title: Glycosylation, activity and secretion of lipoprotein lipase in cultured brown adipocytes of newborn mice. Effect of tunicamycin, monensin, 1-deoxymannojirimycin and swainsonine.
abstract: The effect of inhibitors on the glycosylation, activity and secretion of lipoprotein lipase was studied in brown adipocytes cultured from newborn mice. Such cells synthesized and secreted active lipoprotein lipase. It is generally accepted that active lipoprotein lipase is a homodimer. Glycosylation of lipoprotein lipase was analysed by PAGE of endoglycosidase H (endo H)-digested subunits of lipoprotein lipase immunoprecipitated from cells incubated for 1-2 h with [35S]methionine. The most prevalent 35S-labelled lipase subunit (Mr 57,000-58,000) in these cells contained endo H-resistant oligosaccharide chains, the next most prevalent contained totally endo H-sensitive chains, and the least prevalent subunit contained partially endo H-sensitive chains. Complete blocking of the glycosylation of lipoprotein lipase with tunicamycin (1 microgram/ml) for 24 h resulted in synthesis of an inactive non-secretable form of lipase with a smaller subunit (Mr 51,000-52,000). Immunofluorescent studies showed that unglycosylated lipase in tunicamycin-treated cells was retained in the endoplasmic reticulum. Cells treated with 1 microM-monensin, an intra-Golgi transport inhibitor, synthesized an active form of lipase which was not secreted, but was retained in the Golgi. The lipase in monensin-treated cells contained only partially or totally endo H-sensitive chains. Blocking either Golgi mannosidase I with 4 mM-1-deoxymannojirimycin or Golgi mannosidase II with 10 microM-swainsonine resulted in production of a form of lipoprotein lipase which was active and secreted, and which contained only endo H-sensitive chains. Our findings demonstrate that core glycosylation of lipoprotein lipase in the endoplasmic reticulum is required for lipase activity and transport from the reticulum, whereas processing of the oligosaccharide chains to endo H-resistant (complex) type chains in the Golgi is not required for either the activity or the secretion of lipoprotein lipase.
authors.affiliation: Endocrinology Section, National Institute of Diabetes, and Digestive and Kidney Disease, National Institutes of Health, Bethesda, MD 20892.
authors.forename: H
authors.initials: H
authors.lastname: Masuno
authors.forename: C J
authors.initials: CJ
authors.lastname: Schultz
authors.forename: J W
authors.initials: JW
authors.lastname: Park
authors.forename: E J
authors.initials: EJ
authors.lastname: Blanchette-Mackie
authors.forename: C
authors.initials: C
authors.lastname: Mateo
authors.forename: R O
authors.initials: RO
authors.lastname: Scow
mesh_terms: D017485:1-Deoxynojirimycin; D002001:Adipose Tissue, Brown / Q000201:enzymology*; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002451:Cell Compartmentation / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006031:Glycosylation; D006493:Heparin / Q000494:pharmacology; D066298:In Vitro Techniques; D008071:Lipoprotein Lipase / Q000378:metabolism*; D051379:Mice; D008817:Mice, Mutant Strains; D008985:Monensin / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017026:Swainsonine; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D000470:Alkaloids; D014415:Tunicamycin; D017485:1-Deoxynojirimycin; D006493:Heparin; D008985:Monensin; D003513:Cycloheximide; D008071:Lipoprotein Lipase; D005944:Glucosamine; D017026:Swainsonine