Synthesis and binding to plant lectins of sulfur-containing analogues of betaGal1,3 alphaGalNAc (T-antigen)

Analogues of the tumor-associated T-antigen (betaGal1,3 alphaGalNAc) containing 4,6-epidithio and 4,6-thietan modifications were synthesized from the alpha-allyl glycoside of betaGal alpha1,3GlcNAc via suitable thiocyanate derivatives. Binding to three leguminous lectins as model systems was investigated in an enzyme-linked lectin assay (ELLA) and IC50 values comparable to the corresponding natural disaccharides T-antigen, lactose and N-acetyllactosamine were found.     

Pentamer is the minimum structure for oligomannosylpeptoids to bind to concanavalin A

Enzyme-linked lectin assay (ELLA) was performed for oligomannosylpeptoids, which were immobilized on microtiter plates through a streptavidin-biotin interaction. The other immobilization methods, a hydrophobic adsorption and a covalent attachment, were found inapplicable to the oligomannosylpeptoids. Penta- and hexamannosylpeptoids with a shorter or longer spacer were found to be significantly recognized by concanavalinA (ConA), while the smaller peptoids showed no bindings. A proportional relationship between the amount of bound ConA and the peptoid density on the microtiter plate was observed, indicating the absence of both cluster and overdense effects that would assist or inhibit the binding increasingly with the ligand density.     

Use of an enzyme-linked lectinsorbent assay to monitor the shift in polysaccharide composition in bacterial biofilms

An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification and characterization of extracellular polysaccharides produced by 1- and 4-day biofilms of 10 bacterial strains isolated from food industry premises. Peroxidase-labeled concanavalin A (ConA) and wheat germ agglutinin (WGA) were used, as they specifically bind to saccharide residues most frequently encountered in biofilms matrices: D-glucose or D-mannose for ConA and N-acetyl-D-glucosamine or N-acetylneuraminic acid for WGA. The ELLA applied to 1- and 4-day biofilms colonizing wells of microtiter plates was able to detect that for Stenotrophomonas maltophilia and to a lesser extent Staphylococcus sciuri, the increase in production of exopolysaccharides over time was not the same for sugars binding with ConA and those binding with WGA. Differences in extracellular polysaccharides produced were observed among strains belonging to the same species. These results demonstrate that ELLA is a useful tool not only for rapid characterization of biofilm extracellular polysaccharides but also, in studies of individual strains, for detection of changes over time in the proportion of the exopolysaccharidic component within the polymeric matrix.     

Protein microarrays to study carbohydrate-recognition events

In order to expand areas in which protein microarrays can be used to solve important biological problems, we have investigated ways in which the technique can be employed for functional glycomics. Initially, our protein microarrays were used for the rapid identification of carbohydrate-binding proteins using trifunctional carbohydrate probes and fluorescent dye-labeled polysaccharides. Glycan probes were selectively bound to the corresponding lectins immobilized on the solid surface. In addition, these microarrays were also employed for profiling of carbohydrates on Jurkat T-cell surfaces. These cells adhered to ConA, RCA(120), SNA and WGA, indicating expression of alpha-Man, Gal, NeuNAcalpha2,6Gal and GlcNAc residues on their surfaces. Furthermore, we determined binding affinities between WGA and carbohydrates by measuring IC(50) values of GlcNAc that inhibited 50% of trivalent GlcNAc binding to WGA immobilized on the solid surface. All the experiments show that protein microarrays can be used to study carbohydrate-recognition events in the field of glycomics.     

Efficient synthesis of spacer-N-linked double-headed glycosides carrying N-acetylglucosamine and N,N'-diacetylchitobiose and their cross-linking activities with wheat germ agglutinin

We describe here an efficient synthetic route to spacer-N-linked double-headed glycosides via a simple two-step procedure. N-Acetylglucosamine (GlcNAc) and N,N'-diacetylchitobiose [(GlcNAc)(2)] were treated with ammonia and the resulting N-beta-glycosylamines were coupled to a series of dicarboxylic acids. Condensation with each dicarboxylic acid proceeded stereoselectively to give the corresponding beta-N-linked double-headed glycoside without the need for any protection/deprotection steps. Interaction of the resulting N-linked double-headed glycosides with wheat germ agglutinin (WGA) were then investigated using a precipitation assay and an optical biosensor based on surface plasmon resonance (SPR). Spacer-N-linked double-headed glycosides bearing GlcNAc and (GlcNAc)(2) were found to be capable of binding and precipitating WGA as divalent ligands. However, the length of the spacer groups between the two terminal sugar residues was found to greatly influence the cross-linking activities with the lectin.     

