Carbohydrate Ligands Research Articles

Switching of bacterial adhesion to a glycosylated surface by reversible reorientation of the carbohydrate ligand.

The surface recognition in many biological systems is guided by the interaction of carbohydrate-specific proteins (lectins) with carbohydrate epitopes (ligands) located within the unordered glycoconjugate layer (glycocalyx) of cells. Thus, for recognition, the respective ligand has to reorient for a successful matching event. Herein, we present for the first time a model system, in which only the orientation of the ligand is altered in a controlled manner without changing the recognition quality of the ligand itself. The key for this orientational control is the embedding into an interfacial system and the use of a photoswitchable mechanical joint, such as azobenzene.

A biophysical study with carbohydrate derivatives explains the molecular basis of monosaccharide selectivity of the Pseudomonas aeruginosa lectin LecB.

The rise of resistances against antibiotics in bacteria is a major threat for public health and demands the development of novel antibacterial therapies. Infections with Pseudomonas aeruginosa are a severe problem for hospitalized patients and for patients suffering from cystic fibrosis. These bacteria can form biofilms and thereby increase their resistance towards antibiotics. The bacterial lectin LecB was shown to be necessary for biofilm formation and the inhibition with its carbohydrate ligands resulted in reduced amounts of biofilm. The natural ligands for LecB are glycosides of d-mannose and l-fucose, the latter displaying an unusual strong affinity. Interestingly, although mannosides are much weaker ligands for LecB, they do form an additional hydrogen bond with the protein in the crystal structure. To analyze the individual contributions of the methyl group in fucosides and the hydroxymethyl group in mannosides to the binding, we designed and synthesized derivatives of these saccharides. We report glycomimetic inhibitors that dissect the individual interactions of their saccharide precursors with LecB and give insight into the biophysics of binding by LecB. Furthermore, theoretical calculations supported by experimental thermodynamic data suggest a perturbed hydrogen bonding network for mannose derivatives as molecular basis for the selectivity of LecB for fucosides. Knowledge gained on the mode of interaction of LecB with its ligands at ambient conditions will be useful for future drug design.

Altered galectin glycosylation: potential factor for the diagnostics and therapeutics of various cardiovascular and neurological disorders.

Galectins are β-galactoside binding mammalian proteins characterized by the presence of a conserved carbohydrate recognition domain, expressed in almost all taxa of living organisms and involved in broad range of significant biological and physiological functions. Previously, we reported the purification and extensive characterization of galectin-1 from goat (Capra hircus) heart. Interestingly, the purified protein was found to have significant level of glycosylation. This intrigued us to evaluate the involvement of glycosylation in relation to protein's structural and functional integrity in its purified form. In the present study, an extensive comparative physicochemical characterization has been performed between the glycosylated and deglycosylated form of the purified protein. Deglycosylation resulted in an enhanced fluorescence quenching and marked reduction in pH and thermal stability of the purified galectin. Exposure to various biologically active chemicals showed significant differences in the properties and stability profile, causing significant deviations from its regular secondary structure in the deglycosylated form. These results clearly indicated enhanced structural and functional stabilization in the glycosylated galectin. The data revealed herein adds a vital facet demonstrating the significance of galectin expression and glycosylation in causation, progression, and possible therapeutics of associated clinical disorders. Our approach also allowed us to define some key interactions between the purified galectin and carbohydrate ligands that could well serve as an important landmark for designing new drug protocols for various cardiovascular and neurological disorders.

Terminal sialic acids on CD44 N-glycans can block hyaluronan binding by forming competing intramolecular contacts with arginine sidechains.

Specific sugar residues and their linkages form the basis of molecular recognition for interactions of glycoproteins with other biomolecules. Seemingly small changes, like the addition of a single monosaccharide in the covalently attached glycan component of glycoproteins, can greatly affect these interactions. For instance, the sialic acid capping of glycans affects protein-ligand binding involved in cell-cell and cell-matrix interactions. CD44 is a single-pass transmembrane glycoprotein whose binding with its carbohydrate ligand hyaluronan (HA), an extracellular matrix component, mediates processes such as leukocyte homing, cell adhesion, and tumor metastasis. This binding is highly regulated by glycosylation of the N-terminal extracellular hyaluronan-binding domain (HABD); specifically, sialic acid capped N-glycans of HABD inhibit ligand binding. However, the molecular mechanism behind this sialic acid mediated regulation has remained unknown. Two of the five N-glycosyation sites of HABD have been previously identified as having the greatest inhibitory effect on HA binding, but only if the glycans contain terminal sialic acid residues. These two sites, Asn25 and Asn120, were chosen for in silico glycosylation in this study. Here, from extensive standard molecular dynamics simulations and biased simulations, we propose a molecular mechanism for this behavior based on spontaneously-formed charge-paired hydrogen bonding interactions between the negatively-charged sialic acid residues and positively-charged Arg sidechains known to be critically important for binding to HA, which itself is negatively charged. Such intramolecular hydrogen bonds would preclude associations critical to hyaluronan binding. This observation suggests how CD44 and related glycoprotein binding is regulated by sialylation as cellular environments fluctuate.

