Purification and characterization of the agglutinins from the sponge Axinella polypoides and a study of their combining sites.
The hemagglutinins from the sponge Axinella polypoides were isolated by affinity chromatography using Sepharose 4B as an absorbent and eluting with DGal. Further separation on DEAE-cellulose and preparative disc electrophoresis on polyacrylamide and agarose gave three fractions. The physicochemical properties and binding specificities of the two main agglutinins were studied. Homogeneity was tested by polyacrylamide electrophoresis and immunoelectrophoresis and by sedimentation analysis. In isoelectric focusing, agglutinin I (mol wt 21 000) showed two bands at pH 3.8 and 3.9. Agglutinin II (mol wt 15 000) showed one 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, Lea, and precursor I blood group substances but to different extents. Inhibition experiments revealed that both agglutinins are inhibited best by terminal nonreducing DGal glycosidically linked beta 1 leads to 6 or by p-nitrophenyl-betaDGal. DGal and DFuc are equally active but about 20 and 12 times less active with agglutinin I and agglutinin II, respectively. DGalNAc and LFuc 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 Axinella polypoides lectins are compared with galactose-specific lectins of different origin and with the aggregation factor system is sponges.
D000373:Agglutinins / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001666:Binding Sites, Antibody; D001789:Blood Group Antigens; D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D005643:Fucose; D005690:Galactose; D006601:Hexoses / Q000032:analysis; D008970:Molecular Weight; D011161:Porifera / Q000276:immunology*
D000373:Agglutinins; D000596:Amino Acids; D001789:Blood Group Antigens; D006601:Hexoses; D005643:Fucose; D002118:Calcium; D005690:Galactose