Author`s personal copy

This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright
Author's personal copy
Toxicon 56 (2010) 1059–1065
Contents lists available at ScienceDirect
Toxicon
journal homepage: www.elsevier.com/locate/toxicon
Immunochemical and biological characterization of monoclonal
antibodies against BaP1, a metalloproteinase from Bothrops asper
snake venom
I. Fernandes a, *, G.G. Assumpção a, C.R.F. Silveira a, E.L. Faquim-Mauro a, I. Tanjoni a,
A.K. Carmona b, M.F.M. Alves b, H.A. Takehara a, A. Rucavado c, O.H.P. Ramos a,
A.M. Moura-da-Silva a, J.M. Gutiérrez c
a
Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, Butantã, CEP 05503-900, São Paulo, SP, Brazil
Departamento de Biofísica, Universidade Federal de São Paulo, SP, Brazil
c
Instituto Clodomiro Picado, Facultad de Microbiologia, Universidad de Costa Rica, San José, Costa Rica
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 May 2010
Received in revised form 19 July 2010
Accepted 22 July 2010
Available online 30 July 2010
BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue
damage associated with envenomations by Bothrops asper, a medically-important species
in Central America and parts of South America. Six monoclonal antibodies (MoAb) against
BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation
constant (Kd), specificity and ability to neutralize BaP1-induced hemorrhagic and
proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The Kds of IgG
MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1
and B. asper venom components but failed to recognize venoms from 27 species of
Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs
tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was
totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic
activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and
partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against
BaP1 may become important tools to understand structure–function relationships of BaP1
and the role of P-I class SVMP in snakebite envenomation.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Monoclonal antibodies
Metalloproteinase
BaP1
Hemorrhage
Snake venom
Neutralizing antibody
1. Introduction
Envenomations by snakes of the genus Bothrops (family
Viperidae) constitute a relevant public health hazard in
Latin America (Fan and Cardoso, 1995; Gutiérrez, 1995).
* Corresponding author. Tel.: þ55 11 37267222x2088/2134, fax: þ55 11
37267222x2134.
E-mail addresses: [email protected] (I. Fernandes),
[email protected] (G.G. Assumpção), [email protected]
(C.R.F. Silveira), [email protected] (E.L. Faquim-Mauro),
[email protected] (I. Tanjoni), [email protected] (A.K. Carmona),
[email protected] (M.F.M. Alves), harumitakehara@butantan.
gov.br (H.A. Takehara), [email protected] (A. Rucavado),
[email protected]
(O.H.P.
Ramos),
[email protected]
(A.M. Moura-da-Silva), [email protected] (J.M. Gutiérrez).
0041-0101/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2010.07.014
Their effects are induced by a variety of venom components, such as myotoxic phospholipases A2 (Gutiérrez and
Lomonte, 1997) and metalloproteinases (Moura da Silva
et al., 2007), among others, provoking prominent local
tissue damage, that is, myonecrosis, blistering, hemorrhage
and edema (Gutiérrez et al., 1989, 2009; Ownby, 1982). The
clinical manifestations develop rapidly after the bite, and
antivenoms, which are usually administered several hours
later, are not completely effective to neutralize the local
effects (Gutiérrez et al., 1998; Warrell, 1992). As a consequence, permanent tissue damage often ensues in patients
bitten by these snakes. An adequate understanding of the
local action of Bothrops snake venoms is necessary for the
development of alternative therapeutic strategies.
Author's personal copy
1060
I. Fernandes et al. / Toxicon 56 (2010) 1059–1065
Snake venom metalloproteinases (SVMP) comprise
a series of zinc-dependent enzymes of varying molecular
mass which are responsible for the hemorrhagic effect
characteristic of viperine and crotaline snake envenomations (reviewed by Gutiérrez and Rucavado, 2000). In
addition, more recent investigations have evidenced that
these enzymes are also involved in the pathogenesis of local
myonecrosis, skin damage, and edema and other reactions
associated with inflammation (Teixeira et al., 2005;
Gutiérrez et al., 2009). Thus, SVMP play a relevant role in
the pathogenesis of venom-induced local tissue damage.
SVMP form, together with the ADAMs (“A Disintegrin
And Metalloproteinase” proteins), the subfamily of reprolysins, since they share a common overall domain organization, although ADAMs have, besides metalloproteinase,
disintegrin-like and cysteine-rich domains, an epidermal
growth factor-like domain, a transmembrane region and
a cytoplasmic tail (Fox and Serrano, 2005). In turn, reprolysins are part of the super family of metzincins, together
with matrix metalloproteinases (MMPs), astacins and serralysins, all of them exhibiting identical zinc-binding
environments.
On the basis of their domain organization, SVMP are
classified in three main groups: 1) P-I, comprising only
the metalloproteinase domain; 2) P-II, having a metalloproteinase domain followed by a disintegrin domain
comprising the classical disintegrins; and 3) PIII, comprising metalloproteinase, disintegrin-like and
cysteine-rich domains. In addition to these domains, some
P-III present a C-type lectin-like subunit (reviewed by Fox
and Serrano, 2008).
