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. 2004 Jan 1;377(Pt 1):215–223. doi: 10.1042/BJ20030818

Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries.

Inn-Ho Tsai 1, Ying-Ming Wang 1, Yi-Hsuan Chen 1, Tein-Shun Tsai 1, Ming-Chung Tu 1
PMCID: PMC1223832  PMID: 12959640

Abstract

Phospholipases A2 (PLA2s) were purified from the Trimeresurus stejnegeri venom obtained from various localities in Taiwan and three provinces in China, by gel filtration followed by reversed-phase HPLC. The precise molecular mass and N-terminal sequence of each PLA2 were determined. In addition to the six previously documented PLA2 isoforms of this species, we identified ten novel isoforms. The venom gland cDNAs of individual specimens of the viper from four localities were used for PCR and subsequent cloning of the PLA2s. The molecular masses and partial sequences of most of the purified PLA2s matched with those deduced from a total of 13 distinct cDNA sequences of these clones. Besides the commonly known Asp49 or Lys-49 PLA2s of crotalid venoms, a novel type of PLA2 with Asn-49 substitution at the Ca2+-binding site was discovered. This type of PLA2 is non-catalytic, but may cause local oedema and appears to be a venom marker of many tree vipers. In particular, we showed that T. stejnegeri displayed high geographic variations of the PLA2s within and between their Taiwanese and Chinese populations, which can be explained by geological isolation and prey ecology. A phylogenetic tree of the acidic venom PLA2s of this species and other related Asian vipers reveals that T. stejnegeri contains venom genes related to those from several sympatric pit vipers, including the genera Tropedolaemus and Gloydius besides the Trimeresurus itself. Taken together, these findings may explain the exceptionally high variations in the venom as well as the evolutionary advantage of this species.

