ABSTRACT
The two major forms of Shiga toxin, Stx1 and Stx2, use the glycolipid globotriaosylceramide (Gb3) as their cellular receptor. Stx1 primarily recognizes the Pk-trisaccharide portion and has three Pk binding sites per B monomer. The Stx2a subtype requires glycolipid residues in addition to Pk. We synthesized analogs of Pk to examine the binding preferences of Stx1 and Stx2 subtypes a to d. Furthermore, to determine how many binding sites must be engaged, the Pk analogues were conjugated to biotinylated mono- and biantennary platforms, allowing for the display of two to four Pk analogues per streptavidin molecule. Stx binding to Pk analogues immobilized on streptavidin-coated plates was assessed by enzyme-linked immunosorbent assay (ELISA). Stx1, but not the Stx2 subtypes, bound to native Pk. Stx2a and Stx2c bound to the Pk analog with a terminal GalNAc (NAc-Pk), while Stx1, Stx2b, and Stx2d did not bind to this analog. Interestingly, the purified Stx2d B subunit bound to NAc-Pk, suggesting that the A subunit of Stx2d interferes with binding. Disaccharide analogs (Galα1-4Gal, GalNAcα1-4Gal, and Galα1-4GalNAc) did not support the binding of any of the Stx forms, indicating that the trisaccharide is necessary for binding. Studies with monoantennary and biantennary analogs and mixtures suggest that Stx1, Stx2a, and Stx2c need to engage at least three Pk analogues for effective binding. To our knowledge, this is the first study examining the minimum number of Pk analogs required for effective binding and the first report documenting the role of the A subunit in influencing Stx2 binding.
INTRODUCTION
Shiga toxin is responsible for the life-threatening condition hemolytic-uremic syndrome (HUS). HUS is characterized by thrombocytopenia, hemolytic anemia, acute renal failure, and neurologic symptoms (1–5). Shiga toxins are members of the AB5 toxin family, consisting of a single A subunit and a pentamer of identical noncovalently associated B subunits. The A subunit mediates an RNA N-glycosidase activity that cleaves an adenine nucleotide from the 28S rRNA, leading to protein synthesis inhibition in the cell and ultimately cellular death (6, 7). The B subunit is the binding part of the toxin; it binds to the cell surface receptor, typically the neutral glycolipid globotriaosylceramide (Gb3), and mediates delivery of the A subunit to the cytoplasm of the cell (8–10). The glycans on Gb3, termed the Pk-trisaccharide, play a major role in Shiga toxin binding.
Two major forms of Shiga toxin, Stx1 and Stx2, share approximately 60% amino acid sequence identity but do not elicit cross-neutralizing antibodies. In addition, Stx2 has a number of subtypes that are over 90% identical. Recognized subtypes of Stx2 include Stx2a through Stx2g (11–13). The highly potent prototype form of Stx2 produced by Escherichia coli O157:H7 is now often called Stx2a for clarity. Stx2a, Stx2c, and Stx2d are closely related and associated with human disease (Fig. 1). Strains of E. coli can express one or more Shiga toxin types, but epidemiological studies have indicated that strains that produce Stx2a are more commonly associated with HUS than are strains that produce Stx1 (14–16). Strains producing only the Stx2c and Stx2d subtypes have occasionally been implicated in the development of hemorrhagic colitis and HUS (17). However, Shiga toxin subtypes differ widely in potency in both cellular and in vivo studies (12, 18–20). While the purified Stx2a and Stx2d subtypes are highly potent in mice (50% lethal doses [LD50s], <10 ng), Stx1, Stx2b, and Stx2c are not potent (LD50s, ≥1,000 ng) (18). In two separate studies, baboons treated with Stx2a displayed more severe disease symptoms than did animals treated with Stx1 (21, 22). Stx2b and Stx2e to Stx2g are less closely related (18, 19) and are rarely isolated from human clinical samples. Stx2e binds preferentially to globotetraosylceramide, as opposed to Gb3, and it causes edema disease in piglets (23–26).
