O-GlcNAcylation of α-Synuclein at Serine 87 Reduces Aggregation without Affecting Membrane Binding

Abstract

The aggregation of neurodegenerative-disease associated proteins can be affected by many factors, including a variety of post-translational 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 were 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 than that for 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 Sulfur-Linked Analogue of O-GlcNAc (S-GlcNAc) Is an Enzymatically Stable and Reasonable Structural Surrogate for O-GlcNAc at the Peptide and Protein Levels

“…Unmodified α -synuclein was recombinantly expressed and purified by RP-HPLC (Figure S10). Unmodified α -synuclein exists as an unstructured random coil in solution, and consistent with our previous results, 14 O-GlcNAcylation at serine 87 did not induce any secondary structure in the protein. Not surprisingly on the basis of our structural data given above, the spectra of α -synuclein(gC87) looked identical to those of both the unmodified and O-GlcNAcylated protein controls, demonstrating that this single-atom substitution has essentially no impact on the secondary structure of α -synuclein in solution (Figure S11).…”

“…14 Separate incubation of these proteins, as well as the O-GlcNAcylated or S-GlcNAcylated β -estrogen receptor peptides, with hOGA showed that while O-GlcNAc can be readily removed the S-GlcNAc modification was completely stable. These data are consistent with previous results obtained by using a bacterial homologue of OGA 19 and show that the hydrolysis of GlcNAc thioglycosides by hOGA observed by Vocadlo and co-workers is most likely confined to more activated leaving groups.…”

“…Notably, we found that the initiating methionine residue was conveniently removed during expression by an endogenous methionine aminopeptidase. 14 Glycopeptide 9 was prepared using standard Fmoc-based solid-phase peptide synthesis on the Dawson aminobenzyol resin that allows the generation of C-terminal peptide thioesters upon linker activation. On-resin deprotection of the acetyl groups on the S-GlcNAc moiety was accomplished using hydrazine before cleavage and purification of the glycopeptide by RP-HPLC (Figure S6).…”

“…As a positive control, the O-GlcNAcylated α -synuclein, α -synuclein(gS87), was prepared using EPL as previously described. 14 Both α -synuclein(gC87) and α -synuclein(gS87) were separately diluted to a concentration of 25 μ M in PBS buffer (pH 7.4) and incubated with hOGA (1 μ M) at 37 °C for ≤72 h. Following incubation, hOGA was denatured and the reaction mixtures were centrifuged to remove any possible aggregates. Following centrifugation, the samples were directly injected onto the HPLC instrument and monitored at 214 nm.…”

O-GlcNAc modification inhibits the calpain-mediated cleavage of α-synuclein

“…27–30 We have previously used synthetic protein chemistry to prepare α-synuclein with O-GlcNAcylation at two of these sites, threonine 72 (T72) and serine 87 (S87). 31,32 Using a variety of biochemical experiments, we found that neither of these modifications inhibit the interaction of α-synuclein with membranes; however, both modifications inhibit protein aggregation, with O-GlcNAcylation at T72 having a stronger inhibitory effect.…”