The Modes of Binding Methyl—α (and β)-D-glucopyranosides and Some of their Derivatives to Concanavalin A—A Theoretical Approach

Abstract

The probable modes of binding of Methyl—α (and β)-D-glucopyranosides and some of their derivatives to concanavalin A have been proposed from theoretical studies. Theory predicts that βMeGlcP can bind to ConA in three different modes whereas α-MeGlcP can bind only in one mode. βMeGlcP in its most favourable mode of binding differs from α-MeGlcP in its alignment in the active-site of the lectin where it binds in a flipped or inverted orientation. Methyl substitution at the C-2 atom of the α-MeGlcP does not significantly affect the possible orientations of the sugar in the active-site of the lectin. Methyl substitution at C-3 or C-4, however, affects the allowed orientations drastically leading to the poor inhibiting power of Methyl-3-O-methyl-α-D-glucopyranoside and the inactivity of Methyl-4-O-methyl-α-D-glucopyranoside. These studies suggest that the increased activity of the α-MeGlcP over β-MeGlcP may be due to the possibility of formation of better hydrogen bonds and to hydrophobic interactions rather than to steric factors as suggested by earlier workers. These models explain the available NMR and other binding studies.     

Carbohydrate-binding specificity of Tetracarpidium conophorum lectin.

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.     

Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom

A lectin in the fruiting bodies of Psathyrella velutina was purified by affinity chromatography on a chitin column and subsequent ion-exchange chromatography. P. velutina lectin (PVL) tends to aggregate irreversibly in buffered saline, but the addition of glycerol (10%, v/v) to lectin solutions was found to prevent aggregate formation. PVL is assumed to occur as a monomer of a polypeptide of Mr = 40,000 as determined by gel filtration and by gel electrophoresis in the presence of sodium dodecyl sulfate. PVL is specific for N-acetylglucosamine (GlcNAc). It was determined by equilibrium dialysis to have four binding sites/polypeptide molecule showing an average intrinsic association constant of K0 = 6.4 x 10(3) M-1 toward this sugar. The binding specificity of the lectin was studied by hemagglutination inhibition assays and by avidin-biotin-mediated enzyme immunoassays using various GlcNAc-containing saccharides. The results indicate that methyl N-acetyl beta-glucosaminide was a slightly better inhibitor than the corresponding alpha-anomer. PVL binds well to oligosaccharides bearing nonreducing terminal beta-GlcNAc linked 1----6 or 1----3 but poorly to those having a 1----4 linkage, such as N-acetylated chito-oligosaccharides. It also binds to the subterminal GlcNAc moiety when it is substituted at the C-6 position but does not interact with the moiety when substituted either at C-3 or C-4. Thus, these results show that PVL is quite different in its binding specificity from other GlcNAc-binding lectins of higher plants since they bind preferentially to beta-GlcNAc in 1----4 linkage and they have a high affinity for chitin oligosaccharides.     

Surface plasmon resonance analysis of antifungal azoles binding to CYP3A4 with kinetic resolution of multiple binding orientations

The heme-containing cytochrome P450s (CYPs) are a major enzymatic determinant of drug clearance and drug-drug interactions. The CYP3A4 isoform is inhibited by antifungal imidazoles or triazoles, which form low-spin heme iron complexes via formation of a nitrogen-ferric iron coordinate bond. However, CYP3A4 also slowly oxidizes the antifungal itraconazole (ITZ) at a site that is approximately 25 A from the triazole nitrogens, suggesting that large antifungal azoles can adopt multiple orientations within the CYP3A4 active site. Here, we report a surface plasmon resonance (SPR) analysis with kinetic resolution of two binding modes of ITZ, and the related drug ketoconazole (KTZ). SPR reveals a very slow off-rate for one binding orientation. Multiphasic binding kinetics are observed, and one of the two binding components resolved by curve fitting exhibits "equilibrium overshoot". Preloading of CYP3A4 with the heme ligand imidazole abolishes this component of the antifungal azole binding trajectories, and it eliminates the conspicuously slow off-rate. The fractional populations of CYP3A4 complexes corresponding to different drug orientations can be manipulated by altering the duration of the pulse of drug exposure. UV-vis difference absorbance titrations yield low-spin spectra and K(D) values that are consistent with the high-affinity complex resolved by SPR. These results demonstrate that ITZ and KTZ bind in multiple orientations, including a catalytically productive mode and a slowly dissociating inhibitory mode. Most importantly, they provide the first example of a SPR-based method for the kinetic characterization of binding of a drug to any human CYP, including mechanistic insight not available from other methods.     

