Cross-linking and amyloid formation by N- and C-terminal cysteine derivatives of human apolipoprotein C-II.
We have investigated the effect of disulfide cross-linking on amyloid formation by human apolipoprotein (apo) C-II. Three derivatives of apoC-II were generated by inserting a cysteine residue on either the N-terminus (C(N)-apoC-II), C-terminus (C(C)-apoC-II), or both termini (C(N)C(C)-apoC-II). Under reducing conditions, all derivatives formed amyloid with a fibrous ribbon morphology similar to that of wild-type apoC-II. Under oxidizing conditions, C(N)- and C(N)C(C)-apoC-II formed a highly tangled network of fibrils, suggesting that the addition of an N-terminal cysteine to apoC-II promotes interfibril disulfide cross-links. Fibrils formed by C(C)-apoC-II under oxidizing conditions were closely packed but less tangled than fibrils formed by the C(N) and C(N)C(C) derivatives. The frequency of closed ring structures was more than doubled for C(C)-apoC-II compared to wild-type apoC-II. The kinetics of fibril formation by all cysteine derivatives was markedly enhanced under oxidizing conditions, suggesting that disulfide cross-linking promotes amyloid formation. Substoichiometric levels of preformed C(N)- and C(C)-apoC-II dimers accelerate amyloid formation by wild-type apoC-II. These data suggest that the N- and C-termini of apoC-II are close together in the amyloid fibril such that covalent cross-linking of either the N or C end of apoC-II promotes nucleation and the "seeding" of fibril growth.
Pham
Chi L L
CL
Russell Grimwade School of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
Hatters
Danny M
DM
Lawrence
Lynne J
LJ
Howlett
Geoffrey J
GJ
D000682:Amyloid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D006801:Humans; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics
D000682:Amyloid; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D003545:Cysteine; D004229:Dithiothreitol