Kim Jaemi, Motomiya Yoshihiro, Ueda Mitsuharu, Nakamura Masaaki, Misumi Yohei, Saito Shiori, Ikemizu Shinji, Misumi Shogo, Ota Kazutoshi, Shinriki Satoru, Kai Hirofumi, Ando Yukio
Department of Molecular Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, Japan.
Ann Clin Biochem. 2008 Sep;45(Pt 5):489-95. doi: 10.1258/acb.2008.008046.
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.
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.
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.
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.
β2-微球蛋白（β2m）已被确定为透析相关淀粉样变性（DRA）的前体蛋白，这是血液透析（HD）患者的一种严重并发症。然而，β2m淀粉样纤维形成的潜在机制仍有待阐明。我们之前在HD患者的淀粉样沉积物中发现了一种C末端未折叠的β2m构象异构体。然而，此前尚未进行直接实验来探讨C末端未折叠的β2m是否易于形成淀粉样纤维。
为了评估C末端氨基酸在β2m诱导的淀粉样形成中的作用，我们生成了六种在C末端区域有氨基酸替代的重组β2m。为了研究它们的构象变化和淀粉样变性，我们测量了重组β2m的圆二色光谱、色氨酸和硫黄素-T（ThT）的荧光强度。为了分析β2m的形态变化，我们对ThT荧光强度升高的样品进行了电子显微镜（EM）观察。我们使用超声处理来增强蛋白质的β2m去稳定性。
与野生型（WT）β2m相比，β2M Trp95Leu和Arg97Ala表现出构象变化并增加了淀粉样变性。通过超声处理，即使在生理溶液中，β2m Trp95Leu和Arg97Ala也比β2m WT产生更多的淀粉样纤维。EM显示，在24小时时，β2m形成了含有典型淀粉样纤维的无定形碎片，此时ThT荧光强度比6小时时低三倍。
β2m C末端的构象变化可能在DRA中起重要作用，并且超声处理有助于分析β2m的淀粉样生成。
