Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper.

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (p > 0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62 µg/g (±2); biomass from cell suspension cultures 83 µg/g (±0.2); culture medium from suspensions 71.75 mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.

When spider and snake get along: fusion of a snake disintegrin with a spider phospholipase D to explore their synergistic effects on a tumor cell

Venoms of spiders and snakes contain toxins extremely active and, thus, provide a natural source for the development of new biotechnological tools. Among the diversity of toxins present in the venom of spiders from genus Loxosceles, the phospholipases D (PLDs) show high hydrolytic activity upon lysophosphatidylcholine (LPC) and sphingomyelin (SM), generating bioactive phospholipids such as cyclic phosphatidic acid (cPA). Since this mediator has been shown to play a major role in complex signaling pathways, including inhibition of tumor cells, the PLDs may hold the key to learn how toxins could be used for therapeutic purposes. However, the strong platelet aggregation of PLDs and their lack of selectivity impose a major limitation. On the other hand, disintegrins present in the venoms of Viperidae snakes are a potent inhibitor of platelet aggregation and possess high affinity and specificity to molecules called integrins that are highly expressed in some tumor cells, such as murine melanoma B16F10. Therefore, disintegrins might be suitable molecules to carry the PLDs to the malignant cells, so both toxins may work synergistically to eliminate these cells. Thus, in this work, a recombinant PLD from Loxosceles gaucho spider was recombinantly fused to a disintegrin from Echis carinatus snake to form a hybrid toxin called Rechistatin. This recombinant toxin was successfully expressed in bacteria, showed binding activity in B16F10 murine melanoma cells and exerted a synergistic cytotoxicity effect on these cells. Therefore, the approach presented in this work may represent a new strategy to explore new potential applications for spider PLDs.

Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in ruralpopulations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced byhyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyperimmune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebiteenvenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have producedrecombinant single chain variable fragment scFv against the venom of the pit viperBothrops asperat high levelsexpressed transiently and stably in transgenic plants andin vitrocultures that is reactive to BaP1 (a metallo-proteinase fromB. aspervenom). The yield from stably transformed plants was significantly (p > 0.05) higherthan the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stabletransformation were: transgenic callus 62µg/g ( ± 2); biomass from cell suspension cultures 83µg/g ( ± 0.2);culture medium from suspensions 71.75 mg/L ( ± 6.18). The activity of scFvBaP1 was confirmed by binding andneutralization of thefibrin degradation induced by BnP1 toxins fromB. neuwiediand by Atroxlysin Ia fromB.atroxvenoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to beused in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms tobe used in human therapy against snakebites.

When spider and snake get along: fusion of a snake disintegrin with a spider phospholipase D to explore their synergistic effects on a tumor cell

Venoms of spiders and snakes contain toxins extremely active and, thus, provide a natural source for the development of new biotechnological tools. Among the diversity of toxins present in the venom of spiders from genus Loxosceles, the phospholipases D (PLDs) show high hydrolytic activity upon lysophosphatidylcholine (LPC) and sphingomyelin (SM), generating bioactive phospholipids such as cyclic phosphatidic acid (cPA). Since this mediator has been shown to play a major role in complex signaling pathways, including inhibition of tumor cells, the PLDs may hold the key to learn how toxins could be used for therapeutic purposes. However, the strong platelet aggregation of PLDs and their lack of selectivity impose a major limitation. On the other hand, disintegrins present in the venoms of Viperidae snakes are a potent inhibitor of platelet aggregation and possess high affinity and specificity to molecules called integrins that are highly expressed in some tumor cells, such as murine melanoma B16F10. Therefore, disintegrins might be suitable molecules to carry the PLDs to the malignant cells, so both toxins may work synergistically to eliminate these cells. Thus, in this work, a recombinant PLD from Loxosceles gaucho spider was recombinantly fused to a disintegrin from Echis carinatus snake to form a hybrid toxin called Rechistatin. This recombinant toxin was successfully expressed in bacteria, showed binding activity in B16F10 murine melanoma cells and exerted a synergistic cytotoxicity effect on these cells. Therefore, the approach presented in this work may represent a new strategy to explore new potential applications for spider PLDs.

Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in ruralpopulations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced byhyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyperimmune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebiteenvenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have producedrecombinant single chain variable fragment scFv against the venom of the pit viperBothrops asperat high levelsexpressed transiently and stably in transgenic plants andin vitrocultures that is reactive to BaP1 (a metallo-proteinase fromB. aspervenom). The yield from stably transformed plants was significantly (p > 0.05) higherthan the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stabletransformation were: transgenic callus 62µg/g ( ± 2); biomass from cell suspension cultures 83µg/g ( ± 0.2);culture medium from suspensions 71.75 mg/L ( ± 6.18). The activity of scFvBaP1 was confirmed by binding andneutralization of thefibrin degradation induced by BnP1 toxins fromB. neuwiediand by Atroxlysin Ia fromB.atroxvenoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to beused in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms tobe used in human therapy against snakebites.

Toxin Fused with SUMO Tag: a new expression vector strategy to obtain recombinant venom toxins with easy tag removal inside the bacteria

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.

Clonagem e expressão de uma fosfolipase D recombinante do veneno da aranha Loxosceles gaucho / Cloning and expression of a recombinant phospholipase D from Loxosceles gaucho spider venom

The venom of the Loxosceles sp spiders comprises a mix of diverse toxins which results in an intense local inflammatory reaction, promotes dermonecrotic skin lesion, platelet aggregation, hemolytic anemia, and acute renal failure in rare cases. Among the toxins found in the venom, the phospholipases D (PLDs) also called dermonecrotic toxins, are the most important and well-studied proteins in the venom, since they assemble the main effects observed in loxoscelism. Despite the importance of these toxins, the biological analyses of PLDs are hampered by their small amount obtained in the venom. Therefore, the strategy of cloning and expression of these toxins in recombinant form in the bacterial system has been widely used and it is currently an important approach to obtain a huge amount of these molecules and to study their biological effects. However, despite the importance of Loxosceles gaucho venom, until now, any PLDs from this specie was isolated, cloned or expressed in a recombinant form. Thereby, in this work, we showed the cloning of cDNA that codifies a PLD from L. gaucho venom gland in pAE plasmid. The recombinant protein, named LgRec1 was produced in E. coli and then purified by IMAC, that resulted in a single band with molecular weight of 32kDa and a yield of 3.2 mg/L for culture medium. Analyzes of biological activity of this toxin showed that is able to promote local reaction (edema, erythema, bruising and pallor) and dermonecrosis; induce platelet aggregation; hydrolyze sphingomyelin and cause hemolysis. Neutralization assays using antibodies produced in rabbits to LgRec1 showed to be effective in inhibiting the local reaction (~ 65%) and dermonecrosis (~ 100%) evoked by whole venom, this indicates a potential use of these antibodies in neutralizing the toxic effects of envenomation. Therefore, the data presented in this work could help us understand the mechanism of action of PLDs in the envenomation caused by Loxosceles spider genus.

O veneno de aranhas Loxosceles compreende uma mistura de diversas toxinas que induz uma intensa reação inflamatória local, promove lesão dermonecrótica, agregação plaquetária, anemia hemolítica e insuficiência renal aguda em casos raros. Entre as várias toxinas encontradas no veneno, as fosfolipases D (FLD), também denominadas de toxinas dermonecróticas, são as mais importantes e mais bem estudadas, uma vez que albergam os principais efeitos observados no loxoscelismo. Apesar de sua importância, a análise biológica de FLDs é dificultada pela quantidade reduzida de veneno obtido. Assim, a estratégia de clonagem e expressão destas toxinas, na forma recombinante em sistema bacteriano, tem sido amplamente empregada e é atualmente uma abordagem muito importante para obter grande quantidade destas toxinas no sentido de estudar os seus efeitos biológicos. Contudo, apesar da importância do veneno de Loxosceles gaucho, até o presente momento nenhuma FLDs desta espécie foi isolada, clonada ou expressa na forma recombinante. Deste modo, mostramos neste trabalho, a clonagem do cDNA que codifica uma FLD da glândula de veneno de L. gaucho em vetor pAE. A proteína recombinante, denominada de LgRec1, foi produzida em E. coli e purificada por IMAC, resultando em uma banda única com massa molecular de 32KDa e rendimento de 3,2 mg/L cultura. Análises de atividade biológica desta toxina mostrou que ela é capaz de promover reação local (edema, eritema, equimose e palidez) e dermonecrose; induzir agregação plaquetária; hidrolizar esfingomielina e promover hemólise. Testes de neutralização utilizando anticorpos produzidos em coelhos contra a LgRec1, mostraram-se eficazes em inibir a reação local (~ 65%) e a dermonecrose (~ 100%) promovidas pelo veneno, indicando um potencial para o uso destes anticorpos na neutralização dos efeitos tóxicos do veneno total. Dessa forma, os dados apresentados neste trabalho poderão colaborar para a compreensão dos mecanismos de ação das FLDs no envenenamento provocado pelas aranhas do gênero Loxosceles.

