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Effects of Low Molecular Weight Sulfated

Galactan Fragments From Botryocladia
Occidentalis on the Pharmacological...

Article in The Protein Journal November 2010

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Protein J (2010) 29:567571
DOI 10.1007/s10930-010-9294-9

BRIEF COMMUNICATION

Effects of Low Molecular Weight Sulfated Galactan Fragments

From Botryocladia Occidentalis on the Pharmacological
and Enzymatic Activity of Spla2 From Crotalus Durissus
Cascavella
M. H. Toyama D. O. Toyama V. M. Torres
G. C. Pontes W. R. L. Farias F. R. Melo S. C. B. Oliveira

F. H. R. Fagundes E. B. S. Diz Filho B. S. Cavada

Published online: 9 November 2010

Springer Science+Business Media, LLC 2010

Abstract Low molecular weight fragments of sulfated Abbreviations

galactans (Boc-5 and Boc-10) from the red algae sPLA2 Secretory phospholipasis A2
Botryocladia occidentalis significantly inhibited Crotalus Boc Sulfated galactan fragment of
durissus cascavella sPLA2 enzymatic activity. Equimolar Botryocladia occidentalis
ratios of sPLA2 to Boc-5 or Boc-10 resulted in allosteric B. occidentalis Botryocladia occidentalis
inhibition of sPLA2. Under the conditions tested, we C. durissus cascavella Crotalus durissus cascavella
observed that both Boc-5 and Boc-10 strongly decreased SDSPAGE sodium dodecyl sulfate
edema, myonecrosis, and neurotoxicity induced by native polyacrylamide gel
sPLA2. electrophoresis
Maldi-Tof Matrix-assisted laser desorption/
Key words Botryocladia occidentalis  Crotalus ionization, time-of-flight mass
durissus cascavella  Secretory phospholipase spectrometer
A2 (sPLA2)  Edema  Myonecrosis CD Circular dichroism
HPLC High-performance liquid
chromatography or high-pressure
liquid chromatography
[14C] 5-HT [14C] 5-hydroxytryptamine
M. H. Toyama  S. C. B. Oliveira  F. H. R. Fagundes  CK Creatine kinase
E. B. S. Diz Filho
UNESP, Campus do Litoral Paulista, Sao Vicente, Brasil

D. O. Toyama
Universidade Presbiteriana Mackenzie, Sao Paulo, Brasil
V. M. Torres  G. C. Pontes  W. R. L. Farias
Departamento de Engenharia de Pesca and/BioMol/BioMar,
UFC, Fortaleza, Ceara, Brasil
F. R. Melo
Hospital Universitario Clementino Fraga Filho (HUCFF)/UFRJ,
Rio de Janeiro, Brasil
S. C. B. Oliveira  F. H. R. Fagundes  E. B. S. Diz Filho (&)
Departamento de Bioqumica Inst. Biologia, Unicamp
Campinas, Sao Paulo, Brasil
e-mail: eduardodizfilho@gmail.com
B. S. Cavada
Departamento de Bioqumica, UFC, Sao Paulo, Brasil
Sulfated polysaccharides are involved in several biological
processes due to their interaction with, and inhibition of,
proteins [25]. Sulfated polysaccharides are found in marine
red algae (Rhodophyta), invertebrates, and sea grass [3]
and [18]. Boc, a sulfated galactan from the red algae
Botryocladia occidentalis (Boc) collected from the Bra-
zilian coast, has been characterized by Farias et al. [5]. Boc
is hydrolyzed to give low molecular weight fragments of
5 kDa (Boc-5) and 10 kDa (Boc-10).
Phospholipase A2 enzymes (PLA2s) are found in high
concentrations in mammalian pancreatic fluids and snake
venoms. Snake venom PLA2s usually possess varying
degrees of enzymatic and toxic activity: some toxic
activities are connected with hydrolytic activity, which

