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Myotoxic phospholipases A2 in Bothrops snake venoms: Effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu
Biochimie 82 (2000) 755−763 © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908400011500/FLA
Myotoxic phospholipases A2 in Bothrops snake venoms: Effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu Silvia H. Andrião-Escarsoa, Andreimar M. Soaresa,b, Veridiana M. Rodriguesa, Yamileth Angulob, Cecília Díazb, Bruno Lomonteb, José M. Gutiérrezb, José R. Giglioa* a
Departamento de Bioquímica, Faculdade de Medicina, Universidade de São Paulo, 14049-900, Ribeirão Preto, SP, Brazil b Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica (Received 13 December 1999; accepted 30 March 2000)
Abstract — Venoms from eight Bothrops spp. were fractionated by ion-exchange chromatography on CM-Sepharose at pH 8.0 for the purification of myotoxins. Chromatographic profiles showed differences regarding myotoxic components among these venoms. B. alternatus, B. atrox and B. jararaca venoms did not show the major basic myotoxic fractions identified in the other venoms. Polyacrylamide gel electrophoresis for basic proteins also showed distinct patterns for these toxins. In vivo, all the isolated myotoxins induced release of creatine kinase due to necrosis of muscle fibers, accompanied by polymorphonuclear cell infiltration, and edema in the mouse paw. In addition, the toxins showed cytotoxic and liposome-disrupting activities in vitro. B. jararacussu bothropstoxins-I (BthTX-I) and II (BthTX-II) were submitted to chemical modifications of: His, by 4-bromophenacyl bromide (BPB) or photooxidation by Rose Bengal (RB); Tyr, by 2-nitrobenzenesulphonyl fluoride (NBSF); and Trp, by o-nitrophenylsulphenyl chloride (NPSC). The myotoxic and cytotoxic activities of BthTX-I, a Lys49 PLA2 homologue, after modification by BPB, RB, NBSF and NPSC, were reduced to 50%, 20%, 75%, 65% and 13%, 0.5%, 76%, 58%, respectively. However, the edema-inducing and liposome-disrupting activities were not significantly reduced by the above modifications. BPB-treated BthTX-II, an Asp49 PLA2 homologue, lost most of its catalytic, indirect hemolytic, anticoagulant, myotoxic and cytotoxic activities. The edema-inducing and liposome-disrupting activities were reduced to 50% and 80%, respectively. Lethality caused by BthTX-I and -II was strongly reduced after treatment with BPB or RB, but only partially with NBSF or NPSC. BthTX-I and -II, both native or modified, migrated similarly in a charge-shift electrophoresis. Antibodies raised against BthTX-I or -II, B. asper Basp-II and the C-terminal 115-129 peptide from Basp-II did not show significant differences in their cross-reactivity with the modified toxins, except with RB photooxidized toxins. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS Bothrops venoms / myotoxins / phospholipases A2 / pharmacological activity / chemical modifications
1. Introduction Phospholipases A2 (PLA2; EC 3.1.1.4) catalyze the Ca2+-dependent hydrolysis of the sn-2 ester bond of phosphoglycerides. They are widely distributed in pancreatic secretions, inflammatory exudates and snake and arthropod venoms [1, 2]. In addition to their normal digestive action, a wide variety of pharmacological activities has been described for venom PLA2s, such as neurotoxic, myotoxic, edematogenic, hypotensive, plateletaggregating, cardiotoxic and anticoagulant activities * Correspondence and reprints: [email protected] Abbreviations: PLA2, phospholipase A2; BthTX-I and II, Bothrops jararacussu myotoxins-I and II; PBS, phosphatebuffered saline; CK, creatine kinase; N, native; M, modified; BPB, 4-bromophenacyl bromide; RB, Rose Bengal; NBSF, 2-nitrobenzenesulphonyl fluoride; NPSC, o-nitrophenylsulphenyl chloride; AMBIC, ammonium bicarbonate buffer; SDS, sodium dodecyl sulfate.
[3–5]. This diverse pharmacological profile has been acquired through evolution by a positive Darwinian selection in the protein-coding exons by an accelerated evolutionary process that has resulted in many variants with diverse pharmacological effects [6]. A number of class II myotoxic PLA2s have been isolated from Bothrops snake venoms [7]. Species from this genus inflict the vast majority of snakebites in Latin America, and acute muscle damage, myonecrosis, is a common finding in these envenomations [8, 9]. Myotoxic PLA2s present in Bothrops venoms are either Asp-49, catalytically-active variants or Lys-49, enzymaticallyinactive homologues [7]. This substitution in the calciumbinding loop renders Lys-49 homologues unable to bind calcium, with the consequent inability of the protein to stabilize the tetrahedral intermediate in the catalytic mechanism. However, despite their lack of enzymatic activity, Lys-49 PLA2s induce myotoxicity by a poorly known mechanism. Thus, comparative studies on Bothrops myotoxic PLA2s and insights in the structure-
756 function relationship of these molecules may help to elucidate the mechanism by which they disrupt membrane integrity. In this study we report a comparative chromatographic analysis of the venoms of eight species of Bothrops, in order to identify the presence of basic myotoxic PLA2s. In addition, chemical modifications of specific residues were performed in Asp-49 and Lys-49 PLA2 variants from B. jararacussu venom, to study the effects of such modifications in enzymatic and pharmacological properties. 2. Materials and methods 2.1. Reagents Venoms were donated by the serpentarium at Faculdade de Medicina de Ribeirão Preto (SP, Brazil) and CEVAPUNESP (Botucatu, SP, Brazil). 4-bromophenacyl bromide (BPB), 2-nitrobenzenesulphonyl fluoride (NBSF) and o-nitrophenylsulphenyl chloride were obtained from Sigma Chemical Co. (St. Louis, USA). 2.2. Purification of toxins Eight lyophilized venoms from Bothrops spp. (200–250 mg) were fractionated on a CM-Sepharose column (2 × 20 cm), which was previously equilibrated with 0.05 M ammonium bicarbonate buffer, pH 8.0 [10–12]. Elution was carried out with a continuous gradient up to a concentration of 0.5 M ammonium bicarbonate. Absorbance of the effluent solution was recorded at a wavelength of 280 nm.
