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University of Southern California Dissertations and Theses
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Interaction of monoclonal antibodies MW1 and PHP1 with huntingtin exon1 protein
(USC Thesis Other)
Interaction of monoclonal antibodies MW1 and PHP1 with huntingtin exon1 protein
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1
Interaction of Monoclonal Antibodies MW1 and
PHP1 with Huntingtin Exon1 protein
A Thesis Presented to the Faculty of the
University of Southern California - Graduate School
In Partial Fulfillment of the Requirements for the Degree of
Master of Science
Biochemistry and Molecular Medicine
By: Rajashree Venkatraman
August 2019
2
Acknowledgements
These two years have flown past in the blink of an eye. I would like to extend my heartfelt gratitude to Dr.
Ralf Langen for being an amazing mentor. I sincerely appreciate your support, guidance and
encouragement. It has been an absolute pleasure to work with you and to be a part of your lab. I would
also like to thank Dr. José Bravo for giving in his valuable time to teach me all the techniques and
procedures for doing my experiments and for always being there to help me troubleshoot them. A big
shout out to my lab members for all their support and for making me feel at home. You guys have taught
me not just lab lessons, but also life lessons that I will always value. I would also like to extend my
gratitude to my other committee members, Dr. Ansgar Siemer and Dr. Tobias Ulmer for their valuable
insights and guidance. A big thank you to the Siemer lab and Ulmer lab members for the all the fun times
and the crazy lunch conversations!! My sincere thanks to my academic advisor Monica Pan, for all her
assistance. Lastly, I would like to thank my family and friends for their endless love, for giving me the
courage to pursue my ambitions and for being my support system. Love you ma and papa, I am what I am
because of you. Can’t wait to see you!!
3
Table of Contents
• Abstract................................................................................................................................ 5
• Introduction ......................................................................................................................... 6
• What is Huntington’s Disease? ................................................................................................ 6
• Huntingtin protein…………………………………………………………………………………………………………………..6
• Protein Aggregation in Huntington's Disease……………………………………………………………………………7
• Antibodies as tools to recognize novel conformation of mutant huntingtin ................................ 7
• PHP1……………………………………………………………………………………………………………………………………….8
• MW1………………………………………………………………………………………………………………………………………8
• EPR Spectroscopy……………………………………………………………………………………………………………………9
• Size Exclusion Chromatography……………………………………………………………………………………………..10
• Results…………………………………………………………………………………………………………………………………11
• Purification of PHP1…………………………………………………………………………………………………………..….11
• Dot Blots……………………………………………………………………………………………………………………………….11
• EPR spectroscopy data…………………………………………………………………………………………………………..12
• DEER Data……………………………………………………………………………………………………………………………..14
• Analysis by solution NMR………………………………………………………………………………………………………15
• MW1…………………………………………………………………………………………………………………………………….16
• Dot Blots……………………………………………………………………………………………………………………………….16
• EPR spectroscopy data…………………………………………………………………………………………………………..16
• DEER Data……………………………………………………………………………………………………………………………..20
• Size Exclusion Chromatography……………………………………………………………………………………………..20
• Discussion……………………………………………………………………………………………………………………………22
• PHP1…………………………………………………………………………………………………………………………………….22
• MW1…………………………………………………………………………………………………………………………………….23
4
• Materials and Method……………………………………………………………………………………………………….27
• Transformation……………………………………………………………………………………………………………………..27
• Protein expression…………………………………………………………………………………………………………………27
• Protein purification……………………………………………………………………………………………………………….27
• PHP1 purification…………………………………………………………………………………………………………………..28
• Dot blots……………………………………………………………………………………………………………………………….28
• Preparation of EPR samples……………………………………………………………………………………………………28
• Size Exclusion Chromatography……………………………………………………………………………………………..29
• Future Directions……………………………………………………………………………………………………………….30
• References………………………………………………………………………………………………………………………….31
5
Abstract
Huntington’s disease is an autosomal dominant neurodegenerative disorder caused by a polyglutamine
expansion in the N terminal region of huntingtin (htt). The mutant protein starts to aggregate and adopt
unique conformations. It is important to know the composition of these unique conformations in order to
develop therapeutics to block the disease progression. In this process, antibodies are being used as a tool
to identify and study these unique conformations. Of the various antibodies used for studying huntingtin,
we have decided to focus on two, MW1 and PHP1. MW1 binds strongly to expanded polyQ region in
monomers whereas PHP1 binds strongly to the PRD region of huntingtin fibrils. The exact binding
stoichiometry of binding is still unknown. My work focuses on mapping on the binding regions of these
antibodies and to study the binding stoichiometry of the antibodies with the protein by using tools like
EPR spectroscopy and size exclusion chromatography.
6
Introduction
What is Huntington’s Disease?
Abnormal poly-glutamine repeats are known to be the cause of neurodegenerative disorders like spinal
and bulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy, and six spinocerebellar
ataxias (Stoyas & La Spada, 2018) that ultimately lead to neuronal death in different regions of the brain
(Todd & Lim, 2013). Of these, Huntington’s disease (HD) is a heritable, autosomal dominant
neurodegenerative disorder (Kim, Chelliah, Kim, Otwinowski, & Bezprozvanny, 2009) with a mean onset
age of 40 years (Ross & Tabrizi, 2010). HD is considered to be a part of a growing group of diseases called
“conformational diseases” which include Alzheimer’s disease, Parkinson’s disease, the prion
encephalopathies etc. (Legleiter et al., 2009).
The genetic defect in HD is caused due to a CAG repeat expansion in the exon 1 of the htt gene, that
translates into an abnormal polyglutamine (polyQ) repeat(>36) in the huntingtin protein (Ko et al., 2018).
