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This article was srcinally published in a journal published byElsevier, and the attached copy is provided by Elsevier for theauthor’s benefit and for the benefit of the author’s institution, fornon-commercial research and educational use including withoutlimitation use in instruction at your institution, sending it to specificcolleagues that you know, and providing a copy to your institution’sadministrator.All other uses, reproduction and distribution, including withoutlimitation commercial reprints, selling or licensing copies or access,or posting on open internet sites, your personal or institution’swebsite or repository, are prohibited. For exceptions, permissionmay be sought for such use through Elsevier’s permissions site at:http://www.elsevier.com/locate/permissionusematerial A u t h o r ' s p e r s o n a l c o p y A novel peptide from the ACEI/BPP-CNP precursor in the venomof Crotalus durissus collilineatus Shigesada Higuchi a,e,1 , Nobuhiro Murayama a , Ken-ichi Saguchi a , Hiroaki Ohi a ,Yoshiaki Fujita a , Nelson Jorge da Silva Jr. b , Rodrigo José Bezerra de Siqueira c ,Saad Lahlou c , Steven D. Aird d, ⁎ a Showa University School of Pharmaceutical Sciences, Shinagawa-ku, Tokyo 142-8555, Japan b Centro de Estudos e Pesquisas Biológicas, Universidade Católica de Goiás, Goiânia, GO 74605-010, Brazil c Departamento de Fisiologia e Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Pernambuco,Cidade Universitária, Recife, PE 50670-901, Brazil d Department of Biology, Norfolk State University, Norfolk, VA 25304, USA e Department of Pharmaceutical Sciences, Sojo University, Ikeda, Kumamoto 860-0082, Japan Received 4 February 2006; received in revised form 17 April 2006; accepted 19 April 2006Available online 28 April 2006 Abstract In crotaline venoms, angiotensin-converting enzyme inhibitors [ACEIs, also known as bradykinin potentiating peptides (BPPs)], are products of agene coding for an ACEI/BPP-C-type natriuretic peptide (CNP) precursor. In the genes from Bothrops jararaca and Gloydius blomhoffii , ACEI/BPPsequences are repeated. Sequencing of a cDNA clone from venom glands of Crotalus durissus collilineatus showed that two ACEIs/BPPs are locatedtogether atthe N-terminus, but without repeats. Anadditional sequence forCNP was unexpectedlyfound atthe C-terminus.Homologousgenes for theACEI/BPP-CNP precursorsuggestthatmostcrotalinevenomscontainbothACEIs/BPPsand CNP.The sequence ofACEIs/BPPsisseparated fromtheCNP sequence by a long spacer sequence. Previously, there was no evidence that this spacer actually coded any expressed peptides. Aird and Kaiser (1986, unpublished) previously isolated and sequenced a peptide of 11 residues (TPPAGPDVGPR) from Crotalus viridis viridis venom. In the present study,analysisofthecDNAclonefrom C.d.collilineatus revealedanearlyidenticalsequenceintheACEI/BPP-CNPspacer.Fractionationofthecrudevenom by reverse phase HPLC (C 18 ), and analysis of the fractions by mass spectrometry (MS) indicated a component of 1020.5 Da. Amino acidsequencing by MS/MS confirmed that C. d. collilineatus venom contains the peptide TPPAGPDGGPR. Its high proline content and paired prolineresidues are typical of venom hypotensive peptides, although it lacks the usual N-terminal pyroglutamate. It has no demonstrable hypotensive activitywhen injected intravenously in rats; however, its occurrence in the venoms of dissimilar species suggests that its presence is not accidental. Evidencesuggests that these novel toxins probably activate anaphylatoxin C3a receptors.© 2006 Elsevier Inc. All rights reserved. Keywords: Angiotensin-converting enzyme inhibitors; Bradykinin-potentiating peptides; Hypotensive peptides; Rattlesnake venoms; Anaphylatoxin C3a; Crotalusdurissus collilineatus ; Crotalus viridis viridi 1. Introduction Five years after the isolation of a kininogenase from thevenom of the South American pitviper, Bothrops jararaca (Henriques et al., 1960), an inhibitor of angiotensin-convertingenzyme (ACE) was isolated from the same venom, when it wasdiscovered t hat venom of that species not only releasedendogenous bradykinin in the prey, but also inhibited itshydrolysis (Ferreira, 1965). The inhibitory activity was attributed to a peptidyl fraction dubbed bradykinin potentiatingfactor (BPF). Ferreira et al. (1970) adopted more sophisticatedchromatographic techniques to show that BPF was actually acombination of nine different peptides. Each peptide wascomposed of 5 – 14 amino acids and was N-terminally blockedwith pyroglutamic acid. All had relatively high proline contents Comparative Biochemistry and Physiology, Part C 144 (2006) 107 – 121www.elsevier.com/locate/cbpc ⁎ Corresponding author. Tel.: +1 757 823 2327. E-mail address:
