Purificação, caracterização e propriedades biológicas de lectinas de equinodermo Holothuria grisea

Abstract

Lectins were detected in protein fractions of sea cucumber Holothuria grisea. Fractions obtained from precipitation with 1.0 vol acetone and supernatant of 2.0 vol. showed hemagglutinating/hemolytic activity for human erythrocytes type A. The first lectin, called HGA, was purified by acetone fractioning followed by TSK gel filtration and ion exchange on DEAE Sepharose chromatographies both on HPLC/FPLC ÂKTA purifier system. The second lectin, called HGL, was isolated from the supernatant fraction of acetone 2.0 vol followed by TSK gel filtration chromatography. These lectins were strongly inhibited by mucin and were not ion-dependent. Their native Mr 16.8 kDa and 228 kDa were estimated for HGL and HGA, respectively. When analyzed by SDS-PAGE showed a Mr of 15.6 kDa (HGL) and 105 kDa (HGA), no changes were observed in the presence of (3-Mercaptoethanol HGA was tested in classical models of nociception and inflammation. HGA was able to inhibit the neutrophils migration into the peritoneal cavity induced by carrageenan by 68% at lmg/kg. In acetic acid-induced writhing test a significant antinociceptive effect was observed by treatment with HGA (0.1, 1 and 10mg/kg) reducing constrictions in 27%, 90% and 84% respectively. In formalise test HGA showed antinociceptive effect only in the inflammatory phase. Nevertheless, in hot-plate test HGA did not show any nociceptive effect. In rota-rod and open-field tests HGA did not alter animal's behavior. The treatment with HGA 10 mg/Kg i.v presented diminished MPO activity (81.6% inhibition) and raised the circulating levels of NO in 50.4% when compared with the group control. HGA has demonstrated the ability to modulate the inflammatory response in models in vivo. HGA is the first marine invertebrate lectin that showed an antiinflammatory effect. Lectin HGL was investigated as its toxicity on artemias together in a comparative way, with aqueous extracts of several marine invertebrates and a lectin with anti-tumour action of the marine sponge C. varians. HGL showed a high toxicity with LC50 of 9.5 mg/ml. The data obtained indicate that the lectins purified and studied in this work possess a potential use as a study tool for better understanding of themechanisms involved in the inflammatory responses and molecular events, specific recognition of pathogens and cytotoxicity. These data had shown the specie Holothuria grisea as a source of lectins and other bioactive molecules with biotechnological potential.