Proteolytic and hemolytic activity in the venom of the lionfish Pterois

Transcripción

REVISTA CUBANA DE CIENCIAS BIOLÓGICAS
http://www.rccb.uh.cu
ARTÍCULO ORIGINAL
Proteolytic and hemolytic activity in the venom of the
lionfish Pterois volitans, an invasive species of Cuban
sea coasts
Actividad hemolítica y proteolítica en el veneno del Pez León Pterois
volitans, especie invasora de las costas cubanas
Lenia Manso, Uris Ros, Gilberto Valdés, Maday Alonso del Rivero,
María E. Lanio y Carlos Álvarez*
Centro de Estudio de Proteínas,
Facultad de Biología, Universidad
de La Habana
* Autor para correspondencia:
[email protected]
Recibido: 2015-02-06
Aceptado: 2015-08-04
ABSTRACT
The Indo-Pacific lionfish Pterois volitans (Family Scorpaenidae) is becoming
rapidly established in Cuban sea waters. Due to its potential adverse effects
on human health and the scarce information on venom composition, the
main purpose of this communication is to report a partial biochemical characterization of its venom. The crude venom extract of lionfish Pterois volitans contents abundant proteins with a molecular weight range between 40
up to 100 kDa and possesses gelatinolytic activity in a wide range of pHs,
been maximal at pHs between 7-9 as well as hemolytic activity which is exerted on rabbit but not on human erythrocytes. Remarkably, both hemolytic
and gelatinolytic activities were abolished after heat treatment (60°C, ~15
min) suggesting the proteinaceous nature of the active entity(ies). Furthermore, the molecule(s) responsible for the gelatinolytic activity in the crude
extract was(were) not able to hydrolyze Suc-(Ala)2- Pro- Phe- pNA , BAPA ,
BAEE, Leu-pNa, Ala-pNa, AAFP, AAFR, Gly-Pro-pNa, classical chromogenic
substrates for serine-, cysteine-, and metalloexo-peptidases. The proteolytic
activity corresponding to the most two active components with molecular
weight lower than 45 kDa were inhibited by EDTA, PMSF, and DTT. Altogether the results obtained indicate that the venom of lionfish contains thermolabile protein(s) with high molecular weight, and exhibits hemolytic and proteolytic activities. Furthermore, the proteolytic activity profile and the effect
of proteinase inhibitors together with the molecular weight of the proteases
suggest the presence of matrix metalloendoproteases in the lionfish venom.
Keywords: venomous fish, hemolytic activity, proteolytic activity, lionfish,
invasive species
RNPS: 2362 • ISSN: 2307-695X • VOL. 4 • N.o 2 • MAYO— SEPTIEMBRE • 2015 • pp. 57-63
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HEMOLYTIC AND PROTEOLYTIC ACTIVITY IN LIONFISH VENOM
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LENIA MANSO ET AL.
RESUMEN
El pez león, Pterois volitans (Familia: Scorpaenidae), oriundo del océano Indo-Pacífico, se ha establecido en las
costas de Cuba. Debido a su potencial efecto adverso en la salud humana y la escasa información que existe sobre la composición de su veneno, el propósito fundamental de esta comunicación es informar los resultados de la
caracterización bioquímica parcial del veneno. El extracto crudo del veneno del pez león contiene abundantes
proteínas con pesos moleculares entre 40 y hasta 180 kDa y posee actividad gelatinolítica en un amplio rango de
valores de pH siendo máxima a pHs entre 7 y 9 así como actividad hemolítica (AH) en eritrocitos de conejo. Dicho
extracto carece de AH en eritrocitos humanos. Es de destacar que ambas actividades son abolidas ante el tratamiento con calor (60°C, ~15 min) lo que sugiere la naturaleza proteica de la(s) entidad(es) involucrada(s). Además, la(s) molécula(s) responsable(s) de la actividad gelatinolítica en el veneno crudo no fueron capaces de hidrolizar Suc-(Ala)2- Pro- Phe- pNA , BAPA , BAEE, Leu-pNa, Ala-pNa, AAFP, AAFR, Gly-Pro-pNa, sustratos cromogénicos clásicos para serino-, cisteíno- y metaloexo-peptidasas. La actividad proteolítica de los dos components más
activos de peso molecular menor de 45 kDa resultó inhibida por EDTA, PMSF y DTT. En conjunto, los resultados
obtenidos indican que el extracto del veneno del pez león contiene proteínas termolábiles de alta masa molecular y exhibe actividad hemolítica y proteolítica. El perfil de actividad proteolítica obtenido y el efecto de los inhibidores de proteasas, así como los pesos moleculares de las proteasas, sugiere la presencia de metaloendoproteasas en el veneno del pez león.
