Proteolytic and hemolytic activity in the venom of the lionfish Pterois
Transcripción
Proteolytic and hemolytic activity in the venom of the lionfish Pterois
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 REVISTA CUBANA DE CIENCIAS BIOLÓGICAS RNPS: 2362 • ISSN: 2307-695X • VOL. 4 • N.o 2 • MAYO— SEPTIEMBRE • 2015 • pp. 57-63 . HEMOLYTIC AND PROTEOLYTIC ACTIVITY IN LIONFISH VENOM 58 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 REVISTA CUBANA DE CIENCIAS BIOLÓGICAS RNPS: 2362 • ISSN: 2307-695X • VOL. 4 • N.o 2 • MAYO— SEPTIEMBRE • 2015 • pp. 57-63 . HEMOLYTIC AND PROTEOLYTIC ACTIVITY IN LIONFISH VENOM 59 LENIA MANSO ET AL. −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. REVISTA CUBANA DE CIENCIAS BIOLÓGICAS RNPS: 2362 • ISSN: 2307-695X • VOL. 4 • N.o 2 • MAYO— SEPTIEMBRE • 2015 • pp. 57-63 . HEMOLYTIC AND PROTEOLYTIC ACTIVITY IN LIONFISH VENOM 60 LENIA MANSO ET AL. 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 REVISTA CUBANA DE CIENCIAS BIOLÓGICAS RNPS: 2362 • ISSN: 2307-695X • VOL. 4 • N.o 2 • MAYO— SEPTIEMBRE • 2015 • pp. 57-63 . HEMOLYTIC AND PROTEOLYTIC ACTIVITY IN LIONFISH VENOM 61 LENIA MANSO ET AL. 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 REVISTA CUBANA DE CIENCIAS BIOLÓGICAS RNPS: 2362 • ISSN: 2307-695X • VOL. 4 • N.o 2 • MAYO— SEPTIEMBRE • 2015 • pp. 57-63 . HEMOLYTIC AND PROTEOLYTIC ACTIVITY IN LIONFISH VENOM 62 LENIA MANSO ET AL. (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. 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