STRUCTURE AND FUNCTION OF A CELL-ASSOCIATED PROTEOGLYCAN

这项建议的长期目标是理解 并确定细胞相关蛋白多糖的添加是如何 糖胺多聚糖侧链会影响这一功能。这个 糖胺多聚糖添加到相关蛋白质中的论证 在基础免疫学和发育过程中引起了人们对 这类岗位的规定及其功能意义-- 翻译修饰。使用人类黑色素瘤模型系统， 黑色素瘤相关蛋白多糖的功能将通过 对其结构的深入研究和操纵。两个蛋白多糖- 阴性黑色素瘤变种将在体外和体内进行测试 软琼脂克隆、细胞黏附和铺展等功能检测 在确定的基质上，以及在裸鼠体内的生长和转移。 其他不能合成蛋白多糖核心蛋白的突变体 或者不能将核心蛋白转化为蛋白多糖形式 也将从诱变的黑色素瘤细胞群中挑选。这个 黑色素瘤蛋白多糖的初级序列将由 从表达文库中分离的cDNA克隆的鉴定 用核心蛋白特异性抗体和最初的cDNAs探针。这个 序列数据将决定结构和结构的组织 功能结构域，如糖胺多聚糖连接位点， 膜相关结构域及其参与蛋白降解的序列 细胞表面形态的释放。部分cdna克隆将用于 分离/构建野生型和诱变型全长黑色素瘤 将被插入哺乳动物体内的蛋白多糖核心蛋白cDNA 表达载体。蛋白多糖阴性变异型黑色素瘤细胞 然后与cdna构建物重组并在 功能分析。通过这种方式，黑色素瘤的功能 蛋白多糖可以在不存在或存在的情况下进行检测 糖胺多聚糖侧链。此外，结构要求 而涉及糖胺多糖添加的调节可以是 已经成立了。这些信息将有助于理解 蛋白糖元作为生物活性分子和蛋白质的关键成分 塑造肿瘤细胞的异常行为。

The long term objective of this proposal is to understand the function of cell-associated proteoglycan and determine how the addition of glycosaminoglycan side chains influences that function. The demonstration of the addition of glycosaminoglycans to proteins involved in basic immunological and development processes has aroused interest in the regulation and functional significance of this type of post- translational modification. Using a human melanoma model system, the function of a melanoma-associated proteoglycan will be addressed through an in depth study and manipulation of its structure. Two proteoglycan- negative melanoma variants will be tested in vitro and in vivo functional assays such as soft agar cloning, cell adhesion and spreading on defined matrices, and growth and metastasis in athymic nude mice. Additional mutants that fail to synthesize the proteoglycan core protein or that are unable to convert the core protein to a proteoglycan form will also be selected from mutagenized melanoma cell populations. The primary sequence of the melanoma proteoglycan will be established by the characterization of cDNA clones isolated from cDNA expression libraries with core protein-specific antibodies and initial cDNA probes. The sequence data will determine the organization of structural and functional domains such as the glycosaminoglycan attachment sites, the membrane-associated domain and the sequences involved in the proteolytic release of cell surface forms. The partial cDNA clones will be used to isolate/construct wild-type and mutagenized full-length melanoma proteoglycan core protein cDNAs that will be inserted into a mammalian expression vector. The proteoglycan-negative variant melanoma cells will then be reconstituted with the cDNA constructs and tested in functional assays. In this way, the function of the melanoma proteoglycan can be examined in the absence and presence of glycosaminoglycan side chains. Moreover, the structural requirements and the regulation involved in glycosaminoglycan addition can be established. This information will contribute to the understanding of proteoglycons as biologically active molecules and as key components in shaping the aberrant behavior of tumor cells.