Palabras clave: peces venenosos, actividad hemolítica, actividad proteolítica, pez león, especie invasora
Abbreviations: AAFP: N-(4-Metoxyphenylazoformyl)-L-phenylalanine); AAFR: N-(4-Metoxyphenylazoformyl)-L-arginine; ala-pNA: L-Alaninep-nitroanilide; BAEE: benzoyl -arginyl- ethyl- ester ; β-ME: β-Mercaptoethanol; BAPA: benzoyl-arginyl-p-nitroanilida×HCl; DTT: Dithiothreitol ;
EDTA: ethylen-2-amino-4-acetic-acid; E64: trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane; Gly-Pro-pNA: glycil-prolyl-p-nitroanilide;
HA: Hemolytic activity; leu-pNA: leucil-p-nitroanilide; MWM: molecular weight markers; PA: Proteolytic activity; PMSF: phenylmethylsulfonyl fluoride; SDS-PAGE: Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; Suc-(Ala)2- Pro- Phe- pNA:
N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide;TBS: Tris-buffered saline; TLCK; N-alpha-tosyl-L-lysinylchloromethylketone.
INTRODUCTION
The species Pterois volitans, commonly known as
lionfish, belong to the Scorpaenidae family and is naturally found in Indo-Pacific tropical sea waters. Since
2008, Chevalier et al. (2008) have reported the increasing presence of the lionfish Pterois volitans in
Cuban waters where this species has become an extensive invader with unpredictable impact on marine
ecosystems (Schofield, 2010). Besides, this fish possesses venomous glands situated in the base of the
dorsal, pelvic, and anal spines. Accidents caused by
lionfish envenomation are usually not lethal but produce edema, intense pain, necrosis at the site of
puncture, neuromuscular and profound cardiovascular affectations commonly associated with the protein
components of this venom (Church and Hodgson,
2002; Gomes et al., 2009).
In contrast with terrestrial animals, venomous fishes
have been by far less studied. In particular, in lionfish
venom only a gelatinolytic protease of about 45 kDa
(Balasubashini et al.2006 a), a proapoptotic peptide of
around 7.6kDa (Balasubashini et al. 2006 b) as well as
a cytolytic protein of approximately 160 kDa deduced
from a nucleotide sequence (Kiriake and Shiomi,
2011) have been reported. In order to obtain insight
into the proteinaceous composition of the venom
produced by the Cuban coasts invader Pterois volitans
here we describe its protein profile as well as its proteolytic (PA) and hemolytic activity (HA).
MATERIAL AND METHODS
Lionfish specimens were collected along the coast of
Havana, and the venom was prepared according to
Church and Hodgson (2002). Briefly, fishes were
killed by cooling and the venomous spines removed
and stored in 10 % glycerol solution at -80ºC until
processed. The spines were thawed and ground in a
chilled mortar in 10% glycerol solution, centrifuged at
7000 g for 15 min at 4oC, the supernatant filtered
through fiberglass and its protein concentration determined by Bradford (1976). Aliquots were stored at
.
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−80ºC until use. The molecular weight profile of protein inlionfish venom was examined by SDS-PAGE
performed according to Laemmli (1970). The proteolytic activity of lionfish crude extract was analyzed by
zymography using 10% SDS-PAGE containing 0.1% copolimerized gelatin as substrate, according to Heussen and Dowdle (1980). The effect of pH on the proteolytic activity was determined using the same method
(Heussen and Dowdle, 1980). Briefly, gels were incubated overnight at room temperature in the following
buffers: 20mM sodium acetate (pH 3, 4 and 5 ), 20
mM sodium citrate (pH 6), 20mM Tris-HCl (7 and 8),
and 20mM glycine (9 and 10). To identify the mechanistic class of the gelatinolytic proteases in the crude
venom, adequate amounts (1 mM) of classical inhibitors: TLCK (for trypsin- like serine-proteases), PMSF
(for trypsin-chymotrypsin- likeserine-proteases), and
E64 (for cysteine-proteases), from Sigma-Aldrich (St
Louis, USA) were preincubated with venom during 10
minutes. The residual gelatinolytic activity of the extract-inhibitor mixture was evaluated as previously
described, by zymography at pH8 in 20mM Tris-HCl
where the activity of crude extract was optimal.
