Binding of 4-methylumbelliferyl beta-D-galactosyl-(1 leads to 3)-N-acetyl-beta-D-galactosaminide to peanut agglutinin. Characterisation and application in substitution titrations

Binding of 4-methylumbelliferyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl) beta-D-galactopyranoside [MeUmb beta Gal(beta 1 leads to 3)GalNAc] to peanut agglutinin was characterized by equilibrium dialysis and by measurement of the increase in ultraviolet absorption or fluorescence of the chromophoric glycoside upon continuous titration with excess of the lectin. All data in the 4-30 degrees C range correspond to delta G = -(26.5 +/- 0.1) kJ mol-1, delta H = -(58.4 +/- 2) kJ mol-1 and delta S = -(107 +/- 8)J mol-1 K-1. Values of the association constants are e.g. K = 2.5 X 10(5) M-1 at 4 degrees C and K = 4.5 X 10(4) M-1 at 25 degrees C. MeUmb beta Gal(beta 1 leads to 3)GalNAc was used as an indicator ligand to determine K values for nonchromophoric carbohydrates by continuous displacement titrations, measuring either fluorescence or difference in absorption of the indicator. The data were analyzed in terms of the general expression for a non-ideal indicator system (as detailed in the appendix). Thus, the values of K are not underestimated. They are K = 4.8 X 10(3) M-1 for methyl alpha-D-galactopyranoside [Me alpha Gal], 2.0 X 10(3) M-1 for methyl beta-D-galactopyranoside [Me beta Gal] and 4.7 X 10(3) M-1 for lactose [Gal(beta 1 leads to 4)Glc], all at 14.5 degrees C. The MeUmb difference absorption spectra resulting from binding of the lectin with MeUmb beta Gal(beta 1 leads to 3)GalNAc and MeUmb beta Gal(beta 1 leads to 4)Glc are larger than for MeUmb beta Gal and MeUmb alpha Gal. These observations are consistent with the extended nature of the combining site of peanut agglutinin.     

Thermodynamic and kinetic studies on the mechanism of binding of methylumbelliferyl glycosides to jacalin.

The binding of Artocarpus integrifolia lectin (jacalin) to 4-methylumbelliferyl (Meumb)-glycosides, Gal alpha Meumb, Gal beta Meumb, GalNAc alpha Meumb, GalNAc beta-Meumb, and Gal beta 3GalNAc beta Meumb was examined by extrinsic fluorescence quenching titration and stopped flow spectrofluorimetry. The binding was characterized by 100% quenching of fluorescence of Meumb-glycosides. Their association constants range from 2.0 x 10(4) to 1.58 x 10(6) M-1 at 15 degrees C. Entropic contribution is the major stabilizing force for avid binding of Meumb-glycosides indicating the existence of a hydrophobic site that is complementary to their methylumbelliferyl group. The second order association rate constants for interaction of these sugars with lectin at 15 degrees C vary from 8.8 x 10(5) to 3.24 x 10(6) M-1 S-1, at pH 7.2. The first order dissociation rate constants range from 2.30 to 43.0 S-1 at 15 degrees C. Despite the differences in their association rate constants, the overall values of association constants for these saccharides are determined by their dissociation rate constants. The second order rate constant for the association of Meumb-glycosides follows a pattern consistent with the magnitude of the activation energies involved therin. Activation parameters for association of all ligands illustrate that the origin of the barrier between binding of jacalin to Meumb-glycosides is entropic, and the enthalpic contribution is small. A correlation between these parameters and the structure of the ligands on the association rates underscores the importance of steric factors in determining protein saccharide recognitions.     

