TENASCIN-X AND THE EHLERS-DANLOS PHENOTYPE

ehers - 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、血管脆弱和伤口愈合不良。这种综合征是由纤维胶原生物合成或沉积缺陷引起的，在EDS亚型中发现了几种胶原基因的突变。然而，导致大多数经典EDS病例的基因仍然未知。最近，PI实验室发现了两例由于tenascin-X（一种大基质糖蛋白）缺乏而患有EDS的无关患者。虽然这一发现证明了任何腱蛋白的第一个已知功能，但尚不清楚n - x突变占EDS的哪一部分，n - x如何导致这种表型，或者n - x的哪些其他功能被n - x和其他腱蛋白之间的冗余所掩盖。这些问题构成了这项建议的基础。在目的1中，将对已知TNX蛋白缺乏的EDS患者、第二组已排除胶原蛋白突变的EDS患者和第三组未选择的EDS患者进行TNX突变分析。将评估培养成纤维细胞分泌TN-X的情况，并通过Southern blotting、PCR和蛋白截断试验鉴定每个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缺乏症的临床认识，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同时相互作用来调节基质沉积。目的2将鉴定整合素和非整合素TN-X受体和TN-X结合蛋白。在这个目的中，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被证明。结合TNX的整合素将通过TNX亲和层析鉴定，然后用抗整合素抗体进行免疫沉淀。非整合素n- x结合蛋白将通过酵母双杂交筛选鉴定。特异性受体参与介导n - x诱导的基质合成和抗粘附将被证明。TN-X与分泌蛋白的相互作用将在固相结合试验中得到验证，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，通过靶向破坏TN-X基因，将在小鼠中重现TN-X缺陷表型。将TN-X缺陷小鼠的结缔组织形态、生物力学特性和伤口愈合与野生型仔鼠进行比较。随后将通过构建TN-X/C双敲除小鼠和TN-X/C/R三敲除小鼠来评估腱蛋白之间的功能冗余。

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.