Bradykinin-related peptides from Phyllomedusahypochondrialis

G.D. Brand a,b, F.C. Krause a, L.P. Silva a, J.R.S.A. Leite a, J.A.T. Melo a, M.V. Prates a,J.B. Pesquero c, E.L. Santos c, C.R. Nakaie c, C.M. Costa-Neto d, C. Bloch Jr.a,*a Laboratorio de Espectrometria de Massa, EMBRAPA, Recursos Geneticos e Biotecnologia, Brasılia, DF 70770-900, Brazilb Programa de pos-graduacao em Biologia Animal, IB, Universidade de Brasılia, Brasılia, DF, BrazilcDepartamento de Biofısica, Escola Paulista de Medicina, UNIFESP, 04023-062 Sao Paulo, BrazildDepartamento de Bioquımica e Imunologia, Faculdade de Medicina de Ribeirao Preto, USP, 14049-900 Ribeirao Preto, Brazil

p e p t i d e s 2 7 ( 2 0 0 6 ) 2 1 3 7 – 2 1 4 6

a r t i c l e i n f o

Article history:

Received 2 March 2006

Received in revised form

11 April 2006

Accepted 11 April 2006

Published on line 22 June 2006

Keywords:

Imaging mass spectrometry

Frog skin

BRP

Phyllomedusa

a b s t r a c t

Bradykinin related peptides (BRPs) present in the water-soluble secretion and freshly

dissected skin fragments of Phyllomedusa hypochondrialis were investigated by mass spectro-

metry techniques. Eighteen BRPs, along with their post-translational modifications, were

characterized in the secretion by de novo MS/MS sequencing and direct MALDI imaging

experiments of the frog skin. These molecules revealed strong sequence similarities to the

main plasma kinin of some mammals and reptiles. Such a diversity of molecules, within the

same peptide family, belonging to a single amphibian species may be related to functional

specializations of these peptides and a variety of corresponding receptors that might be

present in a number of different predators. Also, a novel analog, [Val]1,[Thr]6-bradykinyl-

Gln,Ser had its biological activity positively detected in cell culture expressing the human

bradykinin B2 receptor and in guinea pig ileum preparations.

# 2006 Elsevier Inc. All rights reserved.

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1. Introduction

Amphibian skin secretions are a rich source of bioactive

molecules which cannot only act as a barrier against

infectious agents but also disrupt the homeostasis of

potential amphibian predators [20,26]. Several families of

opioid and neurotransmitter-like peptides present in anuran

skin are capable of inducing hypotension, hallucination,

smooth-muscle contractions, and changes in the ingestion

behavior of snakes, birds and mammals [17,19,21]. A common

target of these peptides is the kallikrein–kinin system,

responsible for maintaining the vascular tone and responses

to tissue injury. In mammals, activation of this system

ultimately leads to the secretion of endogenous pro-inflam-

* Corresponding author at: Laboratorio de Espectrometria de Massa,Biologico, Final W5, Asa Norte, Brasılia, DF 70770-900, Brazil. Tel.: +55

E-mail address: cbloch@cenargen.embrapa.br (C. Bloch Jr.).

0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserveddoi:10.1016/j.peptides.2006.04.020

matory peptides such as bradykinin (BK), which exerts its

effects via the activation of the BK B2 receptor, inducing

arteriolar dilation, nociceptor hypersensitivity and gastro-

intestinal smooth-muscle contraction [3,10,21]. It is now well

documented that anuran skin secretions contain large

amounts of bradykinin related peptides (BRPs) that potentiate

the effects of endogenous BK of predatory species, possibly

altering their vascular and gastrointestinal functions [6–

8,15,18]. Since amphibian BRPs are molecules secreted in the

skin, it has been suggested that they are in fact acting as part

of a defense strategy rather than as endogenous hormones

like their BK mammalian analogs [9]. Consistent with this

idea is the apparent lack of a kallikrein–kinin system in the

plasma of amphibians [9].

EMBRAPA, Recursos Geneticos e Biotecnologia, Estacao Parque61 3448 4636; fax: +55 61 3340 3658.

