p e p t i d e s 2 8 ( 2 0 0 7 ) 5 1 5 – 5 2 3

Isolation and characterization of a novel bradykininpotentiating peptide (BPP) from the skin secretion ofPhyllomedusa hypochondrialis

Katia Conceicao a, Katsuhiro Konno a, Robson Lopes de Melo a, Marta M. Antoniazzi b,Carlos Jared b, Juliana M. Sciani c, Isaltino M. Conceicao c, Benedito C. Prezoto c,Antonio Carlos Martins de Camargo a, Daniel C. Pimenta a,*a LETA (Laboratorio Especial de Toxinologia Aplicada), Center for Applied Toxinology (CAT/CEPID), Instituto Butantan,

Avenida Vital Brasil 1500, Sao Paulo, SP 05503-900, BrazilbDepartmento de Biologia Celular, Instituto Butantan, Avenida Vital Brasil 1500, Sao Paulo, SP 05503-900, Brazilc Laboratorio de Farmacologia, Instituto Butantan, Avenida Vital Brasil 1500, Sao Paulo, SP 05503-900, Brazil

a r t i c l e i n f o

Article history:

Received 28 August 2006

Received in revised form

2 October 2006

Accepted 3 October 2006

Published on line 13 November 2006

Keywords:

Phyllomedusa hypochondrialis

Bradykinin-potentiating peptide

Mass spectrometry

De novo sequencing

Natural peptides

Secretion

Bradykinin

a b s t r a c t

Bradykinin potentiating peptides (BPPs) from Bothrops jararaca venom were first described in

the middle of 1960s and were the first natural inhibitors of the angiotensin-converting

enzyme (ACE). BPPs present a classical motif and can be recognized by their typical

pyroglutamyl (Pyr)/proline rich sequences presenting, invariably, a proline residue at the

C-terminus. In the present study, we describe the isolation and biological characterization of

a novel BPP isolated from the skin secretion of the Brazilian tree-frog Phyllomedusa hypo-

chondrialis. This new BPP, named Phypo Xa presents the sequence Pyr-Phe-Arg-Pro-Ser-Tyr-

Gln-Ile-Pro-Pro and is able to potentiate bradykinin activities in vivo and in vitro, as well as

efficiently and competitively inhibit ACE. This is the first canonical BPP (i.e. Pyr-Aaan-Gln-

Ile-Pro-Pro) to be found not only in the frog skin but also in any other natural source other

than the snake venoms.

# 2006 Elsevier Inc. All rights reserved.

avai lab le at www.sc iencedi rec t .com

journal homepage: www.elsev ier .com/ locate /pept ides

1. Introduction

Bradykinin (BK), which was first discovered by Rocha e Silva

in 1949 [42], can be described as the hydrolysis product of

low-molecular-weight (LMW) kininogen by tissue kallikrein,

or by a few venoms [11,12]. Among its classical and most

* Corresponding author. Tel.: +55 11 3726 1024; fax: +55 11 3726 1024.E-mail address: dcpimenta@butantan.gov.br (D.C. Pimenta).

Abbreviations: ACN, acetonitrile; BK, bradykinin; BPP, bradykinin pmetry; FRET, fluorescence energy transfer resonance; ACh, acetylcholpeptide; CIF, collision induced fragmentation; Pyr, pyroglutamic acid0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserveddoi:10.1016/j.peptides.2006.10.002

studied effects, bradykinin is able to induce contraction of

guinea-pig ileum in vitro and lower the blood-pressure in

vivo [3]. Moreover, bradykinin has been associated to several

physiological processes such as the inflammatory responses

and induction of nociception and hyperalgesia [5]. Interest-

ingly, it was found that there existed a factor in the venom of

otentiating peptide; TFA, trifluoroacetic acid; MS, mass spectro-ine; ACE, angiotensin converting enzyme; BRP, bradykinin related

.

p e p t i d e s 2 8 ( 2 0 0 7 ) 5 1 5 – 5 2 3516

Bothrops jararaca, which was able to potentiate the biological

actions of bradykinin [5,17–20], specifically the guinea-pig

ileum contraction. Moreover, this factor (or, more specifi-

cally: these peptides, as later characterized and denomi-

nated bradykinin potentiating peptides, or BPPs) could

inhibit the enzymatic activity of angiotensin-converting

enzyme (ACE), a cytoplasmatic membrane peptidase of

endothelial cells responsible for the conversion of angio-

tensin I to angiotensin II [36–38]. Indeed, the history of BK

isolation and characterization has long been closely related

to the use of potentiating factors. Kato and Suzuki [27,28],

Ferreira et al. [16], and Ondetti et al. [37] have isolated

different oligopeptides with bradykinin potentiating activity

from the venoms of Agkistrodon halys blomhoffii and B.

jararaca.

