Cleavage of normal and pathological forms of alpha-synuclein by neurosin in vitro.
Neurosin is one of the serine proteases predominantly expressed in the central nervous system. Neurosin is presumed to play an important role in the degradation of alpha-synuclein (alpha-syn), since a previous study showed that neurosin degrades alpha-syn, inhibits polymerization of alpha-syn in vitro, and exists in Lewy bodies. However, the details of alpha-syn degradation by neurosin are little known. We investigated neurosin-mediated cleavage of alpha-syn by immunoblotting and liquid chromatography-ion trap mass spectrometry (LC/MS/MS). We also compared alpha-syn degradation by neurosin between phosphorylated and non-phosphorylated forms of alpha-syn, and between mutant and wild-type alpha-syn. Neurosin cleaved alpha-syn at specific sites. The major cleavage site was localized between Lys80 and Thr81 within the NAC region (E61 to V95), which is important for alpha-syn aggregation, and accordingly may preclude alpha-syn polymerization. Meanwhile, alternative, minor forms of processing also occur. They conserve the NAC region with truncation of the C-terminal region, and accordingly may contribute to alpha-syn polymerization. Phosphorylated alpha-syn was more resistant to degradation by neurosin than non-phosphorylated alpha-syn. The A30P mutant was more resistant to degradation than the wild-type and other alpha-syn mutants. This resistance to neurosin-mediated degradation of phosphorylated alpha-syn and the A30P mutant, which are, respectively, posttranslational and genetic factors related to the development of Parkinson's disease (PD), provides supporting evidence that neurosin is involved in the pathogenesis of PD.
Kasai
Takashi
T
Department of Neurology, Research Institute for Geriatrics, Kyoto Prefectural University of Medicine, Kyoto 602-0841, Japan.
Tokuda
Takahiko
T
Yamaguchi
Nozomi
N
Watanabe
Yoshihisa
Y
Kametani
Fuyuki
F
Nakagawa
Masanori
M
Mizuno
Toshiki
T
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D007610:Kallikreins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D051844:alpha-Synuclein; C103551:KLK6 protein, human; D007610:Kallikreins