Uridine binding by Zn(II) macrocyclic complexes: diversion of RNA cleavage catalysts

Zn(II) complexes of 1-oxa-4,7,10-triazacyclododecane (12[ane]N3O), 1,5,9-triazacyclododecane (12[ane]N3), and 1-hydroxyethyl-1,4,7-triazacyclononane (9[ane]N3OH) promote cleavage of the RNA analogue, 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) at pH 8.0, I=0.10 M (NaCl), 25 degrees C with second-order rate constants of 8.9x10(-3), 9.0x10(-3), and 3.3x10(-3) M-1 s-1, respectively. Cleavage of HpPNP by these catalysts is inhibited by uridine with inhibition constants (Ki) of 1.2, 0.46, and 45 mM, respectively, under these conditions. Binding constants derived from these inhibition constants are 2-200-fold larger than those for binding of related Zn(II) complexes to phosphate diesters under similar conditions, suggesting that uridine sequences in RNA will inhibit Zn(II)-catalyzed cleavage by competing with phosphate diester binding sites. Further studies are carried out that utilize pH-potentiometric titrations to monitor uridine binding to five Zn(II) macrocyclic complexes in aqueous solution at 25 degrees C, I=0.10 M (NaCl). The data are consistent with binding of the Zn(II) complexes to the N3-deprotonated form of uridine to give log KU.-values of 5.29, 4.57, 4.56, 3.47, and 2.65 for the Zn(II) complexes of 12[ane]N3, 12[ane]N4, 12[ane]N3O, 15[ane]N3O2, and 9[ane]N3OH, respectively (12[ane]N4=1,4,7,10-tetraazacyclododecane, 15[ane]N3O2=1,4-dioxa-7,10,13-triazacyclopentadecane). For the five Zn(II) complexes studied, there is a linear relationship between uridine anion binding constants and hydroxide binding constants.     

Selectivity among two lectins: probing the effect of topology, multivalency and flexibility of "clicked" multivalent glycoclusters

The design of multivalent glycoconjugates has been developed over the past decades to obtain high-affinity ligands for lectin receptors. While multivalency frequently increases the affinity of a ligand for its lectin through the so-called "glycoside cluster effect", the binding profiles towards different lectins have been much less investigated. We have designed a series of multivalent galactosylated glycoconjugates and studied their binding properties towards two lectins, from plant and bacterial origins, to determine their potential selectivity. The synthesis was achieved through copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) under microwave activation between propargylated multivalent scaffolds and an azido-functionalised carbohydrate derivative. The interactions of two galactose-binding lectins from Pseudomonas aeruginosa (PA-IL) and Erythrina cristagalli (ECA) with the synthesized glycoclusters were studied by hemagglutination inhibition assays (HIA), surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). The results obtained illustrate the influence of the scaffold's geometry on the affinity towards the lectin and also on the relative potency in comparison with a monovalent galactoside reference probe.     

Synthesis of dodecavalent fullerene-based glycoclusters and evaluation of their binding properties towards a bacterial lectin

Multivalency is playing a major role in biological processes and particularly in lectin-carbohydrate interactions. The design of high-affinity ligands of lectins should provide molecules capable of interfering with these biological processes and potentially inhibit bacterial or viral infections. Azide-alkyne "click" chemistry was applied to the synthesis of dodecavalent fullerene-based glycoclusters. The conjugation could be efficiently performed from alkyne or azide functions on either partners (i.e. hexakis-fullerene adduct or glycoside). PA-IL is a bacterial lectin from the opportunistic pathogen Pseudomonas aeruginosa and is involved in the recognition of glycoconjugates on human tissues. The glycoclusters obtained were evaluated as ligands of PA-IL and for their potential for competing with its binding to glycosylated surfaces. The affinities measured by hemagglutination inhibition assay (HIA), enzyme-linked lectin assay (ELLA), and surface plasmon resonance (SPR) displayed a significant "glycoside cluster effect" with up to a 12,000-fold increase in binding when comparing a monovalent carbohydrate reference probe with a dodecavalent fullerene-based glycocluster, albeit with some differences depending on the analytical technique.     

