Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family

Because of their roles in controlling the activity of several bioactive pep tides, members of the neprilysin family of zinc metallopeptidases have been identified as putative targets for the design of therapeutic agents. Prese ntly, six members have been reported, these are: neprilysin, endothelin-con verting enzyme (ECE)-1 and ECE-2, the Kell blood group protein, PHEX (produ ct of the phosphate-regulating gene with homologies to endopeptidase on the X chromosome) and X-converting enzyme (XCE). In order to identify new memb ers of this important family of peptidases, we designed a reverse transcrip tase-PCR strategy based on conserved amino acid sequences of neprilysin, EC E-1 and PHEX. We now report the cloning from mouse testis of a novel nepril ysin-like peptidase that we called NL1. NL1 is a glycoprotein that, among t he members of the family, shows the strongest sequence identity with nepril ysin. However, in contrast with neprilysin and other members of the family which are type II integral membrane proteins, NL1 was secreted when express ed 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-te rminus of the transmembrane domain. The recombinant enzyme exhibited nepril ysin-like peptidase activity and was efficiently inhibited by phosphoramido n and thiorphan, two inhibitors of neprilysin. Northern blot analysis and i n 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 process es related to fertility.