Purification and characterization of the agglutinins from the sponge Aaptos papillata and a study of their combining sites

Abstract

The lectins from the sponge Aaptos papillata were isolated by affinity chromatography using polyleucyl blood group A + H substances from hog stomach linings as an absorbent and eluting with 3 M MgCl2. Further separation on diethylaminoethylcellulose and preparative disc electrophoresis on polyacrylamide gave the three fractions, Aaptos lectins I, II, and III. They were essentially homogenous in polyacrylamide electrophoresis and sedimentation analysis: a small second component was seen in lectins I and II in immunoelectrophoresis at high concentration. The S20,W0 values for Aaptos lectins I, II, and III were 3.5, 6.0, and 5.5. By electrophoresis in sodium dodecyl sulfate with an without beta-mercaptoethanol Aaptos lectin I showed two bands corresponding to molecular weights of 12 000 and 21 000; Aaptos lectins II and III gave only one band of molecular weight of 16 000. In isoelectric focusing, Aaptos lectin I showed bands at pH 4.7 and 5.4 and in the range between 6.8 and 7.6, while Aaptos lectins II and III were almost identical with bands at pH 3.8, 4.7 to 4.9, and 5.3. Aaptos lectin I differed from II and III in amino acid composition but the latter two were very similar. They contained no significant carbohydrate. Aaptos lectin I reacted best with blood group substances with terminal nonreducing N-acetyl-D-glucosamine residues precipitating about two-thirds of the lectin N added while blood group substances with terminal nonreducing DGalNAc were almost inactive. However, Aaptos lectin II was completely precipitated by blood group substances and glycoproteins containing terminal DGalNAc, DGlcNAc, or sialic acid residues. Aaptos lectin III had a precipitation pattern similar to Aaptos lectin II. DGlcNAc but not DGalNAc inhibited precipitation of Aaptos lectin I by blood group substances and N, N', N'', N'''-tetraacetylchitotetraose was the best inhibitor and was 2000 times more active than DGlcNAc. Precipitin reactions with Aaptos lectin II were inhibited by equal amounts of DGlcNAc and by sialic acid which were four times more potent than DGalNAc. N,N',N''-triacetylchiotriose was the best inhibitor and was 13 times better than DGlcNAc. At 37 degrees C three to four times higher amounts of inhibitor were necessary to inhibit precipitation of Aaptos lectin II than were needed at 4 degrees C, indicating higher affinity of blood group substances for Aaptos lectin II with increasing temperature. Aaptos lectin I was precipitated by the monofunctional hapten p-nitrophenyl-alphaDGalNAc, while p-nitrophenyl-betaDGalNAc did not precipitate and was a good inhibitor. Both phenomena indicate involvement of hydrophobic bonds.