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Blood Group Antigen Recognition by Escherichia coli Heat-labile Enterotoxin
doi:10.1016/j.jmb.2007.05.064
J. Mol. Biol. (2007) 371, 754–764
Blood Group Antigen Recognition by Escherichia coli Heat-labile Enterotoxin Åsa Holmner 1,2 ⁎, Glareh Askarieh 2 , Mats Ökvist 2 and Ute Krengel 1,2 ⁎ 1
Department of Chemistry and Bioscience, Chalmers University of Technology, PO box 462, SE-40530 Göteborg, Sweden 2
Department of Chemistry, University of Oslo, PO box 1033 Blindern, NO-0315 Oslo, Norway
In a number of bacterial infections, such as Helicobacter pylori, Campylobacter jejuni and Vibrio cholerae infections, a correlation between the severity of disease and blood group phenotype of infected individuals has been observed. In the present investigation, we have studied the molecular basis of this effect for enterotoxigenic Escherichia coli (ETEC) infections. ETEC are non-invasive bacteria, which act through second messenger pathways to cause diarrhea. It has been suggested that the major virulence factor of ETEC from human isolates, i.e. the human heat-labile enterotoxin (hLT), recognizes certain blood group epitopes, although the molecular basis of blood group antigen recognition is unknown. The 2.5 Å crystal structure of the receptor-binding B-subunit of hLT in complex with the blood group A antigen analog GalNAcα3(Fucα2)Galβ4(Fucα3)Glcβ provides evidence of a previously unknown binding site in the native toxin. The structure reveals the molecular interactions underlying blood group antigen recognition and suggests how this protein can discriminate between different blood group epitopes. These results support the previously debated role of hLT in the blood group dependence of ETEC infections. Similar observations regarding the closely related cholera toxin in V. cholera infections are also discussed. © 2007 Elsevier Ltd. All rights reserved.
*Corresponding authors
Keywords: blood-group recognition; bacterial toxins; clinical/epidemiological data; protein-carbohydrate interactions; X-ray crystal structure
Introduction Bacterial infections have historically killed more people than any other disease and the annual burden of diarrheal diseases alone is estimated to three million deaths, according to the World Health Present address: Å. Holmner, Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden. Abbreviations used: CCP4, Collaborative Computational Project 4; CT, cholera toxin; CTB, cholera toxin B-pentamer; ETEC, enterotoxigenic Escherichia coli; Fuc, L-fucose; Gal, D-galactose; GalNAc, 2′-N-acetyl galactosamine; Glc, D-glucose; LT, heat-labile enterotoxin; LTB, heat-labile enterotoxin B-pentamer; hLTB, B-pentamer of human isolates of heat-labile enterotoxin; PBS, phosphate-buffered saline; PDB, Protein Data Bank; r.m.s.d., root-mean-square difference/deviation; WHO, world health organization; LPS, lipopolysaccharide(s). E-mail addresses of the corresponding authors: [email protected]; [email protected]
Organization (WHO). Cholera, which is caused by the human pathogen Vibrio cholerae, gives rise to the most severe form of secretory diarrhea, whereas the most frequently isolated enteropathogen is enterotoxigenic Escherichia coli (ETEC), accounting for 210 million diarrhea episodes annually. When infected with certain V. cholerae strains, people of blood group O phenotype are more prone to develop severe symptoms than people of blood group A, B or AB phenotype.1–4 A similar correlation has been found for ETEC infections, although the epidemiological evidence in this case is contradictory. For reasons not yet understood, one ETEC study shows a clear blood group correlation,5 whereas two others find no such association.1,6 The blood group ABO antigens are fucosylated carbohydrate structures carried on both glycolipids and glycoproteins. They are widely distributed throughout the body and are found also in the small intestine, which is the site of cholera and ETEC infections. In this tissue, blood group antigens are found on the intestinal epithelium as glycoproteins and glycolipids.7–9 In addition, people belonging to
0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
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Blood Group Recognition in ETEC Infections
the secretor phenotype express blood group antigens also in secretions, such as saliva and gastric mucosa.10–12 The shortest ABO blood group antigen, termed H, is characterized by the addition of Fucα2 to the terminal β-galactosyl residue of tissue specific glycan precursors. People with blood group O only carry the H antigen in its unmodified form, while for people of blood group A and B phenotype, the H antigen is further modified by the addition of either GalNAcα3 or Galα3 to generate the A and B antigens, respectively. It is widely accepted that the dense glycan layer lining the respiratory, gastric and urinary tracts serves to protect the body from invasion of foreign pathogens13 and it has been suggested that the human ABO polymorphism has evolved for the same purpose.12,14 For cholera and ETEC infections several attempts have been made to explain the molecular basis of blood group dependence and the role of ABH antigens in disease development. The risk of infection has earlier been related to the primary steps of infection, such as colonization and mucosal adherence.15,16 However, it has later become increasingly clear that only the severity of disease and not the risk of infection is correlated with the observed blood group dependence.1,2,4 This points to the significance of later steps in pathogenesis, such as the elaboration of the major virulence factors, i.e. cholera toxin (CT) and heatlabile enterotoxin (LT). CT and LT are structurally and