Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol
Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol
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DOI：
10.1042/0264-6021:3430001
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发表时间：
1999-10-01
影响因子：
4.1
通讯作者：
Vance, DE
中科院分区：
文献类型：
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作者：
Lehner, R;Vance, DE
最近从猪肝脏中纯化了一种微体三酰基甘油水解酶（TGH） [Lehner and Verger (1997) Biochemistry 36, 1861-1868]。为了进一步了解TGH的功能，我们从大鼠肝脏cDNA文库中克隆了一段编码TGH的cDNA，并生成了稳定表达大鼠TGH的McArdle RH7777大鼠肝癌细胞系。从cDNA序列衍生的推定蛋白包含一个可切割的信号序列和一个存在于所有已知脂肪酶中保守的五肽基序（GXSXG）中的催化位点丝氨酸残基。与对照细胞相比，转染tgh的细胞在预先标记的三酰基甘油储存的耗散率方面增加了2倍。因此，TGH能够水解储存的三酰基甘油。相比之下，标记的磷脂酰胆碱在载体和tgh转染的细胞中周转率相似。在转染tgh的细胞中进行的研究表明，细胞内分泌的三酰甘油池的利用率约为。比载体转染细胞高30%。而对照组和转染tgh的细胞分泌的磷脂酰胆碱基本相同，转染tgh的细胞分泌的磷脂酰胆碱也大致相同。与对照细胞相比，在极低密度脂蛋白密度范围内的载脂蛋白B100水平增加了25%。结果表明微粒体TGH积极参与细胞质三酰基甘油储存的动员，其中一些可用于脂蛋白组装。
A microsomal triacylglycerol hydrolase (TGH) was recently purified from porcine liver [Lehner and Verger (1997) Biochemistry 36, 1861-1868]. To gain further insight into the function of TGH, we have cloned a cDNA encoding TGH from a rat liver cDNA library and generated McArdle RH7777 rat hepatoma cell lines that stably express the rat TGH. The putative protein derived from the cDNA sequence contains a cleavable signal sequence and a catalytic site serine residue present within a pentapeptide motif (GXSXG) that is conserved in all known lipases. TGH-transfected cells showed a 2-fold increase, compared with control cells, in the rate of depletion of prelabelled triacylglycerol stores. Thus, TGH is capable of hydrolysis of stored triacylglycerol. In contrast, the rate of turnover of labelled phosphatidylcholine was similar in both the vector- and TGH-transfected cells. Studies in TGH-transfected cells demonstrated that utilization of intracellular triacylglycerol pools for secretion was approx. 30 % higher than in vector-transfected cells. Whereas phosphatidylcholine secretion was essentially the same in control and TGH-transfected cells, TGH-transfected cells also secreted an approx. 25 % greater mass of triacylglycerol into the medium and had increased levels of apolipoprotein B100 in the very-low-density lipoprotein density range compared with control cells. The results suggest that the microsomal TGH actively participates in the mobilization of cytoplasmic triacylglycerol stores, some of which can be used for lipoprotein assembly.