In the non-HD population, this CAG sequence is repeated 9 to 35 times(Kremer et al., 1994). The age of
onset of the disease is inversely proportional to the length of the CAG expansion. There are cases wherein
juvenile onset is associated with HTT carrying about 75 or more CAG repeats (Lee et al., 2012). The disease
is characterized by motor, cognitive, and psychiatric symptoms (Khoshnan, Ko, & Patterson, 2002). The
neuropathology of HD is characterized by the dysfunction and death of specific neurons within the brain
(Ross & Tabrizi, 2010), especially in the striatum (Saudou & Humbert, 2016). Cellular studies have
indicated that htt is mostly localized in aggregates in the nucleus (Khoshnan et al., 2002). These aggregates
contain fragments of mHTT protein, including those composed of only the first exon of Htt, the region
where the polyglutamine repeat is located (Darrow et al., 2015). These aggregates are linked to impaired
cell signaling, neuroinflammation and neurodegeneration (Ko et al., 2018).
Huntingtin protein
The wild type htt protein is 348kDa and is encoded by the htt gene present on the short arm of
chromosome 4. It is ubiquitously expressed in most human tissues (Saudou & Humbert, 2016) but is found
in high levels in the brain (Darrow et al., 2015). Some of the functions associated with the wild type htt
include controlling vesicular trafficking, regulating ciliogenesis, coordination in cell division, mediating
endocytosis, vesicle recycling, endosomal trafficking and many more (Saudou & Humbert, 2016).
The htt protein consists of 67 exons of which exon 1 is widely studied as it contains the CAG mutation.
Exon 1 consists of the polyQ repeat preceded by the N17 region and followed by PRD region (Saudou &
Humbert, 2016). It was seen that an overexpression of Httex1 with expanded polyQ repeats alone is
sufficient to cause the disease in mice (Mangiarini et al., 1996). Recent studies suggest that the N17 and
7
PRD region greatly influence mHTT aggregation and biology (Shen et al., 2016). The N17 domain harbors
multiple sites for several post-translational modifications, is known to interact with chaperones (Shen et
al., 2016) which may alter the folding of mHTTx1. The PRD of mHTT is also described as the most dynamic
epitope in fibrils which may contribute to misfolding and formation of new conformations. The PRD is also
known to interact with various cellular proteins, which may control the formation of various assemblies
in vivo (Ko et al., 2018).
Aggregation of huntingtin in HD
The aggregation of mutant htt leads to the formation of intermediate prefibrillar oligomers (Sontag et al.,
2012) that ultimately give rise to fibrils and fibrillar oligomers which are the end products of the misfolded
protein (Isas, Langen, & Siemer, 2015) and (Sontag et al., 2012).There are various mechanisms proposed
for the aggregation of mHTT. In vitro biophysical studies suggest that Httex1 aggregation, although polyQ
dependent, occurs through a two-step mechanism where the N17 domain forms homo-oligomeric species
which then undergoes conformational changes to form fibrils that grow through monomer addition. Other
studies suggest that the N17 interaction with the polyQ domain influences the structure of Httex1 in ways
that favor the population of aggregation-prone conformational intermediates (Deguire et al., 2018).
Figure.1. Genetic mutation that causes Huntington’s Disease. The CAG trinucleotide repeats when exceed above
36 is responsible for causing the disease. Reference: https://hdsa.org/what-is-hd
8
Antibodies as tools to recognize the novel conformations of mutant httex1
Although there has been substantial progress in detecting early signs and diagnosing motor symptom
manifest HD, there is a dire need to develop HD-specific symptomatic treatment options and effective
intervention to slow progression or prevent onset of disease (Deb, Frank, & Testa, 2017). Identification of
new targets, strategies for drug discovery, and therapeutic approaches are now gaining momentum in
clinical management of HD (Ross & Tabrizi, 2010). An interesting approach to comprehend the disease
pathogenesis would be to identify molecules that block the toxic effects of huntingtin or the consequences
of it binding to other proteins. These molecules might also serve as potential therapeutics. (Khoshnan et
al., 2002).
Biophysical approaches have provided some valuable insights into the structures of the various oligomeric
species, but their exact structures still remain unclear. In this pursuit, antibodies are being examined as a
powerful tool for detecting the novel conformations or oligomeric states of aggregated protein (Legleiter
et al., 2009). Antibodies that can recognize specific conformations such as “prefibrillar oligomeric forms”
and “fibrillar forms” of amyloids, independent of its primary protein sequence are important tools to
detect the presence of protein conformers (Sontag et al., 2012).
Figure.2. Toxic species of Huntingtin. A monomer having a Q length more than 36 aggregates to form oligomers,
protofibrils and fibrils. These species are known to be associated with the disease pathology.
9
Previous research has shown that antibodies recognizing amyloid fibrils usually do not react with the
natively folded protein from which they are formed indicating that these antibodies recognize a novel
conformational epitope (Sontag et al., 2012). Several antibodies display conformational dependent
interactions with amyloids, aggregation intermediates or natively folded precursor proteins (Legleiter et
al., 2009). This suggests that amyloid oligomers and soluble fibrils share a common structure that is
distinct from other forms of the protein like monomers (Sontag et al., 2012). Antibodies have been
developed specifically for oligomers and fibrils which display common structural motifs associated with
amyloid diseases, independent of the peptide sequence from which they are formed, strongly suggesting
a common mechanism of aggregation and toxicity (Legleiter et al., 2009).
PHP1
PHP1 is a monoclonal antibody that displays high reactivity to unbundled huntingtin fibrils and
comparatively less binding to monomers and bundled fibrils. Epitope mapping has shown that PHP1 binds
to the regions between the proline rich domain of htt (Ko et al., 2018). Cell culture studies also indicate
that PHP1 binds to high molecular weight fibrils of HD mice. Electron microscopy studies have revealed
that PHP1 binds to mHTT assemblies associated with myelin sheath. An EPR experiment showed that
when PHP1 was included in fibril assembly assays, it significantly attenuated the reduction in EPR
amplitude in a concentration dependent manner (Ko et al., 2018).