In Central America, southern Mexico and regions of
northern South America, most snakebite envenomations
are caused by Bothrops asper, a large and widely distributed
species in tropical rainforests and in altered areas devoted
to agriculture and cattle raising (Warrell, 2004; Sasa, 2009;
Angulo and Lomonte, 2009). Envenoming by B. asper is
characterized, among other clinical features, by severe local
tissue damage, often associated with permanent disability
and sequelae (Gutiérrez, 1995; Warrell, 2004; Gutiérrez
et al., 2009). Local pathology induced by this venom
involves muscle necrosis, hemorrhage, edema, and blistering, a complex series of events mediated predominantly
by venom phospholipases A2 and SVMP (Gutiérrez and
Lomonte, 1995; Gutiérrez and Rucavado, 2000; Gutiérrez
et al., 2009). Among several SVMPs isolated from B. asper
venom (Angulo and Lomonte, 2009), BaP1 is a 22.7 kDa P-I
SVMP comprising a single chain of 202 amino acids that
shows highest sequence identity with SVMP isolated from
the venoms of snakes of the subfamily Crotalinae. The
amino acid sequence and the crystal structure of this
enzyme have been described (Watanabe et al., 2003;
Lingott et al., 2009). BaP1 exerts multiple tissuedamaging activities, including hemorrhage, myonecrosis,
dermonecrosis, blistering, and edema (Gutiérrez et al.,
1995; Rucavado et al., 1995, 1998; Jiménez et al., 2008).
The structure–function relationships of P-I SVMP remain
unclear, as there are representatives of this class of enzymes
that induce hemorrhagic activity whereas others are unable
to provoke microvascular disruption, despite the fact that
they degrade extracellular matrix components in vitro
(Gutiérrez et al., 2005). Monoclonal antibodies constitute
highly useful tools to investigate the structural determinants of toxicity in venom. The objective of this study was to
produce and characterize monoclonal antibodies against
BaP1, and to analyze their ability to neutralize BaP1-induced
hemorrhage and its catalytic activity. These antibodies will
be useful for investigating structure–function relationships
associated with various toxic effects in a single SVMP.
2. Materials and methods
2.1. Animals and venom/enzymes
BALB/c and Swiss mice (18–20 g) were provided by the
Instituto Butantan animal house. All procedures were
approved by Ethical Committee for Animal Research of
Instituto Butantan (382/07).
Bothrops jararaca and Bothrops neuwiedi venoms were
provided by M.F.D. Furtado from the Herpetology Laboratory of the Instituto Butantan, while B. asper venom was
provided by Instituto Clodomiro Picado, Costa Rica. The
venoms corresponded to pools obtained from many specimens and were lyophilized and stored at 20 C. BaP1 was
purified as previously described (Gutiérrez et al., 1995;
Rucavado et al., 1998). The P-I SVMPs moojeni protease A
(MPA), insularinase and neuwiedase were isolated from the
venoms of Bothrops moojeni, Bothrops insularis and B. neuwiedi, respectively, as previously described (Assakura et al.,
1985; Rodrigues et al., 2000; Modesto et al., 2005).
2.2. Preparation of polyclonal antiserum
A group of 6 BALB/c mice were injected by the intraperitoneal (i.p.) route with 10 mg BaP1 emulsified in
complete Marcol/Montanide adjuvant. Boosters of 10 mg
BaP1 in incomplete Marcol/Montanide adjuvant were
administered two weeks after each immunization. Animals
were bled seven days after the last booster and the serum
was obtained after clotting and centrifugation (4 C, 10 min,
800 g). This immune mouse serum (IMS) was used as
positive control.
2.3. Production and purification of monoclonal antibodies
(MoAbs)
MoAbs were produced as described by Köhler and
Milstein (1975), with modifications. Popliteal lymph node
cells from BALB/c mice immunized with BaP1 were fused
with SP2-O cells (2:1) using polyethylene glycol 4000
(MERCK). Hybrids were selected in RPMI 1640 medium
plus 3% HAT (hypoxanthine 10 mM, aminopterin 40 mM and
thymidine 1.6 mM) (GibcoBRL) containing 10% FCS (GibcoBRL) at 37 C and 5% CO2. The supernatant fluids were
screened for species-specific antibodies by ELISA, as
described in 2.7. Antibody-secreting cells were expanded
and cloned twice at limiting dilution. The MoAbs contained
in culture supernatants were purified by affinity chromatography on protein-A Sepharose (Pharmacia) equilibrated
in TBS buffer, pH 8.5. The proteins were eluted in 0.2 M
glycine/HCl buffer, 0.15 M NaCl, pH 2.8, and dialyzed in TBS.
Author's personal copy
I. Fernandes et al. / Toxicon 56 (2010) 1059–1065
Homogeneity was assessed by SDS-PAGE using 12%
acrylamide.
2.4. Isotyping
The heavy chain isotype was determined by ELISA, using
monoclonal antibodies against different mouse immunoglobulin classes and subclasses.
