Purification and Characterization of a Novel Rhamnose/Fucose-Specific Lectin from the Hemolymph of Oak Tasar (Antheraea proylei J.) Silkworm

BACKGROUND: Lectins are proteins or glycoproteins of non-immune origin which bind specifically but reversibly to carbohydrates or glycoconjugates. They play a crucial role in various biological processes including host defense mechanism, inflammation and metastasis. Therefore, there is an expanding scientific emphasis on purification and characterization of novel lectins possessing different useful biological properties. OBJECTIVE: The present investigation is concerned with purification and characterization of a novel lectin from the hemolymph of oak tasar (Antheraea proylei J.) silkworm. METHODS: The lectin was purified from the hemolymph by a procedure involving successive steps of hemocyte-free hemolymph preparation, ammonium sulfate (0-40%) fractionation and affinity chromatography on a column of Sephadex G-50 covalently coupled with L-rhamnose. It was then characterized by various physic-chemical methods including SDS-PAGE, gel filtration, hemagglutination assay, hemagglutination inhibition assay and tandem mass spectrometry (LC-MS/MS) coupled with Mascot sequence matching software (Matrix Science). RESULTS: The lectin was purified to electrophoretic homogeneity from the silkworm hemolymph and was found to be a monomeric protein with a native molecular weight of 39.5 kDa. It was specifically inhibited by L-rhamnose and D-fucose, the former being sixteen times more inhibitory than the latter. The hemagglutinating activity further was characterized by independency of metal ion, optimum at pH 7-7.5 and thermal stability with t1/2 of 60 degrees C. Analysis with tandem mass spectrometry coupled with Mascot sequence matching software confirmed the purified lectin to be a protein not purified and characterized earlier. CONCLUSION: A novel rhamnose/fucose-specific lectin was purified to electrophoretic homogeneity from hemolymph of oak tasar (Antheraea proylei J.) silkworm. The lectin was found to be a monomeric protein with a native molecular weight of 39.5 kDa. Its activity was found to be independent of metal ion, optimum at pH 7-7.5 and characterized by thermal stability with t1/2 of 60 degrees C. Analysis with tandem mass spectrometry coupled with Mascot sequence matching software confirmed the purified lectin to be a protein not characterized earlier.