Gb3 Binding Research Articles

Cell Function Research of β-Trefoil Lectins from Mytilidae

Two novel β-trefoil lectins, MytiLec-1 and SeviL were found from mussels in the coast of Yokohama and Nagasaki. MytiLec-1 was purified from gill and mantle of Mytilus galloprovincialis. It was consisted of 149 amino acid residues and there was no similarity with any other proteins when it was discovered. We advocate for this "Mytilectin" as a new protein family because of their novelty of its primary structure and homologues were also found in other mussels. Glycan array analysis revealed that MytiLec-1 specifically bound to the Gb3 and Gb4 glycan which contained the α-galactoside. MytiLec-1 caused the apoptosis against the Burkitt's lymphoma cells through the interaction of Gb3 express in their cell surface. On the other hand, SeviL obtained from gill and mantle of Mytilisepta virgata showed the specific binding against GM1b, asialo GM1 and SSEA-4 which are known as glycosphingolipid glycan including the β-galactoside. In addition, SeviL was identified as R type lectin by confirmation of QXW motif within its primary structure. Messenger RNA of SeviL like R type lectins was also found among the musssels including Mytilus galloprovincialis. SeviL also showed the apoptosis against asialo GM1 expressing cells. To apply the anticancer lectin as a novel molecular target drug, primary structure of MytiLec-1 was analyzed to enhance the stabilization of confirmation by computational design technique. It was succeeded to produce a monomeric artificial β-trefoil lectin, Mitsuba-1 without losing the Gb3 binding ability. Comparison of biological function between Mitsuba-1 and MytiLec-1 is also described in this study.

Structural Basis for the Specific Neutralization of Stx2a with a Camelid Single Domain Antibody Fragment.

Background: Shiga toxin-producing Escherichia coli (STEC) are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. The Shiga toxins are the main virulence factors and divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans. Results: An immune library of nanobodies (Nbs) was constructed after immunizing an alpaca with recombinant Shiga toxin-2a B subunit (rStx2aB), to retrieve multiple rStx2aB-specific Nbs. The specificity of five Nbs towards rStx2aB was confirmed in ELISA and Western blot. Nb113 had the highest affinity (9.6 nM) and its bivalent construct exhibited a 100-fold higher functional affinity. The structure of the Nb113 in complex with rStx2aB was determined via X-ray crystallography. The crystal structure of the Nb113–rStx2aB complex revealed that five copies of Nb113 bind to the rStx2aB pentamer and that the Nb113 epitope overlaps with the Gb3 binding site, thereby providing a structural basis for the neutralization of Stx2a by Nb113 that was observed on Vero cells. Finally, the tandem-repeated, bivalent Nb1132 exhibits a higher toxin neutralization capacity compared to monovalent Nb113. Conclusions: The Nb of highest affinity for rStx2aB is also the best Stx2a and Stx2c toxin neutralizing Nb, especially in a bivalent format. This lead Nb neutralizes Stx2a by competing for the Gb3 receptor. The fusion of the bivalent Nb1132 with a serum albumin specific Nb is expected to combine high toxin neutralization potential with prolonged blood circulation.

Polar Versus Non-polar Local Ordering at Mobile Sites in Proteins: Slowly Relaxing Local Structure Analysis of (15)N Relaxation in the Third Immunoglobulin-Binding Domain of Streptococcal Protein G.

We developed recently the slowly relaxing local structure (SRLS) approach for studying restricted motions in proteins by NMR. The spatial restrictions have been described by potentials comprising the traditional L = 2, K = 0, 2 spherical harmonics. However, the latter are associated with non-polar ordering whereas protein-anchored probes experience polar ordering, described by odd-L spherical harmonics. Here we extend the SRLS potential to include the L = 1, K = 0, 1 spherical harmonics and analyze (15)N-(1)H relaxation from the third immunoglobulin-binding domain of streptococcal protein G (GB3) with the polar L = 1 potential (coefficients c0(1) and c1(1)) or the non-polar L = 2 potential (coefficients c0(2) and c2(2)). Strong potentials, with ⟨c0(1)⟩ ∼ 60 for L = 1 and ⟨c0(2)⟩ ∼ 20 for L = 2 (in units of kBT), are detected. In the α-helix of GB3 the coefficients of the rhombic terms are c1(1) ∼ c2(2) ∼ 0; in the preceding (following) chain segment they are ⟨c1(1)⟩ ∼ 6 for L = 1 and ⟨c2(2)⟩ ∼ 14 for L = 2 (⟨c1(1)⟩ ∼ 3 for L = 1 and ⟨c2(2)⟩ ∼ 7 for L = 2). The local diffusion rate, D2, lies in the 5 × 10(9)-1 × 10(11) s(-1) range; it is generally larger for L = 1. The main ordering axis deviates moderately from the N-H bond. Corresponding L = 1 and L = 2 potentials and probability density functions are illustrated for residues A26 of the α-helix, Y3 of the β1-strand, and L12 of the β1/β2 loop; they differ considerably. Polar/orientational ordering is shown to be associated with GB3 binding to its cognate Fab fragment. The polarity of the local ordering is clearly an important factor.

