Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Jan 1;337(Pt 1):133–140.

Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum.

A J Verhoeven 1, B P Neve 1, H Jansen 1
PMCID: PMC1219946  PMID: 9854035

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (200.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bamberger M., Glick J. M., Rothblat G. H. Hepatic lipase stimulates the uptake of high density lipoprotein cholesterol by hepatoma cells. J Lipid Res. 1983 Jul;24(7):869–876. [PubMed] [Google Scholar]
  2. Barth J. D., Jansen H., Kromhout D., Reiber J. H., Birkenhager J. C., Arntzenius A. C. Progression and regression of human coronary atherosclerosis. The role of lipoproteins, lipases and thyroid hormones in coronary lesion growth. Atherosclerosis. 1987 Nov;68(1-2):51–58. doi: 10.1016/0021-9150(87)90093-1. [DOI] [PubMed] [Google Scholar]
  3. Ben-Zeev O., Stahnke G., Liu G., Davis R. C., Doolittle M. H. Lipoprotein lipase and hepatic lipase: the role of asparagine-linked glycosylation in the expression of a functional enzyme. J Lipid Res. 1994 Sep;35(9):1511–1523. [PubMed] [Google Scholar]
  4. Berryman D. E., Mulero J. J., Hughes L. B., Brasaemle D. L., Bensadoun A. Oligomeric structure of hepatic lipase: evidence from a novel epitope tag technique. Biochim Biophys Acta. 1998 Feb 17;1382(2):217–229. doi: 10.1016/s0167-4838(97)00150-7. [DOI] [PubMed] [Google Scholar]
  5. Breedveld B., Schoonderwoerd K., Verhoeven A. J., Willemsen R., Jansen H. Hepatic lipase is localized at the parenchymal cell microvilli in rat liver. Biochem J. 1997 Jan 15;321(Pt 2):425–430. doi: 10.1042/bj3210425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burkhardt J. K., Argon Y. Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing. J Cell Sci. 1989 Apr;92(Pt 4):633–642. doi: 10.1242/jcs.92.4.633. [DOI] [PubMed] [Google Scholar]
  7. Busch S. J., Barnhart R. L., Martin G. A., Fitzgerald M. C., Yates M. T., Mao S. J., Thomas C. E., Jackson R. L. Human hepatic triglyceride lipase expression reduces high density lipoprotein and aortic cholesterol in cholesterol-fed transgenic mice. J Biol Chem. 1994 Jun 10;269(23):16376–16382. [PubMed] [Google Scholar]
  8. Busch S. J., Martin G. A., Barnhart R. L., Jackson R. L. Heparin induces the expression of hepatic triglyceride lipase in a human hepatoma (HepG2) cell line. J Biol Chem. 1989 Jun 5;264(16):9527–9532. [PubMed] [Google Scholar]
  9. Chang S., Borensztajn J. Hepatic lipase function and the accumulation of beta-very-low-density lipoproteins in the plasma of cholesterol-fed rabbits. Biochem J. 1993 Aug 1;293(Pt 3):745–750. doi: 10.1042/bj2930745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Doms R. W. Oligomerization and protein transport. Methods Enzymol. 1990;191:841–854. doi: 10.1016/0076-6879(90)91051-7. [DOI] [PubMed] [Google Scholar]
  11. Doolittle M. H., Wong H., Davis R. C., Schotz M. C. Synthesis of hepatic lipase in liver and extrahepatic tissues. J Lipid Res. 1987 Nov;28(11):1326–1334. [PubMed] [Google Scholar]
  12. Ehnholm C., Greten H., Brown W. V. A comparative study of post-heparin lipolytic activity and a purified human plasma triacylglycerol lipase. Biochim Biophys Acta. 1974 Jul 26;360(1):68–77. doi: 10.1016/0005-2760(74)90180-5. [DOI] [PubMed] [Google Scholar]
