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.
| Salimath, P V; Spiro, R C; Freeze, H H (1995) Identification of a novel glycosaminoglycan core-like molecule. II. Alpha-GalNAc-capped xylosides can be made by many cell types. J Biol Chem 270:9164-8 |
| Spiro, R C; Freeze, H H; Sampath, D et al. (1991) Uncoupling of chondroitin sulfate glycosaminoglycan synthesis by brefeldin A. J Cell Biol 115:1463-73 |