1061
antibody anti-Taenia crassiceps; Espindola et al., 2002), for
1 h at 37 C. After incubation, the mixture (100 mL) was
centrifuged and injected i.d. into the dorsal skin of Swiss
mice. Mice were killed 3 h after injection, the skin was
removed and the extent of the hemorrhagic spots determined by multiplying the largest diameter by its perpendicular. Results are shown in cm2 SD. Normal mouse
serum (NMS) or isotype control (IC) was used as negative
control for all experiments.
2.5. Measurement of dissociation constants
2.9. Proteolysis of a synthetic substrate by BaP1
The dissociation constants (Kd) of antigen–antibody
interactions were determined under equilibrium conditions according to the method described by Friguet et al.
(1985). Dilutions of different MoAbs, selected in the linear
part of the ELISA titration curves (linear regression analysis
was performed to assess the linearity of the curve), were
incubated overnight at 4 C with various concentrations of
BaP1. The concentration of free MoAb was determined by
ELISA. For this, aliquots (100 mL) of incubation medium
were transferred into the wells of microtiter plates coated
with BaP1 (2 mg/mL) and allowed to react for 30 min. The
plates were washed with PBS-Tween and the subsequent
steps of ELISA were performed according to the general
procedure. Dissociation constants were deduced from
Scatchard plots.
2.6. Dot-blotting
For dot blots, venom samples (2 mg/mL) from different
species or isolated SVMPs, diluted in PBS, without previous
fractionation or treatment (native form), were dotted on
nitrocellulose membranes. Some samples were denatured
by boiling (3 min) in Tris-buffer containing 1% SDS or
denaturated and reduced (R) with 2-mercaptoethanol (2ME). After blocking with skimmed milk at 5%, membranes
were incubated with the solutions containing antibodies
followed by incubation with sheep IgG anti-mouse IgG
labeled with horseradish peroxidase (1:2000). Immunodetection signals were visualized by addition of 0.05% 4chloro-1-naphthol in 15% methanol (v/v), in presence of
0.03% H2O2 (v/v).
2.7. ELISA
ELISA was carried out according to Theakston et al.
(1977). Briefly, plates were coated with venoms (2 micrograms/well ) or class P-I SVMPs (neuwiedase, insularinase,
MPA or BaP1) and, after blocking with 3% bovine serum
albumin, monoclonal antibodies were added. Antigen–
antibody reaction was detected by addition of anti-mouse
IgG-peroxidase conjugate and ortho-phenylenediamine
(1 mg/mL, Sigma) and H2O2 as enzyme substrates.
The catalytic activity of BaP1 (4.4 1011 M) was tested
using the fluorescence resonance energy transfer (FRET)
peptide, Abz-LVEALYQ-EDDnp (Abz ¼ ortho-aminobenzoic
acid; EDDnp ¼ N-[2,4-dinitrophenyl] ethylenediamine) as
substrate (amino acid sequence based on the insulin
b chain). Briefly, the assays were carried out at 37 C with
different concentrations of the fluorogenic peptide in
100 mM Tris–HCl buffer containing 50 mM NaCl, pH 7.0.
The hydrolysis was continuously followed in a Hitachi
F-2000 fluorimeter by measuring the fluorescence at
lex ¼ 320 nm and lem ¼ 420 nm, following the procedure
previously described (Chagas et al., 1990). The slope was
converted into micromols of substrate hydrolyzed/min
based on a calibration curve obtained from the complete
hydrolysis of the peptide. Kinetic parameters were calculated by nonlinear regression analyses of initial velocities of
substrate hydrolysis using the Grafit computer program
(Leatherbarrow, 2001). The inhibition of BaP1 by 1,10phenanthroline (10 mM) was determined under the same
conditions using Abz-LVEALYQ-EDDnp as substrate.
2.10. Inhibition of BaP1 enzymatic activity by monoclonal
antibodies
MABaP1 were incubated with BaP1 (2:1 molar ratio) for
15 min at 37 C before addition of the fluorogenic peptide.
Enzymatic activity was then estimated as described above.
2.11. Statistics
All analyses were carried out in triplicates with results
obtained from a minimum of two independent experiments. The significance of the differences of two mean
values was analyzed by the Student’s t-test. When more
than two experimental groups were compared, the significance of the differences was determined by ANOVA, followed by Tukey test.
3. Results
3.1. Production and characterization of the monoclonal
antibodies (MoAbs)
2.8. Neutralization of BaP1-induced hemorrhage
The ability of the different MoAbs to neutralize BaP1induced hemorrhage was estimated by incubating one
Minimum Hemorrhagic Dose (MHD) (35 mg) of BaP1 or B.
asper venom (20 mg) with IgG purified from hybridomas
(1.5:1 molar ratio) or with isotype control (monoclonal
Fusion of myeloma SP2-O cells with popliteal lymphocytes of mice immunized with BaP1 resulted in 91
hybridomas of which 19 secreted antibodies against BaP1
by ELISA. We selected 10 hybridomas with the highest O.D.
(>1.0) to be cloned and recloned to ensure monoclonality. A
total of 6 stable immortalized clones secreting anti-BaP1
Author's personal copy
1062
I. Fernandes et al. / Toxicon 56 (2010) 1059–1065
antibodies were obtained. These MoAbs were designated as
MABaP1-2, -3, -6, -7, -8 and -10.
