Mishima Tomonori, Ohkuri Takatoshi, Monji Akira, Kanemaru Takaaki, Abe Yoshito, Ueda Tadashi
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
J Mol Biol. 2009 Oct 2;392(4):1033-43. doi: 10.1016/j.jmb.2009.07.078. Epub 2009 Aug 6.
Many proteins form amyloid-like fibrils in vitro under partially or highly unfolding conditions. Recently, we showed that the residual structure in highly unfolded state is closely related to amyloid fibril formation in hen lysozyme. Thus, to better understand the role of the residual structure on amyloid fibril formation, we focused on AL amyloidosis, which results from the extracellular deposition of monoclonal immunoglobulin light-chain variable domains (V(L)s) as insoluble fibrils. We examined the relationship between the residual structure and amyloid fibril formation on three lambda6 recombinant V(L) (rVlambda6) proteins, wild type, Jto, and Wil. Although rVlambda6 proteins are highly unfolded in pH 2, (15)N NMR transverse relaxation experiments revealed nonrandom structures in regions, which include some hydrophobic residues and a single disulfide bond, indicating the existence of residual structure in rVlambda6 proteins. However, the residual structure of Wil was markedly disrupted compared with those of the other proteins, despite there being no significant differences in amino acid sequences. Fibrillation experiments revealed that Wil had a longer lag time for fibril formation than the others. When the single disulfide bond was reduced and alkylated, the residual structure was largely disrupted and fibril formation was delayed in all three rVlambda6 proteins. It was suggested that the residual structure in highly unfolded state has a crucial role in amyloid fibril formation in many proteins, even pathogenic ones.
许多蛋白质在部分或高度展开的条件下于体外形成淀粉样纤维。最近，我们发现高度展开状态下的残余结构与溶菌酶的淀粉样纤维形成密切相关。因此，为了更好地理解残余结构在淀粉样纤维形成中的作用，我们聚焦于AL淀粉样变性，它是由单克隆免疫球蛋白轻链可变区（V(L)s）作为不溶性纤维在细胞外沉积所致。我们研究了野生型、Jto和Wil这三种λ6重组V(L)（rVλ6）蛋白的残余结构与淀粉样纤维形成之间的关系。尽管rVλ6蛋白在pH 2时高度展开，但(15)N NMR横向弛豫实验揭示了包括一些疏水残基和一个二硫键的区域存在非随机结构，表明rVλ6蛋白中存在残余结构。然而，尽管氨基酸序列没有显著差异，但与其他蛋白相比，Wil的残余结构明显被破坏。纤维形成实验表明，Wil形成纤维的延迟时间比其他蛋白更长。当单一二硫键被还原并烷基化时，所有三种rVλ6蛋白的残余结构都被大大破坏，纤维形成延迟。这表明高度展开状态下的残余结构在许多蛋白质甚至致病性蛋白质的淀粉样纤维形成中起着关键作用。
