Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion

Abstract

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 ϳ 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., M. Doolittle, S. Santamarina-Fojo, and A. L. White. Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion.

Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion

“…Cultures were labeled overnight in medium [SFM or DMEM containing BSA/linoleate (0.5 mg/ml, 0.79% linoleic acid) and insulin (5 g/ml)] containing one-tenth the normal concentration of methionine and cysteine and Expre 35 S 35 S label (125 Ci/ml). Medium and cell lysates were collected and immunoprecipitated as described previously (18). Bands on autoradiographs were quantified by densitometric scanning as previously described (18).…”

“…Medium and cell lysates were collected and immunoprecipitated as described previously (18). Bands on autoradiographs were quantified by densitometric scanning as previously described (18).…”

“…HL and LPL activities were measured in medium containing heparin (10 U/ml) conditioned by overnight incubation with transfected cells. The assays were per formed as previously described (18,24), using as substrate emulsions of radiolabeled triolein prepared by sonication. Samples were assayed in duplicate and values obtained were normalized either to total cell protein determined by bicinchoninic acid assay (Pierce, Rockford, IL) or to the relative mass of HL in the culture medium determined by immunoprecipitation of medium labeled overnight as described above.…”

“…In addition, to control for the possibility of remaining plasmid contamination, RNA prepared from cells transfected with hHL-SK was included as this plasmid would give a positive signal in the RPA, but would not give rise to mRNA. HL mRNA levels were quantifi ed by densitometry (18), and normalized to ␤ -actin mRNA levels.…”

“…We are interested in determining the molecular mechanisms that regulate the secretion and activity of human HL (hHL). Previous results suggested an important, although nonessential, role for calnexin in hHL secretion (18). In the current study we investigated the effect of cld on the secretion of hHL protein mass and activity.…”