Animal cells synthesize a complex pattern of oligosaccharides on their glycoconjugates. In a single cell, glycoproteins may demonstrate site- specific glycosylation, controlled microheterogeneity of the oligosaccharide at each site, and differential glycosylation relative to the oligosaccharides found on glycosphingolipids. Since the configuration of oligosaccharides at cell surfaces is presumed to provide individuality of cellular function and behavior, these glycosylation reactions must be exquisitely controlled; however, our understanding of the many elements involved in regulating animal cell glycosylation is severely limited. The objective of this research is to understand more about the precise role of glycosyltransferases in the regulation of glycoconjugate biosynthesis in animal cells. This will be accomplished by monitoring the structural changes in glycoconjugates (neoglycans) as a result of a single genetic event, namely, the introduction of a "new" glycosyltransferase gene into a "wild type" background. Chinese hamster ovary (CHO) cells will be used as "naive" recipients for the stable expression of cloned cDNAs encoding a murine (alpha1,3)galactosyltransferase and three human fucosyltransferases: the blood group H (alpha1,2)fucosyltransferase, the Lewis blood group (Fuc to GlcNAc-alpha1,3/4)fucosyltransferase, and the (Fuc to GlcNAcalpha1,3)galactosyltransferase. The structural features of metabolically radiolabeled glycoconjugates synthesized by these cell lines will be determined and compared to parental-cell oligosaccharides. To determine if specific glycoproteins in these cell lines are preferentially glycosylated with neoglycans, metabolically radiolabeled glycoproteins will be resolved by SDS-PAGE gels and subjected to partial structural characterization of their neoglycans. The CHO cell transferrin receptor and Lysosome Associated Membrane Proteins (LAMPs), known to exhibit different oligosaccharide side chains, will be used as "reporter glycoproteins" to determine if and to what degree their N-linked oligosaccharides are modified by the newly expressed glycosyltransferase. The degree to which there is a direct relationship between transcript levels, specific enzyme activities, and the relative amounts of neoglycan synthesized by transfected cells will be determined by analyses of the metabolically radiolabeled glycoconjugates in cell lines constructed so that the dosages of glycosyltransferase genes are increased. These studies will provide important new information on the precise role of glycosyltransferases in terminal glycosylation reactions, as well as, contribute to defining factors that orchestrate the complex oligosaccharide patterns expressed by animal cells.
NIH ExPorter
NIH ExPorter