.

p e p t i d e s 2 7 ( 2 0 0 6 ) 2 1 3 7 – 2 1 4 62138

Since the first report describing the presence of BK in the

skin secretion of Rana temporaria, several structural variants of

BKs have been identified in other anuran species. The present

work describes the characterization of eighteen bradykinin

related peptides isolated from the skin secretions of the tree

frog Phyllomedusa hypochondrialis. Mass spectrometric strate-

gies were used to determine their complete primary structures

and identify their post-translational modifications (PTMs). In

addition, the peptides’ direct spatial distribution in the frog

skin was established by matrix-assisted laser desorption/

ionization mass spectrometry (MALDI-MS) imaging [4,23]. The

biological activity of one novel analog, [Val]1,[Thr]6-bradyki-

nyl-Gln,Ser, was also determined in cell cultures expressing

the human B2 receptor and in guinea pig smooth-muscle

preparations.

2. Material and methods

2.1. Amphibians

Frog skin secretions were obtained from adult specimens of P.

hypochondrialis, taken from the cerrado region of Brazil. Frogs

were captured according to the Instituto Brasileiro do Meio

Ambiente e dos Recursos Renovaveis, IBAMA, under the

license number 240/2005-CGFAU (process no. 034/06-COFAN).

2.2. Peptide purification

Frog secretion was obtained by mild electric stimulation of the

skin glands of P. hypochondrialis and collected in distilled water

as a soluble extract. The extract was filtered by gravity through

filter paper, frozen, and lyophilized (Centrivap Concentrador,

Labconco, Kansas City, MO, USA). Peptide separation was

performed by injecting the crude extract onto a semi-

preparative Vydac 218 TP 510 (Hesperia, CA, USA) reverse-

phase chromatographic column in a HPLC system (Class LC-

10VP from Shimadzu Corp., Kyoto, Japan). Peptides were

purified using a linear gradient from 0 to 70% acetonitrile

containing 0.1% trifluoroacetic acid. RP-HPLC experiments

were monitored at two different wavelengths (216 and

280 nm), and fractions were manually collected and subse-

quently lyophilized. When necessary, the isolated fractions

were submitted to further steps of purification using Vydac 218

TP 54 and GE Healthcare C2C18 columns with optimized

gradients. Sample purity and mass analyses were evaluated by

mass spectrometry.

2.3. Mass spectrometry analysis and de novo sequencing

Peptides were analyzed by MALDI-TOF/TOF on an ABI 4700

Proteomics Analyzer (Applied Biosystems, Framingham,

MA, USA), an Ultraflex II (Bruker Daltonics, Germany) and

a Q-TOF Ultima API (Micromass, UK). For the MALDI-MS

analyses, samples were mixed with a saturated matrix

solution of a-cyano-4-hydroxycinnamic acid (1:3) and

spotted on a sample plate. The MS and the MS/MS spectra

were carried out in the reflector mode with external

calibration, using the 4700 Standard kit (Applied Biosys-

tems) and Peptide Calibration Standard—Starter Kit 4

(Bruker Daltonics). Peptide de novo sequencing was per-

formed by precursor ion fragmentation using argon as

collision-induced dissociation gas in both spectrometers.

Electrospray ionization (ESI) analyses were performed in a

Q-TOF Ultima API mass spectrometer operating in W mode.

The sample dissolved in a mixture consisting of water/

methanol/acetic acid (50:50:0.1) was applied at 2 mL/min.

The capillary and cone voltages were set at 2.8 kV and 30 V,

respectively. For the MS/MS experiments, collision cell

voltage was set to 30–70 eV. CapLC (Micromass, Manchester,

UK) analysis was performed using a microbore Symmetry1

C18 5 mm (0.32 � 150) column at a constant flow rate of 1 mL/

min using a gradient spanning from 5 to 100% of reagent B in

400 min. The mobile phases were a mixture of water/

acetonitrile/trifluoroacetic acid, in the proportions of

90:10:0.1 and 10:90:0.1, for reagents A and B, respectively.

Mass detection was performed in ESI+ mode on a Q-TOF

Ultima API (Waters, UK) operating in W mode using the DDA

Survey acquisition mode.

2.4. N-terminal sequencing

Amino acid sequencing was performed by the automated

EDMAN degradation method on a PPSQ-23 protein peptide

sequencer (Shimadzu Co., Japan).

2.5. Determination of [Hyp]3-phyllokinin sulphation

[Hyp]3-phyllokinin was incubated with D2O/D-methanol/D-

acetic acid (50:50:0.1) for 10 min, which is a sufficient time for

full H/D exchange (data not shown). MS/MS experiments were

conducted using the previously described parameters for the

Q-TOF Ultima API MS analysis.

