Molecular and structural basis for Lewis glycan recognition by a cancer-targeting antibody.

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.

Glycan-dependent cell adhesion mechanism of Tc toxins.

Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.

Study of glycosylation of prostate-specific antigen secreted by cancer tissue-originated spheroids reveals new candidates for prostate cancer detection.

Prostate-specific antigen (PSA) is the most frequently used biomarker for the screening of prostate cancer. Understanding the structure of cancer-specific glycans can help us improve PSA assay. In the present study, we analysed the glycans of PSA obtained from culture medium containing cancer tissue-originated spheroids (CTOS) which have similar characteristics as that of the parent tumour to explore the new candidates for cancer-related glycoforms of PSA. The glycan profile of PSA from CTOS was determined by comparing with PSA from normal seminal plasma and cancer cell lines (LNCaP and 22Rv1) using lectin chromatography and mass spectrometry. PSA from CTOS was mostly sialylated and the content of Wisteria floribunda agglutinin reactive glycan (LacdiNAc) was similar to that of PSA derived from seminal plasma and 22Rv1. Conversely, concanavalin A (Con A)-unbound PSA was definitely detected from the three cancer origins but was almost negligible in seminal PSA. Two novel types of PSA were elucidated in the Con A-unbound fraction: one is a high molecular weight PSA with highly branched N-glycans, and the other is a low molecular weight PSA without N-glycans. Furthermore, the existence of Lewis X antigen group on PSA was indicated. These PSAs will be candidates for new cancer-related markers.

Enzymatic Sequence Analysis of N-Glycans by Exoglycosidase Cleavage and Mass Spectrometry: Detection of Lewis X Structures.

Enzymatic sequencing of oligosaccharides provides structural information on sequence of monosaccharides and type of linkage within the oligosaccharide chain. This data can be obtained by stepwise enzymatic digestion of a single, isolated oligosaccharide using individual or mixtures of specific exoglycosidases. N-glycans have to be fractionated from mixtures prior to sequence analysis to assign this type of structural information to a specific glycan. Enzymatic sequencing can as well be applied to oligosaccharide mixtures to evaluate the occurrence of distinct oligosaccharide motives of functional and/or structural interest.Here we describe the application of enzymatic sequence analysis to a mixture of N-glycans released from α1-acid glycoprotein. The experimental conditions are optimized for detection of possible Lewis X structures after stepwise exoglycosidase digestion by MALDI-TOF mass spectrometry. However, the described method is generally applicable to analyze other structural properties of N-glycans using (respective) specific exoglycosidases.

Convergent synthesis of tetra- and penta-saccharide fragments of dimeric Lewis X.

The convergent synthesis of tetra- and penta-saccharide fragments of the TACA dimeric Lex is described. The synthetic strategy relied on the preparation of a protected GlcNTCA-(1,3)-Gal-(1,4)-GlcNAc trisaccharide diol free at O-3 of both glucosamine residues. Key steps in the preparation of this diol involved glycosylation at O-4 of N-acetylglucosamine using activation of a trichloroacetimidate with BF3·Et2O at 40 °C, removal of the non-reducing end O-3' chloroacetate with thiourea, and glycosylation with a N-trichloroacetamido glucosamine trichloroacetimidate donor. After conversion to the diol acceptor, the trisaccharide was selectively fucosylated at the nonreducing end under NIS/TMSOTf activation, or di-fucosylated under CuBr2/Bu4NBr activation. The protected tetra- and pentasaccharides were then efficiently deprotected under dissolving metal conditions and the nonreducing end glucosamine residues were N-acetylated during the reaction work up. The deprotected compounds will be used as soluble competitors to characterize the epitopes recognized by anti-polymeric Lex antibodies.

Structural Studies of Fucosylated N-Glycans by Ion Mobility Mass Spectrometry and Collision-Induced Fragmentation of Negative Ions.