123
568
hydrolyses the fatty acid from the sn-2 position of the
phospholipid, while others (such as Lys49 myotoxins) are
not. Pharmacological and biological activities of PLA2s
seem to be largely dependent on catalysis, but also rec-
ognition of the pharmacological site is also very important
[24]. The pharmacological activities of snake venom
PLA2s vary widely and include neurotoxic, cardiotoxic,
hemolytic, myonecrotic, anticoagulant, convulsant, hypo-
tensive, contractile, and edema-inducing activities [16]
and [22].
Here, we investigated the ability Boc-5 and Boc-10 to
interact with and modulate the catalytically active PLA2
enzyme from C. durissus cascavella.
Whole C. durissus cascavella venom was fractioned as
described by Toyama et al. [21], and the resulting sPLA2
was termed sPLA2 Col. Purity was evaluated by Tricine
SDS-PAGE and MALDI-TOF mass spectrometry as
described by Toyama et al. [23]. Boc-5 and Boc-10 were
supplied by Prof. Mourao (Universidade Federal do Rio de
Janeiro).
PLA2 activity was measured using a chromogenic sub-
strate (4-nitro-3-octanoyloxy-benzoic acid, ENZO LIFE
SCIENCE, Plymouth meeting, PA, USA), as described by
Lima et al. [13]. Absorbance was measured using a Spec-
tramax 340 multiwell plate reader (Molecular Devices,
Sunnyvale, CA, USA). Purified sPLA2 was incubated with
equimolar amounts of Boc-5 or Boc-10 (n = 12) following
the procedure of Oliveira et al. [17]. At concentrations
below 1 nmol, both Boc-5 and Boc-10 diminished the
enzymatic activity of sPLA2 in a dose-dependent manner.
Maximum inhibition was observed at 1.25 nmol/mL
(Fig. 1a). Thus, we chose equal molar concentrations of
Boc-5 or Boc-10 to sPLA2 for further pharmacological
studies.
The results show that heparin induces an increase in
enzymatic activity of PLA2, i.e. heparin general point of
view could be considered a kind of positive modulator of
enzymatic activity of PLA2 isolated from snake venom.
Boc The isolated algae has some similarities with heparin,
which is a sulfated molecule glycosaminoglycan (GAG)
composed of negatively charged disaccharide repeats. Yet
the results of enzyme activity clearly show that Boc can
act as both an inhibitor and a stimulator of enzyme
activity, depending on the concentration used to be incu-
bated with PLA2. At low concentrations of Boc 5 or 10
there is a significant decrease of enzyme activity and high
concentrations observed an increase in enzyme activity.
Hepariana heparin like compounds and usually do not
affect the enzymatic activity of PLA2 [14] and [2], but
either heparin can significantly affect the Boc and different
forms of sPLA2 enzymatic activity of Crotalus durissus
cascavella, as shown in this work. The results obtained
with the PLA2 from Crotalus durissus cascavella not
123
M. H. Toyama et al.
extrapolated to other PLA2 without proper study. 14:02
did not observe the same effect as presented here were
probably due to the type of PLA2 used, type of test and
others. Under the same conditions, heparin acted as an
allosteric activator of sPLA2 (Fig. 1b) by increasing the
affinity of sPLA2 for its substrate (Fig. 1b). Each point
represents the data obtained from analysis of 12 mea-
surements and asterisks indicates P \ 0.05 compared to
the native sPLA2.
Circular dichroism (CD) spectroscopy of protein sam-
ples was conducted as described by Iglesias et al. [11].
Nine scans were taken for each sample and all spectra were
corrected by subtraction of buffer blanks. The CD spectra
of sPLA2 and sPLA2 with Boc-5 or Boc-10 did not show
significant differences (Fig. 1c), suggesting that Boc-5 and
Boc-10 did not affect secondary structure of the protein.
HPLC molecular exclusion chromatography of protein
samples was done as described by Oliveira et al. [17] and
indicated a molecular mass of approximately 5.0 and
10.0 kDa for isolated Boc-5 and Boc-10, respectively,
30.0 kDa for isolated sPLA2, and 60 kDa for PLA2 and
Boc-5 or Boc-10 (Fig. 1d).
Edema assays were performed following the protocols
of Toyama et al. [20] and Fonseca et al. [7]. Subplantar
injection of sPLA2 (10 lg/site) induced dose-dependent rat
paw edema characterized by rapid and transient swelling
within 60 min and showed an edema value of 1.66
0.12 mL (n = 4). Boc-5 or Boc-10 induced a maximum
edema of 0.24 0.03 mL (n = 4), significantly different
from that induced by sPLA2, further, when incubated with
sPLA2 at the same mass ratio, Boc-10 virtually abolished
edema (0.42 0.03 mL, n = 4, P \ 0.05), whereas Boc-5
only strongly reduced the sPLA2-induced edema
(0.72 0.07 mL, n = 4, P \ 0.05), (Fig. 2a).
Mast cells were collected from 4 to 6 male Wistar rats
(200300 g) by a 10-mL KrebsRinger phosphate solution
(pH 7.3) wash of the peritoneal cavity. Degranulation was
measured by release of [14C] 5-hydroxytryptamine ([14C]
5-HT, 40 nCi/mL) upon stimulation. All values were cor-
rected for the spontaneous release of [14C] 5-HT in the
absence of stimulus. sPLA2 at 10 lg induced a significant
release of [14C]5-HT, whereas Boc-5 or Boc-10 had no
significant activity at 10 lg (n = 5, Fig. 2b). Boc-5 or
Boc-10 incubated with sPLA2 strongly reduced native
sPLA2-induced mast cell degranulation (n = 5, P \ 0.05)
(Fig. 2b).
Skin edema was measured as described by Camara et al.
[4]. The plasma extravasation induced by Boc-5 or Boc-10
(5 lg/site) was 8.3 2.4 lL; (n = 5, P \ 0.05) and
4.2 0.6 lL (n = 5, P \ 0.05), respectively (Fig. 2c),
whereas Boc-5 or Boc-10 with sPLA2 showed plasma
extravasation of 31.6 3.8 lL (n = 5, P \ 0.05) and
22.4 5.4 lL (n = 5, P \ 0.05), respectively. Thus, both
Effects of Low Molecular Weight Sulfated Galactan Fragments 569

