Molecular cloning and expression of human leukotriene C4 synthase.
Molecular cloning and expression of human leukotriene C4 synthase.
复制标题
人白三烯C4合酶的分子克隆和表达。
DOI：
10.1073/pnas.91.21.9745
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Peter C. Isakson
中科院分区：
文献类型：
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作者：
D. J. Welsch;D. P. Creely;S. Hauser;Karl J. Mathis;G. Krivi;Peter C. Isakson
摘要
白三烯-C4 合酶（LTC4S；EC 2.5.1.37）催化肽白三烯生物合成的关键步骤，这在哮喘的发病机制中很重要。基于先前报道的人 LTC4S 的部分氨基酸序列，生成了针对合成肽的抗体 [Nicholson, D.W.、Ali, A.、Vailncourt, J.P.、Calaycay, J.R.、Mumford, R.A.、Zamboni, R.J. & Ford-Hutchinson, A. W. (1993) Proc。国家。阿卡德。科学。 USA 90, 2015-2019] 和从 THP-1 细胞中获得的特异性结合的去污剂溶解的 LTC4S，证实所发表的序列与酶活性相关。使用基于部分蛋白质序列的含肌苷寡核苷酸从 THP-1 细胞中分离出 LTC4S 的 679 bp cDNA。该 cDNA 包含一个开放阅读框，编码 150 个氨基酸的蛋白质 (M(r) = 16,568)，计算得出的 pI 值为 11.1。推导的蛋白质序列主要由疏水性氨基酸组成；水病分析预测由两个亲水环连接的三个跨膜结构域。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-转移酶没有同源性。 LTC4S被发现与5-脂氧合酶激活蛋白最相似（31%同一性，53%相似性），这是另一种参与白三烯生物合成的蛋白质。活性酶在细菌、昆虫和哺乳动物细胞中表达，如在含有 LTA4 和还原型谷胱甘肽的孵育混合物中 LTC4 的生物合成所示。人LTC4S的克隆和表达为更好地了解肽白三烯生物合成中的关键酶提供了基础。
Abstract
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.