O-GlcNAcylation of Î±-Synuclein at Serine 87 Reduces Aggregation

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O-GlcNAcylation of #-synuclein at serine 87 reduces aggregation without affecting membrane binding Yuka E. Lewis, Ana Galesic, Paul M. Levine, Cesar A De Leon, Natalie Lamiri, Caroline K Brennan, and Matthew R. Pratt ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00113 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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ACS Chemical Biology

O-GlcNAcylation of α-synuclein at serine 87 reduces aggregation without affecting membrane binding. Yuka E. Lewis,† Ana Galesic,† Paul M. Levine,† Cesar A. De Leon,† Natalie Lamiri,† Caroline K. Brennan,† and Matthew R. Pratt*,†,§ †

§

Department of Chemistry and Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089-0744 ABSTRACT: The aggregation of neurodegenerative-disease associated proteins can be affected by many factors, including a variety of posttranslational modifications. One such modification, O-GlcNAcylation, has been found on some of these aggregation prone proteins, including α-synuclein, the major protein that plays a causative role in synucleinopathies like Parkinson’s disease. We previously used synthetic protein chemistry to prepare α-synuclein bearing a homogeneous O-GlcNAc modification at threonine 72 and showed that this modification inhibits protein aggregation. However, the effects of the other eight O-GlcNAcylation sites that have been identified was unknown. Here, we use a similar synthetic strategy to investigate the consequences of this modification at one of these sites, serine 87. We show that O-GlcNAcylation at this site also inhibits α-synuclein aggregation but to a lesser extent that the same modification at threonine 72. However, we also find that this modification does not affect the membrane-binding properties of α-synuclein, which differentiates it from phosphorylation at the same site. These results further support the development of therapies that can elevate O-GlcNAcylation of α-synuclein to slow the progression of Parkinson’s disease.

The addition of the monosaccharide N-acetyl-glucosamine, or O-GlcNAc modification (Figure 1A), occurs in plants and animals and has been found on hundreds of proteins in the cytosol, nucleus, and mitochondria.1,2 The sugar is added to the side chain hydroxyl groups of serine and threonine residues of substrate proteins by the enzyme O-GlcNAc transferase (OGT) and can be rendered dynamic through subsequent removal by O-GlcNAcase (OGA). There are several lines of evidence that implicate the misregulation of O-GlcNAcylation in neurodegenerative diseases.3,4 Mice with neuron-specific deletion of OGT have hyperphosphorylated tau and suffer from locomotor defects before dying within 10 days of birth.5 Decreased global levels of O-GlcNAcylation has also been observed in the brains of patients who succumbed to Alzheimer's disease (AD).6 Furthermore, increasing the levels of OGlcNAcylation in a mouse model of AD with a smallmolecule inhibitor of OGA slowed the pace of neurodegeneration,7 and the same small-molecule reduced the amount of hyperphosphorylated tau in healthy rats.8 Notably, several of the proteins that form the toxic aggregates that are hallmarks of neurodegenerative diseases are directly modified by OGlcNAcylation, which can result in inhibition of protein aggregation. For example, Vocadlo and co-workers enzymatically O-GlcNAcylated the AD-associated protein tau in a heterogeneous fashion and showed that this modified protein had a decreased propensity for aggregation.7 We previously used a synthetic strategies to build site-specifically O-GlcNAcylated α-synuclein peptides and the full-length protein, the major aggregating protein in Parkinson’s disease (PD), and found that the modification completely blocks its aggregation.9,10 α-Synuclein is a small (140 amino acids) protein that is highly enriched in pre-synaptic neurons of the central nervous system,11 where it appears to be involved in vesicle remodeling

and trafficking.12 When in contact with membranes, the protein forms an extended α-helix that can induce membrane bending,13-15 while it exists as predominantly an unstructured monomer in solution and the cytosol. In PD and other synucleinopathies, however, α-synuclein is found in aggregates that have the features of the β-sheet rich fibers that are common to all amyloid proteins.16 Solid-state NMR and EPR experiments have defined the region of α-synuclein that forms the core of these aggregates to be approximately residues 61-95.17-19 Several proteomics studies from both neurons and erythrocytes, which also have high concentrations of α-synuclein, have identified nine different in vivo sites of O-GlcNAcylation (Figure 1B).20-23 This modification being present at many sites is not necessarily surprising as OGT prefers to modify unstructured regions of proteins. As mentioned above, we previously used a synthetic protein chemistry strategy to investigate the consequences of O-GlcNAcylation at α-synuclein T72.10 We found that modification at T72 completely blocks in vitro the first step of the aggregation process, termed nucleation, and had a slight inhibitory effect on the second elongation step that generates larger fiber structures. Here, we continue to explore the effects of O-GlcNAcylation on the biophysical properties of α-synuclein. More specifically, we chose to focus on modification at residue S87. Notably, this serine is mutated to glutamine in rodent α-synucleins, and mouse synuclein was recently found to inhibit the aggregation of the human protein.24 Furthermore, S87 is also a site of phosphorylation, and both the enzymatically phosphorylated protein and the phosphomimetic mutation S87E inhibit α-synuclein aggregation, although phosphorylation at this site also inhibits membrane binding.25 In a similar strategy to our previous effort, we used chemical ligation to prepare α-synuclein with OGlcNAcylation at S87 and found that this modification also