Sequence alignment of Stx1 and Stx2 subtypes. Sequence alignment was done by using NCBI BLAST. Dots indicate identity, and dashes indicate absent amino acids. (A) Alignment of the C-terminal 20 of the 297 amino acids of the A subunit, corresponding to the region that traverses the B pentamer and influences receptor binding. (B) Alignment of the B subunits. Numbers correspond to the amino acid positions in the mature polypeptide of Stx2a.
Since Stx1 and Stx2a display indistinguishable RNA N-glycosidase activities in cell-free systems, differences in binding properties have been proposed to account for the difference in toxicity between Stx1 and Stx2a (14, 27–30). However, studying the fine details of receptor binding is challenging. Unlike typical protein-carbohydrate interactions, where a single binding site engages a single glycan, binding of Shiga toxin is the sum of multiple interactions, originating from two distinct mechanisms, protein-glycan interactions at the binding site and avidity, or the ability to enhance binding by simultaneously engaging multiple binding sites on the toxin. For example, Stx1 incubated with a single Pk-trisaccharide displays a dissociation constant (Kd) in the millimolar range, and in some studies, binding to Stx2 was 10-fold less avid (31–33). In contrast, both toxins bind to synthetic Pk glycoclusters (e.g., Starfish and Daisy) (34), linear polymers of Pk-trisaccharide (35), and the silicon-based dendrimers of the trisaccharide (36) in the micro- and nanomolar ranges. In practice, weak affinity at the binding site can be masked by the engagement of multiple binding sites, making it difficult to define the best toxin receptor. Even though these synthetic receptor mimics have been designed to act as competitive inhibitors and block Shiga toxin from binding to its natural cellular receptors, none have yet proven to be effective in clinical trials (37–39), possibly because the more potent toxin, Stx2a, does not have a high affinity for Pk. In addition, cellular toxicity occurs in the picomolar range (18), suggesting that in vitro systems do not replicate all aspects of in vivo interactions.
More is known about how Stx1 interacts with its receptor than about how Stx2 interacts with its receptor. The crystal structure of Stx1 revealed 3 Pk-trisaccharide binding sites per monomer, for a theoretical total of 15 sites in the pentamer (40). However, each of the three binding sites displays a different affinity for Pk and it is possible that some sites prefer a glycan other than Pk. This is supported by a recent study where Gb3 mixed with other neutral glycolipids bound Stx1 and Stx2a better than did Gb3 alone (33). Most studies examine binding to only a single Pk mimic and thus assess primarily the contribution of the five identical sites with the highest affinity for the receptor mimic being examined, leaving unexplored the potential contribution of engagement of the other binding sites.
In contrast to Stx1, it is not clear how many binding sites are present on the Stx2 B monomer. Stx2 binds Pk-trisaccharide weakly, and recent studies suggest that Stx2a recognizes portions of the ceramide of Gb3 in addition to Pk and prefers to bind to Gb3 in the presence of cholesterol (33, 41, 42). In addition, while most, if not all, of the binding of Stx1 is thought to be mediated by the B pentamer, the C terminus of the A subunit of Stx2 is displayed on the binding face of the B pentamer and can influence binding. For example, proteolytic removal of the two C-terminal amino acids (GE) of Stx2d has been reported to increase the potency of crude toxin extracts (20) and confer a modest increase in potency on purified, elastase-treated Stx2d (18).
In this study, we synthesized a series of synthetic glycoconjugates designed to independently assess the contributions of binding site affinity and avidity (Fig. 2). Pk-trisaccharide and NAc-Pk-trisaccharide analogs of Gb3 can differentiate between Stx1 and Stx2a (43). Di- and trisaccharides with and without an N-acetyl group on the terminal or penultimate galactose unit allow for understanding of the contributions of individual glycans in the trisaccharide. Mono- and biantennary constructs allow for understanding of the role of avidity, with streptavidin display playing a key role in avidity assessment. Streptavidin has four biotin binding sites, but previous studies have demonstrated that a single molecule of Shiga toxin can access only Pk mimics immobilized in the two adjacent biotin binding sites because of spatial constraints (44). Thus, monoantennary constructs immobilized on streptavidin display two Pk analogues while biantennary constructs display four (Fig. 2A). In addition, mixing of different Pk analogues for display on streptavidin can also provide insight into synergistic interactions between binding sites with different binding preferences. In this study, we used these constructs to examine the binding preferences of the Shiga toxin subtypes commonly associated with human disease and their purified B subunits. We found that Stx2 subtypes display significant differences in glycoconjugate preferences, and these studies have important implications for the design of receptor mimics for diagnostics and therapeutics.
