Insights into cardiovascular effects of proline-rich oligopeptide (Bj-PRO-10c) revealed by structure-activity analyses: Dissociation of antihypertensive and bradycardic effects

Article abstract of DOI:10.1007/s00726-013-1630-x

We have previously reported that the proline-rich decapeptide from Bothrops jararaca (Bj-PRO-10c) causes potent and sustained antihypertensive and bradycardic effects in SHR. These activities are independent of ACE inhibition. In the present study, we used the Ala-scan approach to evaluate the importance of each amino acid within the sequence of Bj-PRO-10c (Pyr¹-Asn ²-Trp³-Pro⁴-His⁵-Pro ⁶-Gln⁷-Ile⁸-Pro⁹-Pro¹⁰). The antihypertensive and bradycardic effects of the analogues Bj-PRO-10c Ala³, Bj-PRO-10c Ala⁷, Bj-PRO-10c Ala⁸ were similar to those of Bj-PRO-10c, whereas the analogues Bj-PRO-10c Ala ², Bj-PRO-10c Ala⁴, Bj-PRO-10c Ala⁵, Bj-PRO-10c Ala⁹, and Bj-PRO-10c Ala¹⁰ kept the antihypertensive activity and lost bradycardic activity considerably. In contrast, Bj-PRO-10c Ala¹ and Bj-PRO-10c Ala⁶ were unable to provoke any cardiovascular activity. In summary, we demonstrated that (1) the Pyr ¹ and Pro⁶ residues are essential for both, the antihypertensive and bradycardic effects of Bj-PRO-10c; (2) Ala-scan approach allowed dissociating blood pressure reduction and bradycardic effects. Conformational properties of the peptides were examined by means of circular dichroism (CD) spectroscopy. The different Ala-scan analogues caused either an increase or decrease in the type II polyproline helix content compared to Bj-PRO-10c. The complete loss of activity of the Pro⁶ → Ala ⁶ mutant is probably due to the fact that in the parent peptide the His⁵-Pro⁶ bond can exist in the cis configuration, which could correspond to the conformation of this bond in the bound state. Current data support the Bj-PRO-10c as a promising leader prototype to develop new agents to treat cardiovascular diseases and its co-morbidities.

Full text of DOI:10.1007/s00726-013-1630-x

Amino Acids (2014) 46:401–413
DOI 10.1007/s00726-013-1630-x
ORIGINAL ARTICLE
Insights into cardiovascular effects of proline-rich oligopeptide
(Bj-PRO-10c) revealed by structure–activity analyses: dissociation
of antihypertensive and bradycardic effects
•
Juliana F. B. Paschoal Juliana Yamaguchi Jose R. R. Miranda
•
•
´
Gustavo Carretero Robson L. Melo Robson A. S. Santos Carlos H. Xavier
•
•
•
•
•
Shirley Schreier Antonio C. M. Camargo Danielle Ianzer
•
Received: 19 September 2013 / Accepted: 25 November 2013 / Published online: 14 December 2013
Ó Springer-Verlag Wien 2013
Abstract We have previously reported that the proline-
rich decapeptide from Bothrops jararaca (Bj-PRO-10c)
causes potent and sustained antihypertensive and brady-
cardic effects in SHR. These activities are independent of
ACE inhibition. In the present study, we used the Ala-scan
approach to evaluate the importance of each amino acid
within the sequence of Bj-PRO-10c (Pyr1-Asn2-Trp3-Pro4-
His5-Pro6-Gln7-Ile8-Pro9-Pro10). The antihypertensive and
bradycardic effects of the analogues Bj-PRO-10c Ala3,
Bj-PRO-10c Ala7, Bj-PRO-10c Ala8 were similar to those
of Bj-PRO-10c, whereas the analogues Bj-PRO-10c Ala2,
Bj-PRO-10c Ala4, Bj-PRO-10c Ala5, Bj-PRO-10c Ala9,
and Bj-PRO-10c Ala10 kept the antihypertensive activity
and lost bradycardic activity considerably. In contrast,
Bj-PRO-10c Ala1 and Bj-PRO-10c Ala6 were unable to
provoke any cardiovascular activity. In summary, we dem-
onstrated that (1) the Pyr1 and Pro6 residues are essential for
both, the antihypertensive and bradycardic effects of Bj-
PRO-10c; (2) Ala-scan approach allowed dissociating blood
pressure reduction and bradycardic effects. Conformational
properties of the peptides were examined by means of cir-
cular dichroism (CD) spectroscopy. The different Ala-scan
analogues caused either an increase or decrease in the type II
polyproline helix content compared to Bj-PRO-10c. The
complete loss of activity of the Pro6 ? Ala6 mutant is
probably due to the fact that in the parent peptide the His5-
Pro6 bond can exist in the cis configuration, which could
correspond to the conformation of this bond in the bound
state. Current data support the Bj-PRO-10c as a promising
leader prototype to develop new agents to treat cardiovas-
cular diseases and its co-morbidities.
J. F. B. Paschoal and J. Yamaguchi should be considered as co-first
authors.
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-013-1630-x) contains supplementary
material, which is available to authorized users.
