This is a competing continuation request for 5 years of research support. The primary focus of the proposed studies is upon the adhesive glycoprotein, laminin, which is a major constituent of a specialized form of the extracellular matrix, the basil lamina. In embryonic tissues the basal lamina serves as an informational determinant during developmental processes whereas in adult tissues it forms a barrier which separates cells from the reticular portion of the extracellular matrix. The properties of the basal lamina are derived, in part, from its content of laminin; this glycoprotein has been implicated in the phenomena of cellular adhesion, spreading, proliferation, neurite outgrowth, and differentiation. It is also of importance in the alterations which occur during carcinomatous cell invasion and metastasis. Laminin binds to other basal lamina constituents and to itself. It also binds to cells by means of integrin and nonintegrin receptors, gangliosides and sulfatides. The biological response of cells to laminin seems to be determined by both amino acid sequences of laminin and its oligosaccharide substituents. objectives of this proposal are to further define the manner in which such oligosaccharides participate in cell spreading and neurite outgrowth.
The specific aims are to determine which oligosaccharides are active, where they are located on the laminin molecule, and how the cells recognize them. To determine which glycosyl moieties participate in the biologic responses, laminin will be fragmented by proteases followed by analysis of the glycopeptides in a cell spreading assay. Glycopeptides which promote the response will be enriched by successive fractionation steps, culminating in selection and identification of the active moieties. Another approach will be to use exoglycosidases on intact laminin, evaluating the loss of specific glycosyl groups in the spreading assay by using the depleted laminin as a cellular substratum. Also to be used as cell substrata are those laminins which have immature glycosyl groups, obtained by employing metabolic inhibitors of oligosaccharide processing. Ideally, laminin obtained by this means will contain a uniform population of glycosyl moieties, minimizing complexity, potentially allowing precise definition of the minimal essential structures needed for the biologic response. Identification of the active oligosaccharides and their attached peptides, in conjunction with the known amino acid sequence of the laminin subunits should define distribution of active oligosaccharides on the laminin molecule. Since Laminin can be fragmented into large specific domains which retain biologic activity it should be possible to predict which fragment(s) are responsible for cell spreading/neurite outgrowth. This prediction can be confirmed by using the large fragments in the biological assay system. To comprehend how cellular recognition of the oligosaccharides occurs, experiments will be done using glycopeptide binding, photo-affinity derivatives, antibody blocking and anti-idiotypic antibodies. This project encompasses the scientific disciplines of biochemistry and cell biology, utilizing contemporary methods to discern the relationships between carbohydrate structure and its biologic activity. The health-relatedness of the research applies to the general area of the role of extracellular matrix in developmental and disease processes.
