Current State Of Bothrops insularisVenom Proteome, A Joint Effort By The Rio de JaneiroProteomic Network

Valente, R.H.

Physiology and Pharmacodynamic Department, Oswaldo Cruz Foundation and Rio de Janeiro Proteomic Network, Rio de Janeiro, Brazil.

Bothropsinsularis is an endemic snake found in Ilha da Queimada Grande, 20 miles from the Brazilian coast. Differently from other bothropic snakes it preys on birds and some invertebrates available in its habitat. Since snake venoms contain a complex mixture of proteins (enzymes, biologically active peptides) and non-proteic components it is thought that evolutionary pressure could have resulted in an adaptation of its venom components providing divergent toxins to better capture the preys. A group from Instituto Butantan, headed by Dr. Paulo L. Ho, is performing a survey of gene expression and diversity in B. insularis venom glands through the generation of expressed sequence tags (ESTs) and putting out a wealth of genomic data. Thus, B. insularis venom seems to be a good model system in order to study toxin expression.

The strategy for the elucidation of the proteome followed the use of preparative 2D-PAGE to separate polypeptide chains. Samples were dissolved in 8-9.5M Urea, 2%(w/v) CHAPS, 20mM Dithiotreitol, 0.002%(w/v) Bromophenol blue and 4-7 IPG buffer or 3-10 Biolyte. The first dimension was performed on 4-7 IPG strips and the second dimension on a 15%T SDS-PAGE under reducing conditions. Several variables were tested in order to identify the best electrophoretic conditions, including: i) removing possible interferents (salts, DNA, RNA etc.) from the venom prior to the 2D; ii) applying different quantities to the immobilized pH gradient strip; iii) loading the venom to the strip by anodic or cathodic cup loading as opposed to in gel rehydration loading; iv) using 3-10 Biolyte instead of 4-7 IPG buffer in rehydration solution and v) using IPG buffer at 0.5 or 1.0% (v/v). Salt removal using trichloroacetic acid precipitation followed by acetone washes did not improve significantly gel quality and neither did the removal of eventual nucleic acids by the use of DNAse and RNAse. Loading less sample (1.0mg of venom as opposed to 1.8 mg) also did not improve gel quality since horizontal streaking and, to a lesser extent, vertical streaking still persisted. Sample loading by anodic cup loading only improved gel quality in the far end basic region while the rest of the gel displayed poor resolution when compared to in gel rehydration sample loading. Cathodic cup loading gave the poorest results. Finally, the use of Biolyte 3-10 did not improve resolution and best results were with 0.5 or 1.0% (v/v) 4-7 IPG buffer.

From the reference gel (1.8 mg, dry weight, venom by in gel rehydration with 0.5% (v/v) 4-7 IPG buffer) major spots were used to generate tryptic hydrolysates for peptide mapping by MALDI-TOF MS and/or sequencing by LC-ESI-ION TRAP MS. Searches in Gene/ ESTs data banks allowed identification and a possible function assignment for the proteins. We were able to identify more than 30 proteins scattered all over our 2D reference gel (with molecular masses ranging from 12-70 kDa and pIs ranging from 4-7), including metalloproteinases, serineproteinases, phospholipases A2, platelet agglutinating inhibiting protein, lectins, platelet glycoprotein Ib-binding protein alpha and beta subunits, vascular endothelial growth factor, convulxin beta and  anticoagulant protein A.

Our results indicate that the horizontal streaking observed in our reference gel is not due to artifacts or bad sample preparation but rather to a large number of protein isoforms and/or glycosylated molecules, which makes snake venom 2D analysis not so straightforward as expected due to the ease of sample preparation.

Supported by: FAPESP and FAPERJ.