Synthesis and assembly of anthrax lethal factor-cholera toxin B-subunit fusion protein in transgenic potato

A DNA encoding the 27-kDa domain I of anthrax lethal factor protein (LF), was linked to the carboxyl terminus of the cholera toxin B-subunit (CTB-LF). The CTB-LF fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated in vivo transformation methods and antibiotic-resistant plants were regenerated. The CTB-LF fusion gene was detected in transformed potato leaf genomic DNA by polymerase chain reaction (PCR)-mediated DNA amplification. Immunoblot analysis with anti-CTB and anti-LF primary antibodies verified the synthesis and assembly of biologically active CTB-LF fusion protein oligomers in transformed plant tuber tissues. Furthermore, the binding of CTB-LF fusion protein pentamers to intestinal epithelial cell membrane receptors measured by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) indicated that the CTB-LF fusion protein made up approx 0.002% of the total soluble tuber protein. Synthesis of CTB-LF monomers and their assembly into biologically active CTB-LF fusion protein pentamers in potato tuber tissues demonstrates the feasibility of using edible plants for production and delivery of adjuvanted LF protein for CTB-mediated immunostimulation of mucosal immune responses against anthrax toxin.     

Inhibition of Escherichia coli heat-labile enterotoxin B subunit pentamer (EtxB5) assembly in vitro using monoclonal antibodies

Heat-labile enterotoxin (Etx) produced by certain strains of Escherichia coli is a major virulence factor related to cholera toxin. Both are hexameric proteins comprising one A-subunit and five B-subunits. The pentameric B-subunit of E. coli has a high affinity for G(M1)-ganglioside receptors on gut epithelial cells and is directly responsible for toxin entry. The pentameric B-subunit (EtxB(5)) is an exceptionally stable protein, being able to maintain its quaternary structure over a wide pH range (2.0- 11.0). However, little is known about the formation of the pentameric structure (EtxB(5)) from newly synthesized B-subunit monomers (EtxB(1)). We previously described and characterized a mAb (LDS47) that was shown to be highly specific for an N-terminal decapeptide region of EtxB(1) (Amin, T., Larkins, A., James, R. F. L., and Hirst, T. R. (1995) J. Biol. Chem. 270, 20143-20150). Here we also describe a mAb (LDS16) with exquisite specificity for pentameric EtxB. In this study, we have used these two mAbs, in combination, to probe the in vitro assembly of EtxB(5) from EtxB(1). EtxB pentamers disassemble in highly acidic conditions, giving rise to monomeric B-subunits that can reassemble if placed in buffers of neutral pH. Using this in vitro assembly model, it was found that at a molar ratio of 1:1; LDS47:EtxB, 50% of reassembly was inhibited, and that this inhibition increased to 90% at a ratio of 2:1. These results infer that the N-terminal decapeptide region (APQSITELCS) defined by the LDS47 antibody is crucial for competent pentameric B-subunit assembly and stabilization.     

Analysis of cholera toxin-ganglioside interactions by flow cytometry

Cholera toxin entry into mammalian cells is mediated by binding of the pentameric B subunit (CTB) to ganglioside GM(1) in the cell membrane. We used flow cytometry to quantitatively measure in real time the interactions of fluorescently labeled pentameric cholera toxin B-subunit (FITC-CTB) with its ganglioside receptor on microsphere-supported phospholipid membranes. A model that describes the multiple steps of this mode of recognition was developed to guide our flow cytometric experiments and extract relevant equilibrium and kinetic rate constants. In contrast to previous studies, our approach takes into account receptor cross-linking, an important feature for multivalent interactions. From equilibrium measurements, we determined an equilibrium binding constant for a single subunit of FITC-CTB binding monovalently to GM(1) presented in bilayers of approximately 8 x 10(7) M(-1) while that for binding to soluble GM(1)-pentasaccharide was found to be approximately 4 x 10(6) M(-1). From kinetic measurements, we determined the rate constant for dissociation of a single site of FITC-CTB from microsphere-supported bilayers to be (3.21 +/- 0.03) x 10(-3) s(-1), and the rate of association of a site on FITC-CTB in solution to a GM(1) in the bilayer to be (2.8 +/- 0.4) x 10(4) M(-1) s(-1). These values yield a lower estimate for the equilibrium binding constant of approximately 1 x 10(7) M(-1). We determined the equilibrium surface cross-linking constant [(1.1 +/- 0.1) x 10(-12) cm(2)] and from this value and the value for the rate constant for dissociation derived a value of approximately 3.5 x 10(-15) cm(2) s(-1) for the forward rate constant for cross-linking. We also compared the interaction of the receptor binding B-subunit with that of the whole toxin (A- and B-subunits). Our results show that the whole toxin binds with approximately 100-fold higher avidity than the pentameric B-subunit alone which is most likely due to the additional interaction of the A(2)-subunit with the membrane surface. Interaction of cholera toxin B-subunit and whole cholera toxin with gangliosides other than GM(1) revealed specific binding only to GD1(b) and asialo-GM(1). These interactions, however, are marked by low avidity and require high receptor concentrations to be observed.     

Two-dimensional structures of the Shiga toxin B-subunit and of a chimera bound to the glycolipid receptor Gb3

The B-subunit of Shiga toxin has been demonstrated as a powerful vector for carrying attached peptides into cells for intracellular transport studies and for medical research. We have investigated the structure of the B-subunit and of a chimera bearing a peptide extension, bound to the membranous lipidic receptor, the globotriaosylceramide (Gb3). Two-dimensional crystals of both B-subunits have been obtained by the lipid layer method and projection maps have been calculated at 8.5A resolution from ice-embedded samples. The B-subunits as the chimera are organized in a pentameric form similar to the X-ray structure of the B-subunit not bound to Gb3. A difference map of both proteins has been calculated in which no density could be attributed to the peptide extension. Cross-correlations with projections of the B-subunit X-ray structure revealed that pentamers in the 2D crystals were oriented with their binding sites pointing to the lipid layer. Thus, it is likely that the peptide extension was disordered and confined to the surface of the pentamer opposite to the Gb3 binding sites. This location confirms the hypothesis that addition of peptide extension to the C-terminus conserves the ability of the modified B-subunit to bind the membranous receptor Gb3.     

Cholera toxin B subunits assemble into pentamers--proposition of a fly-casting mechanism

The cholera toxin B pentamer (CtxB(5)), which belongs to the AB(5) toxin family, is used as a model study for protein assembly. The effect of the pH on the reassembly of the toxin was investigated using immunochemical, electrophoretic and spectroscopic methods. Three pH-dependent steps were identified during the toxin reassembly: (i) acquisition of a fully assembly-competent fold by the CtxB monomer, (ii) association of CtxB monomer into oligomers, (iii) acquisition of the native fold by the CtxB pentamer. The results show that CtxB(5) and the related heat labile enterotoxin LTB(5) have distinct mechanisms of assembly despite sharing high sequence identity (84%) and almost identical atomic structures. The difference can be pinpointed to four histidines which are spread along the protein sequence and may act together. Thus, most of the toxin B amino acids appear negligible for the assembly, raising the possibility that assembly is driven by a small network of amino acids instead of involving all of them.     

HIV-1 gp120 V3 cholera toxin B subunit fusion gene expression in transgenic potato

A cDNA fragment encoding the V3 loop of human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp120 was fused to the cholera toxin B subunit gene (CTB-gp120) and transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB-gp120 fusion gene was detected in genomic DNA from transformed potato leaves by PCR DNA amplification. Synthesis and assembly of the CTB-gp120 fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-gp120 fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA results indicated that CTB-gp120 fusion protein made up 0.002-0.004% of the total soluble tuber protein. Synthesis of CTB-gp120 monomers and their assembly into biologically active oligomers in transformed potato tuber tissues demonstrates for the first time the expression of HIV-1 gp120 in plants and emphasizes the feasibility of using edible plant-based vaccination for protection against HIV-1 infection.     

Novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits

The B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) are structurally and functionally related. However, the carbohydrate binding specificities of the two proteins differ. While both CTB and LTB bind to the GM1 ganglioside, LTB also binds to N-acetyllactosamine-terminated glycoconjugates. The structural basis of the differences in carbohydrate recognition has been investigated by a systematic exchange of amino acids between LTB and CTB. Thereby, a CTB/LTB hybrid with a gain-of-function mutation resulting in recognition of blood group A and B determinants was obtained. Glycosphingolipid binding assays showed a specific binding of this hybrid B-subunit, but not CTB or LTB, to slowly migrating non-acid glycosphingolipids of human and animal small intestinal epithelium. A binding-active glycosphingolipid isolated from cat intestinal epithelium was characterized by mass spectrometry and proton NMR as GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)Glc NAcbeta3Galbeta4Glc NAcbeta3Galbeta4Glcbeta1Cer. Comparison with reference glycosphingolipids showed that the minimum binding epitope recognized by the CTB/LTB hybrid was Galalpha3(Fucalpha2)Galbeta4(Fucalpha3)GlcNAc beta. The blood group A and B determinants bind to a novel carbohydrate binding site located at the top of the B-subunit interfaces, distinct from the GM1 binding site, as found by docking and molecular dynamics simulations.     

Glycolipid Binding Preferences of Shiga Toxin Variants

The major virulence factor of Shiga toxin producing E. coli, is Shiga toxin (Stx), an AB₅ toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. The two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h) significantly differ in toxicity. The exact reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to play a role. Previous studies used enzyme linked immunosorbent assay (ELISA) to study binding of Stx1 and Stx2a toxoids to glycolipid receptors. Here, we studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) in the presence of cell membrane components such as phosphatidylcholine (PC), cholesterol (Ch) and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all Stx holotoxins significantly increased in the presence of PC and Ch. Previously, Stx2a has been shown to form a less stable B-pentamer compared to Stx1. However, its effect on glycolipid receptor binding is unknown. In this study, we showed that even in the absence of the A-subunit, the B-subunits of both Stx1 and Stx2a were able to bind to the glycolipids and the more stable B-pentamer formed by Stx1 bound better than the less stable pentamer of Stx2a. B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for binding of Stx1 than Stx2a.     

Synthesis and assembly of a cholera toxin B subunit SHIV 89.6p Tat fusion protein in transgenic potato

A cDNA encoding the simian-human immunodeficiency virus (SHIV 89.6p) Tat regulatory element protein was fused to the c-terminus of the cholera toxin B subunit gene (ctxB-tat) and introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods. The fusion gene was detected in the genomic DNA of transformed potato leaf cells by PCR DNA amplification. Synthesis and assembly of the CTB-Tat fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-Tat fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). Based on the ELISA results, CTB-Tat fusion protein made up about 0.005-0.007% of total soluble tuber protein or approximately 4.6mg per 100g potato tuber tissue. The synthesis and assembly of CTB-Tat monomers into biologically active oligomers in transformed potato tuber tissues demonstrates the feasibility of using viral pathogen antigens synthesized in edible plants for mucosal immunization against HIV-1 infection.     

Thermodynamic identification of stable folding intermediates in the B-subunit of cholera toxin

