The Diversity of Bioactive Proteins in Australian Snake Venoms

Jeffrey J. Gorman,Tristan P. Wallis,Martin F. Lavin,Geoff W. Birrell,Paul P. Masci,John de Jersey,Stephen T.H. Earl

doi:10.1074/mcp.m600419-mcp200

Abstract

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from <7 to over 150 kDa and in pI from 3 to >10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of post-translational modification). Venom proteins and their post-translational modifications were characterized using specific antibodies, phosphoprotein- and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A2 and alpha-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5'-nucleotidases, cysteine-rich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.

Highlights

Australian elapid snakes are among the most venomous in the world
Protein components of the venoms from the 18 species listed in Table I were initially separated by 1D SDS-PAGE and subsequently silver-stained (Fig. 1)
A prominent band of 30 –35 kDa corresponding in size to the heavy chain of Factor Xa (FXa)-like protease was observed in several venoms including the Notechis, Pseudonaja, and Oxyuranus species

Summary

Introduction

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5؅-nucleotidases, cysteinerich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics. The 1461amino acid FVa-like non-enzymatic subunit of Pseutarin C has been shown to share 50% sequence identity and significant structural homology with human FVa [15] Another hemostasis-related family of proteins identified in Australian snake venom is the textilinin family of plasmin inhibitors [16]. Six isoforms of textilinin have been identified in P. textilis venom gland-derived cDNA [7]

Results

Discussion

Conclusion