FIGURE 4.

A, transglutaminase 1–3 and 5 elicit a promiscuous cross-linking of α-synuclein (α-Syn) in solution, as evidenced by the formation of cross-linked oligomers not fully separated by size on SDS-PAGE. 250 μg/ml α-synuclein was exhaustively reacted with recombinant human TGs in solution. Electrophoretic separation of 1 μg/lane transglutaminase-modified α-synuclein was visualized by immunoblotting. Low (10 μg/ml) α-synuclein concentrations favor intramolecular cross-linking, as shown by the dispersion of α-synuclein electrophoretic mobility toward faster moving products (B) and multiple cross-linked glutamine and lysine residues identified in the transglutaminated products by mass sequencing after digestion with trypsin and Glu-C endoprotease (C). Mass peaks were sequenced by electrospray ionization-tandem mass spectrometry and shown in a deconvoluted format. Only transglutaminated product peaks are labeled. The cross-linked residues are listed in D. Residues utilized for cross-linking are shown in superscript, and the neglected ones are shown in subscript. Single letter code for amino acids is used.