Surface Ligand Density Switches Glycovesicles between Monomeric and Multimeric Lectin Recognition

Carbohydrate-protein interactions define a multitude of cellular recognition events. We present herein synthetic glycovesicles as cell-surface mimics in order to switch the nature of lectin recognition. The covalent glycovesicles, constituted with diacetylene monomers of various ligand densities at their surfaces, are prepared through photo-polymerization. Vesicles with sparsely imbedded ligands engage in a lectin interaction leading to the formation of a dense, crosslinked multimeric complex. On the other hand, vesicles with many ligands, or completely covered with them, switch the lectin interaction to form a fully soluble monomeric complex, without crosslinking. Nanomolar dissociation constants govern these interactions, as assessed by a ligand-displacement assay. The study demonstrates the switching nature – between monomeric and multimeric – of the interaction as a function of ligand density in the vesicles; the results are directly relevant to understanding such a phenomenon occurring at cell surfaces.     

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.     

Lipid exchanges between dioctadecyldimethylammonium bromide monolayer and vesicles in the subphase

Studies on the interaction of lipid monolayer with bilayer structures, such as vesicles, are relatively scarce in the literature. In order to ascertain whether these structures interact for cationic dioctadecyldimethylammonium bromide (DODAB) monolayers at the aqueous surfaces of 0, 0.05, and 0.5 mmol L⁻¹ DODAB vesicle dispersions, differential scanning calorimetry (DSC) and surface film balance experiments were carried out. DSC results confirmed the presence of unilamellar vesicles in the subphase, while changes in the monolayer surface pressure–area per molecule (π–A) isotherm profile yielded by the presence of DODAB vesicle in the subphase revealed monolayer-vesicle interactions as a result of monomer exchanges between the monolayer and the vesicles with stronger effects at the higher vesicle concentration investigated.     

Human Macrophage Galactose-Type Lectin (MGL) Recognizes the Outer Core of Escherichia coli Lipooligosaccharide

Carbohydrate–lectin interactions intervene in and mediate most biological processes, including a crucial modulation of immune responses to pathogens. Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. Herein, a novel molecular interaction between the human macrophage galactose-type lectin (MGL) and the lipooligosaccharide (LOS) of Escherichia coli strain R1 is described. Saturation transfer difference NMR spectroscopy analysis, supported by computational studies, demonstrated that MGL bound to the purified deacylated LOS_R1 mainly through recognition of its outer core and established crucial interactions with the terminal Galα(1,2)Gal epitope. These results assess the ability of MGL to recognise glycan moieties exposed on Gram-negative bacterial surfaces.     

Novel hyperbranched glycomimetics recognized by the human mannose receptor: quinic or shikimic acid derivatives as mannose bioisosteres

The mannose receptor mediates the internalization of a wide range of molecules or microorganisms in a pattern recognition manner. Therefore, it represents an attractive entry for specific drug, gene, or antigen delivery to macrophages and dendritic cells. In an attempt to design novel effective synthetic mannose receptor ligands, quinic and shikimic acid were selected as putative mannose mimics on the basis of X-ray crystallographic data from the related rat mannose-binding lectin. As the mannose receptor preferentially binds to molecules displaying several sugar residues, fluorescein-labeled cluster quinic and shikimic acid derivatives with valencies of two to eight were synthesized. Their mannose receptor mediated uptake was assayed on monocyte-derived human dendritic cells by cytofluorimetric analysis. Mannose-receptor specificity was further assessed by competitive inhibition assays with mannan, by confocal microscopy analysis, and by expression of the mannose receptor in transfected Cos-1 cells. Constructs derived from both quinic and shikimic acid were efficiently recognized by the mannose receptor with an optimum affinity for the molecules with a valency of four. As a result, commercially available quinic and shikimic acids appear as stable mannose bioisosteres, which should prove valuable tools for specific cell delivery.     

The Characteristic Structure of Anti‐HIV Actinohivin in Complex with Three HMTG D1 Chains of HIV‐gp120

