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

Abstract

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 Galalpha1-->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 Pkactive Galalpha1-->4Gal, was the best. It was 7.4-fold less active than melibiose (Galalpha1-->6Glc). PA-IL has a preference for the alpha-anomer in decreasing order as follows: Galalpha1-->6 >Galalpha1-->4 >Galalpha1-->3. Of the monosaccharides studied, the phenylbeta derivatives of Gal were much better inhibitors than the methylbeta derivative, while only an insignificant difference was found between the Galalpha 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 Galalpha1-->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.

Role of LecA and LecB Lectins in Pseudomonas aeruginosa -Induced Lung Injury and Effect of Carbohydrate Ligands

“…for P. aeruginosa (15). LecA is a D-galactose-binding lectin with high affinity for terminal glycans presenting an ␣-galactose residue at the nonreducing end (4,18). The LecB lectin has a high affinity for L-fucose and its derivatives (24,35).…”

Defining the Carbohydrate Specificities of AplysiaGonad Lectin Exhibiting a Peculiar d-Galacturonic Acid Affinity

“…The Microtiter Plate Lectin-Enzyme Binding Assay (ELLSA)-ELLSA was performed according to the procedures of Duk et al (10) or as described previously (12,13). The volume of each reagent applied to the plate was 50 l/well, and all incubations, except for coating, were performed at 20°C.…”

“…In the present study, we defined the glycan affinity of this lectin by both enzyme-linked biotin/avidin-mediated microtiter plate lectin assay (ELLSA) and also by examination of the inhibition of AGL-glycan interaction (10,11). The great advantage of this method is that the amount of lectin and glycoform required is about 1/10 to 1/1000 of that required for the quantitative precipitin assay (12,13). The results show that the carbohydrate affinity hierarchy of this lectin can be regarded as: tri-GalUA␣134 to mono-GalUA Ͼ branched and/or clusters of E(Gal␣134Gal), I(Gal␤133GlcNAc), and/or II(Gal␤134 GlcNAc) Ͼ Ͼ monomeric E and I Ͼ II, although GalNAc is inactive.…”

RapA2 Is a Calcium-binding Lectin Composed of Two Highly Conserved Cadherin-like Domains That Specifically Recognize Rhizobium leguminosarum Acidic Exopolysaccharides

“…The binding of RapA2 to EPS was further analyzed by means of a binding inhibition assay using a modified ELISA technique (38). Microtiter wells coated with EPS of R. leguminosarum bv.…”