Different from the relatively small number of putative

endogenous BPPs encoded at the level of DNA, the snake

venom gland, not only from B. jararaca but also from other

snakes species, produces a large variety of these molecules

[17,24,25,45,53]. Surprisingly, peptides with BK-potentiating

activity have also been generated by limited proteolysis of

seric proteins [51], hemoglobin [25,39,54], milk [23,31], or

wheat germ [34]. However, the most important characteristic

of the classical BPPs is that they are not BK analogs or agonists;

they function by a different, yet to be characterized, mechan-

ism other than a synergic action and/or of BK proteolysis

prevention [33,52].

Also noteworthy, is that BKs and bradykinin related

peptides (BRPs) are found in several organisms other than

mammals, including invertebrates [47], but so far no BPPs

(with their classical motif: <E-Xn-Q-I-P-P, <E being pyroglu-

tamic acid) have been found in any other species than snakes.

The reason why BKs and BRPs are present in wasp venoms

[29], amphibian skin secretions [6,10] or crustacean [41] is not

clear yet. Maybe, finding BPPs in other secretions and venoms

may have the same reason and is not likely to be such an odd

event.

Erspamer et al. [14] once described the Phyllomedusinae frog

sub-family as ‘‘huge storehouses of bioactive peptides’’,

whose skin secretions are among the ones with the highest

degree of peptide (<5 kDa) complexity. One representative

member of this subfamily is the tigerleg monkey tree-frog

Phyllomedusa hypochondrialis [13]. Several studies have demon-

strated that stress or injury result in the secretion of the

granular gland contents onto the skin surface of this

amphibian. This event is mediated by the sympathetic

nervous system and involves the contraction of the surround-

ing myoepithelial cells [15,30].

Amphibian skin peptides have been the subject of intense

research interest for many years from both academic and

pharmaceutical groups due to their potential applications in

biophysical research, biochemical taxonomy and in lead

compound development for new pharmaceuticals [7,50]. In

the present work, we describe the isolation, biochemical

characterization, amino acid sequencing and biological effects

of a novel bradykinin potentiator peptide (BPP), named Phypo

Xa, isolated from the skin of P. hypochondrialis. This is the first

canonical BPP (i.e. Pyr-Aaan-Gln-Ile-Pro-Pro) to be found not

only in the frog skin secretions but also in any other natural

source than the snake venoms.

2. Materials and methods

2.1. Animals

Male Wistar rats (220–290 g) and female guinea-pigs (300 and

400 g) were bred at Instituto Butantan, Sao Paulo, SP, Brazil.

The animals had had free access to water and food, and were

kept under a 12 h light–dark cycle. Procedures involving

animals and their care have been conducted in accordance

with the Guidelines for the Use of Animals n Biochemical

Research [21]. This study was approved by ‘Comissao De Etica

No Uso De Animais, Instituto Butantan’, under protocol

number 261/06.

2.2. Collection of the specimens

Specimens (n = 12) of P. hypochondrialis were collected at

Angicos, in the state of Rio Grande do Norte, Brazil. The

tree-frogs were kept alive in the biotherium of the Department

of Cellular Biology Institute Butantan, Sao Paulo, Brazil. The

tree frogs were collected according to the Brazilian Environ-

mental Agency (IBAMA (Instituto Brasileiro do Meio Ambiente

e dos Recursos Naturais Renovaveis)) under the license

02027.023238/03-91.

2.3. Drugs and reagents

Sep-Pak C-18 cartridges are from Waters Co. Milford, MA, USA;

Fmoc-amino acids were purchased from Calbiochem–Nova-

biochem Corp., San Diego, CA, USA. Acetonitrile (HPLC grade)

were purchased from J.T. Baker (Phillipsburg, NJ, USA).

