OLIGOSACCHARIDE ASSEMBLY ON RECOMBINANT PROTEINS

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The long-range goal of this research is to identify some of the factors that govern the nature of glycan assembly on glycoproteins, with emphasis on specificity controls dictated by different cells and by the protein substrate. Since our previous investigations have suggested that recombinant (r) human plasminogen (HPg), a key zymogen of the fibrinolytic system, may contain structural determinants for the types of glycans assembled on its sole N-linked glycosylation site, emphasis has been placed on studies with this protein. Five specific aims are proposed: (1) to investigate the temporal nature of the appearance of different types of N- and O-linked glycans on r-HPg, as well as on r- urokinase (UK), that are expressed in several r-baculovirus-infected lepidopteran insect cell lines; (2) to evaluate whether temporal processing of oligosaccharides on r-HPg occurs in a virally-infected mammalian cell line; (3) to determine whether the stimulations of Man6- mannosidase and Nacetylglucosaminyl-transferase-1 (GlcNAc-TI) activities in baculovirus-infected insect cells are general properties of their glycosylation machinery; (4) to assay the enzymatic activities of GlcNAc- TII, GlcNAc-TIII, mannosidase II, alpha-fucosyltransferase, beta- galactosyltransferase, and alpha-sialyltransferases, as well as transferases important for O-linked glycosylation of r-HPg, in subcellular fractions from noninfected, r-virus infected, and control- virus infected insect cells; and (5) to purify the Man6-mannosidase for further characterization, and to purify GlcNAc-TI for characterization, cloning and evaluation of the molecular basis of its stimulation in baculovirus-infected cells. The design of this project involves determination of glycan structures on r-proteins that have been expressed in different virally-infected cell systems. The activities of key glycoprocessing enzymes will be also be studied in these same infected cell lines. The results obtained will allow development of strategies to control the nature of the glycosylation of rglycoproteins so that they will closely resemble their natural states, a factor particularly important when therapeutic use of such proteins is contemplated. Since the glycans present on proteins may affect their structures, functions, immunogenicities, turnover characteristics, and/or binding to cellular determinants, among other properties, the ability to understand how to control the nature of the glycans present will be a most valuable scientific asset.