
5.5.9 Aggregation reactions
Synthetic or recombinant wild-type or α-synuclein(gT72) were aliquoted and ly-
ophilized. Lyophilized proteins were resuspended in reaction buffer (10 mM NaH2PO4,
0.05% NaN3, pH 7.4) to the appropriate concentration (50 µM), and all material was dis-
solved using a bath sonicator. For mixtures of recombinant α-synuclein and α-
synuclein(gT72), 50, 25, or 10% α-synuclein(gT72) material was aliquot into the same
tube prior to lyophilization and resuspended the same as pure samples. Reactions were
centrifuged for 15 min. at 14,000 x g to remove any debris or aggregated material. The
supernatant was then split into triplicate reaction in 1.5 mL conical centrifuge tubes
(VWR). Reactions were then incubated in a Thermomixer F1.5 orbital shaker at 1000
rpm, 37 °C for seven days. Sample aliquots for analysis at t0 were removed prior to heat-
ing to 37 °C.

5.5.10 Circular Dichroism
Circular dichroism spectra were collected on a Jasco J-815 CD Spectrometer.
Sample aliquots were diluted to 7.5 µM α-synuclein with reaction buffer containing no
NaN3. Spectra were collected from 250-195 nm with a 0.1 nm data pitch, 50 nm min
-1

