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.     

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.     

Isothermal titration calorimetric studies on the binding of deoxytrimannoside derivatives with artocarpin: implications for a deep-seated combining site in lectins

The carbohydrate binding specificity of the seed lectin from Artocarpus integrifolia, artocarpin, has been elucidated by the enzyme-linked lectin absorbent assay [Misquith, S., et al (1994) J. Biol. Chem. 269, 30393-30401], wherein it was demonstrated to be a Man/Glc specific lectin with high affinity for the trisaccharide present in the core of all N-linked oligosaccharide chains of glycoproteins. As a consequence of this characterization, the binding epitopes of this trisaccharide, 3, 6-di(alpha-D-mannopyranosyl)-D-mannose, for artocarpin were investigated by isothermal titration calorimetry using its monodeoxy as well as Glc and Gal analogues. The thermodynamic data presented here implicate 2-, 3-, 4-, and 6-hydroxyl groups of the alpha(1-3) Man and alpha(1-6) Man residues, and the 2- and 4-OH groups of the central Man residue, in binding to artocarpin. Nevertheless, alpha(1-3) Man is the primary contributor to the binding affinity, unlike other Man/Glc binding lectins which exhibit a preference for alpha(1-6) Man. In addition, unlike the binding reactions of most lectins reported so far, the interaction of mannotriose involves all of its hydroxyl groups with the combining site of the lectin. Moreover, the free energy and enthalpy contributions to binding of individual hydroxyl groups of the trimannoside estimated from the corresponding monodeoxy analogues show nonlinearity, suggesting differential contributions of the solvent and protein to the thermodynamics of binding of the analogues. Thus, this study not only provides evidence for the extended site recognition of artocarpin for the trimannoside epitope but also suggests that its combining site is best described as a deep cleft as opposed to shallow indentations implicated in other lectins.     

Structure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism

The seeds of jack fruit (Artocarpus integrifolia) contain two tetrameric lectins, jacalin and artocarpin. Jacalin was the first lectin found to exhibit the beta-prism I fold, which is characteristic of the Moraceae plant lectin family. Jacalin contains two polypeptide chains produced by a post-translational proteolysis which has been shown to be crucial for generating its specificity for galactose. Artocarpin is a single chain protein with considerable sequence similarity with jacalin. It, however, exhibits many properties different from those of jacalin. In particular, it is specific to mannose. The structures of two crystal forms, form I and form II, of the native lectin have been determined at 2.4 and 2.5 A resolution, respectively. The structure of the lectin complexed with methyl-alpha-mannose, has also been determined at 2.9 A resolution. The structure is similar to jacalin, although differences exist in details. The crystal structures and detailed modelling studies indicate that the following differences between the carbohydrate binding sites of artocarpin and jacalin are responsible for the difference in the specificities of the two lectins. Firstly, artocarpin does not contain, unlike jacalin, an N terminus generated by post-translational proteolysis. Secondly, there is no aromatic residue in the binding site of artocarpin whereas there are four in that of jacalin. A comparison with similar lectins of known structures or sequences, suggests that, in general, stacking interactions with aromatic residues are important for the binding of galactose while such interactions are usually absent in the carbohydrate binding sites of mannose-specific lectins with the beta-prism I fold.     

Crystal structure of Pterocarpus angolensis lectin in complex with glucose,sucrose, and turanose

The crystal structure of the Man/Glc-specific seed lectin from Pterocarpus angolensis was determined in complex with methyl-alpha-d-glucose, sucrose, and turanose. The carbohydrate binding site contains a classic Man/Glc type specificity loop. Its metal binding loop on the other hand is of the long type, different from what is observed in other Man/Glc-specific legume lectins. Glucose binding in the primary binding site is reminiscent of the glucose complexes of concanavalin A and lentil lectin. Sucrose is found to be bound in a conformation similar as seen in the binding site of lentil lectin. A direct hydrogen bond between Ser-137(OG) to Fru(O2) in Pterocarpus angolensis lectin replaces a water-mediated interaction in the equivalent complex of lentil lectin. In the turanose complex, the binding site of the first molecule in the asymmetric unit contains the alphaGlc1-3betaFruf form of furanose while the second molecule contains the alphaGlc1-3betaFrup form in its binding site.     

