MOLECULAR BIOLOGY OF PROCOLLAGEN TRANSPORT AND SECRETION

这一建议审查了第一和第三类假设， 前胶原分子含有转运信号和/或结合 决定因素：1）分子的运动， 粗面内质网（RER），到高尔基体，到细胞表面;和 2)前胶原与细胞外基质的结合 （ECM）。 实验方法是将真核细胞 具有正常和诱变的人I型和III型DNA的细胞， 然后分析转录、合成、跨细胞转运， 在体外瞬时和长时间的分泌和基质产生中， 术语表达系统。 这些研究将需要：a） 前体蛋白1（I）和前α 1的完整cDNA的构建 (III)通过探测一个特殊的cDNA成纤维细胞文库， 代表胶原蛋白5'和中间部分的cDNA 基因，然后切割并拼接回收的cDNA 必要时的片段; B）引入选择的突变 （缺失、插入、碱基替换）插入cDNA中， 前α 1（I）、前α 2（I）和前α 1（III）。 这将涉及 鉴定用于切除和插入的有用限制性位点， 和用于定点诱变的寡核苷酸拷贝; c） 将所述cDNA插入有效表达载体中;和d） PC 12细胞、HeLa细胞、COS细胞、 淋巴细胞谱系，以及某些有用的突变小鼠和人类 成纤维细胞系和菌株，作为转染受体， 各种分析。 通过这种方式，我们希望定义域 在前胶原分子中， 和纤维形成。 我们预计，完整的cDNA和 本计划中开发的转染系统将有助于 研究胶原蛋白生物学其他方面的研究人员。 这些 研究应该增加我们对蛋白质分泌的理解， 特别是前胶原分泌。 鉴于 胶原沉积在形态发生和创伤中的中心作用 修复，这些研究应该对许多生物医学问题产生影响。

This proposal examines the hypothese that type I and III procollagen molecules contain transport signals and/or binding determinants that govern: 1) the molecules' movement from rough endoplasmic reticulm (RER), to Golgi, to cell surface; and 2) the incorporation of procollagen into the extracellular matrix (ECM). The experimental approach is to transfect eukaryotic cells with normal and mutagenized human type I and III DNAs and to then analyze transcription, synthesis, transcellular transport, secretion and martix production in the in vitro transient and long- term expression systems. These studies will require: a) the construction of complete cDNAs for proalphs1 (I) and proalpha1 (III) by probing a special cDNA fibroblast library enriched for cDNAs representative of the 5' and middle portions of collagen genes, and then cutting and splicting the recovered cDNA segments as necessary; b) introduction of selected mutations (deletions, insertions, base replacements) into the cDNAs for proalpha1 (I), proalpha2 (I) and proalpha1 (III). This will involve identifying useful restriction sites for excisions and insertions, and oligonucleotide copying for site-directed mutagenesis; c) insertion of the cDNAs into efficient expression vectors; and d) evaluation of PC12 cells, HeLa cells, COS cells, cells of lymphocytic lineage, and certain useful mutant mouse and human fibroblast lines and strains, as transfection recipients for the various analyses. In this manner, we hope to define domains within the procollagen molecules that govern transport, secretion and fibrillogenesis. We anticipate that the complete cDNAs and transfection systems developed in this program will be useful to investigators examining other aspects of collagen biology. These studies should increase our understanding of protein secretion in general, and procollagen secretion in particular. Given the central role of collagen deposition in morphogenesis and wound repair, these studies should impact on many biomedical problems.