MW1
MW1 is another monoclonal antibody that strongly binds to the expanded polyQ region compared to wild
type huntingtin. There were two hypotheses proposed that explain the interaction of the antibody with
the protein. According to the ‘toxic threshold model’, the expanded polyQ region undergoes a
conformational transition resulting in a unique conformation that can be recognized by an antibody (Nagai
et al., 2007). Evidence for this model was provided by an anti poly Q antibody 3B5H10 that recognized a
single epitope with a distinct pathologic conformation of soluble expanded polyQ (Miller et al., 2011).
In contrast, another model that was proposed was the ‘linear lattice model’. According to this model, the
expanded polyQ sites contains more epitopes compared with the normal polyQ, resulting in higher avidity
for bivalent proteins like antibodies (Bennett et al., 2002). Several experiments like gel filtration
chromatography, dynamic light scattering and SAXS data have shown that MW1 interacts with htt in a
linear lattice mode of recognition (Owens, New, West, Bjorkman, 2015). The crystal structure analysis of
variable region of MW1 with the polyQ region also suggests that scFv-MW1 binds to an extend coil-like
structure of expanded polyQ that is in line with the linear lattice model, where expanded polyQ increases
the number of binding sites within the polyQ tract (Li et al., 2007).
10
But the questions that remain unanswered are, where exactly in the polyQ region does MW1 bind and,
most importantly, what is the binding stoichiometry of MW1-Httex1 complex and their molecular weights.
Knowing the binding stoichiometry will help in understanding how the antibody interacts with the protein.
Similarly, with PHP1, it is still unclear as to where the antibody binds within the PRD and its binding affinity
with various mHttex1 oligomer species. To answer these questions, we have used tools like EPR
spectroscopy and size exclusion chromatography.
EPR Spectroscopy
To study the interaction of antibodies with the htt protein we used a technique called ‘site directed spin
labelling’ coupled with EPR spectroscopy. This technique allows monitoring the backbone structure of
proteins. For SDSL, a cysteine mutation is introduced in the desired position in the protein’s primary
sequence. This is followed by the covalent attachment of a nitroxide spin label to the cysteine residue.
This side chain contains a free electron that can be detected in a magnetic field. EPR spectroscopy can be
used to study the mobility of the spin label (Margittai & Langen, 2008). The overall mobility of the spin
label is determined three factors, namely the (1) motion of the label in relation to the peptide backbone,
(2) variations of the α-carbon backbone, and (3) rotational motion of the entire protein or peptide (Klug
& Feix, 2008). Attachment of the label to an unfolded site in the protein results in an EPR spectrum with
three sharp and narrowly spaced lines with a large amplitude. Attachment of the spin label to an
immobilized site on the contrary, results in broad lines, large separation of outer lines, and a decrease in
amplitude (Margittai & Langen, 2008).
Size Exclusion Chromatography
Size Exclusion Chromatography is a technique used for separating proteins based on their molecular
weight. Separation of molecules is achieved using a porous matrix to which the molecules have different
degrees of access (Hagel, 2001). The exclusion limit which is defined as the smallest-sized protein molecule
that is excluded from the pores of the matrix—is the most important factor while selecting a matrix for
an experiment. The matrix which usually consists of Sephacryl or Superdex resin, is packed into a
chromatographic column, the sample mixture applied, and the separation achieved by passing an aqueous
buffer (the mobile phase) through the column. Large analytes that are excluded from the pores will pass
through the spaces between the particles and will appear first in the eluate. Smaller analytes will be
distributed between the mobile phase inside and outside the particles and will therefore pass through the
column at a slower rate, hence appearing last in the eluate. The protein zones eluted are detected by a
UV monitor connected to the system and fractions are collected for future use (Hagel, 2001).
11
Results
PHP1
I would like to acknowledge Dr. Ali Khoshnan from Caltech for providing the PHP1 ascites.
Purification of PHP1
PHP1 was purified from mouse ascites using protein A affinity chromatography. Protein A is produced
by Staphylococcus aureus and has a very high affinity for Igg immunoglobulins. On performing SDS PAGE
to check for the purity, a clean band as observed at approximately 150 KDa for PHP1.
Dot Blots
Figure.4. Dot Blots with PHP1. For the dot blots, 1.5µl of the protein was blotted onto the nitrocellulose
membrane and incubated with PHP1 at a 1:5000 dilution. A secondary antibody conjugated with HRP
at a 1:10,000 dilution was used for visualization.
Figure 3. SDS PAGE. 10µl of purified PHP1 was run along
with MW1 and 2B7 as controls on an SDS PAGE gel at 200V
for 45 minutes.
12
To check for the reactivity of purified PHP1, dot blots were performed with the recombinant Httex1. The
dot blots revealed that PHP1 bound to Httex1 with a strong preference to fibrils compared to monomers.
PHP1 also bound to htt tetramers but with less reactivity compared to monomers. PHP1 did not bind to
htt tetramers which lacked the PRD region. PHP1 did not bind to α-synuclein monomers and fibrils
indicating its specificity to the huntingtin protein. PHP1 bound very strongly to the α-synuclein-PRD fusion
protein fibrils compared to α-synuclein-PRD monomer protein. This indicates that PHP1 is specific to the
PRD region.