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Reaction specificity and thermodynamics of interactions of small substrates and ligands with the 1,4-beta-glucan cellobiohydrolase II

The 1,4-beta-glucan cellobiohydrolase II (CBH II) from Trichoderma reesei QM 9414 catalyses the hydrolysis of the 4-methylumbelliferyl beta-D-glycosides derived from cellotriose, cellotetraose and cellopentaose [MeUmb(Glc)n; n = 3 - 5]. The reaction has been followed by quantitative high-performance liquid chromatography. Specific activity for cellobiose removal at apparent substrate saturation were determined as (0.8 +/- 0.2) min-1 for MeUmb(Glc)3 and (9 +/- 2) min-1 for MeUmb(Glc)4. The enzyme showed a deviant specificity with MeUmb(Glc)5 as substrate. Two chromophoric products were formed simultaneously [MeUmb(Glc)3 and MeUmb(Glc)2] with turn-over numbers (17 +/- 4) min-1 and (21 +/- 6) min-1, respectively. Methylumbelliferyl beta-glucoside (MeUmbGlc) and the corresponding cellobioside [MeUmb(Glc)2] were used in equilibrium binding experiments. Both ligands yielded one binding site per molecule of Mr = 54000 upon forced flow dialysis (diafiltration). The association constants found were in fair agreement with those determined from MeUmb fluorescence quenching titrations. Quenching was total at all temperatures investigated for MeUmb(Glc)2, whereas for MeUmbGlc it increased from 80% to 100% between 2 degrees C and 20 degrees C. The association constants fitted linear van't Hoff plots in both cases. MeUmb(Glc)2 and MeUmbGlc were also used as indicator ligands to determine the association constants and thermodynamic parameters of several non-chromophoric ligands of CBH II. The binding of glucose increased the affinity for MeUmb(Glc)2 whereas it displaced MeUmbGlc from its complex. A putative binding site of the CBH II containing four subsites can be proposed. The thermodynamic data for methyl beta-D-glucopyranoside and cellobiose as ligands also point at an extended binding site.     

Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.     

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.     

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Binding of small ligands to the 1,4-beta-glucan cellobiohydrolase II and influence of glucose on their affinity

Binding onto cellobiohydrolase II from Trichoderma reesei of glucose, cellobiose, cellotriose, derivatized and analogous compounds, is monitored by protein-difference-absorption spectroscopy and by titration of ligand fluorescence, either at equilibrium or by the stopped-flow technique. The data complete earlier results [van Tilbeurgh, H., Pettersson, L. G., Bhikhabhai, R., De Boeck, H. and Claeyssens, M. (1985) Eur. J. Biochem. 148, 329-334] indicating an extended active center, with putative subsites ABCD. Subsite A specifically complexes with beta-D-glucosides and D-glucose; at 25 degrees C the latter influences the concomitant binding of other ligands at neighbouring sites. For several ligands this cooperative effect for binding (at 0.33 M glucose and temperature range 4-37 degrees C) was characterized by a substantial increase of the enthalpic term (delta delta H = -35 kJ mol-1). Glucose (0.33 M) decreases the association and dissociation rate parameters of 4-methylumbelliferyl beta-D-cellobioside by one order of magnitude: k+ = (3.6 +/- 0.5) x 10(-5) M-1 s-1 versus (5.1 +/- 0.1) x 10(-6) M-1 s-1 (in the absence of glucose) and k- = (1.3 +/- 0.1) s-1 versus (14.0 +/- 0.3) s-1. As deduced from substrate-specificity studies and inhibition experiments, subsite B interacts with terminal non-reducing glucopyranosyl residues of oligomeric ligands and substrates, whereas catalytic (hydrolytic) cleavage occurs between C and D. Association constants 10-100 times higher than those for cellobiose or its glycosides were observed for D-glucopyranosyl-(1----4)-beta-D-xylopyranose and cellobionolactone derivatives, suggesting 'transition-state'-type binding for these ligands at subsite C. Although subsite D can accomodate a bulky chromophoric group (MeUmb) its preference for a glucosyl residue is reflected in the lower binding enthalpy of cellotriose (-34 kJ mol-1) as compared to cellobiose (-28.3 kJ mol-1) and MeUmb(Glc)2 (-11.6 kJ mol-1). This model indicates that oligomeric ligands (substrates) interact through cooperativity of their subunits at the extended binding site of cellobiohydrolase II.     

