Determinação da estrutura primária e avaliação preliminar dos efeitos inflamatórios de uma lectina de Canavalia bonariensis lindl

Abstract

Lectins are (glycol) proteins that bind specifically and reversibly to carbohydrates. These proteins, in particular those from plant, are important tools in glicobiochemistry and glycobiology. Canavalia bonariensis Lindl is a species of Leguminosae family, Papilionoideae subfamily, tribe Phaseoleae, subtribe Diocleinae, native of the southern region of the country. In this work was realized the analysis of the primary sequence of CaBo (Canavalia bonariensis lectin) and preliminary evaluation of the pro-inflammatory effects of the lectin. The purification of CaBo was realized through affinity chromatographic and the process was monitored by SDS-PAGE and hemagglutinating activity. It was observed that the purified lectin is characterized by an electrophoretic profile that consists of a higher band with approximately 29 kDa, and two bottom bands with apparent molecular mass of 14 and 12 kDa. CaBo was tested for the thermostability of their hemagglutinating activity after incubation for one hour at different temperatures, losing activity only at 80° C after one hour. Regarding its stability at different pH, CaBo was stable in a pH range between 7.0 and 9.0. The CaBo activity was also affected after serial dilution in the presence of the chelating agent EDTA and it was recovered significantly after addition of CaCl2 and MnCl2, proving to be dependent of divalent metal cations. The analysis by mass spectrometry indicated that CaBo has an α chain with molecular mass of 25512 kDa and and two subunits (β and γ chains) with molecular mass of 12999 Da and 12537 Da, respectively. The primary sequence of the lectin was determined by a combination of tandem mass spectrometry and molecular biology, by the translation of cDNA isolated from the lectin’s gene. CaBo presented high sequencial similarity with lectins from the same subtribe and the phylogenetic evaluation indicated that the protein must be the most primitive among lectins from the same gender. CaBo showed inflammatory activity in the rat paw edema model, and it is necessary to realize others test in order to elucidate the biological effect of the lectin.