Hydrolysis of Insect Neuropeptides by an Angiotensin-Converting Enzyme From the Housefly, Musca domestica1

Peptides, Vol. 18, No. 1, pp. 47–52, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00

PII S0196-9781(96)00232-X

Hydrolysis of Insect Neuropeptides by an Angiotensin-Converting Enzyme From the Housefly, Musca domestica 1 N. S. LAMANGO,* R. J. NACHMAN,† T. K. HAYES,‡ A. STREY‡ AND R. E. ISAAC* 2 *Department of Biology, University of Leeds, Leeds LS2 9JT, UK †Veterinary Entomology Research Unit, Food and Animal Protection Research Laboratory, United States Department of Agriculture, 2881 F and B Road, College Station, TX 77845 ‡Laboratories for Invertebrate Neuroendocrine Research, Department of Entomology, Texas A&M University, College Station, TX 77843 Received 4 March 1996; Accepted 30 May 1996 LAMANGO, N. S., R. J. NACHMAN, T. K. HAYES, A. STREY AND R. E. ISAAC. Hydrolysis of insect neuropeptides by an angiotensin-converting enzyme from the housefly, Musca domestica. PEPTIDES 18(1) 47–52, 1997.—The presence in insect tissues of peptides with structural similarities to angiotensin I and to bradykinin, the two best known substrates of mammalian angiotensinconverting enzyme, has not been reported. As part of our study to identify potential substrates for insect angiotensin-converting enzyme, we have investigated the susceptibility of a number of known insect peptide hormones and neurotransmitters to hydrolysis by Musca domestica angiotensin-converting enzyme. Insect peptides belonging to the red pigment-concentrating hormone, leucokinin, locust tachykinin, and depolarizing peptide families were hydrolyzed by housefly angiotensin-converting enzyme, whereas proctolin and crustacean cardioactive peptide were not substrates. Cus-DP II, LK I, LK II, and Lom-TK I were all cleaved at the penultimate C-terminal peptide bond to release a dipeptide amide as a major fragment with Km values of 94 { 11, 634 { 81, and 296 { 35 mM for Cus-DP II, LK I, and Lom-TK I, respectively. The ability of insect angiotensin-converting enzyme to hydrolyze C-terminally amidated peptides in vitro might be of functional significance because the enzyme has been localized to neuropile regions of the insect brain and is present in the hemolymph of houseflies. q 1997 Elsevier Science Inc. Musca domestica

Angiotensin-converting enzyme

Neuropeptide metabolism

ANGIOTENSIN-CONVERTING enzyme (EC 3.4.15.1, peptidyl dipeptidase A, ACE) is a Zn 2/ metallopeptidase responsible in mammals for the conversion of angiotensin I (ANG I) to angiotensin II (ANG II) and the inactivation of bradykinin (BK) by removing C-terminal dipeptides (6). The central role of ACE in both the formation of a vasoconstrictor (ANG I) and the inactivation of a vasodilator (BK) has been successfully exploited in the development of ACE inhibitors as antihypertensive drugs (15). ACE can also act in vitro as an endopeptidase hydrolyzing C-terminally amidated peptides to release either a dipeptide amide or a tripeptide amide (6). A dipeptide amide is cleaved from cholecystokinin (4), [Leu 5 ]enkephalinamide and [Met 5 ]enkephalinamide (7), whereas substance P is hydrolyzed independently at two peptide bonds to release a tripeptideamide and a dipeptideamide, with the former predominating (2,8,18,24). LH-RH is a very unusual in vitro substrate for ACE, because an N-terminal tripeptide is cleaved in addition to a C-terminal tripeptide amide (19). The broad substrate specificity and widespread distribution of ACE in mammalian tissues suggests