The anti‐HIV lectin actinohivin (AH) specifically interacts with HMTG (high‐mannose‐type glycan), which is attached to the glycoprotein gp120 of HIV‐1 in a process in which the three branched mannotriose chains (D1, D2, and D3) of HMTG exhibit different binding affinities, it being estimated that that of D1 is the strongest, that of D3 is weaker, and that of D2 is undetectable. These properties have been ascribed to the stereochemical differences in linkages between the second and the third mannose residues of the three chains. In order to clarify the interaction geometry between AH and the major target D1, an X‐ray determination of the crystal structure of AH in complex with D1—which is α(1,2)mannotriose composed of three mannose (Man) residues linked together only by α(1,2) bonding—has been performed. In each of the three D1‐binding pockets of AH, two Man residues of D1 are accommodated at zones 1 and 2 in the pocket, in the same way as those found in the α(1,2)mannobiose‐bound AH crystals. However, an OMIT map shows poor densities at both ends of the two residues. This suggests the existence of positional disorder of D1 in the pocket: the two zones are each occupied by two Man residues in two different modes, with mode A involving the Man1 and Man2 residues and mode B the Man2 and Man3 residues. In each mode, D1 is stabilized by adopting a double‐bracket‐shaped conformation through C?H ??? O interactions. In mode B, however, the Man1 residue, which is the most sensitive residue to AH binding, protrudes wholly into the solvent region without contacts with AH. In mode A, in contrast, the Man3 residue interacts with the essential hydrophobic amino acid residues (Tyr and Leu conserved between the three pockets) of AH. Therefore, mode A is likely to be the one that occurs when whole HMTG is bound. In this mode, the two hydroxy groups (O3 and O4) of the Man2 residue are anchored in zone 2 by four hydrogen bonds with Asp, Asn, and Tyr residues of AH. In addition, it has been found that an isolated water molecule buried in the hydrophobic long loop bridges between Asp of AH and the hydroxy group of Man2 through hydrogen bonds. The most interesting feature is found in the interaction of the Man1 and Man3 residues with AH. All eight hydroxy groups of the two residues are completely exposed in the solvent region, whereas their hydrophobic parts make contacts with a Leu residue and two Tyr residues so that the shape of D1 and the surface of AH fit well over a wide area. These structural characteristics are potentially useful for development of AH to produce more effective antiretroviral drugs to suppress the infectious expansion of HIV/AIDS and to help expedite an end to the HIV/AIDS pandemic in the near future.     

Electronic and Steric Control of n→π* Interactions: Stabilization of the α-Helix Conformation without a Hydrogen Bond

The preferred conformations of peptides and proteins are dependent on local interactions that bias the conformational ensemble. The n→π* interaction between consecutive carbonyls promotes compact conformations, including the α-helix and polyproline II helix. In order to further understand the n→π* interaction and to develop methods to promote defined conformational preferences through acyl N-capping motifs, a series of peptides was synthesized in which the electronic and steric properties of the acyl group were modified. Using NMR spectroscopy, van't Hoff analysis of enthalpies, X-ray crystallography, and computational investigations, we observed that more electron-rich donor carbonyls (pivaloyl, iso-butyryl, propionyl) promote stronger n→π* interactions and more compact conformations than acetyl or less electron-rich donor carbonyls (methoxyacetyl, fluoroacetyl, formyl). X-ray crystallography indicates a strong, electronically tunable preference for the α-helix conformation, as observed directly on the φ and ψ torsion angles. Electron-donating acyl groups promote the α-helical conformation, even in the absence of the hydrogen bonding that stabilizes the α-helix. In contrast, electron-withdrawing acyl groups led to more extended conformations. More sterically demanding groups can promote trans amide bonds independent of the electronic effect on n→π* interactions. Chloroacetyl groups additionally promote n→π* interactions through the interaction of the chlorine lone pair with the proximal carbonyl π*. These data provide additional support for an important role of n→π* interactions in the conformational ensemble of disordered or unfolded proteins. Moreover, this work suggests that readily incorporated acyl N-capping motifs that modulate n→π* interactions may be employed rationally to promote conformational biases in peptides, with potential applications in molecular design and medicinal chemistry.     

Kinetic Properties of Carbohydrate–Lectin Interactions: FimH Antagonists

The lectin FimH is terminally expressed on type 1 pili of uropathogenic Escherichia coli (UPEC), which is the main cause of urinary tract infections (UTIs). FimH enables bacterial adhesion to urothelial cells, the initial step of infection. Various mannose derivatives have been shown to antagonize FimH and are therefore considered to be promising therapeutic agents for the treatment of UTIs. As part of the preclinical development process, when the kinetic properties of FimH antagonists were examined by surface plasmon resonance, extremely low dissociation rates (k_off) were found, which is uncommon for carbohydrate–lectin interactions. As a consequence, the corresponding half‐lives (t_1/2) of the FimH antagonist complexes are above 3.6 h. For a therapeutic application, extended t_1/2 values are a prerequisite for success, since the target occupancy time directly influences the in vivo drug efficacy. The long t_1/2 value of the tested FimH antagonists further confirms their drug‐like properties and their high therapeutic potential.     

