MUCIN GLYCOPROTEIN CONFORMATION

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The goals of this proposal are to elucidate the molecular interactions and structures that give rise to the highly extended conformations of the heavily O-glycosylated domains in mucins and mucin-like glycoproteins. Such O-glycosylated domains, by stiffening and extending the peptide core, contribute to the viscoelastic properties of mucins which protect the surfaces of the respiratory, digestive and urogenital tracts. Similar heavily O-glycosylated domains are also found in the extracellular portions of several membrane- associated glycoproteins. These domains act to tether globular enzymes or receptors above the cell surface and as cell surface coatings which may modulate immune surveillance and recognition. Molecular dynamics modeling and NMR approaches will be used to determine the conformations and dynamics of a series of mucin and recombinant DNA-derived O-linked glycopeptides of different primary structures and glycosylation patterns. The role of peptide sequence, in particular the presence of Gly and Pro, and the clustering of glycosylated Ser and Thr will be studied with regard to their effect on glycopeptide conformation, chain extension and stiffness. These studies will allow us to determine specifically how glycosylation affects peptide conformation and the roles of carbohydrate-peptide and carbohydrate-carbohydrate interactions. These results will lead to a better understanding of carbohydrate-protein interactions and eventual improvements in the computer modeling of glycoproteins. The effects of solution environment and the possibility of inter-chain carbohydrate-carbohydrate interactions may also be examined. These studies are an integral part of our long range goals of determining both the molecular basis of the physical properties of mucins and the molecular processes involved in the hydration and secretion of mucins. Our major experimental objectives are to: 1) Characterize and perform NM conformational analyses on a series of short O-linked glycopeptides derived from partially deglycosylated porcine submaxillary mucin, 2) To perform molecular modeling and molecular dynamics calculations for comparison, and 3) to use recombinant DNA techniques to express additional O-linked glycopeptides of unique peptide sequences, unavailable from PSM, in transfected Chinese hamster ovary (CHO) cells.