Membrane invagination induced by Shiga toxin B-subunit: from molecular structure to tube formation.

The bacterial Shiga toxin is composed of an enzymatically active A-subunit, and a receptor-binding homopentameric B-subunit (STxB) that mediates intracellular toxin trafficking. Upon STxB-mediated binding to the glycolipid globotriaosylceramide (Gb3) at the plasma membrane of target cells, Shiga toxin is internalized by clathrin-dependent and independent endocytosis. The formation of tubular membrane invaginations is an essential step in the clathrin-independent STxB uptake process. However, the mechanism by which STxB induces these invaginations has remained unclear. Using a combination of all-atom molecular dynamics and Monte Carlo simulations we show that the molecular architecture of STxB enables the following sequence of events: the Gb3 binding sites on STxB are arranged such that tight avidity-based binding results in a small increment of local curvature. Membrane-mediated clustering of several toxin molecules then creates a tubular membrane invagination that drives toxin entry into the cell. This mechanism requires: (1) a precise molecular architecture of the STxB binding sites; (2) a fluid bilayer in order for the tubular invagination to form. Although, STxB binding to the membrane requires specific interactions with Gb3 lipids, our study points to a generic molecular design principle for clathrin-independent endocytosis of nanoparticles.

The Dramatic Modulatory Role of the 2'N Substitution of the Terminal Amino Hexose of Globotetraosylceramide in Determining Binding by Members of the Verotoxin Family

Although globotetraosylceramide (Gb4) is only recognized by a single member of the verotoxin family namely, the pig edema disease toxin (VT2e), removal of the acetyl group from the terminal N-acetyl hexosamine of Gb4 to generate the free amino sugar containing species (aminoGb4) results in the generation of a glycolipid preferentially recognized by all members of the verotoxin family (i.e., VT1, VT2, VT2c, and VT2e). GT3, a site-specific mutant of VT2e, in which Gb4 recognition is lost but Gb3 binding is retained, also binds aminoGb4. We have now compared the binding of VT1, VT2, VT2e, and GT3 to a series of aminoGb4 derivatives using a TLC overlay technique. DimethylaminoGb4 is bound by VT1 and VT2 but not VT2e or GT3; formylaminoGb4 binds all toxins but poorly to VT2 and preferentially VT2e; trifluoroacetylaminoGb4 binds only VT2e and GT3; isopropylaminoGb4 binds VT1 and poorly to VT2; benzylaminoGb4 binds all four toxins. Thus, there is a marked distinction between the permissible amino substitutions for VT1 and VT2e binding. GT3 is a hybrid between these in that, according to the substitution, it behaves similarly either to VT1 or to VT2e. For each species, GT3 does not however, show a hybrid binding between that of VT1 and VT2e. Analysis of the binding as a function of pH shows opposite effects for VT1 and VT2e: decreased pH increases VT1, but decreases VT2e receptor glycolipid binding.

The Crystal Structure of Shiga Toxin Type 2 with Bound Disaccharide Guides the Design of a Heterobifunctional Toxin Inhibitor

Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB5 toxin, gains entry into human cells via the glycosphingolipid receptor Gb3. We have determined the first crystal structure of a disaccharide analog of Gb3 bound to the B5 pentamer of Stx2a holotoxin. In this Gb3 analog,-GalNAc replaces the terminal-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in theB5 pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb3 trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.