Capture of Uropathogenic E. coli by Using Synthetic Glycan Ligands Specific for the Pap‐Pilus

Biotinylated mono‐ and biantennary di‐/trisaccharides were synthesized to evaluate their ability to capture E. coli strains that express pilus types with different receptor specificities. The synthesized biotinylated di‐/trisaccharides contain Galα(1→4)Gal, Galα(1→4)GalNHAc, GalNHAcα(1→4)Gal, Galα(1→4)Galβ(1→4)Glc and GalNHAcα(1→4)Galβ(1→4)Glc as carbohydrate epitopes. These biotinylated oligosaccharides were immobilized on streptavidin‐coated magnetic beads, and incubated with different strains of live E. coli. Capturing ability was assessed by using a luciferase assay that detects bacterial ATP. The trisaccharides containing Galα(1→4)Galβ(1→4)Glc and the disaccharides containing Galα(1→4)Gal as the epitopes exhibited strong capturing ability for uropathogenic E. coli strains with the pap pilus genotype, including CFT073, J96 and J96 pilE. The same ligands failed to capture E. coli strains with fim, prs, or foc genotypes. Uropathogenic CFT073 was also captured moderately by biantennary disaccharides containing a GalNHAc moiety at the reducing end; however, other saccharides containing GalNHAc at the nonreducing end did not capture the CFT073 strain. These synthetic glycoconjugates could potentially be adapted as rapid diagnostic agents to differentiate between different E. coli pathovars.     

A Simple Glycolipid Mimic of the Phosphatidylinositol Mannoside Core from Mycobacterium tuberculosis Inhibits Macrophage Cytokine Production

Glycolipids from Mycobacterium tuberculosis have a profound impact on the innate immune response of the host. Macrophage‐inducible C‐type lectin (Mincle) is a pattern‐recognition receptor that has been shown to bind trehalose dimycolate (TDM) from the mycobacterium and instigate intracellular signalling in the immune cell. There are structural similarities between the structures of TDM and phosphatidyl inositol mannoside (PIM). We thus hypothesized that these latter structures might also modulate an immune response in a similar manner. To test this, we synthesized a series of new mannose derivatives modified with fatty esters at the 6‐position and assessed the release of inflammatory cytokines in human U937 macrophages under the induction of lipopolysaccharides (LPS) after glycolipid treatment. The results showed that the amount of two major cytokines—tumour necrosis factor (TNF)‐α and interleukin (IL)‐6—released from LPS‐stimulated U937 cells decreased significantly when compared to a control upon treatment with the prepared glycolipids, thus indicating a reduction in cytokine production by the macrophages.     

Rapid Screening of Lectins for Multivalency Effects with a Glycodendrimer Microarray

Multivalency is an important phenomenon in protein–carbohydrate interactions. In order to evaluate glycodendrimers as multivalent inhibitors of carbohydrate binding proteins, we displayed them on a microarray surface. Valencies were varied from 1 to 8, and corrections were made for the valencies so that all surfaces contained the same amount of the sugar ligand. Five different carbohydrates were attached to the dendrimers. A series of fluorescent lectins was evaluated, and for each of them a binding profile was obtained from a single experiment showing both the specificity of the lectin for a certain sugar and whether it prefers multivalent ligands or not. Very distinct binding patterns were seen for the various lectins. The results were rationalized with respect to the interbinding distances of the lectins.     

Effects of the Surface Densities of Glycoclusters on the Determination of Their IC50 and Kd Value Determination by Using a Microarray

Pseudomonas aeruginosa (PA) is an opportunistic bacterium involved in 10–30 % of nosocomial diseases. It causes severe lung injury to cystic fibrosis patients, often leading to patient death. PA strains are multidrug resistant, thus making the design of new therapeutics a challenge for public health. One promising therapeutic option is to design glycoclusters that target the virulence factor of PA. LecA is a galactose‐specific lectin that might be involved in adhesion and biofilm formation by PA. The DNA‐directed immobilization (DDI) microarray is a powerful tool for screening and understanding of structure–activity relationships between glycoclusters and lectins. High‐throughput and multiplexed analysis of lectin–glycocluster interactions on a DDI microarray allows measurement of IC₅₀ and dissociation constant (K_d) values with minute amounts of material. In order to study the robustness of the DDI microarray in determination of IC₅₀ and K_d values, the impact of glycocluster surface density was investigated. The data obtained show that measured IC₅₀ values were influenced by glycocluster surface density: as the density of glycoclusters increases, the measured IC₅₀ values increase too. In contrast, the measured K_d values were not affected by glycocluster surface density, provided that the experimental conditions allow interaction between glycocluster and lectin at single‐molecule level (no surface cluster effect).     