Laboratory-deionized water was produced with Milli-Q water

purifying system (Millipore, Bedford, USA); a-cyano-4-hydro-

xycinnamic acid, iodoacetamide, NaI, molecular mass stan-

dards (ProteoMass Kit), acetylcholine hydrochloride, Somatic

ACE (rabbit lung) and bradykinin acetate were purchase from

Sigma–Aldrich (St Louis, MO, USA). Sodium pentobarbital and

sodium heparin from Roche Laboratories (Brazil) and angio-

tensin II from Ciba Laboratories (Switzerland). Angiotensin I

was synthesized at the Universidade Federal de Sao Paulo

(Department of Biophysics, Sao Paulo, SP, Brazil).

2.4. Skin secretion obtainment and solid phase extraction

Glandular secretions were obtained from adult specimens of P.

hypochondrialis that were submerged in a Beaker containing

deionized water and manually and gently compressed. This

solution was lyophilized and stored at �20 8C. Pooled lyophi-

lized secretions from 12 animals (male and female) were

dissolved in deionized water and centrifuged at 5000 � g for

20 min (room temperature) the supernatant was pre-purified by

solid phase extraction (SPE) using Sep–Pak, C18 cartridges

(Waters Corporation, Milford, MA, USA). A single aliquot of 3 mg

diluted in 3 mL of 0.1% TFA was loaded and a elution of 80%

acetonitrile containing 0.1% TFA was performed.

2.5. Peptide purification

Reversed-phase binary HPLC system (Akta, Amersham) was

used to the sample separation. The SPE eluted fraction was

p e p t i d e s 2 8 ( 2 0 0 7 ) 5 1 5 – 5 2 3 517

loaded in a Shimadzu C18 column (Shim-Pack 5 m, 4.6 mm �250 mm) in a two-solvent system: (A) trifluoroacetic acid

(TFA)/H2O (1:1000) and (B) TFA/acetonitrile (ACN)/H2O

(1:900:100). The column was eluted at a flow rate of 1.0 mL/

min with a 10–80% gradient of solvent B over 60 min. The HPLC

column eluates were monitored by their UV absorbance at

214 nm. For peptides purification, further chromatographic

steps were necessary, using the same column with optimized

gradients over 60 min.

2.6. Mass spectrometry

Molecular mass analyses of the fractions and purified peptides

were performed on a Q-TOF Ultima API (Micromass, Manche-

ster, UK), under positive ionization mode and/or by MALDI-

TOF mass spectrometry on an Ettan MALDI-TOF/Pro system

(Amersham Biosciences, Sweden), using a-cyano-4-hydroxy-

cinnamic acid as matrix.

2.7. ‘De novo’ peptide sequencing

Mass spectrometric ‘de novo’ peptide sequencing was carried

out in positive ionization mode on a Q-TOF Ultima API fitted

with an electrospray ion source (Micromass, Manchester, UK).

The samples were dissolved into a mobile phase of 50:50

aqueous formic acid 0.1 %/acetonitrile and directly injected

(10 mL) using a Rheodyne 7010 sample loop coupled to a LC-10A

VP Shimadzu pump at 20 mL/min, constant flow rate. The

instrument control and data acquisition was conducted by

MassLynx 4.0 data system (Micromass, Manchester, UK) and

experiments were performed by scanning from a mass-to-

charge ratio (m/z) of 50–1800 using a scan time of 2 s applied

during the whole infusion. The mass spectra corresponding to

each signal from the total ion current (TIC) chromatogram

were averaged, allowing an accurate molecular mass deter-

mination. External calibration of the mass scale was

performed with NaI. For the MS/MS analysis, collision energy

ranged from 18 to 45 eV and the precursor ions were selected

under a 1 � (m/z) window.