functionally related and consist of one enzymatically active A-subunit anchored in the center of a homo B-pentamer.17–19 The A-subunit displays ADP-ribosyl transferase activity whereas the B-pentamers, CTB and LTB, anchor the toxin to carbohydrate receptors present on the target cells.19–21 In addition to significant homology in sequence and structure, the two toxins share the primary cellular receptor in the small intestine, i.e. the GM1 ganglioside.21 GM1 is believed to induce CT and LT endocytosis and is found to be critical for targeting the toxins to the intracellular compartment, which is required to elicit a cellular response.22–24 However, at least for LT there seem to be GM1-independent routes of intoxication, since blocking of this receptor does not completely inhibit the cellular response typical for the enzymatic activity of the toxin.25,26 In differentiated HT29 cells, for example, the secretory response caused by LT can only be inhibited by simultaneously blocking both GM1 and blood group A active glycoconjugates, which points to a possible functional role of blood group antigens also in ETEC infections.27 Recombinantly expressed CTB and LTB from human isolates, i.e. hLTB, have indeed been shown to bind blood group antigens, with preference for A and B-epitopes, and it has been hypothesized that these antigens might disturb the action of the toxin by interfering with binding to GM1 in the small intestine.28–31 However, the ability of CTB to recognize these receptors is controversial. Furthermore, the well-conserved GM1 binding site is believed to be ganglioside-specific and cannot accommodate
the fucosylated blood group antigens according to computer modeling studies.32 Consequently, the molecular basis for blood group antigen recognition in CTB and hLTB has remained unclear. In a recent investigation of a CTB/ hLTB toxin hybrid in complex with a blood group A type 2 antigen analog, we identified a potential binding site for blood group ligands, which is clearly distinct from the primary GM1 binding site.33 This hybrid showed an enhanced binding affinity for blood group A and B antigens compared to the parent toxins,32 an affinity that has later been re-created by introducing the CTB residue Asn in LTB at position 4 (Ser in hLTB).33 Here, we have solved the structure of hLTB in complex with the same ligand, confirming the presence of a second binding site in the native toxin. The crystal structure provides an explanation to why the toxin binds to blood group A and B, rather than H antigens and brings further insight into the role of blood group antigens in ETEC and cholera infections.
Results Final crystallographic model Recombinant hLTB in complex with the pentasaccharide GalNAcα3(Fucα2)Galβ4(Fucα3)Glcβ crystallized in space group P21212 and there are two homo-pentamers, i.e. two copies of the biological unit, in the asymmetric unit. Each of the protomers is composed of a five-stranded antiparallel β-sheet as well as two helices, which together form the center of the doughnut-shaped pentamer. Despite the modest resolution, the data are of high quality and the two B-pentamers are characterized by clear electron density. Only a few solvent exposed regions, mainly loop regions 53–60, are poorly ordered. This is also observed in other reported CTB and LTB structures crystallized in the absence of ligands of the primary GM1 receptor site. The blood group binding site, however, is well-defined and the carbohydrate ligand could be unambiguously modeled into all ten crystallographically independent sites (Figure 1), with the exception of subunit I, for which Fucα3 and Glcβ are characterized by weak electron density. The final crystallographic model (R/Rfree = 18.2/23.9; Table 1) contains 1012 amino acid residues, 280 water molecules and ten pentasaccharides. Root-mean-square deviations (r.m.s.d.) from ideal geometry are 0.011 Å and 1.34° for bond lengths and bond angles, respectively (see Table 1). Details of blood group antigen recognition The ligand used in the present study is the pentasaccharide GalNAcα3(Fucα2)Galβ4(Fucα3) Glcβ, which is closely related to the blood group A type 2 pentasaccharide GalNAcα3(Fucα2)Galβ4 (Fucα3)GlcNAcβ; the only difference being the substitution of the GlcNAc group at the reducing
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Blood Group Recognition in ETEC Infections
Figure 1. (a) Surface representation of the hLT B-pentamer. The blood group A antigen analog GalNAcα3(Fucα2) Galβ4(Fucα3)Glcβ is shown in red stick representation. A model of the GM1 pentasaccharide (black) is depicted for comparison. (b) Well-defined electron density (2Fo−Fc map, at 1σ) of the pentasaccharide, at the interface of two B-subunits (in green and blue, respectively) The Figure was generated with PyMOL: DeLano, W.L. [http://www.pymol.org].
end of the blood group determinant by an unmodified Glc residue. The blood group A analog was found to bind to a new binding pocket, which is clearly distinct from the primary GM1 binding site. Whereas GM1 binding occurs at what is considered the bottom of the B-pentamer, i.e. the side facing the target cell upon ligand binding and endocytosis, the blood group antigens bind at the periphery of the ringshaped structure (Figure 1(a)). All ten ligand binding sites of the final model are crystallographically unique and display subtle differences with respect to the topology of the ligand and the amino acid residues constituting the binding pocket. However, the essential interaction partners and most of the contacts are well conserved and have served as a basis for the structural analysis. In subunits D and L, there are some additional contacts caused by the crystal packing, but these are not discussed further.