We also evaluated the effect of the following compounds on the crude venom gelatinolytic activity: Ca2+
(5mM), Pefabloc (5mM), PMSF (5mM), DTT (5mM),
EDTA (5mM) and ortophenantroline (1mM). They
were included in the buffer in which the zymogram
was incubated (20 mMTris-HCl, pH 8). In addition we
evaluated the specificity of cleavage of gelatinases in
Pterois volitans extract using the following chromogenic substrates: Suc-(Ala)2- Pro- Phe- pNA , BAPA ,
BAEE, Leu-pNa, Ala-pNa, AAFP, AAFR, Gly-Pro-pNa,
from Sigma-Aldrich (St Louis, USA) which recognize
different types of proteases (Reytor et al., 2011), see
table 1 for details.Hemolytic activity (HA) against human and rabbit erythrocytes was evaluated turbidimetrically at 600 nm at room temperature (22 ± 2ºC)
as previously described (Martínez et al., 2001). In
short, erythrocyte suspensions were prepared using
pooled fresh red blood cells, washed and resuspended in physiological TrisHCl-buffered saline (TBS, 145
mM NaCl, 10 mM Tris–HCl, pH 7. 4). The cell suspension was diluted to an absorbance of 0.1 at 600 nm.
The crude venom was two-fold serially diluted in saline buffer and the reaction was started by adding the
same volume of cell suspension to each well (200 µl
final volume).
RESULTS AND DISCUSSION
The protein composition of the crude venom as evaluated by SDS PAGE showed numerous protein bands
with molecular weight ranging from 40 to 100 kDa
(figure 1A). These molecular characteristics are in
agreement with those reviewed by Gomes et al.,
(2009) for the crude-venom of other fishes from the
Scorpaenidae family, to which the lionfish belongs.
Upon incubation with β-ME inter-chain disulfide
bonds were reduced as revealed by the disappearance of the 66 and 104 kDa bands, reinforcement of
the 45 kDa molecular specie(s), and appearance of
some other bands in the electrophoregram (figure
1A). This result evinces, as described for the venom of
other fishes of the Scorpaenidae family, the presence
of multimeric proteins stabilized by covalent interactions (Garnier et al., 1995; Karmakar et al., 2004; Poh
et al., 1991). On the other hand, the venom extract
was heated at different temperatures and centrifuged
(1000 g, 15 min) in order to eliminate those proteins
denatured as a consequence of the heat treatment.
The loss of bands as visualized by SDS-PAGE, insofar
A
B
Figure 1: Protein composition of lionfish crude venom determined by
SDS-PAGE. A) Treatment with the reducing agent β-ME. Lane 1:
without β-ME. Lane 2: with β-ME. B) Protein profile of the supernatant after heat treatment. Lane 1: 37oC, 15 min; Lane 2: 37oC, 30
min; Lane 3: 37oC, 60 min Lane 4: 60oC, 15 min. Assay conditions:
gel concentration (10 %), protein applied (100 µg). The gel was
stained with Coomassie Blue.
Figura 1: Composición protéica del veneno crudo de pez león, determinado por SDS-PAGE. A) Tratamiento con el agente reductor βME. Carril 1: sin β-ME. Carril 2: con β-ME. B) Perfil protéico del
sobrenadante luego de tratamiento térmico. Carril 1: 37oC, 15 min;
Carril 2: 37oC, 30 min; Carril 3: 37oC, 60 min Carril 4: 60oC, 15 min.
Condiciones del ensayo: concentración de gel (10 %), proteína aplicada (100 µg). El gel fue teñido con Azul Coomassie.
.
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Table 1. Chromogenic substrates and proteases inhibitors used in enzymatic and inhibition assays in order to identify the mechanistic class of the proteases in Pterois volitans crude venom.
Tabla 1. Sustrato cromogénico e inhibidores de proteasas usados en los ensayos enzimáticos y de inhibición para identificar las
clases mecanísticas de las proteasas en el veneno crudo de Pterois volitans.
Mechanistic class of proteases evaluated (protease type)
Serine-proteases (Trypsin-like)
Serine-proteases (Chymotrypsin-like)
Chromogenic substrates/ Inhibitors
BAPA/ (TLCK, PMSF and Pefabloc)
Suc-(Ala)2- Pro- Phe- pNA/ (PMSF and Pefabloc)
Cysteine-proteases (Papain-like)
BAPA/ (E64, DTT
Metaloexo-peptidases (Aminopeptidase-like)
Metaloexo-peptidases(Carboxipeptidase- Alike)
Leu-pNA, Ala-pNA/ (ortophenantroline, EDTA)
AAFP/ (ortophenantroline, EDTA)
Metaloexo-peptidases (Carboxipeptidase- Blike)
AAFR/ (ortophenantroline, EDTA)
Dipeptidil-peptidase IV
Gly-Pro-pNA/ non evaluated
Esterase
BAEE/ non evaluated
as the incubation temperature increases, indicates
that lionfish venom comprises thermolabile proteins
that become denaturated and insolubilized by incubation at 60oC during 15 min (figure1B). Similarly, it has
been described the presence of thermolabile proteins
for other fishes in the Scorpaenidae family (Gomes et
al., 2009). As the best of our knowledge, this is the
first report of the protein profile of the crude venom
of lionfish and the presence of thermolabile and multichain proteins stabilized by disulfide bonds in the
venom of this species.