Biotinylated monoantennary and biantennary glycoconjugates. (A) Monoantennary (top) and biantennary (bottom) biotinylated constructs. Cartoons represent glycoconjugate display on streptavidin. (B) Glycan structures. Cartoon representations use the standard symbol nomenclature proposed by the Consortium for Functional Glycomics: open circles, galactose (Gal); open squares, N-acetylgalactosamine (GalNAc); black circles, glucose (Glc).
MATERIALS AND METHODS
Glycoconjugates.The synthesis of the biotinylated glycoconjugates used in this study has been described previously (45). Biotinylated polyethylene glycol (PEG; molecular weight, 3,400) was purchased from Laysan Bio (Arab, AL).
Antibodies.Rabbit polyclonal antibodies to Stx1 and Stx2 were obtained from Meridian Bioscience (Cincinnati, OH). Peroxidase-conjugated goat anti-rabbit IgG was purchased from MP Biomedicals (Solo, OH). Chicken IgY against the Stx2 B subunit and peroxidase-conjugated rabbit anti-chicken IgY were purchased from Lampire Biological Laboratories (Ottsville, PA). A mouse monoclonal antibody against the Stx2 A subunit (11E10) was obtained from the National Cell Culture Center, and a peroxidase-conjugated goat anti-mouse antibody was purchased from ICN Biomedicals (Aurora, OH).
Shiga toxin holotoxins.Shiga toxin-containing supernatants were produced as previously described (43, 46). Toxin concentrations were determined by quantitative Western blot assays with mouse monoclonal antibody 11E10 to the Stx2 A subunit and a rabbit polyclonal antibody to Stx1. The purified Stx1 and Stx2 A subunits were used as standards in Western blot assays. Toxin activities and concentrations were verified in a protein synthesis inhibition assay with luciferase-conjugated Vero monkey kidney cells.
Purified B subunit.The Stx2 B wild-type (WT), Stx2 B Q40L mutant, and Stx2d B WT subunits were purified as previously described (47). Protein purity was verified by a single band at 8 kDa, corresponding to the molecular mass of a B subunit monomer on a Coomassie-stained SDS-PAGE gel. The concentrations of the B subunits were determined by bicinchoninic acid protein assay.
Binding assays and ELISAs.For enzyme-linked immunosorbent assay (ELISA) studies, biotinylated glycoconjugates were dissolved in phosphate-buffered saline (PBS) and added in excess (6.25 μM) to commercially available high-capacity (125 pM) streptavidin-coated microwell plates (Thermo Scientific), and binding was allowed to proceed for 2 h at room temperature. The wells were washed three times with PBS (pH 7.4) containing 0.1% bovine serum albumin and 0.5% Tween 20 and incubated with Shiga toxin containing supernatants at 37°C for 1 h. The maximum toxin concentration used was 1.40 μM. Toxin binding was detected with rabbit polyclonal antibodies (against Stx1 and Stx2) and peroxidase-conjugated goat anti-rabbit IgG. QuantaBlu fluorogenic peroxidase substrate (Pierce) was used to develop the plates, and the values were determined with a fluorometer. The same protocol was used in the purified Stx2B subunit binding study, except that chicken IgY and peroxidase-conjugated rabbit anti-chicken IgY were used as the primary and secondary antibodies to detect binding. Binding curve determination and analysis were performed by Prism 5.0 (GraphPad Software, La Jolla, CA).