Structure-activity relationships of progesterone derivatives that bind to the digitalis receptor: modifications in A and B rings

A number of progesterone derivatives, having a 17 alpha-acetoxy group and various functions at C-3 and C-6, interact at the cardiac glycoside (CG) binding site, using [3H]ouabain in a radioligand binding assay (RBA) with membranes from dog myocardium. We now report on results of structure-activity studies concerned with modification of the A and B rings as they influence potency in the RBA. Some progesterone derivatives with 5 alpha- or 5 beta-stereochemistry show weak receptor competing activity. Among the congeners highest potency is associated with the presence of C-4 or C-4,6 unsaturation and a C-6 substituent (CH3, Cl, Br) whose importance appears to reside in its steric rather than electronic character. The C-3 function may be carbonyl, 3 beta-hydroxy or 3 beta-acetoxy when associated with C-4 or C-4,6 unsaturation. In compounds with other substituents that promote activity, C-6 alpha substitution with -CH3, -Cl, or -Br strongly enhances activity; -F, -OCH3, carbonyl, or the unsubstituted compound promotes weak binding; and -OC2H5, -OAc, -OCOOCH3, or -OH eliminates binding activity. Receptor interaction with the double bond at C-4, but not C-5, appears to be particularly important for binding. The most potent analog identified thus far is chlormadinone acetate (17 alpha-acetoxy-6-chloropregna-4,6-diene-3,20-dione), which has 1/20 the potency of ouabain in the RBA. Studies to determine optimal structural requirements for CG-receptor binding by these hormonal steroid congeners, in conjunction with appropriate biological assays, may provide insight into the nature of a putative endogenous counterpart, lead to a better understanding of the mode of action of the CG and yield CG-like compounds with superior therapeutic properties.     

Computer modelling approach to study the modes of binding of alpha- and beta-anomers of D-galactose, D-fucose and D-glucose to L-arabinose-binding protein

The modes of binding of alpha- and beta-anomers of D-galactose, D-fucose and D-glucose to L-arabinose-binding protein (ABP) have been studied by energy minimization using the low resolution (2.4 A) X-ray data of the protein. These studies suggest that these sugars preferentially bind in the alpha-form to ABP, unlike L-arabinose where both alpha- and beta-anomers bind almost equally. The best modes of binding of alpha- and beta-anomers of D-galactose and D-fucose differ slightly in the nature of the possible hydrogen bonds with the protein. The residues Arg 151 and Asn 232 of ABP from bidentate hydrogen bonds with both L-arabinose and D-galactose, but not with D-fucose or D-glucose. However in the case of L-arabinose, Arg 151 forms hydrogen bonds with the hydroxyl group at the C-4 atom and the ring oxygen, whereas in case of D-galactose it forms bonds with the hydroxyl groups at the C-4 and C-6 atoms of the pyranose ring. The calculated conformational energies also predict that D-galactose is a better inhibitor than D-fucose and D-glucose, in agreement with kinetic studies. The weak inhibitor D-glucose binds preferentially to one domain of ABP leading to the formation of a weaker complex. Thus these studies provide information about the most probable binding modes of these sugars and also provide a theoretical explanation for the observed differences in their binding affinities.     

Carbohydrate binding specificity of immobilized Allomyrina dichotoma lectin II

The carbohydrate binding specificity of Allomyrina dichotoma lectin II was investigated by analyzing the behavior of various complex type oligosaccharides and human milk oligosaccharides on an A. dichotoma lectin II-agarose column. Basically, the lectin interacts with the Gal beta 1----4GlcNAc group. Substitution of their terminal galactose residues by Neu5Ac alpha 2----6 will enhance their affinity to the lectin. By contraries, substitution at the C-2 or C-3 position of their terminal galactose with other sugars including sialic acid deprives their affinity to the lectin. With this characteristic, the immobilized lectin column can be used to separate complex type oligosaccharides with the Neu5Ac alpha 2----6Gal beta 1----4GlcNAc group from their isomeric oligosaccharides with the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc group, where Neu5Ac is N-acetylneuraminic acid.     