Insect neuropeptides

that ACE might have physiological roles in addition to controlling blood pressure, water, and salt balance through the metabolism of ANG I and BK (5,6). Mammalian somatic ACE is composed of two very similar domains, each containing a dipeptidyl carboxypeptidase catalytic site. The active sites display different substrate preferences and different dissociation constants for ACE inhibitors, suggesting the possibility of different roles for each active site (20). Recently the hemoregulatory peptide N-acetyl-Ser-Asp-Lys-Pro has been identified as a possible ACE substrate and it is specifically cleaved in vitro by the N-domain active site of human ACE (16,17). ACE might also function in the brain to inactivate as yet unidentified peptide neurotransmitters at postsynaptic sites in the striatonigral pathway of the mammalian brain (1). A dipeptidyl carboxypeptidase with very similar properties to mammalian ACE was recently identified in the housefly, Musca domestica ( 12,13 ) . This first report of an invertebrate ACE was significant because it provided evidence for an evolutionary ancient ACE gene. The cloning and analysis of a

1 Taken in part from a paper presented at a satellite symposium on Insect Neuropeptides during the Seventh Annual Neuropeptide Conference, February 1–6, 1996, Breckenridge, CO. 2 Requests for reprints should be addressed to Dr. R. E. Isaac.

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LAMANGO ET AL. TABLE 1 RELATIVE RATES OF METABOLISM OF INSECT PEPTIDES (200 mM) BY Musca domestica ACE

Peptide

Primary Structure

Relative Hydrolysis (% of Maximum)

LK I LK II Lom-TK I Lom-TK II Allatostatin I SchistoFLRFamide Pea-MP I Cam-HTH Lom-AKH I RPCH Proctolin CCAP Cus-DP I Cus-DP II

Asp-Pro-Ala-Phe-Asn-Ser-Trp-Gly-NH2 Asp-Pro-Gly-Phe-Ser-Ser-Trp-Gly-NH2 Gly-Pro-Ser-Gly-Phe-Tyr-Gly-Val-Arg-NH2 Ala-Pro-Leu-Gly-Phe-Tyr-Gly-Val-Arg-NH2 Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2 Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH2 pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2 pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2 pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2 Arg-Tyr-Leu-Pro-Thr Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2 Asn-Pro-Phe-His-Ser-Trp-Gly-NH2 Asn-Asn-Ala-Asn-Val-Phe-Tyr-Pro-Trp-Gly-NH2

100 80 81 86 72 70 22 15 20 63 0 0 * *

Initial rates of hydrolysis were determined by measuring the reduction of the parent peptide under conditions that resulted in less than 25% hydrolysis. Results are expressed relative to the rate of hydrolysis of LK I and are the mean of four determinations (SEM õ { 5%). * Hydrolysis was observed but the use of a lower substrate concentration did not permit the results to be expressed in a comparative manner.

FIG. 1. The hydrolysis of insect neuropeptides by Musca domestica ACE. HPLC chromatograms (UV detector set at 214 nm) of the peptide fragments generated on incubation of peptide (70–300 mM) with 0.11 mg of ACE as described in the Method section. (A) Cus-DP II: peak 1, Trp-Gly-NH2 . (B) LK I: peak 1, Trp-Gly-NH2 . (C) LK II: peak 1, Trp-Gly-NH2 . (D) Lom-TK I: peak 1, Val-Gly-NH2 ; peak 2, Gly-Pro-Ser-Gly-Phe [Lom-TK I(1–5)]; peak 3, Gly-Pro-Ser-Gly-Phe-Tyr-Gly [Lom-TK I(1–7)]. In all chromatograms P indicates elution of the initial peptide substrate. Peak contents were identified by FAB-MS analysis (see the Method section).

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HOUSEFLY ANGIOTENSIN-CONVERTING ENZYME

49

TABLE 2 CLEAVAGE OF SELECTED INSECT PEPTIDES BY MUSCA DOMESTICA ACE Peptide

Sites of Hydrolysis

Km

Cus-DP II

Asn-Asn-Ala-Asn-Val-Phe-Tyr-Pro-Trp-Gly-NH¤

≤94 6 11 mM

LK-I

Asp-Pro-Ala-Phe-Asn-Ser-Trp-Gly-NH¤

634 6 81 mM

LK-II

Asp-Pro-Gly-Phe-Ser-Ser-Trp-Gly-NH¤

—

Lom-TK I

Gly-Pro-Ser-Gly-Phe-Tyr-Gly-Val-Arg-NH¤

296 6 35 mM

Primary hydrolysis sites are denoted by ( ) whereas established secondary hydrolysis sites are indicated by ( ). Km and Vmax values were obtained using a curve-ˆtting computer program (P.ˆt, Biosoft, UK).

