Secondary literature sources for the CLECT domain

For each of the hand selected primary papers associated with the CLECT domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Distribution patterns of neoglycoprotein-binding sites (endogenous lectins) and lectin-reactive glycoconjugates during cartilage and bone formation in human finger.

Zschabitz A, Gabius HJ, Krahn V, Michiels I, Schmidt W, Koepp H, Stofft E
Acta Anat (Basel). 1995; 154: 272-82

The distribution of endogenous lectins, visualized by labelled neoglycoproteins, and of defined oligosaccharide structures, reactive with plant lectins, during fetal development of the fingers was analyzed in sections of human 3- to 8-month-old fetal specimens. Chondrogenesis as well as ossification were correlated with characteristic modulations in the expression of both glycoligand-binding molecules and characteristic carbohydrate structures. Occurrence of xylose-specific receptors was judged to be an early sign of cartilage development. Similarly, alpha-mannosyl residues that had been attached to labelled carrier proteins were strongly bound by the extracellular matrix already during early stages of finger maturation. Staining intensity for heparin gradually increased during chondrogenesis, whereas affinity for mannose showed a stage-related decline. Binding of mannose-6-phosphate was confined to hypertrophied cartilage of primary ossification centers. Accessible binding sites for terminal N-acetylneuraminic acid and N-acetylgalactosamine moieties were detected only in osteoid. In addition to monitoring the sugar-binding capacity, presence and developmental regulation of distinct carbohydrate structures were also assessed. PSA and SBA enabled the demonstration of an abrupt loss of staining affinity in the zone of maturing hypertrophic cartilage. Succinylated WGA proved to be an apparently useful marker of evolving bone tissue. GSL-II binding was restricted to chondroclasts and osteoclasts. The findings of this investigation are consistent with the supposed role of glycoconjugate-lectin interactions in cartilage and bone development.

Mammalian fetuin-binding proteins sarcolectin, aprotinin and calcyclin display differences in their apparent carbohydrate specificity.

Zeng FY, Gabius HJ
Biochem Int. 1992; 26: 17-24

The interferon antagonist sarcolectin, the protease inhibitor aprotinin and calcyclin whose expression is regulated by growth stimulation in quiescent fibroblasts display sialic acid-dependent binding to fetuin, visualized by solid-phase assays using biotinylated fetuin. The potential functional importance of this property prompted its comparative characterization with an array of neutral or negatively charged sugars, sulfated polysaccharides and sialoglycoproteins as inhibitors of binding of biotinylated fetuin to the immobilized proteins. The results revealed that this activity of sarcolectin and calcyclin is nearly unaffected by charge-free carbohydrates in contrast to aprotinin, calcyclin exhibits a notable affinity for Neu5Gc and together with aprotinin for phosphorylated sugars, and that sarcolectin's binding is affected to the highest extent by sulfated sugars relative to aprotinin and calcyclin.

Analysis of sequence variation among legume lectins. A ring of hypervariable residues forms the perimeter of the carbohydrate-binding site.

Young NM, Oomen RP
J Mol Biol. 1992; 228: 924-34

Twelve plant lectins from the Papilionoideae subfamily were selected to represent a range of carbohydrate specificities, and their sequences were aligned. Two variability indices were applied to the aligned sequences and the results were analysed using the three-dimensional structures of concanavalin A and the pea lectin. The areas of greatest variability were located in the carbohydrate-binding site region, forming a perimeter around a well-conserved core. These residues are inferred to be specificity determining, in the manner of antibodies, and the most variable position corresponded to Tyr100 in concanavalin A, a known ligand contact residue. In addition to the five peptide loops known to form the binding site from crystallographic studies, a sixth segment with variable residues was located in the binding-site region, and this may contribute to oligosaccharide specificity. In their overall composition, the lectin sites resemble those of the sugar-transport proteins rather than antibodies. The prospects for modelling lectin binding sites by the methods used for antibodies were also assessed.

Neoglycoprotein-liposome and lectin-liposome conjugates as tools for carbohydrate recognition research.

Yamazaki N, Kodama M, Gabius HJ
Methods Enzymol. 1994; 242: 56-65

[Carbohydrate recognition system of legume lectins]

Yamamoto K
Tanpakushitsu Kakusan Koso. 1992; 37: 1820-9

[Carbohydrate-protein interactions]

Yamamoto K
Tanpakushitsu Kakusan Koso. 1994; 39: 1241-51

[Structure and function of lectins and lectin-carbohydrate interactions]

Yamamoto K
Seikagaku. 1994; 66: 1111-29

A guide for carbohydrate specificities of lectins.

Wu AM, Sugii SJ, Herp A
Adv Exp Med Biol. 1988; 228: 819-47

Tissue carbohydrate identification by the use of lectins.

West KP, Platts HA, Fletcher A, Walker F
J Clin Pathol. 1982; 35: 239-40

The C-type lectin superfamily in the immune system.

Weis WI, Taylor ME, Drickamer K
Immunol Rev. 1998; 163: 19-34

Protein-carbohydrate interactions serve multiple functions in the immune system. Many animal lectins (sugar-binding proteins) mediate both pathogen recognition and cell-cell interactions using structurally related Ca(2+)-dependent carbohydrate-recognition domains (C-type CRDs). Pathogen recognition by soluble collections such as serum mannose-binding protein and pulmonary surfactant proteins, and also the macrophage cell-surface mannose receptor, is effected by binding of terminal monosaccharide residues characteristic of bacterial and fungal cell surfaces. The broad selectivity of the monosaccharide-binding site and the geometrical arrangement of multiple CRDs in the intact lectins explains the ability of the proteins to mediate discrimination between self and non-self. In contrast, the much narrower binding specificity of selectin cell adhesion molecules results from an extended binding site within a single CRD. Other proteins, particularly receptors on the surface of natural killer cells, contain C-type lectin-like domains (CTLDs) that are evolutionarily divergent from the C-type lectins and which would be predicted to function through different mechanisms.

Molecular mechanisms of complex carbohydrate recognition at the cell surface.

Weis WI, Quesenberry MS, Taylor ME, Bezouska K, Hendrickson WA, Drickamer K
Cold Spring Harb Symp Quant Biol. 1992; 57: 281-9

Cell-surface carbohydrate recognition by animal and viral lectins.

Weis WI
Curr Opin Struct Biol. 1997; 7: 624-30

Many animal and viral lectins are specific for monosaccharides found in particular glycosidic linkages, or for larger oligosaccharide structures. Recent crystal structures of complexes between these proteins and receptor fragments have provided insights into the recognition of linkage isomers and oligosaccharide conformation.

Specificity of C-glycoside complexation by mannose/glucose specific lectins.

Weatherman RV, Mortell KH, Chervenak M, Kiessling LL, Toone EJ
Biochemistry. 1996; 35: 3619-24

The binding of the mannose/glucose specific lectins from Canavalia ensiformis (concanavalin A) and Dioclea grandiflora to a series of C-glucosides were studied by titration microcalorimetry and fluorescence anisotropy titration. These closely related lectins share a specificity for the trimannoside methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, and are a useful model system for addressing the feasibility of differentiating between lectins with overlapping carbohydrate specificities. The ligands were designed to address two issues: (1) how the recognition properties of non-hydrolyzable C-glycoside analogues compare with those of the corresponding O-glycosides and (2) the effect of presentation of more than one saccharide recognition epitope on both affinity and specificity. Both lectins bind the C-glycosides with affinities comparable to those of the O-glycoside analogues; however, the ability of both lectins to differentiate between gluco and manno diastereomers was diminished in the C-glycoside series. Bivalent norbornyl C-glycoside esters were bound by the lectin from Canavalia but only weakly by the lectin from Dioclea. In addition to binding the bivalent ligands, concanavalin A discriminated between C-2 epimers, with the manno configuration binding more tightly than the gluco. The stoichiometry of binding of the bivalent ligands to both di- and tetrameric lectin was two binding sites per ligand, rather than the expected 1:1 stoichiometry. Together, these results suggest that concanavalin A may possess more than one class of carbohydrate binding sites and that these additional sites show stereochemical discrimination similar to that of the previously identified monosaccharide binding site. The implications of these findings for possible in vivo roles of plant lectins and for the use of concanavalin A as a research tool are discussed.

Cell calcium signalling induced by endogenous lectin carbohydrate interaction in the Jurkat T cell line.

Walzel H, Hirabayashi J, Kasai K, Brock J, Neels P
Glycoconj J. 1996; 13: 99-105

The effects of the beta-galactoside-binding lectin from human placenta (HPL14) on intracellular calcium concentration ([Ca2+]i) were examined in the human Jurkat T cell line. The lectin induces a concentration dependent increase in [Ca2+]i. This calcium signalling effect is clearly mediated through complementary cell surface galactoglycoconjugates because it can be blocked by beta-galactosides. The observed Ca2+ - response involves both the release of calcium from intracellular stores and a calcium influx from the extracellular space. It is sustained in the presence of 1 mM extracellular calcium whereas it becomes transient when the influx of extracellular calcium was blocked by calcium chelation to EGTA. Voltage-sensitive calcium channel blockers like verapamil and prenylamine were without effect on the action of HPL14. Protection of the sugar binding activity of HPL14 in the absence of a thiol-reducing reagent by carboxamidomethylation (CM-HPL14) or by substitution Cys2 with serine (C2S) results in lectin proteins with considerably decreased calcium signalling efficiency. The recombinant lectin (Rec H) and the mutant protein obtained by substitution of highly conservative Trp68 with tyrosine (W68Y) induce lower levels of [Ca2+]i compared to wild type lectin.

Lectins.

Vijayan M, Chandra N
Curr Opin Struct Biol. 1999; 9: 707-14

Lectins - carbohydrate-binding proteins involved in a variety of recognition processes - exhibit considerable structural diversity. Three new lectin folds and further elaborations of known folds have been described recently. Large variability in quaternary association resulting from small alterations in essentially the same tertiary structure is a property exhibited specially by legume lectins. The strategies used by lectins to generate carbohydrate specificity include the extensive use of water bridges, post-translational modification and oligomerization. Recent results pertaining to influenza and foot-and-mouth viruses further elaborate the role of lectins in infection.

Humoral lectins in the scorpion Vaejovis confuscius: a serological characterization.

Vasta GR, Cohen E
J Invertebr Pathol. 1984; 43: 226-33

Animal lectins as self/non-self recognition molecules. Biochemical and genetic approaches to understanding their biological roles and evolution.

Vasta GR, Ahmed H, Fink NE, Elola MT, Marsh AG, Snowden A, Odom EW
Ann N Y Acad Sci. 1994; 712: 55-73

In recent years, the significant contributions from molecular research studies on animal lectins have elucidated structural aspects and provided clues not only to their evolution but also to their multiple biological functions. The experimental evidence has suggested that distinct, and probably unrelated, groups of molecules are included under the term "lectin." Within the invertebrate taxa, major groups of lectins can be identified: One group would include lectins that show significant homology to membrane-integrated or soluble vertebrate C-type lectins. The second would include those beta-galactosyl-specific lectins homologous to the S-type vertebrate lectins. The third group would be constituted by lectins that show homology to vertebrate pentraxins that exhibit lectin-like properties, such as C-reactive protein and serum amyloid P. Finally, there are examples that do not exhibit similarities to any of the aforementioned categories. Moreover, the vast majority of invertebrate lectins described so far cannot yet be placed in one or another group because of the lack of information regarding their primary structure. (See Table 1.) Animal lectins do not express a recombinatorial diversity like that of antibodies, but a limited diversity in recognition capabilities would be accomplished by the occurrence of multiple lectins with distinct specificities, the presence of more than one binding site, specific for different carbohydrates in a single molecule, and by certain "flexibility" of the binding sites that would allow the recognition of a range of structurally related carbohydrates. In order to identify the lectins' "natural" ligands, we have investigated the interactions between those proteins and the putative endogenous or exogenous glycosylated substances or cells that may be relevant to their biological function. Results from these studies, together with information on the biochemical properties of invertebrate and vertebrate lectins, including their structural relationships with other vertebrate recognition molecules, are discussed.

Sialic acids as ligands in recognition phenomena.

Varki A
FASEB J. 1997; 11: 248-55

The sialic acids are acidic monosaccharides typically found at the outermost ends of the sugar chains of animal glycoconjugates. They potentially can inhibit intermolecular and intercellular interactions by virtue of their negative charge. However, they can also act as critical components of ligands recognized by a variety of proteins of animal, plant, and microbial origin (sialic acid binding lectins). Recognition can be affected by specific structural variations and modifications of sialic acids, their linkage to the underlying sugar chain, the structure of these chains, and the nature of the glycoconjugate to which they are attached. Presented here is a summary of the various proteins that can recognize and bind to this family of monosaccharides, comparing and contrasting the structural requirements and mechanisms involved in binding. Particular attention is focused on the recently evolving information about sialic acid recognition by certain C-type lectins (the selectins), I-type lectins (e.g., CD22 and sialoadhesin), and a complement regulatory protein (the H protein). The last two instances are examples of the importance of the side chain of sialic acids and the effects of natural substitutions (e.g., 9-O-acetylation) of this part of the molecule.

Mutational analysis of the sugar-binding site of pea lectin.

Van Eijsden RR, De Pater BS, Kijne JW
Glycoconj J. 1994; 11: 375-80

Comparison of x-ray crystal structures of several legume lectins, co-crystallized with sugar molecules, showed a strong conservation of amino acid residues directly involved in ligand binding. For pea (Pisum sativum) lectin (PSL), these conserved amino acids can be classified into three groups: (I) D81 and N125, present in all legume lectins studied so far; (II) G99 and G216, conserved in almost all legume lectins; and (III) A217 and E218, which are only found in Vicieae lectins and are possibly determinants of sugar-binding specificity. Each of these amino acids in PSL was changed by site-directed mutagenesis, resulting in PSL molecules with single substitutions: for group I D81A, D81N, N125A; for group II G99R, G216L; and for group III A217L, E218Q, respectively. PSL double mutant Y124R; A126S was included as a control. The modified PSL molecules appeared not to be affected in their ability to form dimeric proteins, whereas the sugar-binding activity of each of the PSL mutants, with the exception of the control mutant (as shown by haemagglutination assays), was completely eliminated. These results confirm the model of the sugar-binding site of Vicieae lectins as deduced from X-ray analysis.

Spectroscopical studies on the structural organization of the lectin discoidin I: analysis of sugar- and calcium-binding activities.

Valencia A, Pestana A, Cano A
Biochim Biophys Acta. 1989; 990: 93-7

One of the common characteristics observed in different families of sugar-binding proteins is the presence of aromatic residues in the proximity of the functional sugar-binding site (Quiocho, F. (1986) Annu. Rev. Biochem. 55, 287-315). This general property has made these proteins a very appropriate subject for studies using intrinsic fluorescence assays. In the present report we have studied the sugar binding activity of the lectin discoidin I, using a fluorescence-monitored titration assay. The galactose binding has been estimated, with an affinity constant of 1.8.10(-7) M-1 in the absence of calcium. In the presence of 1 mM Ca2+, the Kd of galactose binding is lowered to 2.7.10(-8) M-1. Calcium binding, by itself, seems to occur as two components with Kd values of 10(-7) and 10(-6) M-1. From these data, and sequence comparison of discoidin I with other lectins, a general model for ligand binding has been proposed in which a sequence from position 176 to 188, together with another region close to an apolar tryptophan residue, most probably Trp-50, would participate in the calcium- and sugar-binding site(s) of this protein.

[Carbohydrate-binding proteins--lectins and glycosidases in plants of Anthurium genus]

Tkachenko VI, Datskiv MZ, Lutsyk MD
Ukr Biokhim Zh. 1993; 65: 29-33

The content of lectins and activity of glycosidases have been estimated in seeds and vegetative organs of 5 strains of plants of Anthurium genus. In seeds of all the investigated strains lectins were detected with the selectivity toward the N-acetyl-galactosamine (minimal inhibitory concentration of sugar was 0.1-0.2 mM) and an anti-A blood group specificity. Lectins of Anthuriums selectively bound O-type glycosidic chains and revealed high affinity toward mucins (salivary or ovary cysts origin). Lectins were not detected in vegetative parts of Anthuriums. In seeds of plants the following glycosidases were detected in the diminishing activity order: alpha-galactosidase, alpha-mannosidase, beta-galactosidase, beta-glucosaminidase.

Mode of molecular recognition of L-fucose by fucose-binding legume lectins.

Thomas CJ, Surolia A
Biochem Biophys Res Commun. 2000; 268: 262-7

Recognition of cell surface carbohydrate moieties by lectins plays a vital role in many a biological process. Fucosyated residues are often implicated as key recognition markers in many cellular processes. In particular, the aspects of molecular recognition of fucose by fucose-bindinglectins UEA 1 and LTA pose a special case because no crystal structure of these lectins is available. The study was conducted to elucidate the process of recognition of l-fucose by UEA1 and LTA by correlating structure-based sequence alignment and other available biochemical/biophysical data. The study points out that the mode of recognition of l-fucose is coordinated by the invariant triad of residues the asparagine 137, glycine 105, and aspartate 87. The major hydrophobic stacking residue in this case is the tyrosine 220. The study also reiterates the key role of the conserved triad of residues in the combining site which is a common feature for all legume lectins whose crystal structures are known.

Analysis and prediction of carbohydrate binding sites.

Taroni C, Jones S, Thornton(2) JM
Protein Eng. 2000; 13: 89-98

An analysis of the characteristic properties of sugar binding sites was performed on a set of 19 sugar binding proteins. For each site six parameters were evaluated: solvation potential, residue propensity, hydrophobicity, planarity, protrusion and relative accessible surface area. Three of the parameters were found to distinguish the observed sugar binding sites from the other surface patches. These parameters were then used to calculate the probability for a surface patch to be a carbohydrate binding site. The prediction was optimized on a set of 19 non-homologous carbohydrate binding structures and a test prediction was carried out on a set of 40 protein-carbohydrate complexes. The overall accuracy of prediction achieved was 65%. Results were in general better for carbohydrate-binding enzymes than for the lectins, with a rate of success of 87%.

Molecular evolution of legume lectins.

Strosberg AD, Lauwereys M, Foriers A
Prog Clin Biol Res. 1983; 138: 7-20

Glycosylation mutants of animal cells.

Stanley P
Annu Rev Genet. 1984; 18: 525-52

Carbohydrate-recognition domains on the surface of phytophagous nematodes.

Spiegel Y, Inbar J, Kahane I, Sharon E
Exp Parasitol. 1995; 80: 220-7

Human red blood cells (HRBC) adhered to preparasitic second-stage juveniles (J2) of Heterodera avenae, Heterodera schachtii, Meloidogyne javanica, Pratylenchus mediterraneus, Rotylenchulus reniformis, and Tylenchulus semipenetrans over the entire nematode body. Binding was conspicuously confined to the head and tail of Longidorus cohni, Xiphinema brevicolle, and Xiphinema index. Binding was Ca2+ and Mg2+ dependent. In contrast, HRBC did not adhere to Anguina tritici, Aphelenchoides subtenius, Ditylenchus dipsaci, M. javanica females, and Panagrellus redivivus, even in the presence of these cations. Incubation of M. javanica J2 with fucose, glucose, N-acetylglucosamine, mannose, or trypsin decreased the intensity of subsequent HRBC binding, while galactose and N-acetylgalactosamine increased binding intensity. HRBC binding was diminished when nematodes were pretreated with trypsin and eliminated when pretreatments with detergents removed the surface coat. HRBC adhered to nylon fibers coated with surface coat extracted from M. javanica J2; binding was Ca2+ and Mg2+ dependent and diminished when the nylon fibers were coated with bovine serum albumin or preincubated with fucose and mannose. These results demonstrate that HRBC adhesion involves carbohydrate moieties of HRBC and corresponding carbohydrate-recognition domains (CRD) distributed in the nematode surface coat. To our knowledge this is the first report of a surface CRD in the phylum Nematoda.

Different architecture of the combining site of the two chicken galectins revealed by chemical mapping studies with synthetic ligand derivatives.