Fig. 1 a Enzyme activity of

sPLA2 of Crotalus durissus
cascavella (1 nmol/mL)
incubated with different
concentrations of Boc-5,
Boc-10, or heparin (from 0 to
2.5 nmol/mL). Each point
represents the mean S.D. of
12 experiments and asterisks
indicate P \ 0.05. b The effect
of substrate concentration on
enzyme activity of native
sPLA2. sPLA2 was pre-
incubated with an equal molar
of Boc-5, Boc-10 or heparin. All
experiments were conducted in
the presence of 1 nmol/mL of
enzyme. Each point represents
the data obtained from analysis
of 12 measurements and
asterisks indicates P \ 0.05
compared to the native sPLA2.
c CD spectra of native sPLA2
and sPLA2 incubated
with Boc-10 or Boc-5. d HPLC
chromatographic profiles native
sPLA2, native Boc-5 and
Boc-10, and sPLA2 incubated
with Boc-5 or Boc-10

Boc-5 and Boc-10 virtually abolished the skin edema
induced by sPLA2 (Fig. 2c).
The biventer cervicis of chicks was prepared as descri-
bed by Ginsborg and Warriner [9]. Contractures due to
exogenously applied acetylcholine (Ach, 55110 mM for
60 s) and KCl (50 mM for 120130 s) were obtained
without nerve stimulation prior to addition of PLA2 or
PLA2 plus Boc-5 or Boc-10, and at the end of the exper-
iment as an additional test for myotoxic and neurotoxic
activities. Isolated sPLA2, at a 10-lg dose had a strong
neurotoxic effect on the chick biventer cervicis prepara-
tion, and sPLA2 plus Boc-10 only decrease the agressive of
the toxin, but does not abolish the neurotoxicity of sPLA,
suggesting that sPLA2 plus Boc-10 only decreases the
affinity of the toxin for the receptors of the chick biventer
nerve. However sPLA2 plus Boc-5 had significantly less
neurotoxicity (n = 4, P \ 0.05, Fig. 2d), suggesting that
sPLA2 plus Boc-5 not only decreases the affinity of the
toxin for the receptors but also their capacity to binding
with the chick biventer nerve, (Fig. 2c).
Release of creatine kinase (CK) from damaged muscle
cells was measured with the Kit 47-UV (SigmaAldrich,
St. Louis, MO, USA), following the method of Fonseca
et al. [6] to measure enzyme activity in mouse plasma.
Mice (1822 g) were injected in the right gastrocnemius
muscle with 50 lL of 0.5 mg/mL native sPLA2 or sPLA2
plus Boc-5 or Boc-10 (n = 4), and the control group
received PBS. Native sPLA2 treatment showed CK
levels of 2645.5 42.3 (n = 4, P \ 0.05), whereas
sPLA2 plus Boc-5 or Boc-10 showed reduced CK levels of
511.5 44.5 (n = 4, P \ 0.05) and 1313.7 15.2
(n = 4, P \ 0.05), respectively (Fig. 2b).
The results are reported as the mean SEM of exper-
iments. Statistical differences between the means were
calculated with an analysis of variance, followed by a
Dunnetts test where several experimental groups were