There is considerable potential for the use of ion mobility mass spectrometry in structural glycobiology due in large part to the gas-phase separation attributes not typically observed by orthogonal methods. Here, we evaluate the capability of traveling wave ion mobility combined with negative ion collision-induced dissociation to provide structural information on N-linked glycans containing multiple fucose residues forming the Lewisx and Lewisy epitopes. These epitopes are involved in processes such as cell-cell recognition and are important as cancer biomarkers. Specific information that could be obtained from the intact N-glycans by negative ion CID included the general topology of the glycan such as the presence or absence of a bisecting GlcNAc residue and the branching pattern of the triantennary glycans. Information on the location of the fucose residues was also readily obtainable from ions specific to each antenna. Some isobaric fragment ions produced prior to ion mobility could subsequently be separated and, in some cases, provided additional valuable structural information that was missing from the CID spectra alone. Graphical abstract ᅟ.

Computational analysis of carbohydrate recognition based on hybrid QM/MM modeling: a case study of norovirus capsid protein in complex with Lewis antigen.

Norovirus is a major pathogen of nonbacterial acute gastroenteritis in humans and animals. Carbohydrate recognition between norovirus capsid proteins and Lewis antigens is considered to play a critical role in initiating infection of eukaryotic cells. In this article, we first report a detailed atomistic simulation study of the norovirus capsid protein in complex with the Lewis antigen based on ab initio QM/MM combined with MD-FEP simulations. To understand the mechanistic details of ligand binding, we analyzed and compared the carbohydrate recognition mechanism of the wild-type P domain protein with a mutant protein. Small structural differences between two capsid proteins are observed on the weak interaction site of residue 389, which is located on the solvent exposed surface of the P domain. To further clarify affinity differences in ligand binding, we directly evaluated free energy changes of the ligand binding process. Although the mutant protein loses its interaction energy with the Lewis antigen, this small amount of energy penalty is compensated for by an increase in the solvation stability, which is induced by structural reorganization at the ligand binding site on the protein surface. As a sum of these opposite energy components, the mutant P domain obtains a slightly enhanced binding affinity for the Lewis antigen. The present computational study clearly demonstrated that a detailed free energy balance of the interaction energy between the capsid protein and the surrounding aqueous solvent is the mechanistic basis of carbohydrate recognition in the norovirus capsid protein.

Embryoglycan: a highly branched poly-N-acetyllactosamine in pluripotent stem cells and early embryonic cells.

Embryonal carcinoma cells, stem cells of teratocarcinomas, are pluripotent stem cells and also prototypes of embryonic stem cells. Embryonal carcinoma cells contain large amounts of a highly branched poly-N-acetyllactosamine called embryoglycan, which has a molecular weight of approximately 10,000 or greater, and is asparagine-linked. This glycan was found by analyses of fucose-labeled glycopeptides, and its characteristics were established by biochemical analyses. The content of embryoglycan progressively decreases during the in vitro differentiation of embryonal carcinoma cells. Embryoglycan is also abundant in mouse embryonic stem cells and preimplantation mouse embryos, and decreases during embryogenesis. Embryoglycan carries a number of carbohydrate markers of murine pluripotent stem cells. Lewis x markers, such as SSEA-1, 4C9 antigen, and binding sites for Lotus tetragonolobus agglutinin are of particular importance. 4C9 antigenicity requires clustering of Lewis x, best accomplished by poly-N-acetyllactosamine branching, whereas SSEA-1 does not. Although in vivo evidence is lacking, these epitopes have been suggested to participate in cell-to-cell and cell-to-substratum adhesion. Other markers on embryoglycan include α-galactosyl antigens such as ECMA-2, and binding sites for Dolichos biflorus agglutinin, the epitope of which is considered to be identical to Sda antigen, namely, GalNAcß1-4(NeuAcα2-3)Galß1-4GlcNAc. While embryoglycan is also present in human teratocarcinoma cells, the carbohydrate markers characterized in human pluripotent stem cells to date are largely carried by glycolipids and keratan sulfate. Information on embryoglycan and markers carried by it may assist in the development of new markers of human pluripotent stem cells and their progenies.

The Hidden Conformation of Lewis x, a Human Histo-Blood Group Antigen, Is a Determinant for Recognition by Pathogen Lectins.

