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

Ann Clin Biochem 2008;45:489-495
© 2008 Association for Clinical Biochemistry



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Original Articles

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}

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 β2min 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 destabilizationof 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 containingtypical amyloid fibrils at 24 hours, when ThT fluorescence intensitywas 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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