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inhibits protein aggregation, albeit to a lesser extent than OGlcNAcylation at T72 or the S87E mutation. However, in contrast to phosphorylation, O-GlcNAcylation had no observable effect on the membrane binding properties of α-synuclein. These results add additional support for the targeting of OGA to raise O-GlcNAcylation levels as a treatment strategy for PD. Furthermore, they demonstrate biophysical differences between O-GlcNAcylation and phosphorylation at the same site, encouraging the further study of the effects of sterics versus charge in the modulation of protein aggregation.

ized by ESI-MS (Supplemental Figures 2 and 3). These proteins were then incubated separately with the recombinant protein thioester 1 to yield the corresponding unmodified or O-GlcNAcylated proteins 7 and 8 (Supplemental Figure 4). In order to generate the α-synuclein proteins with no mutations, the cysteine residues at the ligation sites were then transformed to the native alanine residues using radical-mediated desulfurization (Supplemental Figure 5). The final products 9 and 10 were then purified by RP-HPLC and characterized by ESI-MS (Figure 2B).

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

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

RESULTS AND DISCUSSION In order to directly test the effect of O-GlcNAcylation at S87 on α-synuclein aggregation and membrane binding, we set out to first prepare the modified protein using expressed protein ligation (EPL). Traditional EPL takes advantage of cysteine residues at the ligation sites through a transthioesterification reaction followed by an S to N acyl shift. α-Synuclein contains no native cysteines, so we decided to introduce these residues at positions 76 and 91 in the primary sequence, which are alanine residues in the native protein. These cysteine residues then enable us to retrosynthetically deconstruct α-synuclein (Figure 2A) into a recombinant protein thioester (1, residues 1-75), a synthetic peptide (2 or 3, residues 76-90), and a recombinant C-terminal fragment (4, residues 91-140). The recombinant protein thioester 1 was prepared by expression of the corresponding intein fusion in E. coli followed by thiolysis. The peptides 2 (unmodified) or 3 (O-GlcNAcylated at S87) were synthesized as the corresponding thioesters by Fmoc-based solid phase peptide synthesis using the Dawson aminobenzyol resin.26 Finally, fragment 4 was prepared by expression in E. coli where the critical N-terminal cysteine residue was generated by the action of an endogenous methionine aminopeptidase. All of these peptide and protein fragments were then purified by RP-HPLC and characterized by ESI-MS (Supplemental Figure 1). Incubation of either peptide 2 or 3 and fragment 4 resulted in formation of the ligation product (Supplemental Figures 2 and 3). At this time, the pH of the buffer was reduced and the N-terminal cysteine protecting-group was removed using methoxylamine and the resulting proteins 5 and 6 were purified by RP-HPLC and character-

With the synthetic proteins in hand, we first compared the unmodified, synthetic α-synuclein to fully recombinant protein. Analysis by circular dichroism (CD) spectroscopy showed that both proteins were unstructured in solution (Supplemental Figure 6A) and dynamic light scattering (DLS) analysis demonstrated that both proteins were monomeric in nature (Supplemental Figure 6B). These two proteins were then subjected in triplicate to aggregation conditions at a concentration of 50 µM for 7 days. Reaction aliquots were removed after 72, 120, and 168 h for visualization of any fiber formation using thioflavin T (ThT) fluorescence (Figure 3A), which demonstrated that synthetic and recombinant αsynuclein aggregated with essentially the same kinetics. To visualize the structure of the fibers that formed, aliquots from the end of the aggregation assay were collected and transmission electron microscopy (TEM) was performed, and both the recombinant and synthetic proteins formed α-synuclein fibers that are consistent with amyloid structures (Figure 3B). These data demonstrate that our synthetic preparation of α-synuclein did not affect its biophysical properties. Next, we set out to determine the consequences of O-GlcNAcylation at S87 using our synthetic protein, termed α-synuclein(gS87). As mentioned above S87 is also a known site of phosphorylation, so we also expressed the S87E mutant of α-synuclein (αsynuclein(S87E), Supplemental Figure 7), as this glutamic acid mutant has been shown to recapitulate some of the effects of phosphorylation at the same site. CD spectroscopy showed that neither O-GlcNAcylation or the S87E mutation induced any secondary structure in α-synuclein (Supplemental Figure 8A), and both proteins were monomeric in nature as deter


