Analysis of the subproteomes of proteinases and heparin‐binding toxins of eight Bothrops venoms

Viperid snakes show the most complex snake‐venom proteomes and offer an intriguing challenge in terms of understanding the nature of their components and the pathological outcomes of envenomation characterized by local and systemic effects. In this work, the venom complexity of eight Bothrops species was analyzed by 2‐DE, and their subproteomes of proteinases were explored by 2‐D immunostaining and 2‐D gelatin zymography, demonstrating the diversity of their profiles. Heparin, a highly sulfated glycosaminoglycan released from mast cells, is involved in anti‐coagulant and anti‐inflammatory processes. Here, we explored the hypothesis that heparin released upon envenomation could interact with toxins and interfere with venom pathogenesis. We first identified the Bothrops venom subproteome of toxins that bind with high‐affinity for heparin as composed of mainly serine proteinases and C‐type lectins. Next, we explored the Bothrops jararaca toxins that bind to heparin under physiological conditions and identified a relationship between the subproteomes of proteinases, and that of heparin‐binding toxins. Only the non‐bound fraction, composed mainly of metalloproteinases, showed lethal and hemorrhagic activities, whereas the heparin‐bound fraction contained mainly serine proteinases associated with coagulant and fibrinogenolytic activities. These data suggest that heparin binding to B. jararaca venom components in vivo has a minor protective effect to venom toxicity.     

Snake venomics of the Brazilian pitvipers Bothrops cotiara and Bothrops fonsecai. Identification of taxonomy markers

We report the proteomic characterization of venom of the pitvipers Bothrops cotiara and Bothrops fonsecai. Crude venoms were fractionated by reverse-phase HPLC, followed by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and CID-MS/MS. Each venom contained around 30 proteins in the range of 7-110 kDa belonging to only 8 (B. cotiara) and 9 (B. fonsecai) families which may target the hemostatic system, albeit distinctly distributed among the two species. B. cotiara and B. fonsecai share medium-sized disintegrins, disintegrin-like/cysteine-rich (DC) fragments, snake venom vascular endothelial growth factor, cysteine-rich secretory proteins, serine proteinases, C-type lectins, l-amino acid oxidase, and Zn(2+)-dependent metalloproteinases. In addition, B. fonsecai expresses a high abundance PLA(2) molecule (13,890 Da), whereas PLA(2) molecules were not detected in B. cotiara's venom. This striking finding is in line with previous biochemical analyses showing the absence of phospholipasic activity in the venom of B. cotiara. The potential adaptive significance of the lack of PLA(2) molecules is enigmatic, and alternative explanations are discussed. B. fonsecai is morphologically extremely similar to B. cotiara. Our comparative proteomic analysis shows that compositional differences between their venoms can be employed as a taxonomy signature for unambiguous species identification independently of geographic origin and morphological characteristics.     

Angiostatin-like molecules are generated by snake venom metalloproteinases

Angiostatin is a plasminogen-derived anti-angiogenic factor composed of its first four kringle structures. This molecule is generated by proteolytic cleavage of plasminogen by some proteolytic enzymes in vitro. Since venoms of viper snakes are a rich source of both serine- and metalloproteinase, we hypothesized that angiostatin-like polypeptides could be generated during the envenomation after snake bites and play a pathophysiological role in the local tissue damage and regeneration. Our results showed that crude venoms from several species of Bothrops snakes were able to generate angiostatin-like polypeptides and purified metalloproteinases but not serine proteinases from Bothrops jararaca and Bothrops moojeni venoms were responsible for their generation in vitro. The putative plasminogen cleavage sites by the crude venoms and purified proteinases were determined by N-terminal amino acid sequencing of the angiostatin-like molecules. Angiostatin-like peptides derived from human plasminogen digestion by jararhagin, a metalloproteinase isolated from B. jararaca venom, inhibited endothelial cell proliferation in vitro. These results indicate that angiostatin-like molecules can be generated upon snakebite envenomations and may account for the poor and incomplete regenerative response observed in the damaged tissue.     