cDNA for a Drosophila melanogaster ACE ( the gene is known as Ance or Race ) revealed a high degree of conservation of the protein structure, especially around the active site region ( 3 ) . In addition, a number of the enzymic properties of ACE have been conserved during evolution ( 3,13,23 ) . Thus, Musca ACE converts ANG I to ANG II, degrades BK, and displays endopeptidase activity towards both substance P and LH-RH ( 13 ) . The insect enzyme is also inhibited by various inhibitors of mammalian ACE and is activated by NaCl ( 12,13 ) . Race ( Ance ) was identified as a putative target gene for the homeobox gene, zerknullt , and was shown to be expressed in all developmental stages ( 22 ) . In late embryogenesis, expression occurs mainly in the midgut and the dorsal vessel ( 22 ) . Drosophila Race ( Ance ) mutants die during larval / pupal development, demonstrating an essential role for ACE ( 22 ) . We believe that understanding the physiological role of insect ACE may lead to the discovery of hitherto unknown, evolutionary conserved functions of ACE in mammals. Therefore, a primary objective of our work is the identification in insects of potential natural substrates of ACE. Although a large number of bioactive peptides have been isolated from various insect species, there have been no reports of the identification of peptides that are structurally related to ANG I and BK. The majority of insect peptides isolated to date have an amidated C-terminus, which is invariably essential for bioactivity and which is likely to confer resistance to attack by carboxypeptidases found in insect tissues (10,14). Our previous study showing that Musca ACE can hydrolyze mammalian peptides that possess an amidated C-terminus suggested that C -terminally amidated insect peptides might also be substrates for insect ACE (13). In the present study we show that a variety of insect neuropeptides are in vitro substrates for ACE, but two peptides [proctolin and crustacean cardioactive peptide (CCAP)] are resistant to hydrolysis. METHOD

Chemicals Adipokinetic hormone ( Lom-AKH I ) and locustatachykinin I and II ( Lom-TK I and II ) were obtained from Peninsula Laboratories Europe ( St Helens, UK ) . Leucokinin I ( LK I ) and II ( LK II ) , proctolin, and captopril were obtained from Sigma Chemical Co. ( Poole, Dorset, UK ) . Red pigment-con-

centrating hormone ( RPCH ) , cockroach myoactive peptide I ( Pea-M I ) , and stick insect hypertrehalosemic hormone ( Cam-HTH ) were obtained from American Peptides Co., Inc. ( Sunnyvale, CA ) . CCAP was obtained from Bachem ( UK ) Ltd. ( Saffron Walden, Essex, UK ) . Culex depolarizing peptides ( Cus-DP I, Cus-DP II, and Cus-DP III ) were synthesized by the Texas Agricultural Station Biotechnology Support Laboratory Peptide Services, College Station, TX. HPLC-grade solvents were obtained from Rathburns Chemicals ( Walkerburn, Scotland, UK ) . Enzyme Musca domestica ACE (Mr 67,000) was purified from a soluble extract of adult flies as described elsewhere (3,13) and yielded enzyme that appeared as a single band by SDS-PAGE. Metabolism of Neuropeptides Unless otherwise stated, peptides (12–750 mM, final concentration) were incubated with Musca ACE in 100 m M Tris-HCl, pH 8.3, in the presence of 10 mM ZnCl2 and 0.3 M NaCl at 377C. Reactions (15 ml) were terminated either by heating to 1007C for 5 min or by the addition of 5 ml of 8% (v/v) TFA, followed by centrifugation (13,000 1 g, Microcentaur) for 2 min and diluted to 100 ml with 0.1% (v/v) TFA before HPLC analysis. HPLC HPLC analysis was performed using a Pharmacia SuperPac Pep-S column (250 1 4 mm, internal diameter) packed with 5 mm ODS particles. Peptides were eluted with 5% acetonitrile in 0.1% (v/v) trifluoroacetic acid for 3 min, followed by a linear increase of the acetonitrile component from 3% to 41% in 11 min (total run time of 14 min). Peptide fragments were collected from HPLC runs and their identities were established using fast atom bombardment-mass spectrometry (FAB-MS) conducted at the Food and Animal Protection Laboratory, USDA (College Station, TX). FAB-MS spectra were obtained by adding HPLCpurified fragments in dimethylformamide (2 ml) to glycerol (1.5 ml) on stainless steel targets (6 1 1.5 mm), followed by bombarment with 8 kV Xe atoms on a VG 70-250 EHF mass spectrometer (VG Analytical, Manchester, UK). Contents of the HPLC peaks were identified by the mass of the parent ion. Km

