Isolation and characterization of invertebrate lectins

Forty five invertebrates samples were gathered from Batangas, cavite, Marinduque and Mindoro [Philippines] and were assayed for the presence of lectin by agglutination using human and animal red blood cells. Of these samples, thirty one were found to contain lectin. Samples with relatively high lectin activity and easy to obtain were further purified and characterized. Ampularia luzonica, Pomacea canaliculata and the two Holothurias were selected for further purification and characterization. The lectin was isolated and purified using conventional protein isolation procedures. The purified lectins were then characterized in terms of the following: blood group specificity, sugar specificity, effect of Ca 2+, molecular weight and number of subunits, protein content and amino acid profile, carbohydrate content and carbohydrate profile, binding specificity and binding constant and biological activity. All four samples were found to be mitogenic and non-blood type and non-blood group specific. A. luzonica lectin is specific for N-acetyl glucosamine while P. canaliculata and the two Holothurias were specific for fetuin, a glucoprotein, glycoprotein containing acetylneuraminic acid. Both A. luzonica and P. canaliculata lectins have low isoelectric points and rich in glutamic acid and aspartic acid. Both lectins also contain mannose and galactose as sugar components of the carbohydrate portion of the molecule. A. luzonica lectin has an estimated MW of 39KD while P. canaliculata has 420 KD with four subunits of the same molecular weight. Binding specificity of the P. canaliculata lectin indicated a monovalent interaction with fetuin. The two Holothurias are both C-type lectins. Holothuria sp. lectin has an estimated MW of 305 KD with 4 subunits of approximately 145, 87, 69 and 44 KD while Holothuria scabra lectin has an estimated MW of 355 KD with 5 subunits of approximately 114, 93, 80, 69 and 49 KD. Both lectins contain glucose and galactose. P. canaliculata and Holothuria sp. are both cytotoxic against mouse cancer cells (Sarcoma T180) and human lung cancer cells (A549). P. canaliculata lectin was demonstrated to enduce release of 1L-alpha and TNF-alpha at 10 ug/ml concentration.