Laboratory of Genes and Development, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
J Mol Biol. 2010 Aug 27;401(4):605-17. doi: 10.1016/j.jmb.2010.06.039. Epub 2010 Jun 25.
FBLN5 encodes fibulin-5, an extracellular matrix calcium-binding glycoprotein that is essential for elastic fibre formation. FBLN5 mutations are associated with two distinct human diseases, age-related macular degeneration (AMD) and cutis laxa (CL), but the biochemical basis for the pathogenic effects of these mutations is poorly understood. Two missense mutations found in AMD patients (I169T and G267S) and two missense mutations found in CL patients (G202R and S227P) were analysed in a native-like context in recombinant fibulin-5 fragments. Limited proteolysis, NMR spectroscopy and chromophoric calcium chelation experiments showed that the G267S and S227P substitutions cause long-range structural effects consistent with protein misfolding. Cellular studies using fibroblast cells further demonstrated that these recombinant forms of mutant fibulin-5 were not present in the extracellular medium, consistent with retention. In contrast, no significant effects of I169T and G202R substitutions on protein fold and secretion were identified. These data establish protein misfolding as a causative basis for the effects of G267S and S227P substitutions in AMD and CL, respectively, and raise the possibility that the I169T and G202R substitutions may be polymorphisms or may increase susceptibility to disease.
FBLN5 编码纤维连接蛋白 5，这是一种细胞外基质钙结合糖蛋白，对弹性纤维的形成至关重要。FBLN5 突变与两种不同的人类疾病有关，即年龄相关性黄斑变性（AMD）和皮肤松弛症（CL），但这些突变的致病作用的生化基础仍知之甚少。在天然状态下分析了在 AMD 患者中发现的两种错义突变（I169T 和 G267S）和在 CL 患者中发现的两种错义突变（G202R 和 S227P）在重组纤维连接蛋白 5 片段中的作用。有限的蛋白水解、NMR 光谱和发色体钙螯合实验表明，G267S 和 S227P 取代导致与蛋白质错误折叠一致的长程结构效应。使用成纤维细胞的细胞研究进一步表明，这些突变型纤维连接蛋白 5 的重组形式不存在于细胞外介质中，这与保留一致。相比之下，I169T 和 G202R 取代对蛋白质折叠和分泌没有显著影响。这些数据将蛋白质错误折叠确立为 AMD 和 CL 中 G267S 和 S227P 取代的影响的致病基础，并提出 I169T 和 G202R 取代可能是多态性或可能增加患病易感性的可能性。