Synthesis of Multivalent Carbohydrate‐Centered Glycoclusters as Nanomolar Ligands of the Bacterial Lectin LecA from Pseudomonas aeruginosa

A family of fifteen glycoclusters based on a cyclic oligo‐(1→6)‐β‐D ‐glucosamine core has been designed as potential inhibitors of the bacterial lectin LecA with various valencies (from 2 to 4) and linkers. Evaluation of their binding properties towards LecA has been performed by a combination of hemagglutination inhibition assays (HIA), enzyme‐linked lectin assays (ELLA), and isothermal titration microcalorimetry (ITC). Divalent ligands displayed dissociation constants in the sub‐micromolar range and tetravalent ligands displayed low nanomolar affinities for this lectin. The influence of the linker could also be demonstrated; aromatic moieties are the best scaffolds for binding to the lectin. The affinities observed in vitro were then correlated with molecular models to rationalize the possible binding modes of these glycoclusters with the bacterial lectin.     

Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy

Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence, robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r(0) was about 0.2, altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited, no labeling is needed. Owing to tryptophan's presence in carbohydrate-binding sites, also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases, glycosidases), this assay procedure can have relevance beyond galectins.     

Computational prediction of monosaccharide binding free energies to lectins with linear interaction energy models

The linear interaction energy (LIE) method to compute binding free energies is applied to lectin‐monosaccharide complexes. Here, we calculate the binding free energies of monosaccharides to the Ralstonia solanacearum lectin (RSL) and the Pseudomonas aeruginosa lectin‐II (PA‐IIL). The standard LIE model performs very well for RSL, whereas the PA‐IIL system, where ligand binding involves two calcium ions, presents a major challenge. To overcome this, we explore a new variant of the LIE model, where ligand–metal ion interactions are scaled separately. This model also predicts the saccharide binding preference of PA‐IIL on mutation of the receptor, which may be useful for protein engineering of lectins. © 2012 Wiley Periodicals, Inc.     

Detection of cytotoxic activity of lectin on human colon adenocarcinoma (Sw480) and epithelial cervical carcinoma (C33-A)

Lectins comprise a heterogeneous class of proteins that recognize the carbohydrate moieties of glycoconjugates with high specificity. Numerous studies have shown that lectins are capable of recognizing specific carbohydrate moieties displayed by malignant cells or tissues. The present work was performed to investigate the effects of tepary bean (Phaseolus acutifolius) lectins on proliferation, colony formation, and alteration of DNA synthesis of human malignant cells. Tepary bean lectin showed dose dependent effects on the inhibition of viability as well as on colony formation in two human malignant cells lines (C33-A, Sw480); By contrast, tepary bean lectin only showed significant effects on DNA synthesis on Sw480 cells. Our results provide evidence of the anti- proliferative and cytotoxic effects of the tepary bean lectins on C33-A and Sw480 cells lines.     

Studying the interaction of carbohydrate-protein on the dendrimer-modified solid support by microarray-based plasmon resonance light scattering assay

Here, a three-dimensional (3D) carbohydrate microarray-based plasmon resonance light scattering (RLS) assay has been established for studying carbohydrate-lectin binding with high selectivity. The 3D carbohydrate microarray is fabricated by immobilizing amino-modified carbohydrates on the home-made fourth-generation (G4) NH(2)-terminated poly(amidoamine) dendrimers (PAMAM)-modified substrate. After marking the carbohydrate-lectin binding events by 13 nm peptide-stabilized gold nanoparticles through the biotin-avidin reaction, the 3D microarray can be directly detected by the RLS scanner without the conventional silver enhancement step. The well defined recognition systems: three monosaccharides (Man-α, Glc-α and Gal-β) with two lectins (Con A and RCA 120), have been chosen here to establish the RLS assay, respectively. Quantitative determination of the surface dissociation constants (K(D,surf)) for surface carbohydrates and lectins has been achieved. In addition, inhibition values (i.e. the inhibition constants (K(i)) and the concentrations of inhibitors required to produce 50% inhibition (IC(50))) for inhibitors in solution are also demonstrated by the saccharide competing assays.     

