Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa

The binding properties of Pseudomonas aeruginosa agglutinin-I (PA-IL) with glycoproteins (gps) and polysaccharides were studied by both the biotin/avidin-mediated microtiter plate lectin-binding assay and the inhibition of agglutinin-glycan interaction with sugar ligands, Among 36 glycans tested for binding, PA-IL reacted best with two glycoproteins containing Gal alpha 1-->4Gal determinants and a human blood group ABO precursor equivalent gp, but this lectin reacted weakly or not at all with A and H active gps or sialylated gps, Among the mammalian disaccharides tested by the inhibition assay, the human blood group P-k active Gal alpha 1-->4Gal, was the best, It was 7.4-fold less active than melibiose (Gal alpha 1-->6Glc). PA-IL has a preference for the a-anomer in decreasing order as follows: Gal alpha-->6 > Gal alpha 1-->4 > Gal alpha 1-->3. Of the monosaccharides studied, the phenyl beta derivatives of Gal were much better inhibitors than the methyl beta derivative, while only an insignificant difference was found between the Gal alpha anomer of methyl-and p-NO2-phenyl derivatives, From these results, it can be concluded that the combining size of the agglutinin is as large as a disaccharide of the alpha-anomer of Gal at nonreducing end and most complementary to Gal alpha 1-->6Glc. As for the combining site of PA-IL toward the beta-anomer, the size is assumed to be less than that of Gal; carbon-6 in the pyranose form is essential, and hydrophobic interaction is important for binding.