BIOCHEMISTRY OF TYPE XVI COLLAGEN

本申请提出研究生物化学、分子和 XVI型胶原蛋白的细胞生物学，这是一种由 cDNA克隆。 长期目标是了解生物学作用 这种新的胶原蛋白。 最近的cDNA克隆编码整个人类 α 1（XVI）链已被分离和表征。 整体 推导的多肽的结构排列显著不同 其他已知的胶原蛋白类型。 然而，这种多肽表现出 某些结构特征， 原纤维相关胶原蛋白，IX、XII和XIV型。 它也类似于 C.角质层胶原两种胶原蛋白都含有类似的 富含半胱氨酸的基序。 为了阐明的生物功能， 对于这种胶原蛋白，我们提出以下具体目标。 (1)制备 和人α 1（XVI）特异性抗体的表征 使用来自cDNA序列的合成肽制备胶原蛋白， 通过在培养的细胞中表达cDNA而产生的重组蛋白。 （二） 培养细胞和组织中XVI型胶原的表征。 的 天然XVI型胶原的分子大小和链组成将 通过使用抗体的免疫印迹和免疫沉淀来测定 上面准备的。 (3)阐明空间和时间表达 的α 1（XVI）胶原蛋白。 细胞内mRNA和蛋白表达 人类和小鼠的组织将通过RNA杂交进行检测 和免疫荧光染色。 (4)阐明 使用蛋白质的α 1（VI）胶原的结构/功能关系 片段、合成肽和重组蛋白， 哺乳动物表达系统。 (5)其他组分的表征 XVI型胶原蛋白链，如果上述研究表明， 是异源三聚体。 (6)小鼠的分离和表征 α 1（XVI）胶原蛋白基因，并使用该基因组片段开发 α 1（XVI）胶原纯合破坏的转基因小鼠 基因

This application proposes to study the biochemistry, molecular and cellular biology of type XVI collagen, a novel collagen discovered by cDNA cloning. the long term goal is to understand the biological role of this new collagen. Recently cDNA clones encoding the entire human alpha1(XVI) chain have been isolated and characterized. The overall structural arrangement of the deduced polypeptide differs significantly from other known collagen types. However, this polypeptide exhibits certain structural features characteristically seen in members of the fibril associated collagen, types IX, XII and XIV. It also resembles the cuticle collagen of C. elegans in that both collagens contain similar cysteine-rich motifs. In order to elucidate the biological function of this collagen, we propose the following specific aims. (1) Preparation and characterization of antibodies specific for the human alpha1(XVI) collagen using synthetic peptides derived from the cDNA sequence and recombinant proteins produced by expressing cDNA in cultured cells. (2) Characterization of type XVI collagen in cultured cells and tissues. The molecular size and chain composition of the native type XVI collagen will be determined by immunoblotting and immunoprecipitation using antibodies prepared above. (3) Elucidation of the spatial and temporal expression of the alpha1(XVI) collagen. The mRNA and protein expression in cells and tissues from humans and mice will be examined by RNA hybridization and immunofluorescence staining. (4) Elucidation of the structure/function relationship of the alpha1(VI) collagen using protein fragments, synthetic peptides and recombinant proteins produced in the mammalian expression system. (5) Characterization of other constituent chain(s) of type XVI collagen if studies proposed above indicate that it is a heterotrimer. (6) Isolation and characterization of the mouse alpha1(XVI) collagen gene and use the genomic fragment to develop transgenic mice with homozygous disruption of the alpha1(XVI) collagen gene.