Estudos de estrutura-função da lectina de sementes de Canavalia virosa com atividade inflamatória e citotóxica: uma potencial ferramenta anticâncer

Abstract

Lectins are proteins that bind specifically to carbohydrates and are capable of forming complexes with molecules containing saccharides without changing the structure of the glycosil ligands. Because of the diverse biological activities displayed by these proteins they have great applicability in biotechnology. In this context, this work reports the obtention of primary and three-dimensional structures of lectin extracted from Canavalia virosa seeds, called ConV, In addition to the analysis of its effects on inflammatory process and as cytotoxic agent for glioma cells. The Canavalia virosa plant belongs to the legume family, subtribe Diocleinae. The largest number of studies with lectins focuses on this family and the subtribe Diocleinae are noteworthy due to the presence of highly studied lectins such as ConA. Lectins from Diocleinae have high structural similarity but differ on the results of biological activities, which may be explained by the structural study of proteins. ConV was purified by a single step by affinity chromatography with Sephadex G-50 matrix. The amino acid sequence of the lectin was obtained by mass spectrometry and showed high similarity to other plant lectins derived from the same subtribe. ConV crystals used for determination of the three dimensional structure were obtained by vapor diffusion method at 293 K and belonged to orthorhombic space group P21221 with a dimer in its asymmetric unit. The structure was solved at 2.53 Å and the methodology chosen to solve the phase problem was the molecular replacement using coordinates of the lectin from Canavalia maritima (PDB: 2CWM). The structure obtained after all the refinements presented Rfactor and Rfree of 18.91% and 24.92% respectively, with no residues in not allowed regions of Ramachandran plot. The obtained structure proved to be very similar to other lectins from the same gender. In inflammatory tests, ConV elicited edema, increasing the animal’s paw volume, and the incubation of lectin with glucose before the application was able to reduce the edematogenic effect indicating the involvement of the carbohydrate recognition domain in this process. ConV showed high toxicity to glioma cells with IC 50 of 58.8 µg/mL, this toxicity is probably caused by the induction of autophagy in these cells, indicating a anticancer potential for the lectin. In silico studies confirmed that ConV interacts preferentially with dimmanosides and trimmanosides, carbohydrates that compose the N-glycans of glycoproteins. This finding corroborates with the hypothesis that the lectin domain interacts with receptors containing mannosides and this contributes to the effects observed in biological activities.