Synthesis of inactive nonsecretable high mannose-type lipoprotein lipase by cultured brown adipocytes of combined lipase-deficient cld/cld mice.
Combined lipase deficiency (cld) is a recessive mutation which causes a severe deficiency of lipoprotein lipase and hepatic lipase activities and lethal hypertriacylglycerolemia within 3 days in newborn mice. The effect of this genetic defect on lipoprotein lipase was studied in primary cultures of brown adipocytes derived from tissue of newborn mice. Cells cultured from cld/cld mice replicated, accumulated triacylglycerol, and differentiated into adipocytes at normal rates. Lipoprotein lipase activity in unaffected cells was detectable on Day 0 of confluence and increased to 1.3 units/mg DNA by Day 6, while that in cld/cld cells was less than 4% of that in unaffected cells on Days 4-6. Unaffected cells released 1.2% of their lipase activity in 30 min in the absence of heparin, and 11% in 10 min in the presence of heparin, whereas cld/cld cells released no lipase activity. cld/cld cells contained 2-3 times as much lipoprotein lipase protein as unaffected cells, and released no lipase protein to the medium. Immunofluorescent lipoprotein lipase was not detectable in unaffected adipocytes unless lipase secretion was blocked with monesin, causing retention of the lipase in Golgi. cld/cld adipocytes, in contrast, contained immunofluorescent lipoprotein lipase distributed in a diffuse reticular pattern, indicating retention of lipase in endoplasmic reticulum. Lipoprotein lipase immunoprecipitated from cells incubated 1-3 h with [35S]methionine was digested with or without endoglycosidase H (endo H) or F, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lipoprotein lipase in unaffected cells (Mr = 56,000-58,000) consisted of three glycosylated forms, of which the most prevalent was endo H-resistant, the next was totally endo H-sensitive, and the least was partially endo H-sensitive. In contrast, lipoprotein lipase in cld/cld cells (Mr = 56,000) consisted of a single, totally endo H-sensitive form. Lipoprotein lipase in both groups of cells contained two oligosaccharide chains. Chromatography studies with heparin-Sepharose indicated that at least some of the lipoprotein lipase in cld/cld cells was dimerized. The findings demonstrate that brown adipocytes cultured from cld/cld mice synthesize lipoprotein lipase with two high mannose oligosaccharide chains, but it is inactive and retained in endoplasmic reticulum. Whether the cld mutation affects primarily processing of oligosaccharide chains of lipoprotein lipase in endoplasmic reticulum, transport of the lipase from the reticulum, or some other process, is to be resolved.
Masuno
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Laboratory of Cellular and Developmental Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Blanchette-Mackie
E J
EJ
Chernick
S S
SS
Scow
R O
RO
D002001:Adipose Tissue, Brown / Q000201:enzymology*; D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D006493:Heparin / Q000494:pharmacology; D006596:Hexosaminidases / Q000494:pharmacology; D007158:Immunologic Techniques; D008071:Lipoprotein Lipase / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D014280:Triglycerides / Q000378:metabolism
D006023:Glycoproteins; D008562:Membrane Glycoproteins; D014280:Triglycerides; C025363:mannoproteins; D006493:Heparin; D008071:Lipoprotein Lipase; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase