Extension of A beta2M amyloid fibrils with recombinant human beta2-microglobulin.
In order to elucidate the pathogenesis of A beta2M amyloidosis, we established an experimental system to study the mechanism of amyloid fibril formation or degradation in vitro. We compared the kinetics of A beta2M amyloid fibril (fA beta2M) extension with native beta2microglobulin (n-beta2M) purified from the urine of a patient suffering from renal insufficiency, with that with recombinant beta2M (r-beta2M) in vitro. n-Beta2M and r-beta2M were incubated with fA beta2M purified from synovial tissues excised from A beta2M amyloidosis patients. The fA beta2M extension reaction could be explained by a first-order kinetic model in both beta2Ms. The extension reaction was greatly dependent on the pH of the reaction mixture and maximum around pH 2.5-3.0 in both beta2Ms. The fA beta2M extended with both beta2Ms assumed the similar helical filament structure, although the fibrils extended with r-beta2M were slightly wider than those extended with n-beta2M and the former fibrils assumed a helical structure more clearly as compared to the latter. In order to obtain pure, unmodified fA beta2M, we next extended fA beta2M repeatedly by the algorithmic protocol with r-beta2M. 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 fA beta2M extended repeatedly with r-beta2M were composed solely of r-beta2M. The use of these r-beta2M and fA beta2M will be advantageous to assess the effects of several amyloid-associated molecules in the formation or degradation of fA beta2M in vitro.
Yamaguchi
I
I
Department of Pathology, Fukui Medical University, Japan.
Hasegawa
K
K
Naiki
H
H
Mitsu
T
T
Matuo
Y
Y
Gejyo
F
F
D000682:Amyloid / Q000737:chemistry / Q000648:ultrastructure* / Q000652:urine; D000686:Amyloidosis / Q000473:pathology*; D006801:Humans; D007676:Kidney Failure, Chronic / Q000209:etiology / Q000652:urine; D007700:Kinetics; D008854:Microscopy, Electron; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*
D000682:Amyloid; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin