Agglutination activities of haemolymph and tissue extracts in scallop Chlamys farreri and purification of mannan-binding lectin from haemolymph

Abstract

Agglutination plays a crucial role in eliminating potential pathogens in marine invertebrates. In this paper, cell-free haemolymph (CFH) and crude extracts from the haemocytes, gills, mantle, hepatopancreas, gonad, kidney and adductor muscle of scallop (Chlamys farreri) were obtained, and the agglutination activities of these extracts were tested separately against mouse blood cells (MBC) and fluorescein isothiocyanate-labelled microbes. Agglutination inhibition assays were carried out with EDTA and saccharides. Finally, a mannan-binding lectin (MBL, one of the main lectins in haemolymph) was purified from CFH by affinity chromatography using a mannan-sepharose column and analysed using native- and denaturing-PAGE. The results showed the CFH could aggregate Vibrio anguillarum, Staphylococcus aureus, Bacillus subtilis, Pichia pastoris and MBC, and the agglutination could be inhibited by DEAE dextran, EDTA, D-mannose-6-phosphate, N-acetyl glucosamine, mannan, zymosan A, chondroitin sulphate, peptidoglycan (PGN) and lipopolysaccharides (LPS); Gill extracts aggregated S. aureus, B. subtilis and P. pastoris, and the agglutination activity could be inhibited by EDTA, N-acetyl galactosamine, mannan, zymosan A, chondroitin sulphate, PGN and LPS; Mantle, hepatopancreas, gonad and kidney extracts could only aggregate B. subtilis, and this could be inhibited by mannan, zymosan A and PGN; however, no agglutination activity was detected using haemocyte or adductor muscle extracts. The results indicate that multiple types of lectins exist in C. farreri, and CFH has the highest agglutination activity. Purified MBL exhibited a molecular mass of 645kDa using native-PAGE and 73kDa using SDS-PAGE.