Background. An interaction between lectins from marine algae and PLA 2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA 2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA 2 and its complex. Results. This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA 2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion. The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules. © 2008 Oliveira et al; licensee BioMed Central Ltd.91 Gabius, H.J., Gabius, S., (1997) Glycoscience: Status and Perspectives, , Weinheim: Chapman and Hall 1Elgavish, S., Shaanan, B., Lectin-carbohydrate interactions: Different folds, common recognition principles (1997) Trends Biochemistry Science, 22, pp. 462-467. , 10.1016/S0968-0004(97)01146-8Boyd, W.C., Almodovar, L.R., Boyd, L.G., Agglutinin in marine algae from human erythrocytes (1966) Transfusion, 6, pp. 82-83Melov, V., Medeiros, D., Rio, F., Casterlar, L., Carvalho, F., Antifungal properties of proteins from the red alga Hypnea musciformis (Wulfen) (1997) Botanica Marina, 40, pp. 281-284Lima, H.C., Costa, F.H.F., Sampaio, A.H., Neves, S.A., Bevebides, N.M.B., Teixeira, D.I.A., Rogers, D.J., Freitas, A.L.P., Induction and inhibition of human lymphocyte transformation by the lectin from the red marine alga Amansia multifida (1998) Journal Applied Phycology, 10 (2), pp. 153-162. , 10.1023/A:1008016731752Munoz, A., Alvarez, O., Alonso, B., Lovo, J., Lectin typing of methicilin-resistant Staphyloccocus aureus (1999) Journal of Medical Microbiology, 48, pp. 495-498. , 10229547Ashraf, M.T., Khan, R.H., Mytogenic lectins (2003) Med Sci Monit, 9 (11), pp. 265-269. , 14586291Liao, W.-R., Lin, J.-Y., Shieh, W.-Y., Jeng, W.-L., Huang, R., Antibiotic activity of lectins from marine algae against marine vibrios (2003) Journal of Ind Microbiology and Biotechnology, 30, pp. 433-439. , 10.1007/s10295-003-0068-7Lima, R.F., Criddle, D.N., Souza, E.P., Sampaio, A.H., Nascimento, K.S., Cavada, B.S., Assreuy, A.M., Red marine alga Bryothamnion triquetrum lectin induces endothelium-dependent relaxation of the rat aorta via release of nitric oxide (2004) J Pharm Pharmacol, 56 (11), pp. 1415-1421. , 10.1211/0022357044616. 15525448Matsubara, K., Sumi, H., Hori, K., Platelet aggregation is inhibited by phycolectins (1996) Experientia, 52 (6), pp. 540-543. , 10.1007/BF01969724. 8698085Mori, T., O'Keefe, B.R., Sowder II, R.C., Bringans, S., Gardella, R., Berg, S., Cochran, P., Boyd, R., Isolation and Characterization of Griffithsin, a Novel HIV-inactivating Protein, from the red alga Griffithsia sp (2005) The Journal of Biological Chemistry, 280 (10), pp. 9345-9353. , 10.1074/jbc.M411122200. 15613479Valentin, E., Lambeau, G., What can venom phospholipases A(2) tell us about the functional diversity of mammalian secreted phospholipases A(2)? (2000) Biochimie, 82, pp. 815-831. , 10.1016/S0300-9084(00)01168-8. 11086212Toyama, M.H., Mancuso, L.C., Giglio, J.R., Novello, J.C., Oliveira, B., Marangoni, S., A quick procedure for the isolation of dimeric piratoxins-I and II, two myotoxins from Bothrops pirajai snake venom. N-terminal sequencing (1995) Biochem Mol Biol Int, 37 (6), pp. 1047-1055. , 8747534Kini, R.K., Iwanaga, S., Structure-function relationships of phospholipases. II: Charge density distribution and the myotoxicity of presynaptically neurotoxic phospholipases (1986) Toxicon, 24 (9), pp. 895-905. , 10.1016/0041-0101(86)90090-5. 