Comparative characterization of Viperidae snake venoms from Perú reveals two compositional patterns of phospholipase A2 expression

Abstract

Snake species within the Bothrops complex (sensu lato) are of medical relevance in Latin America, but knowledge on their venom characteristics is limited, or even unavailable, for some taxa. Perú harbors 17 species of pit vipers, within the genera Bothrops, Bothriechis, Bothrocophias, Porthidium, Crotalus, and Lachesis. This study compared the venoms of twelve species, through chromatographic and electrophoretic profiles, as well as proteolytic and phospholipase A₂ (PLA₂) activities. Also, proteomic profiles were analyzed for nine of the venoms using a shotgun approach. Results unveiled conspicuous differences in the expression of venom PLA₂s among species, six of them presenting scarce levels as judged by RP-HPLC profiles. Since most species within the bothropoid lineage possess venoms with high to intermediate abundances of this protein family, our findings suggest the existence of a phenotypic duality in the expression of venom PLA₂s within the Bothrops (sensu lato) complex. Bothrops barnetti and Bothrocophias andianus venoms, very scarce in PLA₂s, were shown to lack significant myotoxic activity, highlighting that the observed variability in PLA₂ expression bears toxicological correlations with effects attributed to these proteins. Finally, an attempt to identify phylogenetic relationships of bothropoid species from Perú presenting low- or high-PLA₂ venom phenotypes showed an interspersed pattern, thus precluding a simple phylogenetic interpretation of this venom compositional dichotomy.

Keywords: Bothropoid; Dichotomy; Phenotype; Phospholipase A2; Snake venom; Viperidae.

Graphical abstract

Fig. 1

SDS-PAGE profiles of venoms from twelve viperid species of Perú, unreduced (A, B) or reduced (C, D). Venoms of some species from other locations are included: Bothrops asper from the Pacific (P) or Caribbean (C) regions of Costa Rica, B. alternatus from Argentina, B. neuwiedi from Uruguay, and B. schlegelii from Costa Rica (CR). An additional sample of B. atrox venom from Perú is indicated with an asterisk. Molecular weight (Mw) markers are indicated in kDa. Proteins were stained with Coomassie brilliant blue G-250. The venom of B. oligolepis could not be analyzed in this technique due to insufficient amount. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

C₁₈ reverse-phase HPLC profiles of venoms from twelve viperid species of Perú. The venom of Bothrops asper from Costa Rica was included as a reference. The dotted light green shaded area indicates the known elution region for PLA₂/PLA₂-like proteins in viperid venoms in this standardized chromatographic gradient. Venoms were divided into two groups, those with evident peaks in such region (enclosed within the blue frame) and those with very minor peaks. For reference to colors, the reader is referred to the web version of this article. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Superposition of the C₁₈ reverse-phase HPLC profiles of the venoms of Bothriechis schlegelii from Costa Rica (CR; green trace) and Perú (blue trace). For reference to colors, the reader is referred to the web version of this article. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Phospholipase A₂ activity of venoms from twelve viperid species of Perú on 4-nitro-3-octanoyloxy-benzoic acid. The venom of Bothrops asper from Costa Rica was included as a reference. Each point represents the mean ± SD of three replicates.

Fig. 5

Proteolytic activity of venoms from twelve viperid species of Perú on azocasein. The venom of Bothrops asper from Costa Rica was included as a reference. Each point represents the mean ± SD of three replicates.

Fig. 6

Comparative evaluation of the myotoxic activity of the venoms of (A)Bothrops barnetti and (B)Bothrocophias andianus (Perú), compared to Bothrops asper (Costa Rica) in mice. Venoms were injected by the intramuscular route into groups of five mice. A control group received an injection of vehicle (PBS) alone. After 3 h the plasma creatine kinase (CK) activity was determined. Each bar represents the mean ± SD (n = 5). No statistical difference (p > 0.05) was observed between CK levels of PBS and B. barnetti venom, or PBS and B. andianus venom. B. asper venom was significantly different from both groups, in both independent experiments.

Fig. 7

Partial phylogenies of bothropoid viperids showing the relationships of species reported to produce venoms with high (pink dots) and low (blue dots) phospholipase A₂ contents. The species from Perú analyzed in the present study are indicated by names in corresponding pink or blue colors. The phylogenetic trees were generated by Carrasco et al. (2016) using a total evidence approach from morphological, ecological, and mtDNA sequences (12 S and 16 S rRNA). Phylogenetic reconstruction included (A) or excluded (B) sequences from ND4 and cytb genes. For reference to colors, the reader is referred to the web version of this article. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Supplemental Fig. S1

RP-HPLC profiles of the venoms of Bothrops ayerbei from Colombia (Mora-Obando et al., 2014b) and Bothrocophias campbelli from Ecuador (Salazar-Valenzuela et al., 2014), showing the large variability in the expression of phospholipase A₂ proteins. These venoms are not included in the phylogenetic trees shown in Fig.7 and therefore their positions and relationships to other bothropoid species cannot be indicated. As indicated in the legend of Fig.2, the dotted light green shaded area indicates the known elution region for PLA₂/PLA₂-like proteins in viperid venoms in this standardized chromatographic gradient. These two species exemplify the evident dichotomy between venoms with “high-" and “low-PLA₂″ phenotypes among bothropoids. For reference to colors, the reader is referred to the web version of this article.