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Selected References

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  1. Chijiwa Takahito, Yamaguchi Yoko, Ogawa Tomohisa, Deshimaru Masanobu, Nobuhisa Ikuo, Nakashima Kinichi, Oda-Ueda Naoko, Fukumaki Yasuyuki, Hattori Shosaku, Ohno Motonori. Interisland evolution of Trimeresurus flavoviridis venom phospholipase A(2) isozymes. J Mol Evol. 2003 Mar;56(3):286–293. doi: 10.1007/s00239-002-2400-7. [DOI] [PubMed] [Google Scholar]
  2. Chow G., Subburaju S., Kini R. M. Purification, characterization, and amino acid sequence determination of acanthins, potent inhibitors of platelet aggregation from Acanthophis antarcticus (common death adder) venom. Arch Biochem Biophys. 1998 Jun 15;354(2):232–238. doi: 10.1006/abbi.1998.0685. [DOI] [PubMed] [Google Scholar]
  3. Creer S., Malhotra A., Thorpe R. S., Chou W. H. Multiple causation of phylogeographical pattern as revealed by nested clade analysis of the bamboo viper (Trimeresurus stejnegeri) within Taiwan. Mol Ecol. 2001 Aug;10(8):1967–1981. doi: 10.1046/j.0962-1083.2001.01332.x. [DOI] [PubMed] [Google Scholar]
  4. Creer Simon, Chou Wen-Hao, Malhotra Anita, Thorpe Roger S. Offshore insular variation in the diet of the Taiwanese bamboo viper Trimeresurus stejnegeri (Schmidt). Zoolog Sci. 2002 Aug;19(8):907–913. doi: 10.2108/zsj.19.907. [DOI] [PubMed] [Google Scholar]
  5. Creer Simon, Malhotra Anita, Thorpe Roger S., Stöcklin Reto S., Favreau Philippe S., Hao Chou Wen S. Genetic and ecological correlates of intraspecific variation in pitviper venom composition detected using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and isoelectric focusing. J Mol Evol. 2003 Mar;56(3):317–329. doi: 10.1007/s00239-002-2403-4. [DOI] [PubMed] [Google Scholar]
  6. Feng Bo, Wu Wei-Jia, Qian Rong, Wang Ke-Yi, Zhou Yuan-Cong. Purification and Characterization of Phospholipase A(2) from the Venom of Snake Trimeresurus stejnegeri Schmidt. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 1996;28(2):201–205. [PubMed] [Google Scholar]
  7. Fukagawa T., Matsumoto H., Shimohigashi Y., Ogawa T., Oda N., Chang C. C., Ohno M. Sequence determination and characterization of a phospholipase A2 isozyme from Trimeresurus gramineus (green habu snake) venom. Toxicon. 1992 Nov;30(11):1331–1341. doi: 10.1016/0041-0101(92)90510-c. [DOI] [PubMed] [Google Scholar]
  8. Fukagawa T., Nose T., Shimohigashi Y., Ogawa T., Oda N., Nakashima K., Chang C. C., Ohno M. Purification, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus gramineus (green habu snake) venom. Toxicon. 1993 Aug;31(8):957–967. doi: 10.1016/0041-0101(93)90255-h. [DOI] [PubMed] [Google Scholar]
  9. Giannasi N., Malhotra A., Thorpe R. S. Nuclear and mtDNA phylogenies of the Trimeresurus complex: implications for the gene versus species tree debate. Mol Phylogenet Evol. 2001 Apr;19(1):57–66. doi: 10.1006/mpev.2001.0899. [DOI] [PubMed] [Google Scholar]
  10. Glenn J. L., Straight R. C. Venom characteristics as an indicator of hybridization between Crotalus viridis viridis and Crotalus scutulatus scutulatus in New Mexico. Toxicon. 1990;28(7):857–862. doi: 10.1016/s0041-0101(09)80008-1. [DOI] [PubMed] [Google Scholar]
  11. Jan Virginie, Maroun R. C., Robbe-Vincent Annie, De Haro Luc, Choumet Valérie. Toxicity evolution of Vipera aspis aspis venom: identification and molecular modeling of a novel phospholipase A(2) heterodimer neurotoxin. FEBS Lett. 2002 Sep 11;527(1-3):263–268. doi: 10.1016/s0014-5793(02)03205-2. [DOI] [PubMed] [Google Scholar]
  12. Janssen M. J., Verheij H. M., Slotboom A. J., Egmond M. R. Engineering the disulphide bond patterns of secretory phospholipases A2 into porcine pancreatic isozyme. The effects on folding, stability and enzymatic properties. Eur J Biochem. 1999 Apr;261(1):197–207. doi: 10.1046/j.1432-1327.1999.00256.x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Malhotra A., Thorpe R. S. A phylogeny of the trimeresurus group of pit vipers: new evidence from a mitochondrial gene tree. Mol Phylogenet Evol. 2000 Aug;16(2):199–211. doi: 10.1006/mpev.2000.0779. [DOI] [PubMed] [Google Scholar]