EPR spectroscopy
From the dot blots it was concluded that PHP1 binds specifically to the PRD region of Httex1. To determine
the binding pocket of PHP1 within the PRD region, EPR spectroscopy was performed with different spin
labelled derivates of Httex1. EPR spectra was obtained by incubating 10µM of Httex1 with increasing
amounts of PHP1. For figure 5, different spin labelled derivates of Httex1 were incubated with 3-fold
excess of PHP1. Negligible spectral changes were observed in the N17 and polyQ regions, but significant
changes were observed in the PRD regions. In the absence of PHP1, the EPR spectra for the various httex1
derivatives had a higher spectral amplitude and sharp spectral lines (black spectra in Figure 5), indicating
Figure.5. Effect of PHP1 binding on EPR spectra of spin labeled Httex1(Q46) derivatives. For the EPR experiments, 10µM of Httex1 and
25 µM of PHP1 was used. The spectra in black is for Httex1 in the absence of PHP1, and the spectra in red is in the presence of PHP1. The
scan width is 100Gauss.
13
that these sites are highly mobile. Whereas, in the presence of PHP1, there was a decrease in spectral
amplitude and broadening of the sharp spectral lines (red spectra in Figure 5) suggesting immobilization
by the antibody leading to a decrease in mobility of the protein. These spectral changes were observed
for certain httex1 derivatives within the PRD region. Thus, it can be concluded that PHP1 immobilizes
Httex1 only at specific positions in the PRD regions. These regions lie in the PRD region between the two
poly proline regions. The residues include positions 76, 81, 86, with strongest immobilization around
positions 81 and 86. There were some spectral changes observed in the N17 and polyQ region in terms of
decrease in spectral amplitude.
To check for the binding affinity of PHP1, the spin labelled derivates of Httex1 were incubated with
increasing amounts of the antibody. Binding saturation was observed around 2-fold amounts of PHP1.
The graph below was plotted by dividing the maximum amplitude value of the EPR spectra obtained for
Httex1- PHP1 complex over Httex1 in the absence of PHP1. The graph below shows that there is a decrease
in the amplitude ratio for positions 76, 81, 86 which is consistent with the spectral data above.
In conclusion, PHP1 strongly immobilizes Httex1 between positions 81 and 86 as shown in the graph
below.
Figure.6. EPR amplitude change of spin Httex1 derivatives in response to PHP1 addition. Httex1(Q46) spin labeled at the
indicated positions were incubated at varying amounts of PHP1 (X-axis denotes the Httex1:PHP1 molar ratio). The amplitudes
are normalized to those of the corresponding Httex1(Q46) derivatives in the absence of antibody, and they are set to 1.
0
0.2
0.4
0.6
0.8
1
1.2
0 0.5 1 1.5 2 2.5 3
Amplitude Ratio
Httex1:PHP1
5 11 35 48 66 71 76 81 86 91 96 101
14
The graph above was plotted by dividing the average maximum spectral amplitude obtained by incubating
3-fold excess of PHP1 with httex1 monomer derivatives over the average maximum spectral amplitude
obtained for httex1 monomer derivatives in the absence of PHP1. The graph shows maximum drop in
amplitude ratio for positions 81 and 86. Thus, based on the dot blots results and the spectral changes
observed for httex1 monomer derivatives 81 and 86, it can be concluded that PHP1 binds strongly to fibrils
and shows maximum immobilization at positions 81 and 86.
DEER Data
To check for structural changes in the PRD region, two Httex1 samples spin labelled at positions 81 and
88, and 75 and 91 were incubated with 3-fold excess of PHP1 and compared with monomers in the
absence of PHP1. No significant changes in distance was observed for positions 81-88, but there was a
significant decrease in the distance for positions 75-91 in the presence of PHP1.
Labeling positions Control (monomer, in
the absence of PHP1)
With PHP1
Q46 81-88 20 Å 27 Å
Q46 75-91 40 Å 22 Å
Figure.7. Normalized EPR amplitude of different Httex1 derivatives spin labeled at the positions
indicated on the x-axis. The amplitude is again normalized to that of the Httex1 derivatives in the
absence of antibody. The data were obtained at a PHP1:Httex1 ratio of 3:1.
15
Analysis by solution NMR
The binding of PHP1 to Httex1(Q7) was monitored by solution NMR at the level of individual backbone
1
H
N
/
15
N nuclei. To maintain observable solution NMR signals, the interaction was monitored at a sub
stoichiometric Httex1(Q7):PHP1 ratio of 9:1. The strong and selective signal reduction mainly in the N17
region indicates its interaction with PHP1. Largest signal reductions were seen in the center of the proline-
rich domain. The C-terminal region of this domain retained some flexibility (higher I/I 0 ratios).
Figure.8. Solution NMR spectroscopy of the Httex1(Q7)-PHP1 antibody interaction. The TROSY-
type HSQC spectrum of 100 M
2
H/
13
C/
15
N-labeled Httex1(Q7) was recorded in the presence of 0
and 10 mM PHP1 in 20 mM NaH 2PO 4/Na 2HPO 4 (pH 7.4), 150 mM NaCl at 10 °C and 700 MHz.
c
16
MW1
I would like to acknowledge Dr. José Bravo for providing the all the graphs and figures for this section. I
have expressed, purified and performed EPR experiments for 8 httex1 derivatives and tested them with
MW1. I have also assisted Dr. Bravo in making samples for the DEER experiment.
Dot blots
To check for the reactivity of MW1, dot blots were performed with monomers and various aggregated
species of Httex1. The dot blots showed that MW1 binds strongly to monomers with little or negligible
binding to fibrils. There is some binding observed for protofibrils.
EPR Data
EPR spectroscopy was performed to check the binding affinity of MW1 with the polyQ region of Httex1.