[email protected] (S.D. Aird). 1 Present Address: Department of Pharmaceutical Sciences, Sojo University,Kumamoto City 860-0082, Japan.1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.cbpc.2006.04.006 A u t h o r ' s p e r s o n a l c o p y and contained a single tryptophan residue. From sequencedifferences, it was clear that this array of peptides could not have been derived from a single precursor. Moreover, none of these peptides was identical to those simultaneously reportedfrom the venom of Gloydius blomhoffii (Kato et al., 1966; Katoand Suzuki, 1969, 1970, 1971).Hypotensive peptides were eventually found to be commonin pitviper venoms. Eight ACEIs/BPPs were isolated from thevenom of Bothrops insularis , an arboreal jararaca endemic tothe Brazilian island of Queimada Grande (Cintra et al., 1990).Ferreira et al. (1992a,b) isolated a peptide (Peptide P) from thevenom of Bothrops jararacussu that potentiated the activity of bradykinin on rat arterial blood pressure and on isolated guinea- pig ileum 2 – 3-fold more effectively than captopril. It was also a potent inhibitor of angiotensin I-converting enzyme. Ferreiraet al. (1995) found a decapeptide in the venom of Agkistrodon p. piscivorus that potentiates the effect of bradykinin on isolatedguinea-pig ileum more effectively than captopril or Peptide P. It also inhibited rat ACE, but had no discernible effect on rat arterial blood pressure. It did not potentiate the activity of bradykinin in relaxing isolated rat duodenum, but did extend the bradykinin-induced relaxation time. Ferreira et al. (1998)isolated and characterized at least three ACEIs/BPPs from Bo-throps neuwiedi venom. These peptides showed bradykinin- potentiating action on isolated guinea-pig ileum. They alsoreduced rat arterial blood pressure, and inhibited ACE. The picture of venom hypotensive peptides was expanded further when forms of CNP were isolated from the venom of the habu( Protobothrops flavoviridis ) (Michel et al., 2000).In addition to the broad array of primary structures, BPP pharmacology has proven to be complex in other regards. Chietal.(1985)reportedthatthebradykinin-potentiatingactivityofa peptide from Gloydius halys venom did not parallel ACE-inhibition activity and suggested that the bradykinin-potentiatingactivity of this ACEI/BPP in an in vitro assay might be due to itsinfluence on the bradykinin receptor rather than its ability toinhibit the kininase. Ferreira et al. (1999) found that ACEIs/BPPsusuallypossesstwodifferentactivities,potentiationofbradykininand ACE inhibition, but exceptions to this rule suggest that thetwo effects may be independent of one another. The picture becamefurthercomplicatedwhenYanoshitaetal.(1999)reportedthat blomhotin, an 11-residue ACEI/BPP from venom of G. blomhoffii , contracts smooth muscle, whereas most other ACEIs/BPPs do not. Because the latter experiments were per-formed using rat stomach fundus strips, it is probable that blomhotin relaxes vascular smooth muscle, an activity that would beconsistentwithotherpharmacologicalactivitiesofACEIs/BPPsand with snake envenomation strategies generally (Aird, 2002).Still other studies examined the gene coding for ACEIs/BPPs(Murayama et al., 1997; Higuchi et al., 1999). A 1.8-kb cDNAclone wasisolatedfroma B.jararaca venomglandcDNAlibrarythat encodes a 256-amino acid precursor for ACEIs/BPPs and aCNP. Seven ACEIs/BPPs are aligned in tandem after thehydrophobic signal peptide sequence, followed by a putativeintervening sequence and a CNP sequence at the C-terminus.The present study reports the sequencing of a cDNA clonefrom venom glands of the central Brazilian rattlesnake ( Crotalusdurissus collilineatus ) (Cdc). As with the B. jararaca clone, the Crotalus clone includes genes for an array of ACEIs/BPPsat the N-terminus with an additional sequence for a CNP at the C-terminus. Additionally,thisclonecodesforapeptidethatisnearly identical to one discovered and sequenced from Crotalus v.viridis (Cvv) venom (Aird and Kaiser, 1986, unpublished). It alsoconfirms the existence of the C. v. viridis peptide (Cvv peptide) of Aird and Kaiser (1986, unpublished). Both peptides depart inseveral significant regards from all ACEI/BPP sequences. 