J. F. B. Paschoal ꢀ J. Yamaguchi ꢀ J. R. R. Miranda ꢀ
R. L. Melo ꢀ A. C. M. Camargo ꢀ D. Ianzer
Special Laboratory of Applied Toxinology—CAT/Cepid,
Butantan Institute, Av. Vital Brasil, 1500, Sao Paulo,
SP 05503-900, Brazil
J. F. B. Paschoal ꢀ J. Yamaguchi ꢀ J. R. R. Miranda
Department of Biochemistry, Federal University of Sao Paulo
(UNIFESP), Sao Paulo, SP, Brazil
Keywords Proline-rich oligopeptide ꢀ Hypertension ꢀ
Ala-scan approach ꢀ Arterial pressure ꢀ Heart rate ꢀ
Circular dicroism
G. Carretero ꢀ S. Schreier
Department of Biochemistry, Institute of Chemistry,
University of Sao Paulo (USP), Sao Paulo, SP, Brazil
Abbreviations
ACE
R. A. S. Santos ꢀ D. Ianzer (&)
Angiotensin-I converting enzyme
Laboratory of Hypertension, Federal University of Minas Gerais
(UFMG), Belo Horizonte, MG, Brazil
e-mail: daianzer@gmail.com
Ala-scan Alanine scan
AsS
Bj
Argininosuccinate synthetase
Bothrops jararaca
C. H. Xavier
Department of Physiological Sciences, Institute of Biological
´ ˆ
Sciences, Federal University of Goias (UFG), Goiania,
Bj-PRO
CD
Bothrops jararaca-proline-rich oligopeptides
Circular dichroism
HR
Heart rate
GO, Brazil
123

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J. F. B. Paschoal et al.
The present work aimed at assessing structure–activity
relationships of Bj-PRO-10c, using a complete alanine scan
(Ala-scan) approach (Cunningham and Wells 1989). In par-
ticular, we studied the cardiovascular parameters of SHR
treated with Bj-PRO-10c and Bj-PRO-10c Ala-scan ana-
logues. Conformational properties of the peptides were
examined by means of circular dichroism (CD) spectroscopy.
i.v.
Intravenous
MAP
MDLA
NO
Mean arterial pressure
Alpha-methyl-DL-aspartic acid
Nitric oxide
PAP
Pyr
Pulsatile arterial pressure
Pyroglutamic acid
SHR
Spontaneously hypertensive rat
Introduction
Materials and methods
Bothrops jararaca (Bj) venom contains a family of proline-
rich oligopeptides (PROs) that are able to potentiate the
activity of bradykinin (Ianzer et al. 2004). Besides being
proline-rich peptides, composed of 5–17 amino acid resi-
dues, these toxins present other structural similarities, such
as pyroglutamic acid and proline residues at N- and
C-terminal positions, respectively (Ianzer et al. 2004;
Zelanis et al. 2010).
Synthesis and purification of peptides
The syntheses of Bj-PRO-10c (Pyr1-Asn2-Trp3-Pro4-His5-
Pro6-Gln7-Ile8-Pro9-Pro10) and its analogues (Table 1)
were performed on an automated PSSM-8 peptide syn-
thesizer (Shimadzu Corp., Kyoto, Japan) by a stepwise
solid-phase method using N-9-fluorenylmethoxycarbonyl
(Fmoc) chemistry (Carmona and Juliano 1996). All Fmoc-
L-amino acids and resins were purchased from Merck
KGaA (Darmstadt, Germany). The following amino acids
were used: Fmoc-L-Ala-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-
L-Gln(Trt)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH,
Fmoc-L-Pro-OH, Fmoc-L-Trp(Boc)-OH, L-Pyr-OH; the
resins employed were Pro-2-Cl-Trt resin (0.45 mmol/g)
and Fmoc-Ala-Wang resin (0.67 mmol/g). Resins were
first conditioned for 20 min in DMF. The Fmoc-amino
acids were then added by a succession of deprotection and
coupling steps. Fmoc group removal was performed
through two consecutive treatments of 10 min each using
piperidine 20 % in DMF. Activation of Fmoc-protected
amino acids (fivefold of excess) was carried out in DMF in
the presence of five equiv of N,N,N0,N0-Tetramethyl-O-
(1H-benzotriazol-1-yl)uronium hexafluorophosphate and
six equiv 4-Methylmorpholine for 5 min following addi-
tion for coupling the deprotected resin for 30 min. After
each step, the resin was washed five times with DMF. Final
cleavage of peptides from the resin and side chain depro-
tections were achieved by treatment with a mixture of
trifluoroacetic acid (TFA) 87.5 %, 1,2-ethanedithiol 2.5 %,
water 5 %, phenol 2.5 % and thioanisole 2.5 % for 2 h at
room temperature. After removal of the resin by filtration
the crude synthetic peptides were precipitated with diethyl
ether and recovered after centrifugation. The crude syn-
thetic peptides were purified by preparative reversed-phase
high-performance liquid chromatography (HPLC) (Shi-
madzu Corp., Japan) on a YMC-Pack ODS column
20 9 150 mm (YMC, Kyoto, Japan), using a linear gra-
dient from 5 to 40 % acetonitrile in 0.1 % TFA, at a flow
rate of 8 ml/min. Buffer elution was removed by freeze
drying, followed by confirmation of identity and purity of
each synthetic peptide fraction by MALDI-TOF mass
spectrometry an Ettan MALDI-TOF/Pro instrument
It has been previously shown that Bj-PRO-10c is part of
the structure of the C-type natriuretic peptide precursor of
the brain and the venom gland of B. jararaca (Hayashi
et al. 2003; Murayama et al. 1997). Following i.v. injec-
tion, its biological effect includes a potent and long-lasting
reduction of the arterial pressure, accompanied by brady-
cardia. Surprisingly, the antihypertensive doses of Bj-PRO-
10c in the spontaneously hypertensive rats (SHRs) do not
affect the angiotensin I-converting enzyme (ACE) activity
in vivo (Ianzer et al. 2007), thus ruling out the classical
hypothesis that the inhibition of ACE by snake venom
peptides is responsible for its antihypertensive activity
(Cushman et al. 1973). Recent results from our laboratory
suggest that the cardiovascular effects of the Bj-PROs go
far beyond the inhibition of ACE (Camargo et al. 2012b).
The cardiovascular effects of Bj-PRO-10c in SHR
involve synergistic actions on the enzyme argininosucci-
nate synthetase (AsS) (Guerreiro et al. 2009) and other
unknown receptor(s) (Guerreiro et al. 2009; Ianzer et al.
