Further characterization of a lectin and its in vivo receptor from Geodia cydonium

From the results of two-dimensional isoelectric focusing, SDS-gel electrophoresis and from immunochemical data it became evident that lectin I and lectin II (corresponding to fractions Geodia I and Geodia II isolated on immobilized lactose) from the sponge Geodia cydonium are apparently identical mixtures of several isolectins, the pI values of their subunits ranging, in contrast to our previous report, from 4.8–7.5. The hypothetical concept of sugar-mediated, specific lectin-lectin interactions (self-recognition) could not be verified by binding of FITC-labelled isolectins (Geodia I) to the lectin subunits, which had been purified by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The concept should also be dismissed on the basis of carbohydrate analyses revealing in contradiction with previous results the exclusive presence of alkali-labile bound tetraglucose on the purified isolectins (1 mol/mol lectin protein). The combining site of the isolectins was shown by a quantitative microprecipitation inhibition assay to be most complementar to oligosaccharides of the β-galactoside series and to interact specifically with particular structural elements of the subterminal sugar(s). Carbohydrates of the anti aggregation receptor, which are assumed to represent the functional ligand of the Geodia-isolectins in vivo, could be demonstrated to have a high affinity for the lectin combining site, exceeding that of the best disaccharide inhibitor, lactose, by five orders of magnitude. A preliminary chemical characterization of the receptor carbohydrate revealed that D-galactose and D-glucose (each approx. 200 mol/mol receptor) are organized in an oligosaccharide, which could be cleaved from the protein by trifluoroacetolysis.