Biological and immunological properties of the venom of Bothrops alcatraz, an endemic species of pitviper from Brazil

Bothrops alcatraz is a new pitviper species derived from the Bothrops jararaca group, whose natural habitat is situated in Alcatrazes Archipelago, a group of marine islands near São Paulo State coast in Brazil. Herein, the biological and biochemical properties of venoms of four adult specimens of B. alcatraz were examined comparatively to a reference pool of Bothrops jararaca venom. Both venoms showed similar activities and electrophoretic patterns, but B. alcatraz venom showed three protein bands of molecular masses of 97, 80 and 38 kDa that were not present in B. jararaca reference venom. The i.p. median lethal dose of B. alcatraz venom ranged from 5.1 to 6.6 mg/kg, while it was 1.5 mg/kg for B. jararaca venom. The minimum hemorrhagic dose of B. jararaca venom was 0.63, whereas 2.28 μg/mouse for B. alcatraz venom. In contrast, B. alcatraz venom was more potent in regard to procoagulant and proteolytic activities. These differences were supported by western blotting and neutralization tests, employing commercial bothropic antivenom, which showed that hemorrhagic and lethal activities of B. alcatraz venom were less effectively inhibited than B. jararaca venom. Such results evidence that B. alcatraz shows quantitative and qualitative differences in venom composition in comparison with its B. jararaca relatives, which might represent an optimization of venom towards a specialized diet.     

Snake population venomics and antivenomics of Bothrops atrox: Paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management

We describe two geographically differentiated venom phenotypes across the wide distribution range of Bothrops atrox, from the Colombian Magdalena Medio Valley through Puerto Ayacucho and El Paují, in the Venezuelan States of Amazonas and Orinoquia, respectively, and S?o Bento in the Brazilian State of Maranh?o. Colombian and Venezuelan venoms show an ontogenetic toxin profile phenotype whereas Brazilian venoms exhibit paedomorphic phenotypes. Venoms from each of the 16 localities sampled contain both population-specific toxins and proteins shared by neighboring B. atrox populations. Mapping the molecular similarity between conspecific populations onto a physical map of B. atrox range provides clues for tracing dispersal routes that account for the current biogeographic distribution of the species. The proteomic pattern is consistent with a model of southeast and southwest dispersal and allopatric fragmentation northern of the Amazon Basin, and trans-Amazonian expansion through the Andean Corridor and across the Amazon river between Monte Alegre and Santarém. An antivenomic approach applied to assess the efficacy towards B. atrox venoms of two antivenoms raised in Costa Rica and Brazil using Bothrops venoms different than B. atrox in the immunization mixtures showed that both antivenoms immunodepleted very efficiently the major toxins (PIII-SVMPs, serine proteinases, CRISP, LAO) of paedomorphic venoms from Puerto Ayacucho (Venezuelan Amazonia) through S?o Bento, but had impaired reactivity towards PLA(2) and P-I SVMP molecules abundantly present in ontogenetic venoms. The degree of immunodepletion achieved suggests that each of these antivenoms may be effective against envenomations by paedomorphic, and some ontogenetic, B. atrox venoms.     

Venom of the crotaline snake Atropoides nummifer (jumping viper) from Guatemala and Honduras: comparative toxicological characterization,isolation of a myotoxic phospholipase A2 homologue and neutralization by two antivenoms

A comparative study was performed on the venoms of the crotaline snake Atropoides nummifer from Guatemala and Honduras. SDS-polyacrylamide gel electrophoresis, under reducing conditions, revealed a highly similar pattern of these venoms, and between them and the venom of the same species from Costa Rica. Similar patterns were also observed in ion-exchange chromatography on CM-Shephadex C-25, in which a highly basic myotoxic fraction was present. This fraction was devoid of phospholipase A₂ activity and strongly reacted, by enzyme-immunoassay, with an antiserum against Bothrops asper myotoxin II, a Lys-49 phospholipase A₂ homologue. A basic myotoxin of 16 kDa was isolated to homogeneity from the venom of A. nummifer from Honduras, showing amino acid composition and N-terminal sequence similar to those of Lys-49 phospholipase A₂ variants previously isolated from other crotaline snake venoms. Guatemalan and Honduran A. nummifer venoms have a qualitatively similar toxicological profile, characterized by: lethal; hemorrhagic; myotoxic; edema-forming; coagulant; and defibrinating activities, although there were significant quantitative variations in some of these activities between the two venoms. Neutralization of toxic activities by two commercially-available antivenoms in the region was studied. Polyvalent antivenom produced by Instituto Clodomiro Picado was effective in the neutralization of: lethal; hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A₂ activities, but ineffective against edema-forming activity. On the other hand, MYN polyvalent antivenom neutralized: hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A₂ activities, albeit with a lower potency than Instituto Clodomiro Picado antivenom. MYN antivenom failed to neutralize lethal and edema-forming activities of A. nummifer venoms.     