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LAMANGO ET AL. and Vmax values were obtained by measuring the initial rates of hydrolysis at different peptide concentrations and by using a curve-fitting program (Pfit, Biosoft, UK). RESULTS

Relative Rates of Hydrolysis of Various Insect Peptides by Musca ACE Rates of hydrolysis were obtained by measuring the rate of depletion of the parent peptide substrates (200 mM, initial concentration) (Table 1). All the peptides except for proctolin and CCAP were degraded by ACE. The rates of hydrolysis of LomTK I, Lom-TK II, LK I, LK II, allatostatin I, SchistoFLRFamide, and RPCH were very similar but three members of the AKH family (Pea-MP I, Cam-HTH, and Lom-AKH I) were hydrolyzed at a much slower rate. Identification of the Primary Cleavage Sites for the Hydrolysis of Cus-DP II, Lom-LK I, Lom-LK II, and Lom-TK I by ACE Peptides generated by the hydrolysis of Cus-DP II, Lom-LK I, Lom-TK II, and Lom-TK I by Musca ACE were resolved by HPLC [Fig. 1(A–D)]. UV (214 nm) absorbing peaks were collected and vacuum dried for analysis by FAB-MS and tryptophan-containing fragments were identified by running parallel chromatograms with the wavelength of the UV detector set at 280 nm. Hydrolysis of Cus-DP II The hydrolysis of Cus-DP II gave rise to the C-terminal dipeptide amide, Trp-Gly-NH2 , as the major fragment, identifying the Pro 8-Trp 9 bond as the initial cleavage site [Fig. 1(A)]. A Km of 94 { 11 mM and a Vmax of 210 { 7 nmol/min/mg of protein ( {SEM, n Å 5) was obtained for the hydrolysis of Cus-DP II [Table 2, Fig. 2(A)]. Hydrolysis of LK I and LK II The major tryptophan-containing fragment formed when either LK I or LK II was incubated with Musca ACE was identified as Trp-Gly-NH2 [Fig. 1(B, C)]. A Km of 634 { 81 mM and a Vmax of 2352 { 157 nmol/min/mg of protein ( {SEM, n Å 5) was obtained for the hydrolysis of LK I [Table 2, Fig. 2(B)]. Hydrolysis of Lom-TK I The C-terminal dipeptide amide, Val-Arg-NH2 , was cleaved from Lom-TK I giving rise to Lom-TK I(1–7) [Fig. 1(D)], which was itself hydrolyzed to Lom TK I(1–5). When the hy-

FIG. 2. Michaelis–Menten plots for the hydrolysis of insect peptides by Musca domestica ACE. The rates of hydrolysis of (A) Cus-DP II, (B) LK I, and (C) Lom-TK I were determined under conditions that resulted in less than 20% degradation of the parent peptide. Initial velocities were determined for Cus-DP II and from LK I from the rates of formation of Trp-Gly-NH2 , which appeared to be the only tryptophan-containing metabolite formed under these assay conditions. Gly-Pro-Ser-Gly-Phe-TyrGly [Lom-TK I(1–7)], a metabolite from the initial cleavage of LomTK I, appeared to be a stable hydrolysis product when the level of hydrolysis of Lom-TK I was kept below 20%. The initial velocities for the hydrolysis of Lom-TK I were determined by measuring the production of Lom-TK I(1–7) by HPLC. All Km and Vmax values were obtained using a curve-fitting computer program (P.fit, Biosoft, UK). One unit of activity is 1 nmol of peptide hydrolyzed per mg of protein.

