Venome of the Brazilian rattlesnake Crotalus durissus terrificus and characterization of a phospholipase A2 inhibitor: a possible adjuvant in the antivenom therapy

Os acidentes ofídicos no Brasil vêm aumentando ano a ano e são considerados um problema negligenciado de saúde pública. O único tratamento eficaz é a administração do soro antiofídico, mas reações adversas estão relacionadas à sua administração. A crotoxina, um complexo de fosfolipases A2 (PLA2), é um dos componentes mais estudados da peçonha crotálica, mas muitos outros componentes são menos conhecidos devido a sua baixa abundância. Portanto, o presente estudo objetivou a análise do transcriptoma da glândula e do proteoma da peçonha da serpente Crotalus durissus terrificus, bem como a caracterização bioquímica e funcional de um inibidor de PLA2 (PLI) recombinante dessa serpente. A biblioteca de cDNA da glândula de peçonha foi construída usando a plataforma Illumina e o seu proteoma foi investigado através de cromatografia líquida de alta resolução acoplada à espectrometria de massas. A integração de ambos os conjuntos de dados permitiu a análise do venoma, sendo que mais de 30 famílias de componentes foram identificadas no transcriptoma, das quais 15 também foram detectadas no proteoma da peçonha. No entanto, poucas famílias (PLA2, SVMP, SVSP e VEGF) são relativamente abundantes e apenas 7 transcritos contribuem com ~82% e ~73% da abundância no transcriptoma e no proteoma, respectivamente. Também foram identificadas sequências de PLIs, que surgem como uma possibilidade de adjuvante na terapia do envenenamento. Esses inibidores são proteínas geralmente encontradas no sangue de serpentes que, recentemente, também têm sido identificadas em glândulas de peçonha. Sendo assim, o gene sintético do inibidor CdtPLI-2 foi expresso em células de Pichia pastoris KM71H em meio BMMY com casaminoácidos 1,5%. A seguir, o PLI recombinante rCdtPLI2 foi purificado do meio de cultura através de diferentes etapas cromatográficas e não foi reconhecido pelo soro anticrotálico comercial. O sequenciamento amino-terminal confirmou sua expressão e sua massa molecular foi determinada por MALDI-TOF (41643,599 Da). A digestão do rCdtPLI2 com PNGase F/alfa-manosidase permitiu análise de sua glicosilação e a retirada total do açúcar deixou a proteína insolúvel. O rCdtPLI2 glicosilado não é capaz de inibir PLA2 ofídicas, mas sua digestão com alfa-manosidase forneceu um inibidor deglicosilado com ação inibitória sobre PLA2 de diferentes peçonhas ofídicas brasileiras, incluindo PLA2 ácidas e básicas. As PLA2 CB-Cdc e Lmr-PLA2 são significativamente inibidas a partir da proporção de 1:10 (inibidor:toxina). Para a CTx-Cdc, grande inibição é vista a partir da proporção 1:5. Além disso, uma análise preliminar in vivo revelou que o rCdtPLI2 deglicosilado pode ser um agente promissor na diminuição do edema causado pela CTx-Cdc. O inibidor (brCdtPLI2) também foi expresso em células de E. coli BL21(DE3)pLysS em meio LB, com indução mantida por IPTG 0,5 mM. Posteriormente, o brCdtPLI2 foi isolado do lisado celular através de diferentes etapas cromatográficas e um ensaio de inibição mostrou que ele inibe a ação enzimática da CB-Cdc, apresentando-se promissor na inibição de outras PLA2 ofídicas e com a vantagem de ausência de glicosilações. Concluindo, esse trabalho forneceu