Survey of immune-related, mannose/fucose-binding C-type lectin receptors reveals widely divergent sugar-binding specificities

C-type lectins (CTLs) are proteins that contain one or more carbohydrate-recognition domains (CRDs) that require calcium for sugar binding and share high degree of sequence homology and tertiary structure. CTLs whose CRD contain EPN (Glu-Pro-Asn) tripeptide motifs have potential to bind mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc) and l-fucose (Fuc), whereas those with QPD (Glu-Pro-Asp) tripeptide motifs bind galactose (Gal) and N-acetylgalactosamine (GalNAc). We report here for the first time a direct comparison of monosaccharide (and some di- and trisaccharides)-binding characteristics of 11 EPX-containing (X = N, S or D) immune-related CTLs using a competition assay and an enzyme-linked immunosorbent assay, and neoglycoproteins as ligand. The EPX CTLs studied are DC-SIGN, L-SIGN, mSIGNR1, human and mouse mannose receptors, Langerin, BDCA-2, DCIR, dectin-2, MCL and MINCLE. We found that: (1) they all bound Man and Fuc; (2) binding of Glc and GlcNAc varied considerably among these lectins, but was always less than Man and Fuc; (3) in general, Gal and GalNAc were not bound. However, dectin-2, DCIR and MINCLE showed ability to bind Gal/GalNAc; (4) DC-SIGN, L-SIGN, mSIGNR1 and Langerin showed enhanced binding of Manα2Man over Man, whereas all others showed no enhancement; (5) DC-SIGN bound Le(x) trisaccharide structure, which has terminal Gal and Fuc residues, more avidly than Fuc, whereas L-SIGN, mSIGNR1, DCIR and MINCLE bound Le(x) less avidly than Fuc. BDCA-2, dectin-2, Langerin, MCL and mannose receptor did not bind Le(x) at all.     

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.     

Surface carbohydrates of Eudiplozoon nipponicum pre- and post-fusion

The development of the monogenean Diplozoon (Nordmann, 1832) (Diplozoidae) necessitates fusion of two larval stages (diporpae) into one double organism. How diporpae find, distinguish and contact each other is unclear, nor is the nature of the stimuli responsible for the dedifferentiation of cells and the formation of new tissues at the site of somatic fusion. Previous studies have implied a role for carbohydrates and glycoproteins in the interactions between helminth parasites and their hosts. Hypothetically, glycoconjugates may also be involved in the establishment of parasite-parasite associations. Changes in the surface saccharide residues during the development of Eudiplozoon nipponicum, a gill ectoparasite of carp (Cyprinus carpio) are described. Flat-fixed specimens and sections of diporpae, juveniles (just-fused) and adult worms were examined following exposure to a panel of 12 FITC-conjugated lectins. All developmental stages exhibited a specific surface binding pattern with ten lectins, indicating that Man/Glc, GlcNAc, Gal and GalNAc are probably present on their surfaces. No reaction was observed with Fuc-specific lectins (UEA-I and LTA). There is evidence that parasite development is accompanied by both qualitative and quantitative changes in the saccharide pattern distribution. The diporpa sucker reacted with nine lectins, excluding BS-II. A very strong binding of PNA, LCA and ConA (Gal and Man/Glc-specific lectins) was observed with the papilla glands of juvenile worms. The role of glandular secretions in this unique fusion process is discussed.     

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.     

Glutaraldehyde cross-linking of lectins to marker enzymes: protection of binding site by specific sugars

The role of bound specific sugars in protecting the sugar binding activity of several galactose binding proteins during their covalent conjugation to horse radish peroxidase by glutaraldehyde-mediated cross-linking was examined by: a) affinity matrix binding of the conjugate, b) enzyme linked lectin assay and c) hemagglutination assay. During conjugation using 1% glutaraldehyde, protection of jack fruit (Artocarpus integrifolia) lectin (jacalin) activity depended on concentration of specific sugar present during conjugation; optimum protection was offered by 50 mM galactose. This indicated the presence of one or more primary groups at the binding site of jacalin, which is (are) essential for sugar binding. On the other hand, such essential amino group(s) was not indicated at the sugar binding site of the peanut lectin, bovine heart galectin or of the human serum anti alpha-galactoside antibody, since exclusion of sugar during their conjugation to HRP did not diminish sugar binding activity. The differential behavior is discussed in the light of reported differences in sugar specificities. Results indicated that sugar mediated blocking of active site may be used in characterization of the latter in lectins.     

Plasticity in the primary binding site of galactose/N-acetylgalactosamine-specific lectins. Implication of the C-H...O hydrogen bond at the specificity-determining C-4 locus of the saccharide in 4-methoxygalactose recognition by jacalin and winged bean (basic) agglutinin I

It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of 4-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The alpha- and beta-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C-H.O-type hydrogen-bond(s) in protein-carbohydrate interactions in general and between the C-4 locus of galactose derivative and the lectins jacalin and WBA I in particular.     

Energetics of galactose- and glucose-aromatic amino acid interactions: implications for binding in galactose-specific proteins

An aromatic amino acid is present in the binding site of a number of sugar binding proteins. The interaction of the saccharide with the aromatic residue is determined by their relative position as well as orientation. The position-orientation of the saccharide relative to the aromatic residue was found to vary in different sugar-binding proteins. In the present study, interaction energies of the complexes of galactose (Gal) and of glucose (Glc) with aromatic residue analogs have been calculated by ab initio density functional (U-B3LYP/ 6-31G**) theory. The position-orientations of the saccharide with respect to the aromatic residue observed in various Gal-, Glc-, and mannose-protein complexes were chosen for the interaction energy calculations. The results of these calculations show that galactose can interact with the aromatic residue with similar interaction energies in a number of position-orientations. The interaction energy of Gal-aromatic residue analog complex in position-orientations observed for the bound saccharide in Glc/Man-protein complexes is comparable to the Glc-aromatic residue analog complex in the same position-orientation. In contrast, there is a large variation in interaction energies of complexes of Glc- and of Gal- with the aromatic residue analog in position-orientations observed in Gal-protein complexes. Furthermore, the conformation wherein the O6 atom is away from the aromatic residue is preferred for the exocyclic -CH2OH group in Gal-aromatic residue analog complexes. The implications of these results for saccharide binding in Gal-specific proteins and the possible role of the aromatic amino acid to ensure proper positioning and orientation of galactose in the binding site have been discussed.     

Secondary sugar binding site identified for LecA lectin from Pseudomonas aeruginosa

The galactose‐specific lectin LecA from Pseudomonas aeruginosa is a target for the development of new anti‐infectious compounds. Sugar based molecules with anti‐adhesive properties present great potential in the fight against bacterial infection and biofilm formation. LecA is specific for oligosaccharides with terminal α‐galactoside residues and displays strong affinity for melibiose (αGal1‐6Glc) with a K_d of 38.8 µM. The crystal structure of LecA/melibiose complex shows classical calcium‐bridged binding of αGal in the primary binding site but also revealed a secondary sugar binding site with glucose bound. This sugar binding site is in close proximity to the galactose binding one, is independent of calcium and mainly involves interactions with a symmetry‐related protein. This discovery would help to the design of new potent inhibitors targeting both binding sites. Proteins 2014; 82:1060–1065. © 2013 Wiley Periodicals, Inc.     

Legume lectins interact with muramic acid and N-acetylmuramic acid

The inhibitory potency of both muramic acid (MurAc) and N-acetylmuramic acid (MurNAc) on various legume lectins, including Glc/Man- and Gal/GalNAc-specific lectins, was investigated by a haemagglutination inhibition technique. Data indicated that many lectins, especially those specific for Glc/Man, specifically interact with MurAc and MurNAc often to a greater extent than with other monosaccharides and their derivatives, such as N-acetylglucosamine (GlcNAc) and sialic acid. Glc/Man-specific lectins were also shown to interact with the muramyl-dipeptide MurNAc-D-Ala-D-isoGln. These interactions could explain why various lectins readily agglutinate some bacterial strains of which cell walls contain peptidoglycans with high amounts of MurNAc.     

