Facile "stop codon" method reveals elevated neuronal toxicity by discrete S87p-α-synuclein oligomers.

Herein, a new method for preparing phosphorylated proteins at specific sites has been applied to α-synuclein (α-Syn). Three different α-Syn species phosphorylated at Serine 87 (S87p-α-Syn), Serine 129 (S129p-α-Syn) and Serine 87/129 (S87p,129p-α-Syn) were prepared through the 'stop codon' method and verified by LC/MS/MS and immunoblotting. Each type of phosphorylated α-Syn was tested for oligomerization trends and cellular toxicity with dopamine (DA), Cu(2+) ions and pyridoxal 5'-phosphate. Aggregation trends induced by DA or DA/Cu(2+) were similar between phosphorylated and non-phosphorylated α-Syn in SDS-PAGE. However, except for the monomer, phosphorylated oligomers showed higher toxicity than the non-phosphorylated α-Syn (Np-α-Syn) oligomers via WST-1 assays when tested on SH-SY5Y human neuroblastoma cells. In particular, S87p-α-Syn and S87p,129p-α-Syn oligomers induced by DA/Cu(2+), showed higher toxicity than did S129p-α-Syn. When α-Syn was treated with pyridoxal 5'-phosphate in the presence of DA or Cu(2+) to determine aggregation effects, high inhibition effects were shown in both non-phosphorylated and phosphorylated versions. α-Syn co-incubated with DA or DA/Cu(2+) showed less cellular toxicity upon pyridoxal 5'-phosphate treatment, especially in the case of DA-induced Np-α-syn. This study supports that phosphorylated oligomers of α-Syn at residue 87 can contribute to neuronal toxicity and the pyridoxal 5'-phosphate can be used as an inhibitor for α-Syn aggregation.