Isolation and characterisation of moult related agglutinins from the serum at intermoult (C) and postmoult (A2) stages of the intertidal mole crab Emerita asiatica

Abstract

Two naturally occurring moult-related hemagglutinins detected in the serum of E. asiatica belonging to intermoult (C) and early postmoult (A2) stages were successfully isolated by affinity chromatography using chitin and ManNAc - Sepharose 6B as affinity matrices, respectively. The bound agglutinins were eluted from their respective matrices to high purity using EGTA, a specific chelator for calcium. The total protein yield was 815 µg/5ml and 217 µg/5ml from C and A2 stage crabs, respectively. The isolated agglutinins showed a 165- and 257- fold increase in specific activity and 68 and 42% recovery from the starting serum samples from intermoult and postmoult crabs, respectively. The agglutinins isolated from both C and A2 stages of E. asiatica appeared as single protein bands in native PAGE and the electropherogram showed distinct differences in velocity of migration among agglutinins from the two different stages of moulting, revealing their purity, electrophoretic homogeneity and identity. In the hemagglutination-inhibition studies performed, the agglutinin isolated from C stage crabs showed a preference for linkages as well as larger binding sites, their activity being inhibited by selective glycoproteins which predominantly contain terminal α-2, 3 or 2, 6 linkages in their oligosaccharide side chains. When subjected to hemagglutination-inhibition assays, the purified agglutinin from A2 stage exhibited a different pattern of sensitivity compared to its native counterpart by being inhibited by hexoses, hexosamines as well as their N-acetyl counterparts thus indicating the novelty of this moult stage specific lectin. Most significantly, while a linkage specific (α-2, 3 or 2, 6) agglutinin detected and isolated from intermoult crabs appear to be found commonly at all the stages of moulting, detection and isolation of a sialic acid specific (either N or O acetylated) only at A2 stage clearly suggest that the later agglutinin may appear at A2 stage alone indicating the confirmed presence of moult specific lectins in the hemolymph of E.asiatica.