Andrião-Escarso et al. tion [12]. CK concentration was determined using 4 µL of plasma according to the manufacturer’s instructions. The enzyme activity was expressed in U/L, and one unit results from the phosphorylation of one µmol of creatine/min at 25 °C. 2.6. Cytotoxic activity This activity was determined on endothelial cells (tEnd) and myoblasts (C2C12) as previously described [18]. Briefly, different concentrations of BthTX-I and II were diluted in culture medium (Dulbecco’s modified Eagle’s medium supplemented with 1% fetal calf serum) and then added to the cells in a total volume of 150 µL/ well. Positive controls consisted in cells treated with 0.1% Triton X-100. After 3 h of incubation, 100 µL of medium was collected for determination of lactic dehydrogenase activity using a colorimetric end-point assay obtained from Sigma Chemical Co. 2.7. Edema Groups of five male Swiss mice (18–22 g) were injected in the subplantar region with 50 µL of BthTX-I and II (100 µg). After different intervals, the edema at the paw was measured using a low pressure spring caliper (Mitutoyo, Japan) [12, 19]. Zero time points were subtracted from all the values and the differences were expressed as media percentage ± S.D. 2.8. Liposome-disrupting activity
Venoms were analyzed by electrophoresis for basic proteins and SDS-PAGE according to Reisfeld et al. [13] and Laemmli [14], respectively.
Negatively-charged liposomes (phosphatidylserine, 63 µmol; dicethylphosphate, 18 µmol; cholesterol, 9 µmol) were obtained from Sigma Chemical Co. The assay was performed according to Díaz et al. [20] by incubating 20 µL of the liposome suspension with 20 µL of BthTX-I or II (in PBS) for 30 min at 37 °C or 4 °C.
2.4. Enzymatic, anticoagulant and hemolytic activities
2.9. Lethal dose 50%
Phospholipase A2 activity was tested using egg yolk as a substrate [15]. Anticoagulant [16] and indirect hemolytic activities [17] were assayed for venoms and isolated toxins.
Lethality induced by BthTX-I and II was evaluated by i.p. injection of 100 µL of different toxin concentrations. This assay was performed in male Swiss mice (18–22 g; n = 6) within 48 h.
2.5. Myotoxic activity
2.10. Chemical modifications
The assay for determination of plasma creatine kinase (CK) activity was carried out using the CK-UV kinetic kit from Sigma Chemical Co. Toxins (2 µg/µL) were injected in 18–22 g male Swiss mice (50 µL, i.m.) (n = 6). Animals used as negative controls were injected with PBS. After 3 and 6 h, a blood sample was collected from the tail in heparin-coated tubes and centrifuged for plasma separa-
Modification of histidine-48 residues with BPB was carried out as previously described [21] using ammonium bicarbonate buffer instead of Tris-HCl. Briefly, 3 mg of toxin were dissolved in 1 mL of 0.1 M ammonium bicarbonate containing 0.7 mM EDTA (pH 8.0) and 150 µL of BPB (0.8 mg/mL) were added. The mixture was incubated for 24 h at 25 °C.
2.3. Biochemical characterization of toxins
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Figure 1. Comparative chromatographic profiles of eight Bothrops venoms on a CMSepharose column (2 × 20 cm). Samples of 200–250 mg were applied and elution was performed with a 0.05–0.5 M ammonium bicarbonate buffer gradient at pH 8.0 at 25 °C. Absorbances were recorded at 280 nm.
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Table I. Phospholipase A2 activity, minimal edematogenic doses (MED, µg ± S.D.) and CK release in plasma (U/l ± S.D.) for the crude venoms and isolated Bothrops myotoxins. For identification of fractions see figure 1. All of them are highly purified myotoxins, except J-I (which contains acidic nonmyotoxic PLA2s), At-VI and Jar-III. Venoms or toxins PLA2 activity MED (µg)b (U/mg)a PBS B. jararacussu J-I BthTX-I BthTX-II B. pirajai PrTX-I PrTX-II PrTX-III B. moojeni MjTX-I MjTX-II B. neuwiedi BnSP-7 B. asper Basp-II B. atrox At-VI B. jararaca Jar-III B. alternatus
0 71.02 119.34 0 37.08 75.04 0 0 38.71 78.37 0 0 78.99 0 82.05 0 107.52 9.34 29.42 0 26.90
3.3 ± 0.2 n.d. n.d. 31.3 ± 1.3 23.8 ± 1.1 n.d. 38.5 ± 1.8 40.5 ± 2.0 25.5 ± 1.7 n.d. 34.6 ± 1.4 32.2 ± 1.5 n.d. 36.8 ± 1.8 n.d. 38.9 ± 1.9 n.d. n.d. n.d. n.d. n.d.
CK (U/l)c 189.53 ± 45.6 3 197.08 ± 219.4 798.01 ± 100.2 2 279.35 ± 172.7 3 556.32 ± 228.6 3 091.40 ± 233.0 2 132.95 ± 193.2 1 787.50 ± 145.0 3 520.20 ± 244.5 2 798.73 ± 186.3 1 759.72 ± 180.0 2 189.91 ± 164.4 3 349.01 ± 253.0 2 056.81 ± 142.8 2 769.98 ± 166.8 2 140.82 ± 187.4 1 307.90 ± 109.7 882.33 ± 176.4 990.59 ± 87.9 649.16 ± 43.1 971.01 ± 56.1
n.d., not determined. a PLA2 activity on egg yolk substrate. Minimal edematogenic dose. c Myotoxicity activity 3 h after injection (50 µg/50 µL). b
Tyrosine residues were modified by treatment with NBSF as previously described [22]. Briefly, 1 µmol of BthTX-I and II (9 µmol of tyrosine) were dissolved in 14 mL 0.1 M Tris-HCl (pH 8.0) and incubated with 9 µmol of NBSF for 20 h at 25 °C. Modification of tryptophan residues was performed according to Takasaki et al. [23]. Briefly, 9 mg of BthTX-I and II were dissolved in 4 mL 50% acetic acid containing 1 mg of NPSC and the reaction was left to proceed for 1 h at 25 °C. Molar extinction coeficients of native (N) and modified (M) toxins were used to follow the reaction. In all cases, excess reagent was removed by ultrafiltration through an Amicon YM-3 membrane, and washed with water or 0.05 M amonium bicarbonate (pH 8.0). 2.11. Charge-shift electrophoresis In order to check the amphiphilic characteristics of the toxins, BthTX-I and II were run in an agarose gel in the presence of anionic, neutral and cationic detergents, as previously described [24].
Figure 2. A. PAGE for basic proteins of the whole venoms and isolated toxins. Lanes: 1, B. asper; 2, Basp-II; 3, B. jararacussu; 4, BthTX-I; 5, BthTX-II; 6, B. moojeni; 7, MjTX-I; 8, MjTX-II; 9, B. pirajai; 10, PrTX-I; 11, PrTX-II; 12, PrTX-III. B. SDSPAGE of the major isolated toxins. Lanes: 1, standard: a) serum albumin (67 000); b) ovalbumin (45 000); c) carbonic anhydrase (29 000); d) cytochrome c (12 380); 2, B. asper Basp-II; 3, B. jararacussu BthTX-I; 4, B. jararacussu BthTX-II; 5, B. moojeni MjTX-I; 6, B. moojeni MjTX-II; 7, B. neuwiedi BnSP-7; 8, B. pirajai PrTX-I; 9, B. pirajai PrTX-II; 10, cytochrome c; and 11, B. pirajai PrTX-III.