Analysis of saccharide binding to Artocarpus integrifolia lectin reveals specific recognition of T-antigen (beta-D-Gal(1----3)D-GalNAc)

The binding of Artocarpus integrifolia lectin to N-dansylgalactosamine (where dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) leads to a 100% increase in dansyl fluorescence with a concomitant blue shift in the emission maximum by 10 nm. This binding is carbohydrate-specific and has an association constant of 1.74 X 10(4) M-1 at 20 degrees C. The lectin has two binding sites for N-dansylgalactosamine. The values of -delta H and -delta S for the binding of N-dansylgalactosamine are in the range of values reported for several lectin-monosaccharide interactions, indicating an absence of nonpolar interaction of the dansyl moiety of the sugar with the combining region of the protein. Dissociation of the bound N-dansylgalactosamine from its complex with the lectin and the consequent change in its fluorescence on addition of nonfluorescent sugars allowed evaluation of the association constant for competing ligands. The thermodynamic parameters for the binding of monosaccharides suggest that the OH groups at C-2, C-3, C-4, and C-6 in the D-galactose configuration are important loci for interaction with the lectin. The acetamido group at C-2 of 2-acetamido-2-deoxygalactopyranose and a methoxyl group at C-1 of methyl-alpha-D-galactopyranoside are presumably also involved in binding through nonpolar and van der Waals' interactions. The T-antigenic disaccharide Gal beta 1----3GalNAc binds very strongly to the lectin when compared with methyl-beta-D-galactopyranoside, the beta(1----3)-linked disaccharides such as Gal beta 1----3GlcNAc, and the beta(1----4)-linked disaccharides, N-acetyllactosamine and lactose. The major stabilizing force for the avid binding of T-antigenic disaccharide appears to be a favorable enthalpic contribution. The combining site of the lectin is, therefore, extended. These data taken together suggest that the Artocarpus lectin is specific toward the Thomsen-Friedenreich (T) antigen. There are subtle differences in the overall topography of its combining site when compared with that of peanut (Arachis hypogaea) agglutinin. The results of stopped flow spectrometry for the binding of N-dansylgalactosamine tot he Artocarpus lectin are consistent with a simple single-step bimolecular association and unimolecular dissociation rate processes. The value of K+1 and K-1 at 21 degrees C are 8.1 X 10(5) M-1 s-1 and 50 s-1, respectively. The activation parameters indicate an enthalpy-controlled association process.     

Structural requirements of carbohydrates to bind Agaricus bisporus lectin

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T- disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.      

Structural basis of oligomannose recognition by the Pterocarpus angolensis seed lectin

The crystal structure of a Man/Glc-specific lectin from the seeds of the bloodwood tree (Pterocarpus angolensis), a leguminous plant from central Africa, has been determined in complex with mannose and five manno-oligosaccharides. The lectin contains a classical mannose-specificity loop, but its metal-binding loop resembles that of lectins of unrelated specificity from Ulex europaeus and Maackia amurensis. As a consequence, the interactions with mannose in the primary binding site are conserved, but details of carbohydrate-binding outside the primary binding site differ from those seen in the equivalent carbohydrate complexes of concanavalin A. These observations explain the differences in their respective fine specificity profiles for oligomannoses. While Man(alpha1-3)Man and Man(alpha1-3)[Man(alpha1-6)]Man bind to PAL in low-energy conformations identical with that of ConA, Man(alpha1-6)Man is required to adopt a different conformation. Man(alpha1-2)Man can bind only in a single binding mode, in sharp contrast to ConA, which creates a higher affinity for this disaccharide by allowing two binding modes.     

Thermodynamic analysis of ligand binding to winged bean (Psophocarpus tetragonolobus) acidic agglutinin reveals its specificity for terminally monofucosylated H-reactive sugars

The sugar-specific binding of N-dansylgalactosamine to WBA II (n = 2; Ka = 5.6 x 10(3) M-1; delta H = -21 kJ.mol-1; delta S = -21.3 J.mol-1.K-1) was utilized in substitution titrations for evaluating the association constants for the interaction of sugars with the lectin. An axial hydroxyl at C-4 and equatorial hydroxyls at C-3 and C-6 as in D-galacto configuration are crucial for binding. Both axial and equatorial hydroxyls are tolerated at C-2. Conformationally akin disaccharides such as lactose, N-acetyllactosamine, Gal beta 1-3GlcNAc, and Gal beta 1-3GalNAc show similar affinities. 2'-Fucosyllactose and H-disaccharide display 146 and 13 times stronger affinity over lactose and galactose, yet fucose by itself is devoid of activity. An interesting feature, noted for the first time, in protein-sugar interactions is the positive entropy change for the binding of 2'-fucosyllactose, suggesting that nonpolar interactions play an important role in stabilization of the lectin-sugar complex. 3-Fucosyllactose, lactodifucotetraose, lacto-N-fucopentaose II and III are inactive, whereas lacto-N-fucopentaose I has 14-fold lower affinity as compared with 2'-fucosyllactose. Conformational analysis indicates that the substitution at subterminal glucose or GlcNAc by L-fucose in either alpha 1-3 or alpha 1-4 linkage leads to its projection so as to sterically hinder the access of 3'-fucosyllactose, lactodifucotetraose, and lacto-N-fucopentaose II and III to the binding site of winged bean agglutinin II. Similarly the projection of alpha 1-3 linked Gal/GalNAc also leads to steric hindrance and hence prevents the binding of blood group A and B reactive sugars. Considering its unique specificity winged bean agglutinin II should be useful in the isolation and characterization of terminally monofucosylated H-reactive oligosaccharides from those that are difucosylated or internally fucosylated.     

Characterization of the carbohydrate moiety of Clerodendron trichotomum lectins. Its structure and reactivity toward plant lectins

Lectins were isolated from fruits and leaves of Clerodendron trichotomum by affinity chromatography on lactamyl-Sepharose. The purified lectins (C. trichotomum agglutinin: CTA) were homogeneous on SDS/polyacrylamide gel electrophoresis, and the carbohydrate moiety was characterized by physicochemical and immunochemical methods. The asparagine-linked oligosaccharides were released by treatment with N-oligosaccharide glycopeptidase (almond, EC 3.5.1.52) of peptic glycopeptides obtained from fruit CTA, and separated by gel filtration and thin-layer chromatography. The structure of the predominant oligosaccharide was determined as Xyl beta 1----2 (Man alpha 1----6)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc by high-performance liquid chromatography, sugar analysis and 1H-NMR spectroscopy. The reactivity of the carbohydrate moiety of CTA toward various lectins was studied. Fruit and leaf CTAs were applied to polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets and detected with horseradish-peroxidase-conjugated lectins. Concanavalin A, lentil lectin, pea lectin, Vicia faba lectin and Ulex europeus agglutinin I, but not wheat germ lectin, bound to fruit CTA. The results indicate new binding properties of these plant lectins: a beta-xylosyl residue substituted at C-2 of the beta-mannosyl residue of N-linked oligosaccharide does not affect the binding with mannose-specific lectins, lentil, pea and Vicia faba lectins can bind to N-linked oligosaccharides containing an alpha-L-fucosyl residue attached to C-3 of the asparagine-linked N-acetyl-D-glucosamine residue, and Ulex europeus agglutinin I can bind to the (alpha 1----3)-linked fucose residue of the N-linked oligosaccharide.     

Mapping of steroids binding to 17 beta-hydroxysteroid dehydrogenase type 1 using Monte Carlo energy minimization reveals alternative binding modes

Crystallographic studies of ligand-protein complexes reveal most preferable ligand binding modes, but do not show less populated modes that may contribute to measurable biochemical and biophysical characteristics of the complexes. In some cases, a ligand may bind a protein in essentially different modes. An example is 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), a steroidogenic enzyme that catalyzes reduction of estrone to estradiol in gonadal and peripheral tissues. The enzyme exhibits a high specificity for estrogens which bind with their C17 atom in the proximity of the NADP(H) cofactor. 17Beta-HSD1 can also bind androgens, but in a reverse binding mode, in which the steroid C3 atom is the closest carbon atom to the cofactor. Here we map the interaction energy of estradiol and dihydrotestosterone binding to 17beta-HSD1. Positions and orientations of the steroids in the ligand-binding tunnel were sampled systematically, and at each combination of these generalized coordinates, the energy was Monte Carlo minimized. The computed maps show energy minima corresponding to the X-ray structures and predict alternative binding modes, in particular, an upside-down orientation in which steroidal face alpha is exposed to protein residues that normally interact with face beta. The methodology can be used for mapping ligand-receptor interactions in various systems, for example, in ion channels and G-protein-coupled receptors that bind elongated ligands in confined space between transmembrane helices.     