3544338Kini, R.M., Evans, H.J., Structure-function relationships of phospholipases. the anticoagulant region of phospholipases A2 (1987) Journal of Biological Chemistry, 262 (30), pp. 14402-14407. , 3117784Maraganore, J.M., Merutka, G., Cho, W., Welches, W., Kezdy, F.J., Heinrikson, R.L., A new class of phospholipases A2 with lysine in place of aspartate 49. Functional consequences for calcium and substrate binding (1984) J Biol Chem, 259 (22), pp. 13839-13843. , 6438084Gutierrez, J.M., Lomonte, B., Phospholipase A2 myotoxins from Bothrops snake venoms (1995) Toxicon, 33 (11), pp. 1405-1424. , 10.1016/0041-0101(95)00085-Z. 8744981Arni, R.K., Ward, R.J., Phospholipase A2- a structural review (1996) Toxicon, 34 (8), pp. 827-841. , 10.1016/0041-0101(96)00036-0. 8875770Hori, K., Matsubara, K., Miyazawa, K., Primary structure of two hemagglutinins from the marine red alga Hypnea japonica (2000) Biochemica et Biophysica Acta, 1474, pp. 226-236Calvete, J.J., Costa, F.H., Saker-Sampaio, S., Murciano, M.P., Nagano, C.S., Cavada, B.S., Grangeiro, T.B., Sampaio, A.H., The amino acid sequence of the agglutinin isolated from the red marine alga Bryothamnion triquetrum defines a novel lectin structure (2000) Cellular and Molecular Life Science, 57 (2), pp. 343-350. , 10.1007/PL00000696Pieterson, W.A., Vidal, J.C., Volwerk, J.J., De Haas, G.H., Zymogen-catalyzed hydrolysis of monomeric substrates and the presence of a recognition site for lipid-water interfaces in phospholipase A2. (1974) Biochemistry, 13, pp. 1455-1460. , 10.1021/bi00704a021. 4819758Volwerk, J.J., De Haas, G.H., (1982) In: Molecular Biology of Lipid-Protein Interactions, , Wiley New York Griffith OH, Jost PCDen, V.B.B., Tessari, M., De Haas, G.H., Verheij, H.M., Boelens, R., Kaptein, R., Solution structure of porcine pancreatic phospholipase A2 (1995) EMBO Journal, 14, pp. 4123-4131. , 7556053Soares, A.M., Andrião-Escarso, S.H., Bortoleto, R.K., Rodrigues-Simioni, L., Arni, R.K., Ward, R.J., Gutiérrez, J.M., Giglio, J.R., Dissociation of enzymatic and pharmacological properties of piratoxins-I and -III, two myotoxic phospholipases A2 from Bothrops pirajai snake venom (2001) Arrchives of Biochemistry and Biophysics, 387 (2), pp. 188-196. , 10.1006/abbi.2000.2244Soares, A.M., Rodrigues, V.M., Homsi-Brandeburgo, M.I., Toyama, M.H., Lombardi, F.R., Arni, R.K., Giglio, J.R., A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (Caissaca) venom: Biochemical characterization, crystallization, myotoxic and edematogenic activity (1998) Toxicon, 36, pp. 503-514. , 10.1016/S0041-0101(97)00133-5. 9637370Lee, W.H., Giotto, M.T.S., Marangoni, S., Toyama, M.H., Polikarpov, I., Garrat, R., Structural Basis for Low Catalytic Activity in Lys49 Phospholipases A2sA Hypothesis: The Crystal Structure of Piratoxin II Complexed to Fatty Acid (2001) Biochemistry, 40, pp. 28-36. , 10.1021/bi0010470. 11141053Rigden, D.J., Hwa, L.W., Marangoni, S., Toyama, M.H., Polikarpov, I., The structure of the D49 phospholipase A2 piratoxin III from Bothrops pirajai reveals unprecedented structural displacement of the calcium-binding loop: Possible relationship to cooperative substrate binding (2003) Acta Crystallization, 59, pp. 255-262Toyama, M.H., De Oliveira, D.G., Beriam, L.O.S., Novello, J.C., Rodrigues-Simioni, L., Marangoni, S., Structural, enzymatic and biological properties of new PLA(2) isoform from Crotalus durissus terrificus venom (2003) Toxicon, 41 (8), pp. 1033-1038. , 10.1016/S0041-0101(03)00085-0. 12875878Hernandez-Oliveira, S., Toyama, M.H., Toyama, D.O., Marangoni, S., Hyslop, S., Rodrigues-Simioni, L., Biochemical, pharmacological and structural characterization of a new PLA2 from Crotalus durissus terrificus (South American rattlesnake) venom (2005) Protein Journal, 24 (4), pp. 233-42. , 10.1007/s10930-005-6718-z. 16283546Landucci, E.C., De Castro, R.C., Toyama, M.H., Giglio, J.R., Marangoni, S., De Nucci, G., Antunes, E., Inflammatory edema induced by the lys-49 phospholipase A(2) homologue piratoxin-i in the rat and rabbit. Effect of polyanions and p-bromophenacyl bromide (2000) Biochemical Pharmacology, 59 (10), pp. 1289-1294. , 10.1016/S0006-2952(00)00248-3. 10736429Boechat, A.L., Paiva, C.S., Franca, F.O., Dos-Santos, M.C., Heparin-antivenom association: Differential neutralization effectiveness in Bothrops atrox and Bothrops erythromelas envenoming (2001) Rev Inst Med Trop Sao Paulo, 43 (1), pp. 7-14. , 11246276Bugs, M.R., Bortoleto-Bugs, R.K., Comelio, M.L., The interaction between heparin and Lys49 phospholipase A2 reveals the natural binding of heparin on the enzyme (2005) International Journal of Biol Macromolecules, 37 (1-2), pp. 21-27. , 10.1016/j.ijbiomac.2005.08.003Kini, R.M., Evans, H.J., A model to explain the pharmacological effects of snake venom phospholipases A2 (1989) Toxicon, 27 (6), pp. 613-635. , 10.1016/0041-0101(89)90013-5. 2665186Lambeau, G., Lazdunski, M., Receptors for a growing family of secreted phospholipases A2 (1999) Trends Pharmacology Science, 20 (4), pp. 162-70. , 10.1016/S0165-6147(99)01300-0Yedgar, S., Cohen, Y., Shoseyov, D., Control of phospholipase A2 activities for the treatment of inflammatory conditions (2006) Biochim Biophys Acta, 11, pp. 1373-1382Triggiani, M., Granata, F., Oriente, A., Gentile, M., Petraroli, A., Balestrieri, B., Secretory phospholipases A2 induce cytokine release from blood and synovial fluid monocytes (2002) Eur J Immunol, 32, pp. 67-76. , 10.1002/1521-4141(200201)32:1<67::AID-IMMU67>3.0.CO;2-3. 11754005Granata, F., Petraroli, A., Boilard, E., Bezzine, S., Bollinger, J., Del Vecchio, L., Gelb, M.H., Triggiani, M., Activation of cytokine production by secreted phospholipase A2 in human lung macrophages expressing the M-type receptor (2005) J Immunol, 174, pp. 464-474. , 15611272Hanasaki, K., Arita, K., Phospholipase A2 receptor: A regulator of biological functions of secretory phospholipase A2 (2002) Prostaglandins Other Lipid Mediat, 69, pp. 71-82. , 10.1016/S0090-6980(02)00022-9Hanasaki, K., Mammalian phospholipase A2: Phospholipase A2 receptor (2004) Biology Pharmaceuthic Bulletin, 27 (8), pp. 1165-1167. , 10.1248/bpb.27.1165Harris, L.K., Franson, R.C., [1-14C]oleate-labeled autoclaved yeast: A membranous substrate for measuring phospholipase A2 activity in vitro (1991) Anal Biochemistry, 193 (2), pp. 191-196. , 10.1016/0003-2697(91)90007-GYang, C.C., Structure-function relationship of phospholipase A2 from snake venoms (1994) J Toxicol, 13 (2), pp. 125-177. , 10.1093/toxsci/23.1.125Soares, A.M., Andriao-Escaso, S.H., Angulo, Y., Lomonte, B., Gutierrez, J.M., Marangoni, S., Toyama, M.H., Giglio, J.R., Structural and functional characterization of myotoxin I, a Lys49 phospholipase A(2) homologue from Bothrops moojeni (Caissaca) snake venom (2000) Arch Biochemistry and Biophysica, 373 (1), pp. 7-15. , 10.1006/abbi.1999.1492Hames, B.D., Rickwood, D., (1983) Gel Electhophoresis of Proteins. a Practical Approach. Washington, pp. 27-33. , IRL PressToyama, M.H., Soares, A.M., Wen-Hwa, L., Polikarpov, I., Giglio, J.R., Marangoni, S., Amino acid sequence of piratoxin-II, a myotoxic lys49 phospholipase A(2) homologue from Bothrops pirajai venom (2000) Biochimie, 82 (3), pp. 245-250. , 10.1016/S0300-9084(00)00202-9. 10863008Holzer, M., MacKessy, S.P., An aqueous endpoint assay of snake venom PLA2 (1996) Toxicon, 34 (10), pp. 1149-1155. , 10.1016/0041-0101(96)00057-8. 8931255