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

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Abstract
The lectins from the sponge A. papillata were isolated by affinity chromatography using polyleucyl blood group A + H substance from hog stomach linings as an absorbent and eluting with 3 M MgCl2. Further separation on DEAE-cellulose and preparative disk electrophoresis on polyacrylamide gave the 3 fractions, Aaptos lectins I, II and III. They were essentially homogeneous in polyacrylamide electrophoresis and sedimentation analysis; a small 2nd component was seen in lectins I and II in immunoelectrophoresis at high concentration. The s20,wO values for Aaptos lectins I, II and III were 3.5, 6.0, and 5.5. By electrophoresis in sodium dodecyl sulfate with and without .beta.-mercaptoethanol Aaptos lectin I showed 2 bands corresponding to MW 12,000 and 21,000; Aaptos lectins II and III gave only 1 band of MW 16,000. In isoelectric focusing Aaptos lectin I showed bands at pH 4.7 and 5.4 and in the range 6.8-7.6, 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 2 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 .apprx.2/3 of the lectin N added while blood group substances with terminal nonreducing DGalNAc were almost inactive. 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 4 times more potent than DGalNAc. N,N'',N"-triacetylchitotriose was the best inhibitor and was 13 times better than DGlcNAc. At 37.degree. C 3-4 times higher amounts of inhibitor were necessary to inhibit precipitation of Aaptos lectin II than were needed at 4.degree. C, indicating higher affinity of blood group substance for Aaptos lectin II with increasing temperature. Aaptos lectin I was precipitated by the monofunctional hapten p-nitrophenyl-.alpha.DGalNAc, while p-nitrophenyl-.beta.DGalNAc did not precipitate and was a good inhibitor. Both phenomena indicated involvement of hydrophobic bonds.