Similar to the GM1 binding site, the blood group recognition site is located at the interface of two Bsubunits, with one of the two B-subunits providing the majority of the contacts to the ligand (Figure 1). The branched pentasaccharide is positioned almost parallel to the protein surface, with Fucα2 entering a shallow extended depression close to the B-subunit interface. It is primarily the terminal trisaccharide, i.e. GalNAcα3(Fucα2)Galβ4, which is bound by direct contacts to the shallow cavity. The central amino acid residues involved in direct hydrogen bonding are Gly45, Thr47, Asn94 and Gln#3 (the hash indicates that the residue belongs to a neighboring B-subunit) (Figure 2(a)). Three other residues, i.e. Tyr18, Ala46 and Phe48, provide extensive hydrophobic contacts and in this way contribute significantly to binding (Table 2). These three residues, together, form a hydrophobic pocket that accommodates the methyl groups of the two
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Blood Group Recognition in ETEC Infections Table 1. Data collection and refinement statistics Space group Unit cell parameters (Å) Resolution (Å) Completeness (%) I/σ Rmerge (%) Redundancy Number of observed reflections Number of unique reflections Reflections in test set (%) Rcryst (%) Rfree (%) r.m.s.d. bond lengths (Å) r.m.s.d. bond angles (°) B-factors (Å2) Ramachandran profile (%)a
Real-space correlation coefficientb Real-space R-factorb PDB id code
a b c
Protein Ligand Water Most favorable Additionally allowed Generously allowed Disallowed
P21212 117.8 167.0 57.5 96.2–2.5 99.0 (94.2) 24.9 (12.4) 3.3 (8.0) 6.1 (3.7) 233,311 38,366 5 18.2 23.9 0.011 1.34 15.3 26.3 24.5 92.2 7.6 0.2 0.0 0.89 0.11 202L
Values in parentheses correspond to the data in the highest resolution shell (2.5–2.6 Å). a According to the program PROCHECK.60 b From the program CNS.52
fucose residues. Tyr18 further plays an important role for the stabilization of the reducing end of the pentasaccharide, as major coordinator of a water network at this site and through strong contacts with the ring structure of Glcβ. Otherwise, there are only few interactions observed at the reducing end of the pentasaccharide (apart from internal carbohydrate– carbohydrate interactions). Consequently, Glcβ as well as Fucα3 are characterized by higher B-factors and somewhat weaker electron density as compared to the terminal trisaccharide. It should be noted that Glcβ is positioned at a water-filled cavity. In particular its 2-OH group, which is modified by an N-acetyl group in pure blood group structures, is highly solvent-accessible and an N-acetyl group could easily be accomodated in the binding site, with no steric interference. In comparison to the reducing end of the pentasaccharide, the terminal trisaccharide and in particular Fucα2 is characterized by exemplary welldefined electron density. Fucα2 forms, as judged from the short distances to its hydrogen bond donors/acceptors, strong hydrogen bonds to three of the four central amino acid residues, i.e. the backbone of Thr47 and Asn94 and the side chain of Gln#3 (Table 2). In addition, there are significant hydrophobic contacts to Phe48 and, to a lesser extent, to Tyr18 and Ala46. This ligand residue alone contributes to 40% of the solvent accessible surface buried upon ligand binding. The monosaccharide determining the blood group A activity, i.e. GalNAcα3, engages in direct hydrogen bonds to Gly45
and Thr47, while Galβ4 forms two hydrogen bonds to Gly45. In addition to the direct interactions, which are well conserved in all or a significant number of the ten individual binding sites, there are a number of water molecules mediating contacts to the ligand both at the reducing and at the non-reducing end of the sugar (Figure 2(a) and Table 2). The water molecules at the reducing end are highly conserved in most high-resolution LTB structures in the Protein Data Bank (PDB) and can therefore be considered as an extension of the protein surface. These watermediated interactions are also preserved in the crystal structure of a CTB/hLTB hybrid, in complex with the same pentasaccharide, which was solved to a higher resolution (1.9 Å).33 Due to the moderate resolution of the hLTB structure presented here, clear and well-ordered electron density is, however, not observed in all of the subunits and water molecules have only been introduced in some of the ten individual sites (Table 2).
