Recent studies have suggested that α-synuclein (α-syn)(molecular weight: 14 kDa) exists predominantly as a tetramer (~58 kDa) in solution. However, a subsequent report has shown that brain- and RBC derived α-syn protein is present as a natively unfolded monomer. Therefore, the native structure of α-syn remains controversial. To determine a higher molecular structure of the α-syn protein in the biological fluid, we analyzed cerebrospinal fluid (CSF) fractionated using ultrafiltration (UF) and size-exclusion chromatography (SEC) with a Superose12 column, as well as Western blot (WB) and ELISA. We also expressed A140C α-syn variant, which forms α-syn dimers, and wild-type α-syn in Escherichia coli (recombinant α-syn proteins), which were then analyzed after fractionation with UF and SEC. Recombinant α-syn proteins were subsequently compared with native α-syn in CSF.
When α-syn was evaluated with SEC columns, α-syn in CSF migrated as 50- to 60-kDa proteins; however, in denaturing gels, they migrated as 15-kDa proteins. The similar result was obtained in recombinant wild-type α-syn. The disulfide-linked dimer (A140C) and oligomer migrated slower than either the unfolded recombinant α-syn or the native α-syn in SEC.
We also confirmed by UF in the presence of 8M urea or PBS. The recombinant α-syn proteins or CSF in these conditions were fractionated by centrifugal UF membrane devices with different molecular weight cut-offs (MWCO): 30 and 100kDa. Neither the unfolded recombinant α-syn nor the CSF α-syn was passed through the 30KDa membrane of both normal and 8M Urea conditions.
These results suggest that α-syn detected in about 60kDa fraction in the SEC was a monomeric protein, implying that α-syn in CSF exists as a monomer, not a tetramer.