Abstract
Glucosidase II is regarded as a resident protein of the endoplasmatic reticulum. The enzyme removes alpha-1-3-linked glucose from high mannose oligosaccharides N-linked to asparagine residues of glycoproteins. Monospecific antibodies raised against the pig kidney enzyme are used to study the metabolism of the enzyme in a rat hepatoma cell line. These antiglucosidase II antibodies specifically immune precipitate glucosidase II as a 100,000-Da species from [35S]methionine-labeled cells. In addition, protein blotting and immune staining of cell extracts from both rat liver and human and rat hepatoma cell lines show identity in apparent Mr (100,000). Glucosidase II synthesized in the presence of tunicamycin is approximately 94,000 Da, indicating the presence of one or more N-linked oligosaccharide chains. Cell-free protein synthesis of rat hepatoma total RNA demonstrates that glucosidase II is synthesized as a slightly higher molecular weight species as compared to the polypeptide synthesized in whole cells in the presence of tunicamycin, indicating that the enzyme has a cleavable signal sequence. Using a pulse-chase protocol, the apparent molecular weight does not change upon longer chase periods. In addition, the 100,000-Da protein remains sensitive to endo-beta-N-acetylglucosaminidase H regardless of prolonged chase periods. The cells incorporate [3H]mannose into the enzyme; after release with endo-beta-N-acetylglucosaminidase H, most of the radioactivity comigrates with Glc1-Man9-GlcNAc on a gel filtration column. Phase separation in Triton X-114 shows a partition between the aqueous and the Triton phase, the major portion being separated in the aqueous phase. In rat hepatoma cells glucosidase II has a half-life of 50 min. This value is not altered if the cells are grown in the presence of monensin nor of methyl-deoxynoijirimycin. However, tunicamycin and low concentrations or primaquine (raising the pH of acidic compartments) causes a 100% increase in half-life of glucosidase II. We conclude that glucosidase II is a hydrophilic, probably not a transmembrane membrane, protein with a short half-life. It is the first example of an oligosaccharide-processing enzyme not being an integral membrane protein.
Highlights
Monospecific antibodies raised againstthe pig kidney oligosaccharides are modifiedby a sequence of reactions, enzyme are used to study thme etabolism of the enzyme generating N-glycans with either high mannose and/or comin a rat hepatoma cell line
Miis synthesized as a slightly higher molecular weight chael and Kornfeld (4)provided kinetic evidence that the species as compared to the polypeptide synthesized in innermost glucose residue is removed at a later time point whole cells in thepresence of tunicamycin, indicating than the outermost two, suggesting that the enzyme can act that theenzyme has acleavable signal sequence
Specific antibodies againstthe native enzymeand the denatured subunit were prepared, and they appeared to cross-react with a similar antigen present riant liver microsomes,as determined by protein blotting combined with an immune-replica technique ( 5 ) .To test the specificity of the antibodyw, e performed binding of the antibodies to total protein extracted fromthe rat hepatoma cell line used in this study and compared the reactivity to total cell extracts from rat liver and the human hepatom ac e l ll i n eH e p G 2 .T oa v o i dp rotei nd egradation tissue and cells were extracted in SDS sample buffer, immediately heated and analyzed by PAGE
Summary
Monospecific antibodies raised againstthe pig kidney oligosaccharides are modifiedby a sequence of reactions, enzyme are used to study thme etabolism of the enzyme generating N-glycans with either high mannose and/or comin a rat hepatoma cell line.
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