Discussion There is evidence that E. coli heat-labile enterotoxin, a close relative to cholera toxin, can bind to certain blood group ABO determinants,26,27,30,31,34 although the molecular basis for this specificity has remained unknown. This is particularly interesting, since the severity of cholera, and probably also ETEC infections, is correlated with the blood group ABO phenotype of the infected individual. The crystal structure of hLTB in complex with a blood group A antigen analog presented here (PDB id code 2O2L) proves that the toxin can in fact bind blood group antigens, and that these epitopes bind to a previously unknown receptor binding site. Discrimination of blood group antigens Based on earlier binding data, hLT binds both A and B antigens, which differ only in the terminal sugar residue.26,30,31 An analysis of the blood group antigen binding site reveals that the GalNAcα3 residue, which defines the blood group A activity, engages in hydrogen bonds mainly with the hydroxyl group on the 3-position and the nitrogen atom of the acetamido group. The terminal sugar residue of the B-antigen, i.e. Galα3, differs from the A-antigen only at the 2-position where the acetamido group is replaced by a hydroxyl group. This hydroxyl will most likely preserve the hydrogen bonds provided by the acetamido nitrogen, which explains why the toxin does not discriminate notably between the A and B epitopes.26,30,31 In contrast, the toxin does not bind the unmodified H antigen at all, or only with negligible affinity. 26,30,31,34 An earlier mutation study has shown that the loss of one single watermediated hydrogen bond to the terminal GalNAcα3 results in a reduced affinity for blood group A antigens.33 Hence, the H-antigen, which lacks the entire terminal sugar residue and all of its interac-
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Blood Group Recognition in ETEC Infections
Figure 2. (a) Detailed stereo-picture of the blood group antigen binding site. Hydrogen bonds are indicated by dashed lines for interactions with optimal geometry. The water molecules (green spheres) are well conserved also in the toxin hybrid structure33 (PDB id code 2NZG). In this structure, the CTB residue Asn4# is in addition engaged in an important water-mediated hydrogen bond to GalNAcα3 of the blood group A-determinant (water molecule not shown). (b) Stereosuperimposition of hLTB (red) with the CTB/hLTB toxin hybrid (green), CTB with El Tor characteristics (blue; featuring Tyr18) and CTB of classical O1 origin (yellow). The pentasaccharide belongs to the hLTB structure. Nearly all amino acid residues important for binding are shared by hLTB, the CTB/hLTB hybrid, and the El Tor model, including Tyr18, which provides important contacts at the reducing end of the ligand (the Figure was generated with PyMOL).
tions, should have a significantly reduced ability to bind to the proposed binding pocket, if binding occurs at all. This is in agreement with previous binding data.30–32 Comparison of LTB and CTB When comparing the Cα trace of the LTB structure with other LTB and CTB structures in the PDB, the molecules superimpose with low r.m.s. deviations (0.4±0.1 Å). The deviation in the blood group binding pocket is even lower (0.2±0.1 Å) reflecting the high sequence conservation in this region (Figure 2(b)). Still, many reports claim that CT fails to recognize blood group determinants, despite a significant sequence and structure homology with hLTB.31,32,34 There are, however, small differences in the sequence of CT from different V. cholerae strains35 (Figure 3). Most studies have reported the use of toxin originating from the classical biotype of serogroup O1. Cholera outbreaks caused by this biotype show a weaker blood group correlation than those caused by serogroup O1 of the El Tor biotype and the more recently discovered serogroup O139 (Bengal).36,37 The toxins produced by the two latter V. cholerae subtypes are identical in amino acid sequence,38 and sequence and structure analyses suggest that these toxin variants are more likely to
bind blood group A antigens than classical CT O1 (Figures 2(b) and 3). First of all, CTB from the El Tor biotype shares with LTB a tyrosine residue at position 18, which has an important role in maintaining a well-conserved water network anchoring the reducing end of the ligand to the binding pocket (Figure 2(a) and (b)). This water network is well defined in the high-resolution CTB/hLTB hybrid structure in complex with the same pentasaccharide ligand and is also present in most high-resolution LTB structures in the PDB.33 Classical CTB of serogroup O1 usually features a histidine residue at this position (Figure 3) and cannot establish the same type of interactions. This is supported by the observation that high-resolution CTB structures from the classical strains are characterized by a different water shell in this area of the binding site. Tyr18 further provides strong contacts to Glcβ at the reducing end of the blood group ligand, which the shorter histidine side-chain most likely cannot match. The other non-conserved residue that is characteristic for El Tor and O139 V. cholerae (Figure 3), Ile47, has different chemical properties compared to Thr47 of the classical Ogawa and Inaba serotypes. Instead of the hydrogen bonds provided by the threonine residue, the isoleucine side-chain may, however, be able to contribute to binding with a number of strong hydrophobic interactions to