RESULTS AND DISCUSSION
Several studies have shown that Stx1 binds to the Pk-trisaccharide (Galα1-4Galβ1-4Glc) portion of Gb3. However, some studies have indicated that Stx2 can bind to the Pk-trisaccharide while in other studies it bound weakly or not at all (27, 32, 48, 49). To further assess Shiga toxin binding preferences, we generated a panel of synthetic biotinylated glycoconjugates (Fig. 2). In previous studies, while Stx1 bound to an analogue displaying the native Pk-trisaccharide, Stx2 did not bind (43, 44). Instead, Stx2 bound to a synthetic variant, NAc-Pk (GalNAcα1-4Galβ1-4Glc), which did not engage Stx1. In this study, synthetic disaccharides were generated to examine the contributions of the individual glycans to binding site recognition. In addition, avidity (the additive effect of engaging multiple binding sites), which is important in Shiga toxin binding, was examined. The disaccharide and trisaccharide Pk analogs were attached to either a monoantennary biotinylated scaffold or a biantennary biotinylated scaffold and immobilized on streptavidin-coated microtiter plates via the biotin moiety. Streptavidin is a tetramer with two closely spaced biotin binding sites on each face of the molecule. While streptavidin has four biotin binding sites, we have demonstrated previously that a single molecule of Shiga toxin can access Pk analogues at only two adjacent biotin binding sites because of spatial constraints (44). Thus, the biantennary glycoconjugates present four potential Pk analogues and the monoantennary glycoconjugates present two potential Pk analogues (Fig. 2A).
Holotoxin binding to biantennary trisaccharides.The binding of Stx1 and Stx2 (Stx2a, -b, -c, and -d) to biantennary Pk and NAc-Pk was examined (Fig. 3, inserts). As reported previously (43, 44), Stx1 bound to Pk but Stx2a and Stx2c did not. Stx2b and Stx2d did not bind to Pk either (Fig. 3A). In addition, consistent with previous studies (43, 44), Stx1 did not bind to NAc-Pk but Stx2a and Stx2c bound to NAc-Pk (Fig. 3B). To determine the apparent dissociation constants (Kds), the binding curves were fitted to a one-site-specific binding model using the Hill coefficient. The apparent Kd of Stx1 binding to Pk was found to be 18 nM (Fig. 3A), whereas the apparent Kds of Stx2a and Stx2c binding to NAc-Pk were found to be 361 and 36 nM respectively (Fig. 3B). The apparent Kd values of the toxins for their preferred receptor analogues fall within the same range (10−7 to 10−9 M) as the Kd values in previous reports (33, 44, 48).
Binding of Stx1 and Stx2 subtypes to biantennary Pk analogues. (A) Binding to biantennary Pk. (B) Binding to biantennary NAc-Pk. Means ± standard errors of the means (error bars) of three independent experiments are shown. RFU, relative fluorescence units. For symbol definitions, see the legend to Fig. 2.
Interestingly, neither Stx2b nor Stx2d bound to NAc-Pk glycoconjugates (Fig. 3B). The B subunit of Stx2b displays several polymorphisms compared to Stx2a, all of which involve the putative Pk binding residues; thus, it was not surprising that Stx2b did not bind to NAc-Pk. On the other hand, the B subunit of the highly potent variant of the Stx2d subtype characterized in this study differs from Stx2a by only a single, conservative amino acid change (I51V) (Fig. 1), which is present on the face of the B subunit away from the receptor-binding interface. However, both Stx2b and Stx2d possess an acidic glutamic acid residue at the C terminus of the A subunit, while Stx2a and Stx2c possess the basic amino acid lysine. The crystal structure of Stx2a shows that the terminal amino acid of the A subunit is displayed on the binding face of the B subunit (19), and this charge change could affect binding.
Purified B subunit binding to biantennary trisaccharides.To determine if the C-terminal region of the A subunit interferes with Stx2d binding, purified B subunits of Stx2a and Stx2d were assessed for binding to NAc-Pk. Contrary to what was seen with the holotoxin, the B subunits of Stx2a and Stx2d bound almost equally to NAc-Pk (Fig. 4), suggesting that the C-terminal region of the Stx2d A subunit interferes with holotoxin binding. Compared to the holotoxin, less binding was observed with the B subunits, likely because pentamerization is required for efficient binding, and the Stx2 B subunit forms a stable pentamer only at concentrations greater than 2 μM (47). In previous studies, the NAc-Pk analogue immobilized on gold nanoparticles was shown to neutralize Stx2d in a cell-based assay (50); however, neutralization of Stx2d was much less efficient than neutralization of Stx2a. The density of glycoconjugate display was much greater on gold nanoparticles, suggesting that avidity could compensate for poor binding site recognition, although presentation could also play a role.
