Extension of Aβ2M amyloid fibrils with recombinant human β2-microglobulin

Abstract

In order to elucidate the pathogenesis ofAβ2M amyloidosis, we established an experimental system to study the mechanism of amyloid fibril formation or degradation in vitro. We compared the kinetics of Aβ2M amyloid fibril (fAβ2M) extension with native β2microglobulin (n-β2M) purified from the urine of a patient suffering from renal insufficiency, with that with recombinant β2M (r-β2M) in vitro. n-β2M and r-β2M were incubated with fβ32Mpurifiedfrom synovial tissues excised from Aβ2M amyloidosis patients. The fA β2M extension reaction could be explained by a first-order kinetic model in both β2Ms. The extension reaction was greatly dependent on the pH of the reaction mixture and maximum around pH 2.5-3.0 in both p2Ms. ThefAβ2M extended with both p2Ms assumed the similar helical filament structure, although the fibrils extended with r-β2M were slightly wider than those extended with n-β2M and the former fibrils assumed a helical structure more clearly as compared to the latter. In order to obtain pure, unmodifiedfAβ2M, we next extendedfA β2M repeatedly by the algorithmic protocol with r-β2M. As the generation of the extended fibrils proceeded, the initial rate of the extension reaction increased. The ultrastructure of fibrils was completely preserved throughout the repeated extension steps. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting revealed that f A P2M extended repeatedly with r-β2Mwere composed solely ofr-β2M. The use of these r-β2M and fAβ2M will be advantageous to assess the effects of several amyloid-associated molecules in the formation or degradation offAβ2M in vitro.