The extracellular matrix is intimately involved in the adhesion, migration, proliferation, and differentiation of cells. One of the major components of the matrix of normal human fibroblasts is a collagen-like glycoprotein with a subunit molecular mass of 140,000 daltons, termed GP140. In the matrix, GP140 contains extensive intermolecular disulfide bonding and co-distributes with fibronectin in fibrillar arrays. Isolated GP140 induces stable cell attachment and may function in cooperation with fibronectin in regulating cell-cell or cell-substratum interactions. Transformed cells synthesize a soluble form of GP140, but fail to assemble this protein into a normal matrix. In recent studies, GP140 was found to be a major component of many human tissues and to be related to type VI collagen. GP140 contains a 64,000 dalton collagenous domain that is unique in the collagen family because of its high degree of intermolecular disulfide bonding and glycosylation. Thus, GP140 appears to be a hybrid molecule with characteristics of both collagens and matrix glycoproteins. The proposed research will investigate the structural/functional relationship of the carbohydrate units of GP140 and the relationships of this carbohydrate to the normal assembly of the matrix as follows: I. Carbohydrate structural analysis of placental GP140. GP 140 will be isolated from placentae, then fragmented into its collagenous and non-collagenous domains. The oligosaccharide units from each domain will be isolated, and subject to carbohydrate structural analysis utilizing the combined techniques of methylation analysis, nuclear magnetic resonance spectroscopy, and sequential enzymatic degradation. II. Microanalysis of glycosylation differences in multiple forms of GP140 from normal and transformed human cells. The various forms of GP140 from cultured cells and tissues will be compared by quantitative lectin blotting and glycopeptide analysis. III. Carbohydrate structural analysis of multiple forms of GP140 from normal and transformed cells. Utilizing the techniques and information gained in Specific Aim I, the differences in glycosylation detected in Specific Aim II will be characterized. IV. The functional role of the carbohydrate units of GP140. Non-glycosylated GP140 will be isolated from tunicamycin-treated cells, then assayed for a series of functions including intermolecular disulfide bonding, interaction with fibronectin, cell attachment, sensitivity to digestion with collagenase and pepsin, and rate of turnover in the matrix. (A)