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.     

Erythrocyte-binding studies on an acidic lectin from winged bean (Psophocarpus tetragonolobus).

An acidic lectin (WBA II) was isolated to homogeneity from the crude seed extract of the winged bean (Psophocarpus tetragonolobus) by affinity chromatography on lactosylaminoethyl-Bio-Gel. Binding of WBA II to human erythrocytes of type-A, -B and -O blood groups showed the presence of 10(5) receptors/cell, with high association constants (10(6)-10(8) M-1). Competitive binding studies with blood-group-specific lectins reveal that WBA II binds to H- and T-antigenic determinants on human erythrocytes. Affinity-chromatographic studies using A-, B-, H- and T-antigenic determinants coupled to an insoluble matrix confirm the specificity of WBA II towards H- and T-antigenic determinants. Inhibition of the binding of WBA II by various sugars show that N-acetylgalactosamine and T-antigenic disaccharide (Thomsen-Friedenreich antigen, Gal beta 1-3GalNAc) are the most potent mono- and di-saccharide inhibitors respectively. In addition, inhibition of the binding of WBA II to erythrocytes by dog intestine H-fucolipid prove that the lectin binds to H-antigenic determinant.     

Topography of the combining region of a Thomsen-Friedenreich-antigen-specific lectin jacalin (Artocarpus integrifolia agglutinin). A thermodynamic and circular-dichroism spectroscopic study.

Thermodynamic analysis of carbohydrate binding by Artocarpus integrifolia (jackfruit) agglutinin (jacalin) shows that, among monosaccharides, Me alpha GalNAc (methyl-alpha-N-acetylgalactosamine) is the strongest binding ligand. Despite its strong affinity for Me alpha GalNAc and Me alpha Gal, the lectin binds very poorly when Gal and GalNAc are in alpha-linkage with other sugars such as in A- and B-blood-group trisaccharides, Gal alpha 1-3Gal and Gal alpha 1-4Gal. These binding properties are explained by considering the thermodynamic parameters in conjunction with the minimum energy conformations of these sugars. It binds to Gal beta 1-3GalNAc alpha Me with 2800-fold stronger affinity over Gal beta 1-3GalNAc beta Me. It does not bind to asialo-GM1 (monosialoganglioside) oligosaccharide. Moreover, it binds to Gal beta 1-3GalNAc alpha Ser, the authentic T (Thomsen-Friedenreich)-antigen, with about 2.5-fold greater affinity as compared with Gal beta 1-3GalNAc. Asialoglycophorin A was found to be about 169,333 times stronger an inhibitor than Gal beta 1-3GalNAc. The present study thus reveals the exquisite specificity of A. integrifolia lectin for the T-antigen. Appreciable binding of disaccharides Glc beta 1-3GalNAc and GlcNAc beta 1-3Gal and the very poor binding of beta-linked disaccharides, which instead of Gal and GalNAc contain other sugars at the reducing end, underscore the important contribution made by Gal and GalNAc at the reducing end for recognition by the lectin. The ligand-structure-dependent alterations of the c.d. spectrum in the tertiary structural region of the protein allows the placement of various sugar units in the combining region of the lectin. These studies suggest that the primary subsite (subsite A) can accommodate only Gal or GalNAc or alpha-linked Gal or GalNAc, whereas the secondary subsite (subsite B) can associate either with GalNAc beta Me or Gal beta Me. Considering these factors a likely arrangement for various disaccharides in the binding site of the lectin is proposed. Its exquisite specificity for the authentic T-antigen, Gal beta 1-3GalNAc alpha Ser, together with its virtual non-binding to A- and B-blood-group antigens, Gal beta 1-3GalNAc beta Me and asialo-GM1 should make A. integrifolia lectin a valuable probe for monitoring the expression of T-antigen on cell surfaces.     

