Role of conformational change in the C-terminus of (β2-microglobulin in dialysis-related amyloidosis

Jaemi Kim; Motomiya, Yoshihiro; Ueda, Mitsuharu; Nakamura, Masaaki; Misumi, Yohei; Saito, Shiori; Ikemizu, Shinji; Misumi, Shogo; Ota, Kazutoshi; Shinriki, Satoru; Hirofumi Kai; Ando, Yukio
September 2008
Annals of Clinical Biochemistry;Sep2008, Vol. 45 Issue 5, p489
Academic Journal
Background: β2-Microglobulin (β2m) has been identified as the precursor protein of dialysis-related amyloidosis (DRA), which is a serious complication for haemodialysis (HD) patients. However, mechanisms underlying β2m amyloid fibril formation remains to be elucidated. We previously demonstrated, in amyloid deposits from HD patients, a conformational isoform of β2m with an unfolded C-terminus. However, no direct experiments have previously been performed to address whether unfolded β2m in the C-terminus may be prone to form amyloid fibrils. Methods: To evaluate roles of C-terminal amino acids in β2m-induced amyloid formation, we generated six types of recombinant β2m with amino acid substitutions in the C-terminal region. To investigate their conformational change and amyloidogenicity, we measured circular dichroism spectra, the fluorescence intensity of tryptophan and thioflavin-T (ThT) of the recombinant β2m. To analyse morphological change of β2m, we performed electron microscopy (EM) on the samples with elevated ThT fluorescence intensity. We used ultrasonication to enhance β2m destabilization of the protein. Results: β2M Trp95Leu and Arg97Ala showed conformational changes and increased their amyloidgenicity compared with β2m wild-type (WT). With ultrasonication, β2m Trp95Leu and Arg97Ala generated more amyloid fibrils than did β2mWT even in physiological solution. EM showed that β2m formed amorphous debris containing typical amyloid fibrils at 24 hours, when ThT fluorescence intensity was three-fold lower than that at six hours. Conclusions: Conformational changes in the C-terminus of β2m may play an important role in DRA and that ultrasonication is useful for analysis of β2m amyloidogenesis.


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