Crystal structure of the jacalin-T-antigen complex and a comparative study of lectin-T-antigen complexes

Thomsen-Friedenreich antigen (Galbeta1-3GalNAc), generally known as T-antigen, is expressed in more than 85% of human carcinomas. Therefore, proteins which specifically bind T-antigen have potential diagnostic value. Jacalin, a lectin from jack fruit (Artocarpus integrifolia) seeds, is a tetramer of molecular mass 66kDa. It is one of the very few proteins which are known to bind T-antigen. The crystal structure of the jacalin-T-antigen complex has been determined at 1.62A resolution. The interactions of the disaccharide at the binding site are predominantly through the GalNAc moiety, with Gal interacting only through water molecules. They include a hydrogen bond between the anomeric oxygen of GalNAc and the pi electrons of an aromatic side-chain. Several intermolecular interactions involving the bound carbohydrate contribute to the stability of the crystal structure. The present structure, along with that of the Me-alpha-Gal complex, provides a reasonable qualitative explanation for the known affinities of jacalin to different carbohydrate ligands and a plausible model of the binding of the lectin to T-antigen O-linked to seryl or threonyl residues. Including the present one, the structures of five lectin-T-antigen complexes are available. GalNAc occupies the primary binding site in three of them, while Gal occupies the site in two. The choice appears to be related to the ability of the lectin to bind sialylated sugars. In either case, most of the lectin-disaccharide interactions are at the primary binding site. The conformation of T-antigen in the five complexes is nearly the same.     

Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcβ1-3Gal/GalNAc-containing and Gal/GalNAcβ1-4GlcNAcβ1-6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcβ1-3Gal, and Galβ1-3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K_d = 3 × 10^− 4 M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.     

Carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus

The seed lectin (DBL) from the leguminous plant Dolichos biflorus has a unique specificity among the members of the legume lectin family because of its high preference for GalNAc over Gal. In addition, precipitation of blood group A+H substance by DBL is slightly better inhibited by a blood group A trisaccharide (GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal) containing pentasaccharide, and about 40 times better by the Forssman disaccharide (GalNAc(alpha1-3)GalNAc) than by GalNAc. We report the crystal structures of the DBL-blood group A trisaccharide complex and the DBL-Forssman disaccharide complex.A comparison with the binding sites of Gal-binding legume lectins indicates that the low affinity of DBL for Gal is due to the substitution of a conserved aromatic residue by an aliphatic residue (Leu127). Binding studies with a Leu127Phe mutant corroborate these conclusions. DBL has a higher affinity for GalNAc because the N-acetyl group compensates for the loss of aromatic stacking in DBL by making a hydrogen bond with the backbone amide group of Gly103 and a hydrophobic contact with the side-chains of Trp132 and Tyr104.Some legume lectins possess a hydrophobic binding site that binds adenine and adenine-derived plant hormones, i.e. cytokinins. The exact function of this binding site is unknown, but adenine/cytokinin-binding legume lectins might be involved in storage of plant hormones or plant growth regulation. The structures of DBL in complex with adenine and of the dimeric stem and leaf lectin (DB58) from the same plant provide the first structural data on these binding sites. Both oligomers possess an unusual architecture, featuring an alpha-helix sandwiched between two monomers. In both oligomers, this alpha-helix is directly involved in the formation of the hydrophobic binding site. DB58 adopts a novel quaternary structure, related to the quaternary structure of the DBL heterotetramer, and brings the number of know legume lectin dimer types to four.     

Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I

The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for alpha-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward alphaGal1-4betaGal-terminating structures and weaker binding to alphaGal1-3betaGal ones in order to determine which human glycoconjugates could play a role in the carbohydrate-mediated adhesion of the bacteria. This was confirmed in vivo by testing the binding of the lectin to Burkitt lymphoma cells that present large amounts of globotriaosylceramide antigen Gb3/CD77/P(k). Trisaccharide moieties of Gb3 (alphaGal1-4betaGal1-4Glc) and isoglobotriaosylceramide (alphaGal1-3betaGal1-4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to alphaGal1-3betaGal1-4Glc trisaccharide has been solved at 1.9-A resolution and revealed how the second galactose residue makes specific contacts with the protein surface. Molecular modeling studies were performed in order to compare the binding mode of PA-IL toward alphaGal1-3Gal with that toward alphaGal1-4Gal. Docking studies demonstrated that alphaGal1-4Gal creates another network of contacts for achieving a very similar affinity, and 10-ns molecular dynamics in explicit water allowed for analyzing the flexibility of each disaccharide ligand in the protein binding site. The higher affinity observed for binding to Gb3 epitope, both in vivo and on glycan array, is likely related to the presentation effect of the oligosaccharide on a surface, since only the Gb3 glycosphingolipid geometry is fully compatible with parallel insertion of neighboring trisaccharide heads in two binding sites of the same tetramer of PA-IL.     

Proteomic analysis of Gal/GalNAc lectin-associated proteins in Entamoeba histolytica

Contact-dependent killing and phagocytosis of target cells by Entamoeba histolytica trophozoites is mediated by the galactose (Gal) and N-acetyl-d-galactosamine (GalNAc)-inhibitable lectin. Previous work has suggested that this lectin functions as part of a signal transduction complex. To identify proteins that might be part of this complex, amebic trophozoites were bound to GalNAc-BSA-labeled magnetic beads and lysed. Bound proteins were eluted from the beads and analyzed by tandem mass spectrometry. Along with the Gal/GalNAc lectin subunits, several cytoskeletal proteins, potential signaling proteins, and a novel transmembrane protein, consistently purified with the GalNAc-BSA beads.     

Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra

Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional X-ray structure of SNA-II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 A and 1.35 A, respectively. In both crystal forms, the SNA-II molecule folds into two linked beta-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is observed at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resolution structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, alpha1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were determined at 1.55 A resolution or better. Binding is observed in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, additional interactions with the protein involve the O6 atom. Analytical gel filtration, small angle X-ray scattering studies and crystal packing analysis indicate that, although some oligomeric species are present, the monomeric species predominate in solution.     

Architecture of the sugar binding sites in carbohydrate binding proteins-a computer modeling study

Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity.     

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.     

Structural basis for langerin recognition of diverse pathogen and mammalian glycans through a single binding site

Langerin mediates the carbohydrate-dependent uptake of pathogens by Langerhans cells in the first step of antigen presentation to the adaptive immune system. Langerin binds to an unusually diverse number of endogenous and pathogenic cell surface carbohydrates, including mannose-containing O-specific polysaccharides derived from bacterial lipopolysaccharides identified here by probing a microarray of bacterial polysaccharides. Crystal structures of the carbohydrate-recognition domain from human langerin bound to a series of oligomannose compounds, the blood group B antigen, and a fragment of β-glucan reveal binding to mannose, fucose, and glucose residues by Ca²⁺ coordination of vicinal hydroxyl groups with similar stereochemistry. Oligomannose compounds bind through a single mannose residue, with no other mannose residues contacting the protein directly. There is no evidence for a second Ca²⁺-independent binding site. Likewise, a β-glucan fragment, Glcβ1-3Glcβ1-3Glc, binds to langerin through the interaction of a single glucose residue with the Ca²⁺ site. The fucose moiety of the blood group B trisaccharide Galα1-3(Fucα1-2)Gal also binds to the Ca²⁺ site, and selective binding to this glycan compared to other fucose-containing oligosaccharides results from additional favorable interactions of the nonreducing terminal galactose, as well as of the fucose residue. Surprisingly, the equatorial 3-OH group and the axial 4-OH group of the galactose residue in 6SO₄-Galβ1-4GlcNAc also coordinate Ca²⁺, a heretofore unobserved mode of galactose binding in a C-type carbohydrate-recognition domain bearing the Glu-Pro-Asn signature motif characteristic of mannose binding sites. Salt bridges between the sulfate group and two lysine residues appear to compensate for the nonoptimal binding of galactose at this site.     

