Purificação, caracterização físico-química, estrutural e biológica de uma lectina de sementes de Canavalia villosa Benth

• Ano de defesa: 2017
• Autor(a) principal: Moreira, Cleane Gomes
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Palavras-chave em Português: Lectina; Canavalia villosa; Espectometria de massas; Cristalografia de raios X; Toxicidade; Hipernocicepção
• Link de acesso: http://www.repositorio.ufc.br/handle/riufc/23676

Presenting important carbohydrate binding properties, lectins are proteins able to decipher the glycocode, and as such, they can be used in bioassays involving, for example, cell-cell communication and cell signaling, as well as protein targeting. In this study, a new mannosespecific lectin from Canavalia villosa seeds (Cvill) was isolated by a single affinity chromatography step in a Sephadex® G-50 column. SDS-PAGE showed the lectin to be composed of three bands, and similar to the other lectins of the subtribe Diocleinae, analysis by mass spectrometry indicated that the lectin C. villosa has three chains (α, β and γ) with masses of 25,647; 12,966 and 12,685 Da, respectively, similarly to the profile of ConA-like lectins specific to mannose. A two-dimensional electrophoresis analysis showed the presence of five isoforms and isoelectric point around pH 5.0. The lectin strongly agglutinated rabbit erythrocytes native and treated with proteolytic enzymes, and presented sugar specificity to α-methyl-D-mannoside and D-mannose, in addition to high stability within a broad range of pH (pH 5,0 a 7,0) and temperature (up to 70 °C). Partial sequence of the protein was obtained by MS-MALDI TOF/TOF covering approximately 41% of the sequence. The protein was crystallized by vapor diffusion method in the presence of α-methyl-D-mannoside. The lectin also showed average toxicity against artemia nauplii and induced paw edema and hypernociception in mice with the participation of the carbohydrate binding site. In tests on C6 lineage of Rattus norvegicus glioma cells, Cvill showed ability to reduce viability and changes in morphology in the cells tested.