Proc. Nati. Acad. Sci. USA Vol. 89, pp. 84-88, January 1992 Biochemistry A peptide-hormone-inactivating endopeptidase in Xenopus laevis skin secretion (metailoendopeptidase/neutral endopeptidase/thermolysin) KRISHNAMURTI DE MORAIS CARVALHO*, CARINE JOUDIOU, HAMADI BOUSSETTA, ANNE-MARIE LESENEY, AND PAUL COHEN Groupe de Neurobiochimie Cellulaire et Moldculaire de l'Universitd Pierre et Marie Curie, Unit6 de Recherche Associ6e 554 au Centre National de la Recherche Scientifique, % Boulevard Raspail, 75006 Paris, France Communicated by I. Robert Lehman, September 16, 1991 ABSTRACT An endopeptidase was isolated from Xenopus Indeed the Ser-Phe dipeptide, or a related motif such as laevis skin secretions. This enzyme, which has an apparent Phe-Phe, Ala-Phe, or His-Phe, is often present near the molecular mass of 100 kDa, performs a selective cleavage at the carboxyl terminus of substances from the bombesin and Xaa-Phe, Xaa-Leu, or Xaa-Ile bond (Xaa = Ser, Phe, Tyr, His, tachykinin families (1). Xaa-Phe, Xaa-Leu, or Xaa-Ile was or Gly) of a number of peptide hormones, including atrial also found frequently at a similar position in other peptide natriuretic factor, substance P, angiotensin H, bradykinin, hormone sequences of higher organisms, notably in atrial somatostatin, neuromedins B and C, and litorin. The peptidase natriuretic factor (ANF). exhibited optimal activity at pH 7.5 and aKm in the micromolar We have purified this enzyme 2029-fold and demonstrate range. No cleavage was produced in vasopressin, ocytocin, that it inactivates ANF by exclusive cleavage of the Ser25- minigastrin I, and [Leu5Jenkephalin, which include in their Phe26 bond and similarly inactivates a number of important sequence an Xaa-Phe, Xaa-Leu, or Xaa-Ile motif. The en- peptide hormones such as somatostatin, bradykinin, neuro- dopeptidase activity was inhibited by divalent cation chelators medins B and C, litorin, substance P, and angiotensin II. and by phosphoramidon only at high concentrations (ICss = 50 jAM), whereas it was insensitive to classical inhibitors of AND chymotrypsin, angiotensin convertase, and serine and cysteine MATERIALS METHODS peptidases, as well as carboxypeptidases. It is hypothesized that Purification Procedure. Exudates were obtained from three this enzyme, which is distinct from neutral endopeptidase (EC X. laevis (Centre National de la Recherche Scientifique, 3.4.24.11), constitutes the prototype of a family of related Montpellier, France), and submitted to Sephadex G-50, metalloendopeptidases that inactivate peptide substrates by DEAE-Sephadex, and aminoethylagarose hydrophobic ab- cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-fle bond. sorption chromatography as in ref. 3. The fractions contain- ing endopeptidase activity were then applied to a Nucleosil A rather limited number ofpeptidases seem to be involved in C4 300-A (8 x 75 mm) HPLC column (SFCC-Shandon, the postsecretory inactivation of peptide hormone messen- Eragny, France) and eluted with 10 mM sodium phosphate/ gers. The importance of these proteolytic mechanisms in 1.5 M sodium chloride buffer, pH 7.4, at a flow rate of 0.5 regulating hormonal action can be demonstrated by the fact ml-min-1, for 10 min followed by a linear gradient from 1.5 to that their effects can be prolonged in vivo or in vitro by 0 M sodium chloride/10 mM sodium phosphate buffer, pH selective inhibitors ofthese enzymes. This has been shown in 7.4, in 10 min. Subsequently, a gradient from 0% to 30% the case of the enkephalin-degrading enzyme neutral en- (vol/vol) methanol in 10 mM sodium phosphate buffer, pH dopeptidase (NEP; enkephalinase, EC 3.4.24.11) and in the 7.4, in 60 min eluted the enzyme, which was recovered at 25% case of angiotensin-converting enzyme (ACE; EC 3.4.15.1). methanol. The purified enzyme was resistant to 30%o meth- Many of these previously identified peptidases cleave with a anol and 1.5 M sodium chloride (90%o of initial activity limited selectivity and sometimes at multiple sites. remained; data not shown). The endopeptidase was com- The skin secretions of Xenopus laevis contain an extraor- pletely separated from the copurifying RXVRG-endopro- dinary number of peptide hormones eliciting a broad spec- tease activity (eluted with 12% methanol). The active frac- trum of biological actions (reviewed in ref. 1). In addition to tions were pooled, concentrated to a 30-,ul final volume, and those substances so far identified exclusively in the batra- analyzed by polyacrylamide gel electrophoresis under either chian skin exudate such as caeruleins, magainins, levitide, nondenaturing or denaturating conditions using the PhastGel PGLa, xenopsin, etc. (1, 2), the secretory glands of these system (Pharmacia) (3, 4). Protein content was evaluated by amphibians can also produce mammalian peptide hormones the method of Bradford (5). and some of their analogs, such