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. 2002 Sep 15;366(Pt 3):971–976. doi: 10.1042/BJ20020092

Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2.

Lucimara Chioato 1, Arthur H C De Oliveira 1, Roberto Ruller 1, Juliana M Sá 1, Richard J Ward 1
PMCID: PMC1222840  PMID: 12079495

Abstract

Bothropstoxin-I (BthTx-I) is a Lys(49)-phospholipase A(2) from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca(2+)-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117-122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys(122), these same substitutions did not significantly alter the Ca(2+)-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca(2+)-independent membrane-damaging activity but, with the exception of Lys(122), had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115-119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A(2), and support a model of a Ca(2+)-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.

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Selected References

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  1. Andrião-Escarso S. H., Soares A. M., Rodrigues V. M., Angulo Y., Díaz C., Lomonte B., Gutiérrez J. M., Giglio J. R. Myotoxic phospholipases A(2) in bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu. Biochimie. 2000 Aug;82(8):755–763. doi: 10.1016/s0300-9084(00)01150-0. [DOI] [PubMed] [Google Scholar]
  2. Baker S. F., Othman R., Wilton D. C. Tryptophan-containing mutant of human (group IIa) secreted phospholipase A2 has a dramatically increased ability to hydrolyze phosphatidylcholine vesicles and cell membranes. Biochemistry. 1998 Sep 22;37(38):13203–13211. doi: 10.1021/bi981223t. [DOI] [PubMed] [Google Scholar]
  3. Braun P., von Heijne G. The aromatic residues Trp and Phe have different effects on the positioning of a transmembrane helix in the microsomal membrane. Biochemistry. 1999 Jul 27;38(30):9778–9782. doi: 10.1021/bi990923a. [DOI] [PubMed] [Google Scholar]
  4. 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. 1998 Oct 15;358(2):343–350. doi: 10.1006/abbi.1998.0853. [DOI] [PubMed] [Google Scholar]
  5. Calderón L., Lomonte B. Inhibition of the myotoxic activity of Bothrops asper myotoxin II in mice by immunization with its synthetic 13-mer peptide 115-129. Toxicon. 1999 Apr;37(4):683–687. doi: 10.1016/s0041-0101(98)00211-6. [DOI] [PubMed] [Google Scholar]
  6. Dijkstra B. W., Drenth J., Kalk K. H. Active site and catalytic mechanism of phospholipase A2. Nature. 1981 Feb 12;289(5798):604–606. doi: 10.1038/289604a0. [DOI] [PubMed] [Google Scholar]
  7. Dijkstra B. W., Kalk K. H., Drenth J., de Haas G. H., Egmond M. R., Slotboom A. J. Role of the N-terminus in the interaction of pancreatic phospholipase A2 with aggregated substrates. Properties and crystal structure of transaminated phospholipase A2. Biochemistry. 1984 Jun 5;23(12):2759–2766. doi: 10.1021/bi00307a035. [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. 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]
  10. Guex N., Peitsch M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis. 1997 Dec;18(15):2714–2723. doi: 10.1002/elps.1150181505. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Heinrikson R. L. Dissection and sequence analysis of phospholipases A2. Methods Enzymol. 1991;197:201–214. doi: 10.1016/0076-6879(91)97146-p. [DOI] [PubMed] [Google Scholar]
  13. Janssen M. J., Burghout P. J., Verheij H. M., Slotboom A. J., Egmond M. R. Introduction of a C-terminal aromatic sequence from snake venom phospholipases A2 into the porcine pancreatic isozyme dramatically changes the interfacial kinetics. Eur J Biochem. 1999 Aug;263(3):782–788. doi: 10.1046/j.1432-1327.1999.00557.x. [DOI] [PubMed] [Google Scholar]
  14. Killian J. A., von Heijne G. How proteins adapt to a membrane-water interface. Trends Biochem Sci. 2000 Sep;25(9):429–434. doi: 10.1016/s0968-0004(00)01626-1. [DOI] [PubMed] [Google Scholar]
  15. Lambeau G., Lazdunski M. Receptors for a growing family of secreted phospholipases A2. Trends Pharmacol Sci. 1999 Apr;20(4):162–170. doi: 10.1016/s0165-6147(99)01300-0. [DOI] [PubMed] [Google Scholar]
  16. Lee B. I., Yoon E. T., Cho W. Roles of surface hydrophobic residues in the interfacial catalysis of bovine pancreatic phospholipase A2. Biochemistry. 1996 Apr 2;35(13):4231–4240. doi: 10.1021/bi9524777. [DOI] [PubMed] [Google Scholar]
  17. Liu X., Wu X., Zhou Y. Identification of key residues responsible for enzymatic and platelet-aggregation-inhibiting activities of acidic phospholipase A2S from Agkistrodon halys Pallas. J Nat Toxins. 2001 Feb;10(1):43–55. [PubMed] [Google Scholar]
  18. 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]
  19. Lomonte B., Pizarro-Cerdá J., Angulo Y., Gorvel J. P., Moreno E. Tyr-->Trp-substituted peptide 115-129 of a Lys49 phospholipase A(2) expresses enhanced membrane-damaging activities and reproduces its in vivo myotoxic effect. Biochim Biophys Acta. 1999 Nov 9;1461(1):19–26. doi: 10.1016/s0005-2736(99)00143-1. [DOI] [PubMed] [Google Scholar]