2.6. Sample preparation for imaging by massspectrometry

Dorsal frog skin was dissected and air-dried under a glass slide

for 10 min. Images of tissue pieces were acquired by light

microscopy and small pieces (1–2 mm) were directly attached

to a MALDI plate. 1 mL of matrix (a-cyano-4-hydroxycinnamic

acid, 10 mg/mL in acetonitrile/water/3% trifluoroacetic acid

5:4:1) was applied with a pipette and spread over the skin

fragment and left to dry at room temperature for 20 min prior

to imaging mass spectrometry (IMS) analyses.

2.7. Imaging by mass spectrometry

Molecular ions profiles were initially generated using a

Voyager DE STR (ABI, Framingham, MA, USA) operating in

positive mode. Automatic scanning steps were accomplished

using the MALDI-MS Imaging Tool 2.2.1 Software for the

Voyager DE STR spectrometer. The pulsed N2 laser was

rastered across the sample surface in 60 mm single steps.

Global spectra were generated for each image from 10 to 15

laser shots at each position in the global maps consisting of

600 spectra. BioMAP 3� Functional Image Analysis Software

was used to generate intensity-based ion maps of the

molecular components present in the skin surface ranging

from 1000 to 4000 Da. Similar conditions were used to perform

p e p t i d e s 2 7 ( 2 0 0 6 ) 2 1 3 7 – 2 1 4 6 2139

MS/MS imaging analysis using the Ultraflex II equipped with

Smartbeam Laser technology and Flex Imaging Software.

2.8. Synthesis of [Val]1,[Thr]6-bradykinyl-Gln,Ser

Val-Hyp-Pro-Gly-Phe-Thr-Pro-Phe-Arg-Gln-Ser was synthe-

sized manually according to the standard Boc/Bzl strategy

[1,2]. Boc (Bzl)-Ser-PAM resin was used as the solid support

and the side-chain protecting groups employed were Bzl (Thr

and Ser) and Tos (Arg). After HPLC purification, the homo-

geneity of the peptide was confirmed by analytical HPLC

(Waters), mass spectrometry on RP-HPLC/MS (Micromass),

and amino acid analysis (Biochrom 20 Plus, Amersham

Biosciences).

2.9. Cell culture and transfections

Chinese hamster ovary (CHO) cells were cultivated in

Dulbecco’s modified Eagle’s medium (DMEM) supplemented

with 10% fetal calf serum and grown in a 5%-CO2 environment.

The expression plasmid containing the BK B2 receptor was

permanently transfected into CHO cells using lipofectin (Life

Technologies, Gaithersburg, MD, USA) as described by the

manufacturer. Cells containing the inserted plasmid were

selected by addition of G418 (500 mg/mL) in DMEM.

2.10. Microphysiometric analysis of the [Val]1,[Thr]6-bradykinyl-Gln,Ser interaction with BK B2 receptors

Cells expressing the B2 receptor were transferred 24 h prior

to the assays to transwells made of polycarbonate mem-

brane with 12 mm diameter and 3 mm pores (5 � 105 cells/

well). The Cytosensor microphysiometer (Molecular Devices,

Sunnyvale CA, USA) was used to measure the acidification

rate in the extracellular microenvironment due to cellular

metabolic activity [5,22]. Cells in the transwell were perfused

with DMEM, pH 7.4, containing BSA 0.1% and 44.4 mM NaCl

using a peristaltic pump, with alternating cycles of perfusion

at 100 mL/min (1 min:40 s) and pauses (20 s). During stimula-

tion the medium was replaced by DMEM containing BK in

concentrations varying from 10�9 to 10�5 M. Experiments

were done in quadruplicate, and the generated dose-

response curves were analyzed by nonlinear regression

analysis using PRISM 3.02 (Graph-Pad Software, San Diego,

CA, USA).

2.11. Smooth muscle assay

Female guinea pigs were killed by decapitation (according to

the Animal Ethics Committee protocol) and the 20-cm

terminal portion of the ileum was removed and washed in

Tyrode solution (NaCl 137 mM, KCl 2.7 mM, CaCl2 1.4 mM,

MgCl2 0.5 mM, NaHCO3 12 mM, NaH2PO4 0.4 mM and D-glucose

5.6 mM). Segments of 3 cm were cut and mounted in 5-mL

chambers containing Tyrode solution at 37 8C and bubbled

with a gas mixture of 95% O2 and 5% of CO2. After a 2-h

equilibration period isometric responses were recorded with a

force-displacement transducer (Hewlett-Packard FTA-100)

and a potentiometric recorder (ECB RB-102). Experiments

were done in duplicate, and the generated dose–response

curves were analyzed by nonlinear regression analysis using

PRISM 3.02 (Graph-Pad Software, San Diego, CA, USA).

