Neprilysin (NEP) 2 is a recently cloned glycoprotein displaying a high degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 subfamily of metalloproteases. Whereas NEP is involved in the metabolism of several bioactive peptides by plasma membranes of various cells, the enzymic properties and physiological functions of NEP2 are unknown. Here we characterize the cell-expression modalities and enzymic specificity of two alternatively spliced isoforms of NEP2 in Chinese hamster ovary and AtT20 cells. In the two cell lines, both isoforms are type II glycoproteins inserted in the endoplasmic reticulum as inactive precursors. Maturation detected by Western-blot analysis of glycosidase digests was cell-specific and more efficient in the endocrine cell line. The enzymic activity of both isoforms semi-purified from AtT20 cells reveals comparable specificities in terms of model substrates, pH optima and inhibitory patterns. NEP2 activity was compared with that of NEP regarding potencies of transition-state inhibitors, modes of hydrolysis, maximal hydrolysis rates and apparent affinities of bioactive peptides. Although all transition-state inhibitors of NEP inhibited NEP2 activity, albeit with different potencies, and many peptides were cleaved at the same amide bond by both peptidases, differences could be observed, i.e. in the hydrolysis of gonadotropin-releasing hormone and cholecystokinin, which occurred at different sites and more efficiently in the case of NEP2. Differences in cleavage of bioactive peptides, in cell-trafficking patterns and in tissue distribution indicate that NEP and NEP2 play distinct physiological roles in spite of their high degree of sequence identity.

Neprilysin 2 (NEP2), a recently identified member of the M13 subfamily of metalloproteases, shares the highest degree of homology with the prototypical member of the family neprilysin. Whereas the study of the in vitro enzymatic activity of NEP2 shows that it resembles that of NEP as it cleaves the same substrates often at the same amide bonds and binds the same inhibitory compounds albeit with different potencies, its physiological role remains elusive because of the lack of selective inhibitors. To aid in the design of these novel compounds and better understand the different inhibitory patterns of NEP and NEP2, the x-ray structure of NEP was used as a template to build a model of the NEP2 active site. The results of our modeling suggest that the overall structure of NEP2 closely resembles that of NEP. The model of the active site reveals a 97% sequence identity with that of NEP with differences located within the S 2 subsite of NEP2 where Ser 133 and Leu 739 replace two glycine residues in NEP. To validate the proposed model, site-directed mutagenesis was performed on a series of residues of NEP2, mutants expressed in AtT20 cells, and their ability to bind various substrates and inhibitory compounds was tested. The results confirm the involvement of the conserved Arg 131 and Asn 567 in substrate binding and catalytic activity of NEP2 and further show that the modifications in its S 2 pocket, particularly the presence therein of Leu 739 , account for a number of differences in inhibitor binding between NEP and NEP2.Neprilysin 2 (SEP and NL-1) is a recently identified type II membrane-bound zinc-dependent metalloprotease (1-3). It is part of the M13 family of metalloproteases, which also comprises neprilysin (NEP, 1 EC 3.4.24.11) (4 -6), the endothelinconverting enzymes, ECE-1 (EC 3.4.24.71) (7) and ECE-2 (8), the Kell blood group antigen (9), the phosphate-regulating neutral endopeptidase on the X chromosome (10) and X-converting enzyme (11). Not only do all of the members of this family of metalloproteases share considerable sequence identities, suggesting a common ancestor as well as a common fold, NEP2, by far the closest homologue of NEP displays over 50% of overall protein sequence identity with NEP, a degree of homology that also suggests a common function (12).The best characterized member of the family, NEP (13, 14), was first identified as a kidney brush-border neutral endopeptidase slowly hydrolyzing [ 125 I]iodoinsulin B chain (15) and rediscovered as an "enkephalinase" of cerebral membranes hydrolyzing the Gly 3 -Phe 4 bond of enkephalins (16). Thereafter, it was shown to hydrolyze a number of biologically active peptides into inactive fragments in vitro, such as tachykinins (17), bradykinin (18), and atrial natriuretic peptides (19,20). The physiological implication of NEP in the inactivation of these messenger peptides was confirmed in vivo by the design and use of selective and potent inhibitors, e.g. thiorphan (21-24). These functions of NEP and the design of specific inhibitors thereof are of ...

Neprilysin (NEP) 2 is a recently cloned glycoprotein displaying a high degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 subfamily of metalloproteases. Whereas NEP is involved in the metabolism of several bioactive peptides by plasma membranes of various cells, the enzymic properties and physiological functions of NEP2 are unknown. Here we characterize the cell-expression modalities and enzymic specificity of two alternatively spliced isoforms of NEP2 in Chinese hamster ovary and AtT20 cells. In the two cell lines, both isoforms are type II glycoproteins inserted in the endoplasmic reticulum as inactive precursors. Maturation detected by Western-blot analysis of glycosidase digests was cell-specific and more efficient in the endocrine cell line. The enzymic activity of both isoforms semi-purified from AtT20 cells reveals comparable specificities in terms of model substrates, pH optima and inhibitory patterns. NEP2 activity was compared with that of NEP regarding potencies of transition-state inhibitors, modes of hydrolysis, maximal hydrolysis rates and apparent affinities of bioactive peptides. Although all transition-state inhibitors of NEP inhibited NEP2 activity, albeit with different potencies, and many peptides were cleaved at the same amide bond by both peptidases, differences could be observed, i.e. in the hydrolysis of gonadotropin-releasing hormone and cholecystokinin, which occurred at different sites and more efficiently in the case of NEP2. Differences in cleavage of bioactive peptides, in cell-trafficking patterns and in tissue distribution indicate that NEP and NEP2 play distinct physiological roles in spite of their high degree of sequence identity.