Comparison of Phylogeny,Venom Composition and Neutralization by Antivenom in Diverse Species of Bothrops Complex

In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB – soro antibotrópico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A₂ reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted.     

Snake venomics of Central American pitvipers: clues for rationalizing the distinct envenomation profiles of Atropoides nummifer and Atropoides picadoi

We report the proteomic characterization of the Central American pitvipers Atropoides nummifer and Atropoides picadoi. The crude venoms were fractionated by reverse-phase high-performance liquid chromatography (HPLC), followed by analysis of each chromatographic fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass fingerprinting, and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of tryptic peptides. Each venom contained a number of bradykinin-potentiating peptides and around 25-27 proteins of molecular masses in the range of 7-112 kDa, belonging to only nine different toxin families (disintegrin, DC fragment, snake venom vascular endothelial growth factor, phospholipases A2, serine protease, cysteine-rich secretory proteins, C-type lectins, L-amino acid oxidase, and Zn2+-dependent metalloproteases), albeit distinctly distributed among the two Atropoides species. In addition, A. nummifer expresses low amounts of a three-finger toxin not detected in the venom of A. picadoi. The major toxins of A. nummifer belong to the PLA2 (relative abundance, 36.5%) and the serine proteinase (22%) families, whereas the most abundant A. picadoi toxins are Zn2+-dependent metalloproteinases (66.4%). We estimate that the similarity of venom proteins between the two Atropoides taxa may be around 14-16%. The high degree of differentiation in the venom proteome among congeneric taxa emphasizes unique aspects of venom composition of related species of Atropoides snakes and points to a strong role for adaptive diversification via natural selection as a cause of this distinctiveness. On the other hand, their distinct venom toxin compositions provide clues for rationalizing the low hemorrhagic, coagulant, and defibrinating activities and the high myotoxic and proteolytic effects evoked by A. nummifer snakebite in comparison to other crotaline snake venoms and the high hemorrhagic activity of A. picadoi.     

Biological and immunological characteristics of the poison of Bothrops cotiara (Serpentes: Viperidae)

Bothrops cotiara is a venomous snake sporadically found in the province of Misiones in Argentina, South of Brazil and Paraguay. Data on the clinics of the envenomation produced by its bite and on its venom are scarce. There is no information on the neutralizing capacity of the antivenoms available. In this study, the lethal potency, hemorrhagic, necrotizing, coagulant and thrombin-like, defibrinogenating, indirect hemolytic and fibrinolytic activities of the venom of B. cotiara specimens from the province of Misiones were determined. The toxic activities were within the range of those described for the other Bothrops species from Argentina, and the electrophoretic and chromatographic studies showed similarities with those described for the other bothropic venoms. The immunochemical reactivity of six South American anti Viper antivenoms (ELISA) have a strong reactivity with all the antivenoms studied. The neutralizing capacity of three of these therapeutic antivenoms against the lethal potency and hemorrhagic, necrotizing, coagulant, thrombin-like and hemolytic activities showed a very close neutralizing capacity. Our data strongly suggest that the antivenoms for therapeutic use available in this area of South America are useful to neutralize the toxic and enzymatic activities of the venom of this uncommon specie of Bothrops.     

Bothrops jararaca venom proteome rearrangement upon neonate to adult transition

The pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. In this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2-DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenal both in terms of the total proteome, as of the glycoproteome. N-glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snake development, the subproteome of metalloproteinases undergoes a shift from a P-III-rich to a P-I-rich profile while the serine proteinase profile does not vary significantly. We also used isobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. The iTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in-depth understanding of venom complexity variation particularly with regard to toxin families that have been associated with envenomation pathogenesis.     