3. Results

3.1. Purification of BKs and primary structure analyses

Samples of the crude skin secretion obtained from P.

hypochondrialis were fractionated by RP-HPLC using a semi-

preparative C18 column and on-line capillary LC/MS/MS

experiments in order to have a comprehensive identifica-

tion/sequencing strategy. Fractions containing peptides iden-

tified as BRPs are indicated in both HPLC and capillary LC

chromatograms in Fig. 1A and B, respectively. Semi-prepara-

tive RP-HPLC fractions, showing the characteristic elution

times of the peptides belonging to the BK family, were further

analyzed by mass spectrometry and fragmented using CID [6–

9,15,18]. Novel analogs were de novo sequenced by MS/MS

experiments and also had their chromatographic fractions

subjected to further purification steps up to homogeneity for

N-terminal Edman degradation and PTMs confirmation.

Sequences were categorized according to their primary

structure similarities based on the main plasma BKs analogs

of some animal groups, and are summarized in Table 1 [9]. MS/

MS spectra for all BRPs can be found as Supplemental material.

3.1.1. Mammalian BK analogsThe peptides [Thr]6-bradykinin, [Hyp]3-bradykinin, and

[Hyp]3,[Thr]6-bradykinin, with measured molecular masses

of [M + H]+ = 1074.56, 1076.63, and 1090.61 Da, respectively,

were identified in the skin extract. These were grouped, along

with the C-terminally cleaved peptide des-Arg-[Thr]6-brady-

kinin, [M + H]+ = 918.48 Da, under the mammalian BKs group.

3.1.2. [Val]1,[Thr]6-bradykinin analogs, or snake BKsThe BRP [Val]1,[Thr]6-bradykinin, [M + H]+ = 1017.54 Da, along

with its cleaved form, des-Arg-[Val]1,[Thr]6-bradykinin,

[M + H]+ = 861.48 Da, and also [Val]1,[Hyp]2,[Thr]6-bradykinin,

[M + H]+ = 1033.54 Da, were identified in the skin secretion of P.

hypochondrialis. The molecules listed above have been pre-

viously described as the main plasma BK in some viperidae

snakes and also in the skin secretion of Rana nigromaculata

[9,13]. However, this is the first report of a hydroxyproline

containing form, such as [Val]1,[Hyp]2,[Thr]6-bradykinin.

Indeed, a variety of BRPs sharing sequence similarities to

these peptides, with characteristic C-terminal extensions,

were also reported for the first time. [Val]1,[Hyp]2,[Thr]6-

bradykinyl-Gln,Ser, [M + H]+ = 1248.69 Da, [Val]1,[Thr]6-brady-

kinyl-Gln,Ser, [M + H]+ = 1232.66 Da and [Val]1,[Hyp]2,[Thr]6-

bradykinyl-Gln, [M + H]+ = 1161.65 Da. Other C-terminally

extended forms, such as [Val]1,[Thr]6-bradykinyl-Val,Asp,

[M + H]+ = 1231.69 Da, and [Val]1,[Hyp]2,[Thr]6-bradykinyl-

Val,Asp, [M + H]+ = 1247.73 were also observed.

3.1.3. PhyllokininsP. hypochondrialis also synthesizes and secretes some of the

phyllokinin variants previously identified in otherPhyllomedusid

species [11,18]. The peptides phyllokinin, [M + H]+ = 1416.70 Da,

[Hyp]3-phyllokinin, [M + H]+ = 1432.70 Da, and [Thr]6-phylloki-

p e p t i d e s 2 7 ( 2 0 0 6 ) 2 1 3 7 – 2 1 4 62140

Fig. 1 – Fractionation of crude skin secretion from P. hypochondrialis. (A) Sample was loaded onto a C18 Vydac 218TP510

(10 mm � 250 mm) column and equilibrated with 0.1% TFA. Elution was performed using an acetonitrile/0.1% TFA solution

at a flow rate of 2.5 mL/min. The absorbance was monitored simultaneously at two wavelengths (216 and 280 nm). (B)

CapLC/MS chromatogram for the frogs skin crude extract in survey mode, using a linear gradient from 5 to 100% of solvent B

in 400 min. In both experiments, encountered BRPs and their corresponding fractions are indicated.

nin, [M + H]+ = 1430.69 Da, all of them in sulphated and non-

sulphated forms, were also sequenced.