Probing the Effects of Cysteine Residues on Protein Adsorption onto Gold Nanoparticles Using Wild-Type and Mutated GB3 Proteins

The role of cysteine residues in the protein binding kinetics and stability on gold nanoparticles (AuNP) was studied using AuNP localized surface plasmon resonance (LSPR) in combination with an organothiol (OT) displacement method. GB3, the third IgG-binding domain of protein G, was used to model protein-AuNP adsorption. While wild-type GB3 (GB30) contains no cysteine residues, bioengineered GB3 variants containing one (GB31) and two (GB32) cysteine residues were also tested. The cysteine content has no significant effect on GB3 binding kinetics with AuNPs, and most protein adsorption occurs within the first few seconds upon protein/AuNP mixing. However, the stability of GB3 on the AuNP surface against OT displacement depends strongly on the cysteine content and the age of the AuNP/GB3 mixture. The GB30 covered AuNPs can be completely destabilized and aggregated by OTs, regardless of the age of the GB30/AuNP mixtures. Long-time incubation of GB31 or GB32 with AuNPs can stabilize AuNPs against the OT adsorption inducted aggregation. This study indicates that multiple forces involved in the GB3/AuNP interaction, and covalent binding between cysteine and AuNP is essential for a stable protein/AuNP complex.

Intracellular trafficking of Shiga‐toxin‐B‐subunit‐functionalized spherulites

Spherulites are multi-lamellar lipidic vesicles that can encapsulate biomolecules and may be used as carriers for drug delivery. STxB (Shiga toxin B-subunit) is known to bind the glycosphingolipid Gb3 (globotriaosyl ceramide), which is overexpressed by various human tumours. After Gb3 binding, the toxin enters the cytoplasm via the retrograde route, bypassing the degrading environment of the late endosomes/lysosomes. STxB is non-toxic and has been identified as a promising tool for drug delivery. So far, applications have relied on direct coupling with therapeutic agents. In the present study, we have investigated the functionalization of spherulites by STxB and the intracellular trafficking of these structures. We demonstrate that STxB-spherulites (ST x B-functionalized spherulites) are internalized into HeLa cells in a receptor-dependent manner. The intracellular distribution was studied by confocal microscopy for lipids, ligand and content. We observed an early separation between spherulites and STxB, leading to a late endosomal/lysosomal localization of lipids and content, whereas STxB remained partially at the plasma membrane. Although recognition of Gb3 is the cause of their specific adhesion to cell membranes, STxB-spherulites do not follow the retrograde transport route. Our results strongly suggest that STxB-spherulites are, at least in part, disrupted at the plasma membrane, leading to lipid and content targeting to the classical endocytic pathway. We discuss how these findings influence the development of innovative delivery strategies.

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 8.5 Å 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.

Differential tissue targeting and pathogenesis of verotoxins 1 and 2 in the mouse animal model

Both verotoxin (VT)1 and VT2 share the same receptor, globotriaosyl ceramide (Gb(3)). Although VT1 is slightly more cytotoxic in vitro and binds Gb(3) with higher affinity, VT2 is more toxic in mice and may be associated with greater pathology in human infections. In this study we have compared the biodistribution of iodine 125 ((125)I)-VT1 and (125)I-VT2 versus pathology in the mouse. (125)I-VT1 whole-body autoradiography defined the tissues targeted. VT1 and VT2 tissue distribution, clearance, and tissue binding sites were compared. The effect of a soluble receptor analogue, adamantylGb(3), on VT2/Gb3 binding and in vivo pathology was assessed. (125)I-VT1 autoradiography identified the lungs and nasal turbinates as major, previously unrecognized, targets, while kidney cortex and the bone marrow of the spine, long bones, and ribs were also significant targets. VT2 did not target the lung, but accumulated in the kidney to a greater extent than VT1. The serum half-life of VT1 was 2.7 minutes with 90% clearance at 5 minutes, while that of VT2 was 3.9 minutes with only 40% clearance at 5 minutes. The extensive binding of VT1, but not VT2, within the lung correlated with induced lung disease. Extensive hemorrhage into alveoli, edema, alveolitis and neutrophil margination was seen only after VT1 treatment. VT1 targeted lung capillary endothelial cells. Identical tissue binding sites (subsets of proximal/distal tubules and collecting ducts) for VT1 and VT2 were detected by toxin overlay of serial frozen kidney sections. Glucosuria was found to be a new marker of VT1- and VT2-induced renal pathology and positive predictor of outcome in the mouse, consistent with VT-staining of proximal tubules. Lung Gb3 migrated on thin-layer chromatography (TLC) faster than kidney Gb(3), suggesting a different lipid composition. AdamantylGb(3), a soluble Gb(3) analogue, competed effectively for Gb3 binding by VT1 and VT2 in vitro. However, the effect in the mouse model (only measured against VT2, due to the lower LD(50), a concentration required for 50% lethality) was to increase, rather than reduce, pathology and further reduce the VT2 serum clearance rate. Additional renal pathology was seen in VT2 + adamantylGb(3)-treated mice. The lung is a preferential (Gb(3)) "sink" for VT1, which explains the relatively slower clearance of VT2 and subsequent increased VT2 renal targeting and VT2 mortality in this animal model.