First demonstration of differential inhibition of lectin binding by synthetic tri- and tetravalent glycoclusters from cross-coupling of rigidified 2-propynyl lactoside

The interplay of mammalian lectins such as galectins with cellular glycoconjugates is intimately involved in crucial reaction pathways including tumor cell adhesion, migration or growth regulation. These clinically relevant functions explain the interest in designing glycoclusters with potent activity to interfere with lectin binding. In view of the perspective for medical applications the following objective arises: to correlate topological factors of ligand display most favorably to reactivity against endogenous lectins. To date, plant agglutinins have commonly been used as models. Properly addressing this issue we first prepared di- to tetravalent clusters from 2-propynyl lactoside under mild oxidative homocoupling conditions and using the Sonogashira palladium-catalyzed cross-coupling reaction with triiodobenzene or pentaerythritol cores. These products were tested for bioactivity in a competitive solid-phase assay using different labeled sugar receptors as probes, i,e. the beta-trefoil mistletoe lectin, the natural lactoside-binding immunoglobulin G fraction from human serum and three mammalian galectins from two subgroups. The lactose headgroups in the derivatives retained ligand properties. Differences in inhibitory capacity were marked between the galectins. In contrast to homodimeric proto-type galectins-1 and -7 significant inhibition of galectin-3 binding with a 7-fold increase in relative potency was observed for the trivalent compound. In comparison, the binding of the beta-trefoil mistletoe agglutinin was reduced best by tetravalent substances The result for galectin-3 was independently confirmed by haemagglutination and cytofluorometric cell binding assays. These data underline the feasibility of galectin-type target selectivity by compound design despite using an identical headgroup (lactose) in synthesis.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells.

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either 125I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10(7) binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37 degrees C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Isothermal titration microcalorimetric method for studying the combined ligand binding with application to the binding of ethylurea and (N,N)dimethylurea on urease

A simple, novel method was introduced for determining equilibrium constants and enthalpies of binding of two different competitive ligands on a macromolecule by isothermal titration microcalorimetry technique. This method was applied to the simultaneous binding of ethylurea (I) and (N,N)dimethylurea (X), on jack-bean urease at pH 7.0 (tris-base; 30 mM) at 27°C. The dissociation equilibrium constants measured by this method were markedly consistent with inhibition constants obtained from assay of enzyme activities in the presence of I and X.     

Neoglycopeptide dendrimer libraries as a source of lectin binding ligands

[structure: see text] A 15 625-membered peptide dendrimer combinatorial library was acylated with an alpha-C-fucosyl residue at its four N-termini and screened for binding to fucose-specific lectins. Dendrimer FD2 (Fuc-alpha-CH2CO-Lys-Pro-Leu)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2 was identified as a potent ligand against Ulex europaeus lectin UEA-I (IC50 = 11 microM) and Pseudomonas aeruginosa lectin PA-IIL (IC50 = 0.14 microM).     

Partial-filling affinity capillary electrophoresis of glycoprotein oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid

Partial-filling affinity capillary electrophoresis has been applied to the simultaneous analysis of interactions between glycoprotein oligosaccharides and certain plant lectins. A lectin solution and a mixture of glycoprotein-derived oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid were introduced to a neutrally coated capillary in this order, and separated by application of a negative voltage. Interaction of a lectin with each oligosaccharide in the mixture was observed as the specific retardation or dissipation of peaks, in addition to the size/charge separation of oligosaccharides by zone electrophoresis in the remainder (≈90%) of the capillary. The strength of the interaction with lectin was controlled by introducing an appropriate volume of lectin solution. Application of various specificities of lectins indicated characteristic migration profiles of the oligosaccharides. Moreover, sequential injection of four lectins (Maachia amurensis mitogen, Sambucus sieboldiana agglutinin, Erythrina cristagalli agglutinin, Aleuria aurantia lectin) induced complete dissipation of complex-type oligosaccharides and enabled specific determination of the presence of high-mannose oligosaccharides without the interference or alteration of the electropherogram in porcine thyroglobulin. This method was also applied to determine the binding constants of ovalbumin-derived oligosaccharides to wheat germ agglutinin.     

