Biochemical and pathological research of acyl-CoA : lysophosphatidic acid acyltransferases.

溶血磷脂酸（LPA）酰基转移酶（LPAAT）是参与甘油脂从头合成的关键酶之一，包括磷脂和三酰基甘油。最近的报道表明，LPAAT可能通过合成磷脂酸（PA），生物活性脂质第二信使，参与信号转导途径和多种疾病，包括癌症，炎症和肥胖的进展。LPAATα在大多数组织中均匀表达；而LPAATβ则有差异表达，在脂肪组织中表达水平最高。据报道，LPAATβ突变可导致先天性全局性脂肪营养不良，这表明β-异构体参与了脂肪细胞中甘油三酯的合成和储存。为了了解LPAAT亚型的生理作用和性质，我们详细研究了人LPAATα和β在Sf9和哺乳动物细胞中表达的酶学。对不同酰基转移酶（LPAATs, sn-…More glycerol-3-phosphate （G3P）酰基转移酶）的氨基酸序列进行比对，发现有4个区域具有很强的同源性（酰基转移酶基序）。我们通过定点诱变检测了这四个基序中高度保守的氨基酸残基在人LPAATα活性中的作用。我们发现H104、D109、F146、R149、E178和R181都参与了LPAAT的催化作用。R149能与碱性氨基酸K交换，而R181不能。这些结果表明酰基转移酶基序似乎在这个酰基转移酶家族中形成了一个重要的催化位点。相比之下，N端或c端结构域的作用尚未确定。基于程序的预测和胰蛋白酶对N端或c端标签的可及性表明，LPAATα或β同工型的c端分别面向细胞质或管腔侧。相反，预测LPAATα和β的n端均面向细胞质侧。LPAATα的n端标签抵抗胰蛋白酶的消化，然而，添加酶的产物PA使它们对胰蛋白酶不稳定。这些结果表明，n端面向胞质侧，但隐藏在膜后，PA诱导构象变化使n端暴露。LPAATα n端结构域（超过预测的第一跨膜结构域）缺失25个氨基酸降低了LPAAT活性，表明这些区域不是必需的，而是影响活性的。在LPAATα的c端区域缺失20个氨基酸增加了活性，表明这些区域不是必需的，而是内在地抑制了活性。综上所述，这些结果表明该酶的n端和c端结构域具有调节作用。少

Lysophosphatidic acid (LPA) acyltransferase (LPAAT) is one of the key enzymes involved in de novo biosynthesis of glycerolipids, including phospholipids and triacylglycerol. Recent reports have suggested that LPAAT may be involved in signal transduction pathways and various diseases including the progression of cancer, an inflammation, and obesity through the synthesis of phosphatidic acid (PA), bioactive lipid second messenger. LPAATα is uniformly expressed throughout most tissues ; whereas LPAATβ is differentially expressed, with the highest level found in adipose tissues. Mutations in LPAATβ are reported to cause congenital generalized lipodystrophy, suggesting that β-isoform is involved in triacylglycerol synthesis and storage in adipocytes. To understand the physiological roles and properties of LPAAT isoforms, we investigated detailed enzymology of human LPAATα and β expressed in Sf9 and mammalian cells. Alignment of amino acid sequences from various acyltransferases (LPAATs, sn- … More glycerol-3-phosphate (G3P) acyltransferase) reveals four regions with strong homology (acyltransferase motifs). We examined the role of the highly conserved amino acid residues in these four motifs in human LPAATα activity by site-directed mutagenesis. We found that the H104, D109, F146, R149, E178, and R181, all play a role in LPAAT catalysis. R149 but not R181 could be exchangeable to basic amino acid K for the activity. T180 could be exchangeable to S. These results suggest that acyltransferase motifs seem to form a catalytically important site in this family of acyltransferases. In contrast, the roles of N- or C-terminal domains have not established. The program-based prediction and accessibility of trypsin to N- or C-terminal tag suggested that C-terminus of LPAATα or β isoforms faces to cytosolic or lumen side, respectively. In contrast, N-terminus of LPAATα and β was predicted that both faced to cytosolic side. N-terminal tag of LPAATα resisted tryptic digestion, however, addition of PA, the product of the enzyme, rendered them labile to trypsin. These results suggest that the N-terminus faces to cytoslic side but hided behind the membranes, and PA induced the conformational changes to expose of the N-terminus. Deletion of 25 amino acids at N-terminal domains of LPAATα (up to predicted first transmembrane domain) reduced the LPAAT activity, suggesting that these regions are not essential but affect for the activity. Deletion of 20 amino acids at C-terminal domains of LPAATα increased the activity, suggesting that these regions are not essential and intrinsically inhibited the activity. Taken together, these results suggested the regulatory roles of N-terminal and C-terminal domains of the enzyme. Less