2007; Camargo et al. 2012b). In the citrulline–nitric oxide
(NO) cycle, AsS is the rate-limiting enzyme to provide
L-arginine, a substrate for the NO synthesis through its
oxidation by the nitric oxide synthase (Flam et al. 2001;
Shen et al. 2005). In vitro, the Bj-PRO-10c activates AsS
and induces NO production in endothelial cells, whereas
in vivo it increases L-arginine plasmatic levels. The partial
reversion of the antihypertensive effect of Bj-PRO-10c by
the alpha-methyl-DL-aspartic acid (MDLA), a specific AsS
inhibitor, reinforces the NO-related mechanism (Guerreiro
et al. 2009). Biodistribution studies showed that Bj-PRO-
10c is also able to cross the blood brain barrier (Silva et al.
2008b), suggesting possible central actions, once it was
observed reduction in the locomotor activity (Ianzer et al.
2007, 2010).
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Insights into cardiovascular effects of proline-rich oligopeptide
403
Table 1 Maximal antihypertensive and bradycardic effects following i.v. injection of Bj-PRO-10c and Bj-PRO-10c Ala-scan analogues
(71 nmol/kg) in SHR and PPII helix content of Bj-PRO-10c and its Ala-scan analogues
Treatment
Amino acid sequence
Basal MAP
(mmHg)
D MAP
(mmHg)
Basal HR
(bpm)
D HR (bpm)
PPII
(%)
Vehicle
–
175
176
168
165
178
167
178
160
162
177
170
169
175
4
3
4
4
6
5
6
6
5
6
5
7
4
-13
1
361
8
-14
4
–
Bj-PRO-10c
Pyr-Asn-Trp-Pro-His-Pro-Gln-Ile-Pro-Pro
Ala-Asn-Trp-Pro-His-Pro-Gln-Ile-Pro-Pro
Pyr-Ala-Trp-Pro-His-Pro-Gln-Ile-Pro-Pro
Pyr-Asn-Ala-Pro-His-Pro-Gln-Ile-Pro-Pro
Pyr-Asn-Trp-Ala-His-Pro-Gln-Ile-Pro-Pro
Pyr-Asn-Trp-Pro-Ala-Pro-Gln-Ile-Pro-Pro
Pyr-Asn-Trp-Pro-His-Ala-Gln-Ile-Pro-Pro
Pyr-Asn-Trp-Pro-His-Pro-Ala-Ile-Pro-Pro
Pyr-Asn-Trp-Pro-His-Pro-Gln-Ala-Pro-Pro
Pyr-Asn-Trp-Pro-His-Pro-Gln-Ile-Ala-Pro
Pyr-Asn-Trp-Pro-His-Pro-Gln-Ile-Pro-Ala
Glu-Asn-Trp-Pro-His-Pro-Gln-Ile-Pro-Pro
-30 3*
-13 2#
-26 3*
-24 2*
-24 4*
-23 4*
-15 2#
-24 2*
-27 4*
-25 4*
-30 1*
-10 6#
367 10
354 10
360 10
367 11
-71 12*
-18 5#
-32 3*, #
-52 12*
-36 3*, #
-44 7*
-27 15#
-43 7*, #
-57 11*
-40 7*, #
-34 9*, #
-39 8 *, #
46
47
48
33
32
55
58
49
60
47
42
nd
Bj-PRO-10c Ala1
Bj-PRO-10c Ala2
Bj-PRO-10c Ala3
Bj-PRO-10c Ala4
Bj-PRO-10c Ala5
Bj-PRO-10c Ala6
Bj-PRO-10c Ala7
Bj-PRO-10c Ala8
Bj-PRO-10c Ala9
Bj-PRO-10c Ala10
Bj-PRO-10c Glu1
357
8
360 10
358 15
356
9
369 17
369 26
357 13
353
5
The maximal changes in MAP and HR were sampled after i.v. injection in each experiment, at the time point they occurred. Data are expressed as
mean SEM, n = 5–7. Bold represents the replacement of the original amino acid residue by Ala
Pyr pyroglutamic acid, nd not determined
* p \ 0.05 compared with vehicle
#
p \ 0.05 compared with Bj-PRO-10c
In vivo assays: blood pressure recording in rats
(Amersham Biosciences, Uppsala, Sweden) and by ana-
lytical, Finnigan Surveyor MSQ Plus Single Quadrupole
LC/MS (Thermo Finnigan, San Jose, CA) and reversed-
phase HPLC in two different solvent systems. Samples
were frozen and then freeze dried (Edwards Freeze Dryer
Super Modulyo Pirani 1001, Thermo Fisher Scientific,
Waltham, MA) for 48 h at -50 °C under vacuum. The
Bj-PRO-10c and its Ala-scan analogues were used with
purity higher than 95 % and dissolved in saline (0.9 %
NaCl) just before use.
Animals
Experiments were carried out in male SHRs (280–350 g)
from CEBIO, University of Sa˜o Paulo. The animals were
bred at the animal facility of the Special Laboratory of
Applied Toxinology, Butantan Institute. The animals had
free access to food and water and were submitted to a
light–dark cycle (12 h each) before the preparation for the
experiments. All animals were handled under ethical con-
ditions according to international rules of animal care,
stated by the International Animal Welfare Recommenda-
tions, and in accordance with the guidelines established by
(1) our local institutional animal welfare committee
(CEUAIB/IBu, protocol 520/08 and CEP/UNIFESP, pro-
tocol 0934/09); (2) EU Directive 2010/63/EU for animal
experiments and (3) Uniform requirements for manuscripts
submitted to biomedical journals.
