Caracterização funcional de uma Cisteíno Catepsina recombinante da formiga cortadeira Atta Sexdens

Abstract

Cutting ants depend on plant leaves to survive, presenting high damage power in agricultural productions, natural and planted forest. The control of these insects through the use of commercial insecticides besides being toxic, it is not selective. Cathepsin L, an enzyme of the cysteine peptidase family, is known to be active in the degradation of embryonic vitellin, in the tissue remodeling, in the reproductive processes and in the cuticle degradation during insect moulting, which make this enzyme an excellent target for the study of promising inhibitors. The cathepsin sequence from Acromyrmex echinator was used to design primers, that together cDNA of the A. sexdens, were used for amplification of the ORF encoding a cathepsin L of the ant A. sexdens by PCR. The ORF called CathL is expressed in the ant developmental stages (larvae, pupae, adult), and is present in parts of the adult insect (head, mesosoma, gaster). The gene was amplified using genomic DNA extracted from the ant's head and used as a template molecule in the PCR, suggesting that the ORF comes from the ant and not from the fungus L. gongylophorus with whom it lives in symbiosis. The ORF was cloned into vector pET32a and the recombinant enzyme AsCathL was expressed in E. coli mainly as inclusion bodies, and through refolding, a purified proenzyme of 52 kDa was obtained with final yield of 5 mg/L. Activation of the protein allowed the removal of the N-terminal pro-enzyme AsCathL on acidic conditions and showed hydrolytic activity in vitro towards the synthetic substrate Z-Phe-Arg-AMC. The enzyme showed higher proteinase activity at pH 4.5. Inhibition assays of the enzymatic activity were carried out using six recombinant sugarcane canacistatins, which demonstrated to be excellent cathepsin inhibitors, with Ki ranging from 2,95 nM to 0,6 nM. The obtained results show perspectives to the pursuit of more knowledge about that cysteine peptidase, an important objective for the studies on the insect control.