Isolation and characterisation of a Salvia bogotensis seed lectin specific for the Tn antigen

A lectin was isolated and characterised from Salvia bogotensis seeds. Removal of the abundant pigments and polysaccharides, which are present in seeds, was an essential step in its purification. Several procedures were assayed and the best suited, including Pectinex treatment, DEAE-cellulose and affinity chromatography, led to a protein being obtained amounting to 18-20mg/100g seeds having high specific agglutination activity (SAA). The lectin specifically agglutinated human Tn erythrocytes and was inhibited by 37mM GalNAc, 0.019mM ovine submaxillary mucin (OSM) or 0.008mM asialo bovine submaxillary mucin (aBSM). Enzyme-linked lectinosorbent assay (ELLSA) revealed strong binding to aOSM and aBSM, corroborating Tn specificity, whereas no binding to fetuin or asialo fetuin was observed. The lectin's monomer MW (38,702Da), amino acid composition, pI, carbohydrate content, deglycosylated form MW, thermal stability and Ca(2+) and Mn(2+) requirements were determined. Evidence of the existence of two glycoforms was obtained. The lectin's specificity and high affinity for the Tn antigen, commonly found in tumour cells, makes this protein a useful tool for immunohistochemical and cellular studies.     

Recognition profile of Bauhinia purpurea agglutinin (BPA)

Bauhinia purpurea agglutinin (BPA) is a Galbeta1-3GalNAc (T) specific leguminous lectin that has been widely used in multifarious cytochemical and immunological studies of cells and tissues under pathological or malignant conditions. Despite these diverse applications, knowledge of its carbohydrate specificity was mainly limited to molecular or submolecular T disaccharides. Thus, the requirement of high density polyvalent or multi-antennary carbohydrate structural units for BPA binding and an updated affinity profile were further evaluated by enzyme-linked lectinosorbent (ELLSA) and inhibition assays. Among the glycoproteins (gps) tested and expressed as 50% nanogram inhibition, the high density polyvalent GalNAcalpha1-Ser/Thr (Tn) and Galbeta1-3/4GlcNAc (I/II) glycotopes present on macromolecules generated a great enhancement of binding affinity for BPA as compared to their monomers. The most potent inhibitors were a Tn-containing gp (asialo OSM) and a I/II containing gp (human blood group precursor gp), which were up to 1.7 x 10(4) and 2.3 x 10(3) times more potent than monovalent Gal and GalNAc, respectively. However, multi-antennary glycopeptides, such as tri-antennary Galbeta1-4GlcNAc, which was slightly more active than II or Gal, gave only a minor contribution. Regarding the carbohydrate structural units studied by the inhibition assay, blood group GalNAcbeta1-3/4Gal (P/S) active glycotopes were active ligands. The overall binding profile of BPA was: high density polyvalent T/Tn and II clusters > Tn-glycopeptides (M.W. <3.0 x 10(3))/Talpha monomer > monovalent P/S > Tn monomer and GalNAc > tri-antennary II > Gal > Man and Glc (inactive). These findings give evidence for the binding of this lectin to dense cell surface T, Tn and I/II glycoconjugates and should facilitate future usage of this lectin in biotechnological and medical applications.     