Specificity of binding of three soluble rat lung lectins to substituted and unsubstituted mammalian beta-galactosides

Binding of a series of mammalian glycoconjugates to three soluble rat lung lectins was determined with a quantitative assay. The three lectins, RL-14.5, RL-18, and RL-29, had a similar apparent affinity for lactose and associated with the same critical determinants, which included positions 4 and 6 of Gal and part of Glc. Derivatization at position 3 of Glc in lactose markedly reduced reactivity with the three lectins. For RL-14.5 and RL-29 the determinant extended specifically to the 3-hydroxyl of Glc which must be equatorial. In contrast, the stereochemical requirements for RL-18 were less specific, and Gal beta 1-3GalNAc bound as well as lactose. For RL-29 activity was markedly enhanced by GalNAc alpha 1-3 substitution on Gal, a modification which had little effect with RL-18 and inhibited binding to RL-14.5. Combinations of these residues in larger oligosaccharides and glycopeptides did not substantially enhance binding above that which might be expected from the sum of the constituent beta-galactoside residues. Although these lectins showed overlapping specificities, their binding properties are sufficiently different to suggest selective interactions with naturally occurring mammalian glycoconjugates.     

酶标白桂木凝集素糖蛋白结合特性的分析

采用辣根过氧化物酶 ( HRP)标记白桂木凝集素 ( AHL) ,应用酶联夹心法及糖竞争抑制实验 ,研究 AHL的糖蛋白结合特性 .研究表明 ,AHL能与两种不同类型的糖蛋白结合 ,一类以胃蛋白酶为代表 ,AHL能以高亲和力与胃蛋白酶结合 .其次能与β-乳球蛋白、牛血清清蛋白结合 ,但结合力依次递减 .AHL也能与透明质酸以较高亲和力相结合 .AHL与胃蛋白酶、β-乳球蛋白、牛血清清蛋白、透明质酸的结合受 Me- Gal的强烈竞争抑制 ,亦受 Me- Man\D- Gal\Raf的抑制 .另一类为Con A,AHL与 Con A的结合受 Me- Man的强烈竞争抑制 ,并受 Me- Glc\D- Man\D- Fru\D- Glc的较强抑制 .各种糖的封闭性抑制实验结果与竞争性抑制实验相似 .提示 AHL上存在 O-糖苷键结合位点 .     

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.     

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.     

Isolation and characterization of <Emphasis Type="Italic">Lentinus edodes</Emphasis> (Berk.) singer extracellular lectins

Lectin preparations have been isolated and purified from the culture liquid of the xylotrophic basidiomycete Lentinus edodes (Berk.) Singer [Lentinula edodes (Berk.) Pegler]. The culture of L. edodes F-249 synthesizes two extracellular lectins different in composition and physicochemical properties. Extracellular lectin L1 from L. edodes is a glycoprotein of mono-subunit structure with molecular weight of 43 kD. L1 is comprised of 10.5 +/- 1.0% (w/w) carbohydrates represented by glucose (Glc). Extracellular lectin L2 is a proteoglycan of mono-subunit structure with molecular weight of 37 kD. L2 is comprised of 90.3 +/- 1.0% (w/w) carbohydrates represented by Glc (73% of the total mass of the carbohydrate moiety of the lectin molecule) and galactose (Gal) (27% of the total mass of the carbohydrate part of the lectin molecule). The content of Asn in L2 is high, i.e. 42% (w/w) of total amino acids. This fact along with the composition of the carbohydrate part of the molecule (Glc + Gal) allows one to assign L2 to N-asparagine-bound proteins. Both lectins are specific to D-Gal and lactose (Lac) at an equal for L1 and L2 minimal inhibiting concentration of these carbohydrates (2.08 mM Gal and 8.33 mM Lac). Other carbohydrates to which the lectins show affinity are different for the two lectins: Rha (4.16 mM) for L1 and Ara (4.16 mM) and mannitol (8.33 mM) for L2. The purified extracellular lectins of L. edodes are highly selective at recognition of definite structures on the surface of trypsinized rabbit erythrocytes and do not react with the erythrocytes of other animals and humans.