  13. Elbein A. D. Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu Rev Biochem. 1987;56:497–534. doi: 10.1146/annurev.bi.56.070187.002433. [DOI] [PubMed] [Google Scholar]
  14. Fuhrmann U., Bause E., Ploegh H. Inhibitors of oligosaccharide processing. Biochim Biophys Acta. 1985 Jun 24;825(2):95–110. doi: 10.1016/0167-4781(85)90095-8. [DOI] [PubMed] [Google Scholar]
  15. Fujiwara T., Oda K., Yokota S., Takatsuki A., Ikehara Y. Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J Biol Chem. 1988 Dec 5;263(34):18545–18552. [PubMed] [Google Scholar]
  16. Groot P. H., van Stiphout W. A., Krauss X. H., Jansen H., van Tol A., van Ramshorst E., Chin-On S., Hofman A., Cresswell S. R., Havekes L. Postprandial lipoprotein metabolism in normolipidemic men with and without coronary artery disease. Arterioscler Thromb. 1991 May-Jun;11(3):653–662. doi: 10.1161/01.atv.11.3.653. [DOI] [PubMed] [Google Scholar]
  17. Hammond C., Braakman I., Helenius A. Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):913–917. doi: 10.1073/pnas.91.3.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hebert D. N., Foellmer B., Helenius A. Glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum. Cell. 1995 May 5;81(3):425–433. doi: 10.1016/0092-8674(95)90395-x. [DOI] [PubMed] [Google Scholar]
  19. Helenius A. How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. Mol Biol Cell. 1994 Mar;5(3):253–265. doi: 10.1091/mbc.5.3.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hide W. A., Chan L., Li W. H. Structure and evolution of the lipase superfamily. J Lipid Res. 1992 Feb;33(2):167–178. [PubMed] [Google Scholar]
  21. Hill J. S., Davis R. C., Yang D., Wen J., Philo J. S., Poon P. H., Phillips M. L., Kempner E. S., Wong H. Human hepatic lipase subunit structure determination. J Biol Chem. 1996 Sep 13;271(37):22931–22936. doi: 10.1074/jbc.271.37.22931. [DOI] [PubMed] [Google Scholar]
  22. Jensen G. L., Bensadoun A. Purification, stabilization, and characterization of rat hepatic triglyceride lipase. Anal Biochem. 1981 May 15;113(2):246–252. doi: 10.1016/0003-2697(81)90073-7. [DOI] [PubMed] [Google Scholar]
  23. Ji Z. S., Lauer S. J., Fazio S., Bensadoun A., Taylor J. M., Mahley R. W. Enhanced binding and uptake of remnant lipoproteins by hepatic lipase-secreting hepatoma cells in culture. J Biol Chem. 1994 May 6;269(18):13429–13436. [PubMed] [Google Scholar]
  24. Kihara S., Wölle J., Ehnholm C., Chan L., Oka K. Regulation of hepatic triglyceride lipase by thyroid hormone in HepG2 cells. J Lipid Res. 1993 Jun;34(6):961–970. [PubMed] [Google Scholar]
  25. Krapp A., Ahle S., Kersting S., Hua Y., Kneser K., Nielsen M., Gliemann J., Beisiegel U. Hepatic lipase mediates the uptake of chylomicrons and beta-VLDL into cells via the LDL receptor-related protein (LRP). J Lipid Res. 1996 May;37(5):926–936. [PubMed] [Google Scholar]
  26. Kuznetsov G., Brostrom M. A., Brostrom C. O. Role of endoplasmic reticular calcium in oligosaccharide processing of alpha 1-antitrypsin. J Biol Chem. 1993 Jan 25;268(3):2001–2008. [PubMed] [Google Scholar]
  27. Laposata E. A., Laboda H. M., Glick J. M., Strauss J. F., 3rd Hepatic lipase. Synthesis, processing, and secretion by isolated rat hepatocytes. J Biol Chem. 1987 Apr 15;262(11):5333–5338. [PubMed] [Google Scholar]
