Inhibitory potential of chemical substitutions at bioinspired sites of β-D-galactopyranose on neoglycoprotein/cell surface binding of two classes of medically relevant lectins

Galactose is the key contact site for plant AB-toxins and the human adhesion/growth-regulatory galectins. Natural anomeric extensions and 3'-substitutions enhance its reactivity, thus prompting us to test the potential of respective chemical substitutions of galactose in the quest to develop potent inhibitors. Biochemical screening of a respective glycoside library with 60 substances in a solid-phase assay was followed by examining the compounds' activity to protect cells from lectin binding. By testing 32 anomeric extensions, 18 compounds with additional 3'-substitution, three lactosides and two Lewis-type trisaccharides rather mild effects compared to the common haptenic inhibitor lactose were detected in both assays. When using trivalent glycoclusters marked enhancements with 6- to 8-fold increases were revealed for the toxin and three of four tested galectins. Since the most potent compound and also 3'-substituted thiogalactosides reduced cell growth of a human tumor line at millimolar concentrations, biocompatible substitutions and scaffolds will be required for further developments. The synthesis of suitable glycoclusters, presenting headgroups which exploit differences in ligand selection in interlectin comparison to reduce cross-reactivity, and the documented strategic combination of initial biochemical screening with cell assays are considered instrumental to advance inhibitor design.     

Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions

Galectins are a family of beta-galactose binding lectins associated with functions such as immunological and malignant events. To study the binding affinity of galectins for natural and artificial saccharides and glycoconjugates we have developed an assay using fluorescence polarization. A collection of fluorescein-conjugated saccharides was synthesized and used as probes with galectins-1 and -3 and the two carbohydrate recognition domains of galectin-4. Direct binding of a fixed probe amount with different amounts of each galectin defined specificity and selectivity and permitted selection of the optimal probe for inhibition studies. Then fixed amounts of galectin and selected probe were used to screen the inhibitory potency of a library of nonfluorescent compounds. As the assay is in solution and does not require separation of free and bound probe, it is simple and rapid and can easily be applied to different unlabeled galectins. As all interaction components are known, K(d) values for galectin-inhibitor interaction can be directly calculated without approximation other than the assumption of a simple one-site competition.     

Assessing the inhibitory potency of galectin ligands identified from combinatorial (glyco)peptide libraries using surface plasmon resonance spectroscopy

Combinatorial (glyco)peptide libraries offer the possibility to define effective inhibitors of protein (lectin)-glycan interactions. If a (glyco)peptide surpasses the inhibitory potency of the free sugar, then the new peptide-lectin contacts underlying the affinity enhancement may guide further rational drug design. Focusing on the adhesion/growth regulatory human galectins 1 and 3, a screening of three combinatorial solid-phase (glyco)peptide libraries, containing Gal(β1-O)Thr, Gal(β1-S)Cys/Gal(β1-N)Asn, and Lac(β1-O)Thr, with the fluorescently labeled lectins had led to a series of lead compounds. To define the inhibitory potency of a selection of resynthesized (glyco)peptides systematically, a surface plasmon resonance-based inhibition assay with immobilized asialofetuin was set up. (Glyco)Peptides with up to 66-fold potency relative to free lactose as inhibitor were characterized. The presence of lactose in the most effective glycopeptides indicated the presence of affinity-enhancing peptide-lectin contacts. In addition to drug design, they may be helpful for fine-structural analysis of the binding sites.     

Crystal structure of the galectin-9 N-terminal carbohydrate recognition domain from Mus musculus reveals the basic mechanism of carbohydrate recognition

The galectins are a family of beta-galactoside-binding animal lectins with a conserved carbohydrate recognition domain (CRD). They have a high affinity for small beta-galactosides, but binding specificity for complex glycoconjugates varies considerably within the family. The ligand recognition is essential for their proper function, and the structures of several galectins have suggested their mechanism of carbohydrate binding. Galectin-9 has two tandem CRDs with a short linker, and we report the crystal structures of mouse galectin-9 N-terminal CRD (NCRD) in the absence and the presence of four ligand complexes. All structures form the same dimer, which is quite different from the canonical 2-fold symmetric dimer seen for galectin-1 and -2. The beta-galactoside recognition mechanism in the galectin-9 NCRD is highly conserved among other galectins. In the apo form structure, water molecules mimic the ligand hydrogen-bond network. The galectin-9 NCRD can bind both N-acetyllactosamine (Galbeta1-4GlcNAc) and T-antigen (Galbeta1-3GalNAc) with the proper location of Arg-64. Moreover, the structure of the N-acetyllactosamine dimer (Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc) complex shows a unique binding mode of galectin-9. Finally, surface plasmon resonance assay showed that the galectin-9 NCRD forms a homophilic dimer not only in the crystal but also in solution.     

