A simple micro-method for determining precise oligosaccharidic specificity of mannose-binding lectins

A simple and inexpensive method was developed to rapidly define the specificity of mannose-specific lectins toward oligomannoside-type structures. The method involved the interaction of a mixture of N-[C-14]-acetylated glycoasparagines, prepared by exhaustive pronase digestion of bovine pancreatic ribonuclease B and N-[C-14]-acetylation with [C-14]-acetic anhydride and containing all the possible oligomannoside-type N-glycans, with the lectin immobilized on Sepharose-4B. After exhaustive desalting, the obtained fractions were separated by high-performance thin-layer chromatography on silica gel plates and visualized by autoradiography with intensifying screen. As an example of the usefulness of this method, the fine specificity of artocarpin, the mannose-specificity lectin isolated from seeds of jackfruit (Artocarpus integrifolia) toward oligomannoside-type structures is presented. On the basis of such a determination, the best oligomannosidic ligand recognized by a mannose-specific lectin can be selected for studies of crystal structures of the lectin in complex with the defined ligand. Furthermore, some of these immobilized lectins, after definition of their precise specificities with the method, could represent valuable tools for the fractionation and characterization of oligomannose-type structures, present in complex mixtures.