Enhanced self-association of mucins possessing the T and Tn carbohydrate cancer antigens at the single-molecule level

Mucins are linear O-glycosylated glycoproteins involved in inflammation, cell adhesion, and tumorigenesis. Cancer-associated mucins often possess increased expression of the T (Galβ1,3GalNAcαThr/Ser) and Tn (GalNAcαThr/Ser) cancer antigens, which are diagnostic markers for several cancers, including colon cancer. We have used AFM based single-molecule forced unbinding under near physiological conditions to investigate the self-interactions between porcine submaxillary mucin (PSM) as well as between PSM analogs possessing various carbohydrates including the T- and Tn-antigen. Distributions of unbinding forces and corresponding force loading rates were determined for force loading rates from 0.18 nN/s to 39 nN/s, and processed to yield most probable unbinding forces f* and lifetimes of the interactions. Parameter f* varied in the range 27 to 50 pN at force loading rates of about 2 nN/s among the various mucins. All mucin samples investigated showed self-interaction, but the tendency was greatest for PSM displaying only the Tn-antigen (Tn-PSM) or a mixture of Tn-, T-antigen, and the trisaccharide Fucα1,2Galβ1,3GalNAc (Tri-PSM). Weaker self-interactions were observed for native PSM (Fd-PSM), which consists of a nearly equal mixture of the longer core 1 blood group A tetrasaccharide (GalNAcα1,3(Fucα1,2)Galβ1,3GalNAcαSer/Thr) and Tn-antigen. The data are consistent with the truncated Tn and T glycans enhancing self-interaction of the mucins. These carbohydrate cancer antigens may, thus, play an active role in the disease by constitutively activating mucin and mucin-type receptors by self-association on cells.     

Crystal Structure of the GalNAc/Gal-Specific Agglutinin from the Phytopathogenic Ascomycete Sclerotinia sclerotiorum Reveals Novel Adaptation of a β-Trefoil Domain

A lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum that shares only weak sequence similarity with characterized fungal lectins has recently been identified. S. sclerotiorum agglutinin (SSA) is a homodimeric protein consisting of two identical subunits of ∼ 17 kDa and displays specificity primarily towards Gal/GalNAc. Glycan array screening indicates that SSA readily interacts with Gal/GalNAc-bearing glycan chains. The crystal structures of SSA in the ligand-free form and in complex with the Gal-β1,3-GalNAc (T-antigen) disaccharide have been determined at 1.6 and 1.97 Å resolution, respectively. SSA adopts a β-trefoil domain as previously identified for other carbohydrate-binding proteins of the ricin B-like lectin superfamily and accommodates terminal non-reducing galactosyl and N-acetylgalactosaminyl glycans. Unlike other structurally related lectins, SSA contains a single carbohydrate-binding site at site α. SSA reveals a novel dimeric assembly markedly dissimilar to those described earlier for ricin-type lectins. The present structure exemplifies the adaptability of the β-trefoil domain in the evolution of fungal lectins.     

Crystal structure of a glutamate/aspartate binding protein complexed with a glutamate molecule: structural basis of ligand specificity at atomic resolution

The crystal structure of a periplasmic l-aspartate/l-glutamate binding protein (DEBP) from Shigella flexneri complexed with an l-glutamate molecule has been determined and refined to an atomic resolution of 1.0 Å. There are two DEBP molecules in the asymmetric unit. The refined model contains 4462 non-hydrogen protein atoms, 730 water molecules, 2 bound glutamate molecules, and 2 Tris molecules from the buffer used in crystallization. The final R_cryst and R_free factors are 13.61% and 16.89%, respectively. The structure has root-mean-square deviations of 0.016 Å from standard bond lengths and 2.35° from standard bond angles.The DEBP molecule is composed of two similarly folded domains separated by the ligand binding region. Both domains contain a central five-stranded β-sheet that is surrounded by several α-helices. The two domains are linked by two antiparallel β-strands. The overall shape of DEBP is that of an ellipsoid approximately 55 Å × 45 Å × 40 Å in size.The binding of ligand to DEBP is achieved mostly through hydrogen bonds between the glutamate and side-chain and main-chain groups of DEBP. Side chains of residues Arg24, Ser72, Arg75, Ser90, and His164 anchor the deprotonated γ-carboxylate group of the glutamate with six hydrogen bonds. Side chains of Arg75 and Arg90 form salt bridges with the deprotonated α-carboxylate group, while the main-chain amide groups of Thr92 and Thr140 form hydrogen bonds with the same group. The positively charged α-amino group of the l-glutamate forms salt bridge interaction with the side-chain carboxylate group of Asp182 and hydrogen bond interaction with main-chain carbonyl oxygen of Ser90. In addition to these hydrogen bond and electrostatic interactions, other interactions may also play important roles. For example, the two methylene groups from the glutamate form van der Waals interactions with hydrophobic side chains of DEBP.Comparisons with several other periplasmic amino acid binding proteins indicate that DEBP residues involved in the binding of α-amino and α-carboxylate groups of the ligand and the pattern of hydrogen bond formation between these groups are very well conserved, but the binding pocket around the ligand side chain is not, leading to the specificity of DEBP. We have identified structural features of DEBP that determine its ability of binding glutamate and aspartate, two molecules with different sizes, but discriminating against very similar glutamine and asparagine molecules.     

