Snake venomics: Proteomic and genomic analysis of Echis ocellatus disintegrins

Juárez, P.¹, Wagstaff, S.², Harrison, R.A.² & Calvete, J.J.¹

1 Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain; 2 Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK

Snake venoms contain a variety compounds affecting many biological functions. The toxic effects of venoms are complex because different components have distinct actions and may, in addition, act in concert with other venom molecules, enhancing their activities or contributing to the spreading of toxins. Venoms of Viperidae and Crotalidae snakes contain proteins that interfere with the coagulation cascade, the normal hemostatic system and tissue repair. We have undertaken the analysis of the protein composition of the venom of Echisocellatus by 2D-SDS-PAGE and RP-HPLC, N-terminal sequencing, MALDI-TOF and MS/MS analysis. The venom proteome is composed of proteins belonging to a few protein families, including enzymes (serine proteinases, Zn²⁺-dependent PI-PIV metalloproteases of the reprolysin family, group II phospholipase A2 isoenzymes) and proteins with no enzymatic activity (disintegrins). Disintegrins selectively block the function of b1 and b3 integrin receptors, and thus establishing structure-function correlations of disintegrins may provide information of structural determinants involved in integrin recognition that may be useful in basic and clinical research.  Disintegrins are divided into five groups according to their polypeptide length and number of disulphide bonds. The first group includes short disintegrins composed of 41-51 residues and 4 disulphide bonds. The second group is formed by the medium-sized disintegrins which contain about 70 amino acids and 6 disulphide bonds.The third group includes the long disintegrins bearing 84 residue polypeptides cross-linked by 7 disulphide bonds. The disintegrin domains of PIII-SVMPs contain about 100 amino acids with 16 cysteine residues involved in the formation of 8 disulphide bonds constitute the fourth group of the disintegrin family. Unlike short, medium and long disintegrins, which are single-chain molecules, the fifth group is composed of homo- and heterodimers. Dimeric disintegrins contain subunits of about 67 residues with 10 cysteines involved in the formation of 4 intra-chain disulphide bonds and 2 interchain cystine linkages. Comparison of the amino acid sequences of disintegrins by multiple sequence alignment and phylogenetic analysis, in conjunction with current biochemical and genetic data, support the view that the different disintegrin subfamilies evolved from a common ADAM scaffold and that structural diversification occurred through disulphide bond engineering. To understand the genomic basis of the accelerated evolution of disintegrins, and the molecular mechanism underlaying their structural diversification, we have started the genomic analysis of cDNAs coding for Echisocellatus disintegrins. Using primers specific for highly conserved disintegrin regions, we have PCR-amplified cDNAs coding for novel monomeric and dimeric disintegrins. These ORFs (350-2000 bp) exhibit conserved structural features, which indicate clues for delineating phylogenetic relationships. Strikingly, many of the cDNA sequences correspond to non-expressed products in the venom, pointing to the existence of a vast evolutionary genomic potential and to regulatory mechanisms for the expression of disintegrins.

Correspondence to: jcalvete@ibv.csic.es or pjuarez@ibv.csic.es