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Blood Group Recognition in ETEC Infections Table 2. Carbohydrate-protein interactions Ligand residue
Direct contacts (Å)
Interaction partner
GalNAc C4
3.1 ± 0.1 (5)a,b 3.1 ± 0.3 (5)a,b 3.2 ± 0.2 (5)b 3.1 ± 0.1 (5)b 3.1 ± 0.2 (10) 3.1 ± 0.2 (10) 3.8 ± 0.2 (10)
Thr47 Oγ1 W1c Thr47 Oγ1 Gly45 O Thr47 Oγ1 Gly45 O Gln#3 Cδ
Gal O3 Gal O4
3.1 ± 0.3 (9)b 2.8 ± 0.3 (10)a
Gly45 O Gly45 O
Fucα2 O2 Fucα2 O3
2.6 ± 0.2 (10) 3.3 ± 0.1 (2)b 3.1 (1) 3.0 ± 0.3 (10) 3.3 ± 0.1 (3)b 2.6 ± 0.2 (10) 3.1 ± 0.2 (10) 3.6 ± 0.1 (10) 3.9 ± 0.2 (10) 3.7 ± 0.2 (10) 3.5 ± 0.2 (10) 3.6 ± 0.2 (10) 3.9 ± 0.1 (5)
Gln#3 Oε1/Nε2 Asn94 N Thr92 Oγ1 Ans94 N Asn94 Oδ1/Nδ2 Thr47 O Thr47 N Asn94 Cγ Tyr18 Cε1/Cε2 Tyr18 Cζ Phe48 Cε1/Cε2 Phe48 Cζ Ala46 Cβ
Glc O2 Glc O6
3.2 ± 0.1 (3)a,b 3.0 ± 0.1 (2)b
Tyr18 Oη W3c
Glc Glc Glc Glc
C1 C2 C3 C5
3.3 ± 0.3 (10) 3.7 ± 0.3 (8)b 3.8 ± 0.3 (9)b 3.9 ± 0.1 (3)b
Tyr18 Oη Tyr18 Oη Tyr18 Oη Tyr18 Oη
Fucα3 C4 Fucα3 C6
3.1 ± 0.1 (3)b 3.8 ± 0.2 (7)b 3.6 ± 0.4 (9)b 3.6 ± 0.3 (9) 3.5 ± 0.2 (9)
Ser44 Oη Gly45 C Ser44 Oγ Ala46 Cα Ala46 Cβ
GalNAc O3 GalNAc O4 GalNAc O7 GalNAc N2
Fucα2 O4 Fucα2 O5 Fucα2 C4 Fucα2 C6
Indirect contacts (Å)
Interaction partner
3.1 ± 0.3 (2)b
Glu#7 Oε1/Oε2
3.2 ± 0.2 (3)b 3.2 ± 0.1 (4)b
Asn89 O Asn94 Oδ1/Nδ2
Listed are hydrogen bonds and van der Waals contacts with distances b 3.4 Å and 4.0 Å, respectively. For water-mediated interactions, only hydrogen bonds (and not van der Waals interactions) are listed, with the following further restrictions: the water molecules are conserved also in the CTB/hLTB hybrid structure and other known LTB structures and the contacts are present in the least 4 of the binding sites (from a total of ten independent sites in the asymmetric unit). (/) Orientation of functional group not known. (#) Residues from neighboring subunit in pentamer. () Number of binding sites, in which the particular interaction is present (). a Unfavorable angle for hydrogen bond (in one or more sites). b Interaction also present in other sites, although with greater distance than the stated cut-off. c Water molecules conserved in the CTB/hLTB toxin hybrid in complex with the same blood group A pentasaccharide (PDB id code 2NGZ).
Fucα2 and the terminal GalNAc N-acetyl group, as deduced from molecular modeling (unpublished data). Apart from the variation between different V. cholerae strains, there are a few notable sequence differences between LTB and CTB. Most importantly, as previously established, the Asn4 residue of CTB (Ser in hLTB) can interact with GalNAcα3 via a water molecule conserved in a significant number of CTB structures and should thus contribute significantly to blood group A antigen binding.33 His94 on the other hand (Asn in LTB) is less likely to affect binding affinity, since the side-chain of histidine should be able to preserve the interaction with the water molecule (W2 in Figure 2(a)) in the water cluster at the reducing end of the ligand (Figure 2). Arg94 of the classical Ogawa serotype may, however, be less compatible with binding blood group antigens.
In summary, as predicted from the sequence of the toxins, we expect variations in binding affinity for different V. cholerae strains. Specifically, we suspect that the toxins belonging to the El Tor biotype and to serogroup O139, which are strongly associated with a blood group dependence of disease outcome, are likely to show enhanced binding to blood group antigens compared to the classical V. cholera O1. The quantitative effect of these substitutions on binding affinity is, however, difficult to judge and this hypothesis remains to be tested. Biological significance of blood group antigen recognition in cholera and ETEC infections The interaction between hLT, presumably also CT, and the blood group A antigen discussed here may help to shed light on the epidemiological relationship pointing to a less severe infection of individuals
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Blood Group Recognition in ETEC Infections
Figure 3. Multiple sequence alignment of hLTB with different CTB strains (residues 1–100). Non-conserved amino acid residues are shown in white. Residues participating in blood group antigen binding are marked with red arrowheads (the Figure was generated with ESPript64).