Carbohydrate Analogue Microarrays for Identification of Lectin‐Selective Ligands

Fifty‐five mono‐ and disaccharide analogues were prepared and used for the construction of microarrays to uncover lectin‐selective ligands. The microarray study showed that two disaccharide analogues, 28′ and 44′ , selectively bind to Solanum tuberosum lectin (STL) and wheat germ agglutinin (WGA), respectively. Cell studies indicated that 28′ and 44′ selectively block the binding of STL and WGA to mammalian cells, unlike the natural ligand LacNAc, which suppresses binding of both STL and WGA to cells.     

Studies of arginine-arene interactions through synthesis and evaluation of a series of galectin-binding aromatic lactose esters

Aromatic lactose 2-O-esters were synthesized and used to probe arene-arginine interactions with the galectin family of proteins. They were found to be low microM inhibitors of galectin-1, -3, and -9N-terminal domain and moderate inhibitors of galectin-7, but not inhibitors of galectin-8N-terminal, which lacks an arginine residue close to the critical, esterified lactose 2-O-position. Molecular modeling of galectins in complex with aromatic lactose 2-O-esters, as well as binding studies with a galectin-3 R186S mutant, confirmed that the inhibitory efficiency of the lactose 2-O-esters was due to the formation of strong interactions between the aromatic ester moieties and the arginine guanidinium groups of galectin-1 and -3. An important common feature shared by galectin-1 and -3 was that the arginines formed in-plane ion pairs with two side-chain carboxylates, which resulted in extended planar pi-electron surfaces that did not require solvation by water; these surfaces were ideal for stacking with aromatic moieties of the ligands. The results provide a basis for the design of lectin inhibitors and drugs that exploit interactions with arginine side-chains via aromatic moieties, which are involved in intramolecular protein salt bridges.     

Acetyl‐α‐d‐mannopyranose‐based cationic polymer via RAFT polymerization for lectin and nucleic acid bindings

Functional cationic polymers carrying mannose moieties were synthesized in a facile manner by employing RAFT polymerization. Initially, a protected carbohydrate based monomer, [2‐(2,3,4,6‐tetra‐O‐acetyl‐α‐d ‐mannopyranosyloxy)ethyl methacrylate (AcManEMA)], was prepared by the O‐glycosylation of 2‐hydroxyethyl methacrylate (HEMA). Subsequently, a macroRAFT agent of poly[2‐(dimethyl)amino ethyl methacrylate] (PDMAEMA) was generated, and a further chain extension polymerization with AcManEMA was carried out in dioxane to form a acetylated mannose cationic diblock copolymer, PDMAEMA‐b‐PAcManEMA. It was attained in high yields and displayed low dispersity (Ð). Acetylated mannose moieties on the polymer were deprotected with sodium methoxide and the amines from the DMAEMA block were protonated to yield a cationic diblock glycopolymer, PDMAEMA‐b‐PManEMA. The cationic property of polymers were characterized by mixing with a negatively charged siRNA duplex and a pDNA, and aggregates of 102 and 233 nm were obtained, respectively. Agarose gel shift assay revealed that the polymers were able to retain the nucleic acids as large polymer complexes. Lectin binding assay proved that the mannose residue on the polymers were only able to bind specifically with ConA. PNA lectin was employed as a control and did not show specific binding. The cationic glycopolymer could be advantageous in targeted nucleic acids delivery in specific cells. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 44947.     

Defining the Interaction of Human Soluble Lectin ZG16p and Mycobacterial Phosphatidylinositol Mannosides

ZG16p is a soluble mammalian lectin that interacts with mannose and heparan sulfate. Here we describe detailed analysis of the interaction of human ZG16p with mycobacterial phosphatidylinositol mannosides (PIMs) by glycan microarray and NMR. Pathogen‐related glycan microarray analysis identified phosphatidylinositol mono‐ and di‐mannosides (PIM1 and PIM2) as novel ligand candidates of ZG16p. Saturation transfer difference (STD) NMR and transferred NOE experiments with chemically synthesized PIM glycans indicate that PIMs preferentially interact with ZG16p by using the mannose residues. The binding site of PIM was identified by chemical‐shift perturbation experiments with uniformly ¹⁵N‐labeled ZG16p. NMR results with docking simulations suggest a binding mode of ZG16p and PIM glycan; this will help to elucidate the physiological role of ZG16p.     