scanning speed, data integration time of 4 sec, 1 nm bandwidth, 1 mm path length with 3
accumulations, at 25 °C.
132
5.5.11 Dynamic light scattering
Light scattering data was collected with a Dynapro Titan temperature controlled
microsampler (Wyatt). Samples taken directly from aggregation reactions (50 µM) at
time 0 h were analyzed with ten 10 s acquisition, at 25 °C, with laser power adjusted to
give an intensity of 2.0E
6
counts sec
-1
. Radii were calculated using a Raleigh sphere ap-
proximation.
5.5.12 Thioflavin T fluorescence
The degree of α-synuclein aggregation was quantified by Thioflavin T fluores-
cence. Samples were prepared by diluting samples from aggregation reaction (final α-
synuclein concentration = 1.25 µM) in 20 µM Thioflavin T in reaction buffer (above).
Samples were diluted, vortexed briefly, and then incubated 2 min before analyzing. Spec-
tra were collected using a NanoLog Spectrofluorometer (Horiba), λex = 450 nm, ex slit =
4 nm, λem = 482, em slit = 3 nm, 10 mm path length, integration time = 0.1 sec, 3 aver-
aged accumulations. Data was measured in triplicate for all aggregation reaction condi-
tions.
5.5.13 Transmission electron microscopy
A 10 µL droplet from each sample was deposited on formvar coated copper grids
(150 mesh, Electron Microscopy Sciences) and allowed to sit for 5 min and then excess
liquid was removed with filter paper. Grids were then negatively stained for 2 min with
133
1% uranyl acetate, washed three times with 1% uranyl acetate, each time removing ex-
cess liquid with filter paper. The grids were desiccated for 48 h in a vacuum desiccator.
Grids were imaged using a JOEL JEM-2100F transmission electron microscope operated
at 200 kV , 60,000x magnification, and an Orius Pre-GIF CCD.
5.5.14 SEC-MALS
Recombinant, wild-type α-synuclein or α-synuclein(gT72) were aggregated at 50
µM as described above. After seven days, aggregation reactions were centrifuged (20,000
x g, 25 °C, 1 h) to remove fibrils and other large aggregates. The supernatants were then
separated using a Shodex KW-802.5 size exclusion chromatography column (mobile
phase 50 mM HEPES, 200 mM Na2SO4, pH 7.5) and light scattering and differential re-
fractive index were measured with an in-line Wyatt Dawn Heleos and Optilab rEX detec-
tors respectively.
5.5.15 Circular Dichroism of α-synuclein in the presence of lipids
CD spectra were obtained using a Jasco J-810 spectropolarimeter at room tem-
perature at a 1:100 protein:lipid molar ratio with vesicles of different degrees of negative
charge. Lipid vesicles were prepared through mixing different ratios of 1-palmitoyl-2-
oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine (POPC) or using 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-RAC-(1-
glycerol)] (POPG). Dried lipid films were resuspended in 10 mM sodium phosphate pH
134
7.4 buffer. Spectra were acquired using a scan rate of 50 nm/minute, bandwidth of 1 nm,
1 sec time response and step resolution of 1 nm. The final spectra were obtained by sub-
tracting away the appropriate blanks.
5.5.16 Transmission electron microscopy of membrane tubulation
O-GlcNAc modified and wild type α-synuclein were incubated with POPG vesi-
cles in a 1:20 protein:lipid molar ratio as previously described(Varkey et al., 2010). 10 µL
of the sample was loaded onto carbon-coated formvar films on copper grids (Electron
Microscopy Sciences) and subsequently stained with 1% uranyl acetate. Negative stain
transmission electron microscopy was performed on a JEOL 1400 transmission electron
microscope accelerated to 100 kV .
5.5.17 Cellular toxicity assay
Cortices from E17 Sprague-Dawley rat embryos were dissociated in a 0.25 %
trypsin 1 mM HEPES, Hank’s balanced salt solution (HBSS) for 15 min; the tissue was
then washed 3 times in fresh HEPES HBSS. At 10 days prior to treatment, the dissociated
neurons were then plated in poly-D-lysine (Sigma) and laminin (Sigma) pre-treated 96-
well plates (Costar, black plate, clear bottom with lid) at a density of 2.5x10
3
cells per
well in supplemented Neurobasal medium (Invitrogen). The Neurobasal medium was
supplemented with 10 ml L
-1
Glutamax (Invitrogen), 1 mg L
-1
gentamicin solution (Invi-
trogen), 20 ml L
-1
B-27 supplement (Invitrogen), and 50 ml L
-1
fetal bovine serum (Invi-
135
trogen) and grown at 37 ℃ under humidified condition in 5% CO2 atmosphere. Four
hours after the neurons were plated, the medium was diluted 1:3 with serum-free supple-
mented Neurobasal medium and again diluted 1:2 with fresh serum-free supplemented
Neurobasal medium after 7 days in vitro. SH-SY5Y cells were grown at 37 ℃ under hu-
midified condition in 5% CO2 atmosphere using DMEM/F12K 1:1 supplemented with
10% FBS. At 48 h prior to the treatment, 1.25 x 10
4
cells per well were plated into 96-
well assay plate (Costar, black plate, clear bottom with lid). Recombinant α-synuclein or
α-synuclein(gT72) were subjected to aggregation conditions (shaking at 1000 rpm, 37
°C) at 50 µM for 7 days. The reactions were then centrifuged (2,000 x g, 25 °C, 1 h) and
the supernatants were removed and lyophilized to dryness. The aggregates/supernatant