Rational design of novel glycomimetics: inhibitors of concanavalin A

A virtual screening approach was used to identify new glycomimetics. The National Cancer Institute Diversity Set was docked into the carbohydrate binding site of the lectin concanavalin A (ConA). The resulting poses were analyzed and 19 molecules were tested for inhibition with an enzyme-linked lectin assay (ELLA). Eight of the 19 molecules inhibited ConA-carbohydrate binding. The two most potent inhibitors have IC(50) values that are an order of magnitude smaller than the monosaccharide methyl alpha-D-mannopyranoside.     

Synthesis of 1-N-glycyl beta-oligosaccharide derivatives. Reactivity of Lens culinaris lectin with a fluorescent labeled streptavidin pseudoglycoprotein and immobilized neoglycolipid.

The lectin from Lens culinaris (lentil) has a binding specificity for glycopeptides bearing 6-O-linked fucose on the reducing terminus on complex-type N-linked oligosaccharides. Lentil lectin therefore provides an excellent example of a carbohydrate binding protein in which high-affinity interactions are dependent on the integrity of the oligosaccharide core structure. We report here the synthesis of the 1-N-glycyl beta-derivative of Gal beta 4GlcNAc beta 2Man alpha 6(Gal beta 4GlcNAc beta 2Man alpha 3)Man beta 4GlcNAc beta 4(Fuc alpha 6)-GlcNAc (Gal-2F) and its subsequent biotinylation and palmitoylation. The biotin derivative when bound to a streptavidin-fluorescein isothiocyanate (FITC) conjugate was able to bind to both concanavalin A (ConA) and lentil lectin affinity columns. In contrast, synthesis of the biotin derivative of the glycamine derivative of Gal-2F and subsequent binding to streptavidin-FITC afforded reactivity to a ConA affinity column but not to a lentil lectin affinity column. Lentil lectin also bound to plastic microtiter plates containing the adsorbed palmitoyl-1-N-glycyl beta-derivative. No binding occurred when the homologous glycamine neoglycolipid was used. These results suggest the 1-N-glycyl beta-derivative of oligosaccharides may have general utility as an intermediate in the synthesis of novel glycoconjugate probes.     

Molecular design of N-linked tetravalent glycosides bearing N-acetylglucosamine,N,N′-diacetylchitobiose and N-acetyllactosamine: Analysis of cross-linking activities with WGA and ECA lectins

Two types of nonspacer- and spacer-N-linked tetravalent glycosides bearing N-acetylglucosamine (GlcNAc), N,N′-diacetylchitobiose [(GlcNAc)₂] and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. The interactions of wheat germ (Triticum vulgaris) agglutinin (WGA) and coral tree (Erythrina cristagalli) agglutinin (ECA) with a series of tetravalent glycosides and related compounds were studied using a hemagglutination inhibition assay, a precipitation assay, double-diffusion test, and an optical biosensor based on surface plasmon resonance (SPR). The tetravalent glycosides were found to be capable of binding and precipitating the lectins as tetravalent ligands. Strong interactions with WGA, due to a combination of multivalency effects and spacer effects, were observed for tetravalent glycosides bearing flexible tandem GlcNAc. The chelate effect leads to large rate enhancement for the tetravalent system with favorable orientation of ligands. Our simple strategy produced multivalent glycosides with strong cross-linking activity for lectin as a specific coagulant.     

An enzyme-linked lectin assay for alpha1,3-galactosyltransferase

UDP-Gal:Galbeta1-4GlcNAc alpha1,3-galactosyltransferase (alpha3GalT) is responsible for the synthesis of carbohydrate xenoantigen Galalpha1-3Galbeta1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of alpha3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Galbeta1-4GlcNAc (acceptor) are incubated with alpha3GalT in the presence of "cold" UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin. The standard curve for correct quantification of alpha3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl-donor UDP-Gal. In accordance with the known specificity of alpha3GalT, no enzymatic conversion of Le(x) into GalalphaLe(x) was observed using this assay. Human alphaGal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive.     

Study of real-time lectin-carbohydrate interactions on the surface of a quartz crystal microbalance

A quartz crystal microbalance (QCM) biosensor system for lectin-carbohydrate interactions has been developed. Yeast mannan was immobilised on polystyrene-coated quartz crystals, and interactions tested with the lectin concanavalin A (Con A). The biosensor could be easily operated, where mannan immobilisation and all binding analyses were performed in real-time using a flow-through system. The apparent binding constant for yeast mannan to Con A was estimated to be 0.4 microM, well in accordance to reported literature values. In addition, the effective concentration values (EC50-values) for a series of mannose/mannoside ligands, acting as competitors to the mannan/Con A interaction, were determined to range from 0.18 to 5.3 mM, in good correlation with a related enzyme-labelled lectin assay (ELLA) protocol.     

Human splenic galaptin: carbohydrate-binding specificity and characterization of the combining site.