Stx2 B subunit binding to NAc-Pk. Means ± standard errors of the means (error bars) of three independent experiments are shown. RFU, relative fluorescence units. For symbol definitions, see the legend to Fig. 2.
We also examined the binding of the Q40L mutant Stx2a B subunit described in a previous report (47). That study demonstrated that the B pentamer of Stx1 is rather stable (Kd = 43 nM) and the B pentamer of Stx2a is much less stable (Kd = 2,300 nM). The stable Stx1 B pentamer has a hydrophobic leucine at the hydrophobic interface of subunits at the B pentamer, and Stx2 instability is due to the presence of a polar glutamine instead of a leucine at the hydrophobic interface. The Q40L mutant Stx2a B subunit, with the destabilizing glutamine substituted for the stabilizing leucine, displayed increased B pentamer stability (Kd = 110 nM). The Q40L mutant form bound better than either the Stx2a or the Stx2d WT B subunit (Fig. 4). Binding of the Q40L mutant B subunit, but not WT Stx2a, is observed at around 1,200 nM, which is well above the Kd for pentamer formation for the Q40L mutant form but below that for the WT Stx2a B subunit. These studies suggest that binding requires the multivalent effect afforded by the pentameric form.
Holotoxin binding to biantennary disaccharides.To investigate the contributions of the three sugar residues of Pk to binding, we examined the binding of a panel of biantennary disaccharides, including Galα1-4Gal, Galα1-4GalNAc, and GalNAcα1-4Gal (Fig. 2B). None of the disaccharides supported toxin binding (data not shown). The crystal structure of Stx1B in complex with the Pk-trisaccharide demonstrates that most of the hydrogen bonding and the stacking interactions occur between the terminal galactose units and the amino acid residues of all three binding sites (40), and binding to glycoconjugates with terminal digalactose (Galα1-4Gal) units was observed in previous studies (8). However, in studies using isothermal titration calorimetry (ITC), Stx1 was shown to bind to the Pk-trisaccharide but not to either Galα1-4Gal or lactose (31). Since ITC examined binding to the free glycans, the contribution of avidity or the ability to improve binding by engaging multiple binding sites was eliminated and the failure to detect binding to the disaccharide by ITC strongly suggests that all three sugar residues, in the appropriate configuration (α or β), contribute to binding affinity. While less information is available about Stx2 binding to synthetic disaccharides, Lingwood and his coworkers have demonstrated that both Stx1 and Stx2 bind to galabiosyl ceramide on thin-layer chromatography (TLC) plates (41). The failure of Stx2 to bind to the biantennary disaccharide glycoconjugates could be due to the difference in the presentation of sugars on the ELISA platform versus TLC plates or due to the fact that fewer hydrogen bonding and hydrophobic interactions can form with glycoconjugates that lack ceramide than with glycoconjugates with galabiosyl ceramide. Nevertheless, failure to detect binding to the biantennary disaccharides indicates that Stx2 also requires three sugar residues for optimal binding on an ELISA platform.
Holotoxin binding to monoantennary di- and trisaccharides.The contribution of avidity to binding was assessed by using the monoantennary glycoconjugates. Stx1 exhibited minimal binding to monoantennary Pk, in contrast to the control biantennary Pk (Fig. 5), and failed to bind to any of the other monoantennary glycoconjugates. Stx2a and the other Stx2 subtypes failed to bind to any of the monoantennary glycoconjugates (data not shown). While the biotinylated biantennary glycoconjugates provide four Pk-trisaccharide analogs for binding via the two streptavidin binding sites, monoantennary glycoconjugates provide only two Pk-trisaccharide analogs for binding. The failure to see binding with the monoantennary glycoconjugates suggests that the holotoxins need to engage more than two Pk-trisaccharide analogs for effective binding and this has been verified by binding studies with mixtures of glycoconjugates as described below.
Stx1 binding to monoantennary Pk analogues. Means ± standard errors of the means (error bars) of three independent experiments are shown. RFU, relative fluorescence units. Biantennary Pk (Pk) served as the positive control. For symbol definitions, see the legend to Fig. 2.