3.2. Analyses of post translational modifications

3.2.1. Hydroxyproline determinationThe mass difference between hydroxyproline (113.048 Da) and

leucine/isoleucine (113.084 Da) residues is 0.036 Da. In proteins

and peptides, these residues cannot be directly resolved using

the resolution of major fragment ion series (y- and b-series)

obtained with the spectrometers available for this work.

Therefore, their precise identification in the investigated

peptides was based on the mass analyses of the main

immonium ions produced after peptide fragmentation in a

series of Q-TOF based experiments, as previously described [25],

and for the first time here with MALDI TOF–TOF data. Fig. 2A

demonstrates that, considering the main proline immonium

([M + H]+ = 70.072) as an internal reference, hydroxyproline

immonium has an experimental [M + H]+ = 86.070 Da (calcu-

lated [M + H]+ = 86.061 Da) while leucine/isoleucine immonium

has an experimental [M + H]+ = 86.110 Da (calculated

[M + H]+ = 86.097 Da). There is also a remarkable difference in

ion intensities, suggesting a preferable ionization for the

hydroxyproline immonium. Fig. 2B demonstrates that for

[Val]1,[Hyp]2,[Thr]6-bradykinyl-Gln,Ser, which was used as a

standard molecule under automatic Edman degradation

experiments, the retention time for the second residue differs

from those expected for proline, leucine or isoleucine, indicat-

ing the presence of a non-common PTH-amino acid absent in

the standard calibration mixture provided by the manufacturer

but compatible with the retention time (5.5 min) of 4-hydro-

xyproline.

3.2.2. Sulphation of phyllokininsTyrosine sulphation ([M + H]+ = 79.957 Da) and phosphorila-

tion ([M + H]+ = 79.966 Da), very common PTMs in proteins, are

p e p t i d e s 2 7 ( 2 0 0 6 ) 2 1 3 7 – 2 1 4 6 2141

Table 1 – BRPs in the skin secretion of P. hypochondrialis

Peptide sequence [M + H]e+ [M + H]t

+

Bradykinin analogs

[Thr]6-bradykinin R P P G F T P F R 1074.56 1074.58

des-Arg-[Thr]6-bradykinin R P P G F T P F 918.48 918.48

[Hyp]3-bradykinin R P P G F S P F R 1076.63 1076.60

[Hyp]3,[Thr]6-bradykinin R P P G F T P F R 1090.61 1090.62

[Val]1,[Thr]6 bradykinin analogs

[Val]1,[Thr]6-bradykinin V P P G F T P F R 1017.54 1017.55

des-Arg-[Val]1,[Thr]6-bradykinin V P P G F T P F 861.48 861.45

[Val]1,[Hyp]2,[Thr]6-bradykinin V P P G F T P F R 1033.54 1033.58

[Val]1,[Thr]6-bradykinyl-Gln,Ser V P P G F T P F R Q S 1232.66 1232.64

[Val]1,[Hyp]2,[Thr]6-bradykinyl-Gln V P P G F T P F R Q 1161.65 1161.64

[Val]1,[Hyp]2,[Thr]6-bradykinyl-Gln,Ser V P P G F T P F R Q S 1248.69 1248.67

[Val]1,[Thr]6-bradykinyl-Val,Asp V P P G F T P F R V D 1231.69 1231.65

[Val]1,[Hyp]2,[Thr]6-bradykinyl-Val,Asp V P P G F T P F R V D 1247.73 1247.68

Phyllokinins

Phyllokinin R P P G F S P F R I Y 1336.74 1336.72

Phyllokinin sulphated R P P G F S P F R I Y (HSO3) 1416.70 1416.68

[Thr]6-phyllokinin R P P G F T P F R I Y 1350.74 1350.73

[Thr]6-phyllokinin sulphated R P P G F T P F R I Y (HSO3) 1430.69 1430.70

[Hyp]3-phyllokinin R P P G F S P F R I Y 1352.74 1352.75

[Hyp]3-phyllokinin sulphated R P P G F S P F R I Y (HSO3) 1432.70 1432.71

P represents hydroxyproline residues; [M + H]e+ and [M + H]t

+ correspond to experimental and theoretical monoisotopic masses, respectively.

rather rare in peptides, existing only in some hormones [8].

