CHARACTERIZATION OF A NEW HUMAN COLLAGEN

VII型胶原在本实验室中被描述为一种独特的 形成反平行二聚体的细胞外基质大分子 然后横向聚集形成称为锚定纤维的结构。 我们推测，二聚体的形成是由氨基-2-甲基-4-氨基-2-氨基-1，3-氨基-2，4-氨基-2，4-氨基-2，4-氨基-2，4-氨基-2，4-氨基- 末端非螺旋结构域(NC-2)。锚固的形态 纤维表明，它们可以稳定外部的附着 上皮细胞与其底层基质基质的连接。这个概念是 更有说服力的是，患有这种疾病的人 以自发性为特征的隐性营养不良大疱性表皮松解症 上皮细胞与间质的分离 锚固原纤维，缺乏可识别的锚固原纤维和III型 胶原蛋白。 III型胶原反平行排列的一个结果是 随后的横向聚集是锚固的两端 原纤维是完全相同的。这些末端含有羧基末端 非螺旋结构域(NC-1)，与上皮基底相互作用 以及我们所描述的“锚定斑块”的结构 是NC-1结构域、IV型胶原和其他 固有的基底膜成分。这些空间关系 提示NC-1结构域参与细胞的形成和稳定。 锚定原纤网。 由于锚定纤维网络中的中断严重 病理后果，重要的是要了解分子 锚定纤维与锚定斑块相互作用的基础 和基底膜。我们建议对该结构进行表征 III型胶原的足够详细的研究，以评估 其结构与其锚定功能有关。我们将测定氨基 使用常规方法对分子的选定部分进行酸序列 生化方法和重组DNA方法。我们将隔离和 鉴定NC-1的亚结构域并评估其二级结构 生物物理技术。同样，我们将确定主要的 NC-2的结构。合成多肽的单抗 模拟NC-2结构将被用来检验NC-2的假设 指导III型二聚反应。我们相信，关于 III型胶原的详细结构将导致 关于特定结构区域如何对 大分子的锚定功能。

Type VII collagen has been characterized in this laboratory as a unique extracellular matrix macromolecule that forms antiparallel dimers that then aggregate laterally to form structures known as anchoring fibrils. We have postulated that dimer formation is mediated by the amino- terminal non-helical domain (NC-2). The morphology of the anchoring fibrils suggest that they may stabilize the attachment of external epithelia to their underlying stromal matrix. This concept is strengthened by the finding that individuals with the disease of recessive dystropic Epidermolysis Bullosa, characterized by spontaneous separation of the epithelia from the stroma through the plane of the anchoring fibrils, lack both identifiable anchoring fibrils and type VII collagen. One consequence of the antiparallel arrangement of type VII collagen and the subsequent lateral aggregation is that the two ends of the anchoring fibril are identical. These ends, containing the carboxy-termininal non-helical domain (NC-1), interact with the epithelial basement membrane and with structures we described as "anchoring plaques" that are aggregates of the NC-1 domains, type IV collagen and perhaps other intrinsic basement membrane components. These spatial relationships suggest that the NC-1 domains mediate the formation and stability of the anchoring fibril network. Since disruptions in the anchoring fibril network have severe pathological consequences, it is important to understand the molecular basis for the interactions of anchoring fibrils with anchoring plaques and with basement membranes. We propose to characterize the structure of type VII collagen in sufficient detail to evaluate he relationship of its structure to its anchoring function. We will determine the amino acid sequence of selected portions of the molecule using conventional biochemical methods and recombinent DNA approaches. We will isolate and identify subdomains of NC-1 and evaluate their secondary structure by biophysical techniques. Similarly, we will determine the primary structure of NC-2. Monoclonal antibodies made to synthetic peptides that mimick NC-2 structure will be used to test the hypothesis that NC-2 directs type VII dimerization. We believe that knowledge of the detailed structure of type VII collagen will lead to the formation of an hypothesis of how specific structural regions contribute to the anchoring function of the macromolecule.