2.8. Peptide synthesis

Synthetic peptide was obtained in automated bench-top

simultaneous multiple solid-phase synthesizer (PSSM 8

system from Shimadzu Co.) using solid phase peptides

synthesis by the Fmoc-Procedure [2]. The peptide was purified

by reversed-phase chromatography (Shim-pack Prep-ODS,

5 m, 20 mm � 250 mm Shimadzu Co.) semi-preparative HPLC,

and the purity and identity of the peptide confirmed by

MALDI-TOF mass spectrometry and by analytical HPLC, in the

same conditions described above.

2.9. Enzymatic assay

Hydrolysis of the FRET substrate at 37 8C in 50 mM HEPES

buffer, containing 200 mM NaCl and 10 mM ZnCl, pH 6.8, was

followed by measuring the fluorescence at lem = 420 nm and

lex = 320 nm in a Hitachi F-2000 spectrofluorometer. The 1 cm

path-length cuvette containing 1 mL of the substrate solution

was placed in a thermostatically controlled cell compartment

for 5 min before the enzyme solution was added and the

increase in fluorescence with time was continuously recorded

for 10 min. The slope was converted into moles of hydrolyzed

substrate per minute based on the fluorescence curves of

standard peptide solutions before and after total enzymatic

hydrolysis. The concentration of the peptide solutions was

determined by colorimetric determination of the 2,4-dinitro-

phenyl group (17,300 M�1 cm�1 extinction coefficient at

365 nm). The enzyme concentration for initial rate determina-

tion was chosen at a level intended to hydrolyze less than 5%

of the substrate present. The pure peptides were assayed as

competitive inhibitor over the hydrolysis of Abz-YRK(Dnp)P-

OH (Km = 5.1 mM, kcat = 246.3 s�1 [1]) by ACE. The Ki values for

competitive inhibition assay were determined according to

[49].

2.10. Biological assays

2.10.1. Potentiation on guinea pig ileumThe bradykinin potentiation assays on guinea pig ileum were

performed according to [44], as follows. After a 24 h fasting

period, segments of approximately 2 cm of the terminal ileum

were removed, cleaned from surrounding tissues, and the

lumens carefully washed with a nutrient solution containing

atropine and diphenhydramine (1 mg/mL) and 138 mM NaCl;

2.7 mM KCl; 1 mM MgCl2; 0.36 mM NaH2PO4; 12 mM NaHCO3;

5.5 mM D-glucose, pH 7.4. Each segment was mounted in a

5 mL chamber containing continuously aerated nutrient

solution at 37 8C. Isometric contractions were recorded by

means of isometric transducers coupled to a recording

system (PowerLab, AD Instruments), under a load of 1.0 g.

Concentration–response curves for bradykinin were obtained

on absence (control) or presence of the pure peptide or

captopril, for calculation of EC50 values. Data were expressed

as percent values of maximum contraction achieved in

control curve.

2.10.2. Bradykinin-potentiation on anaesthetized rat blood

pressureAssays were performed as describe by Hayashi et al. [22].

Briefly, male Wistar rats (280–350 g) were anaesthetized

with sodium pentobarbitone (60 mg/kg intraperitonealy).

Tracheotomies were performed and polyethylene cannulas

(PE-50) inserted into the right carotid arteries and external

right jugular veins, to measure the mean arterial pressure

(MAP) and the administration of drug solutions, respec-

tively. The animals received heparin (200–300 U intrave-

nously) and MAP were continuously recorded with a Stathan

Gould physiological transducer (Cleveland, OH) coupled to a

Gould WindGraf1 930 polygraph (0.25 mm/min paper speed

and 0–200 mmHg calibration scale). After MAP stabilization,

the responses to i.v. injections of Bk (1.7–3.4 mg/kg),

angiotensin I (AI) and angiotensin II (AII) (0.3–0.6 mg/kg)

and acetylcholine (Ach) (0.3 mg/kg) dissolved in saline

solution at 0.9% were recorded. Pure peptide (150–400 mg/

kg) was assayed on in vivo inhibition of ACE activity

(convertion of AI to AII and inactivation of BK), as follows:

Bk, AI and AII were injected before and after (5, 10, 20 and

30 min) Pure peptide, and the hypotensive or hypertensive

responses were recorded and compared. The initial effects

p e p t i d e s 2 8 ( 2 0 0 7 ) 5 1 5 – 5 2 3518

(provoked by Bk and AI and AII) were considered as 100%

(control).