The structural stability and domain structure of the pentameric B-subunit of cholera toxin have been measured as a function of different perturbants in order to assess the magnitude of the interactions within the B-subunits. For these studies, temperature, guanidine hydrochloride (GuHCl), and pH were used as perturbants, and the effects were measured by high-sensitivity differential scanning calorimetry, isothermal reaction calorimetry, fluorescence spectroscopy, and partial protease digestion. At pH 7.5 and in the absence of any additional perturbants, the thermal unfolding of the B-subunit pentamer is characterized by a single peak in the heat capacity function centered at 77 degrees C and characterized by a delta Hcal of 328 kcal/mol of B-subunit pentamer and delta Hvh/delta Hcal of 0.3. Lowering the pH down to 4 or adding GuHCl up to 2 M results in a decrease of the calorimetric enthalpy with no significant effect on the van't Hoff enthalpy. The transition enthalpy decreases in a sigmoidal fashion with pH, with an inflection point centered at pH 5.3. Isothermal titration calorimetric studies as a function of pH also report a transition centered at pH 5.3 and characterized by an enthalpy change of 27 kcal/mol of B-subunit pentamer at 27 degrees C. Below this pH, the enthalpy change for the unfolding transition is reduced to approximately 100 kcal/mol of B-subunit pentamer. Similar behavior is obtained with GuHCl. In this case, a first transition is observed at 0.5 M GuHCl and a second one at 3 M GuHCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholera toxin and Escherichia coli enterotoxin B-subunits inhibit macrophage-mediated antigen processing and presentation: evidence for antigen persistence in non-acidic recycling endosomal compartments

Cholera toxin (Ctx) and the closely related Escherichia coli heat-labile enterotoxin (Etx) not only act as mediators of diarrhoeal disease but also exert potent immunomodulatory properties on mammalian immune systems. The toxins normally exert their diarrhoeagenic effects by initiating receptor-mediated uptake into vesicles that enter a retrograde trafficking pathway, circumventing degradative compartments and targeting them to the trans-Golgi network (TGN) and endoplasmic reticulum. Here, we examine whether receptor-mediated binding and cellular entry by the toxin B-subunits also lead to concomitant changes in uptake and trafficking of exogenous antigens that could contribute to the potent immunomodulatory properties of these toxins. Treatment of the macrophage (J774.2) cell line with Etx B-subunit (EtxB) resulted in EtxB transport to the TGN and also led to the formation of large, translucent, non-acidic, EtxB-devoid vacuoles. When exogenous antigens were added, EtxB-treated cells were found to be proficient in both internalization of ovalbumin (OVA) and phagocytosis of bacterial particles. However, the internalized OVA, instead of trafficking along a lysosome-directed endocytic pathway via acidified endosomes, persisted in a non-acidic, light-density compartment that was distinct from the translucent vacuoles. The rerouted OVA did not co-localize with the endosomal markers rab5 or rab11, nor with EtxB, but was retained in a transferrin receptor-positive compartment. The failure of OVA to enter the late endosomal/lysosomal compartments correlated with a striking inhibition of OVA peptide processing and presentation to OVA-responsive CD4+ T-cells. CtxB also modulated OVA trafficking and inhibited antigen presentation. These findings demonstrate that the B-subunits of Ctx and Etx alter the progression of exogenous antigens along the endocytic processing pathway, and prevent or delay efficient epitope presentation and T-cell stimulation. The formation of such 'antigen depots' could contribute to the immunomodulatory properties of these bacterial virulence determinants.     

Primary structure of cholera toxin B-subunit

The primary structure of cholera toxin B-subunit, responsible for the binding of the toxin to cell surfaces, has been elucidated. The polypeptide contains 103 amino acid residues and one intra-chain disulfide bridge between Cys 9 and Cys 86. The molecular weight is calculated to be 11,637, 15–20% higher than the values estimated by physicochemical methods. This value is consistent with a structure containing five moles of B-subunits per mole of cholera toxin.     