2.12. Enzyme-immunoassay 96-well plates were coated overnight with 0.2 µg/well BthTX-I and II [25]. Cross-reactivity of these toxins was determined using antibodies raised against Bothrops jararacussu BthTX-I and II and B. asper myotoxin II (whole molecule and the C-terminal peptide 115–129). Normal serum and crotamine were used as negative controls.
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Table II. Summarized biological effects of Bothrops jararacussu N or M-BthTX-I and II. Myotoxin BthTX-I BPB-BthTX-I RB-BthTX-I NBSF-BthTX-I NPSC-BthTX-I BthTX-II BPB-BthTX-II RB-BthTX-II NBSF-BthTXII NPSC-BthTXII
DL50 (mg/kg)
Myotoxicitya (%)
Edemab (%)
Cytotoxicityc (%)
Liposome disrupting activityd (%)
PLA2 activitye (U/mg)
8.5 ± 0.85 40.2 ± 0.38 40.8 ± 0.45 24.5 ± 0.48 15.2 ± 0.71 7.0 ± 0.81 41.8 ± 0.39 42.1 ± 0.32 14.6 ± 0.65 14.8 ± 0.71
100 49.3 19.2 73.3 62.7 100 0.5 0.2 23.8 48.5
125 ± 3.8 110 ± 4.2 98 ± 2.6 118 ± 3.2 121 ± 2.7 155 ± 5.7 74 ± 1.9 76 ± 3.4 121 ± 4.6 127 ± 4.4
100 ± 1 13 ± 2 0.5 ± 0.02 76 ± 1 58 ± 3 95 ± 2 1 ± 0.02 0.5 ± 0.02 1 ± 0.02 65 ± 1
84.3 81.1 81.5 79.6 80.1 95.8 78.2 76.4 80.5 79.8
0 0 0 0 0 37.09 0.05 5.32 18.56 19.44
a Values 3 h after inoculation of BthTX-I and II (100 µg). b Values 30 min after inoculation of BthTX-I and II (100 µg). c Values for doses of 20 µg of BthTX-I and II. d Values for doses of 2.5 µg of BthTX-I and II incubated at 37°C. e Values for doses of 50 µg of BthTX-I and II.
3. Results Chromatographic fractionation of eight Bothrops venoms on CM-Sepharose revealed different PLA2 myotoxins in all the venoms except B. atrox, B. jararaca and B. alternatus (figure 1). B. jararacussu BthTX-II and B. pirajai PrTX-III, which are Asp49-PLA2, showed moderate catalytic activity whereas the other PLA2s tested, which are Lys49 isoforms, showed extremely low or no activity at all when tested on phospholipids present on egg yolk (table I). Figure 2 shows the electrophoretic patterns of the venoms and isolated toxins. All venoms induced the release of creatine kinase (CK) after 3 h of injection, due to a direct action on muscle by their myotoxins (table I) and some of them also by indirect action (6 h after injection) by the effect of proteases on the blood vessels (results not shown). Table I also shows the minimal edema-inducing dose (MED, defined as µg of protein able to induce 30% of edema) of some of the venoms. Histopathological examination showed severe myonecrosis 24 h after myotoxin inoculation (results not shown). These alterations were characterized by the onset of intense edema and loss of muscle cell structure. Myonecrosis was accompanied by leukocyte infiltration, and no lesion of capillary vessels, veins or arteries was observed. After chemical modification of BthTX-I and II, amino acid composition showed that one His or two Tyr residues were modified by BPB and NBSF, respectively (results not shown). Photooxidation by RB was able to modify other residues as well. Both toxins, which are highly toxic when injected i.p., decreased their lethal activity after chemical modification of their amino acid residues with BPB and RB (table II). Chemical modification also altered other activities. The enzymatic activity of N-BthTX-II, for example, which was very low when compared with the activity of acidic
PLA2s present at the same venom (see J-I, table I), was almost completely abolished by BPB and partially modified by RB, NBSF and NPSC. Regarding the myotoxic effects, both N-BthTX-I and N-BthTX-II induced muscle damage and release of intracellular CK as can be seen in figure 3. After alkylation with BPB, BthTX-I lost around 50% of its activity whereas BthTX-II lost all of it. Photooxidation by RB, on the other hand, reduced in a high extent the release of CK while NBSF and NPSC were partially effective reducing around 25% and 35% of the activity, in the case of BthTX-I and 70% and 50%, in the case of BthTX-II, respectively. The cytotoxic activity of BthTX-I and II was almost completely abolished by treatment with BPB and RB. In addition, NBSF was also able to strongly reduce this activity in BthTX-II (figure 4). The other modifications also affected the activity but to a lesser extent (figure 4). The edema-inducing effect of BthTX-II was also partially inhibited by the treatment with the alkylating reagents (figure 5) whereas the ability to disrupt liposomes (table II) and the electrophoresis mobility on the chargeshift system practically did not change (results not shown). Figure 6 shows the effect of the different chemical modifications on the hemolytic and anticoagulant activities of BthTX-II. It can be seen that even when BPB was the most effective reagent altering these activities, most of the treatments had some effect. No significant alterations in the structure, however, were determined by comparing the immunogenicity of the modified and native toxins, except for a small decrease (20%) observed in the crossreactivity after toxin photooxidation, when antibodies against B. asper MT-II and its peptide 115–129 were tested (results not shown).
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Figure 3. Myotoxic activity of Bothrops jararacussu BthTX-I (A) and II (B) in mice. Time course of the plasma creatine kinase (CK) increased after the intramuscular injection of 100 µg/50 µL of PBS, native or BPB, RB, NBSF and NPSC modified toxins. Results are presented as means ± S.D. (n = 6).
Figure 4. Cytotoxic effect of BthTX-I (A) and II (B) and chemically-modified variants on C2C12 myoblasts/myotubes in culture. Doses of 20 µg of native and modified myotoxin were incubated with cells for 3 h at 37 °C, and the release of lactate dehydrogenase (LDH) into the supernatant was quantified. Results represent mean ± S.D. (n = 3).
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Figure 5. Edema induced by native or modified BthTX-I (A) and II (B) (100 µg/50 µL) in the paw of 18–22 g male Swiss mice. PBS was used as a control. Each bar represents the mean ± S.D. (n = 5).
