Smallridge R S, Whiteman P, Doering K, Handford P A, Downing A K
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
J Mol Biol. 1999 Feb 26;286(3):661-8. doi: 10.1006/jmbi.1998.2536.
Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins. We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor beta-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+ affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.
钙结合表皮生长因子样结构域（cbEGF）存在于许多细胞外蛋白中，包括原纤蛋白-1、Notch-3、蛋白S、因子IX和低密度脂蛋白（LDL）受体，这些蛋白具有多种功能。导致这些蛋白内氨基酸变化的基因突变分别与马方综合征（MFS）、伴有皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病（CADASIL）、蛋白S缺乏症、乙型血友病和家族性高胆固醇血症有关。其中一些突变破坏了钙与cbEGF的结合，强调了钙在这些蛋白中的关键功能作用。我们使用二维核磁共振（NMR）和荧光技术测定了来自人原纤蛋白-1的一个cbEGF对（cbEGF12-13）内两个位点的钙结合亲和力。原纤蛋白-1是一种镶嵌蛋白，含有43个cbEGF结构域，主要排列为串联重复。我们的结果表明，cbEGF12-13对中的cbEGF13位点具有从原纤蛋白-1研究的任何cbEGF中最高的钙亲和力。对这些以及先前报道的来自原纤蛋白-1的钙结合数据的比较分析表明，通过N端cbEGF结构域的连接，cbEGF13的亲和力提高了70多倍。相比之下，将分离的cbEGF32与连接到转化生长因子β结合蛋白样结构域（TB6-cbEGF32）时的钙结合进行比较，发现该异源结构域对未观察到相同的增强作用。综上所述，这些结果表明原纤蛋白-1 cbEGF的Ca2+亲和力可被与其N端相连的结构域类型显著调节。cbEGF12-13对位于原纤蛋白-1中最长的连续cbEGF区域内，该区域的一些突变与最严重的新生儿型MFS有关。该区域中cbEGF结构域13和14的亲和力明显高于原纤蛋白-1的C端区域。这种增加的亲和力可能对原纤蛋白组装成10-12nm的结缔组织微原纤维很重要，和/或可能有助于微原纤维网络的生物力学特性。