Binding of holotoxins to glycoconjugate mixtures.Recent binding studies have demonstrated that mixtures of glycoconjugates can support better binding than a single glycoconjugate can (33, 44, 51). Stx1 possesses three nonequivalent binding sites for Pk-trisaccharide per monomer of B pentamer (40), and glycoconjugate mixtures could enhance overall binding by allowing each binding site to engage its preferred glycoconjugate. Alternatively, the presence of glycoconjugate mixtures could result in decreased overall binding because of competition between preferred and suboptimal receptor mimics at a single binding site. We assessed Shiga toxin binding in the presence of its preferred glycoconjugate mixed with an equal amount of another glycoconjugate.
Binding preferences of Stx1.Biantennary Pk was mixed at a 1:1 ratio with other glycoconjugates or PEG (as a negative control), and binding to Stx1 was assessed (Fig. 6A and B). As observed previously (44), little to no binding was observed when biantennary Pk was mixed with PEG (Fig. 6A), which does not support binding by itself (34, 44). Similarly, we observed weak binding to monoantennary Pk (Fig. 5), which also results in the presentation of two Pk analogues, suggesting that two Pk analogues are not sufficient to support binding. However, mixtures of biantennary Pk with other glycoconjugates supported binding to various degrees. Binding to the glycoconjugate mixtures was calculated at the highest Stx1 concentration tested, by setting binding to unmixed biantennary Pk at 100% (Fig. 6B). The most binding (72%) was observed when biantennary Pk was mixed with monoantennary Pk, a mixture that should result in the presentation of, on average, three Pk analogues per streptavidin molecule. These results suggest that the engagement of at least three Pk-trisaccharides is critical for Stx1 binding and the presence of the fourth Pk-trisaccharides causes only a modest increase in binding.
(A) Stx1 binding to biantennary Pk and mixtures. RFU, relative fluorescence units. (B) Pk analogues were mixed with each of the glycoconjugates shown in the bar graph, and percent binding was determined as a percentage of the binding of Stx1 to the indicated biantennary Pk analog in the absence of other analogues at the highest toxin concentrations tested (145 μM). Means ± standard errors of the means (error bars) of three independent experiments are shown. Statistical analysis with a Student t test comparing each mixture with Pk was performed (*, P < 0.05). The zigzag line represents PEG. For symbol definitions, see the legend to Fig. 2.
While less effective than monoantennary Pk, NAc-Pk could also provide additional binding contacts when mixed with biantennary Pk (52%), and monoantennary NAc-Pk (51%) supported equivalent levels of binding when mixed with Pk (Fig. 6A). The ability of biantennary NAc-Pk to support binding of Stx1 in the presence of Pk was reported previously (44). This study suggests that only one NAc-Pk is engaged instead of two NAc-Pk receptor mimics because there is no significant difference in Stx1 binding to biantennary Pk mixed with biantennary or monoantennary NAc-Pk.
The monoantennary disaccharides also seem to provide additional binding contacts for Stx1 but are not as effective as the Pk or NAc-Pk-trisaccharide. Monoantennary Galα1-4Gal (Fig. 6A and B, Gal-Gal mono) supported 46% binding. Two disaccharides with a GalNAc residue, GalNAcα1-4Gal (35%) and Galα1-4GalNAc (35%), supported less binding than did the disaccharide Galα1-4Gal, suggesting that galactose at both positions is preferable to GalNAc at either position (Fig. 6A and B). These results are consistent with the crystal structure of Stx1 with Pk, where contact is made primarily with the two terminal galactose residues (40). Overall, these results demonstrate that a minimum of three Pk analogues need to be engaged for the binding of Stx1. When two native Pk-trisaccharides are present, monoantennary trisaccharide and disaccharide Pk analogues can provide additional binding contacts with Stx1, with native Pk being more effective than other Pk analogues.