Different spin labelled derivatives of two monomeric proteins having different Q lengths, Q25 and Q46,
were tested with MW1. EPR spectra was obtained by incubating 20µM of Httex1 with increasing amounts
of the antibody. For figure 8, different spin labelled derivates of Q25 Httex1 were incubated with 3-fold
excess of MW1. No significant spectral changes were observed in the N17 and PRD, but significant spectral
changes were observed in the polyQ region. In the absence of MW1, the EPR spectra for the various httex1
derivatives have a higher spectral amplitude and sharp spectral lines (black spectra in Figure 10) indicating
that these sites are highly mobile. Whereas, in the presence of MW1, there is a decrease in spectral
amplitude and broadening of the sharp spectral lines (orange spectra in Figure 10) suggesting
immobilization by the antibody leading to a decrease in mobility of the protein. These spectral changes
Figure.9. Dot Blots with MW1. For the dot blots, 1.5µl of the protein was
blotted onto the membrane and incubated with MW1 at a 1:5000 dilution.
A secondary antibody conjugated with HRP at a 1:10,000 dilution was used
for visualization. Acknowledgements: Dr. José Bravo.
17
were observed for certain httex1 derivatives within the polyQ region. Thus, from EPR spectroscopy it can
be concluded that the binding pocket for MW1 lies between residues 25 and 35. To check for the binding
affinity of MW1, the spin labelled derivates of Q25 Httex1 were incubated with increasing amounts of
MW1. Binding saturation is observed around 2-fold amounts of MW1. The graph shows that there is a
decrease in the amplitude ratio for positions 25,35 which is consistent with the spectral data.
Similarly, for Q46, different spin labelled derivatives of Httex1 were incubated with increasing amounts of
MW1. EPR spectra was obtained by incubating 20µM of Httex1 with increasing amounts of MW1. For
Figure 11, different spin labelled derivates of Httex1 were incubated with 3-fold excess of MW1. As with
Q25, no significant spectral changes were observed in the N17 and PRD region, but significant spectral
changes were observed in the polyQ region. In the absence of MW1, the EPR spectra for the various httex1
derivatives have a higher spectral amplitude and sharp spectral lines (black spectra in Figure 11) indicating
that these sites are highly mobile. Whereas, in the presence of MW1, there is a decrease in spectral
amplitude and broadening of the sharp spectral lines (orange spectra in Figure 11) immobilization by the
antibody leading to a decrease in mobility of the protein. These spectral changes were observed for
certain httex1 derivatives within the polyQ region. Thus, from EPR spectroscopy it can be concluded that
the binding pocket for MW1 lies between residues 25 and 60. To check for the binding affinity of MW1,
Figure.10. Effect of MW1 binding on EPR spectra of spin labeled Httex1(Q25) derivatives. For the EPR experiments, 20µM
of Httex1 and 60 µM of MW1 was used. The spectra in black is for Httex1 in the absence of MW1, and the spectra in blue,
orange and green are in the presence of PHP1. The scan width is 100Gauss. Acknowledgements: Dr. Jose Bravo.
18
the spin labelled derivates of Q46 Httex1 were incubated with increasing amounts of MW1. Binding
saturation is observed around 2-fold amounts of MW1. The graph shows that there is a decrease in the
amplitude ratio between positions 25 and 60 which is consistent with the spectral data.
For figure 12 and 13, different spin labelled derivates of Q25 and Q46 Httex1 were incubated with 3-fold
excess of MW1. The graphs in these figures were plotted by dividing the average maximum spectral
amplitude obtained by incubating 3-fold excess of MW1 with httex1 monomer derivatives over the
average maximum spectral amplitude obtained for httex1 monomer derivatives in the absence of MW1.
There is a significant drop in the spectral amplitude ratio between positions 25 and 60 for Q46 httex1
derivatives and between positions 25 and 35 for Q25 httex1 derivatives. Thus, it can be concluded that
MW1 immobilizes Httex1 in the polyQ region, between the residues 25 and 35 for Q25 and positions 25
and 60 for Q46.
Figure.11. Effect of MW1 binding on EPR spectra of spin labeled Httex1(Q46) derivatives. For the EPR experiments, 20µM of
Httex1 and 60 µM of MW1 was used. The spectra in black is for Httex1 in the absence of MW1, and the spectra in blue, orange
and green are in the presence of PHP1. The scan width is 100Gauss. Acknowledgements: Dr. Jose Bravo.
19
Figure.12. Normalized EPR amplitude of different Httex1 derivatives spin labeled at the
positions indicated on the x-axis. The amplitude is again normalized to that of the Httex1
derivatives in the absence of antibody. The data were obtained at a MW1:Httex1 ratio of
3:1. Acknowledgements: Dr. José Bravo.
Figure.13. Normalized EPR amplitude of different Q25 Httex1 derivatives spin labeled at the
positions indicated on the x-axis. The amplitude is again normalized to that of the Httex1
derivatives in the absence of antibody. The data were obtained at a MW1:Httex1 ratio of 3:1.
Acknowledgements: Dr. José Bravo.
10 20 30 40 50 60
0.0
0.2
0.4
0.6
0.8
1.0
Normalized Amplitude
Residue Number Httex1-Q25
20
DEER Data
For DEER spectroscopy, monomeric Httex1 with a Q length of 46 which was spin labelled at various
positions was incubated with 3-fold excess of MW1 and compared with monomeric Httex1 in the absence
of the antibody. Significant changes in distance were observed for positions in the table below.
Labeling pair
Httex1(Q46)
Distance in
Httex1(Q46)
Distance with
MW1
Fully extended*
25R1/35R1 ~20 Å (broad
distribution)
34 Å (narrow
distribution)
35 Å
(10 amino acids
x3.5Å/amino
acid)
35R1/45R1 ~23 Å (broad
distribution)
34 Å (narrow
distribution)
35 Å
25R1/45R1 ~31 Å (broad
distribution)
> 50 Å 70 Å
30R1/40R1 ~20 Å (broad
distribution)
34Å (narrow
distribution)
35Å
30R1R1/50R1 ~32 Å (broad
distribution)
~59 Å 70 Å
40R1/50R1 20 Å (broad
distribution)
34 Å 35 Å
Gel filtration data
Gel filtration experiments using a Superdex 200 (GE Healthcare) column were used to obtain the size of
the various Httex1-MW complexes. Toward this end, we incubated 10 M Httex1 of different Q-lengths
with either 0.5 or 1 equivalents of MW1. The elution profiles were then expected molecular weight. The
Trx-Httex1 fusion protein used for these studies eluted around 48 kDa, consistent with its apparent larger
size on SDS gels. The most pronounced size increases were seen for MW1 in the presence of Httex1(Q46).