Solis D, Romero A, Kaltner H, Gabius HJ, Diaz-Maurino T
J Biol Chem. 1996; 271: 12744-8

The detailed comparison of the carbohydrate-binding properties of related galectins from one organism can be facilitated by the application of an array of deliberately tailored methyl beta-lactoside derivatives. Focusing on chicken due to its expression of two galectins as a model for this approach, the combining-site architecture of the lectin from adult liver (CL-16) is apparently homologous to that previously observed for bovine galectin-1 (Solis, D., Jimenez-Barbero, J., Martin-Lomas, M., and Diaz-Maurino, T. (1994) Eur. J. Biochem. 223, 107-114). Besides preservation of the key interactions and minor differences, the lectin from adult intestine (CL-14) is able to accommodate an axial HO-3 at the glucose moiety. Homology-based modeling enabled us to tentatively attribute the observed differences to a slightly different orientation of pivotal side chains in the binding pocket due to distinct substitutions of amino acid residues in the variable region within the carbohydrate-recognition domain. Thus, the results suggest overlapping but distinct ranges of potential ligands for the two chicken lectins and provide new information on their relationship to mammalian galectins. The described approach is suggested to be of relevance to design pharmaceuticals with enhanced selectivity to a certain member within a family of related lectins.

Probing hydrogen-bonding interactions of bovine heart galectin-1 and methyl beta-lactoside by use of engineered ligands.

Solis D, Jimenez-Barbero J, Martin-Lomas M, Diaz-Maurino T
Eur J Biochem. 1994; 223: 107-14

The binding of different synthetic monodeoxy, O-methyl and fluorodeoxy derivatives of methyl beta-lactoside to galectin-1 from bovine heart has been studied to probe the role of hydrogen bonding in the recognition and binding. The energetic contributions of the hydroxyl groups of methyl beta-lactoside directly involved in the interaction have been estimated and the nature of the protein residues involved has been predicted on the basis of the free energy data. Interpretations of the results have been sustained by molecular modeling of the three-dimensional structure of the sugars in solution. One side of the disaccharide molecule is not involved (HO-6 and HO-2') or only marginally involved (HO-3') in hydrogen bonding. Moreover, O-methylation at these positions causes an enhancement of the binding, suggesting favourable interactions of the methyl groups which may come into contact with hydrophobic residues at the periphery of the combining site. Hydrogen-bonding interactions are almost exclusively restricted to the other side of the molecule: the C-4' and C-6' hydroxyl groups act as donors of the strongest hydrogen bonds to charged groups of the lectin, while the C-3 hydroxyl group participates in a strong hydrogen bond with a neutral group. The results also suggest that the N-acetyl NH group in N-acetyllactosamine, as well as the hydroxyl group at position C-2 in methyl beta-lactoside, are involved in a polar interaction with neutral groups of the combining site. This hydrogen-bonding pattern contrasts markedly with that previously reported for the two galactose-specific Ricinus communis lectins. The recognition of different epitopes of the same basic structure underlies the differences in the oligosaccharide-binding specificities of galectin-1 and the R. communis lectins.

Hydrogen-bonding pattern of methyl beta-lactoside binding to the Ricinus communis lectins.

Solis D, Fernandez P, Diaz-Maurino T, Jimenez-Barbero J, Martin-Lomas M
Eur J Biochem. 1993; 214: 677-83

The binding of O-methyl and fluorodeoxy derivatives of methyl beta-lactoside to the Ricinus communis toxin (RCA60) and agglutinin (RCA120) was studied in order to determine the donor/acceptor relationships of the hydrogen bonds between the hydroxyl groups of methyl beta-lactoside and the binding sites of the lectins. Free energy contributions of the hydrogen bonds at each position have been estimated from these data and from those previously reported for the monodeoxy derivatives [Rivera-Sagredo, A., Solis, D., Diaz-Maurino, T., Jimenez-Barbero, J. & Martin-Lomas, M. (1991) Eur. J. Biochem. 197, 217-228; Rivera-Sagredo, A., Jimenez-Barbero, J., Martin-Lomas, M., Solis, D. & Diaz-Maurino, T. (1992) Carbohydr. Res. 232, 207-226]. The nature of the groups of the lectins involved in hydrogen bonding has been predicted on the basis of the free energy data. Analysis of the results indicates that both the C-3' and C-4' hydroxyl groups act as hydrogen-bond donors to charged groups of both RCA60 and RCA120. The C-6' and probably also the C-2' hydroxyl groups participate both as donors and as acceptors of two hydrogen bonds with neutral groups of the lectins. And finally, the C-6 hydroxyl group possibly acts as a donor of a weak hydrogen bond to a neutral group in RCA60, but not in RCA120. The results provide a molecular basis to explain some features of the binding specificity of the lectins. Comparison of RCA60 binding data with the recently refined X-ray crystal structure of the RCA60-lactose complex shows similarities but also some discrepancies that can be attributed to the marked influence of the pH on the carbohydrate-lectin interaction.

Conformational analysis of biantennary glycans and molecular modeling of their complexes with lentil lectin.

Sokolowski T, Peters T, Perez S, Imberty A
J Mol Graph Model. 1997; 15: 37-42

Some mannose-binding legume lectins show higher affinity for fucosylated glycans than for glycans without fucose. These lectins possess a secondary binding site. Owing to the possibility of additional fucose binding, oligosaccharides adopt different conformations depending on whether they contain fucose or not. To study these conformational differences, complexes of fucosylated and unfucosylated glycans with Lens culinaris lectin have been modeled. Starting points were X-ray structures of lentil lectin and complexes of the homologous Lathyrus ochrus lectin. The SYBYL molecular modeling package with the TRIPOS force field was used. Two different models were built, displaying in both a network of hydrogen bonds between the saccharide and the binding site. Furthermore, to compare the free and bound ligand, conformational analysis in the free state has been performed. A complete analysis of all possible disaccharide fragments has been performed using the MM3 force field. A CICADA analysis employing the same force field was carried out to study the complete oligosaccharide. Low-energy conformers found by CICADA were clustered in conformational families and analyzed in terms of flexibility and rotational barriers. All values of glycosidic torsion angles are in the range as calculated by MM3 for the disaccharides.

Occurrence and characterization of lectins in actinomycetes.

Singh J, Ahluwalia J, Kamboj SS, Singh S
J Basic Microbiol. 1993; 33: 207-11

25 species of actinomycetes were tested for the occurrence of lectins. Using a battery of normal and desialized erythrocytes, each species was screened for 3 types of lectin activity i.e. surface bound, extracellular and intracellular. As many as 13 species showed one or more types of activity; some of them were characterized with regard to their biological action spectrum and sugar specificity.

Prolactin alters the expression of integumental glycoconjugates in the red-spotted newt, Notophthalmus viridescens.

Singhas CA, Ward DL
Anat Rec. 1993; 236: 537-46

Prolactin (PRL)-mediated changes in the texture and secretory activity of the skin in adult red-spotted newts may involve alterations in the distribution and/or expression of structural and secretory epidermal glycoconjugates. To explore this possibility, skin samples were obtained from groups of conditioned animals that had received injections of either ovine prolactin or amphibian saline over a 14-day period. Glycoconjugates within the epidermis and cutaneous glands were examined by means of lectin histochemistry using a panel of eight HRP-labelled lectins. PRL increased levels of sialic acid and n-acetylglucosamine in the stratum corneum. In contrast, glycoconjugates containing fucose, galactose, n-acetylgalactosamine, and galactose-(1,3)-n-acetylgalactosamine were decreased by PRL within both glands and epidermis. These results suggest that the integumental effects associated with prolactin in the red-spotted newt are mediated, at least in part, through the alteration of epidermal and glandular glycoconjugates.

Interactions of carbohydrates and lectins with complement.

Sim RB, Malhotra R
Biochem Soc Trans. 1994; 22: 106-11

Lectins: endogenous carbohydrate-binding proteins from vertebrate tissues: functional role in recognition processes?

Simpson DL, Thorne DR, Loh HH
Life Sci. 1978; 22: 727-48

Carbohydrate binding properties of the stinging nettle (Urtica dioica) rhizome lectin.

Shibuya N, Goldstein IJ, Shafer JA, Peumans WJ, Broekaert WF
Arch Biochem Biophys. 1986; 249: 215-24

The interaction of the stinging nettle rhizome lectin (UDA) with carbohydrates was studied by using the techniques of quantitative precipitation, hapten inhibition, equilibrium dialysis, and uv difference spectroscopy. The Carbohydrate binding site of UDA was determined to be complementary to an N,N',N"-triacetylchitotriose unit and proposed to consist of three subsites, each of which has a slightly different binding specificity. UDA also has a hydrophobic interacting region adjacent to the carbohydrate binding site. Equilibrium dialysis and uv difference spectroscopy revealed that UDA has two carbohydrate binding sites per molecule consisting of a single polypeptide chain. These binding sites either have intrinsically different affinities for ligand molecules, or they may display negative cooperativity toward ligand binding.

Lectins--proteins with a sweet tooth: functions in cell recognition.

Sharon N, Lis H
Essays Biochem. 1995; 30: 59-75

Lectins, non-enzymic proteins that bind mono- and oligosaccharides reversibly and with high specificity, occur widely in nature. They come in a variety of sizes and shapes, but can be grouped in families with similar structural features. The combining sites of lectins are also diverse, although they are similar in the same family. The specificities of lectins are determined by the exact shape of the binding sites and the nature of the amino acid residues to which the carbohydrate is linked. Small changes in the structure of the sites, such as the substitution of only one or two amino acids, may result in marked changes in specificity. The carbohydrate is linked to the protein mainly through hydrogen bonds, with added contributions from van der Waals contacts and hydrophobic interactions. Coordination with metal ions may occasionally play a role too. Microbial surface lectins serve as a means of adhesion to host cells of viruses (e.g. influenza virus), bacteria (e.g. E. coli) and protozoa (e.g. amoeba): a prerequisite for the initiation of infection. Blocking the adhesion by carbohydrates that mimic those to which the lectins bind prevents infection by these organisms. The way is thus open for the development of anti-adhesive therapy against microbial diseases. Lectin-carbohydrate mediated interactions between leucocytes and endothelial cells are the first step in the recirculation of lymphocytes and in the migration of neutrophils to sites of inflammation. Such interactions may also feature highly in the formation of metastases. Studies of these processes are expected to lead to the development of carbohydrate-based anti-adhesion drugs for the treatment of inflammatory diseases as well as cancer.

Legume lectins--a large family of homologous proteins.

Sharon N, Lis H
FASEB J. 1990; 4: 3198-208

More than 70 lectins from leguminous plants belonging to different suborders and tribes have been isolated, mostly from seeds, and characterized to varying degrees. Although they differ in their carbohydrate specificities, they resemble each other in their physicochemical properties. They usually consist of two or four subunits (25-30 kDa), each with one carbohydrate binding site. Interaction with carbohydrates requires tightly bound Ca2+ and Mn2+ (or another transition metal). The primary sequences of more than 15 legume lectins have been established by chemical or molecular genetic techniques. They exhibit remarkable homologies, with a significant number of invariant amino acid residues, among them most of those involved in metal binding. The 3-dimensional structures of the legume lectins are similar, too, and are characterized by a high content of beta-sheets and a lack of alpha-helix. The location of the metal and carbohydrate binding sites, established unequivocally in concanavalin A by high resolution X-ray crystallography, appears to be the same in the other legume lectins. Several of the lectin genes have been cloned and expressed in heterologous systems. This opens the way for the application of molecular genetics to the investigation of the atomic structure of the carbohydrate binding sites of the lectins, and of the relationship between their structure and biological activity. The new approaches may also provide information on the mechanisms that control gene expression in plants and on the role of lectins in nature.

Lectin-carbohydrate complexes of plants and animals: an atomic view.

Sharon N
Trends Biochem Sci. 1993; 18: 221-6

Lectins are a structurally diverse class of proteins, their only common features being the ability to bind carbohydrates specifically and reversibly, and to agglutinate cells. Some, however, can be grouped together into distinct families, such as those of the legumes or the cereals that are structurally similar, or the C-type (Ca(2+)-dependent) animal lectins that contain homologous carbohydrate recognition domains. Recent high-resolution X-ray crystallographic studies have revealed the structures of the sugar complexes of over half a dozen lectins. These studies demonstrate that the combining sites of lectins are also structurally diverse, although they may be similar in the same family.

When lectin meets oligosaccharide.

Sharon N
Nat Struct Biol. 1994; 1: 843-5

Imparting exquisite specificity to peanut agglutinin for the tumor-associated Thomsen-Friedenreich antigen by redesign of its combining site.

Sharma V, Vijayan M, Surolia A
J Biol Chem. 1996; 271: 21209-13

Lectins from legumes constitute one of the most thoroughly studied families of proteins, yet the absence of a rigorous framework to explain their carbohydrate binding specificities appears to have prevented a rational approach to alter their ligand binding activity. Studies reported here deal with the redesign of the recognition propensity of peanut agglutinin (PNA), an important member of the family. PNA is extensively used as a tool for recognition of the tumor-associated Thomsen-Friedenrich antigen (T-antigen; Galbeta1-3GalNAc) on the surfaces of malignant cells and immature thymocytes. PNA also recognizes N-acetyllactosamine (LacNAc; Galbeta1-4GlcNAc), which is present at the termini of several cell-surface glycoproteins. The crystal structure of the PNA-lactose complex revealed, in addition to the expected interactions with the residues constituting the binding site, the presence of leucine 212 at a position close enough to be in steric contact with the acetamido group on LacNAc. We report here two leucine mutants, one to asparagine (L212N) and the other to alanine (L212A), that exhibit distinct preference for T-antigen and N-acetyllactosamine, respectively. Carbohydrate binding studies reveal that mutant L212N does not recognize LacNAc at high concentrations, thus making it an exquisitely specific cell-surface marker compared with its wild-type counterpart.

Analyses of carbohydrate recognition by legume lectins: size of the combining site loops and their primary specificity.

Sharma V, Surolia A
J Mol Biol. 1997; 267: 433-45

Recognition of cell-surface carbohydrates by lectins has wide implications in important biological processes. The ability of plant lectins to detect subtle variations in carbohydrate structures found on molecules, cells and organisms have made them a paradigm for protein-carbohydrate recognition. Legume lectins, one of the most well studied family of plant proteins, display a considerable repertoire of carbohydrate specificities owing perhaps to the sequence hypervariability in the loops constituting their combining site. However, lack of a rigorous framework to explain their carbohydrate binding specificities has precluded a rational approach to alter their ligand binding activity in a meaningful manner. This study reports an extensive analysis of sequences and structures of several legume lectins and shows that despite the hypervariability of their combining regions they exhibit within a significant pattern of uniformity. The results show that the size of the binding site loop D is invariant in the Man/Glc specific lectins and is possibly a primary determinant of the monosaccharide specificities of the legume lectins. Analyses of size and sequence variability of loops reveal the existence of a common theme that subserves to define their binding specificities. These results thus provide not only a framework for understanding the molecular basis of carbohydrate recognition by legume lectins but also a rationale for redesign of their ligand binding propensities.

Pertussis toxin has eukaryotic-like carbohydrate recognition domains.

Saukkonen K, Burnette WN, Mar VL, Masure HR, Tuomanen EI
Proc Natl Acad Sci U S A. 1992; 89: 118-22

Bordetella pertussis is bound to glycoconjugates on human cilia and macrophages by multiple adhesins, including pertussis toxin. The cellular recognition properties of the B oligomer of pertussis toxin were characterized and the location and structural requirements of the recognition domains were identified by site-directed mutagenesis of recombinant pertussis toxin subunits. Differential recognition of cilia and macrophages, respectively, was localized to subunits S2 and S3 of the B oligomer. Despite greater than 80% sequence homology between these subunits, ciliary lactosylceramide exclusively recognized S2 and leukocytic gangliosides bound only S3. Substitution at residue 44, 45, 50, or 51 in S2 resulted in a shift of carbohydrate recognition from lactosylceramide to gangliosides. Mutational exchange of amino acid residues 37-52 between S2 and S3 interchanged their carbohydrate and target cell specificity. Comparison of these carbohydrate recognition sequences to those of plant and animal lectins revealed that regions essential for function of the prokaryotic lectins were strongly related to a subset of eukaryotic carbohydrate recognition domains of the C type.

Insights into carbohydrate recognition by Narcissus pseudonarcissus lectin: the crystal structure at 2 A resolution in complex with alpha1-3 mannobiose.

Sauerborn MK, Wright LM, Reynolds CD, Grossmann JG, Rizkallah PJ
J Mol Biol. 1999; 290: 185-99

Carbohydrate recognition by monocot mannose-binding lectins was studied via the crystal structure determination of daffodil (Narcissus pseudonarcissus) lectin. The lectin was extracted from daffodil bulbs, and crystallised in the presence of alpha-1,3 mannobiose. Molecular replacement methods were used to solve the structure using the partially refined model of Hippeastrum hybrid agglutinin as a search model. The structure was refined at 2.0 A resolution to a final R -factor of 18.7 %, and Rfreeof 26.7 %.The main feature of the daffodil lectin structure is the presence of three fully occupied binding pockets per monomer, arranged around the faces of a triangular beta-prism motif. The pockets have identical topology, and can bind mono-, di- or oligosaccharides. Strand exchange forms tightly bound dimers, and higher aggregation states are achieved through hydrophobic patches on the surface, completing a tetramer with internal 222-symmetry. There are therefore 12 fully occupied binding pockets per tetrameric cluster. The tetramer persists in solution, as shown with small-angle X-ray solution scattering. Extensive sideways and out-of-plane interactions between tetramers, some mediated via the ligand, make up the bulk of the lattice contacts.A fourth binding site was also observed. This is unique and has not been observed in similar structures. The site is only partially occupied by a ligand molecule due to the much lower binding affinity. A comparison with the Galanthus nivalis agglutinin/mannopentaose complex suggests an involvement of this site in the recognition mechanism for naturally occurring glycans.

A novel mode of carbohydrate recognition in jacalin, a Moraceae plant lectin with a beta-prism fold.

Sankaranarayanan R, Sekar K, Banerjee R, Sharma V, Surolia A, Vijayan M
Nat Struct Biol. 1996; 3: 596-603

Jacalin, a tetrameric two-chain lectin (66,000 Mr) from jackfruit seeds, is highly specific for the tumour associated T-antigenic disaccharide. The crystal structure of jacalin with methyl-alpha-D-galactose reveals that each subunit has a three-fold symmetric beta-prism fold made up of three four-stranded beta-sheets. The lectin exhibits a novel carbohydrate-binding site involving the N terminus of the alpha-chain which is generated through a post-translational modification involving proteolysis, the first known instance where such a modification has been used to confer carbohydrate specificity. This new lectin fold may be characteristic of the Moraceae plant family. The structure provides an explanation for the relative affinities of the lectin for galactose derivatives and provides insights into the structural basis of its T-antigen specificity.

Studies on the carbohydrate binding sites of the hemolytic lectin CEL-III isolated from the marine invertebrate Cucumaria echinata.

Sallay I, Hatakeyama T, Yamasaki N
Biosci Biotechnol Biochem. 1998; 62: 1757-61

The binding of carbohydrates to the hemolytic lectin CEL-III isolated from the marine invertebrate Cucumaria echinata was studied. Equilibrium dialysis data suggest that CEL-III has two carbohydrate-binding sites with equal affinity. The binding of specific carbohydrates to CEL-III induces a decrease in the fluorescence intensity at 339 nm and the shift of the fluorescence emission maximum to a wavelength shorter by 3 nm, owing to the change in the environment of tryptophan. By analyzing the change in the fluorescence intensity at 339 nm as a function of the concentration of carbohydrates, the association constants for binding of individual carbohydrates to CEL-III were calculated. The results indicate that GalNAc, lactulose, and lactose are bound by CEL-III with fairly high affinity among the carbohydrates tested. The pH-dependence profile of the association constant of lactose suggests that CEL-III binds carbohydrates with highest affinity around pH 5.0. Modification of CEL-III with N-bromosuccinimide produces an oxidized derivative, in which four tryptophan residues/mol were oxidized and had no hemolytic activity. However, two out of these four tryptophans escaped from the modification in the presence of specific saccharides and the resulting derivative retained fairly high hemolytic activity.

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

Sajjan SU, Patanjali SR, Surolia A
Indian J Biochem Biophys. 1997; 34: 76-81

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.

Medicinal chemistry based on the sugar code: fundamentals of lectinology and experimental strategies with lectins as targets.

