Molecular, genetic, and pharmacological studies have shown that neprilysin (also called NEP) catabolizes amyloid beta peptides (A beta) in healthy conditions. However, in Alzheimer disease (AD), A beta accumulates forming senile plaques in brain parenchyma and amyloid deposition around blood vessels. In this study, we tested at cellular level the relationship between neprilysin and A beta in human healthy and AD brain. Our results provided evidence for declining levels of neprilysin in AD brains as compared to healthy controls in parallel with increasing deposition of A beta. In hippocampus of AD individuals we observed a significant down-regulation of neprilysin expression in pyramidal neurons, consistent with the possibility that neprilysin controls the level of A beta accumulation and plaque formation in this area. In the cortex and leptomeninges, neprilysin was expressed in the smooth muscle cells of blood vessels. In sections from AD patients we observed a clear inverse relationship between neprilysin and A beta peptide levels in the vasculature, implicating neprilysin in cerebral amyloid angiopathy.

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.

Members of the M13 family of zinc metalloendopeptidases have been shown to play critical roles in the metabolism of various neuropeptides and peptide hormones, and they have been identified as important therapeutic targets. Recently, a mouse NL1 protein, a novel member of the family, was identified and shown to be expressed mainly in the testis as a secreted protein. To define its physiological role(s), we used a gene targeting strategy to disrupt the endogenous murine Nl1 gene by homologous recombination and generate Nl1 mutant mice. The Nl1 ؊/؊ mice were viable and developed normally, suggesting that zygotic expression of Nl1 is not required for development. However, Nl1؊/؊ males produced smaller litters than their wild-type siblings, indicating specific male fertility problems. Reduced fertility may be explained by two impaired processes, decreased egg fertilization and perturbed early development of fertilized eggs. These two phenotypes did not result from gross anatomical modifications of the testis or from impaired spermatogenesis. Basic sperm parameters were also normal. Thus, our findings suggest that one of the roles of NL1 in mice is related to sperm function and that NL1 modulates the processes of fertilization and early embryonic development in vivo.A wide variety of biologically active peptide hormones, neuropeptides, and regulatory peptides are proteolytically activated or inactivated by zinc metalloproteases (58, 59). The M13 family of zinc metalloproteases attracts much interest because its members are involved in the metabolism of several regulatory peptides of the mammalian nervous, cardiovascular, inflammatory, and immune systems as well as in the mineralization process (8,56,57,59). For these reasons, several members of this family of peptidases have been selected as targets for therapeutic intervention by inhibitors or pharmacological agents.NL1 (also called SEP or NEP2) is the newest member of the M13 family of zinc metallopeptidases (19,25,42), which also includes neprilysin (neutral endopeptidase 24.11; also called NEP and CALLA) (reviewed in reference 11), the endothelinconverting enzymes 1 and 2 (ECE-1 and ECE-2) (reviewed in reference 55), PHEX (formerly PEX; a phosphate regulating gene with homologies to endopeptidases on the X chromosome) (reviewed in reference 54), the Kell blood group protein (reviewed in reference 35), and ECEL1/DINE (endothelinconverting enzyme-like 1/damage-induced neuronal endopeptidase) (29, 60, 61). Nl1 cDNA was simultaneously cloned by reverse transcription-PCR technology with degenerate oligonucleotide primers based on conserved sequence similarity between members of the family and mRNA isolated from mouse and rat testis or from Ece-1 Ϫ/Ϫ embryos (19,25,42). Finally, a clone coding for a putative human homologue has been obtained from a central nervous system cDNA library (4).As observed for other members of the M13 family of peptidases, the Nl1 cDNA encodes a type II transmembrane glycoprotein encompassing a short N-terminal cytoplasmic tail, a single tran...

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.

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