Cyclic neoglycodecapeptides: how to increase their inhibitory activity and selectivity on lectin/toxin binding to a glycoprotein and cells

Protein (lectin/toxin)–glycan interaction can be clinically harmful so that the design of inhibitors has become an aim. Cyclic decapeptides are suited as rigid carriers for carbohydrate derivatives. We herein document the bioactivity of sugar headgroups covalently attached to this carrier for the cases of five proteins, i.e. a potent biohazardous plant agglutinin, a leguminous model lectin and three adhesion/growth‐regulatory human lectins. They represent the different types of topological organization within the galectin family. The relative inhibitory activities of glycoclusters with the three ligands (galactose, lactose and the disaccharide of the Thomsen‐Friedenreich antigen) reflected the affinity of free carbohydrates, hereby excluding an impairment of binding activity by chemical derivatization and conjugation. Headgroup tailoring is thus one route to optimize activity and selectivity of cyclopeptide‐based glycoclusters. The increase of ligand density from tetra‐ to hexadecavalency added a second route. The plant toxin and tandem‐repeat‐type galectin‐4 were especially sensitive to this parameter change. Strategically combining solid‐phase assays for screening with analysis of lectin binding to cells in different systems revealed efficient inhibition by distinct glycoclusters, thereby protecting cells from lectin association. Cyclic neoglycodecapeptides thus warrant further study as lectin‐directed pharmaceuticals. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Galectins bind to the multivalent glycoprotein asialofetuin with enhanced affinities and a gradient of decreasing binding constants

Our previous isothermal titration microcalorimetry (ITC) studies of the binding of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) showed negative binding cooperativity that was due to the carbohydrate ligands and not the proteins [Dam, T. K., et al. (2002) Biochemistry 41, 1351-1358]. The negative cooperativity was associated with the decreasing functional valence of the carbohydrates upon progressive binding of their epitopes. The present study also shows negative cooperativity in the ITC binding data of asialofetuin (ASF), a glycoprotein that possesses nine LacNAc epitopes, to galectin-1, -2, -3, -4, -5, and -7, and truncated, monomer versions of galectin-3 and -5, which are members of a family of animal lectins. Although the observed K(a) values for binding of ASF to the galectins and two truncated forms are only 50-80-fold greater than that of LacNAc, analysis of the data in terms of the relationship between the observed macroscopic free energy of binding and the decreasing microscopic free energies of binding of the epitopes shows that the first LacNAc epitope of ASF binds with approximately 6000-fold higher affinity than the last epitope. Thus, the microscopic binding constants of the galectins for the first epitope(s) of ASF are in the nanomolar range, with a gradient of decreasing binding constants of the remaining epitopes. The results indicate that the above galectins bind with fractional, high affinities to multivalent glycoproteins such as ASF, independent of the quaternary structures of the galectins. These findings have important implications for the binding of galectins to multivalent carbohydrate receptors.     

Hydrodynamic Properties of Human Adhesion/Growth-Regulatory Galectins Studied by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy is applied on homologous human lectins (i.e., adhesion/growth-regulatory galectins) to detect influence of ligand binding and presence of the linker peptide in tandem-repeat-type proteins on hydrodynamic properties. Among five tested proteins, lactose binding increased the diffusion constant only in the cases of homodimeric galectin-1 and the linkerless variant of tandem-repeat-type galectin-4. To our knowledge, the close structural similarity among galectins does not translate into identical response to ligand binding. Kinetic measurements show association and dissociation rate constants in the order of 1 to 10³ M⁻¹ s⁻¹ and 10⁻⁴ s⁻¹, respectively. Presence of the linker peptide in tandem-repeat-type protein leads to anomalous scaling with molecular mass. These results provide what we believe to be new insights into lectin responses to glycan binding, detectable so far only by small angle neutron scattering, and the structural relevance of the linker peptide. Methodologically, fluorescence correlation spectroscopy is shown to be a rather simple technical tool to characterize hydrodynamic properties of these proteins at a high level of sensitivity.     

