C-terminal truncation is a prominent post-translational modification of human erythrocyte α-synuclein.

α-Synuclein (α-Syn) is a protein related to synucleinopathies with high expression in the central nervous system and erythrocytes which are a major source of peripheral α-Syn. Recent reports have suggested the presence of α-Syn within extracellular vesicles (EVs) derived from erythrocytes, potentially contributing to the pathogenesis of synucleinopathies. While Lewy bodies, intracellular inclusions containing aggregated α-Syn, are prominently observed within the brain, their occurrence in peripheral neurons implies the dissemination of synucleinopathy pathology throughout the body via the propagation of α-Syn. In this study, we found erythrocytes and circulating EVs obtained from plasma contained α-Syn, which was separated into four major forms using high-resolution clear native-PAGE and isoelectric focusing. Notably, erythrocyte α-Syn was classified into full-length and C-terminal truncated forms, with truncation observed between Y133 and Q134 as determined by LC-MS/MS analysis. Our finding revealed that C-terminally truncated α-Syn, which was previously reported to exist solely within the brain, was also present in erythrocytes and circulating EVs obtained from plasma.

Post-translational modification of lysine residues in erythrocyte α-synuclein.

α-Synuclein is a protein linked to various synuclein-associated diseases ('synucleinopathies'), including Parkinson's disease, dementia with Lewy Bodies and multiple system atrophy, and is highly expressed in the central nervous system and in erythrocytes. Moreover, α-synuclein-containing erythrocyte-derived extracellular vesicles may be involved in the pathogenesis of synucleinopathies and their progression across the blood-brain barrier. Several post-translational modifications of α-synuclein have been reported in brain inclusions, including S129 phosphorylation, but fewer have been found in erythrocytes. In this study, we analysed the post-translational modifications of erythrocyte α-synuclein using liquid chromatography-mass spectrometry. We found that all lysine residues in the α-synuclein protein could be modified by acetylation, glycation, ubiquitination or SUMOylation but that phosphorylation, nitration and acylation were uncommon minor post-translational modifications in erythrocytes. Since the post-translational modification of lysine residues has been implicated in both membrane association and protein clearance, our findings provide new insight into how synucleinopathies may progress and suggest possible therapeutic strategies designed to target α-synuclein.