MoAbs belong to three different isotypes, as determined
by ELISA (Table 1). The dissociation constants (Kd) of
antigen–antibody interactions were determined according
to the method described by Friguet et al. (1985). MABaP1
had Kd in the 109 range, indicating a high affinity to BaP1
(Table 1).
3.2. Antigen recognition by the different MABaP1
First, we attempted to verify whether recognition of
epitopes by MABaP1 is dependent of native-like conformations. As shown in Fig. 1, all MABaP1 recognized only the
antigen in its native form, failing to react with BaP1 after
heat denaturation or treatment with reducing agents, thus
evidencing that epitopes require native-like structures to
be recognized by MABaP1. In contrast, polyclonal antibodies were able to react with BaP1 after denaturation or
reduction, albeit the immunodetection signal was very
weak when compared to that of the native antigen (Fig. 1).
The specificity of anti-BaP1 MoAbs was then assessed.
MABaP1–7 antibody recognized P-I SVMPs isolated from
other venoms of Bothrops snakes such as insularinase, MPA
or neuwiedase when assayed by dot blot (Fig. 2). In addition, the ability of MABaP1–7 to recognize B. asper and B.
neuwiedi venoms, and BaP1 and neuwiedase enzymes were
then quantified by ELISA. High titres of antibodies were
obtained with crude venoms as well as with the homologous (BaP1) and heterologous (neuwiedase) enzymes. The
differences in antibody titres were within two dilution
steps (Table 2). The other antibodies have not been tested.
On the other hand, when MoAbs were tested by dot
blotting for reactivity with venoms from snakes of
different species of various families, they recognized B.
asper, B. moojeni and B. neuwiedi venoms but did not
recognize the following venoms: B. jararaca, Bothrops
erythromelas, Bothrops jararacussu, Bothrops atrox, Crotalus
durissus terrificus, Crotalus atrox, Crotalus adamanteus,
Crotalus vegrandis, Calloselasma rhodostoma, Agkistrodon
contortrix, Trimeresurus albolabris, Bitis arietans, Bitis caudalis, Vipera ammodytes, Phylodryas olfersii, Phylodryas
patagonensis, Naja mossambica, Naja melanoleuca, Pseudechis porphyriacus, Notechis scutatus, Hoplocephalus stephensii, Oxyuranus microlepidotus and Micrurus lemniscatus
(not shown). In contrast, when immunoreactivity of the
Table 1
Isotyping and dissociation constants of MoAbs against BaP1.
MABaP1
Isotypeb
3
6
7
8
2 and 10
IgG2b
IgG1
IgG1
IgG1
IgM
Antibody reactivitya (M)
2.4
8.2
5.9
3.7
109
109
109
109
ND
ND ¼ Not determined.
a
Minimal molar concentration that reacts with venoms in a typical
ELISA assay.
b
The heavy chain isotype was determined by ELISA, using monoclonal
antibodies against different mouse immunoglobulin classes and
subclasses.
polyclonal antibody raised against BaP1 was assessed, the
following venoms were recognized in dot blot analysis: B.
asper, B. moojeni, B. neuwiedi, B. jararacussu, B. atrox, C.
durissus terrificus, C. atrox, C. adamanteus, T. albolabris and
A. contortrix (not shown).
3.3. Neutralization of BaP1-induced hemorrhage by MoAbs
The ability of the different MoAbs to neutralize BaP1 or
B. asper venom-induced hemorrhage was estimated in
Swiss mice injected with 1 Minimum Hemorrhagic Dose
(MHD) BaP1 (35 mg) or B. asper venom (20 mg) previously
incubated (molar ratio 1.5:1) with IgG purified from
hybridomas. MABaP1 clones 3, 6 and 8 completely
neutralized BaP1-induced hemorrhage while clone 7 did
not neutralize this activity (Fig. 3). None of the MABaP1 was
able to inhibit B. asper venom-induced hemorrhage (not
shown).
3.4. Neutralization by MoAbs of BaP1-induced enzymatic
activity on a synthetic substrate
The ability of the different MoAbs to neutralize BaP1induced proteolytic activity was also estimated using AbzLVEALYQ-EDDnp peptides in fluorimetric assays. MABaP1–3
and 6 totally neutralized this activity while, in the case of
clone 8, neutralization was only partial, and clone 7 failed to
inhibit proteolytic activity of BaP1 (Table 3).
4. Discussion
In this work we have obtained and characterized six
different MoAbs against BaP1, a 22.7 kDa SVMP from
B. asper venom. These MoAbs were designated Mouse
against BaP1 (MABaP1) and showed Kd in the nanomolar
range, indicating high affinities to BaP1. Regarding their
isotype, two MABaP1 are IgM, three are IgG1 and one is
IgG2b. All MoAbs recognized only native BaP1 and B. asper
venom. Immunoreactivity was abrogated after reduction
with 2-mercaptoethanol, indicating that antibodies recognize conformational epitopes that are disulfide bonddependent. BaP1 has six Cys residues involved in three
disulfide bridges (Cys 117–Cys 197, Cys 159–Cys 181, Cys
157–Cys 164) which contribute to the overall structure
consisting of a major subdomain (residues 1–152),
comprised of four a-helices and a five-stranded b-sheet,
and a minor subdomain, which is formed by a single a-helix
and several loops, and contains the active site cleft
(Watanabe et al., 2003).