Rudiger H, Siebert HC, Solis D, Jimenez-Barbero J, Romero A, von der Lieth CW, Diaz-Marino T, Gabius HJ
Curr Med Chem. 2000; 7: 389-416

Theoretical calculations reveal that oligosaccharides are second to no other class of biochemical oligomery in terms of coding capacity. As integral part of cellular glycoconjugates they can serve as recognitive units for receptors (lectins). Having first been detected in plants, lectins are present ubiquitously. Remarkably for this field, they serve as bacterial and viral adhesins. Following a description of these branches of lectinology to illustrate history, current status and potential for medicinal chemistry, we document that lectins are involved in a wide variety of biochemical processes including intra- and intercellular glycoconjugate trafficking, initiation of signal transduction affecting e. g. growth regulation and cell adhesion in animals. It is thus justified to compare crucial carbohydrate epitopes with the postal code ensuring correct mail routing and delivery. In view of the functional relevance of lectins the design of high-affinity reagents to occupy their carbohydrate recognition domains offers the perspective for an attractive source of new drugs. Their applications can be supposed to encompass the use as cell-type-selective determinant for targeted drug delivery and as blocking devices in anti-adhesion therapy during infections and inflammatory disease. To master the task of devising custom-made glycans/glycomimetics for this purpose, the individual enthalpic and entropic contributions in the molecular rendezvous between the sugar receptor under scrutiny and its ligand in the presence of solvent molecules undergoing positional rearrangements need to be understood and rationally exploited. As remunerative means to this end, cleverly orchestrated deployment of a panel of methods is essential. Concerning the carbohydrate ligand, its topological parameters and flexibility are assessed by the combination of computer-assisted molecular-mechanics and molecular-dynamics calculations and NMR-spectroscopic measurements. In the presence of the receptor, the latter technique will provide insights into conformational aspects of the bound ligand and into spatial vicinity of the ligand to distinct side chains of amino acids establishing the binding site in solution. Also in solution, the hydrogen-bonding pattern in the complex can be mapped with monodeoxy and monofluoro derivatives of the oligosaccharide. Together with X-ray crystallographic and microcalorimetric studies the limits of a feasible affinity enhancement can be systematically probed. With galactoside-binding lectins as instructive mo del, recent progress in this area of drug design will be documented, emphasizing the general applicability of the outlined interdisciplinary approach.

Lectin-carbohydrate interactions in disease. T-cell recognition of IgA and IgD; mannose binding protein recognition of IgG0.

Rudd P, Fortune F, Lehner T, Parekh R, Patel T, Wormald M, Malhotra R, Sim R, Dwek R
Adv Exp Med Biol. 1995; 376: 147-52

Two disease associated lectin-carbohydrate interactions have been studied. (1) A T-cell surface lectin which binds IgA1 and IgD is expressed on CD4+ and CD8+ T-lymphocytes in a number of diseases including systemic lupus erythematosus, rheumatoid arthritis (RA), Behcet's disease and IgA nephropathy. We have demonstrated that calcium independent binding to this receptor is mediated by the O-linked disaccharide Gal beta 3GalNAc which is associated with the hinge regions of both IgA1 and IgD. (2) In rheumatoid arthritis the proportion of IgG0 glycoform populations lacking terminal galactose increases. We have shown that terminal GlcNAc residues on oligosaccharides in the Fc region of IgG0 can bind to the C-type lectin, serum mannose binding protein, and thus activate the classical complement pathway. This provides a mechanism of activation of the complement system not available to the other classes of IgG glycoforms.

Conjugation of lectins with fluorochromes: an approach to histochemical double labeling of carbohydrate components.

Roth J, Binder M, Gerhard UJ
Histochemistry. 1978; 56: 265-73

Methodical investigations on the coupling of lectins (Con A, LcL, WGA, RcA) to tetramethylrhodamine isothiocyan ate (TRITC) are reported. 20-microgram of TRITC per mg of lectin were found to be the optimal amount of TRITC for the conjugation. With this fluorochrome: protein ratio conjugates were produced which resulted in a specific and brilliant fluorescence in tissue staining. The optimally conjugated lectins were separated on DEAE-Sephadex-A 50. Using two different lectins which were conjugated with TRITC or FITC, respectively, a double labeling of different lectin-binding sites in tissue sections was achieved.

Gold-conjugated arabinogalactan-protein and other lectins as ultrastructural probes for the wheat/stem rust complex.

Rohringer R, Chong J, Gillespie R, Harder DE
Histochemistry. 1989; 91: 383-93

Arabinogalactan-protein (AGP, "beta-lectin") was isolated from leek seeds, tested for specificity, conjugated with gold colloids, and used as a cytochemical probe to detect beta-linked bound sugars in ultrathin sections of wheat leaves infected with a compatible race of stem rust fungus. Similar sections were probed with other gold-labeled lectins to detect specific sugars. AGP-gold detected beta-glycosyl in all fungal walls and in the extrahaustorial matrix. Other lectin gold conjugates localized galactose in all fungal walls except in walls of the haustorial body. Limulus polyphemus lectin bound only to the outermost layer of intercellular hyphal walls of the fungus. Binding of these lectins was inhibited by their appropriate haptens and was diminished or abolished in specimens pretreated with protease, indicating that the target substances in the tissue were proteinaceous or that polysaccharides possessing affinity to the lectin probes had been removed by the enzyme from a proteinaceous matrix by passive escape. Binding of Lotus tetragonolobus lectin was limited to the two outermost fungal wall layers but was not hapten-inhibitable. Limax flavus lectin, specific for sialic acids, had no affinity to any structure in the sections. In the fungus, the most complex structure was the outermost wall layer of intercellular hyphal cells; it had affinity to all lectins tried so far, except to Limax flavus lectin and to wheat germ lectin included in an earlier study. In the host, AGP and the galactose-specific lectins bound to the inner domain of the wall in areas not in contact with the fungus. At host cell penetration sites, affinity to these lectins often extended throughout the host wall, confirming that it is modified at these sites. Pre-treatment with protease had no effect on lectin binding to the host wall. After protease treatment, host starch granules retained affinity to galactose-specific lectins, but lost affinity for AGP.

Reexamination of the carbohydrate binding stoichiometry of lima bean lectin.

Roberts DD, Goldstein IJ
Arch Biochem Biophys. 1984; 230: 316-20

The carbohydrate binding stoichiometry of lima bean lectin component III was reexamined using equilibrium dialysis and quantitative affinity chromatography following limited chemical modification. Equilibrium dialysis employing methyl[2-14C]benzamido-2-deoxy-alpha-D-galactopyranoside as ligand demonstrated that the lectin tetramer bound 4 mol of sugar with Kassoc = 1.44 +/- 0.13 X 10(3) M-1 (T = 5 degrees C, pH 7.0, ionic strength 0.1). The previous report of two sites/tetramer [Bessler, W. and Goldstein, I. J. (1974) Arch. Biochem. Biophys. 165, 444] appears to be the result of partial inactivation of the lectin due to oxidation of essential thiol groups. Following limited chemical modification of the thiol groups by methyl methanethiosulfonate, multiple intermediate forms with reduced affinity for Synsorb A were obtained. The number and hemagglutinating activities of these intermediates provided further support for the presence of four carbohydrate binding sites on lima bean lectin component III.

Effect of carbohydrate and metal ion binding on the reactivity of the essential thiol groups of lima bean lectin.

Roberts DD, Goldstein IJ
J Biol Chem. 1984; 259: 903-8

A free sulfhydryl group previously has been shown to be required for carbohydrate binding to the lectin from lima bean (Phaseolus lunatus) (Gould, N. R. and Scheinberg, S. L. (1970) Arch. Biochem. Biophys. 141, 607-613). Modification of this group by sulfhydryl reagents was specifically inhibited by D-GalNAc. We have further examined the reactivity of sulfhydryl groups in lima bean lectin with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) as a probe for carbohydrate and metal ion binding. The 4 thiol groups in tetrameric lima bean lectin component III gave identical kinetics for reaction with Nbs2 involving formation of a weak noncovalent complex between Nbs2 and the lectin. The pH-independent reactivity of the thiol groups at neutral pH suggested that the thiols may exist as ion pairs with a nearby ionized group. Carbohydrate ligands were competitive inhibitors of thiol modification. The thiol groups on all 4 subunits of lima bean lectin were completely and reversibly protected by carbohydrate binding. The ability of carbohydrates to inhibit thiol modification correlated with their potency as inhibitors in a precipitin inhibition assay. The best inhibitors were the oligosaccharides alpha-D-GalNAc-(1 leads to 3)[alpha-L-fucose-(1 leads to 2)]beta-D-Gal(1 leads to R) and alpha-D-GalNAc-(1 leads to 2)beta-D-Gal(1 leads to R). Apparent thermodynamic parameters for binding of several carbohydrates were determined by measuring the temperature dependence of thiol protection. Removal of the bound metal ions Ca2+ and Mn2+ following dialysis into EDTA inactivated the lectin and increased the reactivity of the thiol groups 60-fold. This conversion was temperature-dependent and could be reversed upon addition of metal ions. The fast-reacting thiol groups were not protected by haptenic sugars from modifications by Nbs2.

X-ray crystal structure of a pea lectin-trimannoside complex at 2.6 A resolution.

Rini JM, Hardman KD, Einspahr H, Suddath FL, Carver JP
J Biol Chem. 1993; 268: 10126-32

The x-ray crystal structure of pea lectin, in complex with a methyl glycoside of the N-linked-type oligosaccharide trimannosyl core, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, has been solved by molecular replacement and refined at 2.6-A resolution. The R factor is 0.183 for all data in the 8.0 to 2.6 A resolution range with an average atomic temperature factor of 26.1 A2. Strong electron density for a single mannose residue is found in the monosaccharide-binding site suggesting that the trisaccharide binds primarily through one of the terminal alpha-linked mannose residues. The complex is stabilized by hydrogen bonds involving the protein residues Asp-81, Gly-99, Asn-125, Ala-217, and Glu-218, and the carbohydrate oxygen atoms O3, O4, O5, and O6. In addition, the carbohydrate makes van der Waals contacts with the protein, involving Phe-123 in particular. These interactions are very similar to those found in the monosaccharide complexes with concanavalin A and isolectin 1 of Lathyrus ochrus, confirming the structural relatedness of this family of proteins. Comparison of the pea lectin complex with the unliganded pea lectin and concanavalin A structures indicates differences in the conformation and water structure of the unliganded binding sites of these two proteins. Furthermore, a correlation between the position of the carbohydrate oxygen atoms in the complex and the bound water molecules in the unliganded binding sites is found. Binding of the trimannose core through a single terminal monosaccharide residue strongly argues that an additional fucose-binding site is responsible for the high affinity pea lectin-oligosaccharide interactions.

Lectin structure.

Rini JM
Annu Rev Biophys Biomol Struct. 1995; 24: 551-77

Lectins comprise a structurally very diverse class of proteins characterized by their ability to bind carbohydrates with considerable specificity. They are found in organisms ranging from viruses and plants to humans and serve to mediate biological recognition events. Although lectins bind monosaccharides rather weakly, they employ common strategies for enhancing both the affinity and specificity of their interactions for more complex carbohydrate ligands. The terms subsite and subunit multivalency are defined to describe the ways in which these enhancements are achieved. Analysis of the X-ray crystal structures of different lectin types serves to illustrate how, in structural terms, subsite and subunit multivalency confer context-specific functional properties.

Carbohydrate-binding sites of plant lectins.

Reeke GN Jr, Becker JW
Curr Top Microbiol Immunol. 1988; 139: 35-58

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

Rao VS, Lam K, Qasba PK
Int J Biol Macromol. 1998; 23: 295-307

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.

Thermodynamic studies of saccharide binding to artocarpin, a B-cell mitogen, reveals the extended nature of its interaction with mannotriose [3,6-Di-O-(alpha-D-mannopyranosyl)-D-mannose].

Rani PG, Bachhawat K, Misquith S, Surolia A
J Biol Chem. 1999; 274: 29694-8

The thermodynamics of binding of various saccharides to artocarpin, from Artocarpus integrifolia seeds, a homotetrameric lectin (M(r) 65, 000) with one binding site per subunit, was determined by isothermal titration calorimetry measurements at 280 and 293 K. The binding enthalpies, DeltaH(b), are the same at both temperatures, and the values range from -10.94 to -47.11 kJ mol(-1). The affinities of artocarpin as obtained from isothermal titration calorimetry are in reasonable agreement with the results obtained by enzyme-linked lectin absorbent essay, which is based on the minimum amount of ligand required to inhibit horseradish peroxidase binding to artocarpin in enzyme-linked lectin absorbent essay (Misquith, S., Rani, P. G., and Surolia, A. (1994) J. Biol. Chem. 269, 30393-30401). The interactions are mainly enthalpically driven and exhibit enthalpy-entropy compensation. The order of binding affinity of artocarpin is as follows: mannotriose>Manalpha3Man>GlcNAc(2)Man(3)>MealphaMan>Man>M analpha6Man> Manalpha2Man>MealphaGlc>Glc, i.e. 7>4>2>1.4>1>0.4>0.3>0.24>0.11. The DeltaH for the interaction of Manalpha3Man, Manalpha6Man, and MealphaMan are similar and 20 kJ mol(-1) lower than that of mannotriose. This indicates that, while Manalpha3Man and Manalpha6Man interact with the lectin exclusively through their nonreducing end monosaccharide with the subsites specific for the alpha1,3 and alpha1,6 arms, the mannotriose interacts with the lectin simultaneously through all three of its mannopyranosyl residues. This study thus underscores the distinction in the recognition of this common oligosaccharide motif in comparison with that displayed by other lectins with related specificity.

The carbohydrate-binding specificity of a highly toxic protein from Abrus pulchellus seeds.

Ramos MV, Teixeira CR, Bomfim LR, Madeira SV, Moreira RA
Mem Inst Oswaldo Cruz. 1999; 94: 185-8

The carbohydrate-binding specificity and molecular modelling of Canavalia maritima and Dioclea grandiflora lectins.

Ramos MV, Moreira Rd, Oliveira JT, Cavada BS, Rouge P
Mem Inst Oswaldo Cruz. 1996; 91: 761-6

The carbohydrate-binding specificity of lectins from the seeds of Canavalia maritima and Dioclea grandiflora was studied by hapten-inhibition of haemagglutination using various sugars and sugar derivatives as inhibitors, including N-acetylneuraminic acid and N-acetylmuramic acid. Despite some discrepancies, both lectins exhibited a very similar carbohydrate-binding specificity as previously reported for other lectins from Diocleinae (tribe Phaseoleae, sub-tribe Diocleinae). Accordingly, both lectins exhibited almost identical hydropathic profiles and their three-dimensional models built up from the atomic coordinates of ConA looked very similar. However, docking experiments of glucose and mannose in their monosaccharide-binding sites, by comparison with the ConA-mannose complex used as a model, revealed conformational changes in side chains of the amino acid residues involved in the binding of monosaccharides. These results fully agree with crystallographic data showing that binding of specific ligands to ConA requires conformational chances of its monosaccharide-binding site.

Sequence and structural determinants of mannose recognition.

Ramachandraiah G, Chandra NR
Proteins. 2000; 39: 358-64

Mannose, an abundant cell surface monosaccharide binds to a diverse set of receptors, which are involved in a variety of important cellular processes. Structural analysis has been carried out on all the proteins containing non-covalently bound mannose as a monosaccharide in the Protein Data Bank, to identify common recognition principles. Proteins, highly specific to mannose, belonging to the super family of bulb lectins, are found to contain a consensus sequence motif QXDXNXVXY, which has been identified to be essential for mannose binding. Analysis of this motif in the crystal structures of bulb lectins has led to the understanding of the contribution of individual residues in mannose recognition. Comparison with other mannose binding proteins, reveals common hydrogen bonding patterns in all of them, despite differences in sequence, overall fold and the substructures at the binding sites of individual proteins. A database analysis also suggests that although the topology of the backbone, as at the binding site in bulb lectins, can generate mannose binding capability in a few other proteins, sequence and disposition of not only the residues in the motif, but also the residues in the neighborhood play a crucial role in allowing that property to be retained.

Carbohydrate specificity and quaternary association in basic winged bean lectin: X-ray analysis of the lectin at 2.5 A resolution.

Prabu MM, Sankaranarayanan R, Puri KD, Sharma V, Surolia A, Vijayan M, Suguna K
J Mol Biol. 1998; 276: 787-96

The structure of basic Winged Bean Agglutinin (WBAI) with two dimeric molecules complexed with methyl-alpha-D-galactopyranoside in the asymmetric unit, has been determined by the molecular replacement method and refined with 2.5 A X-ray intensity data. The polypeptide chain of each monomer has the characteristic legume lectin tertiary fold. The structure clearly defines the lectin-carbohydrate interactions. It reveals how the unusually long variable loop in the binding region endows the lectin with its characteristic sugar specificity. The lectin forms non-canonical dimers of the type found in Erythrina corallodendron lectin (EcorL) even though glycosylation, unlike in EcorL, does not prevent the formation of canonical dimers. The structure thus further demonstrates that the mode of dimerisation of legume lectins is not necessarily determined by the covalently bound carbohydrate but is governed by features intrinsic to the protein. The present analysis and our earlier work on peanut lectin (PNA), show that legume lectins are a family of proteins in which small alterations in essentially the same tertiary structure lead to wide variations in quaternary association. A relationship among the non-canonical modes of dimeric association in legume lectins is presented.

Cell surface carbohydrates and lectins in early development.

Poirier F, Kimber S
Mol Hum Reprod. 1997; 3: 907-18

Current knowledge on the development regulation of cell surface carbohydrates and lectins in mammalian embryos is summarized. Much of this data comes from observations on mouse embryos but information on the human embryo is included where it is available. Over the last few years, numerous studies have indicated that carbohydrates play a critical role in the cell-cell interactions of the pre- and peri-implantation embryo. Functional tests suggest a role for terminal fucosylated Galbeta1-3/4GlcNAc structures in the early steps of implantation. We also now have clear evidence for the expression of lectins in the trophectoderm just prior to implantation. Mouse mutants have been generated which lack particular enzymes involved in glycosylation or particular lectins, but so far they have not been informative about the role of glycoconjugates or lectins in the very early embryo.

[Vicia graminea lectin or Vicia unijuga lectin-binding (Vgu) glycoproteins as new tumor-associated substances]

Ohkuma S
Yakugaku Zasshi. 1993; 113: 95-113

The purification and serological and chemical properties of Vicia graminea lectin (VGA) and Vicia unijuga lectin (VUA) were described, and then the binding-specificity of anti-M and -N antibodies and both the lectins was discussed in this review. On the basis of the facts that Vgu glycoproteins which react with either VGA or VUA but not with anti-M and -N antibodies and were not detected in human normal organ tissues and sera, were separated and identified as mucin-type glycoproteins with very high molecular weights from human cancerous organ tissues, ascitic fluids of cancer patients and cyst fluids of human ovarian cystadenoma in malignant, it was concluded that Vgu glycoproteins are new tumor-associated substances.

A novel developmental stage-specific lectin of the basidiomycete Pleurotus cornucopiae.

Oguri S, Ando A, Nagata Y
J Bacteriol. 1996; 178: 5692-8

A novel lectin was isolated from mycelia of the basidiomycete Pleurotus cornucopiae grown on solid medium. The lectin was purified to homogeneity by mucin-Sepharose affinity chromatography. The molecular mass of the lectin was 40 kDa under reducing conditions, but the subunits were polymerized through disulfide bridges under physiological conditions. Hemagglutinating activity of this lectin was completely inhibited by 2-mercaptoethanol, indicating that the multimer is active. The activity was also inhibited by EDTA, and restored by CaCl2. N-Acetyl-D-galactosamine was the most potent hapten inhibitor. N-terminal amino acid sequence analysis revealed that the mycelial lectin was different from the fruit body lectin of this organism. The mycelial lectin appeared prior to fruit body formation and disappeared during the formation of fruit bodies. The lectin was localized on the surface of solid-medium-grown mycelia, and only dikaryotic, and not monokaryotic, mycelia produced the lectin. These results suggest that the appearance of this lectin is associated with fruit body formation.

Evaluation of fluorescence polarization method for binding study in carbohydrate-lectin interaction.

