Comparative Biochemical Studies Of Myotoxic Phospholipase A2 From Bothrops Venom

Venoms from Bothrops jararacussu, Bothrops asper, Bothrops atrox, Bothrops pirajai, Bothrops moojeni, Bothrops alternatus and Bothrops (Bothriopsis) bilineata were fractionated using a simplified procedure based on ion-exchange chromatography on CM-Sepharose at pH 8.0 or reverse phase HPLC. The resulting elution profiles showed important differences in the myotoxin content of these venoms. The venoms from B. alternatus, B. atrox and Bothriopsis bilineata did not contain the major myotoxin found in the other venoms. The amino acid sequence of the first 50 residues of the N-terminal region of the PLA2-like myotoxins showed a homology of 90-96% with other bothropic myotoxins. All of the myotoxins isolated induced rat paw edema, increased the level of plasma creatine kinase and produced myonecrosis together with polymorphonuclear cell infiltration.83179186Hoge, A.R., Romano Hoge, S.A.R.W.L., (1978) Mem. Inst. Butantan, 42-43, pp. 373-496Chang, L.-S., Kuo, K.-W., Lin, S.-R., Chang, C.-C., (1994) J. Protein Chem., 13, pp. 641-648Shimohigashi, Y., Tani, A., Matsumoto, H., Nakashima, K.-I., Yamaguchi, Y., Oda, N., Takano, Y., Ohno, M., (1995) J. Biochem. (Tokyo) Biochem., 118, pp. 1037-1044Ogawa, T., Nakashima, K.-I., Nobushima, I., Deshimaru, M., Shimohigashi, Y., Fukumaki, Y., Sakaki, Y., Ohno, M., (1996) Toxicon, 34, pp. 1229-1236Dennis, E.A., (1994) Biol. Chem., 269, pp. 13057-13060Araújo, H.S., White, S.P., Ownby, C.L., (1996) Toxicon, 34, pp. 1237-1242Kini, R.M., Iwanaga, S., (1986) Toxicon, 24, pp. 895-905Arni, R.K., Ward, R.J., (1996) Toxicon, 34, pp. 827-841Mancuso, L.C., Correa, M.M., Vieira, C.A., Cunha, O.A.B., Lachat, J.J., Selistre, H.S.A., Ownby, C.L., Giglio, J.R., (1995) Toxicon, 33, pp. 615-626Soares, A.M., Rodrigues, V.M., Homsi-Brandeburgo, M.I., Toyama, M.H., Lombard, F.R., Arni, A.K., Giglio, J.R., (1998) Toxicon, 36, pp. 503-514Gutiérrez, J.M., Lomonte, B., (1995) Toxicon, 33, pp. 1405-1424Fletcher, J.E., Humbert, M., Wieland, S.J., Gong, Q.H., Jiang, M.S., (1996) Toxicon, 34, pp. 1301-1311Cho, W., Kezdy, F.J., (1991) Methods Enzymol., 197, pp. 75-79Holzer, M., Mackessy, S.P., (1996) Toxicon, 35, pp. 1149-1155Marangoni, S., Toyama, M.H., Arantes, E.C., Giglio, J.R., Da Silva, C.A., Carneiro, E.M., Gonçalves, A.A., Oliveira, B., (1995) Biophys. Acta, 1243, pp. 309-314Higgins, D.G., Sharp, P.M., (1989) Comput. Appl. Biosci., 5, pp. 151-153Araújo, A.L., Radvanyi, F., Bon, C., (1994) Toxicon, 32, pp. 1069-1081Fukagawa, T., Nose, T., Shimohigashi, Y., Ogawa, T., Oda, N., Nakashima, K.I., Chang, C.C., Ohno, M., (1993) Toxicon, 31, pp. 957-967Homsi-Brandeburgo, M.I., Queiroz, L.S., Santo-Neto, H., Rodrigues-Simioni, L., Giglio, J.R., (1988) Toxicon, 26, pp. 615-627Nakai, M., Nakashima, K.I., Ogawa, T., Shimohigashi, Y., Hattori, S., Chang, C.C., Ohno, M., (1995) Toxicon, 33, pp. 1469-1478Toyama, M.H., Soares, A.M., Vieira, C.A., Novello, J.C., Oliveira, B., Giglio, J.R., Marangoni, S., (1998) J. Protein Chem., 17, pp. 713-718Soares, A.M., Anzaloni Pedrosa, L.H., Fontes, M.R.M., Da Silva, R.J., Giglio, J.R., (1998) J. Venom. Anim. Toxins, 4. , in pressDe Azevedo W.F., Jr., Ward, R.J., Gutierrez, J.M., Arni, R.K., (1998) Toxicon, pp. 1395-1406Francis, B., Gutiérrez, J.M., Lomonte, B., Kaiser, I.I., (1991) Arch. Biochem. Biophys., 284, pp. 352-359Toyama, M.H., Mancuso, L.C., Giglio, J.R., Novello, J.C., Oliveira, B., Marangoni, S., (1995) Biochem. Mol. Biol. Inter., 37, pp. 1047-1055Heirikson, R.L., Trueger, E.T., Kein, P.S., (1977) J. Biol. Chem., 252, pp. 4913-4921Lomonte, B., Gutiérrez, J.M., Mata, E., (1985) Toxicon, 23, pp. 807-813Cintra, A.C.O., Marangoni, S., Oliveira, B., Giglio, J.R., (1993) J. Protein Chem., 12, pp. 57-64Kaiser, I.I., Gutiérrez, J.M., Plummer, D., Aird, S.D., Odell, G.V., (1990) Arch. Biochem. Biophys., 278, pp. 319-325Gowda, V.T., Schmidt, J., Middlebrook, J.L., (1994) Toxicon, 32, pp. 665-67