carrying these blood group epitopes. However, despite the identification and delineation of this previously unknown molecular interaction, the mechanism by which these antigens influence the disease process is largely unknown. The small intestine, which is the site for cholera and ETEC infections, has a rich glycoconjugate flora, and blood group ABH antigens are found on both glycolipids and glycoproteins in the gastric epithelium.7–9 In addition, people of the secretor phenotype carry blood group epitopes on mucins constituting the protective mucosa.10–12 It is thus plausible to believe that people expressing blood group A and/or B epitopes in the target tissue, and in particular in the protective mucus layer,13,39 would be somewhat protected from severe intoxication and experience milder symptoms than individuals lacking these, i.e. people of the blood group O phenotype. However, several issues are still in apparent contradiction to this hypothesis and need to be further evaluated. First of all, recombinant LT binds to blood group antigens with higher affinity than CT, whereas cholera shows significantly stronger blood group dependence than ETEC infections, according to epidemiological data. In this context it is important to take into account that in vitro studies are traditionally performed using recombinantly expressed LT(B), which is not a biologically relevant form of the toxin. In vivo, CT is secreted solubly whereas LT was recently shown to be transported to the host cells in association with vesicles40,41 by binding surface lipopolysaccharides (LPS).42 This difference is not due to CTs inability to bind LPS in general as CT is able to bind to E. coli LPS, but rather due to the different chemical characteristics of ETEC and Vibrio LPS, which in contrast to the former are phosphorylated at the Kdo core.43 LT has the ability to simultaneously bind to LPS and host GM1-receptors,42 but it is still unclear to what extent this holds true for the blood group antigens. Interestingly, it has been proposed that the LPS binding site is located in the same general region as the blood group site in LT.43 If this is indeed the case, this could explain why CT is more susceptible to competition by blood-group
ligands (as inferred from clearer epidemiological evidence), since there is no additional competition with LPS. Another potentially critical issue concerns the possible competition of blood group ligands with GM1. The affinity of the toxins for GM1 is roughly in the nanomolar range44–46 and the affinity for blood group antigens seems to be significantly lower, as judged from qualitative binding studies.33 For this reason and on the basis of the location of the two distinct binding sites, the two receptors are unlikely to compete on equal terms. Blood group antigens are on the other hand more abundant than GM1 in the small intestine and could still slow down the process of GM1-directed endocytosis, especially if the competing epitopes are situated in the protective mucus layer, which represents the first line of defense against pathogens or injury. This hypothesis is in agreement with the observation that the prevalence of nonsecretors among people suffering from severe cholera is significantly higher than expected for the general population.47 Finally, A-antigens in the epithelial cells of the human small intestine were recently suggested to act as functional receptors for LT,27 which is in apparent contradiction to the epidemiological reports suggesting low disease prevalence in blood group A individuals. However, the distribution of functional blood group A receptors in this tissue, i.e. epitopes allowing internalization and activation of the enzymatic toxin A-subunit, is still unknown. Blood group O individuals might still attract a higher toxin dose due to the total lack of binding epitopes competing with GM1. It is also possible that non-secretors of blood group A phenotype (i.e. individuals, who do not express blood group A antigens on the secreted glycoproteins constituting the protecting and constantly shedding mucus layer) are at slightly higher risk of developing severe symptoms. This remains to be tested, since there are no epidemiological studies performed, which document both the blood group phenotype and secretor status of the affected individuals simultaneously.
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Blood Group Recognition in ETEC Infections
Concluding remarks Based on the crystal structure of hLTB in complex with a blood group A antigen analog presented here, the molecular basis for blood group antigen recognition of heat-labile enterotoxin is finally revealed. The structure explains how the toxin can discriminate between different ABH antigens and strengthens the hypothesis that hLT, and possibly CT, are responsible for the blood group dependence observed for cholera and ETEC infections. However, despite the identification and characterization of this previously unknown molecular interaction, the mechanism by which these antigens affect the disease process remains unclear and several issues need further clarification. In particular, there is an obvious need to assess epidemiological investigations with respect to secretor status, since it is currently unknown if the observed correlation between blood group phenotype and severity of disease also holds for nonsecretors. In this context, it is of considerable interest to investigate if blood group antigen binding influences toxin interaction with the primary cellular receptor GM1 and, in the case of hLT, if (as we suspect) blood group antigen binding is conflicted by LPS-binding during vesicle driven toxin transport. Second, the distribution of blood group antigens and in particular functional glycoconjugates in the human small intestinal epithelium should be further investigated in order to decide if blood group antigens can indeed act as functional receptors in this tissue. The above issues clearly need further clarification; however, the present evaluation points to a possible explanation for the previously observed correlation between blood group phenotype and severity of disease. All reviewed data are consistent with the hypothesis that toxin binding is prevented or slowed down by binding to A or B antigens on the protecting mucus layer, while the shorter H antigen of blood group O individuals, which lacks the terminal Gal or GalNAc residue, is unable to perform the same task and, hence, predisposes blood group O individuals to severe disease.
