Ann Beaumont

Neutral endopeptidase-24.11 inhibitors: from analgesics to antihypertensives?

A limited number of ectoenzymes appear to be involved in inactivating circulating regulatory peptides. The widely distributed angiotensin-converting enzyme controls the concentration of angiotensin II in the blood, thereby limiting its vasoconstrictor effects. Inhibitors of this enzyme, such as captopril and enalapril, are clinically used as antihypertensives. Neutral endopeptidase-24.11 is known to be involved in inactivating opioid peptides in the CNS and much research has focused on the use of its inhibitors in manipulating endogenous pain-control mechanisms. Recent evidence that this metallopeptidase inactivates atrial natriuretic peptide, summarized here by Bernard Roques and Ann Beaumont, has led to a re-evaluation of the potential use of its inhibitors as novel diuretics and antihypertensive agents.

Intramolecular Processing of Prothermolysin

Thermolysin, an extracellular zinc endopeptidase from Bacillus thermoproteolyticus, is synthesized as a pre-proenzyme and the prosequence has been shown to assist the refolding of the denatured enzyme in vitro and to inhibit enzyme activity (O’Donohue, M. J., and Beaumont, A. (1996)J. Biol. Chem. 271, 26477–26481). To determine whether prosequence cleavage from the mature enzyme is autocatalytic and if so, whether it is an intermolecular or intramolecular process, N-terminal histidine-tagged prothermolysin was expressed inEscherichia coli. Although partial processing to mature enzyme occurred, most of the proenzyme was recovered intact from inclusion bodies. This was then solubilized in guanidinium hydrochloride, immobilized on a cobalt-containing resin, and after dialysis against renaturation buffer, was quantitatively transformed to mature enzyme. However, when a mutation was introduced into the mature sequence to inactivate thermolysin, the proenzyme was not processed either in vivo or in vitro. In addition, mutated prothermolysin was not processed by exogenous thermolysin under a variety of experimental conditions. The results demonstrate that thermolysin maturation can proceed via an autocatalytic intramolecular pathway.

Neutral endopeptidase 24.11 and angiotensin converting enzyme like activity in calla positive and calla negative lymphoid cells

Taking advantage of the recently demonstrated identity of common acute lymphoblastic leukemia antigen (CALLA) and neutral endopeptidase EC.24.11 (NEP) the presence of this ectoenzyme on lymphoid cells has been reassessed using highly sensitive assays (cleavage of [3H]-D-Ala2-leucine-enkephalin and binding of the inhibitor [3H]HACBO-Gly. NEP activity was found not only on already classified CALLA + ve cells but also on numerous cells (including mature B and polyclonal T cells) previously considered as CALLA-ve. This suggests that CALLA/NEP is expressed all along the differentiation pathway in B and T cell lineage. Moreover substantial ACE-like activity was also detected in three tested cells, all of the pre-B phenotype. The availability of specific inhibitors for these enzymes should help clarify their role in cell-differentiation.

Presence of a histidine at the active site of the neutral endopeptidase-24.11

Diethylpyrocarbonate treatment of the neutral endopeptidase (EC 3.4.24.11) inhibits both catalytic activity and binding of the inhibitor [3H]-N(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]-glycine. The loss of activity can be reversed by hydroxylamine and almost completely prevented by the competitive inhibitor phenylalanyl-leucine suggesting the presence, as in thermolysin, of a histidine residue at the active site. Butanedione treatment also reduces both catalytic activity and [3H] inhibitor binding. Phenylalanyl-leucine completely protects from the butanedione induced loss of activity, providing further evidence for an essential arginine at the active site. In contrast, the tyrosine modifying agent N-acetylimidazole has no apparent effect on enzyme activity.

Increase of neutral endopeptidase-24.11 with cellular density and enzyme modulation with an inhibitor on human Reh6 cell line.

Neutral endopeptidase (EC 3.4.24.11, NEP) is an ectoenzyme, identified as the common acute lymphoblastic leukemia antigen (CALLA, CD10). This enzyme is involved in the inactivation of regulatory peptides such as enkephalins and atrial natriuretic peptide and its expression on the cell surface is therefore essential. NEP levels have been measured under different conditions on leukemic cell lines. NEP activity per cell was found to increase during the cell growth of Reh6 and CEM cells, a cell-cell contact mechanism being suggested by experiments using Transwell cell chambers. The same process was not observed with ICIG-7 fibroblasts. The numbers of enzymatic sites was also found to be selectively modulated by treatment with 0.1 microM N-[3-(R,S)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]glycine (HACBOGly), a potent (Ki = 1.4 nM) and specific inhibitor of NEP. A maximal 13% decrease in sites was observed after 8 hr incubation, this effect disappearing after 12 hr. This weak but specific negative modulation was not observed with a compound, chemically related to HACBOGly, which has a 10,000-fold lower inhibitory potency. The modulation was inhibited by low temperature or monensin treatment and could be brought about by an internalization of the enzyme, compensated for by an increased biosynthesis or by the sequestration of NEP in a non-membranous compartment.