Different binding property of verotoxin-1 and verotoxin-2 against their glycolipid receptor, globotriaosylceramide.

We determined the binding of verotoxin-1 (VT1) and verotoxin-2 (VT2) against globotriaosylceramide (Gb3) by a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA). Ethanolic solution of Gb3 containing cholesterol and phosphatidylcholine was passively adsorbed onto the wells of microtiter plate, and Gb3-bound VT1 and VT2 were detected by anti-VT1 and anti-VT2 mAbs, respectively. Although both VT1 and VT2 reacted with Gb3 in a concentration dependent manner, terminal galactose requirement for Gb3 binding was also different from each other. Pretreatment of VT1 showed the inhibitory effect on the binding of VT2 to Gb3, while the VT2-pretreatment showed no inhibitory effect on VT1 binding to Gb3. This was not due to the replacement of Gb3-bound VT2 with post-treated VT1. These results suggest that the binding sites of VT1 and VT2 on Gb3 are not identical to each other.

The identification of three biologically relevant globotriaosyl ceramide receptor binding sites on the Verotoxin 1 B subunit.

The Verotoxin 1 (VT1) B subunit binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). Receptor-binding specificity is associated with the terminally linked Galalpha(1-4) Galbeta disaccharide sequence of the receptor. Recently, three globotriose (Galalpha[1-4] Galbeta [1-4] Glcbeta) binding sites per B-subunit monomer were identified by crystallography. Two of these sites (sites I and II) are located adjacent to phenylalanine-30. Site I was originally predicted as a potential Gb3 binding site on the basis of sequence conservation, and site II was additionally predicted based on computer modelling and receptor docking. The third (site III) was also identified by crystallography and is located at the N-terminal end of the alpha-helix. To determine the biological significance of sites II and III, and to support our previous findings of the significance of site I, we examined the binding properties and cytotoxicity of VT1 mutants designed to block Gb3 binding at each site selectively. The Scatchard analysis of saturation-binding data for each mutant revealed that only the amino acid substitutions predicted to affect site I (D-17E) or site II (G-62T) caused reductions in the binding affinity and capacity of VT1 for Gb3. Similarly, those mutations at sites I and II also caused significant reductions in both Vero and MRC-5 cell cytotoxicity (by seven and five logs, respectively, for G-62T and by four and two logs, respectively, for D-17E). In contrast, the substitution of alanine for W-34 at site III did not reduce the high-affinity binding of the B subunit, despite causing a fourfold reduction in the receptor-binding capacity. The corresponding mutant W-34A holotoxin had a two-log reduction in cytotoxicity on Vero cells and no statistically significant reduction on MRC-5 cells. We conclude that the high-affinity receptor binding most relevant for cell cytotoxicity occurs at sites I and II. In contrast, site III appears to mediate the recognition of additional Gb3 receptor epitopes but with lower affinity. Our results support the significance of the indole ring of W-34 for binding at this site.

Localization of the binding site for modified Gb3 on verotoxin 1 using fluorescence analysis.

Verotoxins (VTs) from Escherichia coli elicit human vascular disease as a consequence of specific binding to globotriaosylceramide (Gb3) receptors on endothelial cell surfaces. Molecular models based on the VT1 crystal structure were used previously to investigate the structural basis for receptor recognition by VT1 and other verotoxins. Interestingly, these model-based predictions of glycolipid binding to VT1 differ somewhat from recently published structural data from cocrystals of the VT1 B-subunit (VT1B) and an analogue of the sugar moiety of Gb3. In this study, fluorescence spectroscopy was used to test model-based predictions of the location of Gb3 binding on the B-subunit pentamer of VT1. Resonance energy transfer was used to calculate the distance from a coumarin probe used to replace the acyl tail of Gb3 and the single tryptophan residue (Trp34) present within each VT1B monomer. The observed energy transfer efficiency (greater than 95%) suggests that these two moieties are approximately 13.3 A apart when a single distance is assumed. This distance is consistent with proposed models for the fit of Gb3 within the "cleft site" of the VT1 B-subunit. When the distances from Trp34 to the other coumarinGb3 molecules (bound to each of the four remaining monomers within the VT1B pentamer) are taken into consideration, it appears likely that the coumarin-modified Gb3 analogue used in this study associates with the previously proposed receptor binding site II of VT1. This is consistent with an observed binding preference of VT2c for coumarinGb3. To provide additional information on the association of Gb3 with the VT1 B-subunit, the influence of Gb3 glycolipid binding on the accessibility of Trp34 to different quenching agents in solution was then examined. Taken together, the data suggest that coumarin-labeled Gb3 preferentially binds to site II on VT1 in a position that is consistent with the previously described molecular models.