Oda Y, Kinoshita M, Nakayama K, Kakehi K
Biol Pharm Bull. 1998; 21: 1215-7

The fluorescence polarization (FP) technique was evaluated to determine molecular interaction between plant lectins and polysaccharides, yeast cells and glycopeptide after labeling the lectins with fluorescein isothiocyanate. Use of Lycoris radiata agglutinin allowed determination of the molecular interactions with large biomolecules containing mannose oligomers and polymers. Another example using a fluorescein-labeled glycopeptide also indicated that use of the FP method would allow easy observation of the molecular interactions on the quantitative base. The present technique is highly sensitive and facile because it does not require any washing procedures before measurement.

Lectin-carbohydrate interaction in the immune system.

Ni Y, Tizard I
Vet Immunol Immunopathol. 1996; 55: 205-23

The immune system consists of various types of cells and molecules that specifically interact with each other to initiate the host defense mechanism. Recent studies have shown that carbohydrates and lectins (carbohydrate-binding proteins) play an essential role in mediating such interactions. Both lectins and carbohydrates are widely distributed in the mammalian tissues as well as in microorganisms. Carbohydrates, due to their chemical nature, can potentially form structures that are more variable than proteins and nucleic acids. Lectins can exist in either soluble or cell-associated form, and although overall structures vary, invariably possess carbohydrate-recognition domains (CRD) with various specificities. The interaction between lectins and carbohydrates have been shown to be involved in such activities as opsonization of microorganisms, phagocytosis, cell adhesion and migration, cell activation and differentiation, and apoptosis. The number of lectins identified in the immune system is increasing at a rapid pace. The development in this area has opened a new aspect in studying the immune system, and at the same time, provided new therapeutic routes for the treatment and prevention of disease.

Carbohydrate-protein interactions in vascular biology.

Nelson RM, Venot A, Bevilacqua MP, Linhardt RJ, Stamenkovic I
Annu Rev Cell Dev Biol. 1995; 11: 601-31

Carbohydrate-protein interactions participate in a wide variety of biological and pathological events. In recent years, particular attention has been paid to the carbohydrate-protein interactions that occur in vascular biology. Sialylated oligosaccharides are ligands of a structurally diverse group of proteins that include the selectins and members of the immunoglobulin superfamily. Various glycosaminoglycans can be recognized by an overlapping set of proteins that include two of the selectins and CD44. Emerging knowledge of carbohydrate-protein interactions in human pathophysiology are discussed.

Fluorescence-labeled synthetic glycopolymers: a new type of sugar ligands of lectins.

Nagata K, Furuike T, Nishimura S
J Biochem (Tokyo). 1995; 118: 278-84

Radical copolymerization of a polymerizable dansyl derivative, N-2-propenyl-(5-dimethylamino)-1-naphthalene sulfonamide, with sugar monomers and acrylamide proceeded smoothly in aqueous solution in the presence of ammonium persulfate and N,N,N',N'-tetramethylethylenediamine and afforded a novel type of water-soluble glycopolymers having fluorescent side-chains. Fluorescence emission spectra of these polymeric sugar-ligands by excitation at 340 nm revealed maxima at 448 and 528 nm. When the glycopolymer carrying galactose residues was saturated with Ricinus communis agglutinin (RCA60), the fluorescence emission maxima at 448 and 528 nm were not shifted significantly, although the fluorescence intensities were decreased by 20 and 14%, respectively. Polymeric sugar-cluster effects drastically enhanced the association constants of galactose residues with RCA60 in the order of 10(8) M-1. The significance for efficient binding of galactose density on the glycopolymer was also demonstrated by using glycopolymers with different degrees of galactose branching.

Sugar-lectin interactions: sugar clusters, lectin multivalency and avidity.

Monsigny M, Mayer R, Roche AC
Carbohydr Lett. 2000; 4: 35-52

Carbohydrate binding properties of banana (Musa acuminata) lectin I. Novel recognition of internal alpha1,3-linked glucosyl residues.

Mo H, Winter HC, Van Damme EJ, Peumans WJ, Misaki A, Goldstein IJ
Eur J Biochem. 2001; 268: 2609-15

Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the alpha-anomeric form of these sugars. Both generate precipitin curves with branched chain alpha-mannans (yeast mannans) and alpha-glucans (glycogens, dextrans, and starches), but not with linear alpha-glucans containing only alpha1,4- and alpha1,6-glucosidic bonds (isolichenan and pullulan). The novel observation was made that banana and plantain lectins recognize internal alpha1,3-linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran. Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear alpha-glucans. This is, the authors believe, the first report of the recognition of internal alpha1,3-glucosidic bonds by a plant lectin. It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch.

[Lectins and recognition of protein ligands]

Miroshnichenko OS
Ukr Biokhim Zh. 1999; 71: 5-9

The general principles of protein-carbohydrate lectin interactions were discussed. Experimental evidences of the molecular mimicry between carbohydrate and protein lectin ligands were analyzed. The interdomain interactions in lectins as well as the interactions between ligands and lectin domains other than carbohydrate-binding domains were considered.

Detection of lectin-sugar interaction by ultraviolet difference spectroscopy.

Matsumoto I, Jinbo A, Kitagaki H, Golovtchenko-Matsumoto AM, Seno N
J Biochem (Tokyo). 1980; 88: 1093-6

It was found that the addition of specific sugars to solutions of several lectins induced ultraviolet difference spectra. The difference spectra of lectins from Lens culinaris, Sophora japonica, Solanum tuberosum and wheat germ have two peaks at 292 nm and 284-287 nm which are characteristic of the tryptophanyl residue. The difference spectrum of Arachis hypogaea agglutinin has two peaks at 285 nm and 279 nm which seem to be characteristic of the tyrosyl residue. In addition to the identification of these amino acid residues as being in or near the sugar binding sites of the lectins, the binding constants of these lectins with the specific sugars can be easily determined from the intensities of the difference spectra at various concentrations of sugars. This method may be useful for the simple and direct determination of the binding constants of lectins with various naturally occurring sugars which have no chromogenic groups.

Possible interaction between animal lectins and bacterial carbohydrates.

Mandrell RE, Apicella MA, Lindstedt R, Leffler H
Methods Enzymol. 1994; 236: 231-54

Specificity mapping of bacterial lectins by inhibition of hemagglutination using deoxy and deoxyfluoro analogs of receptor-active saccharides.

Magnusson G, Hultgren SJ, Kihlberg J
Methods Enzymol. 1995; 253: 105-14

[Use of lectins in light-microscopic histochemistry (methodologic aspects)]

Lutsik AD, Detiuk ES
Arkh Anat Gistol Embriol. 1987; 92: 74-89

Collectin-43 is a serum lectin with a distinct pattern of carbohydrate recognition.

Loveless RW, Holmskov U, Feizi T
Immunology. 1995; 85: 651-9

Collectin-43 (CL-43) is a C-type serum lectin and a member of the collectin family of soluble proteins that are effector molecules in innate immunity. We have investigated the binding specificity of CL-43 using as model systems a panel of structurally defined oligosaccharides in the form of neoglycolipids, and several glycoproteins derived from the complement glycoprotein C3 during activation of the complement cascade. A specificity is revealed towards fucose as part of the Lea oligosaccharide sequence, and towards mannose as found on high mannose-type chains. These are features shared with other serum collectins, conglutinin and mannan-binding proteins; a major difference is the lack of detectable binding by CL-43 to N-glycosidic oligosaccharides terminating in N-acetylglucosamine. CL-43 has a unique pattern of reactivity towards high mannose-type oligosaccharides on the two glycosylation sites of C3 and derived glycoproteins: it binds to C3c (not bound by conglutinin and mannan-binding protein) but not to hydrolysed C3 [C3(H2O)], C3b or iC3b immobilized on microwells (all bound by conglutinin but not by mannan-binding protein). When these glycoproteins are sodium dodecyl sulphate (SDS)-treated and immobilized on nitrocellulose, CL-43 (but not conglutinin nor mannan-binding protein) binds strongly to C3(H2O), iC3b and C3c. The salient conclusions are, first, that there are remarkable positive or negative effects of carrier protein on oligosaccharide presentation and these differ for the individual collectins. Second, the different though partially overlapping binding patterns among the collectins may be important for their function as circulating effector molecules with broad surveillance capabilities.

Legume lectin structure.

Loris R, Hamelryck T, Bouckaert J, Wyns L
Biochim Biophys Acta. 1998; 1383: 9-36

The legume lectins are a large family of homologous carbohydrate binding proteins that are found mainly in the seeds of most legume plants. Despite their strong similarity on the level of their amino acid sequences and tertiary structures, their carbohydrate specificities and quaternary structures vary widely. In this review we will focus on the structural features of legume lectins and their complexes with carbohydrates. These will be discussed in the light of recent mutagenesis results when appropriate. Monosaccharide specificity seems to be achieved by the use of a conserved core of residues that hydrogen bond to the sugar, and a variable loop that determines the exact shape of the monosaccharide binding site. The higher affinity for particular oligosaccharides and monosaccharides containing a hydrophobic aglycon results mainly from a few distinct subsites next to the monosaccharide binding site. These subsites consist of a small number of variable residues and are found in both the mannose and galactose specificity groups. The quaternary structures of these proteins form the basis of a higher level of specificity, where the spacing between individual epitopes of multivalent carbohydrates becomes important. This results in homogeneous cross-linked lattices even in mixed precipitation systems, and is of relevance for their effects on the biological activities of cells such as mitogenic responses. Quaternary structure is also thought to play an important role in the high affinity interaction between some legume lectins and adenine and a series of adenine-derived plant hormones. The molecular basis of the variation in quaternary structure in this group of proteins is poorly understood.

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

Loris R, De Greve H, Dao-Thi MH, Messens J, Imberty A, Wyns L
J Mol Biol. 2000; 301: 987-1002

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.

Carbohydrate binding activities of Bradyrhizobium japonicum: IV. Effect of lactose and flavones on the expression of the lectin, BJ38.

Loh JT, Ho SC, Wang JL, Schindler M
Glycoconj J. 1994; 11: 363-70

BJ38 is a galactose/lactose-specific lectin (M(r) approximately 38,000) found at one pole of Bradyrhizobium japonicum. It has been implicated in mediating the adhesion of the bacteria to soybean roots, leading to the establishment of a nitrogen-fixing symbiosis. When the ligand lactose is added to cultures of the bacteria for at least 1 h prior to harvesting the cells for BJ38 isolation, the yield of the protein was found to be elevated in a dose-dependent fashion. Half maximal stimulation was observed at approximately 50 microM; the effect was saturated at approximately 1 mM, where a 10-fold higher yield of BJ38 was obtained. Saccharides with a lower affinity for BJ38 than lactose yielded a correspondingly smaller induction effect when compared at a concentration of 1 mM. The higher level of BJ38 induced by lactose is also manifested by an elevated amount of BJ38 detectable at the cell surface and by a higher number of B. japonicum cells adsorbed onto soybean cells. Surprisingly, the induction of BJ38 expression seen with lactose was also observed with certain, but not all, flavonoids that induce the nod genes of the bacteria; genistein mimicked the induction observed with lactose, whereas luteolin failed to stimulate BJ38 production.

Carbohydrate binding activities of Bradyrhizobium japonicum: unipolar localization of the lectin BJ38 on the bacterial cell surface.

Loh JT, Ho SC, de Feijter AW, Wang JL, Schindler M
Proc Natl Acad Sci U S A. 1993; 90: 3033-7

A polyclonal antiserum generated against the Bradyrhizobium japonicum lectin BJ38 was characterized to be specifically directed against the protein. Treatment of B. japonicum cells with this antiserum and subsequent visualization with transmission electron microscopy and both conventional and confocal fluorescence microscopy revealed BJ38 at only one pole of the bacterium. BJ38 appeared to be organized in a tuft-like mass, separated from the bacterial outer membrane. BJ38 localization was coincident with the attachment site for (i) homotypic agglutination to other B. japonicum cells, (ii) adhesion to the cultured soybean cell line SB-1, and (iii) adsorption to Sepharose beads covalently derivatized with lactose. In contrast, the plant lectin soybean agglutinin labeled the bacteria at the pole distant from the bacterial attachment site. These results indicate that the topological distribution of BJ38 is consistent with a suggested role for this bacterial lectin in the polar binding of B. japonicum to other cells and surfaces.

The molecular mechanism of sulfated carbohydrate recognition by the cysteine-rich domain of mannose receptor.

Liu Y, Misulovin Z, Bjorkman PJ
J Mol Biol. 2001; 305: 481-90

The mannose receptor (MR) binds foreign and host ligands through interactions with their carbohydrates. Two portions of MR have distinct carbohydrate recognition properties. One is conferred by the amino-terminal cysteine-rich domain (Cys-MR), which plays a critical role in binding sulfated glycoproteins including pituitary hormones. The other is achieved by tandemly arranged C-type lectin domains that facilitate carbohydrate-dependent uptake of infectious microorganisms. This dual carbohydrate binding specificity enables MR to bind ligands by interacting with both sulfated and non-sulfated polysaccharide chains. We previously determined crystal structures of Cys-MR complexed with 4-SO(4)-N-acetylglucosamine and with an unidentified ligand resembling Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]). In continued efforts to elucidate the mechanism of sulfated carbohydrate recognition by Cys-MR, we characterized the binding affinities between Cys-MR and potential carbohydrate ligands using a fluorescence-based assay. We find that Cys-MR binds sulfated carbohydrates with relatively high affinities (K(D)=0.1 mM to 1.0 mM) compared to the affinities of other lectins. Cys-MR also binds Hepes with a K(D) value of 3.9 mM, consistent with the suggestion that the ligand in the original Cys-MR crystal structure is Hepes. We also determined crystal structures of Cys-MR complexed with 3-SO(4)-Lewis(x), 3-SO(4)-Lewis(a), and 6-SO(4)-N-acetylglucosamine at 1.9 A, 2.2 A, and 2.5 A resolution, respectively, and the 2.0 A structure of Cys-MR that had been treated to remove Hepes. The conformation of the Cys-MR binding site is virtually identical in all Cys-MR crystal structures, suggesting that Cys-MR does not undergo conformational changes upon ligand binding. The structures are used to rationalize the binding affinities derived from the biochemical studies and to elucidate the molecular mechanism of sulfated carbohydrate recognition by Cys-MR. Copyright 2001 Academic Press.

Structural basis for the recognition of carbohydrates by human galectin-7.

Leonidas DD, Vatzaki EH, Vorum H, Celis JE, Madsen P, Acharya KR
Biochemistry. 1998; 37: 13930-40

Knowledge about carbohydrate recognition domains of galectins, formerly known as S-type animal lectins, is important in understanding their role(s) in cell-cell interactions. Here we report the crystal structure of human galectin-7 (hGal-7), in free form and in the presence of galactose, galactosamine, lactose, and N-acetyl-lactosamine at high resolution. This is the first structure of a galectin determined in both free and carbohydrate-bound forms. The structure shows a fold similar to that of the prototype galectins -1 and -2, but has greater similarity to a related galectin molecule, Gal-10. Even though the carbohydrate-binding residues are conserved, there are significant changes in this pocket due to shortening of a loop structure. The monomeric hGal-7 molecule exists as a dimer in the crystals, but adopts a packing arrangement considerably different from that of Gal-1 and Gal-2, which has implications for carbohydrate recognition.

Involvement of water in host-guest interactions.

Lemieux RU, Delbaere LT, Beierbeck H, Spohr U
Ciba Found Symp. 1991; 158: 231-45

As predicted by inhibition studies the X-ray crystal structure of the complex formed between the tetrasaccharide alpha-L-Fuc(1----2)-beta-D-Gal(1----3) [alpha-L-Fuc-(1----4)]-beta-D-GlcNAc- OMe (Leb-OMe) and the lectin IV of Griffonia simplicifolia (GS-IV) shows three hydroxyl groups (referred to as the polar key) hydrogen bonded within the combining site and flanked by hydrophobic surfaces. Apart from OH-6 of the beta-D-GlcNAc unit, the six other hydroxyl groups reside at or near the periphery of the combining site. Linear enthalpy-entropy compensation is observed for complex formation with monodeoxy and other derivatives of Leb-OMe involving one of these six hydroxyl groups. Decreases in both the thermodynamic parameters (- delta H 0 and - delta S 0) are largest when a hydroxyl group is in contact with water at the periphery of the combining site. The experimental evidence indicates that the binding reactions involve very similar if not identical changes in the conformations of both the lectin and the ligands; it is therefore proposed that the enthalpy-entropy compensations arise because water molecules hydrogen bonded to the amphiphilic surfaces of the unbound oligosaccharide and the protein are more mobile (higher entropy content) and less strongly hydrogen bonded than are water molecules in bulk solution. Monte Carlo simulations of the hydration of Leb-OMe appear to support this idea. In accordance with this proposal the association of complementary amphiphilic molecular surfaces from aqueous solution is driven by the release of the water molecules from both non-polar and polar regions of the amphiphiles to form stronger hydrogen bonds in bulk water. In the case of highly amphiphilic molecules such as the oligosaccharide Leb-OMe the negative contributions to entropy change dominate positive contributions that may arise from hydrophobic effects. The GS-IV(Leb-OMe)2 complex is stabilized by the hydrogen-bonding networks involving an asparate, an asparagine and a serine residue within the combining site and the above-mentioned key hydroxyl groups. Improved packing of the molecules may also be involved.

Biochemistry of carbohydrate-protein interaction.

Lee YC
FASEB J. 1992; 6: 3193-200

Recognition of glycoconjugates is an important event in biological systems, and is frequently in the form of carbohydrate-protein interactions. To thoroughly understand these interactions, well-defined carbohydrate ligands must be available. Naturally derived glycoconjugates can be highly purified, and their structures (including conformational structures) can be elucidated to provide such ligands. This requires highly effective methods of separation, such as various forms of high-performance liquid chromatography. Alternatively, structurally well-defined glycoconjugates can be synthesized for this purpose. These include conjugates of carbohydrate derivatives to proteins, lipids, and nonbiological carriers and polymers. The efficacy of these conjugates is amply demonstrated in the studies of carbohydrate-binding proteins from animals. Hepatic carbohydrate receptors, requiring calcium for binding, recognize only the terminal sugar residues. Although different sugar specificities are manifested by different species, there is some commonality in the requirement of the substituents of the sugar rings. Clustering of the target sugars in proper geometric arrangement greatly enhances the binding by these proteins. Some other animal carbohydrate-binding proteins, however, may require penultimate sugars for optimal binding.

Sweet and shapely: ligands for animal lectins.

Lee YC
Biochem Soc Trans. 1993; 21: 460-3

Major lectin of alligator liver is specific for mannose/L-fucose.

Lee RT, Yang GC, Kiang J, Bingham JB, Golgher D, Lee YC
J Biol Chem. 1994; 269: 19617-25

We surveyed the calcium-requiring (C-type) lectins in alligator liver. The major lectin purified by affinity chromatography was termed alligator hepatic lectin (AHL) and was found to be specific for mannose/L-fucose. AHL contained approximately equal amounts of 21- and 23-kDa bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding characteristics of AHL were similar to those of hepatic lectins of other classes in that 1) only terminal monosaccharide was recognized, and 2) the affinity increased exponentially when neoglycoproteins containing increasing numbers of mannose or L-fucose were used as ligand. However, unlike mammalian and chicken hepatic lectins, which exist as hexamers in Triton-containing solutions, AHL was present mainly as monomers, although small amounts of dimer and higher oligomers were present in equilibrium. Mannose-binding proteins and mannose-specific lectins of macrophages bind N-acetylmannosamine, glucose, and N-acetylglucosamine in addition to mannose, indicating that the nature and orientation of the C-2 substituent are not important to these lectins. In contrast, AHL shows a strict requirement for the presence of an axial hydroxyl group at the C-2 position (i.e. mannose).

Lectin-carbohydrate interactions: fine specificity difference between two mannose-binding proteins.