  20. Maraganore J. M., Heinrikson R. L. The lysine-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. Relation of structure and function to other phospholipases A2. J Biol Chem. 1986 Apr 15;261(11):4797–4804. [PubMed] [Google Scholar]
  21. Nelson R. M., Long G. L. A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal Biochem. 1989 Jul;180(1):147–151. doi: 10.1016/0003-2697(89)90103-6. [DOI] [PubMed] [Google Scholar]
  22. Núez C. E., Angulo Y., Lomonte B. Identification of the myotoxic site of the Lys49 phospholipase A(2) from Agkistrodon piscivorus piscivorus snake venom: synthetic C-terminal peptides from Lys49, but not from Asp49 myotoxins, exert membrane-damaging activities. Toxicon. 2001 Oct;39(10):1587–1594. doi: 10.1016/s0041-0101(01)00141-6. [DOI] [PubMed] [Google Scholar]
  23. Ownby C. L., Selistre de Araujo H. S., White S. P., Fletcher J. E. Lysine 49 phospholipase A2 proteins. Toxicon. 1999 Mar;37(3):411–445. doi: 10.1016/s0041-0101(98)00188-3. [DOI] [PubMed] [Google Scholar]
  24. Prijatelj P., Copic A., Krizaj I., Gubensek F., Pungercar J. Charge reversal of ammodytoxin A, a phospholipase A2-toxin, does not abolish its neurotoxicity. Biochem J. 2000 Dec 1;352(Pt 2):251–255. doi: 10.1042/0264-6021:3520251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rufini S., Cesaroni P., Desideri A., Farias R., Gubensek F., Gutiérrez J. M., Luly P., Massoud R., Morero R., Pedersen J. Z. Calcium ion independent membrane leakage induced by phospholipase-like myotoxins. Biochemistry. 1992 Dec 15;31(49):12424–12430. doi: 10.1021/bi00164a018. [DOI] [PubMed] [Google Scholar]
  26. Scott D. L., White S. P., Otwinowski Z., Yuan W., Gelb M. H., Sigler P. B. Interfacial catalysis: the mechanism of phospholipase A2. Science. 1990 Dec 14;250(4987):1541–1546. doi: 10.1126/science.2274785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Selistre de Araujo H. S., White S. P., Ownby C. L. cDNA cloning and sequence analysis of a lysine-49 phospholipase A2 myotoxin from Agkistrodon contortrix laticinctus snake venom. Arch Biochem Biophys. 1996 Feb 1;326(1):21–30. doi: 10.1006/abbi.1996.0042. [DOI] [PubMed] [Google Scholar]
  28. Sumandea M., Das S., Sumandea C., Cho W. Roles of aromatic residues in high interfacial activity of Naja naja atra phospholipase A2. Biochemistry. 1999 Dec 7;38(49):16290–16297. doi: 10.1021/bi9921384. [DOI] [PubMed] [Google Scholar]
  29. VAN DEENENL, DE HAAS G. H. THE SUBSTRATE SPECIFICITY OF PHOSPHOLIPASE A. Biochim Biophys Acta. 1963 Oct 22;70:538–553. doi: 10.1016/0006-3002(63)90792-3. [DOI] [PubMed] [Google Scholar]
  30. Ward R. J., Alves A. R., Ruggiero Neto J., Arni R. K., Casari G. A SequenceSpace analysis of Lys49 phopholipases A2: clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity. Protein Eng. 1998 Apr;11(4):285–294. doi: 10.1093/protein/11.4.285. [DOI] [PubMed] [Google Scholar]
  31. Ward R. J., Monesi N., Arni R. K., Larson R. E., Paço-Larson M. L. Sequence of a cDNA encoding bothropstoxin I, a myotoxin from the venom of Bothrops jararacussu. Gene. 1995 Apr 24;156(2):305–306. doi: 10.1016/0378-1119(95)00099-r. [DOI] [PubMed] [Google Scholar]
  32. Ward R. J., de Oliveira A. H., Bortoleto R. K., Rosa J. C., Faça V. M., Greene L. J. Refolding and purification of Bothropstoxin-I, a Lys49-phospholipase A2 homologue, expressed as inclusion bodies in Escherichia coli. Protein Expr Purif. 2001 Feb;21(1):134–140. doi: 10.1006/prep.2000.1353. [DOI] [PubMed] [Google Scholar]
  33. Ward Richard J., Chioato Lucimara, de Oliveira Arthur H. C., Ruller Roberto, Sá Juliana M. Active-site mutagenesis of a Lys49-phospholipase A2: biological and membrane-disrupting activities in the absence of catalysis. Biochem J. 2002 Feb 15;362(Pt 1):89–96. doi: 10.1042/0264-6021:3620089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yu B. Z., Rogers J., Tsai M. D., Pidgeon C., Jain M. K. Contributions of residues of pancreatic phospholipase A2 to interfacial binding, catalysis, and activation. Biochemistry. 1999 Apr 13;38(15):4875–4884. doi: 10.1021/bi982215f. [DOI] [PubMed] [Google Scholar]
  35. da Silva Giotto M. T., Garratt R. C., Oliva G., Mascarenhas Y. P., Giglio J. R., Cintra A. C., de Azevedo W. F., Jr, Arni R. K., Ward R. J. Crystallographic and spectroscopic characterization of a molecular hinge: conformational changes in bothropstoxin I, a dimeric Lys49-phospholipase A2 homologue. Proteins. 1998 Mar 1;30(4):442–454. doi: 10.1002/(sici)1097-0134(19980301)30:4<442::aid-prot11>3.0.co;2-i. [DOI] [PubMed] [Google Scholar]
  36. de Oliveira A. H., Giglio J. R., Andrião-Escarso S. H., Ito A. S., Ward R. J. A pH-induced dissociation of the dimeric form of a lysine 49-phospholipase A2 abolishes Ca2+-independent membrane damaging activity. Biochemistry. 2001 Jun 12;40(23):6912–6920. doi: 10.1021/bi0026728. [DOI] [PubMed] [Google Scholar]

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