Circular dichroism (CD) spectroscopy
CD spectra were acquired at room temperature in a Jasco
J-720 spectropolarimeter. Samples were placed in 0.1 mm
optical length quartz cells. The final spectra resulted from
the average of six scans, after subtracting the spectrum
obtained under the same conditions of a sample without
peptide. The peptides, whose concentration ranged
between 160 and 205 lM, were dissolved in isotonic sal-
ine. Except for Bj-PRO-10c Ala3, whose concentration was
determined by weighing, the peptides concentration was
determined spectrophotometrically by measuring the
absorption of tryptophan (e280 = 5,690 M-1 ꢀ cm-1). CD
spectra were scanned from 190 to 260 nm at 50 nm ꢀ s21
using a 2-nm slit. The CD spectra are reported as mean
Arterial pressure measurements
The cardiovascular parameters, pulsatile arterial pressure
(PAP), mean arterial pressure (MAP) and heart rate (HR)
were monitored by a solid-state strain gauge transducer
connected to a computer through a data acquisition system
(MP 100; BIOPAC Systems Inc., USA). The PAP, MAP
residue ellipticity ([h]) in degrees cm2 ꢀ dmol-1
.
123

404
J. F. B. Paschoal et al.
and HR were monitored jointly during experiments in
different monitor channels and recorded in the computer
hard disk for later analysis.
Figures 1 and 2 present the changes in arterial pressure
and heart rate, respectively, produced by the i.v.
bolus injection of Bj-PRO-10c and Ala-scan analogues
(71 nmol/kg). There were slight changes in MAP and HR
following injection of vehicle which can be attributed to
the fact that the experimental procedure was performed in
free-behaving animals (Figs. 1a, 2a). In these cases, it is
not unlike to observe discrete variations in the cardiovas-
cular parameters during similar experimental conditions
(Ianzer et al. 2007).
The assays for evaluate the effects of Bj-PRO-10c ana-
logues on cardiovascular parameters of SHR were per-
formed as described by Ianzer et al. (Ianzer et al. 2007).
Briefly, 20 h before the experiment, under anesthesia with
0.05 ml of 10 % ketamine and 2 % xylazine (1:1) intra-
peritoneally, a polyethylene catheter (PE-10 connected to
PE-50) was introduced into the abdominal aorta through a
femoral artery for measurements of cardiovascular
parameters and into a femoral vein for intravenous (i.v.)
injection. After recovery from anesthesia, the rats were
kept in individual cages with free access to water and chow
until the end of the experiments.
As expected and previously described (Ianzer et al.
2007), Bj-PRO-10c caused potent and long-lasting antihy-
pertensive and bradycardic effects in SHR. Changes in
MAP were evident after 15 min, achieving amplitudes that
ranged between -20 and -27 mmHg after 180 min of
administration. These changes were maintained until the
end of the observation period (360 min) (Fig. 1b). HR falls
were seen from 25 min past injection and reached a sus-
tained bradycardia until the end of the experiment,
achieving -52 bpm 270 min following i.v. injection
(Fig. 1b). The monitoring of cardiovascular parameters of
SHR following i.v. injection of the Bj-PRO-10c Ala-scan
analogues (71 nmol/kg) showed that most of these peptides
were able to reduce MAP and HR. However, the ampli-
tudes of the antihypertensive and bradycardic effects were
different for each peptide (Figs. 1, 2).
The cardiovascular parameters were monitored for 1 h
before drug administration (baseline period). After this
period, i.v. bolus injection of Bj-PRO-10c and its Ala-scan
analogues (71 nmol/kg) or vehicle (NaCl 0.9 %) in a total
volume of 0.5 ml was made (n = 5–7). The cardiovascular
parameters were monitored continuously for 6 h after drug
administration. MAP and HR values were sampled for 30-s
periods every 5 min during the entire recording period. The
choice of dose (71 nmol/kg) was based on the effects of
Bj-PRO-10c found in a dose–response curve (0.47–71
nmol/kg) in SHR (Ianzer et al. 2007).
Comparisons with the group injected with vehicle
revealed that the Bj-PRO-10c Ala1 did not significantly
alter the MAP and HR of SHR (Figs. 1c, 2c). The Bj-PRO-
10c Ala2 caused gradual reduction in MAP, reaching
-25 mmHg after 200 min of administration, which was
sustained until the end of the recording (Fig. 1d). Bj-PRO-
10c Ala2 caused a slight reduction in HR. The bradycardic
effect (about -20 bpm) was clear 75 min following i.v.
injection, remaining stable until the end of the experi-
mental period (Fig. 2d).
Statistical analysis
The time-course changes were calculated from the differ-
ence between basal average value (5 min average sampled
immediately before i.v. injection) and value of every 5 min
post injection time. The maximal changes in MAP and HR
were sampled after i.v. injection in each experiment, at the
time point they occurred.
Comparisons were made by Student’s unpaired t test or
two-way ANOVA with Bonferroni post-test when appro-
priate. GraphPad Prism 5.0, GraphPad Software, Incorpo-
ration software program (USA) was used in all statistical
analysis. The criterion for statistical significance was set at
p \ 0.05.
The time-course of MAP changes evoked by Bj-PRO-10c
Ala3 was significantly different from the one elicited by the
vehicle (Fig. 1e). In addition, Bj-PRO-10c Ala3 slightly
reduced the heart rate. The average reduction reached
-40 bpm, close to end of experimental period (Fig. 2e).
The antihypertensive effect of the Bj-PRO-10c Ala4
followed a different time course. In general, the changes
caused by Bj-PRO-10c were evident after 15 min. Other-
wise, the reduction in MAP caused by Bj-PRO-10c Ala4
was late, starting around 180 min after injection. The
amplitude of the Bj-PRO-10c Ala4 effects on MAP reached
approximately -17 mmHg 260 min following i.v. injec-
tion, which was kept until the end of the record (Fig. 1f).
Despite causing a negative chronotropy smaller than that
evoked by Bj-PRO-10c, the Bj-PRO-10c Ala4 caused a
sustained bradycardia (-25 bpm), also starting at 180 min
(Fig. 2f).