Lee RT, Shinohara Y, Hasegawa Y, Lee YC
Biosci Rep. 1999; 19: 283-92

Two types of rat mannose-binding proteins (MBPs), MBP-A (serum type) and MBP-C (liver type), have similar binding specificity for monosaccharide and similar binding site construct according to the X-ray structure, but exhibit different affinity toward natural oligosaccharides and glycoproteins. To understand the basis for this phenomenon, we used cloned fragment of MBP-A and -C (entire carbohydrate-recognition domain and a short connecting piece) that exists as stable trimers in various binding studies. Binding of a number of mannose-containing di- and tri-saccharides and high-mannose type oligosaccharides indicated that MBP-C has an extended binding area of weak interaction with the second and the third mannose residues, whereas MBP-A recognizes just a single mannose residue. In addition, MBP-C has a weak secondary binding site some 25 A away from the primary site. These findings explain the higher affinity of MBP-C for natural high-mannose type oligosaccharides as compared to MBP-A. A huge affinity differential manifested by natural glycoproteins (e.g., inhibitory potency of thyroglobulin is approximately 200 fold higher for MBP-C than for MBP-A in a solid-phase assay) may be due to steric hindrance experienced by MBP-A in the competition assay, and suggests different arrangement of subunit in the MBP trimers.

Further studies on the binding characteristics of rabbit liver galactose/N-acetylgalactosamine-specific lectin.

Lee RT, Myers RW, Lee YC
Biochemistry. 1982; 21: 6292-8

The affinity of various carbohydrates for the galactose/N-acetylgalactosamine-specific lectin of the rabbit liver was assessed by determining the effect of these carbohydrates on the binding of [125I]asialoorosomucoid (125I-ASOR) by the lectin. To obtain the concentration of the inhibitor that causes 50% reduction in the 125I-ASOR binding (I50), we carried out inhibition assays with fixed concentrations of 125I-ASOR and the purified, detergent-solubilized lectin, while the concentrations of the inhibitors were varied. The concentrations of the 125I-ASOR and the lectin were chosen such that the I50 value obtained closely approximates the dissociation constant of the inhibitor. Previously, we had shown that equatorial 2-hydroxyl (or acetamido), equatorial 3-hydroxyl, and axial 4-hydroxyl groups of a D-galactopyranosyl (or 2-acetamido-2-deoxy-D-galactopyranosyl) residue in the neoglycoprotein ligand participate in the binding to the lectin [Stowell, C. P., Lee, R. T., & Lee, Y. C. (1980) Biochemistry 19, 4904-4908; Lee, R. T. (1982) Biochemistry 21, 1045-1050]. In this study, we demonstrate that the methylene group (C-6) and certain aglycons also contribute to the binding. The presence of an unsaturated group such as C = NH at the gamma position to the anomeric carbon enhances the binding of an equatorially oriented aglycon. In addition, there seems to be a nonspecific hydrophobic interaction between some aglycons and the lectin binding site. Thus altogether five groups (aglycon, 2-OH or 2-NHAc, 3-OH, 4-OH, and 6-CH2-) in a galactopyranoside (or N-acetylgalactosaminide) have been shown to participate in lectin-ligand interactions. However, not all five groups are absolutely necessary for binding, since significant binding to the liver lectin occurs when only four of these groups are present.

The photoreactive carbohydrate probe, a novel method for detection of lectins.

Lauc G, Barisic K, Zanic T, Flogel M
Eur J Clin Chem Clin Biochem. 1995; 33: 933-7

One of the main difficulties in the research of lectins is the absence of an adequate technique for their specific and routine detection. Here, we present a photoreactive carbohydrate-probe which could help to overcome this problem. The probe was comopsed by joining four segments: (i) a carbohydrate moiety, (ii) the digoxigenin tag, (iii) the photoreactive cross-linker and (iv) the lysyl-lysine backbone. After incubation with lectins in the dark, the probe can be activated and cross-linked to the lectins by illumination. The result is a lectin with covalently incorporated digoxigenin tag. Such a labelled lectin can be easily identified using anti-digoxigenin antibodies in a Western blot technique. This method is of high specificity and sensitivity and enables direct detection of lectins in complex mixtures, even whole cell homogenates.

Gel-immobilized heparin-binding lectin as sensitive sensor for certain groups of charge-bearing carbohydrates.

Koopmann J, Hocke J, Gabius HJ
Biol Chem Hoppe Seyler. 1993; 374: 1029-32

The specificity of lectins to carbohydrate moieties in principle enables them to serve as sensors for sugars with ligand properties. However, experimental systems and parameters to measure this interaction need to be defined. On the basis of knowledge about temperature-sensitive volume changes of gels, composed of acrylamide derivatives, and about the influence of presence of charge-bearing groups within the gel on this behavior, we covalently immobilized a human heparin-binding lectin into a gel matrix. Besides the lectin-carrying derivative N-isopropylacrylamide and N,N'-methylenebisacrylamide are the monomeric constituents of the polymer. The lectin has been attached to divinyl sulfone-activated N-hydroxymethylacrylamide. Several anionic sugar moieties are added to the solution, covering the gel pieces, and the mechanical response of the individual gel slices in dependence to stepwise temperature increases is automatically recorded with an electronic transducer at a sensitivity of 5 mV/microns. Only carboxyl group-containing sugar moieties like glucuronic acid notably reduce the extent of the temperature-dependent gel shrinking as indicator for a protein-carbohydrate interaction. The individual slices are reuseable, emphasizing practical applications. This sensitive and automated assay concept with the covalently immobilized heparin-binding protein is supposed to be adaptable to other groups of lectins with specificity to anionic sugars like sialic acid-binding proteins.

Sugar binding specificities of anti-H(O) lectins disclosed by use of fucose-containing human milk oligosaccharides as binding inhibitors.

Konami Y, Tsuji T, Toyoshima S, Matsumoto I, Osawa T
Chem Pharm Bull (Tokyo). 1989; 37: 729-31

The binding to normal and sialidase-treated human erythrocytes of six 125I-labeled lectins [Ulex europeus lectin I (UEA-1) and II (UEA-II), Laburnum alpinum lectins I (LAA-I) and II (LAA-II), and Cytisus multiflorus lectins I (CMA-I) and II (CMA-II)], was studied in detail. Quantitative inhibition assays of the lectin binding to the cells were also performed with various human milk oligosaccharides as inhibitors. Based on a comparison of the inhibition constants of the inhibitors thus obtained with the association constants of the lectins to the cells, the relative activities of cell surface blood group antigens toward the lectins are discussed.

Structural basis of galactose recognition by C-type animal lectins.

Kolatkar AR, Weis WI
J Biol Chem. 1996; 271: 6679-85

The asialoglycoprotein receptors and many other C-type (Ca2+-dependent) animal lectins specifically recognize galactose- or N-acetylgalactosamine-terminated oligosaccharides. Analogous binding specificity can be engineered into the homologous rat mannose-binding protein A by changing three amino acids and inserting a glycine-rich loop (Iobst, S. T., and Drickamer, K. (1994) J. Biol. Chem. 269, 15512-15519). Crystal structures of this mutant complexed with beta-methyl galactoside and N-acetylgalactosamine (GalNAc) reveal that as with wild-type mannose-binding proteins, the 3- and 4-OH groups of the sugar directly coordinate Ca2+ and form hydrogen bonds with amino acids that also serve as Ca2+ ligands. The different stereochemistry of the 3- and 4-OH groups in mannose and galactose, combined with a fixed Ca2+ coordination geometry, leads to different pyranose ring locations in the two cases. The glycine-rich loop provides selectivity against mannose by holding a critical tryptophan in a position optimal for packing with the apolar face of galactose but incompatible with mannose binding. The 2-acetamido substituent of GalNAc is in the vicinity of amino acid positions identified by site-directed mutagenesis (Iobst, S. T., and Drickamer, K. (1996) J. Biol. Chem. 271, 6686-6693) as being important for the formation of a GalNAc-selective binding site.

Mechanism of N-acetylgalactosamine binding to a C-type animal lectin carbohydrate-recognition domain.

Kolatkar AR, Leung AK, Isecke R, Brossmer R, Drickamer K, Weis WI
J Biol Chem. 1998; 273: 19502-8

The mammalian hepatic asialoglycoprotein receptor, a member of the C-type animal lectin family, displays preferential binding to N-acetylgalactosamine compared with galactose. The structural basis for selective binding to N-acetylgalactosamine has been investigated. Regions of the carbohydrate-recognition domain of the receptor believed to be important in preferential binding to N-acetylgalactosamine have been inserted into the homologous carbohydrate-recognition domain of a mannose-binding protein mutant that was previously altered to bind galactose. Introduction of a single histidine residue corresponding to residue 256 of the hepatic asialoglycoprotein receptor was found to cause a 14-fold increase in the relative affinity for N-acetylgalactosamine compared with galactose. The relative ability of various acyl derivatives of galactosamine to compete for binding to this modified carbohydrate-recognition domain suggest that it is a good model for the natural N-acetylgalactosamine binding site of the asialoglycoprotein receptor. Crystallographic analysis of this mutant carbohydrate-recognition domain in complex with N-acetylgalactosamine reveals a direct interaction between the inserted histidine residue and the methyl group of the N-acetyl substituent of the sugar. Evidence for the role of the side chain at position 208 of the receptor in positioning this key histidine residue was obtained from structural analysis and mutagenesis experiments. The corresponding serine residue in the modified carbohydrate-recognition domain of mannose-binding protein forms a hydrogen bond to the imidazole side chain. When this serine residue is changed to valine, loss in selectivity for N-acetylgalactosamine is observed. The structure of this mutant reveals that the beta-branched valine side chain interacts directly with the histidine side chain, resulting in an altered imidazole ring orientation.

Assay and characterization of carbohydrate binding by the lectin discoidin I immobilized on nitrocellulose.

Kohnken RE, Berger EA
Biochemistry. 1987; 26: 3949-57

Discoidin I is the most abundant galactose binding lectin produced by the cellular slime mold Dictyostelium discoideum and has been implicated in cell-substratum adhesion. We have developed an assay of carbohydrate binding activity utilizing binding of 125I-asialofetuin to discoidin I, or to other lectins, immobilized on nitrocellulose. Among the proteins examined, only lectins exhibited the ability to bind asialofetuin. Specificity of asialofetuin binding was demonstrated by competition with monosaccharides, which inhibited binding consistent with the known sugar specificity of the lectins examined. Experiments with fetuin and derivatives differing in their oligosaccharide structure indicated a requirement for terminal galactosyl residues for probe binding to discoidin I. We have used this assay to characterize the carbohydrate binding behavior of discoidin I. The extent of asialofetuin binding to discoidin I was dependent on the concentrations of both lectin and ligand. Interpretation of equilibrium binding data suggested that, under saturating conditions, 1 mol of oligosaccharide was bound per mole discoidin I monomer. Furthermore, discoidin I in solution and discoidin I on nitrocellulose were equally effective at competing for soluble asialofetuin, suggesting that immobilization had no effect on the carbohydrate binding behavior of discoidin I. Binding was strongly inhibited by ethylenediaminetetraacetic acid; both Ca2+ and Mn2+ could overcome that inhibition, but Mg2+ could not. Preincubation of discoidin I at 60 degrees C stimulated asialofetuin binding 2-fold by increasing the affinity, while preincubation at higher temperatures resulted in a complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Modular organization of carbohydrate recognition domains in animal lectins.

Kishore U, Eggleton P, Reid KB
Matrix Biol. 1997; 15: 583-92

In spite of the great diversity of animal lectins, a common characteristic is their ability to bind sugars by means of discrete, modular carbohydrate recognition domains, CRDs. Three different groups of animal lectins-galectins, P-type and C-type lectins- have different types of CRDs which they arrange in a number of combinations, in three dimensions, in order to increase the affinity for oligosaccharides associated with glycoconjugates. The necessity of combining multiple CRDs in a native lectin molecule in order to increase the affinity for multiple ligands is of great importance physiologically, since many of the carbohydrate structures associated with proteins exist in a variety of different conformations. Recent work has clarified the structural basis for carbohydrate recognition by some of these lectins.

Homogeneous enzyme-linked binding assay for studying the interaction of lectins with carbohydrates and glycoproteins.

Kim B, Cha GS, Meyerhoff ME
Anal Chem. 1990; 62: 2663-8

A simple and rapid homogeneous enzyme-linked binding assay method for studying lectin-carbohydrate interactions is described. The method is based on the homogeneous inhibition of appropriate enzyme-saccharide conjugates by specific carbohydrate-binding lectins. In the presence of carbohydrate structures recognized by the lectins, enzyme activity is regained in an amount of proportional to the concentration of carbohydrate. The new method can be used to rapidly assess the relative carbohydrate specificity of the various lectins and for the selective analytical detection of simple saccharides and complex glycoproteins. Indeed, when Jacalin lectin is used in conjunction with a malate dehydrogenase-galactose conjugate, selective measurement of human IgA (immunoglobulin A) at microgram per milliliter levels in less than 10 min is possible. The potential for using this analytical methodology for determining changes in the carbohydrate structure of intact recombinant glycoproteins is also discussed.

Strength in numbers: non-natural polyvalent carbohydrate derivatives.

Kiessling LL, Pohl NL
Chem Biol. 1996; 3: 71-7

Many processes mediated by protein-carbohydrate interactions involve multivalent low-affinity binding, which is inherently difficult to study. New structural templates for the generation of multivalent carbohydrate displays have recently been developed, and tailored multivalent saccharide derivatives can now be used to study and modulate a wide variety of biological recognition events.

Lectin-carbohydrate interactions in immunoregulation.

Kery V
Int J Biochem. 1991; 23: 631-40

Further characterization of glucose-specific peanut root lectin (PRA II).

Kalsi G, Das HR, Das RH, Babu CR
Indian J Biochem Biophys. 1993; 30: 400-4

Amino acid analysis of PRA II, a glucose-specific lectin isolated from 7 day-old peanut seedling roots shows that this lectin is rich in glycyl (103 per mole) and seryl residues (59 per mole), and poor in essential amino acids, the acidic amino acid content is higher than the basic amino acids and that its amino acid composition differs from its seed counterpart (PNA), although neither of the lectins contains cystein. PRA II has two carbohydrate binding sites per molecule as determined by equilibrium dialysis. Modifications of the specific amino acid residues of the lectin with group specific reagents indicate that hydroxyl group of tyrosine is involved in the binding of carbohydrate to PRA II.

Manganese(II) in concanavalin A and other lectin proteins.

Kalb AJ, Habash J, Hunter NS, Price HJ, Raftery J, Helliwell JR
Met Ions Biol Syst. 2000; 37: 279-304

Conglutinin and CL-43, two collagenous C-type lectins (collectins) in bovine serum.

Jensenius JC, Laursen SB, Zheng Y, Holmskov U
Biochem Soc Trans. 1994; 22: 95-100

Structural requirements of carbohydrates to bind Agaricus bisporus lectin.

Irazoqui FJ, Vides MA, Nores GA
Glycobiology. 1999; 9: 59-64

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

Molecular modelling of protein-carbohydrate interactions. Understanding the specificities of two legume lectins towards oligosaccharides.

Imberty A, Perez S
Glycobiology. 1994; 4: 351-66

By means of a series of new molecular modelling tools, the conformational behaviour of mannose-containing di- and trisaccharides bound to either concanavalin A or Lathyrus ochrus isolectin I (LOLI) has been assessed. Tools for estimating and analysing either the 'rigid' or the 'relaxed' potential energy surfaces, representing the conformational space available for carbohydrates once interacting with lectins, are reported for the first time. Restrictions of conformational space are predicted to occur with different magnitudes, depending on the nature of the glycosidic linkages, as well as the size of the carbohydrates. Results from these molecular modelling studies are compared to existing structural data. Not only could the observed conformations and orientations of carbohydrates in crystalline lectin-oligosaccharides complexes be reproduced, but several other likely situations were also predicted to occur. Entropy calculations have been performed for comparison with experimental thermodynamics data. The results of the stimulation can also help giving an explanation of some observed affinity constants at the molecular level.

Mac-2: a versatile galactose-binding protein of mammalian tissues.

Hughes RC
Glycobiology. 1994; 4: 5-12

Mac-2 is a member of the S-(soluble) lectin family. Its identification and isolation from a wide variety of cell types and tissues suggest a diversity of roles in various biological systems. The key points to be made are that Mac-2, and the S-lectins in general, by virtue of their recognition of a variety of carbohydrate structures expressed on different glycoproteins, are well placed to exert discrete biological effects according to the distribution of those glycoproteins in tissues and their differential patterns of glycosylation according to developmental status and cell type. In this regard, the lectins are fundamentally different in character to other effector molecules that in general bind to specific receptors to trigger single signal transduction events.

The galectin family of mammalian carbohydrate-binding molecules.

Hughes RC
Biochem Soc Trans. 1997; 25: 1194-8

Carbohydrate binding activities of Bradyrhizobium japonicum. III. Lectin expression, bacterial binding, and nodulation efficiency.

Ho SC, Wang JL, Schindler M, Loh JT
Plant J. 1994; 5: 873-84

In previous studies, evidence that the Bradyrhizobium japonicum lectin, designated BJ38, mediated the observed carbohydrate-specific binding activities of the bacteria, including the saccharide-specific adhesion to soybean root cells was presented. In the present study, it is found that both B. japonicum, as well as the purified BJ38, bind predominantly to young emergent root hairs of soybean roots and, to a much lesser extent, to the root cap, mature root hairs, epicotyl or hypocotyl regions. Thus, the region of preferential binding for both the bacteria and the isolated lectin coincide with the region of the soybean root most susceptible to B. japonicum infection. The importance of bacterial binding for the nodulation process was studied by comparing the nodulation efficiency of binding-deficient mutants N4 and N6 to the wild-type. These mutants had been shown to be defective in carbohydrate recognition, as represented by their diminished ability to bind to soybean roots. BJ38 was immunolocalized to one pole of the cell surface of wild-type B. japonicum, but no surface labeling could be detected on either mutant. Moreover, both N4 and N6 showed a substantial decrease in nodulation activity, relative to the wild-type. These results provide additional evidence that the carbohydrate binding activity of B. japonicum, most probably mediated by BJ38, may play an important role(s) in the initial phases of the infection process.

Developmentally regulated expression of surface carbohydrate residues on larval stages of the avian schistosome Trichobilharzia szidati.

Horak P
Folia Parasitol (Praha). 1995; 42: 255-65

Except other functions, surface saccharide residues on trematode larvae are supposed either to be the targets of the intermediate (molluscan) and final host immune systems, or to represent candidates for molecular mimicry. Therefore, changes in surface saccharide patterns during the development of the avian schistosome Trichobilharzia szidati were characterized. Whole parasite larval stages and their tissue sections were examined using FITC- conjugated lectins. Marked surface differences were found among larval stages (miracidia, mother sporocysts, daughter sporocysts, cercariae, schistosomula). Staining by some lectins reflected known ultrastructural changes of the outer tegument. Reaction of lectins with cercarial embryos was almost negative. In case of other developmental stages, binding of at least one member from each carbohydrate-specificity group of lectins (Man/Glc-, GlcNAc-, Gal/GalNAc- and Fuc-specific) occurred. One exception is represented by mother and daughter sporocysts which practically failed to react with Fuc-specific lectins. Besides other lectins which recognized larval surfaces, alpha-L-fucose-specific lectins (LTA, UEA-1) and (GlcNAc beta 1-->4)n-specific WGA bound very strong to certain stages. The comparison of mature intrasporocystic cercariae with those emerged from snails brought the indication that some snail glycosylated molecules adhere to the surface of schistosome larvae or that emerged cercariae express some new carbohydrate epitopes under changed environmental conditions. The result partially supports the theory of parasite mimicry/ masking strategies and immune evasion in the host.

[Galectins: decoder of glyco-code]

Hirabayashi J, Kasai K
Seikagaku. 1995; 67: 1366-87

An assay for lectin activity using microtiter plate with chemically immobilized carbohydrates.

Hatakeyama T, Murakami K, Miyamoto Y, Yamasaki N
Anal Biochem. 1996; 237: 188-92

A simple microtiter plate assay for lectins or carbohydrate-binding proteins was developed. The method utilizes carbohydrates immobilized in the wells of the microtiter plate containing primary amino groups on their surface. After incubation of the lectins, bound proteins are measured by the protein assay using the colloidal gold solution. When the binding of Ricinus communis agglutinin, concanavalin A, and wheat germ agglutinin was measured using the microtiter plate wells coated with lactose, mannose, or N-acetylglucosamine, binding of the lectins according to their known specificity was observed. Inhibition experiments with various carbohydrates also demonstrated that the specificity of lectins for different carbohydrates could be determined quantitatively. Since there is no need for modification of the lectins, such as biotinylation or conjugation with marker enzymes, the carbohydrate-binding ability of intact proteins can be easily determined by this method. When gel filtration fractions from the extract of the marine invertebrate Cucumaria echinata were subjected to this assay, different carbohydrate-binding activities were observed with different elution profiles, suggesting that this assay could also be widely applicable for the simultaneous detection of lectins from various sources.

Carbohydrate-binding properties of the hemolytic lectin CEL-III from the holothuroidea Cucumaria echinata as analyzed using carbohydrate-coated microplate.

Hatakeyama T, Miyamoto Y, Nagatomo H, Sallay I, Yamasaki N
J Biochem (Tokyo). 1997; 121: 63-7

The carbohydrate-binding properties of the hemolytic lectin CEL-III from the Holothuroidea Cucumaria echinata were studied using the microplate assay system which we have recently developed [Hatakeyama et al. (1996) Anal. Biochem. 237, 188-192]. When the binding of CEL-III to lactose covalently immobilized on a microplate was examined using colloidal gold solution, the binding was detected with as little as 1 microgram/ml protein. Affinity of several carbohydrates to CEL-III was assessed by means of an inhibition experiment using the lactose-coated plate and it was found that N-acetylgalactosamine has the highest affinity for CEL-III, followed by lactose and lactulose. Examination of the binding of CEL-III to the lactose-coated plate at various pH values and temperatures revealed that the affinity is higher in the acidic pH region and at lower temperatures. From the Ca(2+)-dependence profile for the binding of CEL-III to the lactose-coated plate, the apparent dissociation constant for Ca2+ was estimated to be 2.3 mM. These results suggested that the carbohydrate-binding properties of CEL-III are closely related to its hemolytic activity, although an additional interaction between the protein and the lipid bilayer, which is enhanced in the alkaline pH region, also seems to be necessary for its hemolytic action.

Oligomerization of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata induced by the binding of carbohydrate ligands.

Hatakeyama T, Furukawa M, Nagatomo H, Yamasaki N, Mori T
J Biol Chem. 1996; 271: 16915-20

The hemolytic lectin CEL-III is a Ca2+-dependent, galactose/GalNAc-specific lectin purified from the marine invertebrate Cucumaria echinata (Holothuroidea). We found that this lectin forms ion-permeable pores in erythrocyte and artificial lipid membranes that have specific carbohydrate ligands on the surface. The hemolytic activity of CEL-III exhibited characteristic pH dependence; activity increased remarkably with pH in the alkaline region, especially above pH 9. When rabbit erythrocyte membrane was examined by immunoblotting using anti-CEL-III antiserum after treatment with CEL-III, the irreversible binding of the CEL-III oligomer increased with pH, indicating that the increase in hemolytic activity at higher pH is associated closely with the amount of oligomer irreversibly bound to the membrane. Surface hydrophobicity of CEL-III, as measured by the fluorescent probe 8-anilino-1-naphthalenesulfonate, increased markedly with the binding of specific ligands such as lactose, lactulose, and N-acetyllactosamine at pH 9-10 in the presence of 1 M NaCl. The enhancement of surface hydrophobicity induced by the binding of carbohydrates was also accompanied by the formation of a CEL-III oligomer, which was found to be the same size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the oligomer that formed in CEL-III-treated erythrocyte membranes. Far-UV circular dichroism spectra of CEL-III and the oligomer revealed a definite difference in secondary structure. These data suggest that the binding of CEL-III to specific carbohydrate ligands on the erythrocyte surface induces a conformational change in the protein, leading to the exposure of a hydrophobic region which triggers oligomerization and the irreversible binding of the protein to the membrane.

Characterization of the carbohydrate binding specificity and kinetic parameters of lectins by using surface plasmon resonance.

Haseley SR, Talaga P, Kamerling JP, Vliegenthart JF
Anal Biochem. 1999; 274: 203-10

An accurate, rapid, and sensitive method for characterizing the carbohydrate binding properties of lectins using a BIAcore apparatus and the detection method of surface plasmon resonance is described. As a model study, the sialic acid binding lectins from Sambucus nigra and Maackia amurensis, which are specific for the epitopes Neu5Ac(alpha2-6)Gal and Neu5Ac(alpha2-3)Gal, respectively, were chosen as suitable candidates. Two systems, one for the analysis of oligosaccharides and the other for glycoproteins, were developed after a rigorous analysis and evaluation of such parameters as binding conditions, buffers, and regeneration conditions. The systems take into account nonspecific binding, using the respective denatured lectin as negative blank, and avoid loss of activity: regeneration of the surface using either 10 mM NaOAc (pH 4.3) buffer (oligosaccharide system) or 20 mM HCl (glycoprotein system). The specificity of the lectins is well illustrated, while the kinetics parameters are shown to be sensitive to subtle changes in the recognized epitopes, and to be affected by steric hindrance. Surface plasmon resonance is a suitable technique for the analysis and characterization of lectins.

The role of weak protein-protein interactions in multivalent lectin-carbohydrate binding: crystal structure of cross-linked FRIL.

Hamelryck TW, Moore JG, Chrispeels MJ, Loris R, Wyns L
J Mol Biol. 2000; 299: 875-83

Binding of multivalent glycoconjugates by lectins often leads to the formation of cross-linked complexes. Type I cross-links, which are one-dimensional, are formed by a divalent lectin and a divalent glycoconjugate. Type II cross-links, which are two or three-dimensional, occur when a lectin or glycoconjugate has a valence greater than two. Type II complexes are a source of additional specificity, since homogeneous type II complexes are formed in the presence of mixtures of lectins and glycoconjugates. This additional specificity is thought to become important when a lectin interacts with clusters of glycoconjugates, e.g. as is present on the cell surface. The cryst1al structure of the Glc/Man binding legume lectin FRIL in complex with a trisaccharide provides a molecular snapshot of how weak protein-protein interactions, which are not observed in solution, can become important when a cross-linked complex is formed. In solution, FRIL is a divalent dimer, but in the crystal FRIL forms a tetramer, which allows for the formation of an intricate type II cross-linked complex with the divalent trisaccharide. The dependence on weak protein-protein interactions can ensure that a specific type II cross-linked complex and its associated specificity can occur only under stringent conditions, which explains why lectins are often found forming higher-order oligomers.

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

Hamelryck TW, Loris R, Bouckaert J, Dao-Thi MH, Strecker G, Imberty A, Fernandez E, Wyns L, Etzler ME
J Mol Biol. 1999; 286: 1161-77

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.

Differential lectin binding of Onchocerca lienalis and Onchocerca ochengi infective larvae following their development in Simulium ornatum s.l.

Hagen HE, Grunewald J, Ham PJ
Trop Med Parasitol. 1994; 45: 13-6

Lectins have been used to investigate species specific differences in carbohydrate moieties on the surface of the infective larvae of two Onchocerca species following their development in Simulium ornatum. Of the seven FITC-labelled lectins used in this study only two, Arachis hypogea (PNA) and Helix pomatia (HPA), bound to the surface of O. lienalis, whereas no lectin binding could be detected on the surface of O. ochengi infective larvae. This indicates that in principal lectins might be a potential tool for the differentiation of more closely related Onchocerca species.

Homogeneous aggregation of the 14-kDa beta-galactoside specific vertebrate lectin complex with asialofetuin in mixed systems.

Gupta D, Brewer CF
Biochemistry. 1994; 33: 5526-30

The galactose-specific 14-kDa family of animals lectins are an evolutionary conserved group of proteins that have been implicated in a wide variety of biological processes including cell proliferation, adhesion, and transformation. The present study demonstrates that the dimeric 14-kDa calf spleen lectin forms homogeneous aggregated cross-linked complexes with asialofetuin, a glycoprotein with multiple carbohydrate chains possessing terminal galactose residues, in the presence of other lectins with similar specificities and cross-linking activities. Several galactose-specific plant lectins also form homogeneous aggregated cross-linked complexes with ASF. These results demonstrate a new source of specificity for the 14-kDa family of vertebrate lectins, namely, the ability to selectively cross-link and aggregate glycoproteins in mixed systems. The results have important biological implications for the interactions of multivalent lectins and glycoconjugates, as well as the thermodynamic interactions of multivalent molecules in general.

Observation of unique cross-linked lattices between multiantennary carbohydrates and soybean lectin. Presence of pseudo-2-fold axes of symmetry in complex type carbohydrates.

Gupta D, Bhattacharyya L, Fant J, Macaluso F, Sabesan S, Brewer CF
Biochemistry. 1994; 33: 5614-22

The tetrameric lectin from Glycine max (soybean) (SBA) has been shown to cross-link and precipitate with N-linked multiantennary complex type oligosaccharides containing nonreducing terminal Gal residues (Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041). In the present study, negative stain electron micrographs of the precipitates of SBA with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal Gal or GalNAc residues show the presence of highly ordered cross-linked lattices for many of the complexes. The precipitates of SBA with a "bisected" and "nonbisected" N-linked biantennary complex type oligosaccharide containing Gal residues at the nonreducing termini show similar two-dimensional patterns. However, the pattern observed for the precipitates of a tetraantennary complex type oligosaccharide with SBA is distinct from those of the two biantennary carbohydrates. Furthermore, the precipitates formed between the lectin and a synthetic O-linked biantennary ("cluster") glycoside with terminal GalNAc residues show a pattern that is different from those above. Four biantennary pentasaccharide analogs of the blood group I antigen containing beta-LacNAc moieties at the 2.3-, 2.4-, 2.6-, and 3.6-positions of the core Gal also showed ordered patterns in their precipitates with SBA. X-ray crystallographic data and mixed quantitative precipitation profiles of binary mixtures of the four analogs demonstrate that each analog possesses a unique cross-linked lattice with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate binding properties of banana (Musa acuminata) lectin II. Binding of laminaribiose oligosaccharides and beta-glucans containing beta1,6-glucosyl end groups.

Goldstein IJ, Winter HC, Mo H, Misaki A, Van Damme EJ, Peumans WJ
Eur J Biochem. 2001; 268: 2616-9

This paper extends our knowledge of the rather bizarre carbohydrate binding poperties of the banana lectin (Musa acuminata). Although a glucose/mannose binding protein which recognizes alpha-linked gluco-and manno-pyranosyl groups of polysaccharide chain ends, the banana lectin was shown to bind to internal 3-O-alpha-D-glucopyranosyl units. Now we report that this lectin also binds to the reducing glucosyl groups of beta-1,3-linked glucosyl oligosaccharides (e.g. laminaribiose oligomers). Additionally, banana lectin also recognizes beta1,6-linked glucosyl end groups (gentiobiosyl groups) as occur in many fungal beta1,3/1,6-linked polysaccharides. This behavior clearly distinguishes the banana lectin from other mannose/glucose binding lectins, such as concanavalin A and the pea, lentil and Calystegia sepium lectins.

The lectins: carbohydrate-binding proteins of plants and animals.

Goldstein IJ, Hayes CE
Adv Carbohydr Chem Biochem. 1978; 35: 127-340

On the carbohydrate binding specificity of lectins: the game is never over.

Goldstein IJ
Indian J Biochem Biophys. 1990; 27: 368-9

The physiological role of plant lectins still remains a mystery. Nevertheless the carbohydrate binding properties of these proteins are a source of great fascination and have been applied to the study of glycoconjugates on cell surfaces and in solution. These matters are discussed.

Knowledge-based modeling of a legume lectin and docking of the carbohydrate ligand: the Ulex europaeus lectin I and its interaction with fucose.

Gohier A, Espinosa JF, Jimenez-Barbero J, Carrupt PA, Perez S, Imberty A
J Mol Graph. 1996; 14: 322-7

Ulex europaeus isolectin I is specific for fucose-containing oligosaccharide such as H type 2 trisaccharide alpha-L-Fuc (1-->2) beta-D-Gal (1-->4) beta-D-GlcNAc. Several legume lectins have been crystallized and modeled, but no structural data are available concerning such fucose-binding lectin. The three-dimensional structure of Ulex europaeus isolectin I has been constructed using seven legume lectins for which high-resolution crystal structures were available. Some conserved water molecules, as well as the structural cations, were taken into account for building the model. In the predicted binding site, the most probable locations of the secondary hydroxyl groups were determined using the GRID method. Several possible orientations could be determined for a fucose residue. All of the four possible conformations compatible with energy calculations display several hydrogen bonds with Asp-87 and Ser-132 and a stacking interaction with Tyr-220 and Phe-136. In two orientations, the O-3 and O-4 hydroxyl groups of fucose are the most buried ones, whereas two other, the O-2 and O-3 hydroxyl groups are at the bottom of the site. Possible docking modes are also studied by analysis of the hydrophobic and hydrophilic surfaces for both the ligand and the protein. The SCORE method allows for a quantitative evaluation of the complementarity of these surfaces, on the basis of molecular lipophilicity calculations. The predictions presented here are compared with known biochemical data.

Typing of halophilic Archaea and characterization of their cell surface carbohydrates by use of lectins.

Gilboa-Garber N, Mymon H, Oren A
FEMS Microbiol Lett. 1998; 163: 91-7

Lectins are important tools for cell typing and for the study of cell surface components. They have been widely used for the analysis of carbohydrates on the surface of many eukaryotic and prokaryotic cells, but they have not yet been exploited in the study of the halophilic Archaea (family Halobacteriaceae), because of the high salinity required for the structural integrity of these microorganisms. We have defined the salt concentration threshold high enough for survival of the Archaea, but sufficiently low for lectins to bind to them. Under these conditions we studied the interactions of a series of lectins, exhibiting different sugar specificities, with diverse halophilic Archaea. Concanavalin A was the most reactive by virtue of its glucose (and mannose) binding. The other lectins varied in their interactions. The results indicate that lectins might be useful probes for both archaeal typing and analysis of their cell surface carbohydrates.

Identification of sugar residues in secretory glycoconjugates of the lining and glandular epithelium of the chick embryo gizzard using lectin histochemistry.

Gheri G, Bryk SG, Sgambati E
Acta Histochem. 1994; 96: 387-97

Using seven horseradish peroxidase-conjugated lectins we studied the distribution of glycoconjugate sugar residues in the lining and glandular epithelium of the gizzard in chick embryos from day 7 to 21 of incubation. At the outer layer of the lining epithelium all investigated sugar residues were present from day 7 onwards, except alpha-L-fucose, which was detected later. From day 9 some cells, which contained granules characterised by D-galactose-(beta 1--->3)-N-acetyl-D-galactosamine and N-acetyl-D-glucosamine, were observed in the inner part of the lining epithelium. Afterwards, although in different time periods, all investigated oligosaccharides were detected in intercellular spaces filled with mucous material. At first, the cells of the anlage of the tubular glands appeared to be characterised by D-glucosamine and alpha-D-mannose only. Afterwards, all the investigated sugar residues were detected. At hatching, the luminal glandular secretion showed all investigated sugar residues, except alpha-L-fucose, which was typical for the early formation of the tubular glands. Our data suggest that during the development of the gizzard three time periods of mucous release can be distinguished.

A lectin histochemical study of gustatory (von Ebner's) glands of the horse tongue.

Gargiulo AM, Pedini V, Ceccarelli P, Lorvik S
Anat Histol Embryol. 1995; 24: 123-6

In the present work, gustatory glands (von Ebner's glands) of the horse tongue were examined by means of five peroxidase-conjugated lectins (PNA, DBA, SBA, UEA I, WGA), with and without prior sialidase digestion, in order to investigate the presence and distribution of carbohydrate residues in secretory cells and duct cells. The most intense staining of secretory cells was observed with PNA after pre-treatment with neuraminidase. This indicates that the terminal trisaccharide sequence sialic acid- (alpha 2-->3, 6) galactosyl (beta 1-->3) N-acetylgalactosamine is the most frequent oligosaccharide chain present in glycoproteins secreted by horse gustatory glands. Secretory cells also contained oligosaccharides with terminal alpha-N-acetylgalactosamine and N-acetylglucosamine, whereas fucose was found in only a few glandular cells. The apical cytoplasm of duct lining cells reacted with all the lectins except WGA.

New insights into the molecular basis of lectin-carbohydrate interactions: a calorimetric and structural study of the association of hevein to oligomers of N-acetylglucosamine.

Garcia-Hernandez E, Zubillaga RA, Rojo-Dominguez A, Rodriguez-Romero A, Hernandez-Arana A
Proteins. 1997; 29: 467-77

Isothermal titration calorimetry was used to characterize thermodynamically the association of hevein, a lectin from the rubber tree latex, with the dimer and trimer of N-acetylglucosamine (GlcNAc). Considering the changes in polar and apolar accessible surface areas due to complex formation, we found that the experimental binding heat capacities can be reproduced adequately by means of parameters used in protein-unfolding studies. The same conclusion applies to the association of the lectin concanavalin A with methyl-alpha-mannopyranoside. When reduced by the polar area change, binding enthalpy values show a minimal dispersion around 100 degrees C. These findings resemble the convergence observed in protein-folding events; however, the average of reduced enthalpies for lectin-carbohydrate associations is largely higher than that for the folding of proteins. Analysis of hydrogen bonds present at lectin-carbohydrate interfaces revealed geometries closer to ideal values than those observed in protein structures. Thus, the formation of more energetic hydrogen bonds might well explain the high association enthalpies of lectin-carbohydrate systems. We also have calculated the energy associated with the desolvation of the contact zones in the binding molecules and from it the binding enthalpy in vacuum. This latter resulted 20% larger than the interaction energy derived from the use of potential energy functions.

Stereochemical metrics of lectin-carbohydrate interactions: comparison with protein-protein interfaces.

Garcia-Hernandez E, Zubillaga RA, Rodriguez-Romero A, Hernandez-Arana A
Glycobiology. 2000; 10: 993-1000

A global census of stereochemical metrics including interface size, hydropathy, amino acid propensities, packing and hydrogen bonding was carried out on 32 x-ray-elucidated structures of lectin-carbohydrate complexes covering eight different lectin families. It is shown that the interactions at primary binding subsites are more efficient than at other subsites. Another salient behavior found for primary subsites was a marked negative correlation between the interface size and the polar surface content. It is noteworthy that this demographic rule is delineated by lectins with unrelated phylogenetic origin, indicating that independent interface architectures have evolved through common optimization paths. The structural properties of lectin-carbohydrate interfaces were compared with those characterizing a set of 32 protein homodimers. Overall, the analysis shows that the stereochemical bases of lectin-carbohydrate and protein-protein interfaces differ drastically from each other. In comparison with protein-protein complexes, lectin-carbohydrate interfaces have superior packing efficiency, better hydrogen bonding stereochemistry, and higher interaction cooperativity. A similar conclusion holds in the comparison with protein-protein heterocomplexes. We propose that the energetic consequence of this better interaction geometry is a larger decrease in free energy per unit of area buried, feature that enables lectins and carbohydrates to form stable complexes with relatively small interface areas. These observations lend support to the emerging notion that systems differing from each other in their stereochemical metrics may rely on different energetic bases.

Structural bases of lectin-carbohydrate affinities: comparison with protein-folding energetics.

Garcia-Hernandez E, Hernandez-Arana A
Protein Sci. 1999; 8: 1075-86

We have made a comparative structure based analysis of the thermodynamics of lectin-carbohydrate (L-C) binding and protein folding. Examination of the total change in accessible surface area in those processes revealed a much larger decrease in free energy per unit of area buried in the case of L-C associations. According to our analysis, this larger stabilization of L-C interactions arises from a more favorable enthalpy of burying a unit of polar surface area, and from higher proportions of polar areas. Hydrogen bonds present at 14 L-C interfaces were identified, and their overall characteristics were compared to those reported before for hydrogen bonds in protein structures. Three major factors might explain why polar-polar interactions are stronger in L-C binding than in protein folding: (1) higher surface density of hydrogen bonds; (2) better hydrogen-bonding geometry; (3) larger proportion of hydrogen bonds involving charged groups. Theoretically, the binding entropy can be partitioned into three main contributions: entropy changes due to surface desolvation, entropy losses arising from freezing rotatable bonds, and entropic effects that result from restricting translation and overall rotation motions. These contributions were estimated from structural information and added up to give calculated binding entropies. Good correlation between experimental and calculated values was observed when solvation effects were treated according to a parametrization developed by other authors from protein folding studies. Finally, our structural parametrization gave calculated free energies that deviate from experimental values by 1.1 kcal/mol on the average; this amounts to an uncertainty of one order of magnitude in the binding constant.

