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

Abstract

Because of their roles in controlling the activity of several bio-active 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.

Transcriptome profiling of the testis reveals genes involved in spermatogenesis and marker discovery in the oriental fruit fly, Bactrocera dorsalis

“…Here, we identified four Nep unigenes in our transcriptome data set: Nep1 , Nep2 , Nep4 and Nep5 . The mRNA of one member of the Nep family, Nep1 , is primarily restricted to round and elongated spermatids, suggesting an important role for the enzyme in reproduction (Ghaddar et al ., ). Nep2 is expressed in the specialized stellate cells of the renal tubules, and in the cyst cells that surround the elongating spermatid bundles in the adult testis of D. melanogaster , suggesting roles for the peptidase in renal function and in spermatogenesis (Thomas et al ., ).…”

Are amyloid‐degrading enzymes viable therapeutic targets in Alzheimer’s disease?

“…1999). Similar strategies developed independently led to the characterization of the same gene product from mouse testis (neprilysin‐like 1) (Ghaddar et al. 2000) and from rat brain (NEP II) (Ouimet et al.…”

Amyloid-Degrading Enzymes as Therapeutic Targets in Alzheimers Disease

“…Several of the NEP-like enzymes are, as yet, orphan peptidases with no recognized peptide substrates. One of the homologues of NEP, namely neprilysin 2 (NEP2, also known as SEP or NL1), was discovered independently by several groups [40][41][42] and in the brain shown to be restricted mainly to developing and differentiated fields of the CNS. Unlike NEP and ECE-1, which are broadly expressed in the CNS and periphery, NEP2 was found to be almost exclusively expressed only in selected populations of neurons and in the spinal cord.…”