Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin

Abstract

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.

Human neprilysin‐2 (NEP2) and NEP display distinct subcellular localisations and substrate preferences

“…3B). This follows the general trend seen in rodent NEP2, whereby phosphoramidon was equally potent against NEP and NEP2, whilst thiorphan was far more effective against NEP [10]. Important substitutions in the active sites of NEP and NEP2 may explain the differences observed in both substrate hydrolysis and inhibitor binding.…”

“…In contrast, previous work on rodent NEP2 found substrate preferences similar to those of NEP [3,10]. Rodent NEP2 efficiently hydrolysed leucine and methionine enkephalin, and bradykinin with specificity constants greater than or equal to NEP [10].…”

“…This has not been reported for the rodent homologues and would appear to be unique to human NEP2. It has been suggested that NEP2 is inactive in the ER and needs to reach the Golgi apparatus to become active [10]. It would therefore seem likely that both NEP2 and NEP2 D exist as an inactive form in the ER, and an active circulating form (NEP2) or an active membrane-bound form (NEP2 D ).…”

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

“…Of a variety of neuropeptides tested, only substance P and angiotensin I were hydrolysed at comparable rates by human NEP and NEP2 (Whyteside and Turner 2008). There are also differences between NEP and NEP2 in terms of sensitivity to the two inhibitors, phosphoramidon and thiorphan, in particular thiorphan being far more effective against NEP than NEP2 (Rose et al 2002;Whyteside and Turner 2008). A threedimensional model of the active site of NEP2 compared with NEP has highlighted potential critical residues involved in specificity differences between these two enzyme (Voisin et al 2004).…”

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24

“…35 Phosphoramidon is a competitive inhibitor and transition-state analog of several soluble ZMPs, particularly the gluzincins thermolysin (M4) 58 and neprilysin (M13). 59 Phosphoramidon also inhibits HsSte24 proteolysis and its binding mode, localized solely within the "ZMP Core" module, is highly conserved among these three families (M4, M13, and M48) of ZMPs. 35 Among soluble gluzincins, the ZMP Core module is only a portion of the entire structure (Figure 2A, left-hand panel).…”