Role of conformational change in the C-terminus of beta2-microglobulin in dialysis-related amyloidosis.
BACKGROUND
Beta(2)-microglobulin (beta(2)m) has been identified as the precursor protein of dialysis-related amyloidosis (DRA), which is a serious complication for haemodialysis (HD) patients. However, mechanisms underlying beta(2)m amyloid fibril formation remains to be elucidated. We previously demonstrated, in amyloid deposits from HD patients, a conformational isoform of beta(2)m with an unfolded C-terminus. However, no direct experiments have previously been performed to address whether unfolded beta(2)m in the C-terminus may be prone to form amyloid fibrils.


METHODS
To evaluate roles of C-terminal amino acids in beta(2)m-induced amyloid formation, we generated six types of recombinant beta(2)m 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 beta(2)m. To analyse morphological change of beta(2)m, we performed electron microscopy (EM) on the samples with elevated ThT fluorescence intensity. We used ultrasonication to enhance beta(2)m destabilization of the protein.


RESULTS
Beta(2)M Trp95Leu and Arg97Ala showed conformational changes and increased their amyloidgenicity compared with beta(2)m wild-type (WT). With ultrasonication, beta(2)m Trp95Leu and Arg97Ala generated more amyloid fibrils than did beta(2)m WT even in physiological solution. EM showed that beta(2)m 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 beta(2)m may play an important role in DRA and that ultrasonication is useful for analysis of beta(2)m amyloidogenesis.
Kim
Jaemi
J
Department of Molecular Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, Japan.
Motomiya
Yoshihiro
Y
Ueda
Mitsuharu
M
Nakamura
Masaaki
M
Misumi
Yohei
Y
Saito
Shiori
S
Ikemizu
Shinji
S
Misumi
Shogo
S
Ota
Kazutoshi
K
Shinriki
Satoru
S
Kai
Hirofumi
H
Ando
Yukio
Y