Synthesis and assembly of a cholera toxin B subunit-rotavirus VP7 fusion protein in transgenic potato

A gene encoding VP7, the outer capsid protein of simian rotavirus SA11, was fused to the carboxyl terminus of the cholera toxin B subunit gene. A plant expression vector containing the fusion gene under control of the mannopine synthase P₂ promoter was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB::VP7 fusion gene was detected in the genomic DNA of transformed potato leaf cells by polymerase chain reaction (PCR) amplification methods. Immunoblot analysis of transformed potato tuber tissue extracts showed that synthesis and assembly of the CTB::VP7 fusion protein into oligomers of pentameric size occurred in the transformed plant cells. The binding of CTB::VP7 fusion protein pentamers to sialo-sugar containing GM1 ganglioside receptors on the intestinal epithelial cell membrane was quantified by enzyme-linked immunosorbent assay (ELISA). The ELISA results showed that the CTB::VP7 fusion protein made up approx 0.01% of the total soluble tuber protein. Synthesis and assembly of CTB::VP7 monomers into biologically active pentamers in transformed potato tubers demonstrates the feasibility of using edible plants as a mucosal vaccine for the production and delivery system for rotavirus capsid protein antigens.     

Ubiquitin fusion enhances cholera toxin B subunit expression in transgenic plants and the plant-expressed protein binds GM1 receptors more efficiently

Developing plant based systems for the production of therapeutic recombinant proteins requires the development of efficient expression strategies and characterization of proteins made in heterologous cellular environment. In this study, the expression of cholera toxin B subunit (CtxB) was examined in the leaves of transgenic tobacco plants. A synthetic gene encoding CtxB was designed for high level expression in plant cells and cloned as ubiquitin (Ub) fusion in a plant expression vector. Tobacco plants were genetically engineered by nuclear transformation to express the CtxB or Ub-CtxB fusion proteins under the control of CaMV35S duplicated enhancer promoter. Functionally active CtxB accumulated in tobacco leaves at 2.5-fold higher level in the Ub-CtxB plants. In the best expressors, CtxB accumulated at 0.9% of the total soluble leaf protein. In both the constructs, molecular mass of the plant-expressed CtxB was 14.6 kDa in contrast to 11.6 kDa for the authentic CtxB. Schiff's test, retention on concanavalin A column and chemical and enzymatic deglycosylation established that the higher molecular mass was due to glycosylation of the CtxB expressed in plant cells. The glycosylated CtxB made in tobacco leaves had higher affinity of binding to the GM1 receptors.     

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.     

Mass production of somatic embryos expressing Escherichia coli heat-labile enterotoxin B subunit in Siberian ginseng

The B subunit of Escherichia coli heat-labile toxin (LTB) is a potent mucosal immunogen and immunoadjuvant for co-administered antigens. In order to produce large scale of LTB for the development of edible vaccine, we used transgenic somatic embryos of Siberian ginseng, which is known as medicinal plant. When transgenic somatic embryos were cultured in 130L air-lift type bioreactor, they were developed to mature somatic embryos through somatic embryogenesis and contained approximately 0.36% LTB of the total soluble protein. Enzyme-linked immunosorbent assay indicated that the somatic embryo-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting the LTB subunits formed active pentamers. Therefore, the use of the bioreactor system for expression of LTB proteins in somatic embryos allows for continuous mass production in a short-term period.     

A cholera toxin B-subunit variant that binds ganglioside G(M1) but fails to induce toxicity

Entry of cholera toxin (CT) into target epithelial cells and the induction of toxicity depend on CT binding to the lipid-based receptor ganglioside G(M1) and association with detergent-insoluble membrane microdomains, a function of the toxin's B-subunit. The B-subunits of CT and related Escherichia coli toxins exhibit a highly conserved exposed peptide loop (Glu(51)-Ile(58)) that faces the cell membrane upon B-subunit binding to G(M1). Mutation of His(57) to Ala in this loop resulted in a toxin (CT-H57A) that bound G(M1) with high apparent affinity, but failed to induce toxicity. CT-H57A bound to only a fraction of the cell-surface receptors available to wild-type CT. The bulk of cell-surface receptors inaccessible to CT-H57A localized to detergent-insoluble apical membrane microdomains (lipid rafts). Compared with wild-type toxin, CT-H57A exhibited slightly lower apparent binding affinity for and less stable binding to G(M1) in vitro. Rather than being transported into the Golgi apparatus, a process required for toxicity, most of CT-H57A was rapidly released from intact cells at physiologic temperatures or degraded following its internalization. These data indicate that CT action depends on the stable formation of the CT B-subunit.G(M1) complex and provide evidence that G(M1) functions as a necessary sorting motif for the retrograde trafficking of toxin into the secretory pathway of target epithelial cells.     

