TENASCIN-X AND THE EHLERS-DANLOS PHENOTYPE

Tenascin-X 和 EHLERS-DANLOS 表型

• 批准号: 6351535
• 负责人: James D BRISTOW
• 金额: $ 25.93万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351535
• 关键词: Ehlers Danlos syndrome; binding proteins; clinical research; extracellular matrix; fibroblasts; gene mutation; gene targeting; genetically modified animals; human subject; immunoprecipitation; integrins; laboratory mouse; phenotype; protein biosynthesis; protein protein interaction; protein structure function; southern blotting; tenascin; tissue /cell culture; wound healing; yeast two hybrid system

The Ehlers-Danlos syndrome (EDS) is a group of connective tissue disorders characterized by hyperextensible skin and joints, vascular fragility, and poor wound healing. The syndrome results from defects in fibrillar collagen biosynthesis or deposition and mutations in several collagen genes are found in EDS subtypes. However, the gene(s) responsible for the majority of classical EDS cases remains unknown. Recently, the PI's laboratory identified two unrelated patients with EDS due to deficiency of tenascin-X (TN-X), a large matrix glycoprotein. While this finding demonstrates the first known function of any tenascin, it is not yet known what fraction of EDS is accounted for by TN-X mutations, how TN-X causes this phenotype, or what other functions of TN-X are obscured by redundancy between TN-X and other tenascins. These questions form the basis of this proposal. In aim 1, TN-X mutational analysis will be performed in EDS patients with known TNX protein deficiency, in a second cohort of EDS patients in whom collagen mutations have been excluded, and a third unselected EDS cohort. TN-X secretion by cultured fibroblasts will be evaluated, and the mutations present on each TN-X mutant allele will be identified by Southern blotting, PCR and the protein truncation test. These data will refine our clinical understanding of TNX deficiency and establish an algorithm for diagnosis. We postulate that TN-X is a matricellular protein that regulates matrix deposition through simultaneous interactions with matrix and cellular receptors. Aim 2 will identify integrin and non-integrin TN-X receptors and TN-X binding proteins. In this aim, specific binding of radiolabeled TN-X to myoblasts and dermal fibroblasts will be demonstrated. TNX binding integrins will be identified by TNX affinity chromatography, followed by immunoprecipitation with anti-integrin antibodies. Non-integrin TN-X binding proteins will be identified through a yeast two-hybrid screen. The involvement of specific receptors in mediating TN-X induction of matrix synthesis and anti-adhesion will be demonstrated. Interaction of TN-X with secreted, proteins will be verified in solid phase binding assays and the role of such interaction in regulation of matrix deposition investigated. In aim 3, the TN-X deficient phenotype will be recapitulated in mice by targeted disruption of the TN-X gene. Morphology, biomechanical properties of connective tissues and wound healing of TN-X deficient mice will be compared with wild type littermates. Functional redundancy among tenascins will be evaluated subsequently by creation of TN-X/C double knockout and TN-X/C/R triple knockout mice.

Ehlers-Danlos综合征（EDS）是一组结缔组织疾病，其特征是皮肤和关节过度伸展、血管脆性和伤口愈合不良。 该综合征是由于纤维状胶原蛋白生物合成或沉积缺陷引起的，并且在EDS亚型中发现了几种胶原蛋白基因的突变。然而，导致大多数经典EDS病例的基因仍然未知。 最近，PI的实验室确定了两个不相关的EDS患者由于缺乏腱生蛋白-X（TN-X），一个大的基质糖蛋白。 虽然这一发现证明了任何腱生蛋白的第一个已知功能，但尚不清楚TN-X突变占EDS的多少比例，TN-X如何导致这种表型，或者TN-X和其他腱生蛋白之间的冗余掩盖了TN-X的其他功能。 这些问题构成了本建议的基础。 在目标1中，将在已知TNX蛋白缺乏的EDS患者中、在已排除胶原蛋白突变的EDS患者的第二队列中以及在第三EDS患者队列中进行TN-X突变分析。 将评价培养的成纤维细胞的TN-X分泌，并通过Southern印迹、PCR和蛋白质截短试验鉴定每个TN-X突变等位基因上存在的突变。 这些数据将完善我们对TNX缺乏症的临床理解，并建立诊断算法。 我们假设TN-X是一种基质细胞蛋白，通过与基质和细胞受体的同时相互作用来调节基质沉积。目的2鉴定整合素和非整合素TN-X受体及TN-X结合蛋白。 在这一目标中，将证明放射性标记的TN-X与成肌细胞和真皮成纤维细胞的特异性结合。 将通过TNX亲和色谱法，然后用抗整联蛋白抗体进行免疫沉淀来鉴定TNX结合整联蛋白。 将通过酵母双杂交筛选鉴定非整联蛋白TN-X结合蛋白。 将证明特异性受体参与介导TN-X诱导基质合成和抗粘附。TN-X与分泌的蛋白质的相互作用将在固相结合试验中验证，并研究这种相互作用在基质沉积调节中的作用。 在目标3中，TN-X缺陷型将通过TN-X基因的靶向破坏在小鼠中重现。 将TN-X缺陷小鼠的结缔组织的形态学、生物力学特性和伤口愈合与野生型同窝小鼠进行比较。随后将通过建立TN-X/C双敲除和TN-X/C/R三敲除小鼠来评估腱生蛋白之间的功能冗余。