4. Discussion Myotoxic PLA2s from Bothrops snake venoms are usually basic proteins, often the last venom components eluting from cation exchange columns using salt gradients at neutral pH [5]. The CM-Sepharose purification procedure described here resulted in the isolation of homogeneous myotoxins from the venoms of B. jararacussu, B. pirajai, B. moojeni, B. neuwiedi and B. asper in a single step. These venoms induced the release of creatine kinase to plasma 3 h after i.m. injection, while those from B. atrox, B. jararaca and B. alternatus showed a low to moderate myotoxicity, only 6 h after injection. The absence of major basic myotoxins in these three venoms, as previously reported [26–28], is confirmed by the low myotoxic activity of the last fractions eluted from the CM-Sepharose, and may explain this delayed response. Myotoxicity may result from a direct action of myotoxins upon the plasma membranes of muscle cells, or it may be due to an indirect vascular degeneration and ischemia caused by venom metalloproteinases [4, 5]. Among the isolated myotoxins, only two are Asp49PLA2 showing catalytic, anticoagulant and indirect hemolytic activities. The remaining ones are Lys49 myotoxins. All of them, but mainly the Asp49 variants,
induced myonecrosis. The higher myotoxic activity of the Asp49 variants, compared to the Lys49 variants, suggests an enhancing action of the catalytic activity upon toxic effects. However, since other important structural differences between these two groups of proteins exist, alternative explanations remain open. Treatment of N-BthTX-I with BPB reduced 45%, 85% and 15% of its myotoxic, cytotoxic and edema inducing activity, respectively, with no significant change in its liposome-disrupting activity. The Tyr and Trp residues of this toxin apparently display a more relevant contribution to the myotoxic and cytotoxic, but not to the edemainducing and liposome-disrupting effects. Díaz et al. [29] and Soares et al. [30] showed that BPB treatment of myotoxin II and BnSP-7, Lys49 myotoxins from B. asper and B. neuwiedi, respectively, reduced 40–55% of its myotoxic activity, but the liposome-disrupting effect was less affected. This same inference may be drawn regarding the almost complete abolishment of the PLA2, anticoagulant, myotoxic and cytotoxic activities of the BPB-treated BthTX-II, while the edema-inducing and liposomedisrupting activities were reduced only 50% and 20%, respectively. It is likely that BthTX-II can lyse liposomes independently of its catalytic activity. These data can be
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Andrião-Escarso et al. independent upon but eventually potentiated by the PLA2 activity. Since however the myotoxic activity of BPBtreated BthTX-I was significantly lower than that of N-BthTX-I, an hypothetic ‘in vivo’ lipolytic activity of this and probably other Lys-49 PLA2s cannot be excluded. Alternatively, modification of this protein by BPB may result in conformational changes affecting its myotoxic mechanism. Photooxidation of both BthTX-I and II by RB modified other residues other than His48. In BthTX-II, the enzymatic, anticoagulant and edema-inducing activities were partially, while the myotoxic and cytotoxic activities were totally abolished. The significant change of the electrophoretic mobility denotes a more drastic structural modification which may be advantageous for the sake of attenuating the lethal effect of the toxin while keeping its antigenicity for the production of antibodies. The data on the modifications of Tyr and Trp residues of BthTX-II reveal a correlation between myotoxic and cytotoxic effects, but less with the catalytic, edemainducing and liposome-rupturing activities, suggesting that these residues are less important for the hydrolytic activity on phospholipids and inflammatory reaction. The finding that N-(native) and M-(modified)-BthTX-I and II were recognised by the antibodies raised against BthTX-I, BthTX-II and C-terminal 115-129 from Basp-II, while the photooxidized forms were only partially recognized, suggests that RB drastically changes the structure of the toxins. Recognition by Basp-II anti-C terminal indicates that this region is antigenically conserved. This region of Basp-II is believed to play an important role in its myotoxic and cytotoxic effects [25, 31–33]. This conclusion may probably be extended to BthTX-I, since Basp-II and BthTX-I have of 98% sequence identity at the C-terminal region. In conclusion, our results show that in BthTX-I, which is catalytically inactive, the His, Tyr and Trp residues play an important role in the myotoxic and cytotoxic activities, but not so in the edema-inducing and liposome-rupturing activities. While for BthTX-II the catalytic activity appears to be relevant to its anticoagulant, myotoxic, cytotoxic and edema inducing effects, it is not for the liposome-disrupting activity. Acknowledgments
Figure 6. Indirect hemolytic (A) and anticoagulant (B) activity of BthTX-II. Results are presented as mean ± S.D. (n = 3).
explained on the basis of a set of pharmacological domains distinct from the catalytic site and apparently
The authors gratefully acknowledge the financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Consejo Nacional de Investigaciones Científicas y Tecnológicas de Costa Rica (FO-013-98), International Foundation for Science (F/2766-1), as well as the skilfull technical assistance of Odete A.B. Cunha, Luiz H. AnzaloniPedrosa and Carlos A. Vieira. This work is part of the Doctoral Thesis of Silvia H. Andrião-Escarso and Andreimar M. Soares at Departamento de Bioquímica, Faculdade de Medicina, Universidade de São Paulo-SP, Brasil.
