Structures of N-Glycans of Bothrops Venoms Revealed as Molecular Signatures that Contribute to Venom Phenotype in Viperid Snakes.

The complexity of snake venoms has long been investigated to explore a myriad of biologically active proteins and peptides that are used for immobilizing or killing prey, and are responsible for the pathological effects observed on envenomation. Glycosylation is the main post-translational modification (PTM) of viperid venoms but currently there is little understanding of how protein glycosylation impacts the variation of venom proteomes. We have previously reported that Bothrops venom glycoproteomes contain a core of components that markedly define their composition and parallel their phylogenetic classification. Here we extend those observations to eight Bothrops species evaluating the N-glycomes by LC-MS as assigned cartoon structures and detailing those structures separately as methylated analogs using ion-trap mass spectrometry (MSn). Following ion disassembly through multiple steps provided sequence and linkage isomeric details that characterized 52 unique compositions in Bothrops venoms. These occurred as 60 structures, of which 26 were identified in the venoms of the Jararaca Complex (B. alcatraz, B. insularis, and B. jararaca), 20 in B. erythromelas, B. jararacussu, B. moojeni and B. neuwiedi venoms, and 22 in B. cotiara venom. Further, quantitative analysis of these N-glycans showed variable relative abundances in the venoms. For the first time a comprehensive set of N-glycan structures present in snake venoms are defined. Despite the fact that glycosylation is not template-defined, the N-glycomes of these venoms mirror the phylogeny cladograms of South American bothropoid snakes reported in studies on morphological, molecular data and feeding habits, exhibiting distinct molecular signatures for each venom. Considering the complexity of N-glycan moieties generally found in glycoproteins, characterized by different degrees of branching, isomer structures, and variable abundances, our findings point to these factors as another level of complexity in Bothrops venoms, features that could dramatically contribute to their distinct biological activities.

Evidence of facultative parthenogenesis in three Neotropical pitviper species of the Bothrops atrox group.

We examined four suspected cases of facultative parthenogenesis in three species of a neotropical lineage of pitvipers of the Bothrops atrox group. Reproduction without mating was observed in captive females of B. atrox, B. moojeni and B. leucurus housed alone for seven years (the two former species) and nine years (the latter one). In addition to the observation of captivity data, we investigated molecularly this phenomenon using heterologous microsatellites. DNA was extracted from the mothers' scales or liver, from embryo and newborn fragments, and yolked ova. Four of the microsatellites showed good amplification using Polymerase Chain Reaction and informative band segregation patterns among each mother and respective offspring. Captivity information, litter characteristics (comparison of the number of newborns, embryos and yolked ova) and molecular data altogether agreed with facultative parthenogenesis predictions in at least three out of the four mothers studied: B. atrox (ID#933) was heterozygous for three out of the four markers, and the sons S1 and S2 were homozygous; B. moojeni (BUT86) was heterozygous for two out of four markers, offspring S1, S3, E2, and E4, and O1 to O6 were homozygous; and B. leucurus (MJJS503) was heterozygous for three out of four markers, and son E1 and O1 were homozygous. B. moojeni (BUT44) was homozygous for all loci analyzed in the mother and offspring, which although not informative is also consistent with parthenogenesis. This study represents the first molecular confirmation of different pitviper species undergoing facultative parthenogenesis among Neotropical endemic snakes.