Use of recombinant endomannosidase for evaluation of the processing of N-linked oligosaccharides of glycoproteins and their oligosaccharide-lipid precursors

Although glucose residues in a triglucosyl sequence are essential for the N -glycosylation of proteins and in their monoglucosyl form have been implica ted in lectin-like interactions with chaperones, their removal is required for the formation of mature carbohydrate units and represents the initial s teps in the glycoprotein processing sequence. In order to provide a probe f or the glucosylation state of newly synthesized glycoproteins obtained from normal or altered cells, we have evaluated the usefulness of recombinant e ndo-alpha-mannosidase employing sodium dodecyl sulfate polyacrylamide gel e lectrophoresis (SDS-PAGE) to monitor the change in molecular mass brought a bout by the release of glucosylated mannose (Glc(1-3)Man), With this approa ch the presence of two triglucosylated-N-linked oligosaccharides in vesicul ar stomatis virus (VSV) G protein formed by castanospermine-treated CHO cel ls or the glucosidase I deficient Lec23 mutant could be clearly demonstrate d and an even more pronounced change in migration was observed upon endoman nosidase 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 c hange in electrophoretic mobility consistent with the presence of monogluco sylated-N-linked oligosaccharides, The finding that endomannosidase also ac ts effectively on oligosaccharide lipids, as assessed by SDS-PAGE or thin l ayer chromatography, indicated that it would be a valuable tool in assessin g the glucosylation state of these biosynthetic intermediates in normal cel ls as well as in mutants or altered metabolic states, even if the polymanno se portion is truncated. Endomannosidase can also be used to determine the glucosylation state of the polymannose oligosaccharides released during gly coprotein quality control and when used together with endo-beta-N-acetylglu cosaminidase H can distinguish between those terminating in a single N-acet ylglucosamine or in a di-N-acetylchitobiose sequence.