Multiple modes of binding enhance the affinity of DC-SIGN for high mannose N-linked glycans found on viral glycoproteins

The dendritic cell surface receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR specifically recognize high mannose N-linked carbohydrates on viral pathogens. Previous studies have shown that these receptors bind the outer trimannose branch Manalpha1-3[Manalpha1-6]Manalpha present in high mannose structures. Although the trimannoside binds to DC-SIGN or DC-SIGNR more strongly than mannose, additional affinity enhancements are observed in the presence of one or more Manalpha1-2Manalpha moieties on the nonreducing termini of oligomannose structures. The molecular basis of this enhancement has been investigated by determining crystal structures of DC-SIGN bound to a synthetic six-mannose fragment of a high mannose N-linked oligosaccharide, Manalpha1-2Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1-6Man and to the disaccharide Manalpha1-2Man. The structures reveal mixtures of two binding modes in each case. Each mode features typical C-type lectin binding at the principal Ca2+-binding site by one mannose residue. In addition, other sugar residues form contacts unique to each binding mode. These results suggest that the affinity enhancement displayed toward oligosaccharides decorated with the Manalpha1-2Manalpha structure is due in part to multiple binding modes at the primary Ca2+ site, which provide both additional contacts and a statistical (entropic) enhancement of binding.     

Human soluble epoxide hydrolase: structural basis of inhibition by 4-(3-cyclohexylureido)-carboxylic acids

X-ray crystal structures of human soluble epoxide hydrolase (sEH) complexed with four different dialkylurea inhibitors bearing pendant carboxylate "tails" of varying length have been determined at 2.3-3.0 A resolution. Similarities among inhibitor binding modes reinforce the proposed roles of Y381 and/or Y465 as general acids that protonate the epoxide ring of the substrate in concert with nucleophilic attack of D333 at the electrophilic epoxide carbon. Additionally, the binding of these inhibitors allows us to model the binding mode of the endogenous substrate 14,15-epoxyeicosatrienoic acid. Contrasts among inhibitor binding modes include opposite orientations of inhibitor binding in the active-site hydrophobic tunnel. Alternative binding orientations observed for this series of inhibitors to human sEH, as well as the binding of certain dialkylurea inhibitors to human sEH and murine sEH, complicate the structure-based design of human sEH inhibitors with potential pharmaceutical applications in the treatment of hypertension. Thus, with regard to the optimization of inhibitor designs targeting human sEH, it is critical that human sEH and not murine sEH be utilized for inhibitor screening, and it is critical that structures of human sEH-inhibitor complexes be determined to verify inhibitor binding orientations that correlate with measured affinities.     

Synthesis and receptor binding of polynuclear organometallic estradiol derivatives

Twelve novel organometallic derivatives of estradiol were synthesized with the aim of utilizing organometallic cold bioprobes as radioisotopic labels substitutes for steroid hormone receptor assays. For this purpose, we envisaged the attachment of several stable cobalt, molybdenum, osmium carbonyl clusters (tetra- and pentanuclear species) at estradiol 17 alpha-, 16 alpha-, 2- or 4-positions. The binding affinity of these new complexes for uterine estradiol receptor has been measured by the competitive binding method. The results show that the 17 alpha-position can tolerate substitution by bulky organometallic groups (especially in the case of cobalt and molybdenum carbonyl clusters). Estradiol derivatives which are functionalized at C-4 and C-16 alpha bind estradiol receptor with reasonable affinity and the RBA values are the same for the complexed and uncomplexed hormones. The 2- position is more sensitive to organometallic substitution and the complexation at the 2- alkyne results in a dramatic decrease of the RBA values. These results show that the attachment of polynuclear moieties in estradiol 17 alpha-, 4- and 16 alpha-, positions gives rise to compounds which are of potential utility in a new non-radioisotopic receptor assay since the metal-carbonyl markers are readily detected by high-sensitivity Fourier-transform infra-red spectroscopy.     