Binding and cross-linking properties of galectins

The galectins are a family of animal lectins that possess similar carbohydrate binding specificities and conserved consensus sequences. The biological properties of mammalian galectins include the regulation of inflammation, cell adhesion, cell proliferation and cell death. Evidence suggests that the biological activities of the galectins are related to their multivalent binding properties since most galectins possess two carbohydrate recognition domains and are therefore bivalent. For example, galectin-1, which is dimeric, binds and cross-links specific glycoprotein counter-receptors on the surface of human T-cells leading to apoptosis [J. Immunol. 163 (1999) 3801]. Different galectin-1 counter-receptors associated with specific phosphatase or kinase activities formed separate clusters on the surface of the cells as a result of the lectin binding to the carbohydrate chains of the respective glycoproteins. Importantly, monovalent galectin-1 is inactive in this system. This indicates that the separation and organization of signaling molecules that result from galectin-1 binding is involved in the apoptotic signal. The separation of specific glycoprotein receptors induced by galectin-1 binding was modeled on the basis of molecular and structural studies of the binding of lectins to multivalent carbohydrates resulting in the formation of specific two- and three-dimensional cross-linked lattices [Biochemistry 36 (1997) 15073]. In this article, the binding and cross-linking properties of galectin-1 and other lectins are reviewed as a model for the biological signal transduction properties of the galectin family of animal lectins.     

Synthesis and molecular recognition of carbohydrate-centered multivalent glycoclusters by a plant lectin RCA120

Water soluble and lectin-recognizable carbohydrate-centered glycoclusters were prepared efficiently by the Huisgen 1,3-cycloaddition reaction of methyl-2,3,4,6-tetra-O-propargyl beta-D-galactopyranoside with 2-azidoethyl glycosides of lactose and N-acetyllactosamine. Their binding by a plant lectin RCA120 was examined by capillary affinity electrophoresis using fluorescence-labeled asialoglycans from human alpha1-acid glycoprotein. The glycoclusters showed 400-fold stronger inhibitory effect than free lactose, manifesting strong multivalency effect.     

Analysis of selected blood and immune cell responses to carbohydrate-dependent surface binding of proto- and chimera-type galectins

Cell surface glycans present docking sites to endogenous lectins. With growing insight into the diversity of lectin families it becomes important to answer the question on the activity profiles of individual family members. Focusing on galectins (-galactoside-binding proteins without Ca²⁺-requirement sharing the jelly-roll-like folding pattern), this study was performed to assess the potency of proto-type galectins (galectins-1 and -7 and CG-16) and the chimera-type galectin-3 to elicit selected cell responses by carbohydrate-dependent surface binding and compare the results. The galectins, except for galectin-1, were found to enhance detergent (SDS)-induced hemolysis of human erythrocytes to different degrees. Their ability to confer increased membrane osmofragility thus differs. Aggregation of neutrophils, thymocytes and platelets was induced by the proto-type galectin-1 but not -7, by CG-16 and also galectin-3. Cell-type-specific quantitative differences and the importance of the fine-specificity of the galectin were clearly apparent. In order to detect cellular responses based on galectin binding and bridging of cells the formation of haptenic-sugar-resistant (HSR) intercellular contacts (an indicator of post-binding signaling) was monitored. It was elicited by CG-16 and galectin-1 but not galectin-3, revealing another level at which activities of individual galectins can differ. Acting as potent elicitor of neutrophil aggregation, CG-16-dependent post-binding effects were further analyzed. Carbohydrate-dependent binding to the neutrophils' surface led to a sustained increase of cytoplasmic Ca²⁺ concentration in a dose-dependent manner. The ability of CG-16 to activate H₂O₂ generation by human peripheral blood neutrophils was primed by the Ca²⁺-ionophor ionomycin and by cytochalasin B. In a general context, these results emphasize that – besides plant lectins as laboratory tools – animal lectins can trigger cell reaction cascades, implying potential in vivo relevance for the measured activities. Within the family of galectins, the activity profiles depend on the target cell type and the individual galectin. Notably, proto-type galectins do not necessarily share a uniform capacity as elicitor.     

Galectin-loaded cells as a platform for the profiling of lectin specificity by fluorescent neoglycoconjugates: a case study on galectins-1 and -3 and the impact of assay setting

The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells. The extent of surface binding of fluorescent neoglycoconjugates depended on the lectin presence and the type of lectin, the nature of the probes' carbohydrate headgroup and the density of unsubstituted beta-galactosides on the cell surface. Using the most frequently studied galectins-1 and -3, application of this assay led to rather equal binding levels for linear and branched oligomers of N-acetyllactosamine. A clear preference of galectin-3 for alpha1-3-linked galactosylated lactosamine was noted. In parallel, a panel of 24 neoglycoconjugates was tested as inhibitors of galectin binding from solution to N-glycans of surface-immobilized asialofetuin. These two assays differ in presentation of the galectin and ligand, facilitating identification of assay-dependent properties. Under the condition of the cell assay, selectivity among oligosaccharides for the lectins was higher, and extraordinary affinity of galectin-1 to 3'-O-sulfated probes in a solid-phase assay was lost in the cell assay. Having introduced and validated a cell assay, the comprehensive profiling of ligand binding to cell-surface-presented galectins is made possible.     