Inhibition of enzymatic and pharmacological activities of some snake venoms and toxins by Mandevilla velutina (Apocynaceae) aqueous extract

Phospholipases A(2) (PLA(2)) are multifunctional proteins which exhibit varied biological activities correlated to the structural diversities of the sub-classes. The crude aqueous extract from subterranean system of Mandevilla velutina, a plant found in Brazilian savanna, was assayed for its ability to inhibit biological activities of several snake venoms and isolated PLA(2)s. The extract induced total inhibition of the phospholipase activity of Crotalus durissus terrificus venom and only partial inhibition of Bothrops venoms. When assayed against purified toxins, the highest efficacy was detected against CB and crotoxin, while almost ineffective against PLA(2)s from the genus Bothrops. Although M. velutina crude extract significantly inhibited the myotoxic activity of C. d. terrificus venom and CB, it produced only partial inhibition of either Bothrops jararacussu venom or its main myotoxins BthTX-I (basic Lys49), BthTX-II (basic Asp49) and BthA-I-PLA(2) (acidic Asp49). The extract exhibited also full inhibition of hemorrhage caused by Bothrops alternatus, Bothrops moojeni and Bothrops pirajai snake venoms, but partial inhibition (90%) of that induced by B. jararacussu venom. The extract was ineffective to inhibit the fibrinogenolytic activity of B. moojeni, B. alternatus and B. pirajai crude venoms, while their caseinolytic activity was only partially inhibited. No inhibition of the anticoagulant activity, although partial reduction of the edema-inducing activity of C. d. terrificus and B. alternatus crude venoms, CB, PrTX-I, BthTX-I and crotoxin was observed. Besides extending survival of mice injected with lethal doses of C. d. terrificus and B. jararacussu venoms, M. velutina extract decreased to 50% the lethality of mice. Extracts of 18 month old micropropagated plants were able to partially neutralize the effect of the crude venoms and toxins.     

Characterization of the snake venoms from seven Brazilian species of Bothrops by FPLC anion-exchange chromatography.

1. The elution profiles and the caseinolytic, myotoxic, coagulant and hemorrhagic activities of the venoms of seven Bothrops species fractionated on a Mono-Q FPLC column were analyzed. 2. Each venom separated into 16-20 peaks, with good reproducibility and the activities were concentrated in virtually discrete regions of the chromatogram. 3. There is a considerable overlap of active proteins in the different species venoms and our results indicate that a venom pool with the species B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. atrox venoms would contain the major active proteins determined in the seven species.     

Neutralization of toxic and enzyme activities of 4 venoms from snakes of Guatemala and Honduras by the polyvalent antivenin produced in Costa Rica

We studied the ability of the polyvalent antivenom produced in Costa Rica to neutralize lethal, hemorrhagic, edema-forming, proteolytic, hemolytic, hyaluronidase and fibrinolytic activities of the venoms of Bothrops asper and B. nummifer from Honduras, and of Agkistrodon bilineatus and Crotalus durissus durissus from Guatemala. Neutralizing ability of antivenom was expressed as ED50 (effective dose 50%), defined as the antivenom/venom ratio at which the activity of the venom is reduced 50%. Antivenom is highly effective in the neutralization of lethal, hemorrhagic, hemolytic, hyaluronidase, and caseinolytic activities of B. asper, B. nummifer, and C. d. durissus venoms. In the case of B. nummifer venom, neutralization of fibrinolytic effect was only partial, whereas this activity was adequately neutralized when studying the venoms of B. asper and C. d. durissus. The venom of A. bilineatus was adequately neutralized by the antivenom, with the only exception of hemolytic effect that was reduced only partially. However, in quantitative terms, a relatively large volume of antivenom was required to neutralize some effects induced by A. bilineatus venom. Regarding edema-forming activity, antivenom neutralized efficiently the venoms of B. asper and A. bilineatus, whereas that of B. nummifer was neutralized only partially; on the other hand, edema induced by the venom of C. d. durissus was not neutralized at all. Immunochemical results indicate a close immunological relationship between venoms of B. asper, B. nummifer and C. d. durissus collected in Honduras and Guatemala with those of the same species collected in Costa Rica. Interspecies comparison, however, showed variation between venoms obtained from different species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemorrhagic, coagulant and fibrino(geno)lytic activities of crude venom and fractions from mapanare (Bothrops colombiensis) snakes