Results
Evaluation of cardiovascular parameters
In order to investigate the role and the importance of each
amino acid residue of the Bj-PRO-10c sequence for the
cardiovascular effects in SHR, we performed an Ala-scan
strategy, in which each residue was systematically replaced
by the small and neutral amino acid, alanine (Alana et al.
2006; Quartara et al. 2000; Corzo et al. 2007).
123

Insights into cardiovascular effects of proline-rich oligopeptide
405
Fig. 1 Time course of MAP changes after administration of Bj-PRO-10c and Bj-PRO-10c Ala-scan analogues (71 nmol/kg) in SHR. The data
are presented as mean SEM, n = 5–7
As shown in Fig. 1g, the antihypertensive effect of Bj-
PRO-10c Ala5 was gradual after 5 min, decreasing to about
-20 mmHg close to the end of experimental period. The
Bj-PRO-10c Ala5 caused slight and transient bidirectional
chronotropic effects during the entire experimental period
(Fig. 2g).
123

406
J. F. B. Paschoal et al.
Fig. 1 continued
Similar to Bj-PRO-10c Ala1, the Bj-PRO-10c Ala6 was
not able to cause important changes in MAP and in HR
when compared with vehicle (Figs. 1h, 2h).
The Bj-PRO-10c Ala7 caused a gradual decrease in
MAP reaching approximately -20 mmHg at 235 min,
which was kept until 360 min (Fig. 1i). This peptide also
promoted a bradycardic effect. The HR reductions were
evident immediately after injection and reached -35 bpm
at 325 min (Fig. 2i).
The antihypertensive effect evoked by Bj-PRO-10c Ala8
was gradual and an average of -22 mmHg was reached at
290 min (Fig. 1j). The Bj-PRO-10c Ala8 caused marked
bradycardia (about -44 bpm), likely initiated 5 min fol-
lowing injection. This effect was observed throughout the
experimental period (Fig. 2j).
The changes in MAP after Bj-PRO-10c Ala9 injection
reached peak values at about 150 min and lasted until the
end of the experimental period (Fig. 2k). The bradycardia
caused by the Bj-PRO-10c Ala9 was discrete and oscilla-
tory, as that observed for Bj-PRO-10c Ala5 (Fig. 2k).
The antihypertensive effect observed after Bj-PRO-10c
Ala10 injection displayed a gradual and stable reduction in
MAP until the end of experimental period, reaching max-
imal averages of -27 mmHg at 270 min (Fig. 1l). In
contrast to other Bj-PRO-10c Ala-scan peptides, Bj-PRO-
10c Ala10 caused transient bidirecional chronotropic
effects. Figure 2l shows the time-course of the effects
evoked by Bj-PRO-10c Ala10 on HR. The fast positive
chronotropy is noteworthy from the fifth minute, showing a
variable profile until 245 min after injection. However,
from the 250-min time point, a small bradycardic effect
was predominant and persisted until the end of the exper-
imental period.
Table 1 presents the mean maximal changes in cardio-
vascular parameters (MAP and HR) caused by the
administration of Bj-PRO-10c and its Ala-scan analogues.
Besides the Bj-PRO-10c Ala-scan analogues, Glu replace-
ment at position 1 (Bj-PRO-10c Glu1) was assessed to
verify whether Pyr is a key residue for Bj-PRO-10c. Bj-
PRO-10c Glu1 did not cause any antihypertensive effect.
Compared to Bj-PRO-10c, the analogue Bj-PRO-10c Glu1
evoked a smaller effect upon HR.
The maximum changes following i.v. administration of
Bj-PRO-10c and analogues at the dose of 71 nmol/Kg
revealed the following: (1) the replacement of pyroglu-
tamic acid and proline residues at positions 1 and 6 (Bj-
PRO-10c Ala1 and Bj-PRO-10c Ala6), respectively, deter-
mined the lack of both antihypertensive and bradycardic
effects; (2) maximal changes in blood pressure caused by
the analogues Bj-PRO-10c Ala2, Bj-PRO-10c Ala3, Bj-
PRO-10c Ala4, Bj-PRO-10c Ala5, Bj-PRO-10c Ala8, Bj-
PRO-10c Ala9 and Bj-PRO-10c Ala10 were statistically
similar than the positive control, Bj-PRO-10c; (3) Bj-PRO-
10c Ala3, Bj-PRO-10c Ala7 and Bj-PRO-10c Ala8 accom-
plished a maximal bradycardia statistically similar to that
found for Bj-PRO-10c, whereas Bj-PRO-10c Ala2, Bj-
123

Insights into cardiovascular effects of proline-rich oligopeptide
407
Fig. 2 Time course of HR changes after administration of Bj-PRO-10c and Bj-PRO-10c Ala-scan analogues (71 nmol/kg) in SHR. The data are
presented as mean SEM, n = 5–7
PRO-10c Ala4, Bj-PRO-10c Ala5, Bj-PRO-10c Ala9 and
Bj-PRO-10c Ala10 presented statistical difference in the
range and direction of HR changes when compared with
Bj-PRO-10c; and (4) none of Ala-scan analogues was more
effective than the Bj-PRO-10c for both antihypertensive
and bradycardic effects.
Some Ala-scan analogues modified the standard
response evoked by Bj-PRO-10c (Table 1). Compared with
123

408
J. F. B. Paschoal et al.
Fig. 2 continued
Bj-PRO-10c (100 %), the active analogues Bj-PRO-10c
Ala10, Ala8, Ala2, Ala9, Ala3, Ala7, Ala4 and Ala5 reached,
respectively, 100, 90.0, 86.7, 83.3, 80.0, 80.0, 80.0 and
76.7 % of the antihypertensive effect of Bj-PRO-10c.