Equilibrium and kinetic analysis of nucleotide binding to the DEAD-box RNA helicase DbpA

The Escherichia coli DEAD-box protein A (DbpA) is an RNA helicase that utilizes the energy from ATP binding and hydrolysis to facilitate structural rearrangements of rRNA. We have used the fluorescent nucleotide analogues, mantADP and mantATP, to measure the equilibrium binding affinity and kinetic mechanism of nucleotide binding to DbpA in the absence of RNA. Binding generates an enhancement in mant-nucleotide fluorescence and a corresponding reduction in intrinsic DbpA fluorescence, consistent with fluorescence resonance energy transfer (FRET) from DbpA tryptophan(s) to bound nucleotides. Fluorescent modification does not significantly interfere with the affinities and kinetics of nucleotide binding. Different energy transfer efficiencies between DbpA-mantATP and DbpA-mantADP complexes suggest that DbpA adopts nucleotide-dependent conformations. ADP binds (K(d) approximately 50 microM at 22 degrees C) 4-7 times more tightly than ATP (K(d) approximately 400 microM at 22 degrees C). Both nucleotides bind with relatively temperature-independent association rate constants (approximately 1-3 microM(-1) s(-1)) that are much lower than predicted for a diffusion-limited reaction. Differences in the binding affinities are dictated primarily by the dissociation rate constants. ADP binding occurs with a positive change in the heat capacity, presumably reflecting a nucleotide-induced conformational rearrangement of DbpA. At low temperatures (<22 degrees C), the binding free energies are dominated by favorable enthalpic and unfavorable entropic contributions. At physiological temperatures (>22 degrees C), ADP binding occurs with positive entropy changes. We favor a mechanism in which ADP binding increases the conformational flexibility and dynamics of DbpA.     

Thermodynamic and kinetic analysis of porphyrin binding to Trichosanthes cucumerina seed lectin.

The interaction of several metallo-porphyrins with the galactose-specific lectin from Trichosanthes cucumeirna (TCSL) has been investigated. Difference absorption spectroscopy revealed that significant changes occur in the Soret band region of the porphyrins upon binding to TCSL and these changes have been monitored to obtain association constants (Ka) and stoichiometry of binding (n). The dimeric lectin binds two porphyrin molecules and the presence of the specific saccharide lactose did not affect porphyrin binding significantly, indicating that the sugar and the porphyrin bind at different sites. The Ka values obtained for the binding of different porphyrins with TCSL at 25 degrees C were in the range of 2 x 10(3)-5 x 10(5) m(-1). Association constants for meso-tetra(4-sulphonatophenyl)porphyrinato copper(II) (CuTPPS), a porphyrin bearing four negative charges and meso-tetra(4-methylpyridinium)porphyrinato copper(II) (CuTMPyP), a porphyrin with four positive charges, were determined at several temperatures; from the temperature dependence of the association constants, the thermodynamic parameters change in enthalpy (DeltaH degrees ) and change in entropy (DeltaS degrees ) associated with the binding process were estimated. The thermodynamic data indicate that porphyrin binding to TCSL is driven largely by a favourable entropic contribution; the enthalpic contribution is very small, suggesting that the binding process is governed primarily by hydrophobic forces. Stopped-flow spectroscopic measurements show that binding of CuTMPyP to TCSL takes place by a single-step process and at 20 degrees C, the association and dissociation rate constants were 1.89 x 10(4) m(-1).s(-1) and 0.29 s(-1), respectively.     

Carbohydrate binding specificity of the Tn-antigen binding lectin from Vicia villosa seeds (VVLB4).

