Plasticity in the Primary Binding Site of Galactose/N-Acetylgalactosamine-specific Lectins

Abstract

The recognition of sugar molecules by carbohydrate-specific proteins, lectins, involves the establishment of an organized set of interactions within the binding site. Hydrogen-bonding interactions are one of the most important factors of molecular recognition in lectinϪsugar interactions, along with van der Waals forces, which, although rather weak (often contributing only a fraction of 1 kcal mol Ϫ1 for each pair of atoms), are frequently numerous and together make a significant contribution to binding (1, 2). Despite the differences in the lectin folds and their modes of sugar binding, the specificity for the recognition of galactose is determined by interactions involving the C-4 locus of the saccharide (1-5). Stereochemical evidence is emerging for a distinct sugar binding specificity-dependent distribution of hydrogen bond donors vis à vis the acceptors in the combining site of lectins (4). The C-4 locus of the monosaccharide within the primary binding site of galactose/N-acetylgalactosamine-specific lectins has hitherto been considered to be absolutely invariant.Though belonging to different families, jacalin, a Moraceae member, and WBA 1 I, a Leguminosae member, both are galactose/N-acetylgalactosamine-specific lectins (6, 7). Whereas jacalin displays a ␤-prism tertiary structural fold wherein a unique post-translationally generated N-terminal glycine residue serves as a critical determinant of galactose specificity (8), WBA I contains a legume lectin fold (9). During the course of mapping and establishing the hydrogen bond donor-acceptor relationship of the primary combining site of galactose-specific lectins (10), unexpectedly, we have discovered that the 4-methoxy derivative of D-galactopyranoside (4-methoxygalactose) binds, with affinities comparable with that of methyl-␣-galactose, to the Moraceae lectin jacalin and the Leguminosae lectin WBA I but not to the related WBA II. EXPERIMENTAL PROCEDURESMaterials-All reagents were of analytical or ultrapure grade. Methyl-␣-galactose was purchased from Sigma. 4-methoxygalactose was synthesized as described, and its purity was checked by melting point, thin-layer chromatography, and high resolution 1 H-NMR at 250 MHz on a Bruker spectrometer (10). Deionized Milli-Q water was used for all studies.Preparation and Analysis of Solutions-All protein as well as sugar solutions were prepared in 20 mM phosphate buffer (pH 7.2) containing 150 mM sodium chloride (PBS). Jacalin (6), WBA I (7, 10), and WBA II (12) were prepared by affinity chromatography as described. Their concentrations were measured spectrophotometrically using ⑀ 280 nm 1%,1 cm ϭ 15.8, 9.37, and 7.7, respectively, determined by weight method as well as from amino acid sequence data. Jacalin is a tetramer, whereas WBA I and WBA II are dimers.ITC Measurements and Analyses-The titration calorimetric measurements and analyses were performed with a Microcal Omega titration calorimeter as described (10,13,14). Aliquots of the sugar solution at 10 -100 times the binding site concentration were added via a 2...

Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose

“…Similar conclusions were drawn with respect to the structure and specificity of the Maclura pomifera agglutinin (MPA) on the basis of the three-dimensional structure of the lectin complexed with the T-antigen disaccharide Galβ1,3GalNAc [13]. More recently, Swaminathan et al [14] reported that the primary binding sites of jacalin and the galactose-specific winged bean lectin exhibit some plasticity. Indeed, both lectins accommodate the bulky 4-O-methoxy-galactose derivative through a novel C-H…O type of hydrogen bond [14].…”

“…As a result, the binding site of jacalin can accommodate epimeric sugars with differently oriented C-2 and C-4 hydroxyl groups and more or less bulky substituents. The presence of an extended site also accounts for the observed specific binding of Neu5Ac and MurNAc to jacalin and for the recently reported binding of 4-Met-Gal to jacalin [14]. Recently, the occurrence of a rather extended carbohydrate-binding site in KMj, the closely related mannose-specific lectin from A. integrifolia, was reported from thermodynamic and calorimetric studies of the lectin complexed to a trimannoside [35,36].…”

“…More recently, Swaminathan et al [14] reported that the primary binding sites of jacalin and the galactose-specific winged bean lectin exhibit some plasticity. Indeed, both lectins accommodate the bulky 4-O-methoxy-galactose derivative through a novel C-H…O type of hydrogen bond [14]. Although these findings have not been interpreted in terms of possible promiscuous specificity, they indicate that the binding sites of these two lectins may accommodate structurally different sugars.…”

Structural Basis for the Energetics of Jacalin–Sugar Interactions: Promiscuity Versus Specificity

“…41,42 A circulating water-bath was employed to help temperature stabilization. The instrument was allowed to equilibrate overnight at 298 K. Aliquots (5-6 ml) of sugar solution were added from the computer-controlled 250 ml rotating syringe, at 396 rpm into the cell containing 1.36 ml of the lectin solution under isothermal conditions.…”

Human hepatocyte functions in a galactosylated membrane bioreactor

“…Studies have revealed that the lectin used in our experiments could specifically bind galactose or N-acetyl-dgalactosamine [37]. FITC fluorescence proved to be relatively high on galactosylated PES membranes, while on native PES no fluorescence signal was detected, so the fluorescence images clearly displayed the real immobilization of the galactose derivatives.…”