Comparisons of maximal HR reduction showed that the
analogues Bj-PRO-10c Ala8, Ala3, Ala5, Ala7, Ala9, Ala4,
Ala10 and Ala2 reached, respectively, 80.3, 73.2, 62.0, 60.6,
56.3, 50.7, 47.9 and 45.1 % of the bradycardic effect of Bj-
PRO-10c. The time-course of the chronotropic effect was
also substantially altered when compared with Bj-PRO-10c
profile (Fig. 2).
negative peak between 200 and 203 nm and a less intense
positive peak between 226 and 228 nm (Fig. 3). In con-
trast, the spectra of Bj-PRO-10c-Ala3, Bj-PRO-10c-Ala4,
and Bj-PRO-10c-Ala10 did not present a positive peak
between 226 and 228 nm; instead, a ‘‘shoulder’’ was
observed in this region (Fig. 4).
The intensity of the positive peak in the 226–228 nm
region varied for the different peptides (Fig. 3). In order to
compare their conformational properties, we used the
equation given by (Kelly et al. 2001) to calculate the per-
centage of PPII helix content in the CD spectra:
%PPII = 100 ([h]MAX
´
? 6,100)/13,700, where [h]MAX is
´
Conformational studies
the maximum value of the molar ellipticity in the
226–228 nm region.
In order to investigate structure–activity relationships, the
peptide conformation was examined in solution (saline) by
means of CD spectroscopy. In solution, very likely, an
equilibrium exists between different conformational states;
it is usually assumed that, upon binding to a ‘‘receptor’’,
this equilibrium will be shifted towards the conformation
that corresponds to the bound state. As expected for pro-
line-rich oligopeptides, the CD spectra of the native pep-
tide and of several of its Ala-scan analogues (Bj-PRO-10c-
Ala1, Bj-PRO-10c-Ala2, Bj-PRO-10c-Ala5, Bj-PRO-10c-
Ala6, Bj-PRO-10c-Ala7, Bj-PRO-10c-Ala8, and Bj-PRO-
10c-Ala9) present features characteristic of type II poly-
proline helix (PPII). These spectra displayed more intense
To extend this analysis to all peptides, the equation was
also applied to the spectra of the analogues Bj-PRO-10c-
Ala3, Bj-PRO-10c-Ala4, and Bj-PRO-10c-Ala10. Since the
positive peak in the 226–228 nm region was absent in these
spectra, the values of [h] at 227 nm were used for the
calculation.
As seen in Table 1, the spectrum of Bj-PRO-10c cor-
responds to 46 % PPII helix. Similar PPII helical contents
were found for Bj-PRO-10cAla1 (47 %), Bj-PRO-10c-Ala2
(48 %), Bj-PRO-10c-Ala7 (49 %), and Bj-PRO-10c-Ala9
(47 %). For the analogues, Bj-PRO-10c-Ala5, Bj-PRO-10c-
Ala6, and Bj-PRO-10c-Ala8 the PPII helix content was
increasingly higher (55, 58, and 60 %, respectively). The
123

Insights into cardiovascular effects of proline-rich oligopeptide
409
Fig. 3 CD spectra of Bj-PRO-10c and Bj-PRO-10c Ala-scan ana-
logues Bj-PRO-10c Ala1, Bj-PRO-10c Ala2, Bj-PRO-10c Ala5,
Bj-PRO-10c Ala6, Bj-PRO-10c Ala7, Bj-PRO-10c Ala8, and Bj-
PRO-10c Ala9 in saline solution. a full spectra, run between 195 and
260 nm; right, b expansion of the 220–240 nm region
Fig. 4 CD spectra of Bj-PRO-10c and Bj-PRO-10c Ala-scan analogues Bj-PRO-10c Ala3, Bj-PRO-10c Ala4 and Bj-PRO-10c Ala10 in saline
solution. a full spectra, run between 195 and 260 nm; b expansion of the 220–240 nm region
PPII helix contents for Bj-PRO-10c-Ala3, Bj-PRO-10c-
Ala4, and Bj-PRO-10c-Ala10 were 33, 32, and 42 %,
respectively.
the choice of the strain (SHR) and dose (71 nmol/kg) used
in the present study. In SHR, the biological activity of
Bj-PRO-10c includes a sustained reduction in MAP
(-30 %) and in HR (-17 %). In normotensive rats, only a
slight reduction in blood pressure was detected after i.v.
injection of Bj-PRO-10c (71 nmol/kg) (Ianzer et al. 2007).
The Ala-scan strategy (Cunningham and Wells 1989)
applied in the present work does not intend to define the
pharmacophore of Bj-PRO-10c as suggested by Peter Gund
(Gund 1977), since this approach is usually applied when
an identified target (receptor or enzyme) is involved in
some biological effect (Alana et al. 2006; Corzo et al.
2007; Quartara et al. 2000). Obviously, the structure–
activity relationship of Bj-PRO-10c depends upon multi-
factorial systems, which regulate the blood pressure and
heart rate (Camargo et al. 2012b).
Discussion
The primary structure of Bj-PRO-10c (Pyr1-Asn2-Trp3-
Pro4-His5-Pro6-Gln7-Ile8-Pro9-Pro10) was found in the
C-Type natriuretic peptide precursor in the venomous
gland of B. jararaca (Murayama et al. 1997). A very
similar protein precursor, containing the Bj-PRO-10c, was
found in the neuroendocrine area of the B.jararaca brain,
suggesting a possible role for this peptide in cardiovascular
homeostasis and hydroelectrolytic balance of the animal
(Hayashi et al. 2003).
The different cardiovascular effects evoked by Bj-PROs
in normotensive and hypertensive rats, as a function of the
administered doses (Ianzer et al. 2007, 2011), determined
In general, for a specific biological activity, peptides must
present a conformation that aligns essential functional
groups in a required spatial orientation. It is important to
123

410
J. F. B. Paschoal et al.
emphasize that proline (Pro) plays a distinct role in the
structure of peptides/proteins (Biedermannova et al. 2008).
The side-chain of proline is cyclized back on to the backbone
amide position, which providing rigidity and a consider-
ably restriction in its conformational freedom compared to
other amino acids (Machado et al. 1993; Moradi et al. 2010).
