Bothrops insularis venomics: a proteomic analysis supported by transcriptomic-generated sequence data

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A2 and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and Mr of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies.

Genetic diversity in populations of the viper Bothrops moojeni Hoge, 1966 in Central Brazil using RAPD markers.

Bothrops moojeni is an abundant venomous snake responsible for most of the snakebite cases in the Central region of Brazil and as a result of the anthropogenic habitat disturbance, such as the increase in extensive farming, the range of B. moojeni has been greatly fragmented. Here, we obtained genomic DNA from a total of 75 snakes belonging to four populations. Genetic variability evaluated for five RAPD primers was low (He = 0.20) and was not spatially structured. We found evidence of significant genetic divergence among B. moojeni populations that were isolated (PhiST values of 0.21 and 0.25), while populations more proximal exhibited less divergence (PhiST values of 0.04 and 0.08). We found only moderate divergence (PhiST value of 0.12) between two populations greatly isolated (851.83 km apart) along with great differentiation (0.24) between two proximal populations (290 km apart). Even though these populations are close to each other, they occur in an urbanized area that is almost completely covered by extensive crops, representing an obstruction to the mobility of this viper. Molecular variance analysis (AMOVA) showed some degree of subdivision in these populations, with a PhiST value of 0.16, significant to the level of 1% by 1000 random permutations. We also performed a Bayesian analysis that confirmed the AMOVA results and found a value of thetaB = 0.14 and an f = 0.27, suggesting a high level of endogamy. This is the first study that characterizes genetic variability for this important species of the Bothrops genus, and our data are of significant importance in terms of classifying populations in relation to their conservational value and management strategies. Thus, given the high levels of population structure found in this case, we recommend sampling as many populations as possible to maximize the genetic variability to be preserved when aiming for in situ conservation. The same should be done to perform samplings toward ex situ conservation.

Bothroalternin, a thrombin inhibitor from the venom of Bothrops alternatus.

Bothroalternin (MW 27 kDa), a new member of the family of C-type lectins is a thrombin inhibitor which was purified from pooled B. alternatus venom by affinity chromatography on PPACK-thrombin-Sepharose, followed by size exclusion and reverse-phase on HPLC columns. Material retained on the affinity column contained proteins with apparent molecular weights ranging from 20 to 60 kDa on SDS-PAGE and inhibited aggregation of rabbit platelets induced by alpha-thrombin (IC50 = 28 microg/ml). A single band of approximately 27 kDa was recognized in Western-blot assays using polyclonal antibodies raised against bothrojaracin, a thrombin inhibitor purified from B. jararaca venom (Zingali et al., 1993). The immunological similarity of this fraction to bothrojaracin was confirmed by ELISA and competitive ELISA. Further purification by size exclusion and reverse-phase on HPLC, produced a single homogenous peak called bothroalternin. This protein was highly homologous to bothrojaracin (95% in its N-terminal sequence-for residues 1 to 25) but displaying lower inhibitory effect on thrombin induced platelet aggregation (Ic50 = 0.19 microg/ml) compared to bothrojaracin (IC50 = 0.06). Altogether, bothroalternin is a new thrombin inhibitor isolated from Bothrops alternatus venom and has been characterized as a bothrojaracin-like protein.

Bothrops jararaca snakes produce several bothrojaracin isoforms following an individual pattern.

More than one isoform of bothrojaracin (BJC), a potent and specific thrombin inhibitor isolated from Bothrops jararaca venom, has been found in individual venoms collected from adult snakes. Variations in snake venom composition have previously been associated with factors such as age, sex, geographic origin, season of the year and diet. In order to obtain further information concerning individual patterns of expression of BJC isoforms, we have analyzed five individual Bothrops jararaca snake venoms collected at the same time from adult female snakes from the same geographic region. As expected, crude venoms showed a similar migration pattern on SDS-PAGE. BJC was purified using a procedure which includes an affinity chromatography step (PPACK-thrombin Sepharose). A slight variation in the amount of BJC obtained from individual venom samples was noticed. Inhibition of thrombin-induced platelet aggregation as well as migration pattern on SDS-PAGE (under reducing and non-reducing conditions) and isoelectric focusing varied considerably among BJC samples from the five snakes. The amino-terminal sequences (residues 1-34) of individual BJC samples were compared with the sequence deduced from isolated cDNAs encoding alpha and beta chains of BJC. A high degree of homology was detected, although some residues differed from one sample to other. Altogether, data confirmed the heterogeneity found for BJC purified from individual snakes. Thus, the results indicate that: (1) individual specimens of Bothrops jararaca have different patterns of BJC isoform expression; and (2) it seems that genetic factors, at least in part, determine the variability found in BJC production.