21
Here we found peaks that ranged from 650 to 2900 kDa. Interestingly, the larger peaks, especially the
peak around 1300 kDa increased in size when more MW1 was added (top trace). These peaks are
surprisingly large and inconsistent with the simple notion that a single Httex1 in isolation binds to multiple
MW1.
Figure.14. Gel Filtration Chromatography for mixtures of MW1 and Httex1 with different Q-lengths. The complexes
were incubated at 1:1 for Httex1Q16, 0.5:1 and 1:1 for both constructs Httex1Q25 and Httex1Q46. The concentration
for Httex1 was 10uM for all experiments. The vertical lines indicate the approximate molecular weights estimated
based on molecular weight standards. Acknowledgements: Dr. José Bravo.
22
Discussion
PHP1
The N17 and PRD region play an import role in htt aggregation. Conformational antibodies have provided
a useful approach to distinguish specific conformations of amyloidogenic proteins, but these
conformations have not yet been defined (Shen et al., 2016). Recent work has shown that PHP1 react with
the peptide sequence QAQPLLPQP within the PRD region of htt (Ko et al., 2018). The experiments
performed above supplements this data. EPR spectroscopy data shows that position 81, a proline residue
and position 86, a lysine residue show maximum immobilization when incubated with PHP1. Httex1
derivatives 35, 48 and 71 show a higher EPR spectra in the presence of PHP1 although a decrease in
amplitude was observed. This was observed after normalization of the data points. This could indicate a
possible manual error during sample preparation.
A bottle brush model for the structure of the huntingtin fibrils was proposed by Isas et. al, 2015. According
to this model, the N17 and the PRD region form the core of the fibrils and the PRD region sticks out from
the core like bristles. Previous data also indicates that the PRD region has the same structure regardless
of whether it is a fibril or a monomer (Pandey et al., 2017). Therefore, the strong binding of PHP1 to fibrils
might be due to entropic reasons rather than structural. PHP1 as two arms for binding, and hence binding
to fibrils would be more favorable compared to monomers, as illustrated in the figure below.
Figure.15. Working model for binding of PHP1. To bind to monomers, PHP1 needs to encounter the PRD region of two monomers,
while in the case of fibrils, binding to PRD region is stronger because of their proximity in the fibrils. Reference: CHDI Research
Agreement.
23
Dot blots with the -synuclein-PRD fusion protein also shows that the bristle density and not a unique
conformation in the PRD region is causing the strong binding. No binding was obtained for -synuclein in
monomeric or fibrillar form. In case of the -synuclein-PRD fusion, however, strong binding could be seen
for fibrils, but not monomers. The signal intensity for the Htt-PRD tetramers is less than that of the
monomers. A higher signal intensity was expected for the Htt-PRD tetramer. This could be because the
tetramers did not bind completely onto the nitrocellulose membrane.
The DEER data indicates that there is a decrease in distance for position 75-91. This suggests that when
PHP1 binds to the PRD region it causes a conformational kink in the structure which causes the PRD region
to bend backwards as illustrated in the figure below.
NMR data indicates that the N17 and polyQ regions appear indirectly affected by PHP1 binding. It is
hypothesized that their mobility is reduced because of PHP1 binding even though this may not be due to
direct contact between PHP1 and Httex1(Q7). EPR data also shows a decrease in amplitude for positions
5, 11, 35, 48. One possibility is that PHP1 may cause the formation of oligomeric species.
MW1
From the dot blots it was seen that MW1 binds strongly to the monomers compared to the fibrils. This is
consistent with the bottle brush model described above. The dot blots reveal that MW1 binds strongly to
monomers compared to fibrils. This result is consistent with the bottle brush model explained above. The
structure of huntingtin exon 1 protein in the preaggregation state, particularly the conformation of the
expanded polyQ repeat, is hypothesized to be critical in understanding the pathogenesis of HD(Li et al.,
2007).
If we compare the EPR data of Q25 and Q46, Q46 shows a stronger binding affinity compared to Q25.
Also, the binding pocket for the Q46 derivatives comprises a large number of amino acid residues
compared to Q25. This suggests that a longer Q length contains more epitopes for the antibody to bind.
Figure.16. Effect of PHP1 on the PRD region of Httex1. When PHP1 binds to the PRD region it induces the formation of a kink which
causes the PRD region to fold backward.
24
Previous research work has also shown that the expanded polyQ region contains multiple epitopes for
MW1 to bind (Li et al., 2007).
The DEER data indicates that when MW1 binds to the polyQ region, it stretched the region. It was initially
speculated that one MW1 could use both of its Fab regions to bind to the same Httex1 molecules. This
would open up the polyQ region and allow an additional MW1 to bind. Ultimately such a complex would
be expected to give rise to a complex with 2 MW1 and one Httex1. The molecular weight for such a
complex would be around 360kDa. But the gel filtration data reveals much larger complexes. To evaluate
the binding stoichiometry of MW1 with Httex1, we performed gel filtration chromatography with various
molar ratios of the antibody and protein.
