Masuno H, Blanchette-Mackie E J, Chernick S S, Scow R O
Laboratory of Cellular and Developmental Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
J Biol Chem. 1990 Jan 25;265(3):1628-38.
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.
联合脂肪酶缺乏症（cld）是一种隐性突变，可导致脂蛋白脂肪酶和肝脂肪酶活性严重缺乏，并在新生小鼠3天内引发致死性高甘油三酯血症。在源自新生小鼠组织的棕色脂肪细胞原代培养物中研究了这种基因缺陷对脂蛋白脂肪酶的影响。从cld/cld小鼠培养的细胞能够正常复制、积累甘油三酯并分化为脂肪细胞。未受影响的细胞在汇合第0天可检测到脂蛋白脂肪酶活性，到第6天增加至1.3单位/毫克DNA，而在第4 - 6天，cld/cld细胞中的该活性不到未受影响细胞的4%。在无肝素的情况下，未受影响的细胞在30分钟内释放其脂肪酶活性的1.2%，在有肝素的情况下10分钟内释放11%，而cld/cld细胞不释放脂肪酶活性。cld/cld细胞所含的脂蛋白脂肪酶蛋白是未受影响细胞的2 - 3倍，且不向培养基中释放脂肪酶蛋白。除非用莫能菌素阻断脂肪酶分泌，导致脂肪酶滞留在高尔基体中，否则在未受影响的脂肪细胞中无法检测到免疫荧光脂蛋白脂肪酶。相比之下，cld/cld脂肪细胞含有呈弥漫网状分布的免疫荧光脂蛋白脂肪酶，表明脂肪酶滞留在内质网中。用[35S]甲硫氨酸孵育1 - 3小时的细胞免疫沉淀的脂蛋白脂肪酶，用或不用内切糖苷酶H（endo H）或F消化，然后通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离。未受影响细胞中的脂蛋白脂肪酶（Mr = 56,000 - 58,000）由三种糖基化形式组成，其中最普遍的是对endo H有抗性的，其次是完全对endo H敏感的，最少的是部分对endo H敏感的。相比之下，cld/cld细胞中的脂蛋白脂肪酶（Mr = 56,000）由单一的、完全对endo H敏感的形式组成。两组细胞中的脂蛋白脂肪酶都含有两条寡糖链。用肝素 - 琼脂糖进行的色谱研究表明，cld/cld细胞中至少一些脂蛋白脂肪酶是二聚体。这些发现表明，从cld/cld小鼠培养的棕色脂肪细胞合成具有两条高甘露糖寡糖链的脂蛋白脂肪酶，但它是无活性的并滞留在内质网中。cld突变是否主要影响内质网中脂蛋白脂肪酶寡糖链的加工、脂肪酶从内质网的转运或其他一些过程，还有待解决。
