A chemically modified preparation of alpha2-macroglobulin binds beta-amyloid peptide with increased affinity and inhibits Abeta cytotoxicity.
Macromolecules that bind beta-amyloid peptide (Abeta) and neutralize Abeta cytotoxicity offer a promising new approach for treating Alzheimer's disease. When the plasma protein, alpha2-macroglobulin (alpha2M), is treated with methylamine (alpha2M-MA), it undergoes conformational change and acquires Abeta-binding activity. In this study, we demonstrate that a chemically stabilized preparation of human alpha2M conformational intermediates (alpha2M-cis-Pt/MA) binds Abeta with greatly increased affinity, compared with alpha2M-MA. alpha2M-cis-Pt/MA was generated by reacting alpha2M with the protein cross-linking reagent, cis-Pt, followed by methylamine. Increased Abeta-binding to alpha2M-cis-Pt/MA was demonstrated by co-migration of radio-iodinated proteins in non-denaturing PAGE, chemical cross-linking, and co-immunoprecipitation. The apparent K(D) for Abeta-binding to alpha2M-cis-Pt/MA was decreased 10-fold, compared with alpha2M-MA, to 29 nm. Native alpha2M demonstrated negligible Abeta-binding, as anticipated. alpha2M-cis-Pt/MA markedly counteracted Abeta-induced C6 cell apoptosis. Essentially complete inhibition of apoptosis was observed even when the Abeta was present at fourfold molar excess to alpha2M-cis-Pt/MA. Under equivalent conditions, alpha2M-MA inhibited apoptosis by 25 +/- 6%. When Abeta and alpha2M-cis-Pt/MA were added to human plasma in vitro, significant binding was detected. No binding was observed when an equivalent concentration of native alpha2M or alpha2M-MA was added to plasma. We propose that alpha2M-cis-Pt/MA is a novel alternative to Abeta-specific antibodies, for studying the efficacy of Abeta-binding agents in vitro and in vivo.
Mettenburg
Joseph M
JM
Department of Pathology, University of California, San Diego, La Jolla, California, USA.
Arandjelovic
Sanja
S
Gonias
Steven L
SL
D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D016923:Cell Death / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007456:Iodine Isotopes / Q000494:pharmacology; D046911:Macromolecular Substances / Q000494:pharmacology; D008744:Methylamines / Q000494:pharmacology; D010455:Peptides; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D012394:Rosaniline Dyes / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000378:metabolism*
D016229:Amyloid beta-Peptides; D003432:Cross-Linking Reagents; D007456:Iodine Isotopes; D046911:Macromolecular Substances; D008744:Methylamines; D010455:Peptides; D012394:Rosaniline Dyes; D000511:alpha-Macroglobulins; C048139:Coomassie blue; C027451:methylamine; D002945:Cisplatin