Comparison of N-Acetyl-Glucosamine to Other Monosaccharides Reveals Structural Differences for the Inhibition of α-Synuclein Aggregation

Abstract

O-GlcNAc modification of the microtubule associated protein tau and α-synuclein can directly inhibit the formation of the associated amyloid fibers associated with major classes of neurodegenerative diseases. However, the mechanism(s) by which this posttranslational modification (PTM) inhibit amyloid aggregation are still murky. One hypothesis is that O-GlcNAc simply acts as a polyhydroxylated steric impediment to the formation of amyloid oligomers and fibers. Here, we begin to test this hypothesis by comparing the effects of O-GlcNAc to other similar monosaccharidesglucose, N-acetyl-galactosamine (GalNAc), or mannoseon α-synuclein amyloid formation. Interestingly, we find that this quite reasonable hypothesis is not entirely correct. More specifically, we used four types of biochemical and biophysical assays to discover that the different sugars display different effects on the inhibition of amyloid formation, despite only small differences between the structures of the monosaccharides. These results further support a more detailed investigation into the mechanism of amyloid inhibition by O-GlcNAc and has potential implications for the evolution of N-acetyl-glucosamine as the monosaccharide of choice for widespread intracellular glycosylation.

Glycation modulates alpha-synuclein fibrillization kinetics: A sweet spot for inhibition

“…Counterintuitively, the smallest agent (MGO) has the largest effect, while the effect of the sugars remains difficult to rationalize by size or chemical structure. A glycosylation study on αSN with similar compounds (GlcNAc, GalNAc, Glc, and Man) showed that even minor chemical differences between these compounds lead to different levels of inhibition of fibril formation as well as formation of different fibrils ( 73 ). It should also be remembered that AGE products are tremendously diverse (comprising hundreds of different compounds, only a few of which are shown in Fig.…”

Methods for Studying Site-Specific O-GlcNAc Modifications: Successes, Limitations, and Important Future Goals

“…We further showed that the PTM is protective against the protein’s cleavage by calpain, 78 and that the anti-aggregation phenotypes imparted by the GlcNAc moiety are unique and not reproduced by other sugars. 79 We also used these techniques to study the effect of O-GlcNAcylation on semisynthetic HMGB1 modified at positions Ser100 and demonstrated the influence O-GlcNAc has on HMGB1-DNA interactions. 80 We showed that the PTM generally enhanced the interactions between the protein and DNA and resulted in error-prone repair of ICL-damaged plasmids in U2OS cell extracts.…”

“…For example, we recently demonstrated that even as subtle of a change as O-GlcNAc to O-GalNAc or O-glucose alters the aggregation behavior of the protein α-synuclein. 79 While incorporation of bona fide O-GlcNAc or even S-GlcNAc through amber suppression in E. coli has been unsuccessful, this should still be a goal of the community. It is possible that different expression systems (e.g., Vmax X2 93 ) or other tRNA/synthetase pairs may overcome the hurdles, and the direct incorporation of either serine or threonine O-GlcNAc by genetic codon expansion would be transformative.…”

Production of Recombinant Alpha-Synuclein: Still No Standardized Protocol in Sight

“…In this comparative study, the authors observed that aSyn extracted by periplasmic lysis and ammonium sulfate precipitation has a higher aggregation propensity, compared to aSyn extracted by boiling, or acid precipitation. However, so far, no study has systematically compared the impact of shorter (5–15 min) heat-treatments that are used in most protocols [ 4 , 19 , 44 , 47 , 51 , 52 ]. In addition, different buffers are used to lyse the bacterial cells [ 4 , 17 , 19 , 37 , 44 , 47 , 51 ], and their effects are largely unknown.…”

“…However, so far, no study has systematically compared the impact of shorter (5–15 min) heat-treatments that are used in most protocols [ 4 , 19 , 44 , 47 , 51 , 52 ]. In addition, different buffers are used to lyse the bacterial cells [ 4 , 17 , 19 , 37 , 44 , 47 , 51 ], and their effects are largely unknown.…”