were resuspended with the appropriate growth medium, followed by bath sonication (20
min) and tip-sonication (20% amplitude, 1 sec pulse duration, 1 sec rest for 14 sec) and
then added to the cells. Cells were monitored using microscope after the treatment. The
ethidium homodimer toxicity assay was performed at t = 60 h. Specifically, 100 µL of 1X
DPBS solution containing 3 µM of Ethidium homodimer (VWR) were added into each
well. The plate was incubated in tissue culture incubator for 40 min before taking read-
ings. The plate was shaken for 10 sec at the highest intensity before fluorescent signal
was measured using Synergy H4 Hybrid reader (BioTek) Excitation = 530 nm, Emission
= 620 nm, Gain = 100, Band width = 20 nm, read height of 5 mm). For SH-SY5Y cells,
the cells were then trypsonized and counted using a Countess II Automated Cell Counter
(Life Technologies) according to the manufacturer’s procedure.
136
5.5.18 In vitro phosphorylation reaction and Western blotting
Either recombinant α-synuclein or α-synuclein(gT72) was subjected to in vitro
phosphorylation with one of three kinases; casein kinase 1 (CK1), polo-like kinase 3
(PLK3), or G protein-coupled receptor kinase 5 (GSK5). Pure, lyophilized proteins were
dissolved at 28 µM in buffer (10 mM dithiothreitol (DTT), 1 mM MgCl2, 50 mM Tris, 1
mM ATP, pH 7.5). These solutions were prepared and split into 49 µL triplicates to which
1 µL of the corresponding kinase was added. Reactions were incubated at 30 °C, shaking
at 500 rpm for 16 h. Reactions were quenched by boiling in 4X SDS loading buffer for 10
min. Samples were loaded at 2.5 ng per well on an 18% Criterion TGX precast gel (Bio-
Rad) and separated by SDS-PAGE at 190 V for 1 h. Proteins were transferred to PVDF
membranes (BioRad) in a Transfer Blot semi-dry transfer cell (BioRad) at 20 V for 1 h.
Membranes were blocked in 5% non-fat milk in Tris buffered saline plus Tween-20
(TBST) for 1 h at room temperature and washed with 3 x 10 mL TBST (10 min). Mem-
branes were then incubate overnight at 4 °C with primary antibodies for either α-
synuclein p-S87 (Ser 87-R, Santa Cruz Biotech., 1:200 dilution) or α-synuclein p-S129
(P-syn/81A, Covance, 1:1000 dilution) in 5% milk in TBST. For a loading control, an
anti-α-synuclein antibody (syn 211, Invitrogen, 1:5000 dilution) was used. Membranes
were washed with 3 x 10 mL TBST (10 min) and then incubated with the appropriate
secondary antibodies at room temperature for 1 h followed by another 3 x 10 mL TBST
wash (10 min). Finally, membranes were incubated with ECL (BioRad) for 3 min and
imaged using a ChemiDoc XRS imaging system (BioRad).
137
5.5.19 Analysis of aggregates by SDS-PAGE and/or Western blotting
Recombinant α-synuclein (25 µM) alone or in the presence of α-synuclein(gT72)
(25 µM) underwent aggregation for 7 days (shaking at 1000 rpm at 37 °C). Reactions
were then centrifuged (20,000 x g, 4 °C, 1 h) and the supernatants were removed and ly-
ophilized. Both the pellets and the lyophilized supernatants were sonicated in freshly pre-
pared 8 M urea, 20 mM HEPES, pH 8.0, followed by boiling for 10 min with 4X SDS
loading buffer. Samples were loaded onto an 18% gel (5 ng/well, BioRad), separated by
SDS-PAGE as described above, and stained with Coomassie brilliant blue 2 hrs, followed
by destaining in 4:5:1 acetic acid, methanol, water overnight. The same samples were
also separated by SDS-PAGE before being transferred to PVDF membrane (Bio-Rad)
using standard Western blotting procedures. Anti-O-GlcNAc blots were blocked in TBST
containing 5% bovine serum albumin (BSA) for 1 h at rt and incubated overnight at 4℃,
then incubated with anti-O-GlcNAc (RL2; Sigma, #MA1-072) at 1:1000 dilution in
blocking buffer for 24 h at 4 ℃. The blots were then washed three times in TBST for 10
min and incubated with the HRP-conjugated anti-mouse (Jackson ImmunoResearch,
#715-035-150) for 1 h in the appropriate blocking buffer at RT. After being washed three
more times with TBST for 10 min, the blots were developed using ECL reagents (Bio-
Rad) and the ChemiDoc XRS+ molecular imager (BioRad).
138
5.5.20 Aggregation reactions with pre-formed fibers
Lyophilized wild-type α-synuclein was resuspended in reaction buffer (10 mM
phosphate, 0.05% sodium azide, pH7.4) to a concentration of 50 µM. After 15 min of
bath sonication, the resuspended protein was incubated at 37 ℃ under continuous shak-
ing (1000 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 sepa-
rated 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 α-synuclein(gT72) monomer were added to give a final concen-
tration of 25 µM fibers and 25 µM monomer. The resulting mixtures were incubated un-
der continuous shaking (1000 rpm) at 37 ℃ in the Eppendorf thermomixer for indicated
times. At each time point, Thioflavin T fluorescence was measured as described above.
139
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Blanco-Canosa, J.B., and Dawson, P.E. (2008). An Efficient Fmoc-SPPS Approach for
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Abstract (if available)