2. Materials and methods 2.1. Materials RestrictionendonucleasesandDNA-modifyingenzymeswere purchased from Takara Shuzo (Kyoto, Japan). Peptides withidentical sequences to the one predicted from the cDNA cloningof Cdc and the one previously known for Cvv were synthesizedon solid phase by the Peptide Institute (Osaka, Japan), andsupplied with the analytical data (amino acid composition andsequence). Venom of C. d. collilineatus was pooled, freeze-driedand kept at − 25 °C until use. Dried venom of C. v. viridis wasobtained from Sigma. Sequanal grade trifluoroacetic acid andHPLC grade acetonitrile were both from Wako, (Tokyo, Japan).All the reagents were analytical grade except otherwise stated. 2.2. Construction and screening of the C. d. collilineatusvenom gland cDNA library Poly(A) + RNA was prepared from the venom glands of asingle specimen of C. d. collilineatus using a Fast Track mRNAisolation kit (Invitrogen, Carlsbad, CA). cDNAwas synthesized,clonedandpackedusingaZAPexpresscDNAsynthesiskitandaZAP-cDNA Gigapack III Gold Packaging Extract (Stratagene).To obtain a specific probe, a 0.29 kb EcoRI/SspI fragment of thecDNAencodingtheACEI/BPP-CNPprecursorpolypeptidefrom B. jararaca was labeled using AlkPhos direct labeling reagents(Amersham Biosciences, Piscataway, NJ). The cDNA libraryfromCdcvenomglandswasspreadontoNZYplates,andplaqueswere transferred onto Hybond-N nylon membranes (AmershamBiosciences). Plaque hybridization was performed using theAlkPhos Direct labeling and detection kit (Amersham Bios-ciences) following the manufacturer's instructions. 2.3. DNA sequencing Nucleotide sequences were determined by the dideoxychain-termination method with a Texas Red-labeled primer and a Hitachi SQ-5500 DNA sequencer using the automatedelectrophoresis detection system (Hitachi). 2.4. Chromatography An LC8020 Model II liquid chromatograph (Toso, Japan)was used to perform HPLC with Superdex 75pg (16×600 mm,Amersham BioSciences, Sweden) and Symmetry300 C 18 5 μ m(4.6×25 mm, Waters, USA) for size exclusion and reversed 108 S. Higuchi et al. / Comparative Biochemistry and Physiology, Part C 144 (2006) 107 – 121 A u t h o r ' s p e r s o n a l c o p y phase chromatography, respectively. In both types of chroma-tography, eluent was monitored at 214 nm. 2.5. Mass spectrometry Mass analysis of custom synthesized peptides and pooledfractions from snake venom were performed on a MicromassQ-TOF Micro mass spectrometer (Waters, USA) in positiveelectrospray ionization mode. Samples previously freeze-driedand dissolved in 0.1% formic acid:acetonitrile (50:50) wereintroduced into the ESI source at a flow rate of 5 μ L/min by aninfusion pump throughout all experiments. The tandem mass(MS/MS) technique was applied to de novo peptide sequencing.Controlled by a MassLynx data system (Waters, USA) coupledto the mass spectrometer, measurements were carried out withthe collision energy fixed at 30 V. The mass scale wascalibrated with [Glu 1 ]-fibrinopeptide B. 2.6. Animals Male Wistar rats (280 – 330 g) were kept under conditions of constant temperature (22±2 °C) with a standard light/dark cycle(12 h light/12 h dark) and free access to food and water. Allanimals were cared for in compliance with the Guide for the Care Fig. 1. Nucleotide and deduced amino acid sequences of cDNAs encoding angiotensin-converting enzyme inhibitors/bradykinin-potentiating peptides (ACEIs/BPPs)and a C-type natriuretic peptide (CNP) precursor from venom glands of Crotalus durissus collilineatus . Nucleotide and amino acid differences between clones areindicated in parentheses. Novel peptide is indicated in black, ACEIs/BPPs and CNP are shaded.109 S. Higuchi et al. / Comparative Biochemistry and Physiology, Part C 144 (2006) 107 – 121 A u t h o r ' s p e r s o n a l c o p y and Use of Laboratory Animals, published by the US NationalInstitutes of Health (NIH Publication 85-23, revised 1996). 2.7. Catheterization procedure Rats were anesthetized intraperitoneally with ( i.p. ) sodium pentobarbital (50 mg/kg) and catheters (PE-10 fused to PE-50)wereimplantedintheabdominalaorta(fortherecordingofarterial blood pressure) and in the inferior vena cava (for drug admin-istration) through the left femoral artery and vein, respectively.These catheters, filled with heparin-saline solution (125 IU/mL),were exteriorized at the dorsal neck level. Postoperatively, the ratswere housed individually in plastic cages and allowed to recover for 48 h before any circulatory experiments. 2.8. Recording of mean aortic pressure and heart rate At the initiation of an experiment, the arterial catheter wasconnected to a blood pressure transducer (Statham P23 ID)coupled to a polygraph recorder; heart rate (HR) was obtainedfrom a cardiotachometer triggered by the pressure pulses. Bothsignals were recorded on a Gilson model 5/6H (MedicalElectronics Inc., Middletown, WI, USA). Mean aortic pressure(MAP) was calculated as diastolic+[(systolic − diastolic)/3]. 