A galactose-binding lectin (galaptin) from human spleen has been purified to homogeneity by affinity chromatography on asialofetuin-Sepharose. The carbohydrate-binding specificity of galaptin has been investigated by analyzing the binding of galaptin to asialofetuin in the presence of putative inhibitors. An enzyme-linked immunosorbent assay (ELISA) was developed that involved adsorption of asialofetuin to microtiter plates. Galaptin bound to asialofetuin was detected with polyclonal rabbit anti-galaptin serum followed by goat anti-rabbit IgG-peroxidase conjugate. The concentrations of inhibitors giving 50% inhibition of galaptin binding relative to controls were graphically determined and normalized relative to galactose or lactose. These analyses revealed that galaptin has a combining site at least as large as a disaccharide. The disaccharides having non-reducing-terminal beta-galactosyl residues linked (1,3), (1,4), and (1,6) to Glc or GlcNAc are better inhibitors than free Gal. GalNAc, either free or glycosidically linked, appears to have no affinity for the lectin. The nitrophenyl galactosides are better inhibitors than methyl galactosides, indicating the occurrence of hydrophobic interactions. The data indicate that OH groups at C-4 and C-6 of Gal and the OH at C-3 of GlcNAc in Gal beta(1,4)GlcNAc are important for lectin sugar interaction. Our data support the hypothesis that endogenous receptors for galaptin are most likely lactosaminoglycan moieties.     

A novel microtiter plate based method for identification of B‐cell epitopes

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B‐cell epitopes in two different proteins using a peptide‐based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15‐mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor‐α, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non‐covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor‐α were added to identify potential linear B‐cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non‐covalent binding although the peptides were in fact present on the non‐covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B‐cell epitopes in protein antigens. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

The use of streptavidin-biotinylglycans as a tool for characterization of oligosaccharide-binding specificity of lectin.

A new rapid and sensitive method for characterizing lectin specificity using streptavidin-biotinylglycans as a tool is presented. This assay is analogous to enzyme immunoassay and takes advantage of the strong, irreversible adsorption of streptavidin to the wells of the chambers of titer plates. A series of streptavidin-biotinylglycans was first coated on a microtiter plate, and then one of six lectins, concanavalin A, wheat germ agglutinin, Phaseolus vulgaris (red kidney bean) erythro-agglutinin, Lens culinaris (lentil) agglutinin, Datura stramoniun agglutinin, or Sambucus nigra (elderberry bark) agglutinin coupled to horseradish peroxidase, was added. After incubation and thorough washing, only the lectin bound to a complementary glycan remained and could be detected and quantified by the peroxidase reaction. It was established that the lectins retained their oligosaccharide-binding specificities after coupling to the peroxidase, that the binding was inhibited by addition of the corresponding sugar inhibitors, and that the color intensity produced by the enzyme reaction is proportional to the amount of lectin-peroxidase bound to biotinylglycan complexed with streptavidin immobilized on the plate. As an example, it was found that the peroxidase-D. stramoniun agglutinin conjugate strongly bound biotinylglycans, GlcNAc3-Man5-R, GalGlcNAc3Man5-R, and GlcNAc3-4Man3-R (R = GlcNAc2-[6-(biotinamido)hexanoyl]-Asn). As little as 10 pmol/ml of lectin was detected. With the growing availability of biotinylglycans, the method should represent a reliable and simple procedure for screening lectin-oligosaccharide recognition qualitatively and quantitatively.     

A solid-phase assay for beta-1,4-galactosyltransferase activity in human serum using recombinant aequorin

We have developed a sensitive and rapid solid-phase assay for the serum enzyme UDPGal:beta-D-GlcNAc beta-1,4-galactosyltransferase (beta 1,4-GT) (EC 2.4.1.38) that employs the recombinant bioluminescent protein aequorin as the enzyme label for product detection. The substrate for beta 1,4-GT is a neoglycoprotein, bovine serum albumin containing covalently attached GlcNAc residues (GlcNAc-BSA), and it was immobilized by adsorption in microtiter plate wells. Serum samples were added to each well along with saturating levels of UDPGal and Mn2+. Galactosylation of the neoglycoprotein acceptor by the serum beta 1,4-GT produces the N-acetyllactosamine derivative Gal beta 1, 4GlcNAc-BSA. The product formed is quantified by adding the biotinylated plant lectin Ricinus communis agglutinin-I, which binds specifically to N-acetyllactosamine, followed by the addition of streptavidin and the biotinylated aequorin. Aequorin produces a flash of light in response to Ca2+ and is detectable to 10(-19) mol in a luminometer. Using this assay, the beta 1,4-GT activity in human serum and the activity of a semipurified beta 1,4-GT are linear with time and serum concentration over a wide range. The reaction is dependent on UDPGal and Mn2+, is highly reproducible with a low background, and can be performed in a few hours. Assays employing aequorin have a wider range of linearity than those employing horseradish peroxidase as an enzyme label. These results demonstrate that the assay for beta 1,4-GT is useful for determining activity in heterogeneous samples and also demonstrate the utility of the recombinant protein aequorin for solid-phase assay methods.     

Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry

The binding affinity and specificity of the mushroom Polyporus squamosus lectin has been determined by the recently developed method of frontal affinity chromatography coupled to electrospray mass spectrometry (FAC/MS). A micro-scale affinity column was prepared by immobilizing the lectin ( approximately 25 microg) onto porous glass beads in a tubing column (9.8 microl column volume). The column was then used to screen several oligosaccharide mixtures. The dissociation constants of 22 sialylated or sulfated oligosaccharides were evaluated against the immobilized lectin. The lectin was found to be highly specific for Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc containing oligosaccharides with K(d) values near 10 microM. The FAC/MS assay permits the rapid determination of the dissociation constants of ligands as well as a higher throughput screening of compound mixtures, making it a valuable tool for affinity studies, especially for testing large numbers of compounds.     

Preparation and some properties of a derivative of wheat germ agglutinin with altered biological activity

An inactive derivative of wheat germ agglutinin, which is a strong activator of blood platelets, was prepared by selective chemical modification of the lectin with cyanogen bromide at acid pH. The derivative was then used as a probe to learn about the initial events in platelet stimulation by physiological agents. Amino acid analysis of the modified lectin confirmed specific cleavage of a methionine residue. Gel filtration studies indicated a molecular weight for the lectin derivative similar to the unmodified lectin. In gel electrophoresis in the presence of sodium dodecyl sulfate, reduced samples of the derivative showed two bands and the main component migrated slightly faster than the native lectin. The derivative retained the capacity to precipitate an antibody to the lectin although at least one of the antigenic sites was lost due to chemical modification. The derivative did not compete with the unmodified lectin for binding to platelets. Unlike the parent lectin, the derivative did not aggregate platelets even at a ten fold higher concentration. Under similar conditions, there were about 1.0 X 10(5) binding sites/platelet for the lectin derivative with an apparent dissociation constant of 1.7 microM compared to 5 X 10(5) sites/cell and a dissociation constant of 0.4 microM for the native lectin. Overnight incubation of platelets or red cells with the derivative in microtiter plates showed about 2-5% agglutinating activity for the derivative compared to the unmodified lectin. Incubation of platelets with the lectin derivative inhibited platelet aggregation by thrombin while aggregation induced by a number of other agents was not significantly affected. This inhibitory effect of the lectin derivative on thrombin-induced platelet aggregation could be readily reversed with GlcNAc. The lectin derivative may be a useful tool to explore the structure-function relationship of cell surface components.     

An Enzyme-Linked Lectin Assay for α1,3-Galactosyltransferase

UDP-Gal:Galβ1-4GlcNAc α1,3-galactosyltransferase (α3GalT) is responsible for the synthesis of carbohydrate xenoantigen Galα1-3Galβ1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of α3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Galβ1-4GlcNAc (acceptor) are incubated with α3GalT in the presence of “cold” UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin. The standard curve for correct quantification of α3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl-donor UDP-Gal. In accordance with the known specificity of α3GalT, no enzymatic conversion of Le^x into GalαLe^x was observed using this assay. Human αGal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive.     

An enzyme-linked lectin binding assay for quantitative determination of lectin receptors

An enzyme-linked lectin binding assay (ELBA) has been developed for the detection of soluble lectin binding substances (receptors) and the determination of their relative affinity for the lectin. The assay is based on competitive binding to enzyme-labeled lectin of a known lectin receptor, bound to a solid phase, and unknown sample receptors. In this paper the assay is exemplified with the mannose/glucose-specific pea lectin, with the glycoprotein ovalbumin as its receptor, and with horseradish peroxidase (EC 1.11.1.7) as the enzyme used for labeling. Also a method was developed for the preparation of peroxidase-labeled lectin. Labeling was started by mixing equimolar amounts of lectin and periodate-oxidized enzyme at pH 4.5 at a final concentration of 10(-4)M, after which conjugation was started by raising the pH to 9.5. This resulted in complete conjugation, after which the product could be diluted 50-500 times for application in ELBA. For the ELBA ovalbumin was adsorbed onto polystyrene microtiter plates. Sample receptors, added together with the enzyme-labeled lectin, inhibited binding of the latter to ovalbumin. Bound enzyme activity was colorimetrically determined after addition of o-phenylenediamine. Relative lectin affinity (KL) was expressed as (formula; see text) in which [X]50% is the concentration of sample receptor necessary to inhibit 50% of the binding of a certain amount of lectin, and [M]50% is the concentration of D-mannose necessary to inhibit 50% binding of the same amount of lectin. With this technique lectin affinity of both monovalent and polyvalent lectin binding substances can be estimated: low KL values mean high lectin affinity.     

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 °C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in ≥50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.