Molecular cloning and expression of human leukotriene-C4 synthase.

Leukotriene-C4 synthase (LTC4S; EC 2.5.1.37) catalyzes the committed step in the biosynthesis of the peptidoleukotrienes, which are important in the pathogenesis of asthma. Antibodies were generated to a synthetic peptide based on the partial amino acid sequence previously reported for human LTC4S [Nicholson, D.W., Ali, A., Vaillancourt, J.P., Calaycay, J.R., Mumford, R.A., Zamboni, R.J. & Ford-Hutchinson, A. W. (1993) Proc. Natl. Acad. Sci. USA 90, 2015-2019] and specifically bound detergent-solubilized LTC4S obtained from THP-1 cells, confirming that the published sequence is associated with enzyme activity. Inosine-containing oligonucleotides based on the partial protein sequence were used to isolate a 679-bp cDNA for LTC4S from THP-1 cells. The cDNA contains an open reading frame that encodes a 150-amino acid protein (M(r) = 16,568) that has a calculated pI value of 11.1. The deduced protein sequence is composed predominantly of hydrophobic amino acids; hydropathy analysis predicts three transmembrane domains connected by two hydrophilic loops. Analysis of the deduced sequence identified two potential protein kinase C phosphorylation sites and a potential N-linked glycosylation site. The amino acid sequence for human LTC4S is unique and shows no homology to other glutathione S-transferases. LTC4S was found to be most similar to 5-lipoxygenase activating protein (31% identity, 53% similarity), another protein involved in leukotriene biosynthesis. Active enzyme was expressed in bacterial, insect, and mammalian cells as shown by the biosynthesis of LTC4 in incubation mixtures containing LTA4 and reduced glutathione. The cloning and expression of human LTC4S provide the basis for a better understanding of this key enzyme in peptidoleukotriene biosynthesis. IMAGES:

白三烯C4合酶（Leukotriene-C4 synthase, LTC4S; EC 2.5.1.37）可催化肽白三烯（peptidoleukotrienes）生物合成途径中的关键限速步骤，该类物质在哮喘的发病机制中发挥重要作用。研究人员基于此前已报道的人LTC4S部分氨基酸序列合成肽段并制备了特异性抗体[Nicholson, D.W., Ali, A., Vaillancourt, J.P., Calaycay, J.R., Mumford, R.A., Zamboni, R.J. & Ford-Hutchinson, A. W. (1993) Proc. Natl. Acad. Sci. USA 90, 2015-2019]，该抗体可特异性结合从THP-1细胞中经去垢剂增溶得到的LTC4S，证实已发表的序列与酶活性相关。基于该部分蛋白序列设计的含肌苷寡核苷酸被用于从THP-1细胞中克隆得到一段长度为679 bp的LTC4S互补DNA（complementary DNA, cDNA）。该cDNA包含一段开放阅读框（open reading frame, ORF），可编码一个由150个氨基酸组成的蛋白质（相对分子质量为16568，理论等电点为11.1）。推导得到的蛋白质序列主要由疏水氨基酸构成；亲水疏水性分析预测其存在3个由两个亲水环连接的跨膜结构域。对推导序列的分析还发现了2个潜在的蛋白激酶C磷酸化位点以及1个潜在的N-连接糖基化位点。人LTC4S的氨基酸序列具有独特性，未发现与其他谷胱甘肽S-转移酶存在同源性。序列比对结果显示，LTC4S与另一种参与白三烯生物合成的蛋白质——5-脂氧合酶激活蛋白（5-lipoxygenase activating protein）的相似度最高（序列同一性为31%，相似性为53%）。通过在细菌、昆虫和哺乳动物细胞中异源表达该基因，在含有白三烯A4（LTA4）与还原型谷胱甘肽（reduced glutathione）的孵育体系中可检测到LTC4的生物合成，证实获得了具有活性的重组酶。人LTC4S的克隆与表达为深入理解肽白三烯生物合成中的这一关键酶提供了重要的研究基础。图像：