The data for Httex1(Q46) can therefore only be explained by the formation of complexes that contain
multiple Httex1 and MW1 molecules. Based on the DEER data we can exclude the possibility that Httex1
molecules come close via oligomerization of the Httex1 moieties. Thus, the antibodies must be bridging
Httex1 together. Much smaller complexes are observed for the shorter Q-lengths. In the case of
Httex1(Q16), binding is weak with significant amounts of free antibody still present (peak at 155 kDa). The
larger peak is at approximately 250 kDa. This molecular weight would be consistent with one MW1
antibody binding to 2 Httex1 proteins. It would be inconsistent, however, with multiple antibodies in the
complex. The same 250 kDa peak is also visible for Httex1(Q25), where it is the dominant peak for
Httex1(Q25):MW1 ratios of 1:0.5. At higher amounts of MW1 (1:1 ratio), a larger complex become
dominant. This complex has an apparent size of 370 kDa. It is likely a complex where 2 MW1 proteins bind
to 2 Httex1 proteins, but we cannot exclude contributions from 2 MW1 and 1 Httex1 proteins either.
Regardless of the exact details, the fundamental difference between the expanded Q-length and the
shorter Q-length proteins lies in the size of the complexes. While the longer Q-length proteins form
complexes with multiple Httex1 molecules bridged together by multiple antibodies, this does not happen
for shorter Q-length.
25
The need to develop effective treatments for HD is of paramount importance today (Tan et al., 2015).
Studying the unique oligomeric assemblies formed by the mutant huntingtin protein by using anti htt
antibodies will play an important role in the development of therapeutics in the future. Also, there is a
great deal of interest in the use of antibodies and intrabodies as potential therapeutic agents to treat HD
and other polyQ disorders (Legleiter et al., 2009). Recent research has also shown that PHP1 blocks fibril
assembly in Httex1 by binding to seeds (Ko et al., 2018). Recently assays have been developed that detect
mutant huntingtin levels in patient biofluids like CSF and blood (Tan et al., 2015). These detection assays,
coupled with the use of anti-huntingtin antibodies like MW1, 2B7, PHP1 will help monitor disease
progression in patients. This information can be used for designing conformational specific drugs to block
disease progression.
Apart from antibodies, there are multiple targeted huntingtin lowering therapies in clinical development.
An intrathecally administered antisense oligonucleotide HTTRx/RG6042, has been shown to lower levels
huntingtin in CSF in patients in the first phase 1/2 clinical trial of a huntingtin-lowering therapy. Another
protein, that is proposed as a potential biomarker is the Neurofilament light protein (NfL), which is the
smallest subunit of neurofilaments and a component of the neuronal cytoskeleton. It is released into CSF
because of neuronal damage and several studies indicate that NfL is increased in the CSF in HD patients
and correlates with clinical severity (Byrne et al., 2018).
Figure.17. Model of MW1 Httex1 interaction. The interaction between MW1 and Httex1 depends on Q-
length. Antibodies are orange and polyQ regions of Httex1 are shown as green sticks. For longer poly Q
regions, several antibodies can bind and thereby non-covalently “crosslink” multiple Httex1 together. One
could envision that repeated assemblies of the model on the left can give rise to larger and larger complexes
with a larger number of Httex1 and MW1 molecules. For short Q-lengths, at most two Httex1 molecules can
be in one complex. A further oligomerization into larger complexes is not possible. Acknowledgements: Dr.
José Bravo.
Short Q-length
Long Q-length
or
Repeating unit
26
Several biophysical tools have also played an important role in understanding Huntington’s disease.
Structural imaging has been the source of the most robust biomarkers for HD. Structural MRI
methodologies have demonstrated changes in the striatum, in terms of strong cross-sectional and
longitudinal changes in its volumes, in both premanifest and manifest HD. Diffusion tensor imaging (DTI)
has revealed abnormalities in neuronal fiber orientation and integrity in white matter and subcortical grey
matter structures in both premanifest and manifest HD (Ross et al., 2014). In conclusion, biomarkers play
an important role in tracking disease progression and in the development of therapies for clinical
management of Huntington’s disease.
27
Materials and Method
Transformation
The BL21DE3 competent cells were made in lab and used for the transformation experiments. Cells were
taken out from the -80°C freezer, placed on ice and allowed to thaw at room temperature. 15µl of
competent cells and 1ul of DNA were mixed in a vial and allowed to incubate on ice for 30 minutes. Heat
shock treatment was given at 42.5°C for 45 seconds and the vial was immediately kept on ice for 2
minutes. 250µl of SOC broth was then added and the suspension was incubated at 37°C for 1hr. 100 µl of
the suspension was plated on LB agarose plates containing ampicillin. The plates were incubated overnight
at 37°C.
Protein Expression
An isolated colony of the transformed cells was inoculated in 25ml of LB media containing ampicillin
(100mg/ml). The culture was incubated at 37°C, 225rpm for 4-5 hours until turbid. This culture was then
expanded into 1L LB media containing ampicillin and incubated at 37°C 190rpm. When the OD of the
culture was around 0.6, it was induced with 1ml IPTG (1M). The incubation temperature was dropped to
16 and the culture was incubated for two nights. The culture was centrifuged at 4000rpm for 25 minutes
4°C and the pellets were stored at -80°C for further use.
Protein purification
Cell pellets were removed from -80°C freezer and allowed to thaw at room temperature. The pellet was
then was suspended in 30ml Lysis buffer (20 mM Tris-HCl, pH 7.4, 300 mM NaCl and 30 mM Imidazole)
with 1200µl Triton (1:4) and 30µl DTT (1M). The pellet was vortexed until completely dissolved. The pellet
mixture was sonicated for 30 seconds on ice(5X) with an interval of 30 seconds between each sonication.