Intracellular targeting of the endoplasmic reticulum/nuclear envelope by retrograde transport may determine cell hypersensitivity to verotoxin via globotriaosyl ceramide fatty acid isoform traffic.

The pentameric B subunit of verotoxin (VT) mediates the attachment to cell surface globotriaosyl ceramide (Gb3) to facilitate receptor-mediated endocytosis of the toxin. In highly toxin-sensitive tumor cells, the holotoxin and VT1 B subunit is targeted intracellularly to elements of the endoplasmic reticulum (ER)/nuclear membrane. In less sensitive cells, the toxin is targeted to components of the Golgi apparatus. We have studied two cell systems: the induced VT hypersensitivity of human astrocytoma cell lines cultured in the presence of sodium butyrate (compared to sodium propionate and capronate) and the increased VT sensitivity of multiple drug-resistant mutants as compared to parental human ovarian carcinoma cells. In both cases, a difference in the intracellular retrograde transport of the receptor-bound internalized toxin to the ER/nuclear envelope, as opposed to the Golgi, correlated with a >1,000-fold increase in cell sensitivity to VT. This change in intracellular routing may be due to sorting of Gb3 fatty acid isoforms, since nuclear targeting was found in turn to correlate with the preferential synthesis of Gb3 containing shorter chain (primarily C16) fatty acid species. We propose that the isoform-dependent traffic of Gb3 from the cell surface to the ER/nuclear membrane provides a new signal transduction pathway for Gb3 binding proteins.

Toxicity and immunogenicity of a verotoxin 1 mutant with reduced globotriaosylceramide receptor binding in rabbits.

The verotoxins (VT1 and VT2), produced by strains of enterohemorrhagic Escherichia coli, have been implicated in the pathogenesis of hemorrhagic colitis and the hemolytic uremic syndrome. To better understand the role of globotriaosylceramide (Gb3) receptor binding by the verotoxins in disease production, we examined the clinicopathologic effects of an intravenously (i.v.) administered verotoxin 1 mutant holotoxin (Phe30Ala) in rabbits. The substitution of alanine for phenylalanine 30 in the VT1 B subunit has been shown previously to reduce both Gb3 binding affinity and capacity in vitro. This reduction in receptor binding corresponded to a 10(5)-fold reduction in the toxic activity of VT1 on a Vero cell monolayer. In this study, purified 125I-labeled Phe30Ala was administered i.v. to rabbits to determine its specific distribution in rabbit tissues. In contrast to the rapid elimination of i.v. administered 125I-VT1 from the bloodstream, 125I-Phe30Ala had a 52-fold-longer half-life in serum and failed to localize preferentially in the gastrointestinal tract and central nervous system (CNS). Rabbits challenged with Phe30Ala at a dose equivalent to 10 times the 50% lethal dose (LD50) of VT1 showed no visible clinical symptoms typical of VT effect after 7 days. Administration of Phe30Ala at a dose equivalent to 100 times the LD50 of VT1, however, caused both clinical and histopathologic features indistinguishable from VT1 toxemia in rabbits, although the onset of symptoms was delayed. Rabbits were immunized with Phe30Ala and challenged i.v. with either 125I-VT1 or 125I-VT2. The specific uptake of 125I-VT1 in the gastrointestinal tract and CNS was totally inhibited in Phe30Ala immune rabbits. Only a partial decrease in target organ uptake was observed in Phe30Ala immune rabbits challenged with 125I-VT2. From this study, we conclude that Gb3 binding is responsible for target organ localization of VT1 and disease production in the rabbit. The ability of Phe30Ala to induce both strong antibody and protective responses against VT1 suggests that VT mutants with reduced receptor binding properties may be useful in vaccine strategies. A further reduction in the toxicity of Phe30Ala would be required for its use as a natural toxoid to protect against human verotoxigenic E. coli infections.