The various MoAbs differ in their ability to inhibit
hemorrhagic and proteolytic activities of BaP1. MABaP1–3,
6 and 8 were able to neutralize BaP1-induced hemorrhagic
activity, whereas clone 7 was not. In agreement, the former
three antibodies neutralized proteolytic activity of BaP1,
albeit only to a partial extent in the case of clone 8. Thus,
there is a clear correlation between the capacity of antibodies to inhibit hemorrhagic and proteolytic activities.
The ability of SVMPs to induce microvessel disruption
leading to hemorrhage depends on their proteolytic
activity, as abrogation of catalysis by chelating agents or
specific inhibitors completely abolishes hemorrhagic
Author's personal copy
I. Fernandes et al. / Toxicon 56 (2010) 1059–1065
1063
Fig. 1. Recognition of conformational epitopes by Monoclonal antibodies against BaP1. Samples of B. asper venom or BaP1 in native form (N), denatured (D) by
boiling in Tris-buffer containing 1% SDS, or denaturated and reduced (R) with 2-mercaptoethanol were dotted on nitrocellulose membranes. After blocking,
membranes were incubated with anti-BaP1 polyclonal antibodies raised in mice (IMS), MABaP1 clones 3, 6, 7 and 8 or isotype control (IC). Antigen–antibody
reaction was detected by addition of anti-mouse IgG-peroxidase followed by the enzyme substrate.
activity as well (Gutiérrez et al., 2005). In contrast,
MABaP1–7, which also reacts with BaP1 with high affinity,
is unable to neutralize hemorrhagic and proteolytic
activities.
MABaP1–7 recognized not only BaP1, but also neuwiedase, insularinase and MPA, three P-I SVMPs isolated
from other Bothrops sp. venoms. These enzymes differ in
their profile of toxicologic activities, since neither of them
induces hemorrhage, and insularinase has procoagulant
activity through activation of prothrombin (Assakura et al.,
1985; Rodrigues et al., 2000; Modesto et al., 2005). Interestingly, therefore, clone 7 is able to react with a set of
SVMPs having different biological effects, evidencing that it
recognizes an epitope common to various types of P-I
SVMPs which is evidently not associated with their ability,
or inability, to induce hemorrhage or coagulation. This
could be a conserved structural epitope. This apparently
conserved epitope may be used in the design of immunization strategies aimed at generating antibodies that react
with a variety of P-I SVMPs from diverse snake venoms. On
the other hand, MABaP1–3, 6 and 8 are likely to recognize
an epitope located in the vicinity of the catalytic site, which
seems to play a role in the ability of some of these enzymes
to induce hemorrhage. It has been proposed that the ability
of P-I SVMPs to induce hemorrhage may depend on
structural features associated with a loop located in the
region of residues 153–176 (Watanabe et al., 2003; Lingott
et al., 2009). Since this loop is close to the enzyme active
site, it is suggested that the epitopes recognized by
MABAP1–3, 6 and 8 are located in the region of the active
site or the neighboring loop.
Despite the fact that various MoAbs were capable of
inhibiting the hemorrhagic activity of BaP1, they failed to
neutralize hemorrhage induced by crude B. asper venom. At
least two highly active hemorrhagic SVMPs of the P-III class
have been isolated from this venom (Angulo and Lomonte,
2009) and they are likely to be predominantly responsible
for the hemorrhagic activity of this venom. The failure of
MoAbs to neutralize this activity suggests that the epitopes
recognized by these antibodies are not present, or inaccessible, in the metalloproteinase domain of these P-III
SVMPs. The comparative immunochemical analysis of the
metalloproteinase domain of P-I and P-III SVMPs deserves
further investigation. Monoclonal antibodies against the PIII SVMP jararhagin were raised in our laboratory and one of
them efficiently neutralized hemorrhage induced by the
isolated SVMP (Tanjoni et al., 2003a) and also by venoms of
several species of Bothrops snakes (Tanjoni et al., 2003b).
The epitope recognized by this neutralizing antibody was
located on the disintegrin-like domain of jararhagin, as
observed by reactivity with recombinant fragments
(Tanjoni et al., 2003a) or by molecular modeling (Mourada-Silva et al., 2008).
In addition to proteolytic and hemorrhagic activities,
BaP1 also induces myonecrosis (Rucavado et al., 1995),
dermonecrosis and blistering (Rucavado et al., 1998;
Jiménez et al., 2008), edema (Gutiérrez et al., 1995),
inflammation (Farsky et al., 2000; Rucavado et al., 2002;
Fernandes et al., 2006) and apoptosis of endothelial cells
(Díaz et al., 2005). Such wide pharmacological spectrum
makes this molecule ideal for structure–function
Table 2
ELISA reactivity of MABaP1–7 to homologous and heterologous
venoms or isolated toxins.
