Chiba Takeshi, Hagihara Yoshihisa, Higurashi Takashi, Hasegawa Kazuhiro, Naiki Hironobu, Goto Yuji
Institute for Protein Research, Osaka University, and CREST, Japan Science and Technology Corp., Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
J Biol Chem. 2003 Nov 21;278(47):47016-24. doi: 10.1074/jbc.M304473200. Epub 2003 Sep 4.
Beta2-microglobulin (beta2-m), a typical immunoglobulin domain made of seven beta-strands, is a major component of amyloid fibrils formed in dialysis-related amyloidosis. To understand the mechanism of amyloid fibril formation in the context of full-length protein, we prepared various mutants in which proline (Pro) was introduced to each of the seven beta-strands of beta2-m. The mutations affected the amyloidogenic potential of beta2-m to various degrees. In particular, the L23P, H51P, and V82P mutations significantly retarded fibril extension at pH 2.5. Among these, only L23P is included in the known "minimal" peptide sequence, which can form amyloid fibrils when isolated as a short peptide. This indicates that the residues in regions other than the minimal sequence, such as H51P and V82P, determine the amyloidogenic potential in the full-length protein. To further clarify the mutational effects, we measured their stability against guanidine hydrochloride of the native state at pH 8.0 and the amyloid fibrils at pH 2.5. The amyloidogenicity of mutants showed a significant correlation with the stability of the amyloid fibrils, and little correlation was observed with that of the native state. It has been proposed that the stability of the native state and the unfolding rate to the amyloidogenic precursor as well as the conformational preference of the denatured state determine the amyloidogenicity of the proteins. The present results reveal that, in addition, stability of the amyloid fibrils is a key factor determining the amyloidogenic potential of the proteins.
β2微球蛋白（β2-m）是一种由七条β链构成的典型免疫球蛋白结构域，是透析相关淀粉样变性中形成的淀粉样纤维的主要成分。为了在全长蛋白的背景下理解淀粉样纤维形成的机制，我们制备了各种突变体，其中在β2-m的七条β链中的每一条上都引入了脯氨酸（Pro）。这些突变在不同程度上影响了β2-m的淀粉样变性潜力。特别是，L23P、H51P和V82P突变在pH 2.5时显著延缓了纤维的延伸。其中，只有L23P包含在已知的“最小”肽序列中，当作为短肽分离时，该序列可以形成淀粉样纤维。这表明最小序列以外区域的残基，如H51P和V82P，决定了全长蛋白中的淀粉样变性潜力。为了进一步阐明突变效应，我们测量了它们在pH 8.0时对天然状态盐酸胍的稳定性以及在pH 2.5时对淀粉样纤维的稳定性。突变体的淀粉样变性与淀粉样纤维的稳定性显著相关，而与天然状态的稳定性几乎没有相关性。有人提出，天然状态的稳定性、向淀粉样变性前体的解折叠速率以及变性状态的构象偏好决定了蛋白质的淀粉样变性。目前的结果表明，此外，淀粉样纤维的稳定性是决定蛋白质淀粉样变性潜力的关键因素。