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Figure 3. O-GlcNAcylation of α-synuclein at S87 inhibits protein aggregation. (A) Synthetic, unmodified α-synuclein aggregates in the same fashion as recombinant protein. Recombinant or unmodified, synthetic α-synuclein were incubated under aggregation conditions (50 µM concentration and agitation at 37 °C) for the indicated lengths of time before analysis by ThT fluorescence (λex = 450 nm, λem = 482 nm). The y-axis shows the fold-increase of fluorescence compared with the corresponding protein at t = 0. Error bars represent ±s.e.m from the mean of biological replicates (n = 3). (B) The structures formed in the same aggregation reactions were visualized by TEM after 168 h. Scale bars, 500 nm. (C) O-GlcNAcylation and a phosphomimetic mutation at S87 inhibit aggregation. Recombinant α-synuclein, αsynuclein(gS87), or α-synuclein(S87E) were subjected to aggregation conditions (50 µM concentration and agitation at 37 °C) for the indicated lengths of time before analysis by ThT fluorescence (λex = 450 nm, λem = 482 nm). The y-axis shows the fold-increase of fluorescence compared with the corresponding protein at t = 0. Error bars represent ±s.e.m from the mean of biological replicates (n = 3), and statistical significance was calculated using a two-tailed Student’s t-test. (D) The same reactions were analyzed by TEM after 168 h. (E) Aliquots from the same reactions were collected and the soluble fractions collected by centrifugation. These soluble proteins were then separated by SDS-PAGE and visualized by Coomassie blue staining. The data is representative of two biological experiments.

mined by DLS (Supplemental Figure 8B). We next simultaneously examined the aggregation of unmodified α-synuclein, α-synuclein(gS87), and α-synuclein(S87E) using a combination of ThT fluorescence, SDS-PAGE analysis, and TEM. First, aggregation reactions at a concentration of 50 µM were again initiated in triplicate. After 48, 72, 120, and 168 hours aliquots were removed and analyzed by ThT fluorescence (Figure 3C). These data showed that α-synuclein(gS87) still aggregated but with slower kinetics than the unmodified protein, and that α-synuclein(S87E) was essentially totally resistant to aggregation, consistent with previously published results. Next, the structure of any aggregates that formed after 168 h was visualized using TEM (Figure 3D and Supple-

mental Figure 9). As expected, the unmodified α-synuclein formed large, regular fibers. In contrast, α-synuclein(S87E) formed only amorphous structures that are consistent with protein precipitation during the preparation of the TEM grids and what could be small, irregular structures. αSynuclein(gS87) formed both shorter fibers and small structures that are similar to those formed by protein precipitation during TEM preparation. As a complementary assay, identical aliquots were first subjected to centrifugation to separate the aggregates from soluble material. The soluble fraction was then concentrated by lyophilization and then subjected to SDS-PAGE and visualized by Coomassie staining (Figure 3E). Consistent with the ThT fluorescence the unmodified



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protein was lost from the soluble fraction to a much higher extent than either α-synuclein(gS87) or α-synuclein(S87E), further demonstrating that both O-GlcNAcylation and the phosphomimetic mutation inhibit aggregation. These data support a model where both O-GlcNAcylation and phosphorylation at S87 have a similar effect on the formation of fibers, namely the inhibition of the formation of fibers and the formation of small, irregular structures. However, the phosphomimetic is more strongly inhibitory compared to OGlcNAcylation at the same site. The aggregation of αsynuclein is known to be concentration dependent. Therefore, we next asked if the inhibitory capacity of O-GlcNAcylation at S87 is dependent on the concentration of the protein, and aggregation reactions were performed at a concentration of 25 µM. As expected, analysis of protein aggregation by ThT fluorescence (Figure 4A) showed that aggregation of unmodified α-synuclein over the same timeframe was reduced by ~5-fold. Again, aggregation mixtures with α-synuclein(S87E) did not give any ThT signal over the course of the reaction. Interestingly, the consequences of O-GlcNAcylation were more significant, suggesting that it could have a reasonable inhibitory effect in neurons where the concentration of α-synuclein has been estimated to be between 10 and 100 µM. We next set out to determine the effects of O-GlcNAcylation on the endogenous function of α-synuclein. As mentioned above, α-synuclein forms an extended α-helix when it comes into contact with negatively charged membranes, and αsynuclein can remodel membranes in isolation and can collaborate with other proteins to alter vesicle trafficking. Notably, α-synuclein that has been enzymatically phosphorylated at S87 has reduced affinity for membranes and therefore may inhibit these functions; however, the pseudo-phosphate mutation S87E had no observable effect.25 To determine if OGlcNAcylation at S87 has consequences on membrane binding, unmodified α-synuclein, α-synuclein(gS87), or αsynuclein(S87E) was incubated with an excess of vesicles for 20 min before measuring the induction of any secondary structure by CD spectroscopy (Figure 4B). As expected from previous results, unmodified protein and α-synuclein(S87E) formed α-helical structures in the presence of the negatively charged lipid vesicles made up of POPG or POPS, and this binding to POPS vesicles can be reduced by in introduction of the zwitterionic lipid POPC. Importantly, these same vesicles have been used extensively for the analysis of α-synuclein membrane interactions.27,28 We also observed no major difference upon O-GlcNAcylation of α-synuclein at S87, indicating that this modification does not have notable effect on the affinity or binding mode of α-synuclein to membranes. Finally, we set out to explore the effect of different substitutions at residue 87 in more detail. More specifically, we chose a further set of mutations (S87A, S87D, S87W, and S87K) and expressed and purified the corresponding α-synuclein proteins (Supplemental Figure 10). We chose S87A as it represents the loss-of-function mutant that cannot be modified and has been used in the past to understand the effect of phosphorylation at this position.25,29 α-Synuclein(S87D) was chosen as it also contains a negative charge, the the exact position of that charge with respect to the protein backbone is altered compared to S87E. The S87W mutation increases the steric bulk of this position similarly to O-GlcNAcylation; however, unlike the carbohydrate tryptophan is largely hydrophobic in nature. Finally, we chose S87K to reverse the charge at this position compared to S87E. Wild-type α-synuclein and each of these