Venoms
Fig. 2. Recognition of homologous and heterologous venoms or isolated class
P-I SVMPs. Venoms (2 mg ) or class P-I SVMPs (neuwiedase, insularinase, MPA
or BaP1) were dotted on nitrocellulose membranes and after blocking,
membranes were incubated with the neutralizing monoclonal antibody
MABaP1–7 (7) or polyclonal anti-BaP1 antibodies (IMS) or normal mouse
serum (NMS). Antigen–antibody reaction was detected by addition of antimouse IgG antibodies conjugated with peroxidase followed by the enzyme
substrate.
ELISA antibody titre
Bothrops asper
Bothrops neuwiedi
1.024
512
Isolated SVMPs
BaP1
neuwiedase
2.048
512
Antibody titres represent the reciprocal of the highest dilution of
MABaP1–7 that results in an absorbance greater than 0.1 at 492 nm,
after reaction with B. asper or B. neuwiedi venoms or BaP1 or neuwiedase
isolated SVMPs coated in ELISA plates (2 mg/mL).
Author's personal copy
1064
I. Fernandes et al. / Toxicon 56 (2010) 1059–1065
structural interaction between MoAbs and their epitopes,
in the case of BaP1, is being pursued in our laboratories.
Acknowledgements
FAPESP, CNPq and INCT-TOX PROGRAM of Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) and Fundação de Amparo a Pesquisa do Estado de
São Paulo, Brazil and PAP (FUNDAP), Vicerrectoría de
Investigación (Universidad de Costa Rica), NeTropica.
Conflict of interest statement
I declare, on behalf of the all authors, that there are no
financial, personal, or professional interests that could be
construed to have influenced the paper.
Fig. 3. Neutralization on venom or BaP1-induced hemorrhage by the
different MoAbs. BaP1 was incubated with PBS (C), MABaP1–3, 6, 7 and 8 or
with the isotype control (IC) and then aliquots of the mixtures, containing
one Minimum Hemorrhagic Dose of the enzyme, were injected in mice, as
described in Materials and methods. Results are presented as the area of the
hemorrhagic spot obtained using two groups of four mice (mean SD).
*p < 0.001 compared to Control group (BaP1).
relationship studies aimed at assessing whether different
regions in the molecule mediate such diverse set of activities or whether they all depend on a single structural
determinant. The set of MoAbs developed in this work
constitute highly useful tools to approach this issue as
were, for instance, MoAbs employed to study structure–
function relationship in the case of jararhagin, a P-III
hemorrhagic SVMP from the venom of B. jararaca, assessing
the molecular regions involved in the different activities of
the toxin (Tanjoni et al., 2010).
MoAbs are important tools for identification of shared
epitopes in a protein family. They react with a specific
region of the protein, and discriminate very subtle immunological differences among proteins from the same group,
such as SVMP. As MABaP1–7, produced in this work,
recognized all P-I SVMP tested, it may be used to characterize antigenic epitopes shared by other P-I SVMP present
in viperid venoms. Likewise, MoAbs may be also used in the
phylogenetic analysis of proteins, as has been performed
with jararhagin (Tanjoni et al., 2003b). The precise
Table 3
Inhibition of BaP1 enzymatic activity on the synthetic
substrate by MABaP1.
Antibody
% Inhibition
Isotype Control
MABaP1–3
MABaP1–6
MABaP1–7
MABaP1–8
10.8
99.5
99.0
0
59.6
BaP1 proteolytic activity was tested (at 37 C) on an internally quenched fluorescent peptide, Abz-LVEALYQ-EDDnp
with amino acid sequence based on the insulin b chain. The
hydrolysis obtained with different concentrations of the
fluorogenic substrate was continuously followed in a Hitachi F-2000 fluorimeter by measuring the fluorescence. For
inhibition assays, BaP1 was incubated with monoclonal
antibodies (2:1 molar ratio) for 15 min at 37 C before
addition of the fluorogenic peptide.
References
Angulo, Y., Lomonte, B., 2009. Biochemistry and toxicology of toxins
purified from the venom of the snake Bothrops asper. Toxicon 54,
949–957.
Assakura, M.T., Reichl, A.P., Asperti, M.C., Mandelbaum, F.R., 1985. Isolation of the major proteolytic enzyme from the venom of the snake
Bothrops moojeni (caissaca). Toxicon 23, 691–706.
Chagas, J.R., Juliano, L., Prado, E.S., 1990. Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins.
Anal. Biochem. 192, 419–425.
Díaz, C., Valverde, L., Brenes, O., Rucavado, A., Gutiérrez, J.M., 2005.
Characterization of events associated with apoptosis/anoikis induced
by snake venom metalloproteinase BaP1 on human endothelial cells.
J. Cell. Biochem. 94, 520–528.
Espindola, N.M., Vaz, A.J., Pardini, A.X., Fernandes, I., 2002. Excretory/
secretory antigens (ES) from in-vitro cultures of Taenia crassiceps
cysticerci, and use of an anti-ES monoclonal antibody for antigen
detection in samples of cerebrospinal fluid from patients with neurocysticercosis. Ann. Trop. Med. Parasitol. 96, 361–368.