High-affinity binding of recombinant human galectin-4 to SO(3)(-)-->3Galbeta1-->3GalNAc pyranoside

Galectin-4 is a member of galectin family and has two carbohydrate recognition domains. Although galectin-4 has been thought to function in cell adhesion, its precise carbohydrate binding specificity has not yet been clarified. We studied the carbohydrate binding specificity of galectin-4 comparatively with that of galectin-3, using surface plasmon resonance, galectin-3- or -4-Sepharose column chromatography and the inhibition assay of their binding to immobilized asialofetuin. Galectin-3 broadly recognized lactose, type 1, type 2, and core 1. The substitution at the C-2 and C-3 position of beta-galactose in these oligosaccharides with alpha-fucose, alpha-GalNAc, alpha-Neu5Ac, or sulfate increased the binding ability for galectin-3, whereas the substitution at the C-4 or C-6 position diminished the affinity. In contrast, galectin-4 had quite weak affinity to lactose, type 1, and type 2 (K(d) congruent with 8 x 10(-4) M). Galectin-4 showed weak binding ability to core 1 and C-2' or -3'-substituted lactose, type 1, and type 2 with alpha-fucose, alpha-GalNAc, or sulfate (K(d) : 5 x 10(-5) approximately 3 x 10(-4) M). Interestingly, the K(d) value, 3.4 x 10(-6) M, of SO(3)(-)-->3Galbeta1-->3GalNAc-O-Bn to galectin-4 at 25 degrees C was two orders of magnitude lower than that of core 1-O-Bn. 3'-Sialylated core 1 had very weak affinity to galectin-4, suggesting that 3'-O-sulfation of core 1 is critical for the recognition. These results suggest that galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.     

The ligand binding specificity and tissue localization of a rat alveolar macrophage lectin

The parameters of the reaction between a rat alveolar macrophage lectin (Mr = 180,000) and its ligands have been examined. The reaction is dependent on Ca2+ over the optimal pH range for binding. The apparent dissociation constant for fucosyl bovine serum albumin, the standard ligand used in these studies, is 1.4 X 10(-10) M. The ligand binding specificity was determined by measurement of the inhibition of binding of fucosyl bovine serum albumin by various glycoproteins and saccharides. D-Mannose, L-fucose, and N-acetyl-D-glucosamine were the most effective inhibitors, and D-galactose was much poorer. The equatorial hydroxyl groups on the C-3 and C-4 of the mannose ring are important in the lectin-ligand interaction, and the axial hydroxyl group on the C-2 contributes to a lesser extent. Immunocytological studies revealed that the lectin isolated from alveolar macrophages is widely distributed in other rat tissues. Hepatocytes are devoid of the lectin, but hepatic Kupffer cells and endothelial cells contain significant amounts. This was confirmed by isolation of the lectin from liver. Spleen and skeletal muscle also contain lectin, but much smaller amounts were found in brain, kidney, and heart muscle.     

Molecular basis of recognition by Gal/GalNAc specific legume lectins: influence of Glu 129 on the specificity of peanut agglutinin (PNA) towards C2-substituents of galactose

The ability to discriminate between galactose and N- acetylgalactosamine, observed in some lectins, is crucial for their biological activity as well as their usefulness as tools in biology and medicine. However, the molecular basis of differential binding of lectins to these two sugars is poorly understood. Peanut agglutinin (PNA) is one of the few galactose-specific legume lectins which does not bind N- acetylgalactosamine at all and is, therefore, ideal for the study of the basis of specificity towards C-2 substituted derivatives of galactopyranosides. Examination of the three-dimensional structure of PNA in complex with lactose revealed the presence of both a longer loop and bulkier residues in the region surrounding the C-2 hydroxyl of the galactopyranoside ring, which can sterically prevent the accommodation of a bulky substituent in this position. One such residue, is a glutamic acid at position 129 which protrudes into the binding site and perhaps directly obstructs any substitution at the C-2 position. Two mutants in bacterially expressed PNA were therefore constructed. These were E129D and E129A, in which Glu129 was replaced by Asp and Ala, respectively. The specificity of the mutants for galactose, galactosamine, and N- acetylgalactosamine was examined through observing the inhibition of hemagglutination and binding of the lectin to immobilized asialofetuin. The results showed that the affinity of E129A and E129D for C-2-substituted derivatives of the galactose varies. The mutant E129D showed significant binding towards N- acetylgalactosamine, suggesting that the residue Glu 129 is crucial in imparting exclusive galactose-specificity upon PNA. This study not only attempts to provide an explanation for the inability of PNA to accommodate C-2-substituted derivatives at its primary subsite, but also seeks to present a basis for engineering lectins with altered specificities.