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mutants were then subjected to aggregation conditions at 25 µM concentration before analysis by ThT fluorescence (Supplemental Figure 11A). In the case of S87A, we found that this mutation was slightly inhibitory to aggregation, while S87D was also inhibitory to a larger extent. In contrast, the S87W and S87K mutations increased the aggregation, with S87K having a larger effect. Together these data indicate that inhibition of aggregation by O-GlcNAcylation and phosphorylation (or the pseudo-phosphate S87E) at this position is not driven by sterics alone but rather by hydrophilicity or negative charge, respectively. Next, we also examined the effect(s) of these mutations on α-synuclein membrane binding by incubating the proteins in excess of POPG vesicles for 20 min before analysis by CD spectroscopy (Supplemental Figure 11B). Interestingly, all of the mutations besides S87D had very little effect on the induction of the α-synuclein α-helix, similar to OGlcNAcylation and S87E. In contrast, α-synuclein(S87D) showed less membrane binding that is quite similar in magnitude to phosphorylation at this residue,25 suggesting that αsynuclein is very sensitive to the exact position of the negative charge at serine 87. Here, we have reported the synthesis of α-synuclein with sitespecific O-GlcNAcylation at residue 87 and show that this modification inhibits protein aggregation. However, this modification is less inhibitory when compared to O-GlcNAcylation at T72, which we previously prepared and investigated. More specifically, α-synuclein(gT72) is completely resistant to aggregation at 50 µM concentration, while α-synuclein(gS87) shows significant aggregation at this concentration and even some aggregation at 25 µM. We speculate that this observation can be explained by the effects of O-GlcNAcylation on the equilibrium between monomeric α-synuclein in solution and protofiber structures that will seed a rapid extension reaction that forms mature fibers. In the case of O-GlcNAcylation at T72 the equilibrium strongly favors monomeric protein, while modification at S87 has a smaller effect that can be overcome at higher monomer concentrations. The site-specific difference are also consistent with a recent NMR structure of the α-synuclein fiber,19 as well as previous structural models that have been developed using EPR and NMR spectroscopy,17,18 as well as cryo-electron microscopy.30 More specifically, T72 lies in the middle of a β-strand in the core structure that makes up the individual monomers in the fiber, while S87 is towards the edge of this core in a potentially more flexible turn between two β-strands that may more readily accommodate the steric bulk of the O-GlcNAcylation. Interestingly, we also find that O-GlcNAcylation is less inhibitory than the pseudo-phosphate S87E mutation. Importantly, our data showing that α-synuclein(S87E) completely blocks aggregation at these concentrations is totally consistent with previous reports.25 Other mutations at S87 ranged from partial inhibition, like S87D, to potentiation (S87W and S87K) of protein aggregation. Again, these observations can be rationalized by the NMR structure.19 The residues that surround S87 in the fiber structure all mostly hydrophobic in nature except for a single glutamic acid at residue 82. Therefore, the negative charge(s) on S87E, S87D, and phosphorylated S87 would strongly promote interactions this area with bulk water, while the uncharged carbohydrate could be more easily desolvated to allow for the formation of the hydrophobic core of the fiber and suggest that the increased steric bulk of O-GlcNAc at S87 is not sufficient to completely block aggregation. Furthermore, the relatively hydrophobic bulk of tryptophan in S87W is well


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tolerated in the aggregate-formation process. Interestingly, the S87K mutation shows the most aggregation, which might be explained through a favorable interaction with the glutamic acid at residue 82 in α-synuclein. This result means that we cannot rule out a charge repulsion to explain the strong inhibition of the longer S87E compared to S87D, as it may be in closer physical proximity to E82.

