Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum.
Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of alpha1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8+/-2.1-fold higher (mean+/-S.D., n=7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3+/-0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose-heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7+/-0. 3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not alpha1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked.
Verhoeven
A J
AJ
Department of Biochemistry, Cardiovascular Research Institute (COEUR), Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.Averhoev@bc1.fgg.eur.nl
Neve
B P
BP
Jansen
H
H
D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D008049:Lipase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008985:Monensin / Q000494:pharmacology; D009844:Oligosaccharides / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D020126:Brefeldin A; D008985:Monensin; D008049:Lipase