After sonication the mixture was centrifuged at 18,500 rpm for 25 minutes at 4°C. The supernatant was
incubated with nickel-nitrilotriacetic acid-agarose beads (Qiagen) equilibrated with lysis buffer for 45
minutes at 4°C on a shaker. The column was mounted and washed with 3 column volumes of Wash buffer
(20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 20 mM Imidazole) wash buffer containing 1M DTT(3X) and twice
with normal Wash buffer. The protein was eluted using 25ml Elution buffer (20 mM Tris-HCl, 300 mM
NaCl, 250 mM Imidazole, pH 7.4). A quick Bradford assay was performed to check for the presence of
protein by mixing 200ul of Bradford reagent with 50ul of protein. The eluted protein was spin labelled
with 14µl MTSL (40mg/ml) and incubated for 45 minutes at RT. After incubation, the protein was diluted
approx. 10 times with Dilution buffer (10mM Tris) and was passed through a HiTrap Q XL column (GE
Healthcare) followed by purification with an AKTA FPLC system (Amersham Pharmacia Biotech) using a
NaCl gradient. The protein eluted at about 300 mM NaCl and was consequently diluted to 20μM with
10mM Tris buffer pH 7.4. PD 10 columns were used to exchange the buffer of the protein sample from
tris to phosphate. After exchanging the buffer, the protein concentration was brought to 10mM.
28
Huntingtin protein constructs having a cysteine mutation in the positions as illustrated in the figure below
were transformed and purified as per the protocol explained above.
Purification of PHP1
Protein A beads were used for the purification of PHP1. The ascites (stored at -80°C) was kept on ice and
allowed to thaw. The ascites(1.5ml) was then mixed with phosphate buffer/soup(10ml) and loaded onto
a protein A column. The flow through was collected and loaded onto the column again. This was repeated
4 times. The column was then washed with PBS(3X). The antibody was eluted with 10ml of 100mM glycine
pH 2.5 and 1ml fractions were collected. Absorption was checked at 280nm using a UV spectrometer. The
sample was diluted with Phosphate buffered saline (20 mM sodium phosphate, pH 7.4, 150 mM NaCl).
SDS PAGE was done to check the purity of the antibody and was stored at 4°C for future use.
Dot blots
1.5µl of the protein sample (14ng/µl) was blotted onto a nitrocellulose membrane. The membrane was
blocked overnight at 4°C in a blocking buffer containing 10% milk powder in TBST (tris buffered saline with
0.1% tween). The membrane was washed with TBST 3 times for 5 minutes each and was incubated with
the primary antibody at a 1:5000 dilution for one hour at room temperature. Again, the membrane was
washed with TBST 3 times and incubated with an HRP conjugated secondary antibody at a 1:10,000
dilution for one hour at room temperature. After washing the membrane with TBST for 3 times, it was
visualized by chemiluminescence staining.
Preparation of EPR samples
Spin labeled samples were consequently diluted to approximately 30 µM and then a PD10 column was
used to buffer exchange it into 20 mM sodium phosphate, pH 7.4, 150 mM NaCl and finally adjust the
protein concentration to 20 µM. A protein concentration 20 µM was used for experiments with MW1 and
a protein concentration of 10 µM was used for PHP1 experiments.
29
MW1 and PHP1 antibodies were concentrated in phosphate buffer (20 mM sodium phosphate, pH 7.4,
150 mM NaCl) to 25µM. Trx-Httex1 (Q46) and MW1 (at ratios 1:0, 1:1, 1:2 and 1:3) and Httex1 (Q25) and
MW1 (at ratios 1:0, 1:1, 1:2 and 1:3) and Trx-Httex1 (Q46) with PHP1 (at ratios 1:0, 1:1, 1:2 and 1:3) were
mixed in Eppendorf tubes and loaded into glass capillaries (0.6-mm inner diameter × 0.84-mm outer
diameter, VitroCom, Mt. Lakes, NJ). EPR spectra were recorded on an X-band Bruker EMX spectrometer
(Bruker Biospin Corporation).
Gel Filtration
Non-equilibrium protein interaction experiments were carried out on a Superdex 200 increase 10/300 GL
gel-filtration column (GE Healthcare) equilibrated in a buffer containing 20 mM Phosphate (pH 7.4) and
150 mM NaCl. A final concentration of 10 µM Trx-Httex1 was used for all experiments, with MW1
concentration varying to create the complexes with final molar ratios of 0.5:1 and 1:1 of each complex.
500 µL was injected and flowed through the column at 0.5 mL/min at room temperature. The absorbance
of the eluent was monitored at 280 and 215 nm. Globular protein standards of known molecular weight
from Bio-Rad were used to calibrate the Superdex-200 gel-filtration column, ranging from 1,350 to
670,000 Da, under nondenaturing conditions; i.e. thyroglobulin, γ-globulin, ovalbumin, myoglobin, and
vitamin B12.
30
Future Directions
1. Perform EPR experiments with Huntingtin fibrils and PHP1.
2. Perform a FRET experiment with huntingtin derivatives in the N17 and polyQ region to investigate
if PHP1 induces oligomerization.
3. Perform Gel filtration experiments with huntingtin monomer and PHP1 to study the binding
affinity.
31
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Abstract (if available)
Abstract
Huntington’s disease is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the N terminal region of huntingtin (htt). The mutant protein starts to aggregate and adopt unique conformations. It is important to know the composition of these unique conformations in order to develop therapeutics to block the disease progression. In this process, antibodies are being used as a tool to identify and study these unique conformations. Of the various antibodies used for studying huntingtin, we have decided to focus on two, MW1 and PHP1. MW1 binds strongly to expanded polyQ region in monomers whereas PHP1 binds strongly to the PRD region of huntingtin fibrils. The exact binding stoichiometry of binding is still unknown. My work focuses on mapping on the binding regions of these antibodies and to study the binding stoichiometry of the antibodies with the protein by using tools like EPR spectroscopy and size exclusion chromatography.
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Venkatrama, Rajashree
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Core Title
Interaction of monoclonal antibodies MW1 and PHP1 with huntingtin exon1 protein
School
Keck School of Medicine
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Master of Science
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Biochemistry and Molecular Medicine
Publication Date
07/29/2019
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