Secretion of rat hepatic lipase is blocked by inhibition of oligosaccharide processing at the stage of glucosidase I.
Rat hepatic lipase is a glycoprotein bearing two N-linked oligosaccharide chains. The importance of glycosylation in the secretion of hepatic lipase was studied using freshly isolated rat hepatocytes. Various inhibitors of oligosaccharide synthesis and processing were used at concentrations that selectively interfere with protein glycosylation. Secretion of hepatic lipase activity was abolished by tunicamycin, castanospermine, and N-methyldeoxynojirimycin. No evidence was found by ELISA or Western blotting for secretion of inactive protein. Inhibition of secretion became apparent after a 30-min lag, corresponding to the time of intracellular transport of pre-existing protein. Simultaneously, intracellular hepatic lipase activity ws depleted. Secretion of hepatic lipase protein and activity was not affected by deoxymannojirimycin and swainsonine. Upon SDS-polyacrylamide gel electrophoresis, hepatic lipase secretion by deoxymannojirimycin- or swainsonine-treated cells showed an apparent Mr of 53 kDa and 55 kDa, respectively, which was distinct from hepatic lipase secreted by untreated cells (Mr = 58 kDa). We conclude that glycosylation and subsequent oligosaccharide processing play a permissive role in the secretion of hepatic lipase. As secretion is prevented by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin, but not by inhibitors of subsequent oligosaccharide trimming, the removal of glucose residues from the high-mannose oligosaccharide intermediate in the rough endoplasmic reticulum appears the determining step.
Verhoeven
A J
AJ
Department of Biochemistry I, Erasmus University Rotterdam, The Netherlands.
Jansen
H
H
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D065089:Glycoside Hydrolase Inhibitors; D006031:Glycosylation; D007212:Indolizines / Q000494:pharmacology; D008049:Lipase / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D009844:Oligosaccharides / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D017026:Swainsonine; D014415:Tunicamycin / Q000494:pharmacology; D000520:alpha-Glucosidases / Q000378:metabolism*; D054836:Indolizidines
D017485:1-Deoxynojirimycin; D000470:Alkaloids; D005944:Glucosamine; D065089:Glycoside Hydrolase Inhibitors; D007212:Indolizines; D008049:Lipase; D009844:Oligosaccharides; D017026:Swainsonine; D014415:Tunicamycin; D000520:alpha-Glucosidases; C042059:glucosidase I; C037806:castanospermine; D054836:Indolizidines; C033424:N-methyldeoxynojirimycin