Bothrops colombiensis venom from two similar geographical locations were tested for their hemostatic functions and characterized by gel-filtration chromatography and SDS-PAGE electrophoresis. The snakes were from Caucagua and El Guapo towns of the Venezuelan state of Miranda. Fibrino(geno)lytic, procoagulant, hemorrhagic, lethal activities, gel-filtration chromatography and SDS-PAGE profiles were analyzed and compared for both venoms. The highest hemorrhagic activity of 5.3 mug was seen in El Guapo venom while Caucagua venom had the lowest LD(50) of 5.8 mg/kg. Both venoms presented similar thrombin-like activity. El Guapo showed a factor Xa-like activity two times higher than Caucagua. Differences were observed in kallikrein-like and t-PA activities, being highest in El Guapo. Caucagua venom showed the maximum fibrin lysis. Both crude venom runs on Sephadex G-100 chromatography gave fraction SII with the high fibrinolytic activity. Proteases presented in SII fractions and eluted from Benzamidine-Sepharose (not bound to the column) provoked a fast degradation of fibrinogen alpha chains and a slower degradation of beta chains, which could possibly be due to a higher content of alpha fibrinogenases in these venoms. The fibrinogenolytic activity was decreased by metalloprotease inhibitors. The results suggested that metalloproteases in SII fractions were responsible for the fibrinolytic activity. The analysis of samples for fibrin-zymography of SII fractions showed an active band with a molecular mass of approximately 30 kDa. These results reiterate the importance of using pools of venoms for antivenom immunization, to facilitate the neutralization of the maximum potential number of toxins.     

BjussuSP-I: a new thrombin-like enzyme isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr=61,000 under reducing conditions and pI approximately 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated serine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca(2+) and Mg(2+)). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against 11 venom samples of Bothrops, 1 of Crotalus, and 1 of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDINEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom.     

Biochemical characterization of the lizard toxin gilatoxin

The Gila monster (genus Heloderma) is the only known lizard to produce and inject a venomous secretion. Little is known about the venom from these lizards, and none of the toxins have been isolated until this time. This paper reports the isolation and characterization of a major lethal toxin (gilatoxin) from the venoms of Heloderma suspectum and Heloderma horridum. Gilatoxins from both species were similar in amino acid composition, electrophoretic mobility, pI, and immunological reactivity. They are acidic proteins possessing molecular weights of 35 000-37 500 and isoelectric points of 4.25 and consist of a single polypeptide chain. Neither is antigenically related to the venoms of snakes. The toxins are devoid of phospholipase A2 activity and proteolytic, hemorrhagic, and hemolytic activities, with lethality being the only biological activity detectably expressed. The toxins appear to be unique and distinct from those of other venomous animals.     

A comparative study of the biological properties of some venoms of snakes of the genus Bothrops (American lance-headed viper).

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 26 samples of venoms from 13 species of Bothrops were determined, and the Sephadex G-75 gel filtration patterns for some of the venoms also examined. 2. The results show that while there are considerable individual variations in the biological activities of many of the Bothrops venoms tested, there are some common characteristics at the genus and species levels. 3. The differences in the biological properties of the Bothrops venoms tested can be used for the differentiation of most Bothrops species examined.     

Profilings of subproteomes of lectin-binding proteins of nine Bothrops venoms reveal variability driven by different glycan types

Snake venom proteomes have long been investigated to explore a multitude of biologically active components that are used for prey capture and defense, and are involved in the pathological effects observed upon mammalian envenomation. Glycosylation is a major protein post-translational modification in venoms and contributes to the diversification of proteomes. We have shown that Bothrops venoms are markedly defined by their content of glycoproteins, and that most N-glycan structures of eight Bothrops venoms contain sialic acid, while bisected N-acetylglucosamine was identified in Bothrops cotiara venom. To further investigate the mechanisms involved in the generation of different venoms by related snakes, here the glycoproteomes of nine Bothrops venoms (Bothrops atrox, B. cotiara, Bothrops erythromelas, Bothrops fonsecai, B. insularis, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi) were comparatively analyzed by enrichment with three lectins of different specificities, recognizing bisecting N-acetylglucosamine- and sialic acid-containing glycoproteins, and mass spectrometry. The lectin capture strategy generated venom fractions enriched with several glycoproteins, including metalloprotease, serine protease, and L- amino acid oxidase, in addition to various types of low abundant enzymes. The different contents of lectin-enriched proteins underscore novel aspects of the variability of the glycoprotein subproteomes of Bothrops venoms and point to the role of distinct types of glycan chains in generating different venoms by closely related snake species.