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763 [19] Lomonte B., Tarkowski A., Hanson L.A., Host response to Bothrops asper snake venom: analysis of edema formation, inflammatory cells, and cytokine release in a mouse model, Inflammation 17 (1993) 93–105. [20] Díaz C., Gutiérrez J.M., Lomonte B., Gené J.A., The effect of myotoxins isolated from Bothrops snake venoms on multilamelar lipossomes, Biochim. Biophys. Acta 1070 (1991) 455–460. [21] Díaz-Oreiro C., Gutiérrez J.M., Chemical modification of histidine and lysine residues of myotoxic phospholipases A2 isolated from Bothrops asper and Bothrops godmani snake venoms, Toxicon 35 (1997) 241–252. [22] Soons K.R., Condrea E., Yang C.C., Rosenberg P., Effects of modification of tyrosines 3 and 62 (63) on enzymatic and toxicological properties of phospholipases A2 from Naja nigricolis and Naja naja atra snake venoms, Toxicon 24 (1986) 679–693. [23] Takasaki A., Sugama A., Yanagita A., Tamiya N., Rowan E.G., Harvey A.L., Effects of chemical modification of PA-11, a phospholipase A2 from the venom of Australian king brown snake, on its biological activities, Toxicon 28 (1990) 107–117. [24] Helenius A., Simons K., Charge shift electrophoresis: single method for distinguishing between amphiphilic and hydrophilic proteins in detergent solution, Proc. Natl. Acad. Sci. USA 74 (1977) 529–532. [25] Calderón L., Lomonte B., Immunochemical characterization and role in toxic activities of region 115-129 of myotoxin II, a Lys49 phospholipase A2 from Bothrops asper snake venom, Arch. Biochem. Biophys. 358 (1998) 343–350. [26] Moura-da-Silva A.M., Cardoso D.F., Tanizaki M.M., Differences in distribution of myotoxic proteins in venoms from Bothrops species, Toxicon 28 (1990) 1293–1301. [27] Moura-da-Silva A.M., Desmond H., Laing G., Theakston R.G.D., Isolation and comparison of myotoxins isolated from venoms of different species of Bothrops snakes, Toxicon 29 (1991) 713–723. [28] Soares A.M., Anzaloni-Pedrosa L.H., Fontes M.R.M., da Silva R.J., Giglio J.R., Polyacrylamide gel electrophoresis as a tool for the taxonomic identification of snakes from the Elapidae and Viperidae families, J. Ven. Anim. Toxins 4 (1998) 137–142. [29] Díaz C., Gutiérrez J.M., Lomonte B., Nunez J., p-bromophenacyl bromide modification of Bothrops asper myotoxin II, a lysine-49 phospholipase A2, affects its pharmacological activities, Toxicon 31 (1993) 1202–1206. [30] Soares A.M., Guerra-Sá R., Borja-Oliveira C.R., Rodrigues V.M., Rodrigues-Simioni L., Rodrigues V., Fontes M.R.M., Lomonte B., Gutiérrez J.M., Giglio J.R., Structural and functional characterization of BnSP-7, a Lysine-49 myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom, Arch. Biochem. Biophys. 378 (2000) 201–209. [31] Lomonte B., Tarkowski A., Bagge U., Hanson L.A., Neutralization of the cytolitic and myotoxic activities of phospholipases A2 from Bothrops asper snake venom by glycosaminoglycans of the heparin/heparan sulfate family, Biochem. Pharmacol. 47 (1994) 1509–1518. [32] Lomonte B., Moreno E., Tarkowski A., Hanson L.A., Maccarana M., Neutralizing interaction between heparins and myotoxin II, a Lys-49 phospholipase A2 from Bothrops asper snake venom, J. Biol. Chem. 269 (1994) 29867–29873. [33] Calderón L., Lomonte B., Inhibition of the myotoxic activity of Bothrops asper myotoxin II in mice by immunization with synthetic 13-mer peptide 115-129, Toxicon 37 (1999) 683–687.
Myotoxic phospholipases A2 in Bothrops snake venoms: Effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu Silvia H. Andrião-Escarsoa, Andreimar M. Soaresa,b, Veridiana M. Rodriguesa, Yamileth Angulob, Cecília Díazb, Bruno Lomonteb, José M. Gutiérrezb, José R. Giglioa* a
Departamento de Bioquímica, Faculdade de Medicina, Universidade de São Paulo, 14049-900, Ribeirão Preto, SP, Brazil b Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica (Received 13 December 1999; accepted 30 March 2000)
Abstract — Venoms from eight Bothrops spp. were fractionated by ion-exchange chromatography on CM-Sepharose at pH 8.0 for the purification of myotoxins. Chromatographic profiles showed differences regarding myotoxic components among these venoms. B. alternatus, B. atrox and B. jararaca venoms did not show the major basic myotoxic fractions identified in the other venoms. Polyacrylamide gel electrophoresis for basic proteins also showed distinct patterns for these toxins. In vivo, all the isolated myotoxins induced release of creatine kinase due to necrosis of muscle fibers, accompanied by polymorphonuclear cell infiltration, and edema in the mouse paw. In addition, the toxins showed cytotoxic and liposome-disrupting activities in vitro. B. jararacussu bothropstoxins-I (BthTX-I) and II (BthTX-II) were submitted to chemical modifications of: His, by 4-bromophenacyl bromide (BPB) or photooxidation by Rose Bengal (RB); Tyr, by 2-nitrobenzenesulphonyl fluoride (NBSF); and Trp, by o-nitrophenylsulphenyl chloride (NPSC). The myotoxic and cytotoxic activities of BthTX-I, a Lys49 PLA2 homologue, after modification by BPB, RB, NBSF and NPSC, were reduced to 50%, 20%, 75%, 65% and 13%, 0.5%, 76%, 58%, respectively. However, the edema-inducing and liposome-disrupting activities were not significantly reduced by the above modifications. BPB-treated BthTX-II, an Asp49 PLA2 homologue, lost most of its catalytic, indirect hemolytic, anticoagulant, myotoxic and cytotoxic activities. The edema-inducing and liposome-disrupting activities were reduced to 50% and 80%, respectively. Lethality caused by BthTX-I and -II was strongly reduced after treatment with BPB or RB, but only partially with NBSF or NPSC. BthTX-I and -II, both native or modified, migrated similarly in a charge-shift electrophoresis. Antibodies raised against BthTX-I or -II, B. asper Basp-II and the C-terminal 115-129 peptide from Basp-II did not show significant differences in their cross-reactivity with the modified toxins, except with RB photooxidized toxins. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS Bothrops venoms / myotoxins / phospholipases A2 / pharmacological activity / chemical modifications
1. Introduction Phospholipases A2 (PLA2; EC 3.1.1.4) catalyze the Ca2+-dependent hydrolysis of the sn-2 ester bond of phosphoglycerides. They are widely distributed in pancreatic secretions, inflammatory exudates and snake and arthropod venoms [1, 2]. In addition to their normal digestive action, a wide variety of pharmacological activities has been described for venom PLA2s, such as neurotoxic, myotoxic, edematogenic, hypotensive, plateletaggregating, cardiotoxic and anticoagulant activities * Correspondence and reprints: [email protected] Abbreviations: PLA2, phospholipase A2; BthTX-I and II, Bothrops jararacussu myotoxins-I and II; PBS, phosphatebuffered saline; CK, creatine kinase; N, native; M, modified; BPB, 4-bromophenacyl bromide; RB, Rose Bengal; NBSF, 2-nitrobenzenesulphonyl fluoride; NPSC, o-nitrophenylsulphenyl chloride; AMBIC, ammonium bicarbonate buffer; SDS, sodium dodecyl sulfate.
