HUMAN AND MURINE MODELS OF TYPE V COLLAGEN DEFICIENCY

描述：(改编自申请人的摘要)-V型胶原是一种 纤维状胶原蛋白亚类中的一员，以少量存在 几乎在所有的非软骨结缔组织中。如所示， 申请人和其他人α1(V)前胶原链单倍性不足 经常导致经典型的Ehler-Danlos综合征(EDS I/II)，它 特点是关节松弛和脱位，严重的皮肤脆性，以及 伤口愈合有缺陷。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 对可获得的前α1(V)链的数量非常敏感 参与纤维形成，还有其他含有α1(V)原的组织 看起来相对不受影响。申请人提出的实验是 旨在阐明V型胶原在调节机械加工中的作用 组织的特性。在第一个目标中，原α1(V)的产物 包含C-前肽突变的表达基因将被分析 自身三聚化或与野生型前α1形成三聚体的能力(V) 链和亲α2(V)链。正常和突变的c-前肽与其结合 伴侣蛋白将通过化学交联法和 免疫沉淀。胶原纤维的形态和沉积 非胶原胶原结合分子将在长期真皮中被测量 成纤维细胞培养。第二个目的是V/XI类型的分子决定因素 链选择将通过三聚实验进行分析，其中完整的 表达前-α1(V)、前-α2 C-原肽的长度或小基因 (V)和前α1(XI)，以及一种新的C-前肽剪接变异体 前-α1(V)将在体外组装，并在细胞内转染后 V/XI型胶原链表达受限的几种细胞类型。 在第三个目标中，纯合子和杂合子col5A1基因敲除小鼠将被 分析组织和组织中特定类型的胶原沉积的变化 皮肤成纤维细胞的长期培养，这在人类中受到严重影响 Col5A1单倍性功能不全，角膜也是如此，甚至看起来都没有 尽管V型胶原在角膜胶原纤维形成中的作用很好 有记录在案。在第四个目标中，纤维形态、生物化学和 Col5A1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型胶原之间的关系 含量、V型/XI型异构体比率、非胶原蛋白含量以及 生物力学特性。

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.