Analysis of Australian elapid snake venoms for the discovery and development of new human therapeutics

Earl, Stephen (2007). Analysis of Australian elapid snake venoms for the discovery and development of new human therapeutics PhD Thesis, School of Medicine, The University of Queensland.

       
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Author Earl, Stephen
Thesis Title Analysis of Australian elapid snake venoms for the discovery and development of new human therapeutics
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2007-07
Thesis type PhD Thesis
Supervisor Martin Lavin
Geoff Birrell
Subjects 11 Medical and Health Sciences
Formatted abstract Australian snake venoms are potent cocktails of bioactive proteins that interfere with several mammalian physiological processes. In many cases, these venom proteins have an extremely stable structure, are very specific in their site of action and act with high efficacy. When compared to exotic snake venoms, Australian snake venom composition has not been extensively studied and thus these venoms are likely to contain as yet undiscovered components. For these reasons, we hypothesise that Australian snake venoms are a potential source of new human pharmaceuticals. Despite significant medical advances over the last few decades, a number of serious health conditions including pain, cancer and stroke remain without adequate pharmaceutical treatments. Hence, pharmaceutical development to treat these conditions is urgently needed and is of great significance to improved health and medical care.

This project has employed a combined approach of transcriptomics through a venom gland cDNA microarray and proteomics via two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to systematically examine the venom components of several Australian elapid snakes. It was designed to identify all venom proteins and from these select novel venom proteins with potential for development as new human therapeutics. The venom gland cDNA microarray employed expression profiling between the venom gland and the liver to identify venom gland specific transcripts. From these, a number of previously known venom proteins were identified, along with new venom proteins and one potential therapeutic candidate, venom nerve growth factor (NGF). When separated by 2-DE, the Australian venoms showed approximately 100- 200 discrete protein spots, varying in molecular weight from 7 to over 100 kDa and pI from 3 to 10. Using MS, approximately 90 % of protein spots were identified. These included previously characterised venom proteins such as phospholipase A2 enzymes, neurotoxins and prothrombin-activating proteins. A number of novel venom proteins were also identified from the proteomic work and two proteins were selected as potential therapeutic candidates, C-type lectin and taicatoxin serine protease inhibitor (TSPI). In addition, the proteomic analysis confirmed that a number of venom proteins contain N-linked carbohydrate groups.

Further transcriptomic strategies such as PCR and rapid amplification of cDNA ends (RACE) were successfully employed to obtain the nucleotide coding sequence for these three candidates from venom gland cDNA. NGF from Oxyuranus scutellatus venom was demonstrated to possess neurite differentiation activity and therefore may have potential application in the treatment of neurodegenerative disorders. Native and recombinant TSPI were found to be potent inhibitors of plasmin and kallikrein and the recombinant protein was shown to have antifibrinolytic effects in whole blood. TSPI may therefore have application as an antibleeding agent or in disorders involving overactive serine proteases such as inflammation. Six distinct isoforms of C-lectin were cloned from the venom gland cDNA of the Australian elapid snakes and an antibody generated against one of these isoforms confirmed the presence of this protein in the venom of several of these snakes. A mannose-binding C-lectin from O. scutellatus venom was demonstrated to bind erythrocytes and it is envisaged that these C-lectins may have potential therapeutic application as new antimicrobial agents.

This work represents the most comprehensive analysis and identification of Australian snake venom proteins. This study has provided important insights into these toxins at the molecular level which may now be utilised both in detection and treatment of snake bites and in the design and development of new human therapeutic agents.

 
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