[3–5]. This diverse pharmacological profile has been acquired through evolution by a positive Darwinian selection in the protein-coding exons by an accelerated evolutionary process that has resulted in many variants with diverse pharmacological effects [6]. A number of class II myotoxic PLA2s have been isolated from Bothrops snake venoms [7]. Species from this genus inflict the vast majority of snakebites in Latin America, and acute muscle damage, myonecrosis, is a common finding in these envenomations [8, 9]. Myotoxic PLA2s present in Bothrops venoms are either Asp-49, catalytically-active variants or Lys-49, enzymaticallyinactive homologues [7]. This substitution in the calciumbinding loop renders Lys-49 homologues unable to bind calcium, with the consequent inability of the protein to stabilize the tetrahedral intermediate in the catalytic mechanism. However, despite their lack of enzymatic activity, Lys-49 PLA2s induce myotoxicity by a poorly known mechanism. Thus, comparative studies on Bothrops myotoxic PLA2s and insights in the structure-
756 function relationship of these molecules may help to elucidate the mechanism by which they disrupt membrane integrity. In this study we report a comparative chromatographic analysis of the venoms of eight species of Bothrops, in order to identify the presence of basic myotoxic PLA2s. In addition, chemical modifications of specific residues were performed in Asp-49 and Lys-49 PLA2 variants from B. jararacussu venom, to study the effects of such modifications in enzymatic and pharmacological properties. 2. Materials and methods 2.1. Reagents Venoms were donated by the serpentarium at Faculdade de Medicina de Ribeirão Preto (SP, Brazil) and CEVAPUNESP (Botucatu, SP, Brazil). 4-bromophenacyl bromide (BPB), 2-nitrobenzenesulphonyl fluoride (NBSF) and o-nitrophenylsulphenyl chloride were obtained from Sigma Chemical Co. (St. Louis, USA). 2.2. Purification of toxins Eight lyophilized venoms from Bothrops spp. (200–250 mg) were fractionated on a CM-Sepharose column (2 × 20 cm), which was previously equilibrated with 0.05 M ammonium bicarbonate buffer, pH 8.0 [10–12]. Elution was carried out with a continuous gradient up to a concentration of 0.5 M ammonium bicarbonate. Absorbance of the effluent solution was recorded at a wavelength of 280 nm.
Andrião-Escarso et al. tion [12]. CK concentration was determined using 4 µL of plasma according to the manufacturer’s instructions. The enzyme activity was expressed in U/L, and one unit results from the phosphorylation of one µmol of creatine/min at 25 °C. 2.6. Cytotoxic activity This activity was determined on endothelial cells (tEnd) and myoblasts (C2C12) as previously described [18]. Briefly, different concentrations of BthTX-I and II were diluted in culture medium (Dulbecco’s modified Eagle’s medium supplemented with 1% fetal calf serum) and then added to the cells in a total volume of 150 µL/ well. Positive controls consisted in cells treated with 0.1% Triton X-100. After 3 h of incubation, 100 µL of medium was collected for determination of lactic dehydrogenase activity using a colorimetric end-point assay obtained from Sigma Chemical Co. 2.7. Edema Groups of five male Swiss mice (18–22 g) were injected in the subplantar region with 50 µL of BthTX-I and II (100 µg). After different intervals, the edema at the paw was measured using a low pressure spring caliper (Mitutoyo, Japan) [12, 19]. Zero time points were subtracted from all the values and the differences were expressed as media percentage ± S.D. 2.8. Liposome-disrupting activity
Venoms were analyzed by electrophoresis for basic proteins and SDS-PAGE according to Reisfeld et al. [13] and Laemmli [14], respectively.
Negatively-charged liposomes (phosphatidylserine, 63 µmol; dicethylphosphate, 18 µmol; cholesterol, 9 µmol) were obtained from Sigma Chemical Co. The assay was performed according to Díaz et al. [20] by incubating 20 µL of the liposome suspension with 20 µL of BthTX-I or II (in PBS) for 30 min at 37 °C or 4 °C.
2.4. Enzymatic, anticoagulant and hemolytic activities
2.9. Lethal dose 50%
Phospholipase A2 activity was tested using egg yolk as a substrate [15]. Anticoagulant [16] and indirect hemolytic activities [17] were assayed for venoms and isolated toxins.
Lethality induced by BthTX-I and II was evaluated by i.p. injection of 100 µL of different toxin concentrations. This assay was performed in male Swiss mice (18–22 g; n = 6) within 48 h.
2.5. Myotoxic activity
2.10. Chemical modifications
The assay for determination of plasma creatine kinase (CK) activity was carried out using the CK-UV kinetic kit from Sigma Chemical Co. Toxins (2 µg/µL) were injected in 18–22 g male Swiss mice (50 µL, i.m.) (n = 6). Animals used as negative controls were injected with PBS. After 3 and 6 h, a blood sample was collected from the tail in heparin-coated tubes and centrifuged for plasma separa-
Modification of histidine-48 residues with BPB was carried out as previously described [21] using ammonium bicarbonate buffer instead of Tris-HCl. Briefly, 3 mg of toxin were dissolved in 1 mL of 0.1 M ammonium bicarbonate containing 0.7 mM EDTA (pH 8.0) and 150 µL of BPB (0.8 mg/mL) were added. The mixture was incubated for 24 h at 25 °C.
2.3. Biochemical characterization of toxins
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Figure 1. Comparative chromatographic profiles of eight Bothrops venoms on a CMSepharose column (2 × 20 cm). Samples of 200–250 mg were applied and elution was performed with a 0.05–0.5 M ammonium bicarbonate buffer gradient at pH 8.0 at 25 °C. Absorbances were recorded at 280 nm.
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Table I. Phospholipase A2 activity, minimal edematogenic doses (MED, µg ± S.D.) and CK release in plasma (U/l ± S.D.) for the crude venoms and isolated Bothrops myotoxins. For identification of fractions see figure 1. All of them are highly purified myotoxins, except J-I (which contains acidic nonmyotoxic PLA2s), At-VI and Jar-III. Venoms or toxins PLA2 activity MED (µg)b (U/mg)a PBS B. jararacussu J-I BthTX-I BthTX-II B. pirajai PrTX-I PrTX-II PrTX-III B. moojeni MjTX-I MjTX-II B. neuwiedi BnSP-7 B. asper Basp-II B. atrox At-VI B. jararaca Jar-III B. alternatus
0 71.02 119.34 0 37.08 75.04 0 0 38.71 78.37 0 0 78.99 0 82.05 0 107.52 9.34 29.42 0 26.90
3.3 ± 0.2 n.d. n.d. 31.3 ± 1.3 23.8 ± 1.1 n.d. 38.5 ± 1.8 40.5 ± 2.0 25.5 ± 1.7 n.d. 34.6 ± 1.4 32.2 ± 1.5 n.d. 36.8 ± 1.8 n.d. 38.9 ± 1.9 n.d. n.d. n.d. n.d. n.d.
