A lectin was purified from the hemolymph of esturarine crab Scylla serrata by successive 40% (NH4)2SO4 precipitation, affinity chromatography on asialo fetuin- Sepharose column, Resource Q anion-exchanger in FPLC system and designated as scyllin-2. Scyllin-2 was a homogeneous monomeric protein of molecular mass 75 kD judged by SDS-PAGE and confirmed by ESI-MS-Q-ToF. Its activity was Ca+2 dependent being maximum at pH 7.5 and at 20 °C. N-terminal sequence of scyllin-2 showed close resemblance to peanut lectin and histidine kinase A. It agglutinated human O, A, B and AB blood group erythrocytes equally well and showed maxi- mum inhibition with α-Gal by hapten-inhibition study. The detailed carbohydrate specificity of scyllin-2 was determined at the macro-molecular level based on the Gal/GalNAc structural units in the mammalian glycoproteins by enzyme-linked lectinosorbent (ELLSA) and inhibition assays. It revealed that scyllin-2 binds specifically to tumor-associated carbohydrate antigens GalNAcα1→Ser/Thr (Tn) and Galβ1→3GalNAcα1→Ser/Thr (Tα). It showed very weak binding with Galβ1→3/4GlcNAc (I/II) glycotopes on glycoproteins and T/Tn covered by sialic acid. Multivalancy of Tn/Tα containing glycoproteins tested resulted in higher binding of 102–104 order than the respective Gal and GalNAc monomer. Scyllin-2 stimulated proliferation of mouse splenocytes. Analysis of expression of receptors for scyllin-2 on HepG2 by flow cytometry showed the binding of FITC-scyllin-2 to HepG2 was 86.51%, which was nearly comparable to Artocarpus lakoocha agglutinin (ALA) (66.41%), another Tn/Tα specific lectin indicating that the glycan structure on HepG2 cell surface shows prevalence of Tn/Tα units. It inhibited proliferation of HepG2 cells (61 µg/ml). The inhibitory effect was comparable to ALA (80 µg/ml). Thus, we have characterized a Tn/T specific invertebrate lectin with biological significance.
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