Figure 4. O-GlcNAcylation at S87 inhibits α-synuclein aggregation without affecting membrane binding. (A) O-GlcNAcylation at S87 is more inhibitory towards aggregation at lower protein concentrations. Recombinant α-synuclein, α-synuclein(gS87), or α-synuclein(S87E) were subjected to aggregation conditions (25 µM concentration and agitation at 37 °C) for the indicated lengths of time before analysis by ThT fluorescence (λex = 450 nm, λem = 482 nm). The y-axis shows the fold-increase of fluorescence compared with the corresponding protein at t = 0. Error bars represent ±s.e.m from the mean of biological replicates (n = 3), and statistical significance was calculated using a two-tailed Student’s t-test. (B) O-GlcNAcylation at S87 has no effect on α-synuclein membrane binding. Recombinant α-synuclein, α-synuclein(gS87), or α-synuclein(S87E) were incubated with an 100-fold excess of

the indicated, preformed vesicles and analyzed using circular dichroism (CD). In the presence of negatively charged vesicles (POPG or POPS), all of the proteins gave essentially indistinguishable CD spectra consistent with the formation of an extended α-helix. The introduction of a zwitterionic lipid (POPC) reduced the α-helix formation equally for both proteins. POPG = 1palmitoyl-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.

In contrast to the effects on protein aggregation, we find that O-GlcNAcylation at S87 has essentially no effect on the ability of α-synuclein to interact with lipid vesicles. This stands in contrast to the observations that have been made with αsynuclein that has been enzymatically phosphorylated at S87.25 Again, these data fit with previous the structural characterization of the α-synuclein extended helix using EPR spectroscopy.13 According to the model developed from these data, S87 lies towards the end of the α-helical structure (residue 90) and close to the lipid-solvent interface near the phosphate headgroups. Therefore, it is not surprising that the doubly-charged phosphorylated S87 would experience more electrostatic repulsion compared to either S87E or O-GlcNAcylation at this position. Notably, however, α-synuclein(S87D) also shows less membrane binding, demonstrating that the exact position of the negative charge may be equally important. This suggests that O-GlcNAcylation may have a smaller effect on the healthy functions of α-synuclein when compared to phosphorylation at the same site. In total, these data add further support for increasing O-GlcNAcylation as a potential therapeutic strategy in PD and highlight the utility of synthetic approaches for testing the consequences of protein posttranslational modifications. EXPERIMENTAL PROCEDURES General. All chemicals and solvents were purchased from commercial venders (Fluka, EMD, Novagen, Sigmα-Aldrich, etc) and used without any additional purification. Growth media (Luria-Bertani, Miller) were prepared and sterilized according to the manufacture protocol. Antibiotics (Kanamycin sulfate, EMD, and Ampicillin sodium salt, EMD) were prepared as stock solution (50 mg mL-1 and 100 mg mL-1 respectively), and stored at -20 ℃. Analytical thin-layer chromatography was performed on 60 Å F254 silica plates with detection by UV light and/or ceric ammonium molybdate (CAM). Agilent Technologies 1200 Series HPLC with Diode Array Detector was used for reverse phase high performance liquid chromatography (RP-HPLC). Unless otherwise noted, the HPLC buffers were, buffer A: water with 0.1% TFA, buffer B: 90% acetonitrile, 10% water with 0.1% TFA. Mass spectra were obtained using API 150EX (Applied Biosystems/MDS SCIEX). Expression of recombinant wild type α-synuclein and mutant α-synuclein proteins. BL21(DE3) E. coli transformed with pRK172 construct containing human wild-type αsynuclein or α-synuclein(S87E) was grown until its OD600 was above 0.6. The culture was induced by addition of 0.5 mM IPTG and incubation for 20 h at 25 ℃. The culture was pelleted by centrifugation at 6,000 rpm. The pellet was lysed by 3 times of freeze and thaw cycle. The resulting lysate was resuspended in lysis buffer (500 mM NaCl, 100 mM Tris, 10 mM beta-mercaptoethanol, 1 mM EDTA, pH 8.0), and heated at 80 ℃ for 10 min. The lysate was allowed to cool down to room temperature before the addition of protease inhibitor cocktail