  28. Leitersdorf E., Stein O., Stein Y. Synthesis and secretion of triacylglycerol lipase by cultured rat hepatocytes. Biochim Biophys Acta. 1984 Jul 6;794(2):261–268. doi: 10.1016/0005-2760(84)90154-1. [DOI] [PubMed] [Google Scholar]
  29. Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Marques-Vidal P., Azéma C., Collet X., Vieu C., Chap H., Perret B. Hepatic lipase promotes the uptake of HDL esterified cholesterol by the perfused rat liver: a study using reconstituted HDL particles of defined phospholipid composition. J Lipid Res. 1994 Mar;35(3):373–384. [PubMed] [Google Scholar]
  31. Martin G. A., Busch S. J., Meredith G. D., Cardin A. D., Blankenship D. T., Mao S. J., Rechtin A. E., Woods C. W., Racke M. M., Schafer M. P. Isolation and cDNA sequence of human postheparin plasma hepatic triglyceride lipase. J Biol Chem. 1988 Aug 5;263(22):10907–10914. [PubMed] [Google Scholar]
  32. McDowell W., Schwarz R. T. Dissecting glycoprotein biosynthesis by the use of specific inhibitors. Biochimie. 1988 Nov;70(11):1535–1549. doi: 10.1016/0300-9084(88)90290-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moore S. E., Spiro R. G. Demonstration that Golgi endo-alpha-D-mannosidase provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins. J Biol Chem. 1990 Aug 5;265(22):13104–13112. [PubMed] [Google Scholar]
  34. Ou W. J., Cameron P. H., Thomas D. Y., Bergeron J. J. Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature. 1993 Aug 26;364(6440):771–776. doi: 10.1038/364771a0. [DOI] [PubMed] [Google Scholar]
  35. Persoon N. L., Sips H. J., Jansen H. Human hepatoma (Hep G2) cultures contain salt-resistant triglyceridase ("liver lipase"). Life Sci. 1986 Mar 17;38(11):1029–1033. doi: 10.1016/0024-3205(86)90237-7. [DOI] [PubMed] [Google Scholar]
  36. Rabouille C., Spiro R. G. Nonselective utilization of the endomannosidase pathway for processing glycoproteins by human hepatoma (HepG2) cells. J Biol Chem. 1992 Jun 5;267(16):11573–11578. [PubMed] [Google Scholar]
  37. Stahnke G., Davis R. C., Doolittle M. H., Wong H., Schotz M. C., Will H. Effect of N-linked glycosylation on hepatic lipase activity. J Lipid Res. 1991 Mar;32(3):477–484. [PubMed] [Google Scholar]
  38. Sultan F., Lagrange D., Jansen H., Griglio S. Inhibition of hepatic lipase activity impairs chylomicron remnant-removal in rats. Biochim Biophys Acta. 1990 Jan 16;1042(1):150–152. doi: 10.1016/0005-2760(90)90071-5. [DOI] [PubMed] [Google Scholar]
  39. Verhoeven A. J., Jansen H. Secretion of rat hepatic lipase is blocked by inhibition of oligosaccharide processing at the stage of glucosidase I. J Lipid Res. 1990 Oct;31(10):1883–1893. [PubMed] [Google Scholar]
  40. Verhoeven A. J., Jansen H. Secretion-coupled increase in the catalytic activity of rat hepatic lipase. Biochim Biophys Acta. 1991 Oct 15;1086(1):49–56. doi: 10.1016/0005-2760(91)90153-9. [DOI] [PubMed] [Google Scholar]
  41. Wölle J., Jansen H., Smith L. C., Chan L. Functional role of N-linked glycosylation in human hepatic lipase: asparagine-56 is important for both enzyme activity and secretion. J Lipid Res. 1993 Dec;34(12):2169–2176. [PubMed] [Google Scholar]
  42. Zambon A., Austin M. A., Brown B. G., Hokanson J. E., Brunzell J. D. Effect of hepatic lipase on LDL in normal men and those with coronary artery disease. Arterioscler Thromb. 1993 Feb;13(2):147–153. doi: 10.1161/01.atv.13.2.147. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES