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. 1997 Sep 15;326(Pt 3):853–859. doi: 10.1042/bj3260853

Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper.

S Lizano 1, B Lomonte 1, J W Fox 1, J M Gutiérrez 1
PMCID: PMC1218742  PMID: 9307037

Abstract

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23-25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23-25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I-IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.

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Selected References

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  1. Ancian P., Lambeau G., Mattéi M. G., Lazdunski M. The human 180-kDa receptor for secretory phospholipases A2. Molecular cloning, identification of a secreted soluble form, expression, and chromosomal localization. J Biol Chem. 1995 Apr 14;270(15):8963–8970. doi: 10.1074/jbc.270.15.8963. [DOI] [PubMed] [Google Scholar]
  2. Borkow G., Gutierrez J. M., Ovadia M. A potent antihemorrhagin in the serum of the non-poisonous water snake Natrix tessellata: isolation, characterization and mechanism of neutralization. Biochim Biophys Acta. 1994 Dec 15;1201(3):482–490. doi: 10.1016/0304-4165(94)90080-9. [DOI] [PubMed] [Google Scholar]
  3. Borkow G., Gutierrez J. M., Ovadia M. Isolation, characterization and mode of neutralization of a potent antihemorrhagic factor from the serum of the snake Bothrops asper. Biochim Biophys Acta. 1995 Oct 19;1245(2):232–238. doi: 10.1016/0304-4165(95)00081-l. [DOI] [PubMed] [Google Scholar]
  4. Bultrón E., Gutiérrez J. M., Thelestam M. Effects of Bothrops asper (terciopelo) myotoxin III, a basic phospholipase A2, on liposomes and mouse gastrocnemius muscle. Toxicon. 1993 Feb;31(2):217–222. doi: 10.1016/0041-0101(93)90289-u. [DOI] [PubMed] [Google Scholar]
  5. Bussolino F., De Rossi M., Sica A., Colotta F., Wang J. M., Bocchietto E., Padura I. M., Bosia A., DeJana E., Mantovani A. Murine endothelioma cell lines transformed by polyoma middle T oncogene as target for and producers of cytokines. J Immunol. 1991 Oct 1;147(7):2122–2129. [PubMed] [Google Scholar]
  6. DOLE V. P. A relation between non-esterified fatty acids in plasma and the metabolism of glucose. J Clin Invest. 1956 Feb;35(2):150–154. doi: 10.1172/JCI103259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Domont G. B., Perales J., Moussatché H. Natural anti-snake venom proteins. Toxicon. 1991;29(10):1183–1194. doi: 10.1016/0041-0101(91)90191-s. [DOI] [PubMed] [Google Scholar]
  8. Díaz C., Gutiérrez J. M., Lomonte B., Gené J. A. The effect of myotoxins isolated from Bothrops snake venoms on multilamellar liposomes: relationship to phospholipase A2, anticoagulant and myotoxic activities. Biochim Biophys Acta. 1991 Dec 9;1070(2):455–460. doi: 10.1016/0005-2736(91)90086-n. [DOI] [PubMed] [Google Scholar]
  9. Díaz C., Lomonte B., Zamudio F., Gutiérrez J. M. Purification and characterization of myotoxin IV, a phospholipase A2 variant, from Bothrops asper snake venom. Nat Toxins. 1995;3(1):26–31. doi: 10.1002/nt.2620030107. [DOI] [PubMed] [Google Scholar]
  10. Fortes-Dias C. L., Fonseca B. C., Kochva E., Diniz C. R. Purification and properties of an antivenom factor from the plasma of the South American rattlesnake (Crotalus durissus terrificus). Toxicon. 1991;29(8):997–1008. doi: 10.1016/0041-0101(91)90082-3. [DOI] [PubMed] [Google Scholar]
  11. Fortes-Dias C. L., Lin Y., Ewell J., Diniz C. R., Liu T. Y. A phospholipase A2 inhibitor from the plasma of the South American rattlesnake (Crotalus durissus terrificus). Protein structure, genomic structure, and mechanism of action. J Biol Chem. 1994 Jun 3;269(22):15646–15651. [PubMed] [Google Scholar]
  12. Francis B., Gutierrez J. M., Lomonte B., Kaiser I. I. Myotoxin II from Bothrops asper (Terciopelo) venom is a lysine-49 phospholipase A2. Arch Biochem Biophys. 1991 Feb 1;284(2):352–359. doi: 10.1016/0003-9861(91)90307-5. [DOI] [PubMed] [Google Scholar]
  13. Gutiérrez J. M., Avila C., Rojas E., Cerdas L. An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon. 1988;26(4):411–413. doi: 10.1016/0041-0101(88)90010-4. [DOI] [PubMed] [Google Scholar]
  14. Gutiérrez J. M., Lomonte B., Chaves F., Moreno E., Cerdas L. Pharmacological activities of a toxic phospholipase A isolated from the venom of the snake Bothrops asper. Comp Biochem Physiol C. 1986;84(1):159–164. doi: 10.1016/0742-8413(86)90183-0. [DOI] [PubMed] [Google Scholar]
  15. Gutiérrez J. M., Lomonte B. Phospholipase A2 myotoxins from Bothrops snake venoms. Toxicon. 1995 Nov;33(11):1405–1424. doi: 10.1016/0041-0101(95)00085-z. [DOI] [PubMed] [Google Scholar]
  16. Gutiérrez J. M., Ownby C. L., Odell G. V. Isolation of a myotoxin from Bothrops asper venom: partial characterization and action on skeletal muscle. Toxicon. 1984;22(1):115–128. doi: 10.1016/0041-0101(84)90144-2. [DOI] [PubMed] [Google Scholar]
  17. Higashino K., Ishizaki J., Kishino J., Ohara O., Arita H. Structural comparison of phospholipase-A2-binding regions in phospholipase-A2 receptors from various mammals. Eur J Biochem. 1994 Oct 1;225(1):375–382. doi: 10.1111/j.1432-1033.1994.00375.x. [DOI] [PubMed] [Google Scholar]
  18. Inoue S., Kogaki H., Ikeda K., Samejima Y., Omori-Satoh T. Amino acid sequences of the two subunits of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis. Sequence homologies with pulmonary surfactant apoprotein and animal lectins. J Biol Chem. 1991 Jan 15;266(2):1001–1007. [PubMed] [Google Scholar]
  19. Ishizaki J., Higashino K., Ohara O., Arita H. Structural characterization of pancreatic group I phospholipase A2 receptor. Adv Prostaglandin Thromboxane Leukot Res. 1995;23:85–87. [PubMed] [Google Scholar]
  20. Kaiser I. I., Gutierrez J. M., Plummer D., Aird S. D., Odell G. V. The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper. Arch Biochem Biophys. 1990 May 1;278(2):319–325. doi: 10.1016/0003-9861(90)90266-2. [DOI] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lomonte B., Gutiérrez J. M. A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo). Toxicon. 1989;27(7):725–733. doi: 10.1016/0041-0101(89)90039-1. [DOI] [PubMed] [Google Scholar]
  23. Lomonte B., Gutiérrez J. M., Carmona E., Rovira M. E. Equine antibodies to Bothrops asper myotoxin II: isolation from polyvalent antivenom and neutralizing ability. Toxicon. 1990;28(4):379–384. doi: 10.1016/0041-0101(90)90075-i. [DOI] [PubMed] [Google Scholar]
  24. Lomonte B., Moreno E., Tarkowski A., Hanson L. A., Maccarana M. Neutralizing interaction between heparins and myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper snake venom. Identification of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecular modeling. J Biol Chem. 1994 Nov 25;269(47):29867–29873. [PubMed] [Google Scholar]
  25. Lomonte B., Tarkowski A., Bagge U., Hanson L. A. Neutralization of the cytolytic and myotoxic activities of phospholipases A2 from Bothrops asper snake venom by glycosaminoglycans of the heparin/heparan sulfate family. Biochem Pharmacol. 1994 Apr 29;47(9):1509–1518. doi: 10.1016/0006-2952(94)90525-8. [DOI] [PubMed] [Google Scholar]
  26. Lomonte B., Tarkowski A., Hanson L. A. Broad cytolytic specificity of myotoxin II, a lysine-49 phospholipase A2 of Bothrops asper snake venom. Toxicon. 1994 Nov;32(11):1359–1369. doi: 10.1016/0041-0101(94)90408-1. [DOI] [PubMed] [Google Scholar]
  27. Ohkura N., Inoue S., Ikeda K., Hayashi K. Isolation and amino acid sequence of a phospholipase A2 inhibitor from the blood plasma of Agkistrodon blomhoffii siniticus. J Biochem. 1993 Apr;113(4):413–419. doi: 10.1093/oxfordjournals.jbchem.a124060. [DOI] [PubMed] [Google Scholar]
  28. Ohkura N., Inoue S., Ikeda K., Hayashi K. Isolation and characterization of a phospholipase A2 inhibitor from the blood plasma of the Thailand cobra Naja naja kaouthia. Biochem Biophys Res Commun. 1994 Apr 29;200(2):784–788. doi: 10.1006/bbrc.1994.1519. [DOI] [PubMed] [Google Scholar]
  29. Ohkura N., Inoue S., Ikeda K., Hayashi K. The two subunits of a phospholipase A2 inhibitor from the plasma of Thailand cobra having structural similarity to urokinase-type plasminogen activator receptor and LY-6 related proteins. Biochem Biophys Res Commun. 1994 Nov 15;204(3):1212–1218. doi: 10.1006/bbrc.1994.2592. [DOI] [PubMed] [Google Scholar]
  30. Ovadia M., Kochva E., Moav B. The neutralization mechanism of Vipera palaestinae neurotoxin by a purified factor from homologous serum. Biochim Biophys Acta. 1977 Apr 25;491(2):370–386. doi: 10.1016/0005-2795(77)90280-x. [DOI] [PubMed] [Google Scholar]
  31. Ovadia M. Purification and characterization of an antihemorrhagic factor from the serum of the snake Vipera palaestinae. Toxicon. 1978;16(6):661–672. doi: 10.1016/0041-0101(78)90194-0. [DOI] [PubMed] [Google Scholar]
  32. Perales J., Villela C., Domont G. B., Choumet V., Saliou B., Moussatché H., Bon C., Faure G. Molecular structure and mechanism of action of the crotoxin inhibitor from Crotalus durissus terrificus serum. Eur J Biochem. 1995 Jan 15;227(1-2):19–26. doi: 10.1111/j.1432-1033.1995.tb20355.x. [DOI] [PubMed] [Google Scholar]
  33. Taylor M. E., Conary J. T., Lennartz M. R., Stahl P. D., Drickamer K. Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains. J Biol Chem. 1990 Jul 25;265(21):12156–12162. [PubMed] [Google Scholar]

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