Materials and Methods Expression and purification Protein for crystallization purposes was obtained from M. Lebens and prepared in variation of a protocol described.33 Crystallization The protein, supplied in PBS buffer, was dialyzed against 10 mM Tris–HCl (pH 7.5) and concentrated to a final concentration of 3.5 mg/ml using a VivaSpin concentrator with 10kD cut-off (Viva Science). Several hours prior to the crystallization set-ups, protein and sugar ligand (Sigma-Aldrich, Sweden) were mixed to a
final molar ratio of 1:10 (the blood group A analog GalNAcα3(Fucα2)Galβ4(Fucα3)Glcβ was used rather than the pure blood group A pentasaccharide GalNAcα3 (Fucα2)Galβ4(Fucα3)GlcNAcβ, since only the former compound was available commercially). Crystals were grown at room temperature using the hanging drop vapor diffusion technique. Successful crystal growth was achieved in 18% (w/v) PEG 1000, 0.1–0.2 M sodium acetate, 5–7% (v/v) glycerol and 0.1 M bis-Tris propane (pH 9.0). The crystals belong to space group P21212, with two homo-pentamers in the asymmetric unit and cell axes of a = 117.8 Å, b = 167.0 Å and c = 57.5 Å. The Matthews coefficient is 2.4 Å3/Da, which corresponds to a solvent content of 48%. Data collection and processing Diffraction data to 2.5 Å data were collected at cryogenic temperature (100 K) at BL911-5, MaxII, Lund, Sweden on a MARCCD detector (600 frames, 0.3° oscillation). The data were processed and scaled using XDS and XSCALE.48–50 Scaling statistics are given in Table 1. Structure determination and refinement The structure was solved by molecular replacement using one LTB pentamer as a search model (PDB id code 1DJR). After rigid body minimization, followed by restrained refinement using Refmac5 in the CCP4 program suite51 and simulated annealing using CNS,52 clear electron density was observed for the ligand in all ten copies of the B-subunits. At this stage, the coordinates of the pentasaccharide were introduced into the structure (taken from the previously determined structure of a CTB/ hLTB hybrid; PDB id code 2NZG). Manual rebuilding using the programs O53 and Coot54 and restrained refinement using Refmac555 followed in an iterative fashion. Ten-fold non-crystallographic symmetry restraints were applied in all refinement steps. In later stages, water molecules were introduced using ARP/wARP.56 TLS refinement was performed with Refmac5 using TLS input parameters calculated by TLSMD.57,58 Each of the five individual subunits of the two pentamers was treated as rigid TLS groups in the following refinement steps. The occupancies of the carbohydrate ligands were refined to match the average B-factors of the coordinating protein residues. In a final step, water molecules were removed/ inserted manually using Coot. The structure was verified by composite simulated annealing OMIT maps calculated with CNS52 and with the validation programs SfCheck59 and Procheck60 of the CCP4 program suite. Refinement statistics are given in Table 1. Sequence alignment and structure comparison The ENTREZ protein sequence database interface was used in combination with BLAST61 and ClustalW62 to obtain and align toxin sequences of various V. cholerae and ETEC origins. Accordingly, proteins are stated with accession codes obtained from the ENTREZ entries; P13811, human LTB; 3CHBH, CTB of Classical O1 Ogawa; AAC34728, CTB of Classical O1 Inaba; CAA53976, CTB of Classical O139; P01556, CTB of El Tor O1 Inaba. Further, representative toxin structures with PBD accession numbers 1XTC (used as model for the El Tor toxin, based on identical sequences in the
762 blood group antigen binding site), 1CHQ (classical CTB O1), 2NZG (CTB/hLTB hybrid) and 2O2L (hLTB) were superimposed using the program Superpose of the CCP4 program suite.63 For a detailed analysis of the blood group binding site, only residues 3–4, 7, 16, 18, 45–47, 92 and 94 were used for the superimposition. R.m.s.d. values were calculated using O53 and the details were analyzed using the program Coot.54
Acknowledgements We thank S. Teneberg, J. Ångström, M. Lebens and R. Friemann for valuable discussions; M. Lebens for providing the protein for crystallization and Y. Cerenius for support at the MAXII synchrotron in Lund. This study has been supported by grants from the Glycoconjugates in Biological Systems program from the Swedish National Foundation for Strategic Research (research position of UK) and the Swedish (621-2003-4057) and Norwegian (171631/V40) Research Councils.