Interactions of the fucose-specific Pseudomonas aeruginosa lectin, PA-IIL, with mammalian glycoconjugates bearing polyvalent Lewis(a) and ABH blood group glycotopes

Pseudomonas aeruginosa Fuc > Man specific lectin, PA-IIL, is an important microbial agglutinin that might be involved in P. aeruginosa infections in humans. In order to delineate the structures of these lectin receptors, its detailed carbohydrate recognition profile was studied both by microtiter plate biotin/avidin-mediated enzyme-lectin-glycan binding assay (ELLSA) and by inhibition of the lectin-glycan interaction. Among 40 glycans tested for binding, PA-IIL reacted well with all human blood group ABH and Le(a)/Le(b) active glycoproteins (gps), but weakly or not at all with their precursor gps and N-linked gps. Among the sugar ligands tested by the inhibition assay, the Le(a) pentasaccharide lacto-N-fucopentaose II (LNFP II, Galbeta1-3[Fucalpha1-4]GlcNAcbeta1-3Galbeta1-4Glc) was the most potent one, being 10 and 38 times more active than the Le(x) pentasaccharide (LNFP III, Galbeta1-4 [Fucalpha1-3]GlcNAcbeta1-3Galbeta1-4Glc) and sialyl Le(x) (Neu5Acalpha2-3Galbeta1-4[Fucalpha1-3] GlcNAc), respectively. It was 120 times more active than Man, while Gal and GalNAc were inactive. The decreasing order of PA-IIL affinity for the oligosaccharides tested was: Le(a) pentaose > or = sialyl Le(a) tetraose > methyl alphaFuc > Fuc and Fucalpha1-2Gal (H disaccharide)>2'-fucosyllactose (H trisaccharide), Le(x) pentaose, Le(b) hexaose (LNDFH I) and gluco-analogue of Le(y) tetraose (LDFT)>H type I determinant (LNFP I)>Le(x) trisaccharide (Galbeta1-4[Fucalpha1-3]GlcNAc) > sialyl Le(x) trisaccharide > Man > Gal, GalNAc, and Glc (inactive). The results presented here, in accordance with the crystal 3D structural data, imply that the combining site of PA-IIL is a small cavity-type best fitting Fucalpha1- with a specific shallow groove subsite for the remainder part of the Le(a) saccharides, and that polyvalent glycotopes enhance the reactivity. The Fuc > Man Ralstonia solanacearum lectin RSL, which resembles PA-IIL in sugar specificity, differs from it in it's better fit to the B and A followed by H oligosaccharides vs. Fuc, whereas, the second R. solanacearum lectin RS-IIL (the structural homologue of PA-IIL) binds Man > Fuc. These results provide a valuable information on PA-IIL interactions with mammalian glycoforms and the possible spectrum of attachment sites for the homing of this aggressive bacterium onto the target molecules. Such information might be useful for the antiadhesive therapy of P. aeruginosa infections.     

