Isolation and partial characterization of brown sea cucumber (Holothuria sp.) lectin by affinity chromatography

A sialic acid specific lectin with mitogenic and cytotoxic activities was isolated from the body wall of brown sea cucumber (Holothuria sp.) by affinity chromatography on fetuin agarose. Lectin activity was detected in the body walls and internal organs of brown, black and white sea cucumbers. Highest agglutination activity was detected in the crude extract of the body wall of brown sea cucumber which was prepared by extraction with phosphate buffered saline (PBS 0.02 M in 0.15 M NaCl, pH 7.2). Inhibition studies showed that Fetuin is the only sugar that prevents interaction of the lectin with erythrocytes. The lectin was further purified by (NH4)2 SO4 precipitation, chromatography on Sephadex G-200 and affinity chromatography on Fetuin Agarose column. The lectin was non-blood type specific since it agglutinated human erythrocytes of all human blood types (A, B, AB, O) and animal erythrocytes (horse and cattle). Also, the lectin was classified as a complete type of lectin because it agglutinated untreated erythrocytes. Trypsinization of the erythrocytes and addition of Ca2+ enhanced the agglutinating activity of the lectin. The lectin is stable at pH 2-12 while maximum agglutinating activity is obtained at pH 10-11. The lectin has an estimated molecular weight of 305 KD by gel chromatography. Electropherogram of SDS-PAGE revealed four bands of approximate molecular weights of 145 KD, 87KD, 69 KD and 44 KD. It is a glycoprotein with 0.15% total sugar content. Glucose, galactose and oligosaccharide residues constitute 6.5%, 10% and 74%, respectively, of its total sugars as determined by High Pressure Liquid Chromatography. Human lymphocytes, upon incubation with Holothuria sp. lectin, were transformed to Lymphoblasts which is indicative of the lectins' mitogenic activity. The lectin likewise exhibited cytotoxicity against mouse (Adonecarcinoma 549) and human lung cancer (Sarcoma 180) cells at 5 ug and 50 ug, respectively