Although their occurrence can be clearly identifiable by MS,

they cannot be distinguished by conventional peptide frag-

mentation, for their masses diverge by only 0.009 Da. Fig. 3

demonstrates the MS strategy used for the differentiation

between sulphation and phosphorilation of [Hyp]3-phylloki-

nin. This is based on the mass differences ([M + H]+ = 79.95 Da)

of the non-sulphated form obtained by fragmenting the

singly-charged sulphated [Hyp]3-phyllokinin, compared with

the same ions using deuterated solvents ([M + D]+ = 79.96 Da).

When a phosphorilated peptide is deuterated, mass differ-

ences arising from phosphoryl groups are incremented by

1 Da, corresponding to hydrogen/deuterium exchange, while

the sulphonyl group does not exchange hydrogen with the

solvent [14].

3.3. Detection and spatial profiling of BRPs on skin

Conventional molecular characterization methodologies,

such as chromatography and mass spectrometry, require

samples to be homogenized prior to analysis, potentially

losing morphologically and physiologically relevant informa-

tion in the process. On the other hand, immunohistochemical

and in situ hybridization methods may retain morphological-

functional aspects but do not permit large-scale analyses. IMS

of frog skin introduces the possibility of imaging molecular

ions [4,23] under almost native conditions, preserving mor-

phological and molecular information, and may represent an

important tool for the investigation of small-sized molecules

and possible PTMs. Indeed, a small dorsal skin fragment of one

specimen of P. hypochondrialis was dissected, as shown in

Fig. 4A and B, mounted onto a MALDI plate, and data were

acquired in the indicated area (Fig. 4B, green box), generating a

global spectra (Fig. 4C) of the components detected in the BRPs

mass range (m/z = 1000–1700 Da). Fig. 4C shows that this

technique is capable of detecting most of the phyllokinins

described herein, probably because these represent the most

abundant kinins in P. hypochondrialis skin, but fails with less

concentrated analogs. Also, it demonstrates that phyllokinins

are found in sulphated and in non-sulphated forms in high

yields under native conditions. The spatial profiles of BRPs

were generated to relate peptide expression to certain skin

sites and co-relate their occurrence to definite areas of the

skin. Indeed, the relation between gland morphological

position and the detection of peptides is demonstrated by

the superimposition of Fig. 4D (a light microscope image),

which constitutes an inset of the imaged area, with the

occurrence of a still unsequenced but identified ion at

m/z = 6121 Da. Therefore, this ion was used as a molecular

reference in order to get an appropriate co-register between

light microscopy and MALDI-IMS (Fig. 4E). Fig. 4F–N shows the

spatial distribution of all detected BRPs in the skin fragment. It

is worth noting that sulphated and non-sulphated phylloki-

nins have very close spatial profiles, suggesting that the same

glands synthesize these molecules.

3.4. Determination of the biological activity of[Val]1,[Hyp]2,[Thr]6-bradykinyl-Gln,Ser

Synthetic [Val]1,[Hyp]2,[Thr]6-bradykinyl-Gln,Ser and human

BK activated the BK B2 receptor expressed in CHO cell culture

and produced smooth-muscle contraction in preparations of

guinea pig ileum in a dose-responsive manner. Fig. 5A

demonstrates that both peptides produce similar maximum

acidification in cells expressing B2 receptors, but their

affinities showed a 10-fold difference, for BK displays an

EC50 of 10 nM, compared to 100 nM for the synthetic analog.

Comparable results were found for guinea pig ileum prepara-

p e p t i d e s 2 7 ( 2 0 0 6 ) 2 1 3 7 – 2 1 4 62142

Fig. 2 – Determination of hydroxyproline. (A) Mass spectra demonstrating that leucine/isoleucine

(immoniums = [M + H]calc+ = 86.097 Da and [M + H]exp

+ = 86.109 Da) and hydroxyproline

(immoniums = [M + H]calc+ = 86.061 Da and [M + H]exp

+ = 86.070 Da) residues can be correctly assigned in BRPs by immonium

mass analysis. (B) N-terminal degradation of the three first cycles of [Val]1,[Hyp]2,[Thr]6-bradykinyl-Gln,Ser demonstrating

that hydroxyproline2 has a different retention time from other standard residues.

tions, where the EC50 for BK is around 60 nM and 100 times

higher for the amphibian analog (Fig. 5B). These indicate that

the [Val]1,[Hyp]2,[Thr]6-bradykinyl-Gln,Ser affinity for the

human B2 receptors is residual and results from the primary

structure identity shared with mammalian BKs.

