Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase)

Malfroy, Bernard; Kuang, Wun-Jing; Seeburg, Peter H.; Mason, Anthony J.; Schofield, Peter R.

doi:10.1016/0014-5793(88)80828-7

Cited by 205 publications

(86 citation statements)

References 16 publications

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“…The purified cDNA insert was subcloned in the pCMV eukaryote expression vector according to the manufacturer's instructions (Stratagene, La Jolla, CA). Sequence verification of the resulting plasmid, called pCMV-hNEP, demonstrated that the amplified NEP sequence corresponded exactly to the published one (24).…”

Section: Methodsmentioning

confidence: 64%

Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways

Wisner

Dufour

Messaoudi³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

160

148

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Mammalian zinc ectopeptidases play important roles in turning off neural and hormonal peptide signals at the cell surface, notably those processing sensory information. We report here the discovery of a previously uncharacterized physiological inhibitor of enkephalininactivating zinc ectopeptidases in humans, which we have named Opiorphin. It is a QRFSR peptide that inhibits two enkephalin-catabolizing ectoenzymes, human neutral ecto-endopeptidase, hNEP (EC 3.4.24.11), and human ecto-aminopeptidase, hAP-N (EC 3.4.11.2). Opiorphin displays potent analgesic activity in chemical and mechanical pain models by activating endogenous opioid-dependent transmission. Its function is closely related to the rat sialorphin peptide, which is an inhibitor of pain perception and acts by potentiating endogenous -and ␦-opioid receptor-dependent enkephalinergic pathways. Here we demonstrate the functional specificity in vivo of human Opiorphin. The pain-suppressive potency of Opiorphin is as effective as morphine in the behavioral rat model of acute mechanical pain, the pin-pain test. Thus, our discovery of Opiorphin is extremely exciting from a physiological point of view in the context of endogenous opioidergic pathways, notably in modulating mood-related states and pain sensation. Furthermore, because of its in vivo properties, Opiorphin may have therapeutic implications.dual neutral endopeptidase͞aminopeptidase N inhibitor ͉ human saliva ͉ pain ͉ peptide mediator ͉ enkephalins Z inc metal ectopeptidases control the receptor-dependent activity of neural and hormonal mediators involved in the regulation of important physiological functions in mammals. They are located at the surface of cells in nervous and systemic tissues and catalyze postsecretory processing or metabolism of neuropeptides and regulatory peptides (1, 2). Prominent among these neuronal and͞or hormonal peptide signals are substance P (SP) and enkephalins, which are implicated in the receptor-dependent modulation of behavioral adaptive responses to stressful or threatening environmental stimuli. They notably regulate spinal processing of nociceptive information and analgesic mechanisms, emotional and͞or motivational responses, anxiety, aggression, and neuroimmune inflammatory phenomena (3-6).Because of the physiological importance and the critical role of zinc ectopeptidases in modulating the functional potency of downstream neuronal and hormonal signals, it is essential to focus on what controls their activity and, as a consequence, the overall regulatory cascade. The discovery of upstream regulators of ectopeptidase activity also is exciting from physiopathological and therapeutic points of view because of the potential for developing new candidate drugs. A brain-specific heptapeptide named spinorphin was isolated and characterized from bovine spinal cord based on its inhibitory activity toward enkephalin-degrading ectoenzymes, such as neutral endopeptidase (NEP; EC 3.4.24.11) and aminopeptidase N (AP-N; EC 3.4.11.2) (7,8). In addition, we characterized rat sial...

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Section: Methodsmentioning

confidence: 64%

Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways

Wisner

Dufour

Messaoudi³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

160

148

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“…This finding raises the question of the existence of such endogenous NEP-ectopeptidase inhibitor in human. And, importantly, sialorphin could spawn a new class of therapeutic compounds, as its target NEP ectopeptidase is highly conserved between species; the rat and human enzymes have high sequence homology (Ն93%) (14,35).…”

Section: Sialorphin An Inhibitor Of the Sp And Me Catabolism By Spinmentioning

confidence: 99%

Sialorphin, a natural inhibitor of rat membrane-bound neutral endopeptidase that displays analgesic activity

Rougeot

Messaoudi

Hermitte

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4 -1 M and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100 -200 g͞kg doses of sialorphin required the activation of -and ␦-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules.

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“…the enkephalins, substance P, atrial natriuretic factor, bradykinin and the chemotactic peptide fMet-Leu-Phe [7,8]. The amino acid sequence of human NEP is identical with that of the common acute lymphoblastic leukemia antigen (CALLA; CD 10) and is highly conserved in different species [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

et al. 1996

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Tripeptide derivatives like 3-carboxypropanoylalanyl-alanyMeucine 4-nitroanilide or 3-carboxypropanoylalanyl-alanyl-phenylalanine 4-nitroanilide are very sensitive substrates for neprilysin (kcat > 102 s-Z; kcatlKm >-106 s -~ "M-~) and are widely employed in investigations of the enzyme. However, these compounds are also good substrates for the serine proteases chymotrypsin and subtilisin (kcat ~ I s-~-34 s-~). By substituting the N-terminal alanine of the substrates with proline, the catalytic efficiency of the enzymic reaction, by the serine proteases, is diminished by 2-3 orders of magnitude, whereas that by neprilysin and thermolysin decreases only slightly. These effects demonstrate that structural alterations in peptide substrates that impair secondary sub-site interactions with one class of peptidases may enhance the selectivity of the substrates towards another class of peptidases.

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Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase)

Cited by 205 publications

References 16 publications

Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways

Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways

Sialorphin, a natural inhibitor of rat membrane-bound neutral endopeptidase that displays analgesic activity

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

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