Comparative biochemical studies of myotoxic phospholipase A(2) from Bothrops venom

Venoms from Bothrops jararacussu, Bothrops asper, Bothrops atrox, Bothrops pirajai, Bothrops moojeni, Bothrops alternatus and Bothrops (Bothriopsis) bilineata were fractionated using a simplified procedure based on ion-exchange chromatography on CM-Sepharose at pH 8.0 or reverse phase HPLC. The resulting elution profiles showed important differences in the myotoxin content of these venoms. The venoms from B. alternatus, B. atrox and Bothriopsis bilineata did not contain the major myotoxin found in the other venoms. The amino acid sequence of the first 50 residues of the N-terminal region of the PLA(2)-like myotoxins showed a homology of 90-96% with other bothropic myotoxins. All of the myotoxins isolated induced rat paw edema, increased the level of plasma creatine kinase and produced myonecrosis together with polymorphonuclear cell infiltration.8317918

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom

An acidic (pI similar to 4.5) phospholipase A(2) (BthA-I-PLA(2)) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an similar to13.7 kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 'SLWQFGKMINYVMJGESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0 Angstrom resolution. These crystals are monoclinic and have unit cell dimensions of a = 33.9, b = 63.8, c = 49.1 Angstrom, and beta = 104.0degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 tithes more active than BthTX-II, a basic D49 myotoxic PLA(2) from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA(2) caused a hypotensive response in the rat and inhibited platelet aggregation, Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA(2), while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies. (C) 2002 Elsevier B.V. All rights reserved

Effects of neutrophil depletion in the local pathological alterations and muscle regeneration in mice injected with Bothrops jararaca snake venom

In order to study the role of neutrophils in the acute local pathological alterations induced by Bothrops jararaca snake venom, and in the process of skeletal muscle regeneration that follows, an experimental model was developed in mice pretreated with either an anti-mouse granulocyte rat monoclonal immunoglobulin G, which induces a profound neutropenia, or an isotype-matched control antibody. B. jararaca venom induced prominent haemorrhage and oedema, but only a moderate myonecrosis. No significant differences were observed in the extent of local haemorrhage, oedema and myonecrosis between neutropenic and control mice, suggesting that neutrophils do not play a determinant role in the acute pathological alterations induced by B. jararaca venom in this experimental model. Moreover, no differences were observed in skeletal muscle regeneration between these two experimental groups. In both the cases, limited areas of myonecrosis were associated with a drastic damage to the microvasculature and a scarce inflammatory infiltrate, with the consequent lack of removal of necrotic debris during the first week, resulting in a poor regenerative response at this time interval. Subsequently, a similar regenerative process occurred in both groups, and by 30 days, necrotic areas were substituted by groups of small regenerating muscle fibres. It is suggested that the drastic effect exerted by B. jararaca venom in the microvasculature precludes an effective access of inflammatory cells to necrotic areas, thereby compromising an effective removal of necrotic debris; this explains the poor regenerative response observed during the first week and the fact that there were no differences between neutropenic and control mice. As neutropenia in this model lasted only 7 days, the successful regenerative process observed at 30 days is associated with revascularization of necrotic regions and with a successful removal by phagocytes of necrotic debris in both groups

Molecular evolution and structure–function relationships of crotoxin-like and asparagine-6-containing phospholipases A(2) in pit viper venoms

Some myotoxic or neurotoxic PLA(2)s (phospholipases A(2)) from pit viper venoms contain characteristic N6 substitutions. Our survey of the venoms of more than ten pit viper genera revealed that N6-PLA(2)s exist only in limited Asian pit vipers of two genera, Protobothrops and Gloydius, and exist as either monomers or the basic subunits of heterodimers in some New World pit vipers. For the newly identified N6-PLA(2)s, the neuromuscular blocking activities were assayed with the chick biventer cervicis neuromuscular tissue, whereas the increased serum creatine kinase level assessed their myotoxicities. The purified N6-PLA(2)s from Protobothrops mangshanensis and Gloydius intermedius saxatilis were found to be presynaptic neurotoxins. In contrast, all N6-PLA(2)s from the venoms of Sistrurus miliarius strackeri, S. m. barbouri, Crotalus viridis viridis, C. lepidus lepidus, Cerrophidion godmani and Bothreichis schlegelii were myotoxins without neurotoxicity even in the presence of crotoxin A. Crotoxin-like complexes were for the first time purified from the venoms of Sitrurus catenatus tergeminus, C. mitchelli mitchelli, C. horridus atricaudatus, C. basiliscus and C. durissus cumanensis. The cDNAs encoding six novel N6-PLA(2)s and subunits of the crotoxin-like complex from S. c. tergeminus were cloned and fully sequenced. Phylogeny analysis showed that two structural subtypes of N6-PLA(2)s with either F24 or S24 substitution have been evolved in parallel, possibly descended respectively from species related to present-day Protobothrops and Gloydius. Calmodulin binds all the N6-PLA(2)s but crotoxin A may inhibit its binding to crotoxin B and to other neurotoxic N6-PLA(2)s. Structure–activity relationships at various regions of the PLA(2) molecules were extensively discussed

Alkylation of Histidine Residues of Bothrops jararacussu Venom Proteins and Isolated Phospholipases A(2): A Biotechnological Tool to Improve the Production of Antibodies

Crude venom of Bothrops jararacussu and isolated phospholipases A(2) (PLA(2)) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) by p-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA(2) native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-inducedmyotoxicity. These results reveal that the chemical modification of the phospholipases A(2) (PLA(2)) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA(2) may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES