Identification of amino groups in the carbohydrate binding activity of winged bean acidic agglutinin

Chemical modification studies reveal that the modification of amino groups in WBA II leads to a complete loss in the hemagglutinating and saccharide binding activities. Since WBA II is a dimeric molecule and contains two binding sites, one amino group in each of the binding sites is inferred to be essential for its activity. The presence of amino group which has a potential to form hydrogen bonded interactions with the ligand, substantiates our observation regarding the forces involved in WBA II-receptor and WBA II-simple sugar interactions.

Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus)

A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha- conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N- dansylgalactosamine and the lectin are consistent with a simple one- step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(- 2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.

Energetics of carbohydrate binding by a 14 kDa S-type mammalian lectin

The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.

Carbohydrate binding specificity of the B-cell maturation mitogen from Artocarpus integrifolia seeds

Artocarpin, a mannose-specific lectin, is a homotetrameric protein (M(r) 65,000) devoid of covalently attached carbohydrates and consists of four isolectins with pI in the range 5-6.5. Investigations of its carbohydrate binding specificity reveal that among monosaccharides, mannose is preferred over glucose. Among mannooligosaccharides, mannotriose (Man alpha 1-3[Man alpha 1-6]Man) and mannopentaose are the strongest ligands followed by Man alpha 1-3Man. Extension of these ligands by GlcNAc at the reducing ends of mannooligosaccharides tested remarkably improves their inhibitory potencies, while substitution of both the alpha 1-3 and alpha 1-6 mannosyl residues of mannotriose and the core pentasaccharide of N-linked glycans (Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc) by GlcNAc or N-acetyllactosamine in beta 1-2 linkage diminishes their inhibitory potencies. Sialylated oligosaccharides are non-inhibitory. Moreover, the substitution of either alpha 1-3 or alpha 1-6 linked mannosyl residues of M5Gn or both by mannose in alpha 1-2 linkage leads to a considerable reduction of their inhibitory power. Addition of a xylose residue in beta 1-2 linkage to the core pentasaccharide improves the inhibitory activity. Considering the fact that artocarpin has the strongest affinity for the xylose containing hepasaccharide from horseradish peroxidase, which differs significantly from all the mannose/glucose-specific lectins, it should prove a useful tool for the isolation and characterization of glycoproteins displaying such structure.

Carbohydrate binding specificity of the basic lectin from winged bean (Psophocarpus tetragonolobus)

The carbohydrate binding specificity of the basic lectin from winged bean (Psophocarpus tetragonolobus) was investigated by quantitative precipitin analysis using blood group A, B, H, Le and I substances and by precipitation inhibition with various mono- and oligosaccharides. The lectin precipitated best with A1 substances and moderately with B and A2 substances, but not with H or Le substances. Inhibition assays of lectin-blood group A1 precipitation demonstration that A substance-derived oligosaccharides having the common structure: d-Ga1NAcα(1 → 3)d-Gal-(β1 → Image ) to a d-Glc, were the best inhibitors and about 8 and 4 times more active than d-Ga1NAc and d-Ga1NAcα(1 → 3)d-Ga1, respectively. A difucosyl A-specific oligosaccharide (A-penta), a monofucosyl (A-tetra) and a non-fucosyl containing (A5 II) oligosaccharide, d-Ga1NAcα(1 → 3)d-Ga1β(1 → 3)d-G1cNAc, had almost the same reactivity, suggesting that the fucose linked to the sub-terminal d-Ga1 or to the third sugar, d-GlcNAc, from the non-reducing end made no contribution to the carbohydrate binding. Although a terminal non-reducing d-Ga1NAc or d-Ga1 residue was indispensible for binding, the lectin bound not only to these terminal non-reducing galactopyranosyl residues, but also showed increased binding to oligosaccharides in which it was bonded to a sub-terminal d-Ga1 joined to a d-GlcNAc residue, as in blood group A or B substances. This defines the site, thus far, as complementary to a disaccharide plus the β linkage to the third sugar (d-Glc or d-GlcNAc) from the non-reducing end. The role of the β(1 → 3) or β(1 → 4) linkage of the sub-terminal non-reducing d-Gal to the d-GlcNAc requires further study.

Influence of glycosidic linkage on the nature of carbohydrate binding in beta-prism I fold lectins: An X-ray and molecular dynamics investigation on banana lectin-carbohydrate complexes

The three crystal structures reported here provide details of the interactions of mannose and the mannosyl-alpha-1,3-mannose component of a pentamannose with banana lectin and evidence for the binding of glucosyl-alpha-1,2-glucose to the lectin. The known structures involving the lectin include a complex with glucosyl-beta-1,3-glucose. Modeling studies on the three disaccharide complexes with the reducing end and the nonreducing end at the primary binding site are also provided here. The results of the Xray and modeling studies show that the disaccharides with an alpha-1,3 linkage prefer to have the nonreducing end at the primary binding site, whereas the reducing end is preferred at the site when the linkage is beta-1,3 in mannose/glucose-specific beta-prism I fold lectins. In the corresponding galactose-specific lectins, however, alpha-1,3-linked disaccharides cannot bind the lectin with the nonreducing end at the primary binding site on account of steric clashes with an aromatic residue that occurs only when the lectin is galactose-specific. Molecular dynamics simulations based on the known structures involving banana lectin enrich the information on lectin-carbohydrate interactions obtained from crystal structures. They demonstrate that conformational selection as well as induced fit operate when carbohydrates bind to banana lectin.

Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry

A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W-102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W-102) in the activity of the lectin. (c) 2015 IUBMB Life, 67(12):943-953, 2015

Modulation of carbohydrates and carbohydrate binding proteins (lectins) during retinoic acid -induced differentiation of murine embryonal carcinoma cells

The current studies were undertaken to examine the effect of retinoic acid (RA)-induced differentiation of the murine embryonal carcinoma cell line, F-9, on the glycosylation of specific cellular glycoproteins and on the expression of two members of the family of endogenous lactoside-binding lectins. It was found that RA-induced differentiation of these cells into cells with the properties of primitive endoderm results in the increased fucosylation of 3 glycoproteins with molecular weights of 175 (gp175), 250 (gp250), and 400 (pg400) kDa. These three fucose-containing glycoproteins can be considered as new markers of differentiation in this system. The increased fucosylation of these glycoproteins preceded the 3-fold increase in fucosyltransferase (FT) activity that was seen upon RA-induced differentiation of these cells, indicating that an increase in fucosyltransferase activity alone cannot explain the increased fucosylation of these glycoproteins.^ The effect of RA and Ch55, a chalcone carboxylic acid with retinoid-like properties, induced differentiation of a variety of murine embryonal carcinoma cell lines on the activities of both FT and sialyltransferase (ST) was examined. The effect of differentiation on the activities of both glycosyltransferases was modulated and most probably is dependent upon the differentiation pathway that is triggered by the retinoids for each of the embryonal carcinoma cell lines.^ Two glycoproteins, Lysosomal Associated Membrane Glycoproteins 1 and 2 (LAMP-1 and LAMP-2) were examined in more detail during the course of RA-induced differentiation of F-9 cells. Both the levels and glycosylation of both glycoproteins are increased following differentiation of these cells. Differentiation results in the increased binding of $\sp{125}$l-labelled L-phytohemagglutinin to bind to LAMP-1 which indicates increased GlcNAc $\beta$1,6 branching of the oligosaccharide side chains.^ We found that RA-induced differentiation of F-9 cells results in the decreased expression of the 34 kDa lectin 24 h after addition of the retinoid to the medium. Additionally, 48 h of RA-treatment results in the increased expression of the 14.5 kDa lectin. By indirect immunofluorescence we were able to colocalize the 14.5 kDa lectin and laminin which suggests that laminin may be a ligand for the lectin in the F-9 cells. (Abstract shortened with permission of author.) ^

Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II

Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.

Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.

Caracterização biofísica e estrutural da metaloproteinase não-hemorrágica do veneno de Bothrops moojeni e da endo-β-glicanase do Bacillus subtilis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCe15A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.

Genetic and functional characterization of the NanA sialidase from Clostridium chauvoei

ABSTRACT: Clostridium chauvoei is the causative agent of blackleg, a wide spread serious infection of cattle and sheep with high mortality. In this study we have analyzed the sialidase activity of the NanA protein of C. chauvoei and cloned the sialidase gene nanA. Sialidase is encoded as a precursor protein of 722 amino acids with a 26 amino acid signal peptide. The mature sialidase has a calculated molecular mass of 81 kDa and contains the carbohydrate binding module 32 (CBM32, or F5/8 type C domain), the sialic acid binding module CBM40 and the enzymatically active sialidase domain found in all pro- and eukaryotic sialidases. Sialidase activity does not require the CBM32 domain. The NanA protein is secreted by C. chauvoei as a dimer. The nanA gene was found to be conserved and sialidase activity was found in C. chauvoei strains isolated over a period of 50 years from various geographical locations. Antiserum directed against a recombinant 40 kDa peptide containing CBM40 and part of the enzymatically active domain of NanA neutralized the secreted sialidase activity of all C. chauvoei strains tested.