title: Use of recombinant endomannosidase for evaluation of the processing of N-linked oligosaccharides of glycoproteins and their oligosaccharide-lipid precursors.
abstract: Although glucose residues in a triglucosyl sequence are essential for the N-glycosylation of proteins and in their monoglucosyl form have been implicated in lectin-like interactions with chaperones, their removal is required for the formation of mature carbohydrate units and represents the initial steps in the glycoprotein processing sequence. In order to provide a probe for the glucosylation state of newly synthesized glycoproteins obtained from normal or altered cells, we have evaluated the usefulness of recombinant endo-alpha-mannosidase employing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to monitor the change in molecular mass brought about by the release of glucosylated mannose (Glc(1-3)Man). With this approach the presence of two triglucosylated-N-linked oligosaccharides in vesicular stomatis virus (VSV) G protein formed by castanospermine-treated CHO cells or the glucosidase I deficient Lec23 mutant could be clearly demonstrated and an even more pronounced change in migration was observed upon endomannosidase treatment of their more heavily N-glycosylated lysosomal membrane glycoproteins. Furthermore, the G protein of the temperature sensitive VSV ts045 mutant was found to be sensitive to endomannosidase, resulting in a change in electrophoretic mobility consistent with the presence of mono-glucosylated-N-linked oligosaccharides. The finding that endomannosidase also acts effectively on oligosaccharide lipids, as assessed by SDS-PAGE or thin layer chromatography, indicated that it would be a valuable tool in assessing the glucosylation state of these biosynthetic intermediates in normal cells as well as in mutants or altered metabolic states, even if the polymannose portion is truncated. Endomannosidase can also be used to determine the glucosylation state of the polymannose oligosaccharides released during glycoprotein quality control and when used together with endo-beta-N- acetylglucosaminidase H can distinguish between those terminating in a single N-acetylglucosamine or in a di-N-acetylchitobiose sequence.
authors.affiliation: Departments of Medicine and Biological Chemistry, Harvard Medical School, and the Joslin Diabetes Center, Boston, MA 02215, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Spiro
authors.forename: R G
authors.initials: RG
authors.lastname: Spiro
mesh_terms: D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D050356:Lipid Metabolism; D008099:Liver / Q000648:ultrastructure; D008247:Lysosomes / Q000737:chemistry; D008361:Mannosidases / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D008361:Mannosidases; D019204:GTP-Binding Proteins