as substance P, corti- Enzyme Assays. Endopeptidase activity was monitored as in coliberin, thyroliberin, and angiotensin I. In the course of ref. 3, using as substrate the diaminobenzylthiocyanate isolating the enzyme responsible for the activation of some X. (DABTC) derivative of [DArg8]kermit, a derivative of kermit laevis skin secretion hormone precursors by cleavage at the (Asp-Val-Asp-Glu-Arg-Asp-Val-Arg-Gly-Phe-Ala-Ser-Phe- Arg-Gly bond of a consensus Arg-Xaa-Val-Arg-Gly (RX- Leu-NH2) that undergoes a single cleavage at the Ser12-Phe13 VRG) sequence, we have detected a contaminating proteo- bond. When other peptide hormones were used as substrates, lytic activity (3). This endopeptidase copurifies with the the conditions were as follows: 1-2 nmol of peptide incubated RXVRG-endoprotease of X. laevis skin secretions and cleaves at the Ser-Phe bond situated on the carboxyl side of Abbreviations: NEP, neutral endopeptidase (EC 3.4.24.11); ACE, the RXVRG consensus sequence in a synthetic peptide (3). angiotensin converting enzyme (EC 3.4.15.1); PHIE, peptide- hormone-inactivating endopeptidase; ANF, atrial natriuretic factor; DABTC, diaminobenzylthiocyanate. The publication costs of this article were defrayed in part by page charge *On leave from: Depto de Fisiologia e Farmacologia da Universidade payment. This article must therefore be hereby marked "advertisement" Federal do CearA (UFC), CX Postal 657, Rua Cel Nunes de Melo in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1127, 60.000-Fortaleza, CE, Brazil. 84 Downloaded by guest on September 27, 2021 Biochemistry: Carvalho et al. Proc. Natl. Acad. Sci. USA 89 (1992) 85 1-5 hr in 100 mM sodium phosphate buffer, pH 7.4, in the Phe-Phe, His-Phe, Gly-Leu, His-Leu, or Tyr-Ile bond of the presence of 10 ng ofpure enzyme in 20 pkl. After heating 10 min tested peptides. Hydrolysis of the Ser25-Phe26 bond of ANF- at 1000C, the resulting products were separated by HPLC (5-28) removed the Phe26-Arg27-Tyr28 tripeptide tail and led using a Nucleosil 5-,um C18 column (146 x 4.5 mm) eluted as to stoichiometric amounts of both the NH2-terminal 5-25 described in the legend of Fig. 2. Substrate and product(s) fragment and this tripeptide (fragments a and b, Fig. 2A). For were monitored by UV absorbance at 220 nm and identified by substance P, the major hydrolytic process occurred at the amino acid analysis using a PicoTag station (Waters) or by Gly9-Leu10 bond (fragments a and c, Fig. 2B) and a minor one amino-terminal sequencing (6). Inhibitors were tested at var- at the Phe7-Phe8 doublet (fragments b and d, Fig. 2B). In the ious concentrations (Table 2), using the routine conditions of case ofangiotensin II only one cleavage was produced, at the the endopeptidase assay (see above) and either ANF-(5-28) or Tyr4-Ile5 bond (fragments a and b, Fig. 2C). Bradykinin was [DArg8]kermit as substrate. Inhibitions were expressed as cleaved at the Gly4-Phe5 bond (fragments a and d, Fig. 2D) percentages of the reference activity measured in the absence with a minor cleavage at the Pro7-Phe8 bond (fragments b and of chemical reagents under the same experimental conditions. c, Fig. 2D). Somatostatin-14 undergoes a single cleavage at The pH profile ofendoprotease activity was obtained by using the Phe6-Phe7 bond situated in the disulfide-linked loop ofthe either [DArg8]kermit or ANF-(5-28) as substrate (2 nmol per tetradecapeptide. This resulted in a single product (Fig. 2E) assay) incubated 1 hr with 10 ng of pure enzyme in a 0.1 M (fragment a was identified by NH2-terminal sequencing). In sodium phosphate buffer adjusted to cover a pH range of 5.8 neuromedin C, as well as in neuromedin B and litorin (data to 8.0 (Fig. 4). Under these conditions enzyme kinetics re- not shown), a major hydrolysis was observed at the His8- mained linear, indicating that the endopeptidase was stable at Leu9 or His8-Phe9 bond (fragments a and d, Fig. 2F) and a the pH values used. Enzyme and inhibition assays were run on minor cleavage at Gly7-His8 (fragments b and c, Fig. 2F). average two or three times for each peptide. Analysis of The pure enzyme exhibited high affinity toward all the products was performed at least in duplicate. cleaved substrates. Km values in the range of 18-63 ,M were Peptides and Chemicals. [DArg8]kermit, ANF-(24-28), measured toward ANF-(5-28), substance P, somatostatin-14, [Ala2IANF-(24-28), and [His25]ANF-(24-28) were prepared by bradykinin, neuromedin C, and angiotensin II (Fig. 3). solid-phase synthesis (7, 8) on a Multisynthesizer NPS 4000 Other peptides containing a Xaa-Phe, Xaa-Leu, or Xaa-Ile (Neosystem, Strasbourg, France), purified by HPLC, and motif, including ocytocin, vasopressin, minigastrin I, checked as described (4, 9). Chemicals and peptide substrates [Leu5]enkephalin, and its amidated derivative were not cleaved or fragments were from Neosystem or Sigma.