Affinity chromatography of glucose-specific lectin using silica-based support

Four silica-based adsorbents were prepared from covalent attachment of four carbohydrates: i. e. maltose, cellobiose, N-acetyl-D -glucosamine and p-aminophenyl-β-D -glucopyranoside, respectively. These adsorbents posses either terminal D -glucose or N-acetyl-D -glucosamine as the ligand on their surfaces with a ligand density ranging from 20 to 29·2 μmol g^?1. The binding of the glucose-specific lectin, concanavalin A (Con A), to the immobilized ligand on the silica surface depended on the configuration of the immobilized glucose and the linkage of the glucose to the support. Con A showed strong affinity for maltose-immobilized silica, which contains terminal α-D -glucose, and p-aminophenyl-β-D-glucopyranoside-immobilized silica. On the other hand, Con A showed no affinity for cellobiose-immobilized silica, which contains terminal β-D -glucose groups, and N-acetyl-D -glucosamine-immobilized silica. The binding constants for the interactions between Con A and immobilized ligands were determined. The columns packed with the resultant affinity adsorbents were then adopted for the purification of Con A from Jack bean meal. As the diluted NaCl extract of Jack bean meal was applied to the column packed with maltose-immobilized silica, a 13·2-fold purification was achieved by stepwise-elution.     

The Tyrosine Gate of the Bacterial Lectin FimH: A Conformational Analysis by NMR Spectroscopy and X‐ray Crystallography

Urinary tract infections caused by uropathogenic E. coli are among the most prevalent infectious diseases. The mannose‐specific lectin FimH mediates the adhesion of the bacteria to the urothelium, thus enabling host cell invasion and recurrent infections. An attractive alternative to antibiotic treatment is the development of FimH antagonists that mimic the physiological ligand. A large variety of candidate drugs have been developed and characterized by means of in vitro studies and animal models. Here we present the X‐ray co‐crystal structures of FimH with members of four antagonist classes. In three of these cases no structural data had previously been available. We used NMR spectroscopy to characterize FimH–antagonist interactions further by chemical shift perturbation. The analysis allowed a clear determination of the conformation of the tyrosine gate motif that is crucial for the interaction with aglycone moieties and was not obvious from X‐ray structural data alone. Finally, ITC experiments provided insight into the thermodynamics of antagonist binding. In conjunction with the structural information from X‐ray and NMR experiments the results provide a mechanism for the often‐observed enthalpy–entropy compensation of FimH antagonists that plays a role in fine‐tuning of the interaction.     

Multivalent presentation of mannose on hyperbranched polyglycerol and their interaction with concanavalin A lectin

We describe the synthesis of multivalent mannose derivatives by using hyperbranched polyglycerols (hPG) as a scaffold with different linker structures. Grafting of protected mannose (Man) units is achieved by using Cu(I) -catalyzed Huisgen click chemistry with either an anomeric azide or propargyl ether onto complementarily functionalized alkyne or azido polymer surfaces. NMR spectroscopy, dynamic light scattering (DLS), IR spectroscopy, size-exclusion chromatography (SEC), and elemental analysis have been used to characterize the hPG-Man compounds. The surface availability and bioactivity of Man-modified polymers were evaluated by using a competitive surface plasmon resonance (SPR)-based binding assay by interactions of the glycopolymers with concanavalin A (Con A), a lectin that binds mannose containing molecules. The results indicated that the novel glycoarchitectures presented in this work are efficient inhibitors of Con A-mannose recognition and resulted in inhibitor concentrations (mean IC(50)) from the micro- to the nanomolar range, whereas the corresponding monovalent mannoside (methyl-Man) requires millimolar concentrations. The results provide an interesting structure-activity relationship for libraries of materials that differ in the linkage of the sugar moiety presented on a biocompatible polyglycerol scaffold.     

Engineering the Ligand Specificity of the Human Galectin-1 by Incorporation of Tryptophan Analogues

Galectin-1 is a β-galactoside-binding lectin with manifold biological functions. A single tryptophan residue (W68) in its carbohydrate binding site plays a major role in ligand binding and is highly conserved among galectins. To fine tune galectin-1 specificity, we introduced several non-canonical tryptophan analogues at this position of human galectin-1 and analyzed the resulting variants using glycan microarrays. Two variants containing 7-azatryptophan and 7-fluorotryptophan showed a reduced affinity for 3’-sulfated oligosaccharides. Their interaction with different ligands was further analyzed by fluorescence polarization competition assay. Using molecular modeling we provide structural clues that the change in affinities comes from modulated interactions and solvation patterns. Thus, we show that the introduction of subtle atomic mutations in the ligand binding site of galectin-1 is an attractive approach for fine-tuning its interactions with different ligands.