Histo-blood group epitopes are fucosylated branched oligosaccharides with well-defined conformations in solution that are recognized by receptors, such as lectins from pathogens. We report here the results of a series of experimental and computational endeavors revealing the unusual distortion of histo-blood group antigens by bacterial and fungal lectins. The Lewis x trisaccharide adopts a rigid closed conformation in solution, while crystallography and molecular dynamics reveal several higher energy open conformations when bound to the Ralstonia solanacearum lectin, which is in agreement with thermodynamic and kinetic measurements. Extensive molecular dynamics simulations confirm rare transient Le(x) openings in solution, frequently assisted by distortion of the central N-acetyl-glucosamine ring. Additional directed molecular dynamic trajectories revealed the role of a conserved tryptophan residue in guiding the fucose into the binding site. Our findings show that conformational adaptation of oligosaccharides is of paramount importance in cell recognition and should be considered when designing anti-infective glyco-compounds.

Schistosoma mansoni α1,3-fucosyltransferase-F generates the Lewis X antigen.

Genetic evidence suggests that the Schistosoma mansoni genome contains six genes that encode α1,3-fucosyltransferases (smFuTs). To date, the activities and specificities of these putative fucosyltransferases are unknown. As Schistosoma express a variety of fucosylated glycans, including the Lewis X antigen Galß1-4(Fucα1-3)GlcNAcß-R, it is likely that this family of genes encode enzymes that are partly responsible for the generation of those structures. Here, we report the molecular cloning of fucosyltransferase-F (smFuT-F) from S. mansoni, as a soluble, green fluorescent protein fusion protein and its acceptor specificity. The gene smFuT-F was expressed in HEK freestyle cells, purified by affinity chromatography, and analyzed toward a broad panel of glycan acceptors. The enzyme product of smFuT-F effectively utilizes a type II chain acceptor Galß1-4GlcNAc-R, but notably not the LDN sequence GalNAcß1-4GlcNAc-R, to generate Lewis X type-glycans, and smFuT-F transcripts are present in all intramammalian life stages.

Total synthesis of Lewis(X) using a late-stage crystalline intermediate.

Herein, we report on a highly efficient synthesis of a crystalline protected Lewis(X) trisaccharide that was converted to Lewis(X) following global deprotection. The trisaccharide was prepared in a highly convergent synthesis (seven steps, longest linear sequence) and in a 38% overall yield using a strategy that involved the regioselective glycosylation of a GlcNAc acceptor with a galactose thioglycoside donor, followed by fucosylation of the remaining free GlcNAc hydroxyl as key steps. The core trisaccharide also has the potential to be converted to other members of the Type-2 Lewis family of antigens due to the orthogonal nature of the protecting groups employed.

Ginsenoside Rg3 suppresses FUT4 expression through inhibiting NF-κB/p65 signaling pathway to promote melanoma cell death.

Abnormal glycosylation is catalyzed by the specific glycosyltransferases and correlates with tumor cell apoptosis. Increased fucosyltransferase IV (FUT4) is seen in many types of cancer, and manipulating FUT4 expression through specific signaling pathway inhibits cell growth and induces apoptosis. NF-κB is known playing a vital role to control cell growth and apoptosis. Ginsenoside Rg3 is an herbal medicine with strong antitumor activity through inhibiting tumor growth and promoting tumor cell death. However, whether Rg3-induced inhibition on tumor development involves reduced NF-κB signaling and FUT4 expression remains unknown. In the present study, we found that Rg3 suppressed FUT4 expression by abrogating the binding of NF-κB to FUT4 promoter through inhibiting the expression of signaling molecules of NF-κB pathway, reducing NF-κB DNA binding activity and NF-κB transcription activity. NF-κB inhibitor (Bay 11-7082) or knocking down p65 expression by p65 siRNA also led to a significant decreased FUT4 expression. In addition, Rg3 induced apoptosis by activating both extrinsic and intrinsic apoptotic pathways. Moreover, in a xenograft mouse model, Rg3 downregulated FUT4 and NF-κB/p65 expression and suppressed melanoma cell growth and induced apoptosis without any noticeable toxicity. In conclusion, Rg3 induces tumor cell apoptosis correlated with its inhibitory effect on NF-κB signaling pathway-mediated FUT4 expression. Results suggest Rg3 might be a novel therapy agent for melanoma treatment.