Lionfish produces one of the most potent and toxic
fish venoms so far described (Haddad Jr. et al., 2004).
This venom exerts cardiovascular, neuromuscular,
and cytotoxic effects that have been associated with
the presence of several proteinaceous toxins and other active components as acetylcholine or noradrenaline and even pore-forming toxins (Church and Hodgson, 2002); however, the presence of pore-forming
activity has not been experimentally demonstrated
since venom did not promote conductance increase
when evaluated in planar lipid membranes (Cohen
and Olek, 1989). The venom of Pterois volitans contains gelatinolytic proteases with different molecular
masses (around 45 and 60 kDa) that show activity
from pH 3-10 , being largest at pH 7-9 (figure 2A). This
result is in contrast with that obtained by Balasubashini et al.,(2006 a) who reported the presence
of a single protein band with proteolytic activity in
the venom of lionfish. Remarkably, the activity found
in this work was abolished after heat treatment (60°C,
15 min) suggesting the proteinaceous nature of the
active entity(ies), (figure2B). This is the first report of
a thermolabile proteolytic activity in this venom.
The entity(ies) responsible for the gelatinolytic activity in the crude extract was/were not able to hydrolyze classical serine, cysteine, or metalloexopeptidases chromogenic substrates (table 1). The proteolytic
activity corresponding to the most two active components with molecular weight lower than 45 kDa were
inhibited by EDTA, PMSF, and DTT; additionally, these
two bands were reinforced when Ca was added to the
incubation medium. These bands can be not attributed to serine or aspartic proteases, since proteins from
these two latter classes are characterized by a lower
molecular weight (around ~ 20-30 kDa) (MEROPS database). Particularly, the maximal gelatinolytic activity was found between pH 7-9 that would preclude
the contribution of aspartic proteases whose optimum pH are typical in the acid pH range. Summarizing: i. the lack of proteolytic activity against the classical chromogenic substrates, ii. the gelatinolytic activity inhibition by EDTA and DTT (Guo-Ping et al. 2010),
iii. the reinforcement of the proteolytic activity bands
by Ca (Kupai et al., 2010), as well as iv. the molecular
weight banding pattern observed in the zymography,
similar to others reported for gelatinolytic activity,
suggest the presence of matrix metalloendoproteases
in this venom (Guo-Ping et al., 2010). Work is in progress in our laboratory in order to get more insights
into the proteases present in lionfish venom
In order to assess the activity of the venom, the loss
of turbidity of a red cell suspension was quantitatively
related to the crude venom HA, which was expressed
as a function of the extract protein concentration
(figure. 2C). The crude venom extract caused rabbit
erythrocytes lysis at protein concentrations in the
range 2.5 to 30 µg.mL-1 in a dose-dependent way. The
.
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Figure 2: Proteolytic and hemolytic activity of the crude venom produced by lionfish. A) Proteolytic activity evaluated by zymography in SDSPAGE gelatin at various pH values, as indicated in each lane. B) Effect of heat treatment (pH 8) on the proteolytic activity. After treatment
both samples were centrifuged and supernatants applied to lanes 1 as 2. Lane 1: Non-treated. Lane 2: 60ºC, 15 min. Assay conditions: gel
(10%), gelatin (1%) protein mass applied (100 µg). The gel was stained with Coomassie Blue. C) Final extent of hemolysis (t=30 min.) caused by lionfish crude venom in rabbit and human erythrocytes. The 100% control for cell lysis was determined by addition of Stichodactyla
helianthus crude venom. D) Typical time-course of lionfish venom extract (45 µg.mL-1) hemolysis of rabbit erythrocytes after heat treatment
at 60ºC, 15 min. (closed squares: HA positive control with Stichodactyla helianthus total extract, in rabbit erythrocytes; closed circles: lionfish
venom extract, in rabbit erythrocytes; open circles: lionfish venom extract, in human erythrocytes). Assay conditions: TBS pH= 7.4, T =25ºC.
