A chemically modified preparation of α2‐macroglobulin binds β‐amyloid peptide with increased affinity and inhibits Aβ cytotoxicity

Macromolecules that bind β‐amyloid peptide (Aβ) and neutralize Aβ cytotoxicity offer a promising new approach for treating Alzheimer's disease. When the plasma protein, α2‐macroglobulin (α2M), is treated with methylamine (α2M‐MA), it undergoes conformational change and acquires Aβ‐binding activity. In this study, we demonstrate that a chemically stabilized preparation of human α2M conformational intermediates (α2M‐ cis ‐Pt/MA) binds Aβ with greatly increased affinity, compared with α2M‐MA. α2M‐ cis ‐Pt/MA was generated by reacting α2M with the protein cross‐linking reagent, cis ‐Pt, followed by methylamine. Increased Aβ‐binding to α2M‐ 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 Aβ‐binding to α2M‐ cis ‐Pt/MA was decreased 10‐fold, compared with α2M‐MA, to 29 n m . Native α2M demonstrated negligible Aβ‐binding, as anticipated. α2M‐ cis ‐Pt/MA markedly counteracted Aβ‐induced C6 cell apoptosis. Essentially complete inhibition of apoptosis was observed even when the Aβ was present at fourfold molar excess to α2M‐ cis ‐Pt/MA. Under equivalent conditions, α2M‐MA inhibited apoptosis by 25 ± 6%. When Aβ and α2M‐ cis ‐Pt/MA were added to human plasma in vitro , significant binding was detected. No binding was observed when an equivalent concentration of native α2M or α2M‐MA was added to plasma. We propose that α2M‐ cis ‐Pt/MA is a novel alternative to Aβ‐specific antibodies, for studying the efficacy of Aβ‐binding agents in vitro and in vivo .