Diversity of metalloproteinases in Bothrops neuwiedi snake venom transcripts: evidences for recombination between different classes of SVMPs.

BACKGROUND: Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and are versatile toxins, targeting many important elements involved in hemostasis, such as basement membrane proteins, clotting proteins, platelets, endothelial and inflammatory cells. The functional diversity of SVMPs is in part due to the structural organization of different combinations of catalytic, disintegrin, disintegrin-like and cysteine-rich domains, which categorizes SVMPs in 3 classes of precursor molecules (PI, PII and PIII) further divided in 11 subclasses, 6 of them belonging to PII group. This heterogeneity is currently correlated to genetic accelerated evolution and post-translational modifications. RESULTS: Thirty-one SVMP cDNAs were full length cloned from a single specimen of Bothrops neuwiedi snake, sequenced and grouped in eleven distinct sequences and further analyzed by cladistic analysis. Class P-I and class P-III sequences presented the expected tree topology for fibrinolytic and hemorrhagic SVMPs, respectively. In opposition, three distinct segregations were observed for class P-II sequences. P-IIb showed the typical segregation of class P-II SVMPs. However, P-IIa grouped with class P-I cDNAs presenting a 100% identity in the 365 bp at their 5' ends, suggesting post-transcription events for interclass recombination. In addition, catalytic domain of P-IIx sequences segregated with non-hemorrhagic class P-III SVMPs while their disintegrin domain grouped with other class P-II disintegrin domains suggesting independent evolution of catalytic and disintegrin domains. Complementary regions within cDNA sequences were noted and may participate in recombination either at DNA or RNA levels. Proteins predicted by these cDNAs show the main features of the correspondent classes of SVMP, but P-IIb and P-IIx included two additional cysteines cysteines at the C-termini of the disintegrin domains in positions not yet described. CONCLUSIONS: In B. neuwiedi venom gland, class P-II SVMPs were represented by three different types of transcripts that may have arisen by interclass recombination with P-I and P-III sequences after the divergence of the different classes of SVMPs. Our observations indicate that exon shuffling or post-transcriptional mechanisms may be driving these recombinations generating new functional possibilities for this complex group of snake toxins.

Diversity of metalloproteinases in Bothrops neuwiedi snake venom transcripts: evidences for recombination between different classes of SVMPs

Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and are versatiletoxins, targeting many important elements involved in hemostasis, such as basement membrane proteins, clottingproteins, platelets, endothelial and inflammatory cells. The functional diversity of SVMPs is in part due to thestructural organization of different combinations of catalytic, disintegrin, disintegrin-like and cysteine-rich domains,which categorizes SVMPs in 3 classes of precursor molecules (PI, PII and PIII) further divided in 11 subclasses, 6 ofthem belonging to PII group. This heterogeneity is currently correlated to genetic accelerated evolution and posttranslationalmodifications. Thirty-one SVMP cDNAs were full length cloned from a single specimen of Bothrops neuwiedi snake,sequenced and grouped in eleven distinct sequences and further analyzed by cladistic analysis. Class P-I and classP-III sequences presented the expected tree topology for fibrinolytic and hemorrhagic SVMPs, respectively. Inopposition, three distinct segregations were observed for class P-II sequences. P-IIb showed the typical segregationof class P-II SVMPs. However, P-IIa grouped with class P-I cDNAs presenting a 100% identity in the 365 bp at their5’ ends, suggesting post-transcription events for interclass recombination. In addition, catalytic domain of P-IIxsequences segregated with non-hemorrhagic class P-III SVMPs while their disintegrin domain grouped with otherclass P-II disintegrin domains suggesting independent evolution of catalytic and disintegrin domains.Complementary regions within cDNA sequences were noted and may participate in recombination either at DNAor RNA levels.