Annals of Clinical Biochemistry > Volume 45, Number 5 > Pp. 489-495

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Original Articles

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

Jaemi Kim¹, Yoshihiro Motomiya², Mitsuharu Ueda³, Masaaki Nakamura⁴, Yohei Misumi⁵, Shiori Saito⁶, Shinji Ikemizu⁷, Shogo Misumi⁸, Kazutoshi Ota⁹, Satoru Shinriki⁹, Hirofumi Kai¹ and Yukio Ando³

¹ Department of Molecular Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811; ² Suiyukai Clinic, Nara; ³ Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811; ⁴ National Institute for Minamata Disease, Kumamoto; ⁵ Department of Neurology; ⁶ Department of Biopharmaceutics; ⁷ Department of Structural Biology; ⁸ Department of Pharmaceutical Biochemistry; ⁹ Department of Oral and Maxillofacial Surgery, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan

Corresponding author: Dr Yukio Ando. Email: andoy709{at}kumamoto-u.ac.jp

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 β₂m WT 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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