A 9 A two-dimensional projected structure of cholera toxin B-subunit-GM1 complexes determined by electron crystallography.

Highly ordered two-dimensional crystals of cholera toxin B-subunit pentamers have been grown by specific interaction with planar lipid films containing monosialoganglioside GM1. Electron diffractograms of frozen-hydrated crystals show diffraction peaks extending to beyond 4 A, while electron images diffract to 8 A. A two-dimensional projected structure of cholera toxin B-subunit-GM1 complex has been calculated at 9 A resolution by combining electron diffraction and image data. Crystals present an approximate pgg projection symmetry, with unit cell dimensions a = 119(+/- 1) A, b = 123(+/- 1) A, gamma = 90 degrees. Each pentameric assembly presents two concentric rings of electron scattering density, separated by an area of lower density. The outer and inner rings are centered at 25 A and and 11 A from the pentamer centre, respectively. The apparent projected density of the outer ring is larger than that of the inner ring. We propose that the outer and inner density rings correspond respectively to the peripheral beta-sheet arrangement and the central alpha-helix barrel, recently identified in the crystal structure of the heat-labile enterotoxin from Escherichia coli.     

High-throughput screening of cell lysates for ganglioside synthesis

A high-throughput screen to detect the synthesis of natural and non-natural gangliosides by cell lysates has been developed and automated. Utilizing the binding specificity of cholera toxin B-subunit for the oligosaccharide moiety of the ganglioside G_M1, the synthesis of sugar–sphingolipid glycosidic linkages was detected using a modified enzyme-linked immunosorbent assay (ELISA)/enzyme-linked lectin assay (ELLA). The screen was optimized and validated for high-throughput screening of cell lysates by evaluating different vectors, promoters, substrates and detection strategies. The extent of ganglioside synthesis was found to be proportional to enzyme concentration and length of incubation time. As a test of the finalized screen efficacy, individual colonies from a saturation mutagenesis library of nucleophile mutants of an endoglycoceramidase were screened to identify the most active enzyme for ganglioside synthesis. This screen should find general application in assaying both glycolipid biosynthesis and glycolipid hydrolysis, as it is highly sensitive and can be used with crude cell extracts.     

No direct binding of the heat-labile enterotoxin of <Emphasis Type="Italic">Escherichia coli</Emphasis> to <Emphasis Type="Italic">E. coli</Emphasis> lipopolysaccharides

A novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits (termed LCTBK) has previously been described, and also the native heat-labile enterotoxin bind to some extent to blood group A/B terminated glycoconjugates. The blood group antigen binding site is located at the interface of the B-subunits. Interestingly, the same area of the B-subunits has been proposed to be involved in binding of the heat-labile enterotoxin to lipopolysaccharides on the bacterial cell surface. Binding of the toxin to lipopolysaccharides does not affect the GM1 binding capacity. The present study aimed at characterizing the relationship between the blood group A/B antigen binding site and the lipopolysaccharide binding site. However, no binding of the B-subunits to E. coli lipopolysaccharides in microtiter wells or on thin-layer chromatograms was obtained. Incubation with lipopolysaccharides did not affect the binding of the B-subunits of heat-labile enterotoxin of human isolates to blood group A-carrying glycosphingolipids, indicating that the blood group antigen site is not involved in LPS binding. However, the saccharide competition experiments showed that GM1 binding reduced the affinity for blood group A determinants and vice versa, suggesting that a concurrent occupancy of the two binding sites does not occur. The latter finding is related to a connection between the blood group antigen binding site and the GM1 binding site through residues interacting with both ligands.