title: Combined venom gland cDNA sequencing and venomics of the New Guinea small-eyed snake, Micropechis ikaheka.
abstract: The venom arsenal of the New Guinea small-eyed snake, Micropechis ikaheka, was investigated by a joint cDNA sequencing and venomics approach. Twenty-seven full-length DNA sequences encoding novel venom proteins were recovered in this study. Using this cDNA dataset we achieved locus-specific resolution for 19 out of the approximately 50 reverse-phase- and SDS-PAGE-separated venom proteins. The venom proteome of M. ikaheka is dominated by at least 29 D49-phospholipase A₂ (PLA₂) and 14 short and long neurotoxins of the three-finger toxin (3FTx) family. These protein classes represent, respectively, 80% and 9.2% of the total venom proteins. Two PIII-metalloproteinase (SVMP) molecules (7.6%), three CRISP isoforms (1.8%), and a single Kunitz-type inhibitor, vespryn, 5'-nucleotidase, serine proteinase and LAO molecules, none of which represents more than 0.7% of the total venom proteome, complete the protein arsenal of M. ikaheka. In concordance with clinical observations, this venom composition points to a central role for post-synaptically-acting neurotoxic toxins in the envenomation strategy developed by this species. PLA₂ molecules represent the main myotoxic components of M. ikaheka venom. In addition, the estimated LD₅₀ for mice of the reverse-phase-isolated 3FTx (0.22 mg/kg) and PLA₂ (1.62 mg/kg) enriched fractions, strongly suggests that these two toxin classes contribute synergistically to venom lethality, with the 3FTxs playing a dominant role. The high structural and functional conservation exhibited by M. ikaheka and Australian elapid venoms may underlay the positive clinical outcomes of envenoming resulting from bites by M. ikaheka that have been documented through the use of bioCSL polyvalent antivenom.
  
  
  CONCLUSIONS
  The poorly understood venom proteome of the New Guinea small-eyed snake, Micropechis ikaheka, a large and powerfully built elapid endemic to Papua New Guinea and Indonesian West Papua province, was investigated through a combined venomics and venom gland transcriptomics approach. Although M. ikaheka accounts for only a small proportion of snakebites on the mainland, 40% of snakebites on Karkar Island are attributed to bites by this snake. Major effects of envenomings include life-threatening post-synaptic neuromuscular blockade resulting in respiratory paralysis, myotoxicity, severe bleeding, hypotension and cardiovascular abnormalities. We have investigated the contribution of 3FTxs and PLA₂ molecules in venom lethality, myotoxicity, and cardiovascular function. Our work provides important correlations between venom composition and its pharmacological activity. In conjunction with the antivenomics work reported in the companion paper, our study may contribute to improve treatment outcomes for snakebite victims of M. ikaheka.
authors.affiliation: School of Medicine & Health Sciences, University of Papua New Guinea, Boroko, NCD, Papua New Guinea.
authors.forename: Owen
authors.initials: O
authors.lastname: Paiva
authors.affiliation: Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain.
authors.forename: Davinia
authors.initials: D
authors.lastname: Pla
authors.affiliation: Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia; Australian Venom Research Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Wright
authors.affiliation: Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia; Australian Venom Research Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia.
authors.forename: Markus
authors.initials: M
authors.lastname: Beutler
authors.affiliation: Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain.
authors.forename: Libia
authors.initials: L
authors.lastname: Sanz
authors.affiliation: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
authors.forename: José María
authors.initials: JM
authors.lastname: Gutiérrez
authors.affiliation: School of Medicine & Health Sciences, University of Papua New Guinea, Boroko, NCD, Papua New Guinea; Australian Venom Research Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia. Electronic address: david.williams@unimelb.edu.au.
authors.forename: David J
authors.initials: DJ
authors.lastname: Williams
authors.affiliation: Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain. Electronic address: jcalvete@ibv.csic.es.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Calvete
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics*; D004546:Elapid Venoms / Q000737:chemistry* / Q000235:genetics*; D017815:Elapidae / Q000235:genetics*; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000235:genetics; D009512:New Guinea; D020543:Proteome / Q000737:chemistry / Q000235:genetics; D012469:Salivary Glands / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D004546:Elapid Venoms; D009498:Neurotoxins; D020543:Proteome
keywords: Micropechis ikaheka; Pharmacological effects of snake venom; Phospholipase A(2); Snake venomics; Three-finger toxin; Venom gland cDNA