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(mini complete EDTA free, Roche). The resulting solution was incubated in ice for 30 min, and the cell debris was pelleted by centrifugation at 15,000 rpm for 30 min at 4 ℃. The pH of the supernatant was adjusted to 3.5 with 1M HCl, and the resulting solution was incubated on ice for additional 30 min. The lysate was cleared by centrifugation (15,000 rpm, 30 min, 4 ℃), and then dialyzed against 3 X 1 L of a degassed 1% acetic acid solution. The dialyzed solution was cleared by centrifugation (6,000 rpm, 15 min, 4 ℃). α-Synuclein was purified on RP-HPLC (40-60% B over 60 min), and lyophilized. The purified protein was characterized by RP-HPLC and ESIMS. Expected mass of wild type α-synuclein is 14,460 Da, and the observed mass was 14,460 ± 3 Da. Expected mass of αsynuclein(S87E) is 14,502 Da, and observed mass was 14,504 ± 1 Da. Expected mass of α-synuclein(S87A) is 14,435 Da, and observed mass was 14,435 ± 1 Da. Expected mass of αsynuclein(S87D) is 14,488 Da, and observed mass was 14,489 ± 1 Da. Expected mass of α-synuclein(S87W) is 14,560 Da, and observed mass was 14,562 ± 2.5 Da. Expected mass of αsynuclein(S87K) is 14,501 Da, and observed mass was 14,503 ± 1 Da. Expression and purification of α-synuclein C-terminal fragment. Transformed BL21(DE3) E. coli with pET42b plasmid containing α-synuclein(C91-140) was expressed and semi-purified by HCl treatment and centrifugation as described above for full-length α-synuclein. α-Synuclein(C91140) was then further purified on RP-HPLC (10-45% B linear gradient over 60 min). The purified fragment was characterized by RP-HPLC and ESI-MS. Expected mass is 5,593 Da, and the observed mass was 5,595 ± 2 Da. Expression and purification of α-synuclein N-terminal thioester. Transformed BL21(DE3) E. coli with pTXB1 plasmid containing α-synuclein(1-75)-AvaDnaE-Histag was grown to OD600 above 0.6. The induction of culture was conducted by addition of 0.5 mM IPTG and incubation for 17 h at 25 ℃. The culture was centrifuged (6,000 rpm, 30 min, 4 ℃) and resuspended with lysis buffer (50 mM phosphate, 5 mM imidazole, 300 mM NaCl, pH 8.0). The cells were lysed by tip sonication (30s/30s ON/OFF cycle, 6 min total, 4 ℃). The lysate was centrifuged (15,000 rpm, 30 min, 4 ℃), and the supernatant was loaded on HisTrap column (GE healthcare). The protein was bound to the column by washing with 5 column volumes (CVs) of buffer A (50 mM phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0), and eluted with 5 CVs of buffer B (50 mM phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0). Elution fractions were pooled and dialyzed against 3X 1 L of degassed buffer C (100 mM phosphate, 150 mM NaCl, 1 mM TCEP, 1mM EDTA pH 7.5). The dialyzed solution was incubated with fresh TCEP (2 mM final concentration) and mercaptoethane sulfonate (MesNa, 200 mM final concentration) for 3 d to generate C-terminal thioester group. The resulting solution was purified on C4 semiprep RP-HPLC (35-55% B over 60 min). The expected mass is 7,686 Da, and the observed mass was 7,686 ± 1 Da. Solid phase synthesis of peptide thioesters. All peptide synthesis was performed manually using protected Dawson linker resin (Millipore). Commercially available side chain and NFmoc protected amino acids (10 equivalents) were activated by the incubation with DIEA (20 equivalents) and HBTU (10 equivalents) for 15 min, and coupled for 1 h 30 min with nitrogen gas agitation. The completion of reaction was confirmed using Kaiser test. If needed, a second coupling was