2.11. Statistical analyses

One-way analysis of variance (ANOVA) followed by Newman–

Keuls test were performed to determine significance of

differences. The significance level was considered as

p < 0.05. Statistical analyses of Bradykinin-potentiation on

anaesthetized rat blood pressure were performed with paired

Student’s t-test. Differences were considered significant when

p < 0.05.

3. Results

3.1. Purification and characterization of the peptide

The lyophilized SPE-P. hypochondrialis crude skin secretion was

fractionated by analytical RP-HPLC, as shown in Fig. 1. Several

peaks along the profile were detected and the arrow indicates

the fraction containing the novel bradykinin potentiating

peptide. The chromatogram presents several other peaks that

are currently under investigation, as well. Once the peak was

chosen, further chromatographic steps were necessary to

purify this peptide, as presented in material and methods

Fig. 1 – RP-HPLC profile of P. hypochondrialis pooled crude skin s

to Section 2. The arrow indicates fraction containing Phypo Xa,

MS profile of the purified peptide and (B) zoomed MALDI-TOF/M

Measured m/z values are typed besides the ions.

section. Purity was assessed by HPLC and MALDI-TOF (Fig. 1,

insets A and B).

3.2. ‘De novo’ peptide sequencing

One aliquot of purified peptide was selected for ‘de novo’

sequencing and processed according to Westermeier and

Naven [48]. Briefly, after cystein bridge reduction and

alkylation by iodoacetamide the obtained peptide was

individually selected for MS/MS analysis and fragmented

by collision with argon (CIF), yielding daughter ion spectra as

presented in Fig. 2, panel A. This routine was repeated until

enough information regarding the peptide sequence was

gathered. The MS/MS spectra were analyzed by the BioLynx

software module of MassLynx 4.0 and manually verified for

accuracy in the amino acid sequence interpretation.

Although de novo sequencing cannot distinguish between

Leu and Ile, we were able to determine that Ile was the

correct amino acid by comparing the retention times of the

purified peptide, with synthetic standards containing either

Leu or Ile at position 8. At 25% B, HPLC isocratic elution,

there is a 2.3 min difference in the retention time of the

analogues (data not shown); therefore, making it possible to

clearly identify the naturally occurring amino acid. The

peptide was sequenced as Pyr-Phe-Arg-Pro-Ser-Tyr-Gln-Ile-

Pro-Pro-OH (<EFRPSYQIPP; Pyr and <E representing

ecretions after solid phase extraction, performed according

comprised between the thick lines. Insets: (A) MALDI-TOF/

S of the peptide presenting the isotopic distribution.

Fig. 2 – Representative ‘De novo’ sequencing of the (A) purified natural peptide and (B) synthetic standard peptide Phypo Xa

in a Q-TOF Ultima API. The fragments shown correspond to some of the a, b and internal fragment daughter ions.

p e p t i d e s 2 8 ( 2 0 0 7 ) 5 1 5 – 5 2 3 519

pyroglutamic acid, observed molecular mass, 1214,617 Da;

theoretical molecular mass, 1214,612 Da) or Phypo Xa as it

will be referred from now on, due to its 10 amino acid

composition as well as its being the first BPP isolated

from P. hypochondrialis. Table 1 shows that Phypo Xa aligns

very well with other 10 amino acid BPPs already described

from other snakes. Moreover, the synthetic Phypo Xa was

fragmented in the same conditions and was able to generate

Table 1 – Aligned 10-amino acid BPPs from differentsources

Peptidesequence

Name MW (Da) Source

<EFRPSYQIPP Phypo Xa 1214.6 Phyllomedusa

hypochondrialis

<ESWPGPNIPP BPP-10a 1074.5 Bothrops jararaca

<ENWPRPQIPP BPP-10b 1214.6 Bothrops jararaca

<ENWPHPQIPP BPP-10c 1195.6 Bothrops jararaca

<ERWPHLEIPP – 1254.6 Crotalus d. terrificus

<EGLPPRPIPP – 1053.6 Agkistrodon halys

blomhoffii

<E.wP..qIPP – – Consensusa

<E represents pyroglutamic acid.a Calculated by Clustal W [46].

a virtually identical CIF spectrum, as presented in Fig. 2,

panel B.