This gives support to data presented here where the structural
changes resulted from the replacement of Pro residues by Ala
at specific position of the Bj-PRO-10c sequence manifest
either through the extent or the nature of biological effects
are affected.
branched residue Ile8 disfavors the PPII helix, the
replacement of this residue by Ala considerably increased
the PPII helix content since this amino acid strongly favors
this conformation (Table 1).
In spite of the similar cardiovascular effects, the con-
formation of Bj-PRO-10c Ala3 differs from that of the
native Bj-PRO-10c. Although tryptophan (Trp) at position
3 weakly contributes to reduce MAP and HR, this amino
acid could be important for an orally active prototype
formulation. In fact, it has been shown that the side chain
of Trp is required for complex formation between the
native decapeptide and b-cyclodextrin (De Sousa et al.
2010). Cyclodextrins are widely used as strategy for
pharmaceutical formulations, because a peptide–cyclo-
dextrin conjugate can survive passage through the stomach
and the small intestine (Uekama et al. 1998; Goldberg and
Gomez-Orellana 2003).
Comparisons of the time-course profile and peak
responses showed that the original proline-rich decapep-
tide, Bj-PRO-10c, was more potent than all generated Ala-
scan peptides in changing the cardiovascular parameters.
The great majority of the individual replacements of amino
acid residues in the Bj-PRO-10c sequence culminates in
reduction or lack of the Bj-PRO-10c effects. To better
understand the biological results, conformational studies
were carried out. It is important to bear in mind that the
conformational studies were performed in solution; there-
fore, the conformations at eventual binding sites were not
trapped. Some points were considered when analyzing the
effect of Ala substitution on peptide conformation: (1) the
propensity of the different amino acids to favor PPII helix
conformation, based on the scale established by Rucker
et al. (Rucker et al. 2003); (2) the fact that aromatic amino
acids tend to favor the cis conformer of proline, thereby
being unfavorable to PPII helix conformation, which
requires Pro in the trans conformation (Rucker et al. 2003).
In this context, it is noteworthy that the aforementioned
factors can account for the observed increase in the PPII
helix content of Bj-PRO-10c-Ala5, Bj-PRO-10c-Ala6, and
Bj-PRO-10c-Ala8.
Group 2—Amino acid residues responsible for the
bradycardic effect: Bj-PRO-10c Ala2, Bj-PRO-10c Ala4,
Bj-PRO-10c Ala5, Bj-PRO-10c Ala9, and Bj-PRO-10c
Ala10 are the peptides which lost part of the ability of Bj-
PRO-10c to cause a remarkable bradycardia. Moreover, the
time-course of the effects also revealed different features.
For example, there was an oscillatory HR profile after i.v.
injection of Bj-PRO-10c Ala4 and Bj-PRO-10c Ala5.
Strikingly, Bj-PRO-10c Ala10 initially evoked slight
tachycardia. Following, a reduction in heart rate was only
observed at end of the experimental period (approximately
4 h after i.v. injection).
No straightforward relationship between structure and
activity was evident for all Ala-scan analogues of this group.
Weak evidence could be suggested, for instance, for the
increase in PPII helix content. It could be related to the
decrease in DHR Bj-PRO-10c Ala2 and Bj-PRO-10c Ala5.
However, better evidence is likely to occur for the replace-
ments of Pro4 and Pro10 by Ala due to the diminishment of
PPII helix content of the resulting peptides. This structural
modification could explain why Ala4 and Bj-PRO-10c Ala10
specifically affect the bradycardic effect observed for the
native peptide. Thus, the presence of proline at positions 4
and 10 of Bj-PRO-10c seems to be crucial for the peptides
activities, suggesting that the ability of these residues to
undergo cis–trans isomerization plays a key role in the
activity of Bj-PRO-10c. In the bound state, the result sug-
gests that the prolinesin the C-terminus of Bj-PRO-10c could
be in the trans configuration. Current data are in agreement
with a previous study that showed a significant conforma-
tional role for the C-terminal prolyl-proline sequence in the
activity of Bj-PRO-9a (Marlborough et al. 1981).
Regarding the maximal cardiovascular effects, we
divided the Ala-scan peptides into three groups, as follows:
Group 1—Amino acid residues that poorly contribute to
the cardiovascular effects: tryptophan at position 3, glu-
tamine at position 7, and isoleucine at position 8 seem to
provide minor contributions to the cardiovascular effects of
Bj-PRO-10c. Despite slight reduction in HR evoked by Bj-
PRO-10c Ala3, Bj-PRO-10c Ala7, and Bj-PRO-10c Ala8,
the time course profile of changes in cardiovascular
parameters and the maximal peak changes in MAP and HR
evoked by Bj-PRO-10c Ala3, Bj-PRO-10c Ala7, and Bj-
PRO-10c Ala8 were statistically similar to those evoked by
Bj-PRO-10c (p = 0.06–0.28). Accordingly, conforma-
tional studies showed that the Ala-scan analogues from this
group showed different PPII helix content when compared
with native peptide. The replacement of Gln7 did not alter
very strongly the PPII helical content. The replacement of
Ile8 for Ala did not alter the bradycardic effect compared
with the native peptide. Although in the native peptide the
Taken together, the results allow us to suggest dissoci-
ation between the effects on blood pressure and on HR
elicited by Bj-PRO-10c. This dissociation was especially
clear, for example, for Bj-PRO-10c Ala10. Considering that
123

Insights into cardiovascular effects of proline-rich oligopeptide
411
Bj-PRO-10c is able to cross the blood–brain barrier (Silva
et al. 2008b), it is worth hypothesizing that the described
heart rate changes caused by Bj-PRO-10c Ala10 and other
analogues of this group may reflect their difficulty to
achieve or to act on the CNS. If this hypothesis proves to
be correct, we might support that the bradycardic effect of
Bj-PRO-10c resulted from a specific central activity of this
peptide. In fact, unloading baroreceptors during an anti-
hypertensive effect, a tachycardia would be expected
(Bunag et al. 1975). Altogether, the residues Asn2, Pro4,
His5, Pro9, and Pro10 are part of an important set of resi-
dues related not only to the vascular but also to the cardiac
effects of Bj-PRO-10c.
