Examination of the structural basis for O(H) blood group specificity by Ulex europaeus Lectin I

The structural basis for carbohydrate specificity of the first lectin from Ulex europaeus (UE-I) is reported. UE-I is a dimeric metalloglycoprotein that binds the H-type 2 human blood group determinant (α-L-Fucα(1[Formula: see text]2)-β;-D-Galβ(1[Formula: see text]4)-β-D-GlcNAcα-), the blood group determinant present on the surface of O-type erythrocytes. The structural characteristics of UE-I involved in carbohydrate recognition have been examined using mass spectrometry (MS) and X-ray diffraction analysis. MS analysis allowed for discrimination between the different primary structures reported for UE-I. To examine the binding of the H-type 2 blood group determinant by UE-I, the methyl glycosides of the fucose monosaccharide (α-L-Fuc-OMe), known to exhibit primary binding specificity, and the H-type 2 trisaccharide (H-type 2-OMe) were, in two separate experiments, co-crystallized into the binding site of UE-I. The UE-I:α-L-Fuc-OMe complex crystallizes in the monoclinic space group P21, with unit cell dimensions a = 71.81, b = 69.00, and c = 119.02 Å, and β = 106.76°. Two UE-I dimers are observed to be present within the asymmetric unit, and the model has been refined to a R-value and RFree of 0.202 and 0.289, respectively, to 2.3 Å resolution. The preliminary model of the UE-I:H-type 2-OMe complex has been refined at 3.0 Å resolution. The UE-I:H-type 2-OMe complex crystallizes in the orthorhombic space group C2221, with unit cell dimensions a = 88.80, b = 164.75, and c = 77.42 Å, and a single UE-I dimer is present within the asymmetric unit. The carbohydrate recognition domain of UE-I has been identified to be comprised of residues Glu44, Thr86, Asp87, Arg102, Ala103, Gly104, Gly105, Tyr106, Ile129, Val133, Asn134, Trp136, Tyr219, and Arg222. Several critical protein-carbohydrate interactions have been identified, including the role of the hydrophobic interaction between the Thr86 side chain and C-5-CH3 of the α-L-Fuc-OMe. The role of these interactions in carbohydrate recognition-binding by UE-I, as well as differences between the observed and previously modeled complexes, are discussed. Key words: Ulex europaeus lectin I, H-type 2 human blood group determinant, protein-carbohydrate interactions, X-ray crystallography, chemical mapping.

Binding of human liver hydrolases by immobilized lectins

The binding of 22 human liver hydrolase activities by immobilized lectins of six different carbohydrate specificities, namely alpha-D-mannose (glucose), D-N-acetylglucosamine, D-N-acetylgalactosamine, L-fucose, alpha-D-galactose and beta-D-galactose, were examined. Differences in binding among these enzymes and within specific enzymes were observed. For example, the neutral forms of alpha-mannosidase and beta-xylosidase were bound by the Ulex europaeus lectin I (specific for L-fucose), whereas the acidic forms were not. Bandierea simplicifolia lectin (specific for alpha-galactose) bound 65% of beta-glucuronidase activity; recycling experiments demonstrated complete binding of the enzyme that had been eluted with the competitor D-galactose and no binding of the fraction that was not initially bound. These results suggested the presence of two forms of this enzyme. Similar data were obtained for acidic beta-galactosidase activity. These experiments may provide the basis for the expanded use of immobilized lectins for purification and characterization of hydrolases and other glycoproteins.