Beyond plant lectin histochemistry: preparation and application of markers to visualize the cellular capacity for protein-carbohydrate recognition.

Gabius HJ, Unverzagt C, Kayser K
Biotech Histochem. 1998; 73: 263-77

Oligosaccharides can store biological information. In this respect, their capacity even outmatches that of oligo- and polymeric structures of nucleotides and amino acids. Protein-carbohydrate interactions are thus considered to be involved in the regulation of diverse cellular activities. Over decades, plant lectins have proven valuable for assessing structural aspects of the enormous variety of carbohydrate epitopes and for monitoring spatially and/or temporally restricted patterns of expression. If the presence of these epitopes and the alterations in their occurrence bear physiological relevance, one reasonable possibility is that the visualized saccharides serve as ligands in an operative protein-carbohydrate recognition system. To support the validity of this hypothesis, receptor sites for a sugar compound must be localized. Carrier-immobilized carbohydrates (neoglycoconjugates) are adequate for this purpose. Chemical synthesis gains access to such probes. In the first stage, the presence of binding sites such as lectins in the tissue is ascertained. The next step toward proving the outlined hypothesis is the application of the first localized then purified endogenous receptors as glycohistochemical markers. It is essential to point out that the fine specificities of plant and animal lectins can differ, although they share an identical monosaccharide specificity. Thus, neoglycoconjugates for localizing sugar ligand-binding proteins and endogenous lectins to detect suitable binding partners are promising probes to enhance our knowledge about the capacities of cells to be engaged in protein-carbohydrate recognition in situ.

Reverse lectin histochemistry: design and application of glycoligands for detection of cell and tissue lectins.

Gabius HJ, Gabius S, Zemlyanukhina TV, Bovin NV, Brinck U, Danguy A, Joshi SS, Kayser K, Schottelius J, Sinowatz F, et al.
Histol Histopathol. 1993; 8: 369-83

Plant and invertebrate lectins are valuable cyto- and histological tools for the localization of defined carbohydrate determinants. The well-documented ubiquitous occurrence of sugar receptors encourages functional considerations. Undoubtedly, analysis of the presence of vertebrate lectins in tissues and cells is required to answer the pertinent and tempting question on the physiological relevance of protein (lectin)-carbohydrate recognition in situ. Carrier-immobilized glycoligands, derived from custom-made chemical synthesis, enable the visualization of respective binding sites. Histochemically inert proteins or synthetic polymers with appropriate functional groups are suitable carrier molecules for essential incorporation of ligand and label. The resulting neoglycoconjugates can track down tissue receptors that are neither impaired by fixation procedures nor blocked by endogenous high-affinity ligands. Lectins, especially the receptors of the tissue under investigation (endogenous lectins), and appropriately tailored immobilized glycoligands or lectin-specific antibodies (when available) are complementary tools to test the attractive hypothesis that diverse, functionally relevant glycobiological processes within or between cells are operative. Concomitant evaluation of both sides of lectin histochemistry, namely lectins as tools and lectins as functionally important molecules in situ, will indubitably render desired progress amenable in our often still fragmentary understanding of the importance of tissue lectin and glycoconjugate expression and its regulation.

Glycohistochemistry: the why and how of detection and localization of endogenous lectins.

Gabius HJ
Anat Histol Embryol. 2001; 30: 3-31

The central dogma of molecular biology limits the downstream flow of genetic information to proteins. Progress from the last two decades of research on cellular glycoconjugates justifies adding the enzymatic production of glycan antennae with information-bearing determinants to this famous and basic pathway. An impressive variety of regulatory processes including cell growth and apoptosis, folding and routing of glycoproteins and cell adhesion/migration have been unravelled and found to be mediated or modulated by specific protein (lectin)-carbohydrate interactions. The conclusion has emerged that it would have meant missing manifold opportunities not to recruit the sugar code to cellular information transfer. Currently, the potential for medical applications in anti-adhesion therapy or drug targeting is one of the major driving forces fuelling progress in glycosciences. In histochemistry, this concept has prompted the introduction of carrier-immobilized carbohydrate ligands (neoglycoconjugates) to visualize the cells' capacity to be engaged in oligosaccharide recognition. After their isolation these tissue lectins will be tested for ligand analysis. Since fine specificities of different lectins can differ despite identical monosaccharide binding, the tissue lectins will eventually replace plant agglutinins to move from glycan profiling and localization to functional considerations. Namely, these two marker types, i.e. neoglycoconjugates and tissue lectins, track down accessible binding sites with relevance for involvement in interactions in situ. The documented interplay of synthetic organic chemistry and biochemistry with cyto- and histochemistry nourishes the optimism that the application of this set of innovative custom-prepared tools will provide important insights into the ways in which glycans can act as hardware in transmitting information during normal tissue development and pathological situations.

Concepts of tumor lectinology.

Gabius HJ
Cancer Invest. 1997; 15: 454-64

Animal lectins.

Gabius HJ
Eur J Biochem. 1997; 243: 543-76

Protein and lipid glycosylation is no longer considered as a topic whose appeal is restricted to a limited number of analytical experts perseveringly pursuing the comprehensive cataloguing of structural variants. It is in fact arousing curiosity in various areas of basic and applied bioscience. Well founded by the conspicuous coding potential of the sugar part of cellular glycoconjugates which surpasses the storage capacity of oligonucleotide- or oligopeptide-based code systems, recognition of distinct oligosaccharide ligands by endogenous receptors, i.e. lectins and sugar-binding enzymes or antibodies, is increasingly being discovered to play salient roles in animal physiology. Having inevitably started with a descriptive stage, research on animal lectins has now undubitably reached maturity. Besides listing the current categories for lectin classification and providing presentations of the individual families and their presently delineated physiological significance, this review places special emphasis on tracing common structural and functional themes which appear to reverberate in nominally separated lectin and animal categories as well as lines of research which may come to fruition for medical sciences.

Carbohydrate-dependent cell adhesion.

Fukuda M
Bioorg Med Chem. 1995; 3: 207-15

Lectin binding to neurites of goldfish retinal explants.

Feldman EL, Heacock AM, Agranoff BW
Brain Res. 1982; 248: 347-54

The new biology of carbohydrates.

Feizi T, Childs RA, Kogelberg H
Glycobiology. 1994; 4: 106-9

Structure of a C-type carbohydrate recognition domain from the macrophage mannose receptor.

Feinberg H, Park-Snyder S, Kolatkar AR, Heise CT, Taylor ME, Weis WI
J Biol Chem. 2000; 275: 21539-48

The mannose receptor of macrophages and liver endothelium mediates clearance of pathogenic organisms and potentially harmful glycoconjugates. The extracellular portion of the receptor includes eight C-type carbohydrate recognition domains (CRDs), of which one, CRD-4, shows detectable binding to monosaccharide ligands. We have determined the crystal structure of CRD-4. Although the basic C-type lectin fold is preserved, a loop extends away from the core of the domain to form a domain-swapped dimer in the crystal. Of the two Ca(2+) sites, only the principal site known to mediate carbohydrate binding in other C-type lectins is occupied. This site is altered in a way that makes sugar binding impossible in the mode observed in other C-type lectins. The structure is likely to represent an endosomal form of the domain formed when Ca(2+) is lost from the auxiliary calcium site. The structure suggests a mechanism for endosomal ligand release in which the auxiliary calcium site serves as a pH sensor. Acid pH-induced removal of this Ca(2+) results in conformational rearrangements of the receptor, rendering it unable to bind carbohydrate ligands.

Chick hepatic lectins: an electron microscopic study on isolated hepatocytes during development.

Falasca L, Mattioli P, Dini L
Biosci Rep. 1991; 11: 257-64

We studied the carbohydrate recognition systems of hepatocytes isolated from 16-day-old embryos, 19-day-old embryos and chicks within 24 h of hatching. We localized and quantified at the ultrastructural level the binding sites for glycoproteins exposing terminal N-acetylglucosamine (GlcNAc), mannose and N-acetylgalactosamine (GalNAc) residues by means of protein-gold complexes. Binding sites specific for GlcNAc and mannose residues are present on hepatocytes from embryos and chicks. On the contrary GalNAc specific binding sites are exclusively observed on cells from 16-day-old embryos. The number and distribution of gold particles on hepatocyte cell surfaces depend on the binding sites and the age considered. We describe a modulation in the number of GlcNAc, and mannose specific receptors present on the cell surface between the embryonal stage and neonatal life.

A role of carbohydrate-carbohydrate interaction in the process of specific cell recognition during embryogenesis and organogenesis: a preliminary note.

Eggens I, Fenderson BA, Toyokuni T, Hakomori S
Biochem Biophys Res Commun. 1989; 158: 913-20

A possible role of cell surface glycoconjugates in cell recognition has been envisioned based on recognition of carbohydrates by cell surface proteins such as endogenous lectins, glycosyltransferases, and hydrolases (refs. 18-22 in text). A new possibility that a specific carbohydrate at the cell surface could be recognized by the same or similar carbohydrate on the counterpart cell surface is now suggested by specific interaction between Lex and Lex, but not between Lex and sialylated or non-substituted type 2 chain. A new hypothesis is hereby proposed for carbohydrate-carbohydrate interactions as recognition signals during embryogenesis and organogenesis.

C-type lectin-like domains.

Drickamer K
Curr Opin Struct Biol. 1999; 9: 585-90

Carbohydrate-recognition domains of C-type (Ca2+-dependent) animal lectins serve as prototypes for an important family of protein modules. Only some domains in this family bind Ca2+ or sugars. A comparison of recent structures of C-type lectin-like domains reveals diversity in the modular fold, particularly in the region associated with Ca2+ and sugar binding. Some of this diversity reflects the changes that occur during normal physiological functioning of the domains. C-type lectin-like domains associate with each other through several different surfaces to form dimers and trimers, from which ligand-binding sites project in a variety of different orientations.

Recognition of complex carbohydrates by Ca(2+)-dependent animal lectins.

Drickamer K
Biochem Soc Trans. 1993; 21: 456-9

Demonstration of carbohydrate-recognition activity in diverse proteins which share a common primary structure motif.

Drickamer K
Biochem Soc Trans. 1989; 17: 13-5

Multiplicity of lectin-carbohydrate interactions.

Drickamer K
Nat Struct Biol. 1995; 2: 437-9

Evolution of Ca(2+)-dependent animal lectins.

Drickamer K
Prog Nucleic Acid Res Mol Biol. 1993; 45: 207-32

Making a fitting choice: common aspects of sugar-binding sites in plant and animal lectins.

Drickamer K
Structure. 1997; 5: 465-8

Comparing sequences of plant and animal lectins reveals that the ability to bind any one type of sugar has evolved several times independently in diverse protein frameworks. Conversely, families of lectins that share common structural features often contain members that recognize different groups of sugars. In the context of this combination of convergent and divergent evolution, our knowledge of the structures of lectin-sugar complexes provides valuable insights into the principles that underlie specific sugar binding.

Ca(2+)-dependent sugar recognition by animal lectins.

Drickamer K
Biochem Soc Trans. 1996; 24: 146-50

Structures of the lectin IV of Griffonia simplicifolia and its complex with the Lewis b human blood group determinant at 2.0 A resolution.

Delbaere LT, Vandonselaar M, Prasad L, Quail JW, Wilson KS, Dauter Z
J Mol Biol. 1993; 230: 950-65

The structures of the fourth lectin isolated from Griffonia simplicifolia (GS4) and its complex with the methyl-glycoside of the Lewis b human blood group determinant (Le(b)-OMe) are reported at high resolution. The native GS4 crystal is isomorphous with the complexed GS4 crystal. The space group is P4(2)2(1)2 with unit cell dimensions a = 78.9 A, c = 89.1 A with one subunit of the lectin (bound to 1 Le(b)-OMe in the complex) in the crystallographic asymmetric unit. The native GS4 structure was solved by the molecular replacement technique and least-squares refined (PROLSQ and X-PLOR). The orientation of the Le(b)-OMe tetrasaccharide in the complex was established from a 2.8 A difference map with coefficients (Fcomplex--Fnative) and calculated phase angles from the native model. Both the final native and complex GS4 models consist of 1904 protein non-hydrogen atoms, one sulfate ion, one Ca ion, one Mn ion and three covalently-bound sugar residues N-linked to Asn18. In addition, the complex model has 47 Le(b)-OMe non-hydrogen atoms. The two structures have 135 water molecules in common in addition to eight and nine unique water molecules in the native and complex structures, respectively. The root-mean-square deviations from ideal bond distances and angles are 0.016 A, 3.2 degrees and 0.016 A, 3.0 degrees, for the native and complexed GS4, respectively. The R index for all unique data from 8 to 2.0 A is 0.187 for the native (19,204 reflections) and 0.181 for the complex (19,212 reflections). The tertiary structure of each subunit is similar to that of other leguminous lectins but the quaternary structure of the molecular dimer is different from that of any other lectin reported to date. The co-ordination about the Ca ion is pentagonal bipyramidal (with 1 long Ca(2+)-oxygen bond) and the co-ordination about the Mn ion is octahedral. Two conserved residues (Asp149 and Ser155) appear to be important because they are hydrogen-bonded to each other and to groups that co-ordinate the Mn ion. There are three cis-peptides in the polypeptide chain; two involve non-proline residues, one of which is homologous with other leguminous lectins and the other is unique to GS4. The two non-proline cis-peptides are located in the carbohydrate-binding site and are important for the specificity of the lectin. The molecular recognition of Le(b)-OMe by GS4 involves both polar and extensive non-polar interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Proceedings: The carbohydrate-binding specificity of the lectin from Vicia faba.

De Clercq A, Van Wauwe JP, Dhaese P, Loontiens FG
Arch Int Physiol Biochim. 1976; 84: 150-1

Binding of multivalent carbohydrates to concanavalin A and Dioclea grandiflora lectin. Thermodynamic analysis of the "multivalency effect".

Dam TK, Roy R, Das SK, Oscarson S, Brewer CF
J Biol Chem. 2000; 275: 14223-30

Binding of a series of synthetic multivalent carbohydrate analogs to the Man/Glc-specific lectins concanavalin A and Dioclea grandiflora lectin was investigated by isothermal titration microcalorimetry. Dimeric analogs possessing terminal alpha-D-mannopyranoside residues, and di-, tri-, and tetrameric analogs possessing terminal 3, 6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside residues, which is the core trimannoside of asparagine-linked carbohydrates, were selected in order to compare the effects of low and high affinity analogs, respectively. Experimental conditions were found that prevented precipitation of the carbohydrate-lectin cross-linked complexes during the isothermal titration microcalorimetry experiments. The results show that the value of n, the number of binding sites on each monomer of the lectins, is inversely proportional to the number of binding epitopes (valency) of each carbohydrate. Hence, n values close to 1.0, 0.50, and 0.25 were observed for the binding of mono-, di-, and tetravalent sugars, respectively, to the two lectins. Importantly, differences in the functional valency of a triantennary analog for concanavalin A and D. grandiflora lectin are observed. The enthalpy of binding, DeltaH, is observed to be directly proportional to the number of binding epitopes in the higher affinity analogs. For example, DeltaH of a tetravalent trimannoside analog is nearly four times greater than that of the corresponding monovalent analog. Increases in K(a) values of the multivalent carbohydrates relative to monovalent analogs, known as the "multivalency effect," are shown to be due to more positive entropy (TDeltaS) contributions to binding of the former sugars. A general thermodynamic model for distinguishing binding of multivalent ligands to a single receptor with multiple, equal subsites versus binding to separate receptor molecules is given.

Structure and biosynthesis of carbohydrate ligands for animal lectins involved in cell adhesion.

Cho MJ, Cummings RD
Biochem Soc Trans. 1994; 22: 378-81

Calorimetric analysis of the binding of lectins with overlapping carbohydrate-binding ligand specificities.

Chervenak MC, Toone EJ
Biochemistry. 1995; 34: 5685-95

The thermodynamics of binding of a system of plant lectins specific for the oligosaccharide methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside have been studied calorimetrically. This system of lectins consists of concanavalin A, the lectin isolated from Dioclea grandiflora, and the lectin from Galanthus nivalis. The group thus contains lectins with similar structures and similar binding properties as well as lectins with different structures but similar binding properties. Concanavalin A and the lectin from Dioclea are highly homologous, while the lectin from Galanthus nivalis shares no sequence homology with either of the legume lectins, although it also binds the mannose trisaccharide tightly. Calorimetric data for oligosaccharide binding to both of the legume lectins suggests that the total binding site comprises a single high-affinity site and an additional extended site. The pattern of binding for the lectin from Galanthus is significantly different. Binding studies with the same saccharides indicate that the lectin has binding sites designed specifically for the 1-->3 and 1-->6 arms of the mannose trisaccharide that are unable to accommodate other saccharides. Enthalpy--entropy compensation was observed for several saccharides as a function of lectin structure. Contributions of solvation effects to the enthalpy of binding and the configurational entropies were determined experimentally. For those systems studied here, solute-solute attractive interactions and configurational entropies were the greatest contributors to enthalpy-entropy compensation. Our studies clearly demonstrate that, despite their common affinity for the mannose trisaccharide, the three lectins bind oligosaccharides very differently.

Variations in lectin localization in different parts of the bovine heart.

Brkovic D, Gabius HJ, Bardosi A
Acta Anat (Basel). 1992; 143: 317-21

In order to study the distribution of endogenous sugar-binding proteins (lectins) in various areas of the adult bovine heart, we used a battery of biotinylated neoglycoproteins. These tools expose carrier-immobilized carbohydrate moieties as ligands for receptor detection. Characteristic staining patterns depending on the type of carbohydrate ligand were observed in all constituents examined. Comparison to data obtained for lectin distribution in the respective areas of the human heart indicate that the localization of certain types of endogenous sugar receptors can exhibit species-dependent variations.

X-ray structure of a (alpha-Man(1-3)beta-Man(1-4)GlcNAc)-lectin complex at 2.1-A resolution. The role of water in sugar-lectin interaction.

Bourne Y, Rouge P, Cambillau C
J Biol Chem. 1990; 265: 18161-5

We describe herein the high resolution refined x-ray structure of a trisaccharide, which is a part of the N-acetyllactosamine type glycan found in the majority of the N-glycosyl-proteins, complexed to the isolectin I. According to the potentials used by Imberty et al. (Imburty, A., Gerber, S., Tran, V., and Perez, S. (1990) Glycoconjugate J. 7, 27-54) the trisaccharide is in a low-energy state. Only one mannose moiety establishes direct hydrogen bonds with the lectin, as it is the case for monosaccharide-lectin complexes. The comparison of our trisaccharide with the one determined in solution by Warin et al. (Warin, V., Baert, F., Fouret, R., Strecker, G., Fournet, B., and Montreuil, J. (1979) Carbohydr. Res. 76, 11-22) shows that both adopt roughly the same conformation. The differences in these two sugar structures allow us to assign the role of water molecules present in the vicinity of our trisaccharide for the stabilization of this sugar-lectin complex.

Interaction of a legume lectin with two components of the bacterial cell wall. A crystallographic study.

Bourne Y, Ayouba A, Rouge P, Cambillau C
J Biol Chem. 1994; 269: 9429-35

We describe herein the refined high resolution x-ray structures of two components of the bacterial cell wall, muramic acid and muramyl dipeptide complexed to isolectin I from Lathyrus ochrus seeds. In both complexes, only the ring hydroxyl oxygen atoms of the bound sugar establish direct hydrogen bonds with isolectin I, as in the case of all the previously determined monosaccharide-lectin complexes. In addition, the lactyl methyl of both components strongly interacts via hydrophobic contacts with the side chains of residues Tyr100 and Trp128 of isolectin I, which could explain the higher affinity of isolectin I for muramic acid as compared with glucose. These 2 residues, however, are not involved in the stabilization of the oligosaccharide-isolectin I complexes. The dipeptide (D-Ala-D-iGln) of the second component is in stacking interaction with the N-acetyl group of glucose and with loop Gly97-Gly98 of isolectin I. In addition to these van der Waals' contacts, the dipeptide interacts with the lectin via well ordered water molecules also. Superposition of the structures of the muramyl dipeptide complex and of the muramic acid complex shows that the glucose ring in the dipeptide compound is tilted by about 15 degrees in comparison with that of muramic acid. The fact that the lactyl group has the same confrontation in both components reveals that the lectin is stereospecific and recognizes only diastereoisomer S of this group, which better fits the saccharide-binding site.

