STRUCTURE AND FUNCTION OF A CELL-ASSOCIATED PROTEOGLYCAN

本提案的长期目标是了解 细胞相关蛋白聚糖，并确定如何添加 糖胺聚糖侧链影响该功能。 的 将糖胺聚糖添加到所涉及的蛋白质中的证明 在基本的免疫学和发育过程中的作用引起了人们的兴趣， 这类职位的规定和职能意义， 翻译修饰 使用人黑素瘤模型系统， 黑色素瘤相关蛋白聚糖的功能将通过 深入研究和操纵其结构。 两种蛋白多糖- 将在体外和体内检测阴性黑素瘤变体 功能测定，如软琼脂克隆、细胞粘附和铺展 以及在无胸腺裸鼠中的生长和转移。 不能合成蛋白聚糖核心蛋白的其他突变体 或不能将核心蛋白转化为蛋白聚糖形式的蛋白质 也选自诱变的黑素瘤细胞群。 的 黑色素瘤蛋白聚糖的一级序列将由 从cDNA表达文库中分离的cDNA克隆的表征 核心蛋白特异性抗体和初始cDNA探针。 的 序列数据将决定结构和 功能结构域，如糖胺聚糖附着位点， 膜相关结构域和参与蛋白水解的序列 释放细胞表面形式。 部分cDNA克隆将用于 分离/构建野生型和诱变全长黑素瘤 蛋白聚糖核心蛋白cDNA将被插入哺乳动物 表达载体 蛋白多糖阴性变异黑色素瘤细胞 然后将用cDNA构建体重建，并在 功能测定 这样一来，黑色素瘤的功能 蛋白聚糖可以在不存在和存在下进行检查， 糖胺聚糖侧链。 此外，结构要求 并且参与糖胺聚糖添加的调节可以是 确立了习 这些信息将有助于了解 蛋白聚糖作为生物活性分子和作为 形成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.