O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease

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 physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting 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 increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

Figure 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. 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.

Figure 2. Semisynthesis of α-synuclein

a, α-Synuclein was retrosynthetically deconstructed into a synthetic thioester-peptide (1), a recombinant protein (2) and a recombinant protein thioester (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 transformed 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 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 molecular mass (14,460 ± 3 Da) in excellent agreement with the predicted weight of 14,460 Da.

Figure 3. 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 semisynthetic route outlined in Fig. 2b by 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, O-GlcNAcylation blocks α-synuclein fiber formation. Recombinant α-synuclein or synthetic α-synuclein or α-synuclein(gT72) (50 μM) were subjected to aggregation conditions (agitation at 37 °C) before analysis by ThT fluorescence (λ_ex = 450 nm, λ_ex = 482 nm) at the indicated time points. y-Axis is fold-change in fluorescence compared to recombinant α-synuclein at t = 0 h. Results are the mean ±s.e.m. of three separate experiments. 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 bar: 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) remains soluble during aggregation. After 7 days, aggregation reactions were also separated by centrifugation, resuspended in 8M urea to disassociate any aggregates, and analyzed by SDS-PAGE and coomassie staining. Essentially all of the recombinant α-synuclein formed insoluble aggregates, 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 approximately 1,000 and 100 kDa were detectable in the recombinant α-synuclein aggregation reaction, but were essentially absent in the α-synuclein(gT72) reaction.

Figure 4. 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. Recombinant α-synuclein or α-synuclein(gT72) 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), both recombinant α-synuclein amd α-synuclein(gT72) gave 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. b, Unmodified α-synuclein and α-synuclein(gT72) bend membranes into tubules equally. Recombinant α-synuclein or α-synuclein(gT72) were incubated with a 20-fold excess of POPG vesicles and analyzed using TEM. Both proteins remodeled the vesicles into tube structures of approximately 30 nm in diameter. 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.

Figure 5. 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 measured with ethidium homodimer fluorescence (λ_ex = 528 nm, λ_ex = 617 nm). Treatment with recombinant α-synuclein resulted in significantly more toxicity than α-synuclein(gT72) in both cells 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 extent than α-synuclein(gT72). Results are the mean ±s.e.m. of three separate experiments. Statistical significance (two-tailed, t-test): *P < 0.05, **P = < 0.01.

Figure 6. O-GlcNAcylated α-synuclein is largely excluded from the protein aggregates

a, Recombinant 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 aggregation conditions (agitation at 37 °C) before analysis by ThT fluorescence (λ_ex = 450 nm, λ_ex = 482 nm) at the indicated time points. y-Axis is fold-change in fluorescence compared to recombinant α-synuclein at t = 0 h. Increasing percentages of α-synuclein(gT72) compared to 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 bar: 500 nm. Increasing percentages of α-synuclein(gT72) compared to recombinant α-synuclein resulted in the formation of fewer overall fiber structures but did not change the gross structures of the fibers formed. c, Recombinant α-synuclein at 50 μM or 25 μM or a 1:1 mixture of recombinant α-synuclein (25 μM) and α-synuclein(gT72) (25 μM) were were subjected to aggregation conditions (agitation at 37 °C) before analysis by ThT fluorescence (λ_ex = 450 nm, λ_ex = 482 nm) at the indicated time points. y-Axis is fold-change in fluorescence compared to 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 bar: 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 significance (two-tailed, t-test): *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.