3.3. Enzymatic assays

We chose to assay a FRET substrate that is equally susceptible to

hydrolysis by both domains of somatic ACE, namely Abz-

YRK(Dnp)P-OH [1]. Phypo Xa was assessed as a competitive

inhibitor for sACE and the calculated Ki value for the inhibition

was 8 nM. Moreover, 10 mM Phypo Xa was incubated with 10 nM

sACE for 24 h, in 50 mM HEPES buffer, containing 200 mM NaCl

and 10 mM ZnCl, pH 6.8, at 37 8C and no hydrolysis product was

observed by HPLC analysis (data not shown).

3.4. Potentiation on guinea pig ileum

As presented by Fig. 3, captopril and Phypo Xa induced a shift

to the left of the bradykinin curve. Captopril significantly

reduced the bradykinin EC50 values in all tested concentra-

tions, 15 nM (EC50 = 9.3 � 3.4 nM), 50 nM (EC50 = 2.8 � 0.9 nM)

and 150 nM (EC50 = 1.8 � 0.6 nM), when compared to the

control EC50 (25.0 � 2.9 nM). Phypo Xa also produced a

significant decrease on the bradykinin EC50 values for the

control curve (13.3 � 5.1 nM), but only at 50 and 150 nM

(EC50 = 2.5 � 1.0 nM and 4.2 � 1.4 nM, respectively). When

used at 15 nM (EC50 = 10.3 � 3.4 nM) Phypo Xa caused no

changes in BK curve.

Fig. 3 – Mean cumulative concentration–response curves for bradykinin (BK) in absence (control) or presence of captopril (A)

or Phypo-Xa (B). Points represent mean W S.E.M. of five experiments.

Fig. 4 – Blood pressure measurements on anesthetized rat. Arrows indicate drug administration as follows: (A) 500 ng BK; (B)

1500 ng BK; (C) 50 ng Ach; (D) 150 ng Ach; (E) 300 ng Ach; (F) 100 ng A-I; (G) 200 ng A-II; (H) 125 mg Phypo Xa; (I) 500 ng BK

(8 min after (H)); (J) 50 ng Ach (14 min after (H)); (K) 100 ng A-I (19 min after (H)); (L) 200 ng A-II (25 min after (H)).

p e p t i d e s 2 8 ( 2 0 0 7 ) 5 1 5 – 5 2 3520

3.5. Bradykinin-potentiation on anaesthetized rat bloodpressure

BK hypotensive response before and after Phypo Xa (or control

drugs) infusion was assessed and the hypotensive response of

one specific dose of Bk prior to Phypo Xa administration was

considered as 100%. Table 2 presents the data regarding the

administration of two doses of Phypo Xa, one control and one

standard drug (captopril). Moreover, Fig. 4 presents a real time

record of the administration of several control drugs and the

effect of Phypo Xa. One can clearly see that Phypo Xa does not

interfere with the effect of Ach (C versus J) or Angiotensin II (G

Table 2 – Vascular effects of the IV administration ofPhypo Xa on anesthetized rats

Drug Timea

0 min(Controlb)

5 min (MAPc

decreased)30 min (MAP

decrease)

0.9% NaCl 100 � 20 5 � 6 N.D.e

Phypo Xa 50 mg 100 � 20 32 � 5 25 � 3

Phypo Xa 100 mg 100 � 20 73 � 6 51 � 10

Captopril 43 mg 100 � 20 59 � 9 N.D.

a Time after 500 ng BK administration.b Normalized data.c Mean arterial pressure.d Percentual decrease, related to the control group; n = 4.e Not determined.

versus L). Nevertheless, besides having a mild transient

hypotensor effect (H), Phypo Xa induces a significant potentia-

tion of Bk hypotension (A versus I) and causes an in vivo

inhibition of the conversion of AI to AII (ACE inhibition) (F

versus K).

In these conditions, a 5 min, 150 mg/kg captopril pretreat-

ment induces to a 59 � 8% potentiation of the Bk-hypotensive

effect (n = 5, p < 0.05). The same dose of Phypo Xa induces to a

potentiation of 33 � 5% (n = 4, p < 0.05) in the Bk-hypotensive

effect and causes a 30 � 3% inhibition of the hypertensive

effect of AI (n = 4, p < 0.05).