4. Discussion

The present work deals with the identification of BRPs

expressed and secreted in the skin of P. hypochondrialis,

focusing on the analyses of PTMs by MS as well as their

incidence under native conditions, as probed by IMS studies

on ions obtained directly from fresh skin segments. The

biological activity of a novel analog [Val]1,[Hyp]2,[Thr]6-

bradykinyl-Gln,Ser was positively detected on cell cultures

expressing the BK B2 receptor and guinea pig illeum prepara-

tions. However, comparisons of relative potency using BK as

the positive control revealed a weaker response of the analog,

possibly due to species-specific receptor targeting. As has

already been suggested, the diversity of the primary structures

of BRPs in frog skin secretions may reflect the pressure exerted

by natural selection for the generation of specific kinins for

each predator [6,9]. Therefore, studies on the structural

diversity of these molecules may shed light on the molecular

events that target kinins towards their specific receptors.

BRPs were arbitrarily classified according to their similarity

to the main plasma kinins of some vertebrates [9]. Molecules

sharing sequence similarity to BK, specifically peptide

sequences with an N-terminal Arg residue, have been

identified as mammalian BKs. Among these, [Thr]6-bradykinin

is present in a number of different frog skin secretions.

p e p t i d e s 2 7 ( 2 0 0 6 ) 2 1 3 7 – 2 1 4 6 2143

Fig. 3 – Determination of [Hyp]3-phyllokinin sulphation. Fragmentation of the precursor ion [M + H]+ = 1432.70 Da causes

[Hyp]3-phyllokinin to generate a peak at [M + H]+ = 1352.75 Da, corresponding to the loss of 79.95 Da. Using deuterated

solvents, [Hyp]3-phyllokinin incorporates 16.23 Da, resulting in a [M + D]+ = 1448.93 Da, that generates a peak 79.96 Da apart

from the first ([M + D]+ = 1368.96 Da) upon its fragmentation. The unchanged mass difference of 79.96 Da corresponds to

sulphation, for phosphoryl groups are incremented by 1 Da upon deuteration.

Although structurally similar to BK, it may be functionally

diverse, as it is found not only in mammals but also as the

main plasma kinin in crocodilians, chelonians, and varanid

lizards [9]. Peptides with the [Val]1,[Thr]6 motif and associated

C-terminal extensions were grouped under the name of snake

BKs, due to the fact that [Val]1,[Thr]6-bradykinin is the main

plasma kinin in rattlesnakes such as Crotalus atrox, and also in

bullsnakes [9,13]. The diversity of snake BRPs found in P.

hypochondrialis points to an equal variety of predators and

probably different receptor subpopulations, but the precise

biological function of the C-terminal extensions shown herein

is an interesting issue to be evaluated. Apart from the

[Hyp]3,[Thr]6-phyllokinin, all of the phyllokinins previously

identified in other Phyllomedusids were also found in the skin

secretion of P. hypochondrialis, and similarly to other species

from the same genera, these analogs represent the most

abundant kinins in their secretions [8,11,12].

Mass spectrometry enables the determination of PTMs in

kinins in a fast and unequivocal way. It has some advantages

over other sequencing methods such as the construction of

cDNA libraries and Edman degradation and could be highly

beneficial if used in conjunction with them. Unfortunately,

although peptide fragmentation patterns are highly informa-

tive, they are often complex to interpret and to extract

meaningful data, especially when dealing with PTMs. The data

presented here demonstrate that hydroxyproline can be

successfully distinguished from leucine/isoleucine residues

in kinins in a single MS/MS experiment. However it should be

noted that peptides containing both residues in their primary

structure might present a mixed population of both immo-

niums. Proline hydroxylation has different functions in

different proteins, ranging from granting greater stability to

sensing oxygen [16]. Its high occurrence in kinins is commonly

attributed to the modulation of peptide-ligand interactions,

but this still remains to be demonstrated.

The discrimination between isobarous modifications such

as peptide phosphorylation and sulphation is also a matter of

great importance. Both modifications are able to modulate

molecular interactions, being common in proteins but less

frequent in peptides. On the other hand, whereas phosphor-

ylation is tightly linked to regulatory events, the role of

sulphation is not clear [16,24]. Amphibian skin peptides often

share substantial identity with mammalian hormones and

these can contain the same PTMs, as in CCK and caeruleins,

both of which are sulphated [6].

The incidence of PTMs in peptides is often correlated with

regulatory events, placing a demand for their identification

and spatial-profiling in situ with little sample handling.

