Transglutaminase-mediated intramolecular cross-linking of membrane-bound alpha-synuclein promotes amyloid formation in Lewy bodies.
The alpha-synuclein immunopositive and chaotrope-insoluble material from human brains with Lewy body pathology was analyzed by mass spectrometry. From the proteinase K-cleavable peripheral fraction of Lewy bodies, which was densely cross-linked by gamma-glutamyl-epsilon-lysine bonds between HspB1 and ubiquitin in a pattern similar to neurofibrillary tangles (Nemes, Z., Devreese, B., Steinert, P. M., Van Beeumen, J., and Fésüs, L. (2004) FASEB J. 18, 1135-1137), 53 proteins were identified. In the core of Lewy bodies only alpha-synuclein was found, and it contained a low amount of intramolecular cross-links between Gln-99 and Lys-58. In vitro cross-linking of alpha-synuclein by transglutaminases 1-3 and 5 produced a heterogeneous population of variably cross-linked alpha-synucleins in solution, which inhibited the aggregation of the protein into amyloid. However, in the presence of phosphatidylserine-rich membranes and micromolar calcium concentrations, the cross-linking by transglutaminases 1, 2, and 5 showed specificity toward the utilization of Gln-99 and Lys-58. As shown by thioflavin T fluorescence monitoring, the formation of this cross-link accelerated the aggregation of native alpha-synuclein. Chemical cross-linking of residues 58-99 triggered amyloid formation, whereas such bonding of residues 99 to 10 was inhibitory. Our findings reveal the pivotal role of membrane attachment and transglutaminase-mediated intermolecular cross-linking for the propagative misfolding and aggregation of alpha-synuclein.
Nemes
Zoltán
Z
Department of Psychiatry, Signaling and Apoptosis Research Group, Hungarian Academy of Sciences, Research Center for Molecular Medicine, University of Debrecen, Debrecen H-4012, Hungary. znemes@dote.hu
Petrovski
Goran
G
Aerts
Maarten
M
Sergeant
Kjell
K
Devreese
Bart
B
Fésüs
László
L
D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D000682:Amyloid / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D005260:Female; D006801:Humans; D016631:Lewy Bodies / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D012995:Solubility; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D051844:alpha-Synuclein / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D000682:Amyloid; D003432:Cross-Linking Reagents; D004151:Dipeptides; D025801:Ubiquitin; D051844:alpha-Synuclein; C001299:epsilon-(gamma-glutamyl)-lysine; D011503:Transglutaminases; D019286:Endopeptidase K