Welsch D J, Creely D P, Hauser S D, Mathis K J, Krivi G G, Isakson P C
Searle Research and Development, Monsanto Company, St. Louis, MO 63198.
Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9745-9. doi: 10.1073/pnas.91.21.9745.
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.
白三烯 - C4 合酶（LTC4S；EC 2.5.1.37）催化肽白三烯生物合成中的关键步骤，肽白三烯在哮喘发病机制中起重要作用。基于先前报道的人 LTC4S 的部分氨基酸序列 [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] 合成了一种肽，并由此产生了抗体，该抗体能特异性结合从 THP - 1 细胞中获得的经去污剂溶解的 LTC4S，证实已发表的序列与酶活性相关。基于部分蛋白质序列的含次黄苷寡核苷酸被用于从 THP - 1 细胞中分离出一个 679 碱基对的 LTC4S cDNA。该 cDNA 包含一个开放阅读框，编码一个 150 个氨基酸的蛋白质（M(r) = 16,568），其计算得到的 pI 值为 11.1。推导的蛋白质序列主要由疏水氨基酸组成；亲水性分析预测有三个跨膜结构域，由两个亲水环连接。对推导序列的分析确定了两个潜在的蛋白激酶 C 磷酸化位点和一个潜在的 N - 连接糖基化位点。人 LTC4S 的氨基酸序列是独特的，与其他谷胱甘肽 S - 转移酶无同源性。发现 LTC4S 与 5 - 脂氧合酶激活蛋白最为相似（同一性为 31%，相似性为 53%），5 - 脂氧合酶激活蛋白是另一种参与白三烯生物合成的蛋白质。在含有 LTA4 和还原型谷胱甘肽的孵育混合物中，LTC4 的生物合成表明活性酶在细菌、昆虫和哺乳动物细胞中均有表达。人 LTC4S 的克隆和表达为更好地理解肽白三烯生物合成中的这一关键酶提供了基础。
