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 Galα1→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 Galα1→4Gal, was the best. It was 7.4-fold less active than melibiose (Galα1→6Glc). PA-IL has a preference for the cα-anomer in decreasing order as follows: Galα1→6 > Galα1→4 > Galα1→3. Of the monosaccharides studied, the phenylβ derivatives of Gal were much better inhibitors than the methylβ derivative, while only an insignificant difference was found between the Galα 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 α-anomer of Gal at nonreducing end and most complementary to Galα1→6Glc. As for the combining site of PA-IL toward the β-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.