The present study allowed us to identify that Bj-PRO-
10c displays two independent cardiovascular activities
(MAP and HR), which was dissociated when distinct Ala-
scan analogues were applied. A second important finding
was that the Pyr1 and Pro6 residues are essential for both
antihypertensive and bradycardic effects in SHR.
Bj-PROs are able to evoke a diverse spectrum of biological
activities (Camargo et al. 2012b). In particular, the biological
activities described for the Bj-PRO-10c include bradykinin
potentiation (Ianzer et al. 2007), inhibition of ACE (Hayashi
et al. 2003), activation, increase in NO and L-Arg production
(Guerreiro et al. 2009), which may modulate the action at the
central and the peripheral nervous system. The hypothesis that
a number of signaling events could happen due to promiscu-
ousinteractionsofthesepeptideswithdomain-bindingsitesof
proteins [PDZ domains, for instance—PDZ domains are
abundant protein interaction modules that often recognize
short amino acid motifs at the C-terminal of target proteins
(Lee and Zheng 2010)], should be considered. In fact, a large
number of biological activities are likely to occur upon
interaction between a particular region in one protein and a
short peptide stretch (Neduva and Russell 2006).
Group 3—Relevant amino acid residues: Bj-PRO-10c
Ala1 and Bj-PRO-10c Ala6 were completely inactive. Since
the CD spectrum of Bj-PRO-10c Ala1 did not differ from Bj-
PRO-10c, the inactivity of Bj-PRO-10c Ala1 could be
explained by its susceptibility to hydrolysis by vascular
aminopeptidases. Peptides are susceptible to proteolytic
degradation in the blood and/or some organs as liver, kid-
neys, and gastrointestinal tract (Camargo et al. 2012a).
However, some Bj-PROs are not susceptible to enzymatic
proteolysis (Silva et al. 2008a, b), which is due, at least in
part, to the presence of pyroglutamate (Pyr) residue at
N-terminal, which can provide metabolic protection against
aminopeptidase action (Isaac et al. 2009). In this regard,
besides the Ala scan approach, we tested Glu replacement
(Bj-PRO-10c Glu1) to ensure whether Pyr is a key residue for
Bj-PRO-10c. The complete lack of antihypertensive effect as
well as a weak bradycardia found in animals treated with
Bj-PRO-10c Glu1 (Table 1) confirmed that cyclization/
blockade of N-terminal conferred by Pyr is essential for
achieving cardiovascular effects of Bj-PRO-10c.
Conclusion
The presented manipulations in the primary structure of the
Bj-PRO-10c allowed improving the knowledge about this
molecule. It showed that Bj-PRO-10c may be a leader
prototype to develop at least two new agents, specifically
driven to treat either specific targets, such as high blood
pressure or tachycardia.
An entirely different reason might explain the absence of
biological activity of Bj-PRO-10c Ala6. It could be related to
the critical changes in structural conformation from the
native peptide. This is further confirmed by the conforma-
tional analysis of Bj-PRO-10c Ala6, which differed from Bj-
PRO-10c. The occurrence of the cis-Pro configuration has
been mostly described for Trp-Pro and Tyr-Pro sequences; it
seems that in Bj-PRO-10c the His5-Pro6 exists preferentially
in this configuration. The replacement of Pro6 by Ala leads to
a great increase in the content of PPII helix. The energy
barrier for cis–trans isomerization of peptide bonds is much
lower for the imino acid Pro than for the other amino acids.
Therefore, replacing Pro6 by Ala would favor an increase in
PPII content, supporting the notion that the His5-Pro6
sequence exists preferentially in the cis configuration. In
such configuration, -X-Ar-Pro-X- sequences (where Ar =
aromatic) give rise to type VI b-turns (Meng et al. 2006;
Thomas et al. 2006). The data suggest that the cis configu-
ration is required at the binding site(s) for both MAP and HR
activities.
Acknowledgments The majority of the in vivo study was used as
part of the requirements for a master’s degree by J.F.B. Paschoal.
This research was supported by Grants provided by Fundac¸a˜o de
`
Amparo a Pesquisa do Estado de Sa
˜o Paulo (CAT/Cepid-FAPESP,
98/14307-9), Conselho Nacional de Desenvolvimento Cientıfico e
´
´
Tecnologico (CNPq) and Coordenac¸a˜o de Aperfeic¸oamento de Pes-
soal de Nıvel Superior (CAPES), Edital Toxinologia—n. 63/2010,
´
AUXPE 1593/2011. G.P.B.C. is recipient of CNPq PhD fellowship.
S.S. is recipient of CNPq research fellowship. The authors would like
´
to acknowledge Beatriz L. Fernandes for critical review, Maria Jose
da Silva and Isaıas Franc¸a da Silva for secretarial assistance, and Vera
´
Pontieri for technical assistance.
Confict of interest The authors declare that they have no conflict of
interest.
Author Contributions Participated in research design: A.C.M.
Camargo and D. Ianzer; Conducted experiments: J.F.B. Paschoal, J.
Yamaguchi, J.R.R. Miranda, C.H. Xavier, G. Carretero and D. Ian-
zer; Contributed new reagents or analytic tools: R.L. Melo, R.A.S.
Santos and S. Schreier; Performed data analysis: D. Ianzer, C.H.
Xavier, G. Carretero and S. Schreier; Wrote or contributed to the
writing of the manuscript: C.H. Xavier, G. Carretero, S. Schreier,
A.C.M. Camargo, and D. Ianzer.
123

412
J. F. B. Paschoal et al.
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