um banco de dados holístico e abre o caminho para investigações e descobertas de futuros agentes farmacológicos ou alvos na terapia antivenenos.Snakebite accidents in Brazil have increased yearly and are considered a neglected public health problem. The only effective treatment is the administration of the snake antivenom, but adverse reactions are related to its administration. Crotoxin, a phospholipases A2 (PLA2) complex, is one of the most studied components in crotalic venom, but many other molecules are less known due to its extremely low abundance. Therefore, this study aimed the analysis of the venom gland transcriptome and the venom proteome of the snake Crotalus durissus terrificus as well as the biochemical and functional characterization of a recombinant PLA2 inhibitor (PLI) from this snake. The venom gland cDNA library was constructed using the Illumina platform and the proteome from this gland was investigated through high resolution liquid chromatography coupled with mass spectrometry. Integration of both datasets allowed the venome analysis, revealing more than 30 protein families in the transcriptome, of which 15 were also detected in the venom proteome. However, few families (PLA2, SVMP, SVSP e VEGF) are relatively abundant and only 7 transcripts correspond to, respectively, ~82% and ~73% of the transcriptome and proteome. Sequences of PLIs have also been identified, which appear as possible adjuvant in the antivenom therapy. These inhibitors are proteins usually found in snakes\' blood, but they gave been detected in venom glands recently. Thus, the CdtPLI-2 synthetic gene was expressed in Pichia pastoris KM71H cells in BMMY with 1.5% casaminoacids medium. Next, the recombinant PLI rCdtPLI2 was purified from the culture medium by different chromatographic steps and it was not recognized by the commercial anticrotalic serum. N-terminal sequencing confirmed its expression, and its molecular mass was determined by MALDI-TOF (41643.599 Da). rCdtPLI2 digestion with PNGase F/alpha-mannosidase allowed the analysis of its glycosylation and the protein became insoluble after total sugar removing. The glycosylated rCdtPLI2 cannot inhibit snake venom PLA2, but its digestion with alpha-mannosidase provided a deglycosylated inhibitor with and inhibitory action on PLA2 from different Brazilian snake venoms including acid and basic PLA2. The PLA2 CB-Cdc and Lmr-PLA2 are significantly inhibited from the ratio 1:10 (inhibitor:toxin). On the other hand, great inhibition is observed from 1:5 ratio for CTx-Cdc. Besides that, an in vivo preliminary analysis revealed that the deglycosylated rCdtPLI2 may be a promising agent in reducing edema caused by CTx-Cdc. The inhibitor (brCdPLI2) was also expressed in E. coli BL21(DE3)pLysS cells in LB medium, with expression induction maintained by 0.5 mM IPTG. Subsequently, brCdtPLI2 was isolated from the cell lysate by different chromatographic steps, and an inhibition assay showed it inhibits the CB-Cdc enzyme action, presenting as a promising molecule in the inhibition of other snake venom PLA2. Moreover, brCdtPLI2 has advantage that there is no sugar content in its structure. Concluding, this study provided a holistic database and paves the way for investigations and discoveries of futures pharmacological agents or targets in antivenom therapies