2.9. Experimental protocol Before each experiment, blood pressure and HR wereallowed to stabilize and were recorded during 15 – 25 minafter i.v. treatment with the peptide or its vehicle (saline). Whensubsequent doses of the peptide were administered, MAP andHR were first allowed to return to their baseline levels, obtained before the first injection of the compound. Two series of experiments were performed as follows. 2.9.1. Series 1 This series of experiments was carried out in anesthetized rats( n =4) to establish a dose – effect relationship. Rats were againanesthetized with sodium pentobarbital (50 mg/kg; i.p. ). Rectaltemperature was kept close to 37 °C by placing the animals on athermostatically controlled table. Each animal received a series of increasingbolus(100 μ L)doses(10,30,100and300 μ g/kg)ofthe peptide (dissolved in sterile isotonic saline) via the i.v. catheter,and time-course of the changes in MAP and HR was recorded. 2.9.2. Series 2 This series of experiments was performed in conscious rats( n =3) in order to establish a dose – effect relationship. Each rat received increasing bolus doses (10, 30, 100 and 300 μ g/kg) asdescribed above. In another set of animals, cardiovascular responses to the peptide (100 and 300 μ g/kg, i.v. ) were alsodetermined in conscious rats ( n =4) that had been pretreated10 min before with the angiotensin-converting enzymeinhibitor, captopril (15 mg/kg, i.v. ). 2.10. In vitro experiments Studies in isolated perfused hearts were performed usinganother set of rats. 2.11. Perfusion model Rats ( n =3) were stunned and killed by cervical dislocation.After thoracotomy, hearts were quickly excised and arrested inice-cold perfusion medium. After removal of lung and fat tissue,hearts were immediately mounted and perfused according to theLangendorff technique at a constant perfusion pressure of 80 mm Hg. Retrograde perfusion of the aorta was achieved withfiltered (Millipore; 0.65 μ m), modified Krebs – Hensleit solution(pH=7.4) containing (in mM) NaCl 129, KCl 5.6, MgCl 2 1.25, NaHCO 3 21, CaCl 2 1.25, NaH 2 PO 4 1.2. Glucose (10 mM) wasadded as a substrate. Sodium pyruvate (2 mM) was added as acosubstrate in order to preserve myocardial performance duringthe 120-min perfusion period. This buffer was continuouslyaerated with 5% CO 2 in oxygen and maintained at 37 °C. Under these conditions the hearts beat spontaneously. 2.12. Recording of cardiac parameters Left ventricular systolic pressure (LVSP) was measuredthrough a cannulated, water-filled latex balloon insertedthrough the atrium into the left ventricle, and connected bya polyethylene catheter to a blood pressure transducer, whichin turn was connected to a Gilson model 5/6 H polygraph(Gilson Medical Electronics Inc., Middletown, WI, USA). HR was obtained from a cardiotachometer triggered by the pressure pulses. The collapsed balloon attached to the trans-ducer was secured in place and filled with water to a volumethat produced a left ventricular diastolic pressure of about 10 mmHg. 2.13. Experimental protocol After perfusing the hearts for an equilibration period of 30 min, baseline measurements of LVSP and HR were made.Thereafter, the time-course of the effects on LVSP and HR Fig. 2. Chromatographicisolationandmass spectrometric analysis of the Crotalus d. collilineatus peptide.A. Fractionswith molecularweight of less than 6000 Da shown by a double-headed arrow (130 – 150 min) were collected on a Superdex 75pg column (1.6×60 cm) equilibrated in 0.2 M ammonium formate (pH 6.7). Flow rate was0.7 mL/min. B. Reverse phase subfractionation of the low molecular weight Superdex fractions on a Symmetry300 C 18 column (5 μ m, 4.6×25 mm). Equilibrating buffer was0.1%aqueoustrifluoroaceticacid(TFA).Elutionwasperformedinalineargradientof0to35%acetonitrilecontaining0.1%TFAin60minataflowrateof1mL/min.Absorbance was monitored at 214 nm. Fractions indicated by a filled rectangular bar on the abscissa were submitted to mass measurements by Q-Tof Micro. C. Massspectrum of the C. d. collilineatus peptide. An isotopic series of [M+2H] 2+ ions having 511.2 Da as a monoisotopic species coincided with the peptide of MW 1021.1 Da predicted from the cDNA sequence analysis. D. MS/MS profile of the fragmentation of the selected ion (511.2 Da [M+2H] 2+ ) and the deduced sequence derived from denovo sequencing based on MassLynks data processing system (Waters, USA). Consistency between the b and y ion series established the sequence TPPAGPDGGPR.110 S. Higuchi et al. / Comparative Biochemistry and Physiology, Part C 144 (2006) 107 – 121