Figura 2: Actividad proteolítica y hemolítica del veneo crudo producido por el pez león. A) Actividad proteolítica evaluado por zimografía en gel SDSPAGE con varios valores de pH, como se indica en cada carril. B) Efecto del tratamiento de calor (pH 8) sobre la actividad proteolítica. Luego del
tratamiento ambas muestras fueron centrifugadas y el sobrenadante aplicado a los carriles 1 y 2. Carril 1: No tratado. Carril 2: 60ºC, 15 min. Condiciones de ensayo: gel (10%), gelatina (1%) masa de proteína aplicada (100 µg). El gel fue teñido con Azul Coomassie. C) Grado final de hemolisis
(t=30 min.) causada por el veneno crudo de pez león en eritrocitos de ratón y humanos. El control del 100 % de la lisis celular fue determinado por la
adición de veneno de veneno crudo de Stichodactyla helianthus. D) Dinámica típica de la hemólisis producida por extracto de veneno crudo de pez
león (45 µg.mL-1) sobre eritrocitos de ratón luego de tratamiento de calor a 60ºC, 15 min. (cuadrados llenos: HA control positivo con extracto total
de Stichodactyla helianthus, en eritrocitos de ratón; círculos rellenos: extracto de veneno de pez león, en eritrocitos de conejo; círculos vacíos: extracto de veneno de pez león, en eritrocitos humanos). Condiciones del ensayo: TBS pH= 7.4, T =25ºC.
HA of lionfish venom was compared with the HA of
the total body extract obtained from the sea anemone Stichodactyla helianthus which contains two poreforming toxins with high HA (in the nM concentration
range) that have been well characterized by our laboratory (Alvarez et al., 2009; Lanio et al., 2001). The HA
of lionfish venom lied below the positive control of
hemolysis induced by Stichodactyla helianthus total
body extract for concentrations lower than 20 μg.mL-1.
Furthermore, the lionfish venom extract was unable
to promote lysis of human erythrocytes (Figure. 2C).
This result is in agreement with those reported by
Shiomi et al., (1989) who demonstrated that Pterois
volitans venom´s HA is highly specific against rabbit
erythrocytes. Since most fish venoms lack phospholipase activity, their hemolytic action has been postulated to be preceded by binding of the lytic component to
a protein receptor on the surface of erythrocytes
.
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(Chhatwal and Dreyer, 1992). However, no further
information is available on this mechanism or the
nature of the lytic component involved. One interesting alternative is that this effect could be attributed to the action of proteases (Khoo et al., 1992), of
the venom that could interact with some membrane
proteins, destabilizing the cell barrier and causing
lysis, as it has been previously claimed for the venom
of the fish Trachinus draco (Chhatwal and Dreyer,
1992) and even the total extract of the sea anemone
Paracondylactis indicus (Adhikari et al., 2007). More
recently, the HA in the venom of the scorpionfish
Scorpaena plumieri was attributed to a pore-forming
protein (Gomes et al., 2013). The possibility of the
presence of pore-forming toxins with this high species
-specificity in lionfish venom is currently under examination in our laboratory. Interestingly, as demonstrated for the proteolytic activity, the HA was abolished
after heat treatment (60°C, 15 min) (Fig. 2D). For the
best of our knowledge, this is the first report of thermolabile hemolytic molecular entit(ies) in the lionfish
venom.
In conclusion this work provides valuable information
about the protein profile and functional activity of the
crude-venom of the lionfish Pterois volitans a species
becoming abundant in the Cuban sea coasts. Lionfish
venom contains proteins of different molecular
weight (40 – 100 kDa), does not hydrolyze classical
chromogenic substrates of serine-, cysteine- or metalloexo-peptidases, and exerts gelatinolytic and hemolytic activity. Both activities are inactivated by heating
(60oC, 15 min) but so far there is no enough evidence
to consider that the HA and PA would reside in the
same molecule. Work is in progress in order to identify, purify, and characterize the components responsible for both activities. These studies should contribute
to understand the complex composition of lionfish
venom.
ACKNOWLEDGEMENTS
The authors thank Pedro Chevalier and Erlan Cabrera from
Cuban National Aquarium for helping with the collection of
the lionfish specimens. This work was partly supported by
CAPES-MES and CNPq-MES (Brazil-Cuba) collaboration projects and Iberoamerican CYTED BIOTOX Network
(212RT0467). U.R is a grantee from IFS (4616), Sweden.
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Shiomi, K.; M. Hosaka, S. Fujita, H. Yamanaka,T. Kikuchi (1989)
Venoms from six species of marine fish: lethal and hemolytic
activities and their neutralization by commercial stonefish antivenom. Mar. Biol. 103:285–289.
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Editor para correspondencia: Dr. Dennis Denis Ávila
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Proteolytic and hemolytic activity in the venom of the lionfish Pterois

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