Novel structures of plant lectins and their complexes with carbohydrates.

Bouckaert J, Hamelryck T, Wyns L, Loris R
Curr Opin Struct Biol. 1999; 9: 572-7

Several novel structures of legume lectins have led to a thorough understanding of monosaccharide and oligosaccharide specificity, to the determination of novel and surprising quaternary structures and, most importantly, to the structural identification of the binding site for adenine and plant hormones. This deepening of our understanding of the structure/function relationships among the legume lectins is paralleled by advances in two other plant lectin families - the monocot lectins and the jacalin family. As the number of available crystal structures increases, more parallels between plant and animal lectins become apparent.

Lectin-carbohydrate interactions studied by a competitive enzyme inhibition assay.

Borrebaeck C, Mattiasson B
Anal Biochem. 1980; 107: 446-50

Insertion of pea lectin into a phospholipid monolayer.

Booij P, Demel RA, de Pater BS, Kijne JW
Plant Mol Biol. 1996; 31: 169-73

Pea lectin (PSL) is a secretory sugar-binding protein, readily soluble in aqueous solutions of low osmolarity. However, PSL also appears to be associated with the plasma membrane at the tip of young pea root hairs. By using the Wilhelmy plate method, we found that PSL can insert into a lipid monolayer. This property appeared to be independent of the sugar-binding ability of the protein. This result suggests that PSL may be directly involved in membrane-mediated interactions with saccharide ligands, for example during root hair infection by symbiotic rhizobia.

Binding and precipitating activities of Erythrina lectins with complex type carbohydrates and synthetic cluster glycosides. A comparative study of the lectins from E. corallodendron, E. cristagalli, E. flabelliformis, and E. indica.

Bhattacharyya L, Haraldsson M, Sharon N, Lis H, Brewer F
Glycoconj J. 1989; 6: 141-50

Erythrina lectins possess similar structural and carbohydrate binding properties. Recently, tri- and tetra-antennary complex type carbohydrates with non-reducing terminal galactose residues have been shown to be precipitated as tri- and tetravalent ligands, respectively, with certain Erythrina lectins [Bhattacharyya L, Haraldsson M, Brewer CF (1988) Biochemistry 27:1034-41]. The present work describes a comparative study of the binding and precipitating activities of four Erythrina lectins, viz., E. corallodendron, E. cristagalli, E. flabelliformis, and E. indica, with multi-antennary complex type carbohydrates and synthetic cluster glycosides. The results show that though their binding affinities are very similar, the Erythrina lectins show large differences in their precipitating activities with the carbohydrates. The results also indicate significant dependence of the precipitating activities of the lectins on the core structure of the carbohydrates. These findings provide a new dimension to the structure-activity relationship of the lectins and their interactions with asparagine-linked carbohydrates.

Lectin-carbohydrate interactions. Studies of the nature of hydrogen bonding between D-galactose and certain D-galactose-specific lectins, and between D-mannose and concanavalin A.

Bhattacharyya L, Brewer CF
Eur J Biochem. 1988; 176: 207-12

The binding of galactose-specific lectins from Erythrina indica (EIL), Erythrina arborescens (EAL), Ricinus communis (agglutinin; RCA-I), Abrus precatorius (agglutinin; APA), and Bandeiraea simplicifolia (lectin I; BSL-I) to fluoro-, deoxy-, and thiogalactoses were studied in order to determine the strength of hydrogen bonds between the hydroxyl groups of galactose and the binding sites of the proteins. The results have allowed insight into the nature of the donor/acceptor groups in the lectins that are involved in hydrogen bonding with the sugar. The data indicate that the C-2 hydroxyl group of galactose is involved in weak interactions as a hydrogen-bond acceptor with uncharged groups of EIL and EAL. With RCA-I, the C-2 hydroxyl group forms two weak hydrogen bonds in the capacity of a hydrogen-bond acceptor and a donor. On the other hand, there is a strong hydrogen bond between the C-2 hydroxyl group of galactose, which acts as a donor, and a charged group on BSL-I. The C-2 hydroxyl group of the sugar is also a hydrogen-bond donor to APA. The lectins are involved in strong hydrogen bonds through charged groups with the C-3 and C-4 hydroxyl groups of galactose, with the latter serving as hydrogen-bond donors. The C-6 hydroxyl group of the sugar is weakly hydrogen bonded with neutral groups of EIL, EAL, and APA. With BSL-I, however, a strong hydrogen bond is formed at this position with a charged group of the lectin. The C-6 hydroxyl groups is a hydrogen-bond acceptor for EIL and EAL, a hydrogen-bond donor for APA and BSL-I, and appears not to be involved in binding to RCA-I. The data with the thiosugars indicate the involvement of the C-1 hydroxyl group of galactose in binding to EIL, EAL, and BSL-I, but not to RCA-I and APA. We have also performed a similar analysis of the binding data of fluoro- and deoxysugars to concanavalin A [Poretz, R. D. and Goldstein, I. J. (1970) Biochemistry 9, 2890-2896]. This has allowed comparison of the donor/acceptor properties and free energies of hydrogen bonding of the hydroxyl groups of methyl alpha-D-mannopyranoside to concanavalin A with the results in the present study. On the basis of this analysis, new assignments are suggested for amino acid residues of concanavalin A [corrected] that may be involved in hydrogen bonding to the sugar.

[Chemistry of lectins and their medical and biological applications]

Beppu M, Toyoshima S, Osawa T, Matsumoto I
Nippon Rinsho. 1980; 38: 2490-500

Blot analysis of glycoconjugates using digoxigenin-labeled lectins: an optimized procedure.

Becker B, Salzburg M, Melkonian M
Biotechniques. 1993; 15: 232-5

Carbohydrate and hydrophobic-carbohydrate recognition sites (CARS and HY-CARS) in solubilized glycosyltransferases.

Basu S, Ghosh S, Basu M, Hawes JW, Das KK, Zhang BJ, Li ZX, Weng SA, Westervelt C
Indian J Biochem Biophys. 1990; 27: 386-95

Six different glycosyltransferases that are active with glycosphingolipid substrates have been purified from Golgi-membranes after solubilization with detergents. It appears that GalT-4(UDP-Gal:GlcNAc-R1 beta 1-4GalT), GalNAcT-2(UDP-Gal:Gal alpha-R2 beta 1-3GalNAcT) and FucT-2(GDP-Fuc:Gal beta GlcNAc-R3 alpha 1-2FucT) are specific for oligosaccharides bound to ceramide or to a protein moiety. These are called CARS (carbohydrate recognition sites) glycosyltransferases (GLTs). On the other hand, GalT-3(UDP-Gal:GM2 beta 1-3GalT), GalNAcT-1(UDP-GalNAc:GM3 beta 1-4GalNAcT) and FucT-3 (GDP-Fuc:LM1 alpha 1-3FucT) recognize both hydrophobic moieties (fatty acid of ceramide) as well as the oligosaccharide chains of the substrates. These GLTs are called HY-CARS (hydrophobic and carbohydrate recognition sites). D-Erythro-sphingosine (100-500 microM) modulates the in vitro activities of these GLTs. Modulation depends on the binding of D-sphingosine to a protein backbone, perhaps on more than one site and beyond transmembrane hydrophobic domains. Control of GLTs by free D-sphingosine was suggested with the concomitant discovery of ceramide glycanase in rabbit mammary tissues. The role of free sphingosine as an in vivo homotropic modulator of glycosyltransferases is becoming apparent.

Mannose-binding plant lectins: Different structural scaffolds for a common sugar-recognition process.

Barre A, Bourne Y, Van Damme EJ, Peumans WJ, Rouge P
Biochimie. 2001; 83: 645-51

Mannose-specific lectins are widely distributed in higher plants and are believed to play a role in recognition of high-mannose type glycans of foreign micro-organisms or plant predators. Structural studies have demonstrated that the mannose-binding specificity of lectins is mediated by distinct structural scaffolds. The mannose/glucose-specific legume (e.g., Con A, pea lectin) exhibit the canonical twelve-stranded beta-sandwich structure. In contrast to legume lectins that interact with both mannose and glucose, the monocot mannose-binding lectins (e.g., the Galanthus nivalis agglutinin or GNA from bulbs) react exclusively with mannose and mannose-containing N-glycans. These lectins possess a beta-prism structure. More recently, an increasing number of mannose-specific lectins structurally related to jacalin (e.g., the lectins from the Jerusalem artichoke, banana or rice), which also exhibit a beta-prism organization, were characterized. Jacalin itself was re-defined as a polyspecific lectin which, in addition to galactose, also interacts with mannose and mannose-containing glycans. Finally the B-chain of the type II RIP of iris, which has the same beta-prism structure as all other members of the ricin-B family, interacts specifically with mannose and galactose. This structural diversity associated with the specific recognition of high-mannose type glycans highlights the importance of mannose-specific lectins as recognition molecules in higher plants.

Spatial differences of endogenous lectin expression within the cellular organization of the human heart: a glycohistochemical, immunohistochemical, and glycobiochemical study.

Bardosi A, Bardosi L, Hendrys M, Wosgien B, Gabius HJ
Am J Anat. 1990; 188: 409-18

Protein-carbohydrate recognition may be involved in an array of molecular interactions on the cellular and subcellular levels. To gain insight into the role of proteins in this type of interaction, surgically removed specimens of human endomyocardial tissue were processed for histochemical and biochemical analysis. The inherent capacity of these sections to bind individual sugar moieties, which are constituents of the carbohydrate part of cellular glycoconjugates, was assessed using a panel of biotinylated neoglycoproteins according to a standardized procedure. Together with appropriate controls, it primarily allowed localization of endogenous lectins. Differences in lectin expression were observed between layers of endocardial tissue, myocardial cell constituents, connective-tissue elements, and vascular structures. The endocardium proved to be positive with beta-galactoside-bearing probes; with neoglycoproteins carrying beta-xylosides, alpha-fucosides, and galactose-6-phosphate moieties; and with probes containing a carboxyl group within the carbohydrate structure, namely sialic acid and glucuronic acid. In contrast, only fucose-and maltose-specific receptors were apparent in the elastic layers of the endocardium. Aside from ascertaining the specificity of the protein-carbohydrate interaction by controls, i.e., lack of binding of the probe in the presence of the unlabelled neoglycoprotein and lack of binding of the labelled sugar-free carrier protein, respective sugar receptors were isolated from heart extracts by using histochemically effective carbohydrates as immobilized affinity ligand. Moreover, affinity chromatography using immobilized lactose as affinity ligand as well as the use of polyclonal antibodies against the predominant beta-galactoside-specific lectin of heart demonstrated that the lactose-specific neoglycoprotein binding was due to this lectin. Remarkably, the labelled endogenous lectin, preferred to plant lectins for detecting ligands of the endogenous lectin, localized ligands in tissue parts where the lectin itself was detected glycohistochemically as well as immunohistologically. This demonstration of receptor-ligand presence in the same system is a further step toward functional assignment of the recorded protein-carbohydrate interaction. Overall, the observed patterns of lectin expression may serve as a guideline to elucidate the precise physiological relevance of lectins and to analyze pathological conditions comparatively.

Conformation, protein-carbohydrate interactions and a novel subunit association in the refined structure of peanut lectin-lactose complex.

Banerjee R, Das K, Ravishankar R, Suguna K, Surolia A, Vijayan M
J Mol Biol. 1996; 259: 281-96

The structure of the complex of the tetrameric peanut lectin with lactose has been refined to an R-value of 16.4% using 2.25 angstroms resolution X-ray diffraction data. The subunit conformation in the structure is similar to that in other legume lectins except in the loops. It has been shown that in the tertiary structure of legume lectins, the short five-stranded sheet plays a major role in connecting the larger flat six-stranded and curved seven-stranded sheets. Furthermore, the loops that connect the strands at the two ends of the seven-stranded sheet curve toward and interact with each other to produce a second hydrophobic core in addition to the one between the two large sheets. The protein-lactose interactions involve the invariant features observed in other legume lectins in addition to those characteristic of peanut lectin. The "open" quaternary association in peanut lectin is stabilised by hydrophobic, hydrogen-bonded and water-mediated interactions. Contrary to the earlier belief, the structure of peanut lectin demonstrates that the variability in quaternary association in legume lectins, despite all of them having nearly the same tertiary structure, is not necessarily caused by covalently bound carbohydrate. An attempt has been made to provide a structural rationale for this variability, on the basis of buried surface areas during dimerisation. A total of 45 water molecules remain invariant when the hydration shells of the four subunits are compared. A majority of them appear to be involved in stabilising loops.

A molecular model of the carbohydrate recognition domain of a rat macrophage lectin and analysis of its binding site.

Bajorath J
J Mol Graph. 1996; 14: 297-301

A three-dimensional model of the carbohydrate recognition domain of a rat macrophage C-type lectin has been constructed by comparative modeling and assessed by inverse folding analysis. Comparative modeling in the presence of low sequence similarity was based on information provided by comparison of X-ray structures and sequence-structure alignments. The sequence-structure compatibility of the model was sound. Its binding site was analyzed in comparison to the X-ray structure of a galactose-specific mutant of the mannose-binding protein. The specificity of the macrophage lectin was discussed in light of mutagenesis data on asialoglycoprotein receptors.

Structural basis for chitin recognition by defense proteins: GlcNAc residues are bound in a multivalent fashion by extended binding sites in hevein domains.

Asensio JL, Canada FJ, Siebert HC, Laynez J, Poveda A, Nieto PM, Soedjanaamadja UM, Gabius HJ, Jimenez-Barbero J
Chem Biol. 2000; 7: 529-43

BACKGROUND: Many plants respond to pathogenic attack by producing defense proteins that are capable of reversible binding to chitin, a polysaccharide present in the cell wall of fungi and the exoskeleton of insects. Most of these chitin-binding proteins include a common structural motif of 30 to 43 residues organized around a conserved four-disulfide core, known as the 'hevein domain' or 'chitin-binding' motif. Although a number of structural and thermodynamic studies on hevein-type domains have been reported, these studies do not clarify how chitin recognition is achieved. RESULTS: The specific interaction of hevein with several (GlcNAc)(n) oligomers has been studied using nuclear magnetic resonance (NMR), analytical ultracentrifugation and isothermal titration microcalorimetry (ITC). The data demonstrate that hevein binds (GlcNAc)(2-4) in 1:1 stoichiometry with millimolar affinity. In contrast, for (GlcNAc)(5), a significant increase in binding affinity is observed. Analytical ultracentrifugation studies on the hevein-(GlcNAc)(5,8) interaction allowed detection of protein-carbohydrate complexes with a ratio of 2:1 in solution. NMR structural studies on the hevein-(GlcNAc)(5) complex showed the existence of an extended binding site with at least five GlcNAc units directly involved in protein-sugar contacts. CONCLUSIONS: The first detailed structural model for the hevein-chitin complex is presented on the basis of the analysis of NMR data. The resulting model, in combination with ITC and analytical ultracentrifugation data, conclusively shows that recognition of chitin by hevein domains is a dynamic process, which is not exclusively restricted to the binding of the nonreducing end of the polymer as previously thought. This allows chitin to bind with high affinity to a variable number of protein molecules, depending on the polysaccharide chain length. The biological process is multivalent.

NMR investigations of protein-carbohydrate interactions: refined three-dimensional structure of the complex between hevein and methyl beta-chitobioside.

Asensio JL, Canada FJ, Bruix M, Gonzalez C, Khiar N, Rodriguez-Romero A, Jimenez-Barbero J
Glycobiology. 1998; 8: 569-77

The specific interaction of hevein with GlcNAc-containing oligosaccharides has been analyzed by1H-NMR spectroscopy. The association constants for the binding of hevein to a variety of ligands have been estimated from1H-NMR titration experiments. The association constants increase in the order GlcNAc-alpha(1-->6)-Man < GlcNAc < benzyl-beta-GlcNAc < p-nitrophenyl-beta-GlcNAc < chitobiose < p-nitrophenyl-beta-chitobioside < methyl-beta-chitobioside < chitotriose. Entropy and enthalpy of binding for different complexes have been obtained from van't Hoff analysis. The driving force for the binding process is provided by a negative DeltaH0which is partially compensated by negative DeltaS0. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 475 accurate protein proton-proton distance constraints after employing the MARDIGRAS program. In addition, 15 unambiguous protein/carbohydrate NOEs were detected. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the highly refined solution conformation of this protein-carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein nOe's were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 20 refined structures was 0.055 nm, while the heavy atom rmsd was 0.116 nm. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to the complex. A comparison of the three-dimensional structure of hevein in solution to those reported for wheat germ agglutinin (WGA) and hevein itself in the solid state has also been performed. The polypeptide conformation has also been compared to the NMR-derived structure of a smaller antifungical peptide, Ac-AMP2.

Synthesis and carbohydrate-binding activity of poly(ethyleneglycol)-Ricinus communis agglutinin I conjugates.

Allen HJ, Sharma A, Matta AK
Carbohydr Res. 1991; 213: 309-19

The synthesis of poly(ethyleneglycol) (PEG)-lectin conjugates was investigated to provide new reagents for evaluation as biological response modifiers. PEG was activated with 1,1'-carbonyldiimidazole (CDI), followed by conjugation with Ricinus communis I (RCAI) lectin. The resulting conjugates were heterodisperse with respect to molecular weight. Carbohydrate-binding activity was retained. The conjugates were separated by affinity chromatography into fractions differing in apparent carbohydrate-binding affinity. Conjugation of RCAI with PEG 4 (mol.wt. 3350) or PEG 6 (mol.wt. 8000) appeared to provide less hindrance of the lectin binding site compared to conjugates prepared with PEG 20 (mol.wt. 20,000). Results of free amine assays indicated that higher ratios of PEG to RCAI in conjugates correlated with loss of low-affinity binding and retention of high-affinity binding. The data showed the feasibility of preparing PEG-lectin conjugates for in vivo use.

Potato lectin - a glycoprotein with two domains.

Allen AK
Prog Clin Biol Res. 1983; 138: 71-85

Histochemistry and cytochemistry of endogenous animal lectins.

Akimoto Y, Imai Y, Hirabayashi J, Kasai K, Hirano H
Prog Histochem Cytochem. 1998; 33: 1-90

Galectins: conservation of functionally and structurally relevant amino acid residues defines two types of carbohydrate recognition domains.

Ahmed H, Vasta GR
Glycobiology. 1994; 4: 545-8

Binding of hydroxylysine-linked saccharides by galaptin, a galactoside-binding animal tissue lectin.

Ahmed H, Allen HJ, DiCioccio RA
Carbohydr Res. 1991; 213: 321-4

Physicochemical characterization of Cajanus cajan lectin: effect of pH and metal ions on lectin carbohydrate interaction.

Ahmad S, Khan RH, Ahmad A
Biochim Biophys Acta. 1999; 1427: 378-84

The association constant of Cajanus cajan lectin for methyl alpha-D-mannopyranoside was studied by equilibrium dialysis method. An attempt was also made to understand the metal ion requirements and to establish that ionizable groups are responsible for lectin-carbohydrate interaction. The N-terminal sequence up to 27 amino acid residues was found to be more than 80% homologous with other mannose-specific legume lectins of the tribe Viceae. Like concanavalin A and pea lectin it also exhibits high affinity for the sugar alpha-methyl mannose and at 37 degrees C the association constant was found to be 1.4x104 M-1. The lectin required one Ca2+ and one Mg2+ per mole and during the lectin sugar interaction two ionizable groups with pK of 3.75 and 8.3 are ionized. Whether the secondary structure is similarly affected with pH changes and presence or absence of metal ion was investigated by circular dichroism studies. Results suggested that changes in carbohydrate binding properties of the Cajanus cajan lectin due to change in pH and addition of metal ions are not accompanied by any significant change in secondary structure.