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

Author(s)

Gisele Adriano Wiezel

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2021-11-19

Examining board members

Eliane Candiani Arantes Braga; Heloisa Sobreiro Selistre de Araújo; Renê de Oliveira Beleboni

• Advisor: Eliane Candiani Arantes Braga

Abstract

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. (AU)

• FAPESP's process: 17/00586-6 - Characterization of a phospholipase A2 inhibitor from the Crotalus durissus terrificus venom gland: a possible adjuvant in the antivenom therapy
• Grantee: Gisele Adriano Wiezel
• Support Opportunities: Scholarships in Brazil - Doctorate