Potential regulation of N-glycosylation precursor through oligosaccharide-lipid hydrolase action and glucosyltransferase-glucosidase shuttle.
The potential role of degradative mechanisms in controlling the level of the dolichyl pyrophosphate-linked Glc3Man9GlcNAc2 required for protein N-glycosylation has been explored in thyroid slices and endoplasmic reticulum (ER) vesicles, focusing on cleavage of the oligosaccharide from its lipid attachment and on the enzymatic removal of peripheral monosaccharide residues. Vesicle incubations demonstrated a substantial release of free Glc3Man9GlcNAc2 (at 30 min approximately 35% of that transferred to protein) which was inhibited in the presence of exogenous peptide acceptor and was sensitive to disruption of membrane integrity by detergent. In thyroid slices glucosylated oligosaccharides terminating in the di-N-acetylchitobiose sequence were also noted and these continued to be formed even during inhibition by puromycin of both protein synthesis and the attendant N-glycosylation. These observations indicated that the oligosaccharide originated from the lipid donor and suggested, together with previously reported similarities in substrate specificity and cofactor requirements, that the oligosaccharyltransferase can carry out in vivo both the hydrolytic and transfer functions. In addition to the release of the intact Glc3Man9GlcNAc2, we also obtained evidence that the lipid-linked oligosaccharide can be modified by the in vivo action of ER glycosidases. Since radiolabeling of the oligosaccharide-lipid in thyroid slices indicated a preferential turnover of the glucose residues, the possible existence of a glucosyltransferase-glucosidase shuttle was explored with the use of castanospermine. In the presence of this glucosidase inhibitor, the formation of under-glucosylated and nonglucosylated oligosaccharides was not observed, even under conditions of energy deprivation in which they accumulate. Glucosidase inhibition in ER vesicle incubations likewise prevented the appearance of incompletely glucosylated oligosaccharide-lipids. Studies employing the mannosidase inhibitor 1-deoxymannojirimycin in thyroid slices furthermore indicated that in vivo removal of at least one mannose residue from the dolichyl pyrophosphate-linked oligosaccharide can occur.
Spiro
M J
MJ
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts.
Spiro
R G
RG
D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002417:Cattle; D002855:Chromatography, Thin Layer; D005959:Glucosidases / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D006031:Glycosylation; D006867:Hydrolases / Q000378:metabolism*; D050356:Lipid Metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D011691:Puromycin / Q000494:pharmacology; D013961:Thyroid Gland / Q000187:drug effects / Q000201:enzymology
D009844:Oligosaccharides; D011691:Puromycin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D005964:Glucosyltransferases; D006867:Hydrolases; D005959:Glucosidases