Indeed, mixed populations of post-translationally modified

and non-modified molecules in the same biological extract

are fairly common raising questions as to whether these exist

in different populations or are the by-product of technical

artifacts. The advent of MALDI-IMS, which generates mole-

cular profiles and two-dimensional ion density maps of

peptide and protein signals directly from the surface of thin

tissue sections, has added a suitable tool, and may represent a

great innovation in the field. Indeed, Fig. 4C demonstrates

that the presence or absence of sulphation in phyllokinins is

not a storage artifact as previously stated for P. sauvageii, since

in the present study it was demonstrated that these kinins are

natively found in sulphated and non-sulphated forms

when directly ionized from the skin of P. hypochondrialis [8].

On the other hand, it does not exclude the possibility that de-

sulphation can be an artifact originating from the preparation

of the sample for mass spectrometry, e.g. matrix deposition.

Since it is highly probable that the P. hypochondrialis

phyllokinin has the same genetic structure as P. sauvageii,

consisting of a single precursor, we can state that phyllokinin

sulphation is either facultative or naturally lost in frog skins.

The only moderate correlation observed between the spatial

p e p t i d e s 2 7 ( 2 0 0 6 ) 2 1 3 7 – 2 1 4 62144

Fig. 4 – MALDI-imaging analysis of P. hypochondrialis skin. (A) Adult specimen of P. hypochondrialis. (B) Light micrograph of a

small skin fragment where the imaged area is indicated. (C) Global spectra of detected ions; those identified as BRPs are

indicated. (D) Inset of the imaged area. (E) Co-localization of glands and the marker molecule (m/z = 6121 Da) used for BRPs

spatial alignment. Mass spectral images of: (F) [Hyp]3-phyllokinin, (G) [Thr]6-phyllokinin, (H) [Hyp]3-phyllokinin sulphated,

(I) [Thr]6-phyllokinin sulphated, (J) [Hyp]3-phyllokinin + [Hyp]3-phyllokinin sulphated, (K) [Thr]6-phyllokinin + [Thr]6-

phyllokinin sulphated, and (L) [Thr]6-bradykinin and (M) [Val]1,[Hyp]2,[Thr]6-bradykinyl-QS.

p e p t i d e s 2 7 ( 2 0 0 6 ) 2 1 3 7 – 2 1 4 6 2145

Fig. 5 – Biological activity of [Val]1,[Hyp]2,[Thr]6-bradykinyl-

Gln,Ser and BK. (A) BK (~) and [Val]1,[Hyp]2,[Thr]6-

bradykinyl-Gln,Ser (&) dose–response acidification rate in

permanently transfected CHO cells expressing the human

B2 receptors. (B) Dose–response contractile effect of

assayed peptides in a guinea pig ileum preparation.

distribution of sulphated and non-sulphated phyllokinins

indicates that some regions either do not have the machinery

used for sulphation or that this modification is lost in

some parts of the skin due to unknown processes. Indeed,

similar hypotheses can be formulated regarding the distribu-

tion of the other BRPs in the skin, indicating the presence of

tissue specialization and/or selectivity in the secretion of

individual BRPs.

The relative potency of the peptide [Val]1,[Hyp]2,[Thr]6-

bradykinyl-Gln,Ser was in agreement with the lower activity of

Val1-substituted analogs as compared to BK in mammalian

smooth-muscle preparations, as depicted in Fig. 5B [6,21]. The

same pattern is observed in the acidification produced by the

activation of B2 receptors expressed in CHO cell cultures

(Fig. 5A). This must be due to the Arg! Val substitution, for

alanine scan studies on BK analogs have demonstrated that

Arg1, Pro2, Gly4, Phe5, Pro7, Phe8 and Arg9 are very important for

mammalian smooth-muscle activation [21]. It is currently

believed that the diversity of BRPs in the skin secretion of P.

hypochondrialis reflects the variability of predators with which

it cohabits [9]. Therefore, the resemblance of [Val]1,[-

Hyp]2,[Thr]6-bradykinyl-Gln,Ser to the main plasma kinin of

colubrid and crotalid snakes indicates that the biological

activity of this peptide would be better evaluated when

compared to [Val]1,[Thr]6-bradykinin using snake smooth-

muscle preparations.

Acknowledgments

The authors wish to thank Dr. Michael Richardson for revising

this manuscript, the Empresa Brasileira de Pesquisa Agrope-

cuaria (EMBRAPA), Recursos Geneticos e Biotecnologia, for

facilities and equipments, CNPq for scholarship funds, and

IBAMA for assistance in the capture of amphibians.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.peptides.

2006.04.020.

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