123
570
Fig. 2 a The effect of sPLA2, sPLA2 incubated with Boc-5 or
Boc-10, Boc-5, and Boc-10 on rat paw edema. All experiments were
done using a sPLA2 concentration of 10 and 10 lg/paw of Boc-5 or
Boc-10. In the sPLA2 incubation, both Boc-5 and Boc-10 were
adjusted to be at the same concentration as sPLA2. The edema is
expressed as the increase in volume (mL) of the injected paw
compared to its basal volume. Each point represents the mean of
animal data; the vertical bars show mean S.D. of 4 experiments
and asterisks indicate P \ 0.05 compared to their respective control
values. b [14C]5-HT release from rat peritoneal mast cells induced by
native sPLA2 (10 lg/mL), Boc-5 or Boc-10 (10 lg/mL), or sPLA2
(10 lg/mL) incubated with an equal mass of Boc-5 or Boc-10.
Results represent the mean S.D. of 5 experiments and asterisks
indicate P \ 0.05 compared to native PLA2. c dose-dependent rat
compared with the control group. The confidence interval
for significance was 5%.
Our results show that, although some slight changes
were observed in especially in the random coils and beta
sheets, Boc-5 and Boc-10 cant induce significantly chan-
ges in the secondary structure of sPLA2 (Fig. 1c). Binding
between native sPLA2 and Boc-5/Boc-10 primarily
involved electrostatic interactions between the acid groups
of Boc-5 and Boc-10 and native sPLA2, similar to the
electrostatic interactions between heparin and myotoxic
sPLA2 described by Landucci et al. [12] and Soares et al.
123
M. H. Toyama et al.
skin edema induced by native PLA2, Boc-5, Boc-10, and sPLA2
incubated with same mass of Boc-5 or Boc-10. Edema was measured
15 min after the intradermal injection of the above agents and
expressed as lL of plasma protein extravasation. Each point
represents the mean of 5 experiments; S.D. is shown by the vertical
bars and asterisks indicate P \ 0.05 compared to native PLA2. d The
neurotoxic effect induced by native sPLA2 or sPLA2 incubated
with Boc-5 or Boc-10. All experiments were conducted using a
sPLA2 concentration of 10 lg/mL and each point represents the
mean S.D. of 4 experiments and asterisks indicate P \ 0.05
compared to native PLA2. e Myotoxic activity evaluated by increase
in plasmatic CK level. Each point represents the mean S.D. of 4
experiments and asterisks P \ 0.05 compared to native PLA2
[19]. Therefore, alteration of the electrostatic interactions
would destabilize binding between Boc-5/Boc-10 and
sPLA2. Acetylation of the lysine or arginine residues or
treatment of sPLA2 with pyridoxal 50 -phosphate would add
negatively charged phosphate groups onto the lysine resi-
dues of the protein surface. In both cases, we observed two
peaks on HPLC molecular exclusion chromatography, one
eluted at 72.6 min and the other after * 90 min. Thus,
both tested treatments suggest that electrostatic interaction
between the basic charged amino acids on sPLA2 and the
acid charged groups of Boc-5 or Boc-10 is the main force
Effects of Low Molecular Weight Sulfated Galactan Fragments
stabilizing the interaction of sPLA2 with both Boc-5 and
Boc-10.
Neither Boc-5 nor Boc-10 alone had any significant
effect on edema, mast cell degranulation, skin edema,
neurotoxic activity, or myotoxicity. However, it is possible
that neither compound has significant pharmacological
action under the conditions used here. Mast cells play an
important regulatory function in skin or paw edema, and
their action should be strongly increased by the presence of
snake venom sPLA2 [15]. Our results showed that, beyond
reducing the enzymatic activity of sPLA2, both Boc-5 and
Boc-10 strongly decreased paw edema induced by sPLA2,
as well mast cell degranulation and skin edema, suggesting
that Boc-5 and Boc-10 inhibited sPLA2-induced edema by
inhibiting the ability of sPLA2 to stimulate mast cell
degranulation [8] and [4].
In the case of neurotoxicity or myonecrosis, only Boc-10
effectively inhibited the effects of sPLA2. This result
suggests that molecular mass or length differences between
Boc-5 and Boc-10 are important for their different
activities.
Our results showed that Boc-5 and Boc-10, despite
structural similarities to heparin, differ from heparin in
terms of modulation of sPLA2 activity. Both Boc-5 and
Boc-10 have a dual mode for modulating the enzymatic
activity of sPLA2, but the action of heparin on sPLA2 from
snake venom remains unclear [1]. It is known that heparin
preferentially targets the C-terminal region of sPLA2, the
location of the myotoxic region [10]. However, this
C-terminal region is not near the enzymatic site of sPLA2,
thus our results suggest that Boc-5 and Boc-10 bind to
another region of sPLA2. Structural analysis of sPLA2
from the C. durissus cascavella showed two molecular
regions that could bind heparin or other anionic com-
pounds, and our results strongly suggest that Boc-5 or
Boc-10 preferentially bind the N-terminal region, which
alters the enzymatic properties of sPLA2 [17].
In conclusion, Boc-5 and Boc-10 have similar molecular
characteristics and could be used as alternatives to heparin
to decrease acute edema. The anti-venom or anti-PLA2
effect of these compounds is dependent on the molecular
weight, chain length, concentration, and purity of these
compounds. Our results support further investigation of the
medical uses of the abundant marine life present along the
Brazilian coastline.
Acknowledgments FAPEPS 2007 54714-3, CNPq 558193/2009-9
for financial support.
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571
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