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HOUSEFLY ANGIOTENSIN-CONVERTING ENZYME drolysis of Lom-TK I was kept below 20%, Lom-TK I(1–7) was the only reaction product, permitting the rate of formation of Lom-TK I(1–7) to be used to determine the initial rates of hydrolysis of Lom-TK I. A Km of 296 { 35 mM and a Vmax of 546 { 33 nmol/min/mg of protein ( {SEM, n Å 5) was obtained for the hydrolysis of Lom-TK I by Musca ACE [Table 2, Fig. 2(C)]. DISCUSSION

The only peptides used in this study that were not degraded by ACE were proctolin and CCAP. Peptides with a C-terminal penultimate proline (e.g., ANG II) are not good substrates for mammalian ACE (6,15) and it is also likely that the presence of a proline residue in the penultimate C -terminal position of proctolin is a contributing factor to the resistance of this insect peptide to hydrolysis by Musca ACE. The fact that CCAP was not metabolized draws comparisons with mammalian atrial natriuretic peptide (ANP), whose breakdown by pig kidney microvillar membranes is almost entirely achieved by endopeptidase 24.11 despite the fact that this tissue is a rich source of ACE and other peptidases (21). CCAP, like ANP, has a single cystine bond that folds away the C-terminus into a conformational structure that probably sterically hinders any productive binding to ACE. The specificity of mammalian ACE towards C -terminally amidated peptides is complex and not always predictable. For example, with substance P, ACE cleaves both a dipeptideamide and a tripeptideamide with the tripeptidase activity predominating (2,8,18,24). Housefly ACE also cleaves the same two bonds of substance P, but with the insect enzyme the dipeptidase activity predominates (13). The hydrolysis of Cus-DP II, LK I, LK II, and Lom-TK I reported in the present study proceeds primarily by cleavage of dipeptideamides, which disrupts the active peptide core and leads to peptide inactivation. The Km of 60 mM for the hydrolysis of Cus-DP II by Musca ACE indicates a strong affinity for this peptide and is similar to the Km for the hydrolysis of

51 ANG I (15–70 mM) (11,20) and the hemoregulatory peptide Nacetyl-Ser-Asp-Lys-Pro (31–39 mM), known physiological substrates of human ACE (17). The presence of an a-amide group on the C-terminal residue of a peptide usually confers resistance to hydrolysis by carboxypeptidases. The observation that a number of insect peptide hormones with amidated C-termini are susceptible to hydrolysis by ACE may therefore be of special significance, especially because high levels of ACE activity have been found in the hemolymph of insects (9). ACE has recently been found in neurosecretory cells and in neuropile regions of the insect brain (Locusta migratoria, Schistocerca gregaria, Neobellieria bullata, and Carausius morosus; personal communication from Dr. L. Schoofs, University of Leuven). This pattern of localization is consistent with a dual role for ACE in the processing of neurosecretory peptides and in the inactivation of peptide neurotransmitters at synapses. A mutation in Drosophila Ance results in larval/pupal lethality and demonstrates an essential role for the peptidyl dipeptidase (22). We have also shown that inhibitors of insect ACE can inhibit growth and development of the lepidopteran insect Manduca sexta (Lamango, Isaac, and Hayes, unpublished data), suggesting that ACE may also have an important role in the metabolism of peptide hormones involved in insect development. In conclusion, we have shown that a number of insect peptides, but not proctolin and CCAP, are susceptible to degradation by insect ACE. However, the unequivocal identification of the physiological substrates of insect ACE requires further work, including the colocalization of neuropeptides with ACE in the insect CNS and the demonstration that ACE inhibitors can alter the normal physiological levels of naturally occurring insect peptides. ACKNOWLEDGEMENTS

The work was supported by a grant from the Science and Engineering Research Council (UK), the United States Department of Agriculture, and the TAMU Biotechnology Support Laboratory.

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