Binding preferences of Stx2 subtypes.The binding of Stx2a (Fig. 7A and B) and Stx2c (Fig. 7C and D) to biantennary NAc-Pk mixed with other glycoconjugates was also assessed. While the binding preferences were similar, in all cases, Stx2c bound better than Stx2a. Similar to Stx1, the monoantennary form of its preferred Pk analogue, NAc-Pk, provided an additional binding contact; however, only about 30% binding was recovered for Stx2a (Fig. 7B), while 77% of Stx2c binding was recovered (Fig. 7D). These results suggest that, like Stx1, Stx2a and Stx2c require a minimum of three trisaccharides for binding.
Stx2 subtype binding to biantennary NAc-Pk and mixtures. Pk analogues were mixed with each of the glycoconjugates shown in the bar graph, and percent binding was determined as a percentage of the value obtained for toxin binding to the indicated biantennary NAc-Pk analog in the absence of other analogues at the highest toxin concentration tested (145 μM). (A, B) Stx2a binding to biantennary NAc-Pk and mixtures. (C, D) Stx2c binding to biantennary NAc-Pk and mixtures. Means ± standard errors of the means (error bars) of three independent experiments are shown. Statistical analysis with a Student t test comparing each mixture with NAc-Pk was performed (*, P < 0.05). Stx2a binding with the mixture of PEG and NAc-Pk was insignificant (data not shown). The zigzag line represents PEG. RFU, relative fluorescence units. For symbol definitions, see the legend to Fig. 2.
The glycoconjugate preferences of the Stx2 subtypes differed from those of Stx1. For both Stx2a and Stx2c, monoantennary NAc-Pk supported binding, suggesting that, like Stx1, these Stx2 subtypes also need to engage three Pk analogues for binding. However, for Stx2a, only 30% binding was observed when biantennary NAc-Pk was mixed with monoantennary NAc-Pk (Fig. 7B), while 77% of Stx2c binding was observed with this mixture (Fig. 7D). In addition, mixtures with native Pk, the nonpreferred analogue, also supported binding. Furthermore, the biantennary form supported more binding than the monoantennary form, suggesting that Stx2a and Stx2c could engage two molecules of native Pk. Unlike Stx1, none of the monoantennary or biantennary disaccharide Pk analogues supported the binding of either Stx2a or Stx2c (data not shown).
In summary, each B subunit monomer of Shiga toxin possesses multiple binding sites for Pk analogues, and each unique binding site is represented five times in the pentamer. Synthetic glycoconjugates immobilized on streptavidin present a robust experimental system to systematically dissect the roles of binding site preferences and binding avidity. We have shown that each form of Shiga toxin displays distinct preferences for the Pk analogues examined in this study. In addition, studies with the biantennary and monoantennary Pk analogues demonstrated that Stx1 and Stx2 subtypes (Stx2a and -c) need to engage at least three Pk analogues for effective binding. This information is critically important, since Pk analogues have been developed to function as therapeutic agents by blocking Shiga toxin binding to its natural cellular receptor (34). Unfortunately, they have not yet proven to be effective inhibitors in clinical trials (37–39). While these synthetic analogs exhibited strong binding affinity for both Stx1 and Stx2a, they bound Stx1 better than the more potent form Stx2a, which could explain their failure in clinical trials.
In this report, we have shown that the different subtypes of Stx2 also display different binding preferences. Most surprising was the failure of the highly potent Stx2d holotoxin to bind to NAc-Pk, even though the purified B pentamer bound to NAc-Pk. This reveals that the C-terminal region of the A subunit could interfere with binding. In addition, there is no obvious relationship between recognition of the synthetic glycan and toxin potency. In previous studies, the lethal dose of either Stx2a or Stx2d for mice was less than 10 ng, whereas the lethal dose of Stx2c was greater than 1,000 ng (18). These studies strongly suggest that factors other than glycan recognition could be important for determining potency. Future studies will examine whether Stx may utilize protein coreceptors.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health grants R01 AI 064893 (A.A.W.) and U01 AI 075498 (A.A.W.) and NSF CAREER CHE-0845005 (S.S.I.).
We thank Sayali Karve and Karen Gallegos for Shiga toxin preparations.
FOOTNOTES
- Received 1 March 2013.
- Returned for modification 28 April 2013.
- Accepted 12 May 2013.
- Accepted manuscript posted online 20 May 2013.
- Copyright © 2013, American Society for Microbiology. All Rights Reserved.
