REGULATION OF MUCIN GLYCOPROTEIN BIOSYNTHESIS

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O-glycosylation represents a crucial functional determinant of mucins, the glycoproteins which endow the mucus coat with its unique protective qualities. However, a critical gap remains in our understanding of the events which regulate the initiation of O-glycosylation. Little is known about how the expression of the protein core of mucin (apomucin) is controlled. We hypothesized that the expression of apomucin is regulated in a tissue-specific manner and that differential glycosylation of variant protein cores contributes to the heterogeneity observed in mucin structure. To test this hypothesis we propose to identify and characterize cDNAs which encode RSMG apomucin. These cDNAs will be used to deduce the amino acid sequence of the apomucin(s) and will be used as a probe to determine the tissue and cellular distribution of transcripts which encode the protein core of the mucin. It is also unclear why some proteins become O-glycosylated whereas others do not become modified. We hypothesize that the sites of O- glycosylation within a protein are directed by molecular signals which are embodied within the information contained in the mRNA encoding the protein core of the glycoprotein. To test this hypothesis we propose to express glycophorin A (a well-characterized O-glycosylated protein), or glutamine/glutamic acid-rich protein (a protein which is devoid of saccharide) in a heterologous cell line (Chinese Hamster Ovary cells) and determine which potential acceptor sites in each recombinant molecule become glycosylated. Specific sequence and conformational domains of glycophorin A and glutamine/glutamic acid-rich protein will be assayed for their ability to promote or inhibit O-glycosylation. These studies will enable us to determine the nature of the signals which direct O-glycosylation. This information would greatly facilitate attempts to engineer bioactive O-glycosylated proteins by recombinant DNA methods. In addition, a more thorough understanding of the normal process of O-glycosylation may provide insight into specific forms of pathology which are thought to be related to aberrations of mucin- glycoprotein biosynthesis.