Discovery of galectin ligands in fully randomized combinatorial one-bead-one-compound (glyco)peptide libraries

The involvement of human lectins (galectins) in disease progression accounts for the interest to design potent inhibitors. Three fully randomized hexa(glyco)peptide libraries were prepared using the portion mixing method combined with ladder synthesis. On-bead screening with fluorescently labelled galectin-1 and -3 yielded a series of lead structures, whose inhibitory activity on carbohydrate-dependent galectin binding was tested in solution by solid-phase and cell assays. The various data obtained define the library approach as a facile route for the discovery of selective (glyco)peptide-based galectin inhibitors.     

Thermodynamic binding studies of galectin-1, -3 and -7

The carbohydrate binding specificities of the galectin family of animal lectins has been the source of intense recent investigations. Isothermal titration microcalorimetry (ITC) provides direct determination of the thermodynamics of binding of carbohydrates to lectins, and has provided important insights into the fine carbohydrate binding specificities of a wide number of plant and animal lectins. Recent ITC studies have been performed with galectin-1, galectin-3 and galectin-7 and their interactions with sialylated and non-sialylated carbohydrates. The results show important differences in the specificities of these three galectins toward poly-N-acetyllactosamine epitopes found on the surface of cells.     

Synthesis of bivalent lactosides and their activity as sensors for differences between lectins in inter- and intrafamily comparisons

The synthesis of nine bivalent lactosides (based on ditriazoles, diamides, a glycocyclophane and an acyclic analogue of the glycocyclophane) and one monovalent lactosyl triazole facilitated the assessment of the sensitivity of plant/animal lectins to this type of ligand display. The inhibitory potency of the compounds was determined in two assays of increasing biorelevance. These were solid-phase and cell binding set-ups. Hereby, the ability of the compounds to inhibit the binding of two plant agglutinins and the entire set of adhesion/growth-regulatory galectins from one organism (chicken) to a glycoprotein or to cell surfaces was systematically evaluated. Differential sensitivities were detected between plant and animal lectins and also between distinct galectin forms within the chicken series. Two of the bivalent probes can be considered as sensors for interlectin differences. Most pronounced were the selectivities of N-glycosyl 1,2,3-triazole derivatives for the chimera-type galectin and its proteolytically truncated version.     

Synthesis of multivalent lactose derivatives by 1,3-dipolar cycloadditions: selective galectin-1 inhibition

Acetylene derivatives of phenylalanine, phenethylamine and the multifunctional unnatural amino acids, phenyl-bis-alanine and phenyl-tris-alanine, were synthesized and functionalized with 2-azidoethyl beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranoside via regioselective copper(I)-mediated 1,3-dipolar cycloaddition to give a panel of mono-, di- and trivalent lactoside derivatives. Evaluation of the compounds as inhibitors against the tumour- and inflammation-related galectin-1, -3, -4N, -4C, -4, -7, -8N and -9N revealed a divalent compound with a Kd value as low as 3.2 microM for galectin-1, which corresponded to a relative potency of 30 per lactose unit as compared to the natural disaccharide ligand lactose. This divalent compound had at least one order of magnitude higher affinity for galectin-1 than for any of the other galectins investigated.     

Thermodynamic binding studies of galectin-1, -3 and -7

The carbohydrate binding specificities of the galectin family of animal lectins has been the source of intense recent investigations. Isothermal titration microcalorimetry (ITC) provides direct determination of the thermodynamics of binding of carbohydrates to lectins, and has provided important insights into the fine carbohydrate binding specificities of a wide number of plant and animal lectins. Recent ITC studies have been performed with galectin-1, galectin-3 and galectin-7 and their interactions with sialylated and non-sialylated carbohydrates. The results show important differences in the specificities of these three galectins toward poly-N-acetyllactosamine epitopes found on the surface of cells. Published in 2004.     

