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

Jennifer C Boedeker,Mark Doolittle,Silvia Santamarina-Fojo,Ann L White

doi:10.1016/s0022-2275(20)33408-8

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 ∼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. J. Lipid Res. 1999. 40: 1627–1635.

Highlights

The addition and endoplasmic reticulum (ER) glucosidase processing of N-linked glycans is essential for the secretion of rat hepatic lipase (HL)
N-linked glycans are added to secretory proteins cotranslationally in the endoplasmic reticulum (ER) lumen, Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]1-propansulfonate; CHO, Chinese hamster ovary cells; CHX, cycloheximide; CST, castanospermine; endo-B-N-acetylglucosaminidase H (endoH), endo-B-N-glucosaminidase H; ER, endoplasmic reticulum; HDL, high density lipoprotein; HL, hepatic lipase; intermediate density lipoproteins (IDL), intermediate density lipoprotein; LPL, lipoprotein lipase; PL, pancreatic lipase; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; TM, tunicamycin; very low density lipoportein (VLDL), very low density lipoprotein
Subtraction of the amount of HL activity secreted in the presence of CHX from the values obtained for control and CST-treated cells allowed us to estimate that CST reduced secretion of HL activity to 44% of control. These values agree very well with the effect of CST on HL protein secretion (Fig. 3A), and suggest that the specific activity of HL secreted in the presence of CST was very similar to that secreted from untreated cells. These results suggest that processing of N-linked glycans by ER glucosidases is not required for human HL secretion, the efficiency of its secretion was significantly reduced in the presence of CST

Summary

Introduction

The addition and endoplasmic reticulum (ER) glucosidase processing of N-linked glycans is essential for the secretion of rat hepatic lipase (HL). In the current study we analyzed the role of N-linked carbohydrate processing and calnexin interaction in human HL secretion from stably transfected Chinese hamster ovary (CHO) cells. To determine the role of N-linked glycans in human HL secretion, pulse-chase experiments were performed in the presence of TM (Fig. 3A).

Results

Conclusion