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performed by incubating 10 equivalents amino acids, 10 equivalents HOBt, and 12 equivalents DCC for 2 h with nitrogen gas agitation. For incorporation of O-GlcNAcylated serine, pentafluorophenyl (PFP) activated O-GlcNAc FmocSerine was synthesized and purified as described previously.9 Two equivalents of this amino-acid were incubated with the peptide resin overnight. Terminal N-Fmoc protecting groups were removed by 20% piperidine (in DMF) incubation for 5 min, followed by 20 min incubation with flesh 20% piperidine. For O-GlcNAc modified peptide, the O-acetyl groups were removed after the completion of the peptide synthesis by treatment of hydrazine hydrate (80% in MeOH) for 30 min twice on resin. Before the cleavage of peptides, the Dawson linker was activated with treatment of para-nitrophenyl chloroformate (5 equivalents in CH2Cl2) for 1 h, followed by incubation with excess DIEA (5 equivalents in DMF) for 30 min. The peptides were cleaved from resin by incubation in cleavage cocktail (95% TFA, 2.5% water, 2.5% TIS) for 4 h, and precipitated in ice-cold diethyl ether overnight. The pellet was then collected by centrifugation (6,000 x g, 30 min, 4 ℃), and resuspended with water/acetonitrile mixture. The solution was lyophilized, resolubilized, and incubated in thiolysis buffer (6 M guanidine-HCl, 200 mM phosphate, 100 mM MesNa, pH 7.5) for 4 h. The desired peptide-thioester was purified on C18 semiprep RP-HPLC (0-30% B over 60 min). Purified peptides were characterized by ESI-MS. The expected mass for unmodified peptide is 1,540 Da, and the observed mass was 1,541 Da. The expected mass for O-GlcNAc modified peptide is 1,743 Da, and the observed mass was 1,742 Da. Unmodified α-synuclein synthesis. Lyophilized peptide thioester (4 mg, 1 equivalent, 4 mM) and C-terminal fragment (26 mg, 2 equivalents) were solubilized in ligation buffer (6 M guanidine-HCl, 300 mM phosphate, 30 mM TCEP, 30 mM MPAA, pH 7.5) and rocked at room temperature. The reaction was monitored by RP-HPLC (10-45% B over 60 min). Once the completion of the ligation reaction was confirmed by RPHPLC and ESI-MS, the reaction mixture was diluted to 2 mM and acidified to pH 4 with HCl. Methoxyamine (100 mM final concentration) was added, and the resulting solution was incubated at room temperature for additional 4 h. The deprotection of thioproline was confirmed by ESI-MS. The product was purified on C18 semiprep RP-HPLC, and lyophilized. Subsequently, the purified and lyophilized product (1 equivalent, 2 mM) and N-terminal thioester (2 equivalents) were resuspended in the same ligation buffer as above. The reaction was rocked at 25 ℃ and monitored by RP-HPLC (25-60% B over 60 min). Once the reaction was completed, the product was purified by C4 semiprep RP-HPLC and lyophilized. Radical catalyzed desulfurization was performed by solubilizing the full length protein at the concentration of 0.75 mg ml-1 in buffer (6 M guanidine-HCl, 300 mM phosphate, 300 mM TCEP, 2.5% v/v ethanethiol, 10% v/v tertbutylthiol, pH 7.0) and addition of radical initiator, VΑ-061 (200 mM in MeOH, 2 mM final concentration). The reaction was incubated at 37 ℃ with constant agitation for 16 h. The product was purified with C4 analytical RP-HPLC (25-60% B over 60 min). The purified unmodified α-synuclein was characterized by RP-HPLC (070% B over 60 min), ESI-MS. The expected mass is 14,460 Da, and the observed mass was 14,461 ± 2 Da. Synthesis of O-GlcNAc modified α-synuclein. Purified OGlcNAc modified peptide thioester (4 mM, 1 equivalent) and α-synuclein C-terminal fragment (2 equivalents) were resus-