Capping and receptor‐mediated endocytosis of cell‐bound verotoxin (shiga‐like toxin) 1: Chemical identification of an amino acid in the B subunit necessary for efficient receptor glycolipid binding and cellular internalization

The glycolipid globotriaosylceramide (Gb3) is the plasma membrane receptor that mediates the internalization of verotoxin (VT1) into susceptible cells by capping and receptor-mediated endocytosis (RME). Internalization of fluorescein isothiocyanate-conjugated holotoxin into Daudi lymphoma cells was found to be slower than the pentameric receptor binding B subunit alone, suggesting that the A subunit may interact with the membrane to compromise the lateral mobility of the receptor bound B subunit. 3-D reconstruction of fluorescent images by confocal microscopy confirmed the complete internalization of holotoxin. VT1 internalization and cytotoxicity was inhibited by monodansyl cadavarine, which supports a role for clathrin coated pits in the RME of VT1. Biotinylation of the B subunit (in contrast to fluorescein labelling) was found to prevent toxin internalization. This effect correlated with reduced binding of Gb3 and reduced cytotoxicity in vitro. By cleavage of the B subunit at the single tryptophan residue, the reduced Gb3 binding and lack of cellular internalization was shown to be due to the biotinylation of lysine 53 in the VT1 B subunit. This residue was not labelled with fluorescein isothiocyanate in the native protein. This conclusion was confirmed by the finding that biotinylation of VT2c (which contains lys 53) prevented glycolipid receptor binding, whereas biotinylation of VT2e (in which lys 53 is substituted by ile) had no effect.

Glycosphingolipid receptor function is modified by fatty acid content. Verotoxin 1 and verotoxin 2c preferentially recognize different globotriaosyl ceramide fatty acid homologues

Verotoxins (VT) are a family of Escherichia coli-derived toxins which have been associated with hemolytic uremic syndrome, the leading cause of acute pediatric renal failure, and hemorrhagic colitis. Verotoxins (VT1 and VT2c) both show terminal gal alpha 1-4gal-dependent binding to globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc-Cer; Gb3), yet VT2c shows a thousandfold lower specific cytotoxic activity in vitro. Our previous studies have shown this discrepancy is a function of the receptor binding B subunit and that VT1/Gb3 binding in a lipid matrix is affected by heterogeneity in the ceramide fatty acid chain length. The influence of the fatty acid composition of Gb3 on the binding of VT1 and VT2c has now been compared using 14 homogeneous semisynthetic Gb3 molecular species of differing fatty acid chain length and degree of saturation from C12 to C24. The binding of verotoxin was quantitated by Scatchard analysis using a solid-phase binding assay in the presence of auxiliary lipids, which may in some respects approximate to receptor function within the plasma membrane of sensitive cells. Differential binding was observed for several of these species in the lipid matrix, indicating that the fatty acid moiety of Gb3 is important for VT binding under such conditions. The short chain fatty acid containing Gb3 (C12 and C14) showed minimal binding. Middle and long chain fatty acid Gb3 homologues (C16, C18, C20, C22, and C24) were effectively recognized by VTs. The presence of an unsaturated fatty acid in Gb3 significantly increased VT binding in all cases. C20:0 and C22:1 containing Gb3 had the greatest capacity to bind VT1. In contrast, C18:0 and C18:1 homologues showed the greatest capacity for VT2c binding (higher than VT1). These results were, in general, reflected in cell cytotoxicity in that receptor-deficient cells reconstituted with C22:1Gb3 were maximally sensitive to VT1 in vitro whereas cells reconstituted with C18:1Gb3 were maximally sensitive to VT2c. VT2c was an ineffective inhibitor of 125I-VT1 binding to C22:1 Gb3 but in contrast, more effective than VT1 to compete binding to C18:1 Gb3. Similarly, VT1 was less effective than VT2c to compete binding of 125I-VT2c to C18:1 but more effective than VT2c to compete for C22:1 Gb3 binding. These results suggest that VT1 and VT2c bind selectively to different but overlapping carbohydrate epitopes on the Gb3 molecule which are differentially available in these Gb3 fatty acid homologues in a lipid environment.(ABSTRACT TRUNCATED AT 400 WORDS)