Spectroscopic investigation on the interaction of ruthenium complexes with tumor specific lectin, jacalin

Several ruthenium complexes are regarded as anticancer agents and considered as an alternative to the widely used platinum complexes. Owing to the preferential interaction of jacalin with tumor-associated T-antigen, we report the interaction of jacalin with four ruthenium complex namely, tris(1,10-phenanthroline)ruthenium(II)chloride, bis(1,10-phenanthroline)(N-[1,10]phenanthrolin-5-yl-pyrenylmethanimine)ruthenium(II)chloride, bis(1,10-phenanthroline)(dipyrido[3,2-a:2′,3′-c]-phenazine)ruthenium(II)chloride, bis(1,10-phenanthroline)(11-(9-acridinyl)dipyrido[3,2-a:2′,3′-c]phenazine)ruthenium(II) chloride. Fluorescence spectroscopic analysis revealed that the ruthenium complexes strongly quenched the intrinsic fluorescence of jacalin through a static quenching procedure, and a non-radiative energy transfer occurred within the molecules. Association constants obtained for the interaction of different ruthenium complexes with jacalin are in the order of 105M−1, which is in the same range as those obtained for the interaction of lectin with carbohydrate and hydrophobic ligand. Each subunit of the tetrameric jacalin binds one ruthenium complex, and the stoichiometry is found to be unaffected by the presence of the specific sugar, galactose. In addition, agglutination activity of jacalin is largely unaffected by the presence of the ruthenium complexes, indicating that the binding sites for the carbohydrate and the ruthenium complexes are different. These results suggest that the development of lectin–ruthenium complex conjugate would be feasible to target malignant cells in chemo-therapeutics.

The carbohydrate-binding promiscuity of Euonymus europaeus lectin is predicted to involve a single binding site.

Euonymus europaeus lectin (EEL) is a carbohydrate-binding protein derived from the fruit of the European spindle tree. EEL was first identified for its erythrocyte agglutinating properties and specificity for B and H blood groups. However, a detailed molecular picture of the structural basis of carbohydrate recognition by EEL remains to be developed. In this study, we performed fluorescence titrations of a range of carbohydrates against EEL. Binding of EEL to a wide range of carbohydrates was observed, including a series of blood group-related carbohydrates, mannosides, chitotriose and sialic acid. Affinity was strongest for carbohydrates with H-related structures and the B trisaccharide. A homology model of EEL was produced from templates identified using the HHPred server, which employs hidden Markov models (HMMs) to identify templates. The HMM approach identified that the best templates for EEL were proteins featuring a ricin B-like (R-type) fold. Separate templates were used to model the core and binding site regions of the lectin. Through the use of constrained docking and spatial comparison with a template ligand, binding modes for the carbohydrate ligands were predicted. A relationship between the experimental binding energies and the computed binding energies of the selected docked poses was determined and optimized. Collectively, our results suggest that EEL utilizes a single site for recognition of carbohydrates terminating in a variety of monosaccharides.

SEAL by NMR: glyco-based selenium-labeled affinity ligands detected by NMR spectroscopy.

We report a method for the screening of interactions between proteins and selenium-labeled carbohydrate ligands. SEAL by NMR is demonstrated with selenoglycosides binding to lectins where the selenium nucleus serves as an NMR-active handle and reports on binding through (77)Se NMR spectroscopy. In terms of overall sensitivity, this nucleus is comparable to (13)C NMR, while the NMR spectral width is ten times larger, yielding little overlap in (77)Se NMR spectroscopy, even for similar compounds. The studied ligands are singly selenated bioisosteres of methyl glycosides for which straightforward preparation methods are at hand and libraries can readily be generated. The strength of the approach lies in its simplicity, sensitivity to binding events, the tolerance to additives and the possibility of having several ligands in the assay. This study extends the increasing potential of selenium in structure biology and medicinal chemistry. We anticipate that SEAL by NMR will be a beneficial tool for the development of selenium-based bioactive compounds, such as glycomimetic drug candidates.

Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.

Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries displayed gene programs for vascular development. HEVs were enriched in genes for immune defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated lymphocyte recruitment including a lymph node HEV-selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.

Glyconanomaterials: Emerging applications in biomedical research.

Carbohydrates constitute the most abundant organic matter in nature, serving as structural components and energy sources, and mediating a wide range of cellular activities. The emergence of nanomaterials with distinct optical, magnetic, and electronic properties has witnessed a rapid adoption of these materials for biomedical research and applications. Nanomaterials of various shapes and sizes having large specific surface areas can be used as multivalent scaffolds to present carbohydrate ligands. The resulting glyconanomaterials effectively amplify the glycan-mediated interactions, making it possible to use these materials for sensing, imaging, diagnosis, and therapy. In this review, we summarize the synthetic strategies for the preparation of various glyconanomaterials. Examples are given where these glyconanomaterials have been used in sensing and differentiation of proteins and cells, as well as in imaging glycan-medicated cellular responses.

The development of new molecular tools containing a chemically synthesized carbohydrate ligand for the elucidation of carbohydrate roles via photoaffinity labeling: Carbohydrate–protein interactions are affected by the structures of the glycosidic bonds and the reducing-end sugar

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate–protein interactions. We investigated whether carbohydrate–lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp–GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.

Receptor-mediated hepatocyte-targeted delivery of primaquine phosphate nanocarboplex using a carbohydrate ligand.

Primaquine phosphate is a drug of choice for the treatment of malarial relapse. However, poor drug concentration in the hepatocytes and dose-related toxicity pose severe limitations. We report a nanocarboplex of primaquine phosphate by a simple in situ process using dextran sulphate as a carbohydrate polymer and pullulan as an asialoglycoprotein receptor ligand. Our aim was to preferentially enhance accumulation of the nanocarboplex in the hepatocytes. The in situ pullulan-anchored primaquine nanocarboplex was prepared by simple addition of a solution of dextran sulphate and pullulan with stabilizer to a measured quantity of primaquine phosphate in a vial, followed by shaking to obtain the primaquine phosphate nanocarboplex ready for injection. The nanocarboplex was characterized and evaluated in vivo for pharmacokinetics and biodistribution in the rat model. Specific uptake by hepatocytes in the liver was also quantified. Increase in t ½ with significant uptake in the RES organ was observed. More importantly, anchoring pullulan favored high liver uptake and preferential accumulation in the hepatocytes with a hepatocytes/nonparenchymal cells ratio of 75:25. The in situ primaquine phosphate nanocarboplex anchored with pullulan provides both a significant technological advantage and the desired targeting to the hepatocytes.

Biosensor for selective detection of E. coli in spinach using the strong affinity of derivatized mannose with fimbrial lectin

Escherichia coli (E. coli) contamination in foods and water resources represents a major threat for human health and the environment. This work exploits the strong affinity of mannose-containing oligosaccharides with the fimbrial lectin of E. coli to design novel biosensors. Modified carbohydrate ligands were synthesized by introducing phenyl residues and aliphatic chains to mannose via reductive amination in order to increase both the affinity and selectivity to E. coli compared to other pathogenic bacteria. The synthesized ligands include p-thiolphenyl aminomannose (PTAM), p-carboxyphenyl aminomannose (PCAM), 1-deoxy-1-aminomannopyranoside (DAMP), glucosamine and low molecular weight chitosan bonded to mercapto undecanoic acid. The structures of the ligands were confirmed using 1H NMR and 1H, 13C, COZY NMR, and ESI/MS. The ligands were immobilized onto gold electrodes and SPR surfaces using-mercaptoundecanoic acid with glycine as deactivating agent. Two detection mechanisms were tested: (i) metal-enhanced electrochemical detection (MED) and (ii) label-free surface plasmon resonance (SPR) detection. The introduction of phenyl residues and aliphatic side groups to the mannose-containing oligosaccharides produced extremely high affinity for E. coli with detection limit of 1cfu/mL. The relative selectivity of these ligands for E. coli, Citrobacter freundii, Staphylococcus epidermidis were 100%, 2.6% and 8.6% respectively. The biosensors were validated using spinach leaves at 3.0cfu/mL. The work provides a generic biosensor for other pathogenic bacteria by enabling multivalent binding, immediate recognition for pathogens as well as inhibition of bacterial growth.