Binding evaluation of isoform 1 fromCratylia mollis lectin to human mammary tissues

The phenomenon of altered carbohydrates in transformed cell surfaces has been studied through histochemical techniques using lectins. Specific binding patterns to normal and transformed mammary tissues were evaluated by Isoform 1 fromCratylia mollis lectin (Cra Iso 1). Protocols using a direct method, incubation of Cra Iso I conjugated to peroxidase (Cra Iso 1-Per) with mammary tissues, followed by diaminobenzidine and hydrogen peroxidase interaction, were performed. Neoplastic tissues, marked by Cra Iso 1, showed a higher intensity of staining than normal ones, in comparison withCanavalia ensiformis lectin, Concanavalin A (Con A), conjugated to peroxidase (Con A-Per). The assay with Cra Iso 1 also indicated a possible utilization of this lectin to characterize normal and transformed mammary cells.     

Biological activity of chitosan–sugar hybrids: specific interaction with lectin

The specific interactions between lectins and chitosan–sugar hybrids, the synthesized chitosan derivatives linking carbohydrate residue to the amino group of chitosan, were investigated. The specific bindings of chitosan‐L ‐fucose (Fuc) hybrid with Ulex europaeus agglutinin I (UEA I, a lectin specific to L ‐Fuc), and chitosan‐N‐acetyl‐D ‐glucosamine (D ‐GlcNAc) hybrid with Concanavalin A (Con A, a lectin specific to D ‐glucose, D ‐mannose and D ‐GlcNAc), were confirmed by a surface plasmon resonance technique. The microscopic observation of Pseudomonas aeruginosa, which was preincubated with the fluorescein isothiocyanate‐labeled chitosan‐L ‐Fuc hybrid, showed bacteria aggregation. The aggregation was thought to be resulted from the specific interaction of the L ‐Fuc residue of the hybrid with PA‐II lectin on the surface of P. aeruginosa. The chitosan‐L ‐Fuc hybrid inhibited P. aeruginosa growth more effectively in comparison with the other hybrids or unmodified chitosan. The enhancement of antimicrobial activity of chitosan‐L ‐Fuc hybrid could be attributed to the specific binding between PA‐II lectin of P. aeruginosa and L ‐Fuc residue of the L ‐Fuc hybrid. Copyright © 2000 John Wiley & Sons, Ltd.     

Synthesis and binding properties of divalent and trivalent clusters of the Lewis a disaccharide moiety to Pseudomonas aeruginosa lectin PA-IIL

The synthesis of oligomeric glycocomimetics has been performed for targeting the Pseudomonas aeruginosa PA-IIL lectin, which is of therapeutical interest for anti-adhesive treatment. The disaccharide alpha-L-Fucp-(1-->4)-beta-D-GlcNAc, which is a high-affinity ligand of the lectin, has been coupled to dimeric and trimeric linkers with various lengths and geometries. A series of linear dimers displayed an efficient clustering effect and a very strong affinity, with a lower dissociation constant of 90 nM. The trimeric compound was less efficient in inhibition assays but displayed high affinity in solution. Titration microcalorimetry and molecular modeling allowed in-depth analysis and rationalization of the binding data. These glycoclusters could act by crosslinking the lectins present on the surface of bacteria and therefore interfere with host recognition or biofilm formation.     

Structural Insights into the Molecular Recognition Mechanism of the Cancer and Pathogenic Epitope,LacdiNAc by Immune-Related Lectins

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we investigated the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAcβ1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Galβ1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions was observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.     