Neurotensin high affinity binding sites and endopeptidase 24.11 are present respectively in the meningothelial and in the fibroblastic components of human meningiomas

The presence of neurotensin receptors and endopeptidase 24.11 (E-24.11) in 16 human meningioma specimens, obtained at surgery, was assessed by measuring the binding of 125I-[tyrosyl3]neurotensin(1-13) (125I-NT) and the inhibitor 3H-N(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl)glycine (3H-HACBO-Gly), for the receptor and enzyme, respectively. E-24.11 activity was also measured. Autoradiography, on the 16 meningiomas, showed that specific 125I-NT labeling (nonspecific labeling was assessed in the presence of excess NT) was exclusively located in the meningothelial regions. In contrast, specific 3H-HACBO-Gly labeling (nonspecific labeling was assessed in the presence of an excess of the E-24.11 inhibitor thiorphan) was exclusively found in fibroblastic regions. No specific labeling of either ligand was found on collagen or blood vessels. In vitro binding assays were performed on membranes of 10 of the 16 meningiomas. In the 4 meningiomas rich in meningothelial cells, 125I-NT specifically bound to one population of sites with Bmax ranging from 57 to 405 fmol/mg protein and Kd around 0.3 nM. These sites share common properties with the brain NT receptor, since the carboxy terminal acetyl NT(8-13) fragment bound to the same sites but with a higher affinity. The carboxy terminal analogue of NT, neuromedin N, also bound to the same sites with a 10-fold lower affinity and the sites were bradykinin and levocabastine insensitive. In the 4 meningiomas rich in fibroblastic cells, 3H-HACBO-Gly specifically bound to one population of sites with Bmax ranging from 251 to 739 fmol/mg protein and Kd around 2.8 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Cleavage of farnesylated COOH-terminal heptapeptide of mouse N-ras by brain microsomal membranes: evidence for a carboxypeptidase which specifically removes the COOH-terminal methionine.

Brain microsomal membranes are capable of sequentially removing Met, Leu and Val from a chemically synthesized COOH-terminal heptapeptide (propionyl-Gly-Ser-Pro-(farnesyl-Cys)-Val-Leu-Met) of mouse N-ras protein. The carboxypeptidase generating Met displays maximum activity at neutral pH and shows high affinity for the farnesylated substrate (Km = 73 microM) as compared to its non farnesylated precursor (Km = 600 microM). The results of inhibitor action suggest that the membrane carboxypeptidase is a novel, probably thiol-dependent, serine type peptidase.

Effects of monoclonal antibodies raised against the common acute lymphoblastic leukemia antigen on endopeptidase-24.11 activity

The common acute lymphoblastic leukemia antigen (CALLA, CD10) has been identified as neutral endopeptidase-24.11 (NEP), a mammalian ectoenzyme involved in the inactivation of regulatory peptides, such as the enkephalins and atrial natriuretic peptide. Twenty monoclonal antibodies directed against the human antigen, were tested for their ability to inhibit the enzymatic activity of the human and rat peptidases expressed by cell lines. Six anti-CALLA antibodies were found to inhibit 50% or more of the hydrolysis of D-Ala2-leucine enkephalin by the neutral endopeptidase present on the human leukemic cell line Reh6 and, to a lesser extent, the hydrolysis of atrial natriuretic peptide. This may indicate that their binding may affect regions of the active site more important for the dipeptidylcarboxypeptidase activity of the enzyme. Only four antibodies cross-reacted with the peptidase from the rat epithelial cell line Rat2, as shown by membrane immunofluorescence, and these also partially inhibited enzyme activity. No antibody was able to inhibit completely the activity of the human and rat enzymes and all the active antibodies appeared to behave as non-competitive inhibitors of substrate cleavage. These monoclonal antibodies could be used in mapping studies of NEP.

An antibody purified with a λGT11 fusion protein precipitates enkephalinase activity

Abstract An antiserum was raised against the neutral endopeptidase “enkephalinase” in guinea pig and used to probe a rabbit kidney cDNA expression library. A positive clone has been isolated and sequenced. The identity of the corresponding fusion protein was ascertained by its ability to select, from the crude antiserum, antibodies which specifically immunoprecipitate neutral endopeptidase enzymatic activity. This approach eliminates the uncertainty inherent to clone identification obtained from oligonucleotide probe derived from a partial sequence of the protein.