Multivalent glycoconjugates as vaccines and potential drug candidates

Pathogens adhere to the host cells during the first steps of infection through multivalent interactions which involve protein–glycan recognition. Multivalent interactions are also involved at different stages of immune response. Insights into these multivalent interactions generate a way to use suitable carbohydrate ligands that are attached to a basic scaffold consisting of e.g., dendrimer, polymer, nanoparticle, etc., with a suitable linker. Thus a multivalent architecture can be obtained with controllable spatial and topology parameters which can interfere with pathogen adhesion. Multivalent glycoconjugates bearing natural or unnatural carbohydrate antigen epitopes have also been used as carbohydrate based vaccines to stimulate an innate and adaptive immune response. Designing and synthesizing an efficient multivalent architecture with optimal ligand density and a suitable linker is a challenging task. This review presents a concise report on the endeavors to potentially use multi- and polyvalent glycoconjugates as vaccines as well as anti-infectious and anti-inflammatory drug candidates.

Nanodiscs and electrospray ionization mass spectrometry: a tool for screening glycolipids against proteins.

Electrospray ionization-mass spectrometry (ESI-MS) is extensively employed to detect and quantify protein-carbohydrate interactions in vitro and is increasingly used to screen carbohydrate libraries against target proteins. However, current ESI-MS methods are limited to carbohydrate ligands that are relatively soluble in water and are, therefore, not generally suitable for studying protein interactions with glycolipids, an important class of cellular receptors. Here, we describe a catch-and-release (CaR)-ESI-MS assay, which exploits nanodiscs (NDs) to solubilize glycolipids and mimic their natural cellular environment, suitable for screening libraries of glycosphingolipids (GSL) against proteins to identify specific interactions and to rank their relative affinities. Using the B subunit homopentamers of cholera toxin and heat labile toxin as model GSL-binding proteins, the CaR-ESI-MS was applied to NDs containing mixtures of gangliosides. The results demonstrate that the CaR-ESI-MS assay can simultaneously detect both high and low affinity GSL ligands using either a library of NDs that each contains one GSL or incorporating a mixture of GSLs into a single ND. Moreover, the relative abundances of the released ligands appear to reflect their relative affinities in solution. Application of the CaR-ESI-MS assay using NDs containing gangliosides extracted from porcine brain led to the discovery of a neolacto GSL as a cholera toxin ligand, highlighting the power of the assay for identifying specific protein-glycolipid interactions from biologically relevant mixtures.

Implications of the E-selectin S128R mutation for drug discovery

The C-type lectin E-selectin mediates the rolling of circulating leukocytes on vascular endothelial cells during the inflammatory process. In numerous studies, the S128R mutation of the E-selectin was associated with cardiovascular and autoimmune diseases. There is evidence that the S128R E-selectin mutation leads to a loss in ligand specificity, thus increasing leukocyte recruitment. Apart from the natural tetrasaccharide ligand sialyl Lewis(x) (sLe(x)), it has previously been proposed that non-fucosylated carbohydrates also bind to S128R E-selectin. To evaluate the therapeutic potential of the antagonism of the E-selectin mutant, ligand specificity was reinvestigated on a molecular basis. We determined the ligand specificity of wild-type and S128R E-selectin in a target-based competitive assay, a glycan array screen and cell-based binding assays under static and flow conditions. Regarding ligand-specificity, the binding properties of S128R E-selectin were identical to those of wt E-selectin, i.e., no mutant-specific binding of 3'-sialyl-N-acetyllactosamine, heparin, fetuin and K562 cells was observed. Additionally, the binding affinities of glycomimetic E-selectin antagonists were identical for wt and S128R E-selectin. Overall, the previous reports on carbohydrate ligand promiscuity of S128R E-selectin could not be confirmed.

Glycan-Functionalized Fluorescent Chitin Nanocrystals for Biorecognition Applications

A new platform based on chitin nanocrystals has been developed for biorecognition applications. TEMPO-oxidized chitin nanocrystals (TCNs) were labeled with a fluorescent imidazoisoquinolinone dye, and simultaneously conjugated with carbohydrate ligands, resulting in dually functionalized TCNs. The biorecognition properties of the nanocrystals were probed with lectins and bacteria, resulting in selective interactions with their corresponding cognate carbohydrate-binding proteins, as visualized by optical, fluorescence, STEM, and TEM imaging. This represents a new approach to multifunctional nanomaterials based on naturally occurring polymers, holding high potential for biomedical applications.

Comparison of human saliva and synthetic histo-blood group antigens usage as ligands in norovirus-like particle binding and blocking assays

Blocking of norovirus-like particle binding to their cellular ligands, histo-blood group antigens with immune sera, is considered a surrogate norovirus neutralization assay. We compared human secretor positive saliva and synthetic biotinylated carbohydrates as a source of histo-blood group antigens in binding and blocking assays. Six norovirus capsid-derived virus-like particles belonging to genogroup I (GI-1-2001 and GI-3-2002) and genogroup II (GII-4-1999, GII-4-2010 New Orleans, GII-4-2012 Sydney and GII-12-1998) noroviruses were produced by a recombinant baculovirus expression system and binding profile to saliva type A, B and O and to synthetic antigens (A trimer, B trimer, H type 1, H type 3, Lewisa and Lewisb) was identified. Good correlation between virus-like particle binding to saliva type A and synthetic A trimer (r = 0.66, p < 0.05) and saliva type B and synthetic B trimer (r = 0.75, p < 0.05) was observed. Binding of each norovirus virus-like particle to the selected histo-blood group antigens was blocked by convalescent sera from NoV-infected subjects or type-specific mouse antisera. Our results support the use of either saliva or synthetic antigens in blocking assay to measure the ability of norovirus antisera to block virus-like particle binding to the carbohydrate ligands.

Biological Evaluation of Multivalent Lewis X–MGL‐1 Interactions

Myeloid C-type lectin receptors (CLRs) expressed by antigen-presenting cells are pattern-recognition receptors involved in the recognition of pathogens as well as of self-antigens. The interaction of carbohydrate ligands with a CLR can trigger immune responses. Although several CLR ligands are known, there is limited insight into CLR targeting by carbohydrate ligands. The weak affinity of lectin-carbohydrate interactions often renders multivalent carbohydrate presentation necessary. Here, we have analyzed the impact of multivalent presentation of the trisaccharide Lewis X (Le(X) ) epitope on its interaction with the CLR macrophage galactose-type lectin-1 (MGL-1). Glycan arrays, including N-glycan structures with terminal Le(X) , were prepared by enzymatic extension of immobilized synthetic core structures with two recombinant glycosyltransferases. Incubation of arrays with an MGL-1-hFc fusion protein showed up to tenfold increased binding to multiantennary N-glycans displaying Le(X) structures, compared to monovalent Le(X) trisaccharide. Multivalent presentation of Le(X) on the model antigen ovalbumin (OVA) led to increased cytokine production in a dendritic cell /T cell coculture system. Furthermore, immunization of mice with Le(X) -OVA conjugates modulated cytokine production and the humoral response, compared to OVA alone. This study provides insights into how multivalent carbohydrate-lectin interactions can be exploited to modulate immune responses.

Expanded potential of seleno-carbohydrates as a molecular tool for X-ray structural determination of a carbohydrate–protein complex with single/multi-wavelength anomalous dispersion phasing

Seleno-lactoses have been successfully synthesized as candidates for mimicking carbohydrate ligands for human galectin-9 N-terminal carbohydrate recognition domain (NCRD). Selenium was introduced into the mono- or di-saccharides using p-methylselenobenzoic anhydride (Tol2Se) as a novel selenating reagent. The TolSe-substituted monosaccharides were converted into selenoglycosyl donors or acceptors, which were reacted with coupling partners to afford seleno-lactoses. The seleno-lactoses were converted to the target compounds. The structure of human galectin-9 NCRD co-crystallized with 6-MeSe-lactose was determined with single/multi-wavelength anomalous dispersion (SAD/MAD) phasing and was similar to that of the co-crystal with natural lactose.

Synthesis of oligosaccharide fragments of the Streptococcus pneumoniae type 14 capsular polysaccharide and their neoglycoconjugates with bovine serum albumin

2-Aminoethyl glycosides of tetra-, hexa- and octasaccharide fragments of the bacteria Streptococcus pneumoniae type 14 capsular polysaccharide were synthesized within a project directed to the development of pneumococcal conjugated vaccine based on synthetic carbohydrate ligands. Squarate method was used to obtain neoglycoconjugates of the synthesized oligosaccharides with bovine serum albumin.