Purification and characterization of the agglutinins from the sponge Axinella polypoides and a study of their combining sites

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Abstract
The hemagglutinins from the sponge A. polypoides were isolated by affinity chromatography using Sepharose 4B as an absorbent and eluting with DGal [D-galactose]. Further separation on DEAE-cellulose and preparative disk electrophoresis on polyacrylamide and agarose gave 3 fractions. The physicochemical properties and binding specificities of the 2 main agglutinins were studied. Homogeneity was tested by polyacrylamide electrophoresis and immunoelectrophoresis and by sedimentation analysis. In isoelectric focusing, agglutinin I (MW 21,000) showed 2 bands at pH 3.8 and 3.9. Agglutinin II (MW 15,000) showed 1 band at pH 3.9. Both agglutinins have a carbohydrate content of about 0.5%, are immunochemically unrelated, and differ in amino acid composition. Both precipitate A1, A2, B, Le3 and precursor I blood group [human] substances but to different extents. Inhibition experiments revealed that both agglutinins are inhibited best by terminal nonreducing DGal glycosidically linked .beta.1 .fwdarw. 6 by p-nitrophenyl-.beta.DGal. DGal and DFuc [D-fucose] are equally active but .apprx. 20 and 12 times less active with agglutinin I and agglutinin II, respectively. DGalNAc and LFuc [L-fucose] were inactive even at much higher concentrations. Both agglutinins have similar specificities and react with the immunodominant determinants of blood group B and Lea but not with A and H substances; in A and H substances, reactivity is with side chains in which .beta.-linked DGal is unsubstituted at the nonreducing terminus. The A. polypoides lectins are compared with galactose-specific lectins of different origin and with the aggregation factor system in sponges.