2,6-branched mannose and the regulation of poly-N-acetyllactosamine biosynthesis in N-linked oligosaccharides of Chinese hamster ovary cells.
Poly-N-acetyllactosamine (PL) sequences (repeating (3Gal beta 1-4GlcNAc beta 1)n) in complex-type N-linked oligosaccharides often occur in branched tri- and tetraantennary chains containing alpha-linked mannosyl residues disubstituted by N-acetyllactosaminyl units at C-2 and C-6 (2,6-branched mannose). We report here our studies on the factors affecting PL biosynthesis and the branching of N-linked oligosaccharides in glycoproteins from Chinese hamster ovary (CHO) cells. For our studies, we utilized a mutant CHO cell line designated Lec8 CHO, which lacks the ability to galactosylate its glycoproteins and consequently synthesizes glycoproteins containing terminal GlcNAc residues and lacking poly-N-acetyl-lactosamine and sialic acid. Glycoproteins in extracts of [3H]glucosamine- or [3H]mannose-labeled Lec8 CHO cells were galactosylated by exogenous beta 1-4-galactosyltransferase and analyzed by chromatography on leukoagglutinating phytohemagglutinin-Sepharose, a lectin reactive with glycoproteins containing 2,6-branched mannosyl residues. Approximately 10% of the radiolabeled glycoproteins were bound, and these were primarily of high molecular mass. Structural analyses of the bound glycoproteins demonstrated that they quantitatively contained 2,6-branched mannose. We then determined whether the "small i" N-acetylglucosaminyl-transferase (iGNT), which initiates PL biosynthesis, could specifically recognize glycoproteins in vitro and whether recognition was dependent on the presence of 2,6-branched mannose. When the galactosylated glycoproteins in extracts of Lec8 CHO cells were incubated with UDP-[3H]GlcNAc, the endogenous iGNT quantitatively added GlcNAc in beta 1-3-linkage to terminal galactosyl residues in the leukoagglutinating phytohemagglutinin-bound glycoproteins. These results demonstrate for the first time that 2,6-branched mannosyl residues are restricted to a subset of CHO glycoproteins and that the iGNT in vitro preferentially recognizes glycoproteins containing the 2,6-branched mannose determinant.
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Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
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D000606:Amino Sugars / Q000096:biosynthesis*; D000818:Animals; D015703:Antigens, CD; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D006801:Humans; D051907:Lysosomal Membrane Proteins; D008358:Mannose / Q000032:analysis*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D013379:Substrate Specificity; D014316:Tritium; D014531:Uridine Diphosphate Galactose / Q000737:chemistry; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000737:chemistry
D000606:Amino Sugars; D015703:Antigens, CD; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D014316:Tritium; D014531:Uridine Diphosphate Galactose; C000458:N-acetyllactosamine; D014537:Uridine Diphosphate N-Acetylglucosamine; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D008358:Mannose