CK (U/l)c 189.53 ± 45.6 3 197.08 ± 219.4 798.01 ± 100.2 2 279.35 ± 172.7 3 556.32 ± 228.6 3 091.40 ± 233.0 2 132.95 ± 193.2 1 787.50 ± 145.0 3 520.20 ± 244.5 2 798.73 ± 186.3 1 759.72 ± 180.0 2 189.91 ± 164.4 3 349.01 ± 253.0 2 056.81 ± 142.8 2 769.98 ± 166.8 2 140.82 ± 187.4 1 307.90 ± 109.7 882.33 ± 176.4 990.59 ± 87.9 649.16 ± 43.1 971.01 ± 56.1
n.d., not determined. a PLA2 activity on egg yolk substrate. Minimal edematogenic dose. c Myotoxicity activity 3 h after injection (50 µg/50 µL). b
Tyrosine residues were modified by treatment with NBSF as previously described [22]. Briefly, 1 µmol of BthTX-I and II (9 µmol of tyrosine) were dissolved in 14 mL 0.1 M Tris-HCl (pH 8.0) and incubated with 9 µmol of NBSF for 20 h at 25 °C. Modification of tryptophan residues was performed according to Takasaki et al. [23]. Briefly, 9 mg of BthTX-I and II were dissolved in 4 mL 50% acetic acid containing 1 mg of NPSC and the reaction was left to proceed for 1 h at 25 °C. Molar extinction coeficients of native (N) and modified (M) toxins were used to follow the reaction. In all cases, excess reagent was removed by ultrafiltration through an Amicon YM-3 membrane, and washed with water or 0.05 M amonium bicarbonate (pH 8.0). 2.11. Charge-shift electrophoresis In order to check the amphiphilic characteristics of the toxins, BthTX-I and II were run in an agarose gel in the presence of anionic, neutral and cationic detergents, as previously described [24].
Figure 2. A. PAGE for basic proteins of the whole venoms and isolated toxins. Lanes: 1, B. asper; 2, Basp-II; 3, B. jararacussu; 4, BthTX-I; 5, BthTX-II; 6, B. moojeni; 7, MjTX-I; 8, MjTX-II; 9, B. pirajai; 10, PrTX-I; 11, PrTX-II; 12, PrTX-III. B. SDSPAGE of the major isolated toxins. Lanes: 1, standard: a) serum albumin (67 000); b) ovalbumin (45 000); c) carbonic anhydrase (29 000); d) cytochrome c (12 380); 2, B. asper Basp-II; 3, B. jararacussu BthTX-I; 4, B. jararacussu BthTX-II; 5, B. moojeni MjTX-I; 6, B. moojeni MjTX-II; 7, B. neuwiedi BnSP-7; 8, B. pirajai PrTX-I; 9, B. pirajai PrTX-II; 10, cytochrome c; and 11, B. pirajai PrTX-III.
2.12. Enzyme-immunoassay 96-well plates were coated overnight with 0.2 µg/well BthTX-I and II [25]. Cross-reactivity of these toxins was determined using antibodies raised against Bothrops jararacussu BthTX-I and II and B. asper myotoxin II (whole molecule and the C-terminal peptide 115–129). Normal serum and crotamine were used as negative controls.
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Table II. Summarized biological effects of Bothrops jararacussu N or M-BthTX-I and II. Myotoxin BthTX-I BPB-BthTX-I RB-BthTX-I NBSF-BthTX-I NPSC-BthTX-I BthTX-II BPB-BthTX-II RB-BthTX-II NBSF-BthTXII NPSC-BthTXII
DL50 (mg/kg)
Myotoxicitya (%)
Edemab (%)
Cytotoxicityc (%)
Liposome disrupting activityd (%)
PLA2 activitye (U/mg)
8.5 ± 0.85 40.2 ± 0.38 40.8 ± 0.45 24.5 ± 0.48 15.2 ± 0.71 7.0 ± 0.81 41.8 ± 0.39 42.1 ± 0.32 14.6 ± 0.65 14.8 ± 0.71
100 49.3 19.2 73.3 62.7 100 0.5 0.2 23.8 48.5
125 ± 3.8 110 ± 4.2 98 ± 2.6 118 ± 3.2 121 ± 2.7 155 ± 5.7 74 ± 1.9 76 ± 3.4 121 ± 4.6 127 ± 4.4
100 ± 1 13 ± 2 0.5 ± 0.02 76 ± 1 58 ± 3 95 ± 2 1 ± 0.02 0.5 ± 0.02 1 ± 0.02 65 ± 1
84.3 81.1 81.5 79.6 80.1 95.8 78.2 76.4 80.5 79.8
0 0 0 0 0 37.09 0.05 5.32 18.56 19.44
a Values 3 h after inoculation of BthTX-I and II (100 µg). b Values 30 min after inoculation of BthTX-I and II (100 µg). c Values for doses of 20 µg of BthTX-I and II. d Values for doses of 2.5 µg of BthTX-I and II incubated at 37°C. e Values for doses of 50 µg of BthTX-I and II.
3. Results Chromatographic fractionation of eight Bothrops venoms on CM-Sepharose revealed different PLA2 myotoxins in all the venoms except B. atrox, B. jararaca and B. alternatus (figure 1). B. jararacussu BthTX-II and B. pirajai PrTX-III, which are Asp49-PLA2, showed moderate catalytic activity whereas the other PLA2s tested, which are Lys49 isoforms, showed extremely low or no activity at all when tested on phospholipids present on egg yolk (table I). Figure 2 shows the electrophoretic patterns of the venoms and isolated toxins. All venoms induced the release of creatine kinase (CK) after 3 h of injection, due to a direct action on muscle by their myotoxins (table I) and some of them also by indirect action (6 h after injection) by the effect of proteases on the blood vessels (results not shown). Table I also shows the minimal edema-inducing dose (MED, defined as µg of protein able to induce 30% of edema) of some of the venoms. Histopathological examination showed severe myonecrosis 24 h after myotoxin inoculation (results not shown). These alterations were characterized by the onset of intense edema and loss of muscle cell structure. Myonecrosis was accompanied by leukocyte infiltration, and no lesion of capillary vessels, veins or arteries was observed. After chemical modification of BthTX-I and II, amino acid composition showed that one His or two Tyr residues were modified by BPB and NBSF, respectively (results not shown). Photooxidation by RB was able to modify other residues as well. Both toxins, which are highly toxic when injected i.p., decreased their lethal activity after chemical modification of their amino acid residues with BPB and RB (table II). Chemical modification also altered other activities. The enzymatic activity of N-BthTX-II, for example, which was very low when compared with the activity of acidic
PLA2s present at the same venom (see J-I, table I), was almost completely abolished by BPB and partially modified by RB, NBSF and NPSC. Regarding the myotoxic effects, both N-BthTX-I and N-BthTX-II induced muscle damage and release of intracellular CK as can be seen in figure 3. After alkylation with BPB, BthTX-I lost around 50% of its activity whereas BthTX-II lost all of it. Photooxidation by RB, on the other hand, reduced in a high extent the release of CK while NBSF and NPSC were partially effective reducing around 25% and 35% of the activity, in the case of BthTX-I and 70% and 50%, in the case of BthTX-II, respectively. The cytotoxic activity of BthTX-I and II was almost completely abolished by treatment with BPB and RB. In addition, NBSF was also able to strongly reduce this activity in BthTX-II (figure 4). The other modifications also affected the activity but to a lesser extent (figure 4). The edema-inducing effect of BthTX-II was also partially inhibited by the treatment with the alkylating reagents (figure 5) whereas the ability to disrupt liposomes (table II) and the electrophoresis mobility on the chargeshift system practically did not change (results not shown). Figure 6 shows the effect of the different chemical modifications on the hemolytic and anticoagulant activities of BthTX-II. It can be seen that even when BPB was the most effective reagent altering these activities, most of the treatments had some effect. No significant alterations in the structure, however, were determined by comparing the immunogenicity of the modified and native toxins, except for a small decrease (20%) observed in the crossreactivity after toxin photooxidation, when antibodies against B. asper MT-II and its peptide 115–129 were tested (results not shown).