Biochemical characterization and in vitro evaluation of the fibroblast activation and the leishmanicide potential of an L-amino acid oxidase (LAAO) from Crotalus durissus terrificus venom

Acidentes causados por animais peçonhentos representam um grave problema de saúde pública, principalmente em áreas de difícil acesso da população ao serviço de saúde. No Brasil, o gênero Crotalus é o gênero de serpente cuja peçonha apresenta o maior índice de letalidade. As L-aminoácido oxidases (LAAOs) estão presentes na peçonha crotálica e são flavoenzimas que catalisam a oxidação de L-aminoácidos, produzindo, concomitantemente, peróxido de hidrogênio e amônia. LAAOs têm demonstrado atividade citotóxica, antimicrobiana, antitumoral, antiparasitária e na agregação plaquetária. Os objetivos desse estudo incluíram o isolamento e a caracterização bioquímica da LAAO de C. d. terrificus, assim como a avaliação de seu potencial leishmanicida e da ativação de fibroblastos. Foram desenvolvidos dois protocolos para isolamento da LAAO. O primeiro consistiu em cromatografias de troca catiônica, filtração molecular e de interação hidrofóbica. O segundo protocolo diferiou do primeiro na terceira etapa (cromatografia de afinidade). Cromatografia de fase reversa da LAAO isolada demonstrou um alto grau de pureza e a separação do cofator FAD. A massa molecular da LAAO foi determinada por espectrometria de massas MALDITOF (58.702,196 Da). A caracterização estrutural dessa LAAO também incluiu a dedução da sua sequência primária e a localização do sítio de glicosilação e das ligações dissulfeto através de espectrometria de massas em equipamentos LC-MS/MS com diferentes tipos de fragmentação (HCD, ETD e EThcD). A sequência primária (498 resíduos) foi obtida após digestão da LAAO com diferentes proteases e o sítio de glicosilação foi localizado na Asn361. Análise por SDS-PAGE da LAAO em condições reduzida e reduzida/deglicosilada mostrou que cerca de 5% da massa da proteína é relativa à presença de açúcar. As ligações dissulfeto (Cys10-Cys171 e Cys331-Cys412) foram localizadas após digestão da enzima em pH ácido e análise por LC-MS/MS. A avaliação qualitativa da especificidade de substratos mostrou preferência por L-aminoácidos hidrofóbicos e, a ordem de especificidade (L-Phe>LLeu> L-Met>L-Trp>L-Ile) foi determinada através da cinética enzimática. A estabilidade da LAAO foi avaliada em diferentes temperaturas, tempos e condições de armazenamento. A enzima mostrou grande perda de atividade ao longo do tempo, sendo que a liofilização e o congelamento a -20 °C inibiram sua atividade completamente. A estabilidade térmica, avaliada pela técnica do Termofluor, mostrou que a LAAO é mais estável na presença de pH ácido, diferentes concentrações de substratos e ausência de NaCl. Promastigotas de Leishmania amazonensis foram estimulados com a LAAO (55 mUAE) e cerca de 30% dos parasitas foram mortos. Fibroblastos L929 também foram estimulados com a LAAO e em baixa concentração da enzima (1,83 mUAE) a viabilidade celular foi próxima de zero. Nas concentrações sem morte celular significativa, a ativação dos fibroblastos foi avaliada através da dosagem de óxido nítrico e citocinas, mas, em nenhum dos casos, houve ativação das células e maior produção desses compostos. Portanto, no presente estudo, foi isolada e caracterizada uma LAAO de C. d. terrificus que apresentou ação contra promastigotas de L. amazonensis e alta citotoxicidade para fibroblastos, sem causar a ativação dessas célulasAcidents caused by venomous animals represent a serious publich health problem, mainly in remote areas where the acess to the health service is difficult. In Brazil, the genus Crotalus is the most lethal genus among the Brazilian snakes. The L-amino acid oxidases (LAAOs) are present in the venom from this genus and they are flavoenzymes that catalyze the oxidation of L-amino acids, producing hydrogen peroxide and ammonia concomitantly. LAAOs have been demonstrating many activities, including cytotoxic, antimicrobial, antitumor, antiparasitic and action on platelet aggregation. The main objectives of this study included the isolation and biochemical characterization of a LAAO from C. d. terrificus, and the evaluation of its leishmanicide potential and the fibroblasts activation. Two protocols were developed to the LAAO isolation from C. d. terrificus venom. First one consists in ionic exchange, gel filtration and hydrophobic interaction chromatographies. The second protocol has a modification in the third step which is affinity chromatography. Reverse-phase chromatography of the isolated LAAO showed high purity degree and the separation of FAD from the enzyme. The LAAO molecular mass was determined by MALDI-TOF mass spectrometry (58,702.196 Da). The structural characterization also included the deduction of primary sequence and the glycosylation site and disulfide bonds determinations through LCMS/ MS with different fragmentation modes (HCD, ETD, EThcD). The primary sequence (498 amino acid residues) was obtained after the LAAO digestion using different proteases. The glycosylation site was located in the Asn361. A SDS-PAGE analysis of reduced LAAO and reduced/deglycosylated LAAO showed that about 5% of the LAAO mass is due to the sugar presence. The disulfide bonds were determined after LAAO digestion at low pH and LC-MS/MS analysis. It showed bonds between Cys10-Cys171 and Cys331-Cys412. The qualitative evaluation of substrate specificity revealed preference for hydrophobic L-amino acids. The specificity order, determined through the kinetics evaluation, is L-Phe>L-Leu>LMet> L-Trp>L-Ile. The LAAO stability was evaluated at different temperatures, timecourse and storage conditions. The enzyme lost its activity over time, and lyophilization and freezing at -20 °C completely inhibited its activity. The thermal stability, evaluated by the Termofluor method, demonstrated that the best LAAO structural stability is achieved at acid pH, different substrate concentrations and at absence of NaCl. Leishmania amazonensis promastigotes were stimulated with LAAO (55 mEAU) and the parasites death was about 30%. The fibroblasts cell line L929 was also stimulated with LAAO, and at low concentration (1.83 mEAU) the cellular viability was close to zero. At lower concentrations, without significative cellular death, the fibroblast activation was evaluated through the nitric oxide e cytokines production, but none of the compounds were released. Therefore, in this study, a LAAO from C. d. terrificus venom was isolated and characterized. Moreover, this enzyme presented leishmanicide against L. amazonensis promastigotes and high cytotoxicity to fibroblasts, without the activation of these cells