Fluorescence studies of the kinetics of binding and removal of metal ions in proteins

Fluorescence-quenching studies involving native protein fluorescence are used to monitor the rates of binding and removal of Hg(II), Cu(II), Ag(I), methylmercury(I), and p-chloro-mercuribenzoate in various protein systems (ovalbumin, bovine serum albumin, myoglobin, lysozyme, and insulin). In some cases, the fluorescence quenching as a function of time can be used to evaluate the rate constants for the binding of a particular metal ion to a protein. In many cases, multiple binding sites with different rate constants can be differentiated. The restoration of fluorescence vs. time on addition of various chelating agents (BAL, EDTA, cysteine and penicilamine) to the metal/protein system can be used to monitor metal ion removal. Multiple binding sites also can be differentiated kinetically in the removal experiments. In some cases, the appearance of multiple steps in the binding or removal or a metal or ion could be explained by small conformational changes. The rates of removal can help in estimating the effectiveness of various reagents as models for drugs in the treatment of heavy-metal poisoning.     

Cell surface carbohydrates of preimplantation embryos as assessed by lectin binding

Preimplantation embryos were obtained from the uteri and oviducts of 2 strains of mice, Swiss CD-1 and B6CBA. After removal of the zona pellucida by treatment with pronase, FITC-lectins were bound to the embryonic cell surfaces at either 4 degrees C or 37 degrees C. Both morula and blastocyst stage embryos bound the following lectins, FITC-ConA, FITC-WGA, FITC-RCAII and FITC-RCAI. No difference in binding was observed between the morula stage and the blastocyst stage within each mouse strain for each specific lectin. However B6CBA embryos bound less FITC-ConA and FITC-WGA than the corresponding Swiss CD-1 embryos. The topographical arrangement of the lectin receptors was observed to differ between 4 degrees C and 37 degrees C for FITC-ConA, FITC-RCAII, and FITC-RCAI. While lectins bound at 4 degrees C showed a pattern of continuous labeling, the same lectin at 37 degrees C showed aggregation of lectin receptors into patches indicating lateral mobility of these receptors within the embryonic cell membranes. In contrast FITC-WGA bound at 4 degrees C and 37 degrees C demonstrated continuous labeling of embryos at both temperatures. FITC-fucose binding protein did not bind to Swiss CD-1 embryos. The invasiveness of trophoblastic cells of mouse blastocysts was studied by culturing isolated embryos without prior enzyme treatment on reconstituted collagen gels. After 4 days in BME containing only glutamine and bovine serum albumin as supplements, the embryos shed their zona pellucida and implanted into the collagen gel as indicated by zones of lysis in proximity to the embryonic cells when analyzed by scanning electron microscopy.     

Fluorescence and absorption spectroscopic studies on the interaction of porphyrins with snake gourd (Trichosanthes anguina) seed lectin

The interaction of several free-base porphyrins and their corresponding copper(II) and zinc(II) derivatives with the galactose-specific lectin from snake gourd (Trichosanthes anguina) seeds has been investigated by absorption and fluorescence spectroscopic techniques. The lectin dimer contains two apparently equivalent binding sites for the porphyrins. Association constants obtained for the interaction of various porphyrins with the lectin are in the range 1.7 x 10(4)-6.2 x 10(5) M(-1), with the metalloporphyrins being seen to have higher affinity for the lectin compared with the free-base analogues. Both positively charged and negatively charged porphyrins bind to snake gourd seed lectin (SGSL) with comparable affinities, suggesting that binding occurs primarily via hydrophobic interactions. Further, binding of porphyrins is found to be largely unaffected by the presence of the sugar ligand, lactose, indicating that the binding sites for the carbohydrate and porphyrin are different. This study thus suggests that the lectin may serve as a receptor for some endogenous non-carbohydrate, hydrophobic ligand in vivo, in addition to the saccharide ligands. It also opens up the possibility of employing the T. anguina lectin in applications such as photodynamic therapy, which involve the use of porphyrins.