Purificação e caracterização parcial de lectina obtida de couve-flor (Brassica oleracea var. Botrytis) e potenciais ações biológicas

• Autor(a) principal: Abranches, Monise Viana
• Data de Publicação: 2013
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/262
• topic: Aglutinina; Cromatrografia; Imunobiologia; Glicobiologia; Carboidrato; Câncer; Resposta Imune; Agglutinin; Cromatrografia; Immunobiology; Glycobiology; Carbohydrate; Cancer; Immune Response; CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL

The lectins are proteins that interact selectively and reversibly with carbohydrates. Protein-carbohydrate interactions are involved in many biological processes, such as mediator of several cellular activities including cell-cell and cell-matrix recognition, control the cellular growing and apoptosis. Moreover, these interactions are implicated in the development of diseases. Additionally, the lectins can promote foreign agents elimination by opsonization and it seems activating the innate immune response. In this study, a lectin of cauliflower has been purified and partially characterized (Brassica oleracea var. Botrytis). In addition, the protein activity against the viability of tumor cells (MDA-MB-231, MCF-7 e HepG2) and on macrophages activation has been assessed too. The lectin purification involved three chromatographic steps (FPLC-affinity on a Hitrap Blue HP column, FPLC-cation exchange on a Hitrap CaptoS column and HPLC-molecular weight exclusion chromatography on a Sephadex 200 HR 10/30 column). The protein molecular mass was estimated by SDS-PAGE under reducing and non-reducing conditions and after PNGase treatment. The protein activity on human and sheep erythrocytes in different temperature and pH conditions, in the presence of divalent cations, and the protein ability to bind different carbohydrate were investigated. The N-terminal amino acid sequence was analyzed by Edman degradation method in the Shimadzu PPSQ-30 Series Protein Sequencer. The viability of tumor cells in the presence of lectin was investigated by MTT assay. Furthermore, it was performed the macrophage phagocytosis assay and it assessed H2O2 and NO- production under influence of lectin. The purification steps have provided a 139-fold purification. The SDS-PAGE analysis in reducing and non-reducing conditions revealed a single band estimated at 34 kDa. It was not observed any difference in the molecular mass when the lectin was previously treated with PNGase. It was noted that the protein was able to cause hemagglutination of human (O and B groups) and sheep erythrocytes. The protein optimal activity was maintained until 60 OC and between pH 7 and pH 8. The lectin presented a binding specificity to complex carbohydrates (fetuin and asialofetuin) and the Mg has improved the lectin hemagglutinating activity. The N-terminal sequence of the encountered lectin was ETRAFREERPSSKIVTIAG. The lectin reduced the viability of the MDA MB 231 and HepG2 bloodlines, but it did not reduce the viability of the MCF-7 cells. In the presence of lectin the phagocytosis was stimulated, as well as H2O2 and NO- production. It is suggested that the studied protein is a new lectin that can inhibit MDA-MB-231 and HepG2 proliferation. In addition, this lectin can also be probed like immunostimulant agent, capable to help the innate immunity, favoring the clearance of non-self-agents.