Fan, H.W., Cardoso, J.L., 1995. Clinical toxicology of snake bites in South
America. In: Meier, J., White, J. (Eds.), Clinical Toxicology of Animal
Venoms and Poisons. CRC Press, Boca Raton, Florida, pp. 667–688.
Farsky, S.H.P., Goncalves, L.R.C., Gutiérrez, J.M., Correa, A.P., Rucavado, A.,
Gasque, P., Tambourgi, D.V., 2000. Bothrops asper snake venom and its
metalloproteinase BaP-1 activate the complement system. Role in
leucocyte recruitment. Mediators. Inflamm. 9, 213–221.
Fernandes, C.M., Zamuner, S.R., Zuliani, J.P., Rucavado, A., Gutiérrez, J.M.,
Terixeira, C.F.P., 2006. Inflammatory effects of BaP1, a metalloproteinase isolated from Bothrops asper snake venom: leukocyte
recruitment and release of cytokines. Toxicon 47, 549–559.
Fox, J.W., Serrano, S.M.T., 2005. Structural considerations of the snake
venom metalloproteinases, key members of the M12 reprolysin
family of metalloproteinases. Toxicon 45, 969–985.
Fox, J.W., Serrano, S.M.T., 2008. Insights into and speculations about snake
venom metalloproteinase (SVMP) synthesis, folding and disulfide
bond formation and their contribution to venom complexity. FEBS J.
275, 3016–3030.
Friguet, B., Chaffote, A.F., Djavadi-Ohaniance, L., Godberg, M.E., 1985.
Measurements of true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J. Immunol.
Methods. 77, 305–319.
Gutiérrez, J.M., Lomonte, B.,1997. Phospholipases A2 myotoxins from Bothrops
snakes venoms. In: Kini, R.M. (Ed.), Venom Phospholipase A2 Enzymes.
Structure, Function and Mechanisms. Wiley, Chichester, pp. 321–352.
Gutiérrez, J.M., Lomonte, B., 1995. Phospholipase A2 myotoxins from
Bothrops snake venoms. Toxicon 33, 1405–1424.
Gutiérrez, J.M., Rucavado, A., 2000. Snake venom metalloproteinases:
their role in the pathogenesis of local tissue damage. Biochimie 82,
841–850.
Gutiérrez, J.M., Chaves, F., Gené, J.A., Lomonte, B., Camacho, Z.,
Schosinsky, K., 1989. Myonecrosis induced in mice by a basic myotoxin isolated from the venom of the snake Bothrops nummifer
(jumping viper) from Costa Rica. Toxicon 27, 735–745.
Gutiérrez, J.M., Leon, G., Rojas, G., Lomonte, B., Rucavado, A., Chaves, F.,
1998. Neutralization of local tissue damage induced by Bothrops asper
(terciopelo) snake venom. Toxicon 36, 1529–1538.
Author's personal copy
I. Fernandes et al. / Toxicon 56 (2010) 1059–1065
Gutiérrez, J.M., 1995. Clinical toxicology of snakebite in Central America.
In: Meier, J., White, J. (Eds.), Clinical Toxicology of Animal Venoms and
Poisons. CRC Press, Boca Raton, Florida, pp. 645–665.
Gutiérrez, J.M., Rucavado, A., Chaves, F., Díaz, C., Escalante, T., 2009.
Experimental pathology of local tissue damage induced by Bothrops
asper snake venom. Toxicon 54, 958–975.
Gutiérrez, J.M., Romero, M., Díaz, C., Borkow, G., Ovadia, M., 1995. Isolation
and characterization of a metalloproteinase with weak hemorrhagic
activity from the venom of the snake Bothrops asper (terciopelo).
Toxicon 33, 19–29.
Gutiérrez, J.M., Rucavado, A., Escalante, T., Díaz, C., 2005. Hemorrhage induced
by snake venom metalloproteinases: biochemical and biophysical
mechanisms involved in microvessel damage. Toxicon 45, 997–1011.
Jiménez, N., Escalante, T., Gutiérrez, J.M., Rucavado, A., 2008. Skin
pathology induced by snake venom metalloproteinase: acute
damage, revascularization, and re-epithelization in a mouse ear
model. J. Invest. Dermatol. 128, 2421–2428.
Köhler, G., Milstein, C., 1975. Continuous cultures of fused cells secreting
antibody of predefined specificity. Nature 256, 495–497.
Leatherbarrow, R.J., 2001. GraFit Version 5. Erithacus Software Ltd.,
Horley, UK.
Lingott, T., Schleberger, C., Gutiérrez, J.M., Merfort, I., 2009. High-resolution crystal structure of the snake venom metalloproteinase BaP1
complexed with a peptidomimetic: insight into inhibitor binding.
Biochemistry 48, 6166–6174.
Modesto, J.C., Junqueira de Azevedo, I.L., Neves-Ferreira, A.G., Fritzen, M.,
Oliva, M.L., Perales, J., Chudzinski-Tavassi, A.M., 2005. Insularinase A,
a prothrombin activator from Bothrops insularis venom, is a metalloproteinase derived from a gene encoding protease and disintegrin
domains. Biol. Chem. 386, 589–600.
