Proteomic and genomic characterisation of venom proteins from Oxyuranus species

The genus Oxyuranus includes three of the largest and most feared Australasian snakes, and are listed within the top ten of the world's most venomous snakes. This genus includes Oxyuranus microlepidotus (inland taipan), Oxyuranus scutellatus scutellatus (coastal taipan) and the subspecies Oxyuranus scutellatus canni (Papuan taipan). Despite comparative differences in the proportions of venom components the Oxyuranus species have been reported to be more closely related to each other than to those of any other large Australian elapid snakes. Nevertheless, differences in the clinical presentation of envenomed patients have been described between species. It has also been shown that the antivenom (produced from O. s. scutellatus) may not be as effective in bite victims of O. microlepidotus or O. s. canni compared to O. s. scutellatus. This project was a comparative study designed to substantially build upon previous research into the venom of Oxyuranus species. The objectives of this study were: firstly, to conduct a comparative study of the composition of venom proteins from the three Oxyuranus species; secondly to clone and characterise venom specific proteins. This study was primarily conducted utilising fundamental proteomic tools including chromatography, one- and two-dimensional gel electrophoresis (2DE) mass spectrometry and N-terminal sequence determination. The third objective was to use a cDNA expression library, constructed using mRNA from a venom gland of O. s. scutellatus, to screen with taipan antivenom in order to isolate nucleotide sequences important within the venom. The venoms from Oxyuranus showed remarkable complexity and stability. It was shown that the three Oxyuranus venoms share strong similarities in the protein patterning of major proteins. O. s. scutellatus and O. s. canni venoms exhibited identical protein patterning throughout electrophoretic, fractionation and immunobinding analysis. The venom similarities, together with previous comparative research using mitochondrial DNA sequencing and morphology studies indicated O. canni is not a subspecies but the same species of O. s. scutellatus. O. microlepidotus on the other hand, though sharing major proteins, revealed patterns distinctly different to O. s. scutellatus through electrophoretic and fractionation examinations. Immunobinding studies showed the major banding differences in O. microlepidotus were antigenic against taipan antivenom (CSL). These results also indicated that diet, feeding and habitat (captive/wild) do not play a major role in contributing to the venom compositions of the taipan, opposing literature stating the contrary in other species. Variation within the venom composition between the species may be due, instead, to the geographic distances and habitat pressures between the species. From these comparisons, an antigenic 300 kDa glycoprotein from O. s. scutellatus was identified using 15% SDS-PAGE and was isolated using size exclusion and affinity chromatography. This protein was comprised of a homo trimer, three subunits of 100kDa joined by disulphide bonds. N-terminal sequence shared homology to a previously identified serine protease. This protein cleaved chromogenic substrate S-2288 and Michaelis-Menten kinetics revealed this protein to undergo allosteric interactions. Results of inhibition profiles, pH optimum and kinetic studies confirmed this to be a trypsin-like serine protease. It was highly sensitive to benzamidine and PMSF and was inhibited only by high concentrations of alkylating and reducing agents NEM and iodoacetamide. Metal chelators 1,10-phenanthroline, EGTA and EDTA showed little inhibition; whereas SDS was the only reagent to completely prevent any activity. Based on this evidence it is proposed all three subunits are required for activity. This protease appeared to be present in the venoms of both O. microlepidotus and O. s. canni and may represent another coagulative enzyme within the venom. Sequencing studies elucidated the nucleotide sequences of a variety of clones from an O. s. scutellatus cDNA library. These clones, translated into full-length protein sequences, included homologous matches to characterised proteins including polymerases, carboxylases, dehydrogenases, Protein disulphide isomerases (PDI's), Heat shock proteins (HSP's) and myosin. Based on the sequences obtained, six PLA2 toxins, two post-synaptic toxins and the complete factor V component of a prothrombin activator (scutelarin) were proposed. This study represents the first report of full-length amino acid sequences of taipan venom proteins; previous studies have been conducted using isolated venom proteins that lack the nucleotide and transit peptide sequences. cDNA and deduced amino acid sequences were compared with those of other snake species. These comparisons conformed to the established primary structures found within the differing peptide classes and, within the putative pre-synaptic neurotoxins, conformed to the evolutionary outline suggested for these sequences