4. Discussion

In this study, the SPE fraction of the crude skin secretion of P.

hypochondrialis was characterized by using RP-HPLC, MALDI-

TOF MS, and bioassay. This strategy led to the identification of

a novel peptide entity in the skin secretion of this tree frog: a

bradykinin potentiating peptide, named here Phypo Xa.

Finding such a novel peptide in the skin secretion of this frog

corroborates our previous assessment that investigating

nature’s selected molecules is the most fruitful source for

the identification of compounds aimed at very specific targets

[25], in our case, the kallikrein–kinin system and its acces-

sories.

The kallikrein–kinin system, having bradykinin lying at

its center, represents a metabolic cascade that, when

activated, triggers the release of vasoactive kinins. This

p e p t i d e s 2 8 ( 2 0 0 7 ) 5 1 5 – 5 2 3 521

complex multiprotein system includes the serine proteases

tissue and plasma kallikreins, which liberate kinins from

high- and low molecular-weight kininogens (HK and LK).

Kinins exert their pharmacological activities by binding

specific receptors, before being metabolized by various

peptidases. The autocrine or paracrine activity of kinins is

regulated by several metallopeptidases, the relative impor-

tance of which varies from one biological medium to the

other. Finally, the regulation of this system by serpins adds

to the complexity of the system, as well as its multiple

relationships with other important metabolic pathways

such as the rennin-angiotensin, coagulation or complement

pathways (for a review on the subject, see the work of [35]).

Nevertheless, bradykinin potentiating peptides (BPPs),

extremely active and widely present on snake venoms,

have not, so far, been found as an endogenous component

of this system, or in any other source than snake venoms.

Despite our own efforts to find a mammal endogenous BPP,

the closest we came to that, was the identification of

mammal peptides that may function like BPPs, but posses a

completely different amino acid sequence, the hemorphins

[26].

The skin secretions of the members of the Phyllomedu-

sinae frog sub-family are widely known to be one the richest

sources of bioactive peptides. However, antimicrobial

peptides (AMPs) and bradykinin-related peptides (BRPs)

are the most commonly found molecules in these amphi-

bians’ skins [4,6,8]. The biological role, relevance and gain

associated to the presence of AMPs on the skin secretion

of these anurans is evident, at least much more evident

than the reason why these animals would secrete so many

BRPs.

Some authors speculate that BKs and BRPs are present in

the skin of these tree frogs for they lack the kallikrein-kinin

system [40,43], and/or that these peptides may act on and/or

potentiate the endogenous BKs action on the cardiovascular

and/or gastrointestinal system of their predators, thus acting

as defensive peptides [9,32]. In view of this, secreting an

additional BPP would greatly potentiate the known biological

effects of the BKs and BRPs already present in the skin

secretion, augmenting, for instance, the proposed defensive

action of these kinins.

This work reports the purification and characterization of

a bradykinin potentiating peptide present in the skin of the

Brazilian monkey tigerleg tree frog P. hypochondrialis. The

peptide, named Phypo Xa have a typical BPP motif, as shown

in Table 2, Moreover, in vivo and in vitro assays were able to

clearly demonstrate that Phypo Xa meets all the functional

requirements for a bioactive BPP. And mostly important,

physiologically, at the level of the frog skin and its

mechanisms of defense, the presence of BPPs can make all

the well documented BKs and BRPs extremely powerful

weapons.

The methodology employed on this work proved to be

extremely efficient for the identification of biologically active

molecules. However, it is crucial to pursue the activity through

the biological assay. The highly sensitive techniques such as

RP-HPLC and MS make it possible to purify and characterize

bioactive peptides from complex mixtures and by using less

amount of crude secretion.

Acknowledgements

The tree frogs were collected according to the Brazilian

Environmental Agency (IBAMA (Instituto Brasileiro do Meio

Ambiente e dos Recursos Naturais Renovaveis)) under the

license number 02027.023238/03-91. Supported by funds

provided by CAT/CEPID, FAPESP, CAPES and CNPq (grant #

303516/2005-4).

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