Abstract Parkinson’s Disease (PD) is the second most prevalent neurodegenerative disease that is characterized by resting muscle tremors and slowing or stiffness of movement. These symptoms are the result of the progressive loss of dopamine producing neurons in a region of the brain called the Substantia nigra pars compacta (SNpc). The SNpc supplies the neurotransmitter dopamine (DA) to the striatum, and loss of DA is the cause of these motor deficits. Progressive neuron loss in PD patients’ brains is also accompanied by the accumulation of intracellular deposits called Lewy bodies (LB). These deposits are a pathological hallmark of the disease and are strongly linked to neuron death. LBs are proteinaceous, and consist mainly of aggregated protein fibers composed of the protein α-synuclein (αSyn). In addition to being the most abundant protein in LBs, αSyn is also genetically linked to PD, with the familial forms of the disease being linked to several mutations to the αSyn gene. Additional copies of the αSyn gene also leads to early onset of PD. While the driving force behind onset of sporadic PD (i.e. non-inherited), which comprises ~95% of cases, is still a mystery, the link between αSyn, it’s aggregated form in LBs, and progressive neuron loss in PD is common factor in all cases. ❧ αSyn is an unusual protein in that it does not fold into a regular structure in solution. It instead exists as a random coil with only weak and transient globular structure. αSyn has a high affinity for cellular lipid membranes and it does adopt a regular, extended, α-helical structure when it associates with membranes or vesicles. This helical structure is strongly linked to the physiological role αSyn plays in neurons where it is involved in neurotransmitter release and maintenance of synaptic vesicles. In the disease-associated state, αSyn monomers aggregate together to form a variety of higher molecular weight structures, including soluble oligomers and long protein fibers. In these aggregates αSyn adopts as fold that is very high in β-sheet content and is very stable to unfolding and degradation. These aggregates have also been shown to be cytotoxic to neuronal cells in a variety of contexts, further strengthening the link between αSyn and PD pathology. ❧ Currently, treatments for PD are limited, with the major treatment being DA replacement therapy to supplement the loss of naturally produced DA. Transplant of new DA producing neurons has also shown some promise, however both of these therapies can only treat the symptoms of the disease, not the underlying problem of αSyn aggregation. The aggregation of proteins such as αSyn is very difficult to target using traditional drug design paradigms, which usually target the function of enzymes or other specific cellular components. Protein aggregates are driven by protein-protein interactions between monomers, and the driving forces for these interactions are the same that drive the correct folding of all other cellular proteins. Thus selectively targeting the protein-protein interactions responsible for disease-associated aggregation is not yet a viable approach. One important aspect of αSyn biology that may make it a better therapeutic target is it’s high level of post-translational modification. Post-translational modifications (PTMs) are chemical modifications that occur on a protein after it has been synthesized, and they take a variety of forms. The key is that most PTMs are installed by enzymes. If a PTM could be identified that effected the aggregation behavior of αSyn, the relevant enzymes for controlling that modification could be targets of study for new therapies. ❧ One PTM of αSyn that has, to date, received little attention from the field is the modification by the O-linked monosaccharide N-acetyl-Glucosamine (O-GlcNAc). This modification has been identified on several sites of αSyn but it’s cellular consequences are still a complete mystery. This particular modification is of interest as it is tightly linked to cell metabolism, which is greatly altered in PD brains, and it has generally shown to help solubilize target substrates and is protective against a variety of cell stressors such as heat shock and oxidative damage. Thus, the focus of the research presented herein was to uncover the biochemical consequence of αSyn O-GlcNAc modification, especially on it’s aggregation behavior. This aim was accomplished through a combination of biochemistry, cell biology, and synthetic protein chemistry. As the results will indicate, modification of αSyn by O-GlcNAc was shown to strongly inhibit both aggregate formation and toxicity in neurons. This result shows great promise for future investigation and suggests O-GlcNAc modification, and the enzymes that regulate it, are possible targets in the search for better PD therapeutics.

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Asset Metadata

Creator Marotta, Nicholas P. (author)

Core Title Uncovering the protective role of protein glycosylation in Parkinson's disease utilizing protein semi-synthesis

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 11/20/2015

Defense Date 10/22/2015

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

Tag alpha-synuclein,glycosylation,native chemical ligation,OAI-PMH Harvest,O-GlcNAc,Parkinson's disease,post-translational modification,semi-synthesis

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

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

Creator Email marotta.np@gmail.com,nmarotta@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-200717

Unique identifier UC11276466

Identifier etd-MarottaNic-4052.pdf (filename),usctheses-c40-200717 (legacy record id)

Legacy Identifier etd-MarottaNic-4052.pdf

Dmrecord 200717

Document Type Dissertation

Format application/pdf (imt)

Rights Marotta, Nicholas P.

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...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA