Handford P, Downing A K, Rao Z, Hewett D R, Sykes B C, Kielty C M
Sir William Dunn School of Pathology, Oxford, United Kingdom.
J Biol Chem. 1995 Mar 24;270(12):6751-6. doi: 10.1074/jbc.270.12.6751.
Human fibrillin-1 is a 350-kDa glycoprotein found in 10-nm connective tissue microfibrils. Mutations in the gene encoding this protein cause the Marfan syndrome, a disease characterized by cardiovascular, ocular, and skeletal abnormalities. Fibrillin-1 has a modular structure that includes 47 epidermal growth factor-like (EGF-like) domains, 43 of which contain a consensus sequence associated with calcium binding. A mutation causing an Asn-2144 --> Ser amino acid change in one of the potential calcium binding residues has been described in a patient with the Marfan syndrome. We have chemically synthesized a wild-type EGF-like domain (residues 2126-2165 of human fibrillin-1) and a mutant EGF-like domain containing the Asn-2144 --> Ser amino acid change and measured calcium binding to each using 1H-NMR spectroscopy. The wild-type domain binds calcium with a similar affinity to isolated EGF-like domains from coagulation factors IX and X; however, the mutant domain exhibits > 5-fold reduction in affinity. Rotary shadowing of fibrillin-containing microfibrils, isolated from dermal fibroblast cultures obtained from the Marfan patient, shows that the mutation does not prevent assembly of fibrillin into microfibrils but does alter the appearance of the interbead region. We have modeled a region of fibrillin-1 (residues 2126-2331) encompassing five calcium binding EGF-like domains, using data derived from the recently determined crystal structure of a calcium binding EGF-like domain from human factor IX. Our model suggests that these fibrillin-1 EGF-like domains adopt a helical arrangement stabilized by calcium and that defective calcium binding to a single EGF-like domain results in distortion of the helix. We propose a mechanism for the interaction of contiguous arrays of calcium binding EGF-like domains within the microfibril.
人原纤蛋白-1是一种350 kDa的糖蛋白，存在于10纳米的结缔组织微原纤维中。编码该蛋白的基因突变会导致马凡综合征，这是一种以心血管、眼部和骨骼异常为特征的疾病。原纤蛋白-1具有模块化结构，包括47个表皮生长因子样（EGF样）结构域，其中43个含有与钙结合相关的共有序列。在一名马凡综合征患者中，已发现一种突变导致潜在钙结合残基之一发生Asn-2144→Ser氨基酸变化。我们化学合成了一个野生型EGF样结构域（人原纤蛋白-1的第2126 - 2165位残基）和一个含有Asn-2144→Ser氨基酸变化的突变型EGF样结构域，并使用1H-NMR光谱法测量了每种结构域与钙的结合情况。野生型结构域与凝血因子IX和X中分离出的EGF样结构域以相似的亲和力结合钙；然而，突变型结构域的亲和力降低了5倍以上。从马凡综合征患者的皮肤成纤维细胞培养物中分离出的含原纤蛋白的微原纤维进行旋转阴影显示，该突变并不阻止原纤蛋白组装成微原纤维，但确实改变了珠间区域的外观。我们利用从最近确定的人因子IX钙结合EGF样结构域晶体结构中获得的数据，对原纤蛋白-1的一个区域（第2126 - 2331位残基）进行了建模，该区域包含五个钙结合EGF样结构域。我们的模型表明，这些原纤蛋白-1的EGF样结构域通过钙形成螺旋排列，单个EGF样结构域的钙结合缺陷会导致螺旋扭曲。我们提出了一种微原纤维内钙结合EGF样结构域连续阵列相互作用的机制。
