HUMAN AND MURINE MODELS OF TYPE V COLLAGEN DEFICIENCY

描述：（改编自申请人的摘要）-V型胶原蛋白是一种 纤维状胶原亚类的一员，少量存在 几乎所有的非软骨结缔组织都有。证明了这 原α 1（V）胶原蛋白链的单倍不足 经常引起经典形式的Ehlers-Danlos综合征（EDS I/II）， 特征是关节松弛和脱位，严重的皮肤脆弱， 伤口愈合不良因此，一些生物力学的完整性 包括皮肤、肌腱和韧带在内的结缔组织似乎 对pro-alpha 1（V）链的数量非常敏感， 参与原纤维形成，而其他含有原α 1（V）的组织 似乎相对不受影响。申请人提出的实验是 旨在阐明V型胶原蛋白在调节机械 组织的性质。在第一个目标中，pro-alpha 1（V）的产物 将分析含有C-前肽突变的表达基因的 自身三聚化或与野生型前-α 1（V）形成三聚体的能力 链和前-α 2（V）链。正常和突变C-前肽与 伴侣蛋白将通过化学交联进行分析， 免疫沉淀。原纤维形状和胶原沉积， 非胶原性胶原结合分子将在长期真皮中测量。 成纤维细胞培养。在第二个目标中，V/Xi型的分子决定簇 链选择将通过三聚实验进行分析，其中完全 表达前-α 1（V）、前-α 2（V）、前-α 3（V）和前-α 4（V）的C-前肽的长度或小基因 (V)和前-α 1（Xi）以及一种新的C-前肽剪接变体， 前-α 1（V）将在体外组装，并在转染后在细胞内组装。 具有V/Xi型胶原蛋白链的受限表达的几种细胞类型。 在第三个目标中，将使用纯合和杂合col 5A 1敲除小鼠， 分析组织中类型特异性胶原沉积的变化， 从皮肤中长期培养成纤维细胞，这在人类中受到严重影响 col 5A 1单倍不足，以及角膜，这似乎是幸免于难，甚至 虽然V型胶原在角膜胶原纤维形成中的作用是好的， 记录在案。在第四个目标中，原纤维形态学、生物化学和 col 5A 1基因缺陷小鼠角膜和真皮的生物力学特性 将进行检查，以确定总V型胶原蛋白之间的关系， 含量、V型/Xi型亚型比率、非胶原蛋白含量，以及 生物力学性能

DESCRIPTION: (Adapted from the applicant's abstract) - Type V collagen is a member of the subclass of fibrillar collagens that is present in minor amounts in virtually all non-cartilaginous connective tissues. As demonstrated by the applicant and others haploinsufficiency of pro-alpha1 (V) collagen chains frequently causes the classical form of Ehlers-Danlos syndrome (EDS I/II) which is characterized by joint laxity and dislocations, severe dermal fragility, and defective wound healing. Therefore, the biomechanical integrity of some connective tissues including skin, tendon, and ligaments appears to be exquisitely sensitive to the quantity of pro-alpha1 (V) chains available to participate in fibrillogenesis, yet other pro-alpha1(V)-containing tissues appear relatively unaffected. Experiments proposed by the applicant are designed to elucidate the role of type V collagen in regulating mechanical properties of tissues. In the first aim the products of pro-alpha1(V) expression genes containing C-propeptide mutations will be analyzed for their ability to self trimerize or to form trimers with wild-type pro-alpha1 (V) chains and pro-alpha2(V) chains. Binding of normal and mutant c-propeptides to chaperone proteins will be analyzed by chemical cross-linking and immunoprecipitation. Fibril shape and deposition of collagens and noncollagenous collagen-binding molecules will be measured in long-term dermal fibroblast cultures. In the second aim the molecular determinants of type V/XI chain selection will be analyzed by trimerization experiments in which full length or minigenes expressing the C-propeptides for pro-alpha1 (V), pro-alpha2 (V), and pro-alpha1 (XI) as well as a novel C-propeptide splice variant of pro-alpha1 (V) will be assembled in vitro and in cellulo after transfection in several cell types having restricted expression of type V/XI collagen chains. In the third aim homozygous and heterozygous col5A1 knockout mice will be analyzed for changes in type-specific collagen deposition in tissues and long-term fibroblast cultures from skin, which is severely affected in human col5A1 haploinsufficiency, and also in cornea, which appears to be spared even though type V collagen's role in collagen fibrillogenesis in cornea is well documented. In the fourth aim fibril morphology, biochemistry, and biomechanical characteristics of cornea and dermis in col5A1-deficient mice will be examined to determine the relationship between total type V collagen content, type V/type XI isoform ratios, noncollagenous protein content, and biomechanical properties.