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pended in the ligation buffer (6 M guanidine-HCl, 300 mM phosphate, 30 mM TCEP, 30 mM MPAA, pH 7.5), and the resulting solution was rocked for 15 h at 25 ℃. The reaction was monitored by RP-HPLC (10-45% B over 60 min). Once the completion of the ligation reaction was confirmed by RPHPLC and ESI-MS, the reaction mixture was diluted to 2 mM and acidified to pH 4 by addition of HCl. Methoxyamine (100 mM final concentration) was added to the solution, and incubated for additional 4 h. Deprotection of thioproline was observed by ESI-MS. The product was purified by RP-HPLC (10-45% B over 60 min) and lyophilized. Subsequently, the lyophilized product (1 equivalent, 2 mM) was dissolved in the ligation buffer with N-terminal thioester (2 equivalents), and rocked at room temperature for 30 h. The reaction was monitored by RP-HPLC. Once the reaction was completed, the product was isolated on C4 analytical RP-HPLC (25-60% B over 60 min). The product fractions were pooled and lyophilized. Finally, radical-catalyzed desulfurization was performed by resuspending full length O-GlcNAc modified α-synuclein with cysteines in desulfurization buffer (6 M guanidine-HCl, 200 mM phosphate, 300 mM TCEP, 2.5% ethanethiol, 10% tertbutylthiol, pH 7.0). The reaction was initiated with the addition of radical initiator, VΑ-061 (200 mM in MeOH, 2 mM final concentration), and the reaction solution was incubated at 37 ℃ with constant agitation in inert gas for 12 h. Desulfurized product was purified on C4 analytical RP-HPLC (25-60% B over 60 min). The purified O-GlcNAc modified αsynuclein was characterized by RP-HPLC and ESI-MS. The expected mass is 14,663 Da, and the observed mass was 14,666 ± 2 Da. Aggregation reaction. Synthetic or recombinant protein was dissolved with bath sonication in a reaction buffer (10 mM phosphate, 0.05% sodium azide, pH 7.4) to make its final concentration at either 50 µM or 25 µM. The solution was centrifuged at 15,000 rpm for 15 min at 4 ℃ to remove any debris, and the supernatant was aliquoted into triplicate reactions. The samples were incubated at 37 ℃ with constant agitation (1,000 rpm) in a Thermomixer F1.5 (Eppendorf) for 7 d. At each time point, solution was aliquoted for ThT analysis. Circular dichroism (CD) spectroscopy. All circular dichroism (CD) spectra were taken with Jasco-J-815 spectrometer at room temperature. Sample aliquots were diluted to 7.5 µM with the aggregation reaction buffer without sodium azide in a 1 mm path length quartz cuvette at 25 ℃. The far UV spectra (195 nm-250 nm) were obtained by averaging three scans with 50 nm min-1 scanning speed, 1 nm bandwidth, a 0.1 nm step size, data integral speed of 4. The buffer readings were subtracted for all samples, and the data were converted into mean residue ellipticity. Dynamic light scattering (DLS). Dynamic light scattering data were obtained with Wyatt Technologies Dynastar. All samples were at t = 0 h of aggregation reaction (50 µM). For all data, an average of 10 scans at 25 ℃ was obtained with laser power adjusted to intensity of 2.6E6 counts sec-1. To calculate radii, Raleigh sphere approximation was used. Thioflavin T (ThT) fluorescence. α-Synuclein aggregation progression was quantified by ThT fluorescence. Samples from the aggregation reaction were diluted to 1.25 µM protein concentration with 20 µM ThT dye in the reaction buffer, followed by brief vortex and incubation for 2 min. Samples in 10 mm path length quartz cuvette were analyzed using NanoLog

spectrofluorometer (Horiba), λex at 450 nm with 4 nm slit, λem at 482 nm with 4 nm slit, data integration time of 0.1 sec, averaging 3 scans. Data were measured in triplicate for all aggregation reaction conditions. Transmission electron microscopy (TEM). At the end of the aggregation reactions, protein solution was diluted to 15 µM by adding the reaction buffer, and the diluted solution (10 µL) was incubated with a formvar coated copper grid (150 mesh, Electron Microscopy Science) for 5 min. Subsequently, the grid was negatively stained with 1% uranyl acetate for 2 min, and washed three times with 1% uranyl acetate. Each time excess liquid was removed with filter paper. The grid was dried for 48 h. Grids were visualized with a JOEL JEM-2100F transmission electron microscope operated at 200 kV, 600,000x magnification and an Orius Pre-GIF CCD. SDS-PAGE Analysis. At each time point, 10 uL of aggregation reaction sample was aliquoted, centrifuged at 20,000 x g for 1 h at 25 ℃. The supernatant was transferred into a new tube and lyophilized to dryness. The lyophilized sample was solubilized in fresh 8M urea 20 mM HEPES buffer (pH 8.0) with subsequent bath sonication for 20 min. The sample was boiled for 10 min with 4X SDS loading buffer and loaded on 4-20% Criterion precast gel (BioRad) and separated by SDSPAGE at 195V. The gel was stained with Coomassie brilliant blue for 30 min, and destained with 1:4:5 acetic acid/water/methanol solution overnight. Circular dichroism (CD) of α-synuclein with lipids. All circular dichroism (CD) spectra were collected with Jasco-J815 spectrometer at room temperature. Samples were prepared by mixing 1:100 ratio of a protein and desired lipid mixture and incubated at room temperature for 20 min. Lipid vesicles were prepared with 1-palmitoyl-2-oleoyl-sn-glycero-3[phospho-RAC-(1-glycerol)] (POPG), or by mixing different ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC). Dried lipid films were solubilized in 10 mM phosphate buffer at pH 7.4 by vortexing. All spectra (190 nm-250 nm) were collected with scan rate of 50 nm min-1, band width of 1 nm, data integration time of 8 s, and a 0.1 nm step resolution. Appropriate buffer spectra were subtracted from the final spectra.

ASSOCIATED CONTENT Supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]

Notes The authors have no competing financial interests.

ACKNOWLEDGMENT This research was supported by the National Institutes of Health (R01GM114537 to M.R.P.). Circular dichroism and dynamic light scattering measurement were performed at the USC Nano Biophysics Core Facility. Transmission electron microscopy of protein fibers was performed at the USC Center for Electron Microscopy and Microanalysis.



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O-GlcNAcylation of Î±-Synuclein at Serine 87 Reduces Aggregation

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