2-Dansylamino-2-deoxy-D-galactose (GalNDns) is a useful fluorescent probe to study the interaction of non-fluorescent sugars with the B4 lectin from Vicia villosa seeds (VVLB4). Binding of the lectin to GalNDns leads to a 5.2-fold increase in Dansyl fluorescence with a concomitant 10 nm blue shift in its emission maximum. The strong binding of GalNDns (Ka = 7.33 x 10(4) M-1 at 20 degrees C) is due to a favourable entropic contribution to the association process. Among the other sugars studied, GalNAc alpha 1-O-Ser followed by Me alpha GalNAc are the best ligands. 2-Deoxygalactose, galactosamine and galactose are 2013, 469 and 130 times weaker ligands, respectively, as compared to GalNAc, whereas GalNDns is about 2.44 times more potent than GalNAc, indicating that substitutions at the C-2 position of GalNAc have a considerable influence on the binding affinities. Equatorial orientation of the hydroxyl group at C-3 and axial orientation at C-4 as in galactose are important for the interaction with VVLB4. The C-6 hydroxyl group is not indispensable. The binding site of the lectin is directed exclusively towards monosaccharides alone. Interestingly enough, despite its preference for Me alpha GalNAc over Me beta GalNAc, in oligosaccharides, the lectin prefers terminal beta-linked GalNAc as compared to the alpha-linked one.     

NMR study of a Lewis(X) pentasaccharide derivative: solution structure and interaction with cations.

The structure and conformation of the synthetic pentasaccharide Gal(beta 1-4){Fuc(alpha 1-3)}GlcNAc(beta 1-3)Gal(beta 1-4)Glc-beta OMe of the Lewis(X) family has been determined by NMR spectroscopy in dimethyl sulfoxide and methanol. In these solvents, the binding constants with calcium have been evaluated as 9.5 and 29.6 M-1, respectively. Study of the interaction sites has been achieved through the use of paramagnetic divalent cations and distance triangulation methods. Two regions have been found, the first one in the vicinity of the fucose unit, the second one closer to the lactose part.     

Carbohydrate binding properties of complex-type oligosaccharides on immobilized Datura stramonium lectin

The carbohydrate binding specificity of Datura stramonium agglutinin was studied by analyzing the behavior of a variety of complex-type oligosaccharides on a D. Stramonium agglutinin-Sepharose column. Oligosaccharides which contain Gal beta 1----4GlcNAc-beta 1----4(Gal beta 1----GlcNAc beta 1----2)Man units are retarded in the column so long as the pentasaccharide unit is not substituted by other sugars. Oligosaccharides which contain unsubstituted Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----4GlcNAc beta 1----2)Man groups and those in which there is at least one Gal beta 1----4GlcNAc repeating unit present on an outer chain bind to the column and are eluted with buffer containing N-acetylglucosamine oligomers. Binding was not affected by the inner core portion of complex oligosaccharides nor by the presence of a bisecting N-acetylglucosamine residue. With these principles in mind, the column can be used as an effective tool for the analysis of complex-type, asparagine-linked sugar chains.     

Characterization of the binding of propionibacterium granulosum to glycosphingolipids adsorbed on surfaces. An apparent recognition of lactose which is dependent on the ceramide structure

The binding properties of a strain of Propionibacterium granulosum derived from human skin was investigated with reference to glycosphingolipids separated on thin layer chromatograms or coated in microtiter wells using externally (125I) and metabolically [( 35S]methionine) labeled bacteria. Binding was found to lactosylceramide (LacCer; Gal beta 1-4Glc beta 1-Cer) but not to glycolipids lacking the lactose sequence (i.e. Glc beta 1-Cer, Gal beta 1-Cer or Gal alpha 1-4Gal beta 1-Cer). In microtiter wells, binding occurred at 50 ng and became half-maximal at the theoretical value for a monomolecular layer of LacCer (i.e. 100-200 ng/well). The bacteria also bound to glycolipids with various substitutions (e.g. GalNAc beta 1-4, Gal beta 1-3GalNAc beta 1-4, Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4, Gal alpha 1-3, GlcNAc beta 1-3, Gal beta 1-3GlcNAc beta 1-3, Gal beta 1-4GlcNAc beta 1-3, and Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3) at the Gal of LacCer, although only those species with GalNAc beta 1-4 or Gal beta 1-3GalNAc beta 1-4 were as active as LacCer itself. Glycolipids with other additions (e.g. Gal alpha 1-4 and NeuAc alpha 2-3) were negative. For unsubstituted LacCer, the binding required either a trihydroxy base or 2-hydroxy fatty acid, specifying the epithelial type of ceramide; LacCer composed of a dihydroxy base and nonhydroxy fatty acid was negative. This is interpreted as indicating that the proper presentation of the binding epitope depends on the ceramide structure. The relevance of this to biological membranes has not yet been established. Neither free lactose (up to 20 mg/ml) nor lactose-bovine serum albumin (5 mg/ml) prevented the binding of bacteria to LacCer, two facts that support the solid-phase binding data demonstrating a low affinity binding and the crucial importance of a particular lactose epitope.     

Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillus circulans

The enzymatic access to nucleotide-activated oligosaccharides by a glycosidase-catalyzed transglycosylation reaction was explored. The nucleotide sugars UDP-GlcNAc and UDP-Glc were tested as acceptor substrates for beta-galactosidase from Bacillus circulans using lactose as donor substrate. The UDP-disaccharides Gal(beta1-4)GlcNAc(alpha1-UDP) (UDP-LacNAc) and Gal(beta1-4)Glc(alpha1-UDP) (UDP-Lac) and the UDP-trisaccharides Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP and Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP) were formed stereo- and regioselectively. Their chemical structures were characterized by 1H and 13C NMR spectroscopy and fast atom bombardment mass spectrometry. The synthesis in frozen solution at -5 degrees C instead of 30 degrees C gave significantly higher product yields with respect to the acceptor substrates. This was due to a remarkably higher product stability in the small liquid phase of the frozen reaction mixture. Under optimized conditions, at -5 degrees C and pH 4.5 with 500 mM lactose and 100 mM UDP-GlcNAc, an overall yield of 8.2% (81.8 micromol, 62.8 mg with 100% purity) for Gal(beta1-4)GlcNAc(alpha1-UDP) and 3.6% (36.1 micromol, 35 mg with 96% purity) for Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP) was obtained. UDP-Glc as acceptor gave an overall yield of 5.0% (41.3 micromol, 32.3 mg with 93% purity) for Gal(beta1-4)Glc(alpha1-UDP) and 1.6% (13.0 micromol, 12.2 mg with 95% purity) for Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP). The analysis of other nucleotide sugars revealed UDP-Gal, UDP-GalNAc, UDP-Xyl and dTDP-, CDP-, ADP- and GDP-Glc as further acceptor substrates for beta-galactosidase from Bacillus circulans.     

Studies of oligosaccharide specificity of perch roe fucolectin,using gold labeled neoglycoproteins

The specificity of perch (Perca fluviatilis) roe fucolectin was studied using the protein dot blot technique, followed by detection with colloidal gold-labeled neoglycoproteins bearing human milk polysaccharides. The strongest binding was noted with the H type 1 pentasaccharide lacto-N-fucopentaose (LNFP I, Fuc alpha 1-2 Gal beta 1-3 GlcNAc beta 1-3 Gal beta 1-4Glc); the interaction with the H type 6 trisaccharide 2'-fucosyllactose (2-FL, Fuc alpha 1-2 Gal beta 1-4 Glc) was weaker. Binding of the perch lectin to the Lewis antigens (associated with tumors and embryonic tissues) was also studied. It was found that the lectin weakly interacted with the hexasaccharide lacto-N-difucohexaose I, Fuc alpha 1-2 Gal beta 1-3[Fuc alpha 1-4]GlcNac beta 1-3 Gal beta 1-4 Glc), but not with Lea, Lec, Lex antigens. Thus, perch roe lectin exhibited pronounced differences in carbohydrate specificity from other fucolectins--a feature that may be used in structural studies and isolation of fucose-containing glycoconjugates.     

Thermodynamic analysis of porphyrin binding to Momordica charantia (bitter gourd) lectin.

