Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion.
The addition and endoplasmic reticulum (ER) glucosidase processing of N-linked glycans is essential for the secretion of rat hepatic lipase (HL). Human HL is distinct from rat HL by the presence of four as opposed to two N-linked carbohydrate side chains. We examined the role of N-linked glycosylation and calnexin interaction in human HL secretion from Chinese hamster ovary (CHO) cells stably expressing a human HL cDNA. Steady-state and pulse-chase labeling experiments established that human HL was synthesized as an ER-associated precursor containing high mannose N-linked glycans. Secreted HL had a molecular mass of approximately 65 kDa and contained mature N-linked sugars. Inhibition of N-linked glycosylation with tunicamycin (TM) prevented secretion of HL enzyme activity and protein mass. In contrast, incubation of cells with the ER glucosidase inhibitor, castanospermine (CST), decreased human HL protein secretion by 60%, but allowed 40% of fully active HL to be secreted. HL protein mass and enzyme activity were also recovered from the media of a CHO-derivative cell line genetically deficient in ER glucosidase I activity (Lec23) that was transiently transfected with a human HL cDNA. Co-immunoprecipitation experiments demonstrated that newly synthesized human HL bound to the lectin-like ER chaperone, calnexin, and that this interaction was inhibited by TM and CST. These results suggest that under normal conditions calnexin may increase the efficiency of HL export from the ER. Whereas a significant proportion of human HL can attain activity and become secreted in the absence of glucose trimming and calnexin association, these interrelated processes are nevertheless essential for the expression of full HL activity.
Boedeker
J C
JC
Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
Doolittle
M
M
Santamarina-Fojo
S
S
White
A L
AL
D000818:Animals; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D014162:Transfection; D000520:alpha-Glucosidases / Q000172:deficiency
D002135:Calcium-Binding Proteins; D002241:Carbohydrates; D037281:Calnexin; D008049:Lipase; C042059:glucosidase I; D000520:alpha-Glucosidases