Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein
Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein
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DOI：
10.1042/0264-6021:3590707
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发表时间：
2001-11-01
影响因子：
4.1
通讯作者：
Young, PR
中科院分区：
文献类型：
--
作者：
Cheng, D;Meegalla, RL;Young, PR
酰基辅酶A：二酰基甘油酰基转移酶（DGAT）是一种整合膜酶，催化二酰基甘油和酰基辅酶A合成三酰基甘油的最后一步。在这里，我们提供了 DGAT 是同源四聚体的实验证据。尽管人 DGAT 蛋白的预测分子量为 55 kDa，但 CHAPS 溶解的重组人 DGAT 在凝胶过滤色谱上以超过 150 kDa 的级分洗脱。膜中的重组 DGAT 与辛二酸二琥珀酰亚胺交联，在 SDS/PAGE 中产生与二聚体 (108 kDa) 和四聚体 (214 kDa) 相对应的条带。最后，当两种不同表位标记形式的 DGAT 共转染到哺乳动物细胞中时，它们可以进行共免疫沉淀。我们从人类脂肪组织 cDNA 文库中克隆了编码 DGAT 新型剪接变体（称为 DGATsv）的 cDNA，该变体包含带有框内终止密码子的 77 nt 未剪接内含子插入片段。这导致了 DGAT 的截短形式，其终止于 Arg-387，从含有推定活性位点的 C 末端删除了 101 个残基。 DGATsv 转染 HEK-293E 细胞时酶失活，但仍能够在交联时形成二聚体和四聚体，表明形成四聚体的能力位于 N 末端区域。当在 HEK-293E 细胞中共表达时，DGATsv 不会抑制全长 DGAT 的活性，表明 DGAT 的亚基独立催化三酰甘油合成。
Acyl-CoA: diacyglycerol acyltransferase (DGAT) is an integral membrane enzyme that catalyses the last step of triacylglycerol synthesis from diacylglycerol and acyl-CoA. Here we provide experimental evidence that DGAT is a homotetramer. Although the predicted molecular mass of human DGAT protein is 55 kDa, CHAPS-solubilized recombinant human DGAT was eluted in fractions over 150 kDa on gel-filtration chromatography. Crosslinking of recombinant DGAT in membranes with disuccinimidyl suberate yielded bands corresponding to the dimer (108 kDa) and the tetramer (214 kDa) in SDS/PAGE. Finally, when two differently epitope-tagged forms of DGAT were co-transfected into mammalian cells, they could be co-immunoprecipitated. From a human adipose tissue cDNA library we cloned a cDNA encoding a novel splice variant of DGAT (designated DGATsv) that contained a 77 nt insert of unspliced intron with an in-frame stop codon. This resulted in a truncated form of DGAT that terminated at Arg-387, deleting 101 residues from the C-terminus containing the putative active site. DGATsv was enzymically inactive when transfected in HEK-293E cells but was still able to form dimer and tetramer on cross-linking, indicating that the ability to form tetramers resides in the N-terminal region. When co-expressed in HEK-293E cells, DGATsv did not inhibit the activity of full-length DGAT, suggesting that the subunits of DGAT catalyse triacylglycerol synthesis independently.