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

Abstract Background Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads’ secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. Methods Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman’s degradation. Results We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. Conclusions We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored

Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom

Abstract Background Lachesis muta rhombeata (Lmr) is the largest venomous snake in Latin America and its venom contains mainly enzymatic components, such as serine and metalloproteases, L-amino acid oxidase and phospholipases A2. Metalloproteases comprise a large group of zinc-dependent proteases that cleave basement membrane components such as fibronectin, laminin and collagen type IV. These enzymes are responsible for local and systemic changes, including haemorrhage, myonecrosis and inflammation. This study aimed the isolation and enzymatic characterization of the first metalloprotease (Lmr-MP) from Lmr venom (LmrV). Methods and results Lmr-MP was purified through two chromatographic steps and submitted to enzymatic characterization. It showed proteolytic activity on azocasein with maximum activity at pH 7.0–9.0. It was inhibited by EDTA (a metal chelator that removes zinc, which is essential for enzymatic activity) and no effect was observed with PMSF, iodoacetic acid or pepstatin (inhibitors of serine, cysteine and aspartyl proteases, respectively). Ca2+, Mg2+ and Ba2+ ions increased its activity, while Al3+, Cu2+, Ni2+ and Zn2+ inhibited it. Additionally, ZnCl2 showed a dose dependent inhibition of the enzyme. Lmr-MP activity was also evaluated upon chromogenic substrates for plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) showing the highest activity on S-2302. The activity in different solutions (5 mM or 50 mM ammonium bicarbonate, pH 7.8; 0.1% trifluoroacetic acid + 50% acetonitrile; phosphate buffer saline, pH 7.4; 50 mM sodium acetate, pH 4.0 or ammonium acetate pH 4.5) was also evaluated and the results showed that its activity was abolished at acidic pHs. Its molecular mass (22,858 Da) was determined by MALDI-TOF and about 90% of its primary structure was verified by high-resolution mass spectrometry using HCD and ETD fragmentations and database search against the sequence of closely related species. It is a novel enzyme which shared high identity with other snake venom metalloproteases (svMPs) belonging to the P-I group. Conclusion The purification procedure achieved a novel pure highly active metalloprotease from LmrV. This new molecule can help to understand the metalloproteases mechanisms of action, the Lachesis envenoming, as well as to open new perspectives for its use as therapeutic tools

Unraveling the structure and function of <i>Cdc</i>PDE: A novel phosphodiesterase from <i>Crotalus durissus collilineatus</i> snake venom

This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. CdcPDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8-8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis(p-nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that CdcPDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that CdcPDE is cytotoxic to human keratinocytes. CdcPDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities