Role of N-linked glycosylation in the secretion and activity of endothelial lipase
Role of N-linked glycosylation in the secretion and activity of endothelial lipase
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DOI：
10.1194/jlr.m400162-jlr200
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发表时间：
2004-11-01
影响因子：
6.5
通讯作者：
Rader, DJ
中科院分区：
文献类型：
--
作者：
Miller, GC;Long, CJ;Rader, DJ
人内皮脂肪酶 (EL) 是甘油三酯脂肪酶基因家族的成员，具有五个潜在的 N 连接糖基化位点，其中两个在脂蛋白脂肪酶和肝脂肪酶中都是保守的。用糖苷酶处理后以及用糖基化抑制剂衣霉素处理表达EL的细胞后，EL的分子量降低，证明EL是糖基化蛋白质。通过天冬酰胺 (Asn) 的定点诱变来检查每个假定的糖基化位点。 Asn-60 的突变显着减少了分泌并略微增加了比活性。 Asn-116 的突变不影响分泌，但增加了比活性。在这两种情况下，这是由于表观 K-m 降低和表观 V-max 增加造成的。 Asn-373 突变不影响分泌，但由于表观 V-max 降低而显着降低比活性。 Asn471 的突变不会导致分泌或比活性降低。 Asn449 的突变不会导致分泌、活性或分子质量发生变化，表明该位点未被利用。检查了以正常水平分泌的突变体介导 LDL 和细胞表面之间桥接的能力。jlr Asn-373 突变体表现出与野生型 EL 相比，桥接减少了 3 倍，而 Asn-116 和 Asn-471 与野生型 EL 相似。
Human endothelial lipase (EL), a member of the triglyceride lipase gene family, has five potential N-linked glycosylation sites, two of which are conserved in both lipoprotein lipase and hepatic lipase. Reduction in molecular mass of EL after treatment with glycosidases and after treatment of EL-expressing cells with the glycosylation inhibitor tunicamycin demonstrated that EL is a glycosylated protein. Each putative glycosylation site was examined by site-directed mutagenesis of the asparagine (Asn). Mutation of Asn-60 markedly reduced secretion and slightly increased specific activity. Mutation of Asn-116 did not influence secretion but increased specific activity. In both cases, this resulted from decreased apparent K-m and increased apparent V-max. Mutation of Asn-373 did not influence secretion but significantly reduced specific activity, as a result of a decrease in apparent V-max. Mutation of Asn471 resulted in no reduction in secretion or specific activity. Mutation of Asn449 resulted in no change in secretion, activity, or molecular mass, indicating that the site is not utilized. The ability of mutants secreted at normal levels to mediate bridging between LDL and cell surfaces was examined.jlr The Asn-373 mutant demonstrated a 3-fold decrease in bridging compared with wild-type EL, whereas Asn-116 and Asn-471 were similar to wild-type EL.