Presence of beta-linked GalNAc residues on N-glycans of human thyroglobulin

Hepatic asialoglycoprotein receptor, which may mediate the clearance of circulating thyroglobulin, is known to have a high affinity for GalNAc. Recently, the receptor has been reported to be present also in the thyroid, implicating interaction with thyroglobulin. Here, mammalian thyroglobulins were analyzed for GalNAc termini by Western blotting with GalNAc-recognizing lectins labeled with peroxidase or (125)I. Wistaria floribunda lectin was found to bind human thyroglobulin and, to some extent, bovine, but not porcine thyroglobulin. After desialylation, the lectin bound all of the thyroglobulins tested. The binding was inhibited by competitive inhibitor GalNAc. Peptide N-glycanase treatment of human desialylated thyroglobulin resulted in the complete loss of reactivity with W. floribunda lectin, indicating that the binding sites are exclusively on N-glycans. The binding sites on human desialylated thyroglobulin were partly sensitive to beta-galactosidase, and the remainder was essentially sensitive to beta-N-acetylhexosaminidase. On the other hand, the binding sites of bovine and porcine desialylated thyroglobulins were totally sensitive to beta-galactosidase. Thus the lectin binds beta-Gal termini, as well as beta-GalNAc. GalNAc-specific Dolichos biflorus lectin also bound human thyroglobulin weakly. In contrast to W. floribunda lectin, desialylation diminished binding, suggesting that these two lectins recognize different GalNAc-terminated structures. Again, the binding was inhibited by GalNAc and by treatment with peptide N-glycanase. These results strongly indicate the presence of distinct GalNAc termini of N-glycans on human thyroglobulin.     

Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site

Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins.The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework.     

Purification,characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells

Background

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.

Methods

Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.

Results

A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.

Conclusions

The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.

General significance

CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.     

Fine carbohydrate recognition of Euphorbia milii lectin

Glycans are key structures involved in biological processes such as cell attachment, migration, and invasion. Information coded on cell-surface glycans is frequently deciphered by proteins, as lectins, that recognize specific carbohydrate topology. Here, we describe the fine carbohydrate specificity of Euphorbia milii lectin (EML). Competitive assays using various sugars showed that GalNAc was the strongest inhibitor, and that the hydroxyl axial position of C4 and acetamido on C2 of GalNAc are critical points of EML recognition. A hydrophobic locus adjacent to GalNAc is also an important region for EML binding. Direct binding assays of EML revealed a stereochemical requirement for a structure adjacent to terminal GalNAc, showing that GalNAc residue is a necessary but not sufficient condition for EML interaction. The capacity of EML to bind epithelial tumor cells makes it a potentially useful tool for study of some over-expressed GalNAc glycoconjugates.     

Structural basis of the carbohydrate specificities of jacalin: an X-ray and modeling study

The structures of the complexes of tetrameric jacalin with Gal, Me-alpha-GalNAc, Me-alpha-T-antigen, GalNAcbeta1-3Gal-alpha-O-Me and Galalpha1-6Glc (mellibiose) show that the sugar-binding site of jacalin has three components: the primary site, secondary site A, and secondary site B. In these structures and in the two structures reported earlier, Gal or GalNAc occupy the primary site with the anomeric carbon pointing towards secondary site A. The alpha-substituents, when present, interact, primarily hydrophobically, with secondary site A which has variable geometry. O-H..., centered pi and C-H...pi hydrogen bonds involving this site also exist. On the other hand, beta-substitution leads to severe steric clashes. Therefore, in complexes involving beta-linked disaccharides, the reducing sugar binds at the primary site with the non-reducing end located at secondary site B. The interactions at secondary site B are primarily through water bridges. Thus, the nature of the linkage determines the mode of the association of the sugar with jacalin. The interactions observed in the crystal structures and modeling based on them provide a satisfactory qualitative explanation of the available thermodynamic data on jacalin-carbohydrate interactions. They also lead to fresh insights into the nature of the binding of glycoproteins by jacalin.