Moura da Silva, A.M., Butera, D., Tanjoni, I., 2007. Importance of snake
venom metalloproteinase in cell biology: effects on platelets,
inflammatory and endothelial cells. Curr. Pharm. Des. 13, 2893–2905.
Moura-da-Silva, A.M., Ramos, O.H., Baldo, C., Niland, S., Hansen, U.,
Ventura, J.S., Furlan, S., Butera, D., Della-Casa, M.S., Tanjoni, I., Clissa, P.
B., Fernandes, I., Chudzinski-Tavassi, A.M., Eble, J.A., 2008. Collagen
binding is a key factor for the hemorrhagic activity of snake venom
metalloproteinases. Biochimie 90, 484–492.
Ownby, C.L., 1982. Pathology of rattlesnake envenomation. In: Tu, A.T. (Ed.),
Rattlesnake Venoms. Their Actions and Treatment. Marcel Dekker, New
York, pp. 163–209.
Rodrigues, V.M., Soares, A.M., Guerra-Sá, R., Rodrigues, V., Fontes, M.R.,
Giglio, J.R., 2000. Structural and functional characterization of neuwiedase, a nonhemorrhagic fibrin(ogen)olytic metalloprotease from
Bothrops neuwiedi snake venom. Arch. Biochem. Biophys. 381, 213–224.
Rucavado, A., Escalante, T., Teixeira, C.F.P., Fernandes, C.M., Díaz, C.,
Gutiérrez, J.M., 2002. Increments in cytokines and matrix
1065
metalloproteinases in skeletal muscle after injection of tissuedamaging toxins from the venom of the snake Bothrops asper.
Mediators. Inflamm. 11, 121–128.
Rucavado, A., Lomonte, B., Ovadia, M., Gutiérrez, J.M., 1995. Local tissue
damage induced by BaP1, a metalloproteinase isolated from Bothrops
asper (terciopelo) snake venom. Exp. Mol. Pathol. 63, 186–199.
Rucavado, A., Núñez, J., Gutiérrez, J.M., 1998. Blister formation and skin
damage induced by BaP1, a haemorrhagic metalloproteinase from the
venom of the snake Bothrops asper. Int. J. Exp. Pathol. 79, 245–254.
Sasa, M., 2009. Natural history of the terciopelo Bothrops asper (Serpentes: Viperidae) in Costa Rica. Toxicon 54, 904–922.
Tanjoni, I., Butera, D., Bento, L., Della-Casa, M.S., Marques-Porto, R.,
Takehara, H.A., Gutiérrez, J.M., Fernandes, I., Moura-da-Silva, A.M.,
2003a. Snake venom metalloproteinases: structure/function relationships studies using monoclonal antibodies. Toxicon 42, 801–
808.
Tanjoni, I., Butera, D., Spencer, P.J., Takehara, H.A., Fernandes, I., Moura-daSilva, A.M., 2003b. Phylogenetic conservation of a snake venom
metalloproteinase epitope recognized by a monoclonal antibody that
neutralizes hemorrhagic activity. Toxicon 42, 809–816.
Tanjoni, I., Evangelista, K., Della-Casa, M.S., Butera, D., Nagalhaes, G.S.,
Baldo, C., Clissa, P.B., Fernandes, I., Eble, J., Moura-da-Silva, A.M., 2010.
Different regions of the class P-III snake venom metalloproteinase
jararhagin are involved in binding to a2b1 integrin and collagen.
Toxicon 55, 1093–1099.
Teixeira, C.F., Chaves, F., Zamunér, S.R., Fernandes, C.M., Zuliani, J.P., CruzHofling, M.A., Fernandes, I., Gutiérrez, J.M., 2005. Effects of neutrophil
depletion in the local pathological alterations and muscle regeneration in mice injected with Bothrops jararaca snake venom. Int. J. Exp.
Pathol. 86, 107–115.
Theakston, R.D.G., Lloyd-Jones, M.J., Reid, H.A., 1977. Micro-ELISA for
detection and assaying snake venom and anti-venom antibody.
Lancet 2, 639–641.
Warrell, D.A., 1992. The global problem of snake bite: its prevention and
treatment. In: Gopalakrishnakone, P., Tan, C.K. (Eds.), Recent
Advances in Toxinology Research, vol. 1. National University of
Singapore, Singapore, pp. 121–153.
Warrell, D.A., 2004. Snakebites in Central and South America: epidemiology, clinical features, and clinical management. In: Campbell, J.
A., Lamar, W.W. (Eds.), The Venomous Reptiles of the Western
Hemisphere, vol. I. Cornell University Press, Ithaca and London, pp.
709–761.
Watanabe, L., Shannon, J.D., Valente, R.H., Rucavado, A., Alape-Girón, A.,
Kamiguti, A.S., Theaskston, R.D., Fox, J.W., Gutiérrez, J.M., Arni, R.K.,
2003. Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple
tissue-damaging activities. Protein. Sci. 12, 2273–2281.