Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family
Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family
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DOI：
10.1042/0264-6021:3470419
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发表时间：
2000-04-15
影响因子：
4.1
通讯作者：
Boileau, G
中科院分区：
文献类型：
--
作者：
Ghaddar, G;Ruchon, AF;Boileau, G
由于它们在控制几种生物活性肽的活性中的作用，锌金属肽酶脑啡肽酶家族的成员已被确定为治疗剂设计的假定靶点。目前已报道了6个成员，它们是：脑啡肽酶、内皮素转换酶（ECE）-1和ECE-2、Kell血型蛋白、PHEX（与X染色体上的内肽酶同源的磷酸调节基因的产物）和X转换酶（XCE）。为了鉴定这个重要的肽酶家族的新成员，我们设计了一种基于脑啡肽酶、ECE-1和PHEX的保守氨基酸序列的逆转录酶-PCR策略。我们现在报告从小鼠睾丸中克隆了一种新的脑啡肽酶样肽酶，我们称之为NL 1。NL 1是一种糖蛋白，在该家族的成员中，其显示出与脑啡肽酶最强的序列同一性。然而，与作为II型整合膜蛋白的脑啡肽酶和该家族的其他成员相比，NL 1在培养的哺乳动物细胞中表达时分泌，这可能是由于枯草杆菌蛋白酶样转化酶在位于跨膜结构域C-末端22个氨基酸残基的弗林蛋白酶样位点处切割。重组酶具有脑啡肽酶样肽酶活性，并能被脑啡肽酶的两种抑制剂phosphoramidon和thiorphan有效抑制。北方杂交和原位杂交结果表明，NL 1 mRNA主要分布在睾丸，特别是圆形和细长的精子细胞。NL 1 mRNA的这种分布表明它可能参与精子形成或其他与生育力相关的过程。
Because of their roles in controlling the activity of several bioactive peptides, members of the neprilysin family of zinc metallopeptidases have been identified as putative targets for the design of therapeutic agents. Presently, six members have been reported, these are: neprilysin, endothelin-converting enzyme (ECE)-1 and ECE-2, the Kell blood group protein, PHEX (product of the phosphate-regulating gene with homologies to endopeptidase on the X chromosome) and X-converting enzyme (XCE). In order to identify new members of this important family of peptidases, we designed a reverse transcriptase-PCR strategy based on conserved amino acid sequences of neprilysin, ECE-1 and PHEX. We now report the cloning from mouse testis of a novel neprilysin-like peptidase that we called NL1. NL1 is a glycoprotein that, among the members of the family, shows the strongest sequence identity with neprilysin. However, in contrast with neprilysin and other members of the family which are type II integral membrane proteins, NL1 was secreted when expressed in cultured mammalian cells, likely due to cleavage by a subtilisin-like convertase at a furin-like site located 22 amino acid residues in the C-terminus of the transmembrane domain. The recombinant enzyme exhibited neprilysin-like peptidase activity and was efficiently inhibited by phosphoramidon and thiorphan, two inhibitors of neprilysin. Northern blot analysis and in situ hybridization showed that NL1 mRNA was found predominantly in testis, specifically in round and elongated spermatids. This distribution of NL1 mRNA suggests that it could be involved in sperm formation or other processes related to fertility.