Owing to the use of porphyrins in photodynamic therapy for the treatment of malignant tumors, and the preferential interaction of lectins with tumor cells, studies on lectin-porphyrin interaction are of significant interest. In this study, the interaction of several free-base and metalloporphyrins with Momordica charantia (bitter gourd) lectin (MCL) was investigated by absorption spectroscopy. Difference absorption spectra revealed that significant changes occur in the Soret band region of the porphyrins on binding to MCL. These changes were monitored to obtain association constants (Ka) and stoichiometry of binding. The tetrameric MCL binds four porphyrin molecules, and the stoichiometry was unaffected by the presence of the specific sugar, lactose. In addition, the agglutination activity of MCL was unaffected by the presence of the porphyrins used in this study, clearly indicating that porphyrin and carbohydrate ligands bind at different sites. Both cationic and anionic porphyrins bind to the lectin with comparable affinity (Ka =10(3)-10(5) m(-1)). The thermodynamic parameters associated with the interaction of several porphyrins, obtained from the temperature dependence of the Ka values, were found to be in the range: DeltaH degrees = -98.1 to -54.4 kJ.mol(-1) and DeltaS degrees =-243.9 to -90.8 J.mol(-1).K(-1). These results indicate that porphyrin binding to MCL is governed by enthalpic forces and that the contribution from binding entropy is negative. Enthalpy-entropy compensation was observed in the interaction of different porphyrins with MCL, underscoring the role of water structure in the overall binding process. Analysis of CD spectra of MCL indicates that this protein contains about 13%alpha-helix, 36%beta-sheet, 21%beta-turn, and the rest unordered structures. Binding of porphyrins does not significantly alter the secondary and tertiary structures of MCL.     

Globotetraosylceramide is recognized by the pig edema disease toxin

The pig edema disease toxin has been shown by a tlc glycolipid binding assay to bind specifically to globotetraosylceramide (Gb4, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer.). Binding was reduced for globotriosylceramide (Gb3, Gal alpha 1-4Gal beta 1-4GlcCer) and more markedly for the Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer). Paragloboside, blood group A glycolipids, glycolipids terminating in Gal NAc beta 1-4Gal-, and glycolipids in which globoside was present as an internal sequence did not bind the toxin. Isogloboside (GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4GlcCer) was efficiently recognized. This toxin is genetically related to the verotoxin (or Shiga-like) family of toxins for which Gb3 has been shown to be the receptor. The difference in susceptibility of cell lines to the cytotoxicity of the pig edema disease toxin and the Shiga and Shiga-like toxins is consistent with the difference in receptor glycolipid binding.     

Enthalpic barriers to the hydrophobic binding of oligosaccharides to phage P22 tailspike protein

The structural thermodynamics of the recognition of complex carbohydrates by proteins are not well understood. The recognition of O-antigen polysaccharide by phage P22 tailspike protein is a highly suitable model for advancing knowledge in this field. The binding to octa- and dodecasaccharides derived from Salmonella enteritidis O-antigen was studied by isothermal titration calorimetry and stopped-flow spectrofluorimetry. At room temperature, the binding reaction is enthalpically driven with an unfavorable change in entropy. A large change of -1.8 +/- 0.2 kJ mol(-1) K(-1) in heat capacity suggests that the hydrophobic effect and water reorganization contribute substantially to complex formation. As expected from the large heat-capacity change, we found enthalpy-entropy compensation. The calorimetrically measured binding enthalpies were identical within error to van't Hoff enthalpies determined from fluorescence titrations. Binding kinetics were determined at temperatures ranging from 10 to 30 degrees C. The second-order association rate constant varied from 1 x 10(5) M(-1) s(-1) for dodecasaccharide at 10 degrees C to 7 x 10(5) M(-1) s(-1) for octasaccharide at 30 degrees C. The first-order dissociation rate constants ranged from 0.2 to 3.8 s(-1). The Arrhenius activation energies were close to 50 and 100 kJ mol(-1) for the association and dissociation reactions, respectively, indicating mainly enthalpic barriers. Despite the fact that this system is quite complex due to the flexibility of the saccharide, both the thermodynamic and kinetic data are compatible with a simple one-step binding model.