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Figure 3. Myotoxic activity of Bothrops jararacussu BthTX-I (A) and II (B) in mice. Time course of the plasma creatine kinase (CK) increased after the intramuscular injection of 100 µg/50 µL of PBS, native or BPB, RB, NBSF and NPSC modified toxins. Results are presented as means ± S.D. (n = 6).
Figure 4. Cytotoxic effect of BthTX-I (A) and II (B) and chemically-modified variants on C2C12 myoblasts/myotubes in culture. Doses of 20 µg of native and modified myotoxin were incubated with cells for 3 h at 37 °C, and the release of lactate dehydrogenase (LDH) into the supernatant was quantified. Results represent mean ± S.D. (n = 3).
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Figure 5. Edema induced by native or modified BthTX-I (A) and II (B) (100 µg/50 µL) in the paw of 18–22 g male Swiss mice. PBS was used as a control. Each bar represents the mean ± S.D. (n = 5).
4. Discussion Myotoxic PLA2s from Bothrops snake venoms are usually basic proteins, often the last venom components eluting from cation exchange columns using salt gradients at neutral pH [5]. The CM-Sepharose purification procedure described here resulted in the isolation of homogeneous myotoxins from the venoms of B. jararacussu, B. pirajai, B. moojeni, B. neuwiedi and B. asper in a single step. These venoms induced the release of creatine kinase to plasma 3 h after i.m. injection, while those from B. atrox, B. jararaca and B. alternatus showed a low to moderate myotoxicity, only 6 h after injection. The absence of major basic myotoxins in these three venoms, as previously reported [26–28], is confirmed by the low myotoxic activity of the last fractions eluted from the CM-Sepharose, and may explain this delayed response. Myotoxicity may result from a direct action of myotoxins upon the plasma membranes of muscle cells, or it may be due to an indirect vascular degeneration and ischemia caused by venom metalloproteinases [4, 5]. Among the isolated myotoxins, only two are Asp49PLA2 showing catalytic, anticoagulant and indirect hemolytic activities. The remaining ones are Lys49 myotoxins. All of them, but mainly the Asp49 variants,
induced myonecrosis. The higher myotoxic activity of the Asp49 variants, compared to the Lys49 variants, suggests an enhancing action of the catalytic activity upon toxic effects. However, since other important structural differences between these two groups of proteins exist, alternative explanations remain open. Treatment of N-BthTX-I with BPB reduced 45%, 85% and 15% of its myotoxic, cytotoxic and edema inducing activity, respectively, with no significant change in its liposome-disrupting activity. The Tyr and Trp residues of this toxin apparently display a more relevant contribution to the myotoxic and cytotoxic, but not to the edemainducing and liposome-disrupting effects. Díaz et al. [29] and Soares et al. [30] showed that BPB treatment of myotoxin II and BnSP-7, Lys49 myotoxins from B. asper and B. neuwiedi, respectively, reduced 40–55% of its myotoxic activity, but the liposome-disrupting effect was less affected. This same inference may be drawn regarding the almost complete abolishment of the PLA2, anticoagulant, myotoxic and cytotoxic activities of the BPB-treated BthTX-II, while the edema-inducing and liposomedisrupting activities were reduced only 50% and 20%, respectively. It is likely that BthTX-II can lyse liposomes independently of its catalytic activity. These data can be
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Andrião-Escarso et al. independent upon but eventually potentiated by the PLA2 activity. Since however the myotoxic activity of BPBtreated BthTX-I was significantly lower than that of N-BthTX-I, an hypothetic ‘in vivo’ lipolytic activity of this and probably other Lys-49 PLA2s cannot be excluded. Alternatively, modification of this protein by BPB may result in conformational changes affecting its myotoxic mechanism. Photooxidation of both BthTX-I and II by RB modified other residues other than His48. In BthTX-II, the enzymatic, anticoagulant and edema-inducing activities were partially, while the myotoxic and cytotoxic activities were totally abolished. The significant change of the electrophoretic mobility denotes a more drastic structural modification which may be advantageous for the sake of attenuating the lethal effect of the toxin while keeping its antigenicity for the production of antibodies. The data on the modifications of Tyr and Trp residues of BthTX-II reveal a correlation between myotoxic and cytotoxic effects, but less with the catalytic, edemainducing and liposome-rupturing activities, suggesting that these residues are less important for the hydrolytic activity on phospholipids and inflammatory reaction. The finding that N-(native) and M-(modified)-BthTX-I and II were recognised by the antibodies raised against BthTX-I, BthTX-II and C-terminal 115-129 from Basp-II, while the photooxidized forms were only partially recognized, suggests that RB drastically changes the structure of the toxins. Recognition by Basp-II anti-C terminal indicates that this region is antigenically conserved. This region of Basp-II is believed to play an important role in its myotoxic and cytotoxic effects [25, 31–33]. This conclusion may probably be extended to BthTX-I, since Basp-II and BthTX-I have of 98% sequence identity at the C-terminal region. In conclusion, our results show that in BthTX-I, which is catalytically inactive, the His, Tyr and Trp residues play an important role in the myotoxic and cytotoxic activities, but not so in the edema-inducing and liposome-rupturing activities. While for BthTX-II the catalytic activity appears to be relevant to its anticoagulant, myotoxic, cytotoxic and edema inducing effects, it is not for the liposome-disrupting activity. Acknowledgments
Figure 6. Indirect hemolytic (A) and anticoagulant (B) activity of BthTX-II. Results are presented as mean ± S.D. (n = 3).
explained on the basis of a set of pharmacological domains distinct from the catalytic site and apparently
The authors gratefully acknowledge the financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Consejo Nacional de Investigaciones Científicas y Tecnológicas de Costa Rica (FO-013-98), International Foundation for Science (F/2766-1), as well as the skilfull technical assistance of Odete A.B. Cunha, Luiz H. AnzaloniPedrosa and Carlos A. Vieira. This work is part of the Doctoral Thesis of Silvia H. Andrião-Escarso and Andreimar M. Soares at Departamento de Bioquímica, Faculdade de Medicina, Universidade de São Paulo-SP, Brasil.
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