  16. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  17. Nakai M., Nakashima K. I., Ogawa T., Shimohigashi Y., Hattori S., Chang C. C., Ohno M. Purification and primary structure of a myotoxic lysine-49 phospholipase A2 with low lipolytic activity from Trimeresurus gramineus venom. Toxicon. 1995 Nov;33(11):1469–1478. doi: 10.1016/0041-0101(95)00090-9. [DOI] [PubMed] [Google Scholar]
  18. Nakashima K., Nobuhisa I., Deshimaru M., Nakai M., Ogawa T., Shimohigashi Y., Fukumaki Y., Hattori M., Sakaki Y., Hattori S. Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5605–5609. doi: 10.1073/pnas.92.12.5605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Renetseder R., Dijkstra B. W., Huizinga K., Kalk K. H., Drenth J. Crystal structure of bovine pancreatic phospholipase A2 covalently inhibited by p-bromo-phenacyl-bromide. J Mol Biol. 1988 Mar 5;200(1):181–188. doi: 10.1016/0022-2836(88)90342-7. [DOI] [PubMed] [Google Scholar]
  20. Rigden Daniel J., Hwa Lee When, Marangoni Sérgio, Toyama Marcos H., Polikarpov Igor. The structure of the D49 phospholipase A2 piratoxin III from Bothrops pirajai reveals unprecedented structural displacement of the calcium-binding loop: possiblerelationship to cooperative substrate binding. Acta Crystallogr D Biol Crystallogr. 2003 Jan 23;59(Pt 2):255–262. doi: 10.1107/s0907444902021467. [DOI] [PubMed] [Google Scholar]
  21. Thomas R. G., Pough F. H. The effect of rattlesnake venom on digestion of prey. Toxicon. 1979;17(3):221–228. doi: 10.1016/0041-0101(79)90211-3. [DOI] [PubMed] [Google Scholar]
  22. Tsai I. H., Chen Y. H., Wang Y. M., Liau M. Y., Lu P. J. Differential expression and geographic variation of the venom phospholipases A2 of Calloselasma rhodostoma and Trimeresurus mucrosquamatus. Arch Biochem Biophys. 2001 Mar 15;387(2):257–264. doi: 10.1006/abbi.2000.2229. [DOI] [PubMed] [Google Scholar]
  23. Tsai I. H., Chen Y. H., Wang Y. M., Tu M. C., Tu A. T. Purification, sequencing, and phylogenetic analyses of novel Lys-49 phospholipases A(2) from the venoms of rattlesnakes and other pit vipers. Arch Biochem Biophys. 2001 Oct 15;394(2):236–244. doi: 10.1006/abbi.2001.2524. [DOI] [PubMed] [Google Scholar]
  24. Tsai I. H., Lu P. J., Wang Y. M., Ho C. L., Liaw L. L. Molecular cloning and characterization of a neurotoxic phospholipase A2 from the venom of Taiwan habu (Trimeresurus mucrosquamatus). Biochem J. 1995 Nov 1;311(Pt 3):895–900. doi: 10.1042/bj3110895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tsai Inn Ho, Wang Ying Ming, Chen Yi Hsuan, Tu Anthony T. Geographic variations, cloning, and functional analyses of the venom acidic phospholipases A2 of Crotalus viridis viridis. Arch Biochem Biophys. 2003 Mar 15;411(2):289–296. doi: 10.1016/s0003-9861(02)00747-6. [DOI] [PubMed] [Google Scholar]
  26. Wang Y. M., Liew Y. F., Chang K. Y., Tsai I. H. Purification and characterization of the venom phospholipases A2 from Asian monotypic crotalinae snakes. J Nat Toxins. 1999 Oct;8(3):331–340. [PubMed] [Google Scholar]
  27. Zhao K., Zhou Y., Lin Z. Structure of basic phospholipase A2 from Agkistrodon halys Pallas: implications for its association, hemolytic and anticoagulant activities. Toxicon. 2000 Jul;38(7):901–916. doi: 10.1016/s0041-0101(99)00193-2. [DOI] [PubMed] [Google Scholar]
  28. Zhong Xiaoyan, Jiao Haomang, Fan Liang, Wu Xiangfu, Zhou Yuancong. Functionally important residues for the anticoagulant activity of a basic phospholipase A(2) from the Agkistrodon halys pallas. Protein Pept Lett. 2002 Oct;9(5):427–434. doi: 10.2174/0929866023408580. [DOI] [PubMed] [Google Scholar]
  29. Zhu H., Dupureur C. M., Zhang X., Tsai M. D. Phospholipase A2 engineering. The roles of disulfide bonds in structure, conformational stability, and catalytic function. Biochemistry. 1995 Nov 21;34(46):15307–15314. doi: 10.1021/bi00046a040. [DOI] [PubMed] [Google Scholar]

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