Evidence for functional importance of usherin/fibronectin interactions in retinal basement membranes
Evidence for functional importance of usherin/fibronectin interactions in retinal basement membranes
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DOI：
10.1021/bi050245u
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发表时间：
2005-08-30
期刊：
影响因子：
2.9
通讯作者：
Cosgrove, D
中科院分区：
文献类型：
--
作者：
Battacharya, G;Cosgrove, D
Usher综合征是一种遗传异质性疾病，其特征是听力丧失伴视网膜色素变性。Usher综合征IIa型是最常见的Usher综合征，占所有病例的一半以上。该基因编码一种180 kDa的基底膜糖蛋白，称为usherin。在这里，我们证明了一个特定的相互作用之间的usherin和纤维连接蛋白在视网膜基底膜。这种相互作用被证实使用生物化学，生物物理和遗传方法。表面等离子体共振分析证实纤连蛋白与usherin具有高亲和力和1：1的化学计量比。使用基于融合肽的免疫共沉淀方法，我们表明，结合纤连蛋白发生在LE结构域的usherin。重组LE结构域特异性肽被工程化，其含有对应于在患有Usher综合征IIa型的人中发现的错义突变的单个氨基酸取代。只有LE结构域d环中的突变才消除LE结构域与纤连蛋白免疫共沉淀的能力。
Usher syndrome is a genetically heterogeneous disorder characterized by hearing loss with retinitis pigmentosa. Usher syndrome type IIa is the most common of the Usher syndromes, accounting for over half of all cases. The gene encodes a 180 kDa basement membrane glycoprotein called usherin. Here, we demonstrated a specific interaction between usherin and fibronectin in retinal basement membranes. This interaction was confirmed using biochemical, biophysical, and genetic approaches. Surface plasmon resonance assay confirmed that fibronectin binding to usherin is of high affinity and 1:1 stoichiometry. Using a fusion peptide-based co-immunoprecipitation approach, we show that binding to fibronectin occurs at the LE domain of usherin. Recombinant LE domain-specific peptides were engineered that contained single amino acid substitutions corresponding to missense mutations found in humans with Usher syndrome type IIa. Only mutations in loop d of the LE domain abolished the ability of the LE domain to co-immunoprecipitate fibronectin.