Biochemical, structural and functional evaluation of a phosphodiesterase from Crotalus durissus collilineatus venom

Author(s)

Isadora Sousa de Oliveira

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2021-06-18

Examining board members

Eliane Candiani Arantes Braga; Wuelton Marcelo Monteiro; Jonas Augusto Rizzato Paschoal; Denise Vilarinho Tambourgi

• Advisor: Eliane Candiani Arantes Braga; Manuela Berto Pucca

Abstract

Snake venoms present a high complexity of components and, in order to elucidate their toxicity mechanisms, it is necessary that they are isolated and characterized, so that their action during envenoming becomes clarified, facilitating the respective symptom treatment. Many minority components from Crotalus durissus collilineatus venom have not yet been studied. Phosphodiesterase (PDE) had already been identified in the venom, but had not been isolated or characterized. PDE enzymes class are responsible for breaking phosphodiester bonds of nucleic acids, ATP, ADP, NAD, NGD, cAMP and cGMP, interfering in physiological or pathological processes, which make them therapeutic targets for various pathologies. Thus, the objectives of this study were the isolation and biochemical, structural and functional characterization of the PDE from C. d. collilineatus venom (CdcPDE). The CdcPDE isolation was performed by combining liquid chromatography techniques, such as molecular filtration, anion and cation exchanges, respectively, with a yield <1%. Through SDS-PAGE, it was possible to observe its migration as a monomer and the presence of glycosylations in the molecule. By mass spectrometry, CdcPDE showed two molecular masses determined by MALDI-TOF, 100 and 105 kDa, and by LC-MS/MS in union with the amino-terminal sequencing, it was possible to determine CdcPDE sequence, consisting by 829 amino acid residues. By in silico analysis, it was possible to estimate their secondary and tertiary structures, as well as their interaction with the bis(p-nitrophenyl) phosphate substrate. Using this same substrate, CdcPDE showed greater enzymatic activity between pH 8 and 8.5, at 37 ºC, and the best temperature to store was 0 °C. Some reducing agents and a metal chelator inhibited the CdcPDE enzymatic activity, suggesting that the disulfide bridges in its structure are essential for the activity, as well as metal ions, which characterizes it as a metalloenzyme. Obtained kinetic parameters are: Km = 0.38 mM, Vmáx = 0.7 &mu;M/s, kcat = 0.14 s-1 and its catalytic efficiency was equal to 0.37 mM.s-1, showing high affinity by the substrate used, as well as, kinetic efficiency, when compared to others snake venom PDE. Other assays demonstrated that CdcPDE reduces/loses enzymatic activity with water loss and in contact with TFA, and its unfolding temperature was 65.71 ºC. By ELISA, it was possible to observe that the anticrotalid serum produced by Instituto Butantan recognizes CdcPDE and, by in silico analysis, 16 possible immunogenic epitopes on the molecule were identified. Finally, it was possible to observe that CdcPDE inhibits platelet aggregation ADP-induced and it is a cytotoxic molecule for human keratinocytes, presenting an IC50 value of 71.65 &mu;g/mL, and this action has never been reported for snake venoms PDE, also suggesting the possibility of local effects of these molecules during the envenoming. Thus, these enzymes can be important molecular tools for research, as well as, for therapy, since they have biochemical functions capable of interfering with both physiological and pathological processes. (AU)

• FAPESP's process: 17/03580-9 - Biochemical, structural and functional evaluation of a phosphodiesterase from Crotalus durissus collilineatus venom
• Grantee: Isadora Sousa de Oliveira
• Support Opportunities: Scholarships in Brazil - Doctorate