Inflammatory cytokines regulate the expression of glycosyltransferases involved in the biosynthesis of tumor-associated sialylated glycans in pancreatic cancer cell lines.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma containing several pro-inflammatory cytokines, which are described to modulate the expression of important genes related to tumor promotion and progression. In the present work we have investigated the potential role of these cytokines in the biosynthesis of tumor-associated carbohydrate antigens such as sialyl-Lewis(x) (SLe(x)) through the regulation of specific glycosyltransferase genes. METHODS: Two human PDAC cell lines MDAPanc-3 and MDAPanc-28 were treated with pro-inflammatory cytokines IL-1ß, TNFα, IL-6 or IL-8, and the content of tumor-associated carbohydrate antigens at the cell membrane was analyzed by flow cytometry. In addition, variation in the mRNA expression of sialyltransferase (ST) and fucosyltransferase (FUT) genes, which codify for the ST and FucT enzymes involved in the carbohydrate antigens' biosynthesis, was determined. The inflammatory microenvironment of PDAC tissues and the expression of Lewis-type antigens were analyzed by immunohistochemistry to find a possible correlation between inflammation status and the presence of tumor-associated carbohydrate antigens. RESULTS: IL-1ß stimuli increased SLe(x) and α2,6-sialic acid levels in MDAPanc-28 cells and enhanced the mRNA levels of ST3GAL3-4 and FUT5-7, which codify for ST and FucT enzymes related to SLe(x) biosynthesis, and of ST6GAL1. IL-6 and TNFα treatments increased the levels of SLe(x) and Le(y) antigens in MDPanc-3 cells and, similarly, the mRNA expression of ST3GAL3-4, FUT1-2 and FUT6, related to these Lewis-type antigens' biosynthesis, were increased. Most PDAC tissues stained for SLe(x) and SLe(a) and tended to be expressed in the tumor samples with a higher presence of inflammatory immune cells. CONCLUSIONS: The inflammatory microenvironment can modulate the glycosylation pattern of PDAC cells, increasing the expression of tumor-associated sialylated antigens such as SLe(x), which contributes to pancreatic tumor malignancy.

Interactions of antitumour Sialyl Lewis X liposomes with vascular endothelial cells.

Recently, we showed that tetrasaccharide selectin ligand SiaLeX provided targeted delivery of liposomes loaded in the bilayer with melphalan lipophilic prodrug to tumour endothelium followed by severe injury of tumour vessels in a Lewis lung carcinoma model. Here, we study the impact of SiaLeX ligand on the interactions of liposomes with human umbilical vein endothelial cells (HUVEC) using flow cytometry, spectrofluorimetry and confocal microscopy. Liposomes composed of egg phosphatidylcholine/yeast phosphatidylinositol/1,2-dioleoyl glycerol ester of melphalan, 8:1:1, by mol, and varying percentages of lipophilic SiaLeX conjugate were labelled with BODIPY-phosphatidylcholine. The increase in SiaLeX content in liposomes led to a proportional increase in their uptake by cytokine-activated cells as opposed to non-activated HUVEC: for 10% SiaLeX liposomes, binding avidity and overall accumulation increased 14- and 6-fold, respectively. The early stages of intracellular traffic of targeted liposomes in the activated cells were monitored by co-localisation with the trackers of organelles. Endocytosis of SiaLeX liposomes occurred mostly via clathrin-independent pathways, which does not contradict the available literature data on E-selectin localisation in the plasma membrane. Using dual fluorescence labelling, with rhodamine-labelled phospholipid and calcein encapsulated at self-quenching concentrations, we found that SiaLeX liposomes undergo rapid (within minutes) internalisation by activated HUVEC accompanied by the disruption of liposomes; non-activated cells consumed a negligible dose of liposomes during at least 1.5h. Our data evidence the selective effect of SiaLeX formulations on activated endothelial cells and indicate their potential for intracellular delivery of melphalan lipophilic prodrug.

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity.

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

Structural characterization of the DC-SIGN-Lewis(X) complex.

Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a C-type lectin highly expressed on the surface of antigen-presenting dendritic cells. DC-SIGN mediates interactions among dendritic cells, pathogens, and a variety of epithelia, myeloid cells, and endothelia by binding to high mannose residues on pathogenic invaders or fucosylated residues on the membranes of other immune cells. Although these interactions are normally beneficial, they can also contribute to disease. The structural characterization of binding geometries is therefore of interest as a basis for the construction of mimetics that can mediate the effects of abnormal immune response. Here, we report the structural characteristics of the interaction of the DC-SIGN carbohydrate recognition domain (CRD) with a common fucosylated entity, the Lewis(X) trisaccharide (Le(X)), using NMR methods. Titration of the monomeric DC-SIGN CRD with Le(X) monitored by 2D NMR revealed significant perturbations of DC-SIGN cross-peak positions in (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra and identified residues near the binding site. Additionally, saturation transfer difference (STD) and transferred nuclear Overhauser effect (trNOE) NMR experiments, using a tetrameric form of DC-SIGN, identified binding epitopes and bound conformations of the Le(X) ligand. The restraints derived from these multiple experiments were used to generate models for the binding of Le(X) to the DC-SIGN CRD. Ranking of the models based on the fit of model-based simulations of the trNOE data and STD buildup curves suggested conformations distinct from those seen in previous crystal structures. The new conformations offer insight into how differences between binding of Lewis(X) and mannose-terminated saccharides may be propagated.

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.

Evidence for two populated conformations for the dimeric Le(x) and Le(a)Le(x) tumor-associated carbohydrate antigens.

The conformational behavior of tumor-associated carbohydrate antigens (TACAs) dimLe(x) and Le(a)Le(x) was studied using a combination of NMR experiments and molecular dynamics simulations. It is shown that within the hexasaccharides, the Le(x) and Le(a) branched trisaccharide fragments adopt the rigid "stacked" conformation known for the isolated trisaccharide antigens. In contrast, the ß-D-GlcNAc-(1→3)-D-Gal glycosidic bond that connects the two Le(x) trisaccharides in dimLe(x), and the Le(a) trisaccharide to the Le(x) trisaccharide in Le(a)Le(x), was found to be very flexible in both hexasaccharides. Our results show that two distinct conformations, differing by the value of the Ψ angle for this glycosidic bond, are populated in solution. While the relative proportions of the two conformations in solution could not be determined accurately, experimental measurements indicate that both conformations are populated in significant amounts.

The three-dimensional structure of the extracellular adhesion domain of the sialic acid-binding adhesin SabA from Helicobacter pylori.

The gastric pathogen Helicobacter pylori is a major cause of acute chronic gastritis and the development of stomach and duodenal ulcers. Chronic infection furthermore predisposes to the development of gastric cancer. Crucial to H. pylori survival within the hostile environment of the digestive system are the adhesins SabA and BabA; these molecules belong to the same protein family and permit the bacteria to bind tightly to sugar moieties Lewis(B) and sialyl-Lewis(X), respectively, on the surface of epithelial cells lining the stomach and duodenum. To date, no representative SabA/BabA structure has been determined, hampering the development of strategies to eliminate persistent H. pylori infections that fail to respond to conventional therapy. Here, using x-ray crystallography, we show that the soluble extracellular adhesin domain of SabA shares distant similarity to the tetratricopeptide repeat fold family. The molecule broadly resembles a golf putter in shape, with the head region featuring a large cavity surrounded by loops that vary in sequence between different H. pylori strains. The N-terminal and C-terminal helices protrude at right angles from the head domain and together form a shaft that connects to a predicted outer membrane protein-like ß-barrel trans-membrane domain. Using surface plasmon resonance, we were able to detect binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X) but not to Lewis(A), Lewis(B), or Lewis(Y). Substitution of the highly conserved glutamine residue 159 in the predicted ligand-binding pocket abrogates the binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X). Taken together, these data suggest that the adhesin domain of SabA is sufficient in isolation for specific ligand binding.

Fluorescent conjugate of sLe(x)-selective bisboronic acid for imaging application.

Carbohydrate-based biomarkers such as sialyl Lewis X are known to correlate with cancer formation and progression. By targeting sialyl Lewis X, we have developed a boronolectin-fluorophore conjugate, which was able to selectively label and image xenograft (sc) tumor. This represents the very first example that a small molecule capable of recognizing a carbohydrate biomarker was used for optical imaging application.