Glycosyldisulfides from dynamic combinatorial libraries as O-glycoside mimetics for plant and endogenous lectins: their reactivities in solid-phase and cell assays and conformational analysis by molecular dynamics simulations

Dynamic combinatorial library design exploiting the thiol-disulfide exchange readily affords access to glycosyldisulfides. In order to reveal lectin-binding properties of this type of non-hydrolyzable sugar derivative, libraries originating from a mixture of common building blocks of natural glycans and thiocompounds were tested against three plant agglutinins with specificity to galactose, fucose or N-acetylgalactosamine, respectively, in a solid-phase assay. Extent of lectin binding to matrix-immobilized neoglycoprotein presenting the cognate sugar could be reduced, and evidence for dependence on type of carbohydrate was provided by dynamic deconvolution. Glycosyldisulfides also maintained activity in assays of increased physiological relevance, that is, using native tumor cells and also adding to the test panel an endogenous lectin (galectin-3) involved in tumor spread and cardiac dysfunction. N-Acetylgalactosamine was pinpointed as the most important building block of libraries for the human lectin and the digalactoside as most potent compound acting on the toxic mistletoe agglutinin which is closely related to the biohazard ricin. Because this glycosyldisulfide, which even surpasses lactose in inhibitory capacity, rivals thiodigalactoside as inhibitor, their degrees of intramolecular flexibility were comparatively analyzed by computational calculations. Molecular dynamics runs with explicit consideration of water molecules revealed a conspicuously high degree of potential for shape alterations by the disulfide's three-bond system at the interglycosidic linkage. The presented evidence defines glycosyldisulfides as biologically active ligands for lectins.     

Expression of galectin-1 and -3 and of accessible binding sites during murine hair cycle

Although protein-carbohydrate interactions are supposed to play key roles in cell adhesion, signalling and growth control. Their exact role in skin physiology has only recently been investigated. The endogenous lectins galectin-1 and galectin-3 have been identified in skin including hair follicles. Here, we analyzed the expression and distribution of these galectins and their binding sites in C57BL/6 mice during hair cycle. The expression of galectin-1 and galectin-3 binding sites was found to be predominantly hair cycle-dependent showing some overlapping to the expression of galectin-1 and -3. The outer root sheath (ORS) expressed galectin-1 binding sites during anagen IV to VI and in early catagen, whereas galectin-1 was expressed from early anagen to late catagen. The ORS expressed galectin-3 binding sites during catagen transition corresponding to a galectin-3 expression during anagen V and catagen. The innermost layer of the ORS expressed galectin-3 binding sites during anagen VI until catagen VIII, but galectin-3 during anagen III to IV and catagen. The inner root sheath (IRS) expressed galectin-3 binding sites only in anagen IV but missed expression of any of the two galectins. The matrix cells expressed galectin-3 binding sites in catagen II-III as well as galectin-3 during anagen V to catagen IV. The present study provides the first evidence for a cycle-related expression of both galectin-1 and -3 and their binding sites during murine hair cycle.     

Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells

Galectins are implicated in a large variety of biological functions, many of which depend on their carbohydrate-binding ability. Fifteen members of the family have been identified in vertebrates based on binding to galactose (Gal) that is mediated by one or two, evolutionarily conserved, carbohydrate-recognition domains (CRDs). Variations in glycan structures expressed on glycoconjugates at the cell surface may, therefore, affect galectin binding and functions. To identify roles for different glycans in the binding of the three types of mammalian galectins to cells, we performed fluorescence cytometry at 4 degrees C with recombinant rat galectin-1, human galectin-3, and three forms of human galectin-8, to Chinese hamster ovary (CHO) cells and 12 different CHO glycosylation mutants. All galectin species bound to parent CHO cells and binding was inhibited >90% by 0.2 M lactose. Galectin-8 isoforms with either a long or a short inter-CRD linker bound similarly to CHO cells. However, a truncated form of galectin-8 containing only the N-terminal CRD bound only weakly to CHO cells and the C-terminal galectin-8 CRD exhibited extremely low binding. Binding of the galectins to the different CHO glycosylation mutants revealed that complex N-glycans are the major ligands for each galectin except the N-terminal CRD of galectins-8, and also identified some fine differences in glycan recognition. Interestingly, increased binding of galectin-1 at 4 degrees C correlated with increased propidium iodide (PI) uptake, whereas galectin-3 or -8 binding did not induce permeability to PI. The CHO glycosylation mutants with various repertoires of cell surface glycans are a useful tool for investigating galectin-cell interactions as they present complex and simple glycans in a natural mixture of multivalent protein and lipid glycoconjugates anchored in a cell membrane.