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9. Björk, S., Breimer, M. E., Hansson, G. C., Karlsson, K.-A. & Leffler, H. (1987). Structures of blood group glycosphingolipids of human small intestine. A relation between the expression of fucolipids of epithelial cells and the AbO, Le and Se phenotype of the donor. J. Biol. Chem. 262, 6758–6765. 10. D'Adamo, P. J. & Kelly, G. S. (2001). Metabolic and immunologic consequences of ABH secretor and Lewis subtype status. Altern. Med. Rev. 6, 390–405. 11. Henry, S., Oriol, R. & Samuelsson, B. (1995). Lewis histo-blood group system and associated secretory phenotypes. Vox Sang. 69, 166–182. 12. Henry, S. M. (2001). Molecular diversity in the biosynthesis of GI tract glycoconjugates. A blood group related chart of microorganism receptors. Transfus. Clin. Biol. 8, 226–230. 13. Moncada, D. M., Kammanadiminti, S. J. & Chadee, K. (2003). Mucin and Toll-like receptors in host defense against intestinal parasites. Trends Parasitol. 19, 305–311. 14. Seymour, R. M., Allan, M. J., Pomiankowski, A. & Gustafsson, K. (2004). Evolution of the human ABO polymorphism by two complementary selective pressures. Proc. Biol. Sci. 271, 1065–1072. 15. Barua, D. & Paguio, A. S. (1977). ABO blood groups and cholera. Ann. Hum. Biol. 4, 489–492. 16. Chaudhuri, A. & De, S. (1977). Cholera and bloodgroups. Lancet, 2, 404. 17. Zhang, R.-G., Scott, D. L., Westbrook, M. L., Nance, S., Spangler, B. D., Shipley, G. G. & Westbrook, E. M. (1995). The three-dimensional crystal structure of cholera toxin. J. Mol. Biol. 251, 563–573. 18. Sixma, T. K., Pronk, S. E., Kalk, K. H., Wartna, E. S., van Zanten, B. A. M., Witholt, B. & Hol, W. G. J. (1991). Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature, 351, 371–377. 19. Merritt, E. A. & Hol, W. G. J. (1995). AB5 toxins. Curr. Opin. Struct. Biol. 5, 165–171. 20. Cassel, D. & Pfeuffer, T. (1978). Mechanism of cholera toxin action: covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system. Proc. Natl Acad. Sci. USA, 75, 2669–2673. 21. Holmgren, J. (1973). Comparison of the tissue receptors for Vibrio cholerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid. Infect. Immun. 8, 851–859. 22. Wolf, A. A., Jobling, M. G., Wimer-Mackin, S., Ferguson-Maltzman, M., Madara, J. L., Holmes, R. K. & Lencer, W. I. (1998). Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia. J. Cell Biol. 141, 917–927. 23. Badizadegan, K., Wolf, A. A., Rodighiero, C., Jobling, M., Hirst, T. R., Holmes, R. K. & Lencer, W. I. (2000). Floating cholera toxin into epithelial cells: functional association with caveolae-like detergent-insoluble membrane microdomains. Int. J. Med. Microbiol. 290, 403–408. 24. Fujinaga, Y., Wolf, A. A., Rodighiero, C., Wheeler, H., Tsai, B., Allen, L. et al. (2003). Gangliosides that associate with lipid rafts mediate transport of cholera and related toxins from the plasma membrane to endoplasmic reticulm. Mol. Biol. Cell, 14, 4783–4793. 25. Holmgren, J., Fredman, P., Lindblad, M., Svennerholm, A.-M. & Svennerholm, L. (1982). Rabbit intestinal glycoprotein receptor for Escherichia coli heatlabile enterotoxin lacking affinity for cholera toxin. Infect. Immun. 38, 424–433.
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26. Galvan, E. M., Roth, G. A. & Monferran, C. G. (1999). Participation of ABH glycoconjugates in the secretory response to Escherichia coli heat-labile toxin in rabbit intestine. J. Infect. Dis. 180, 419–425. 27. Galvan, E. M., Roth, G. A. & Monferran, C. G. (2006). Functional interaction of Escherichia coli heat-labile enterotoxin with blood group A-active glycoconjugates from differentiated HT29 cells. FEBS J. 273, 3444–3453. 28. Bennun, F. R., Roth, G. A., Monferran, C. G. & Cumar, F. A. (1989). Binding of cholera toxin to pig intestinal mucosa glycosphingolipids: relationship with the AbO blood group system. Infect. Immun. 57, 969–974. 29. Monferran, C. G., Roth, G. A. & Cumar, F. A. (1990). Inhibition of cholera toxin binding to membrane receptors by pig gastric mucin-derived glycopeptides: differential effect depending on the ABO blood group antigenic determinants. Infect. Immun. 58, 3966–3972. 30. Barra, J. L., Monferran, C. G., Balanzino, L. E. & Cumar, F. A. (1992). Escherichia coli heat-labile enterotoxin preferentially interacts with blood group Aactive glycolipids from pig intestinal mucosa and Aand B-active glycolipids from human red cells compared to H-active glycolipids. Mol. Cell. Biochem. 115, 63–70. 31. Balanzino, L. E., Barra, J. L., Galvan, E. M., Roth, G. A. & Monferran, C. G. (1999). Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with glycoconjugates from rabbit intestinal brush border membranes: Relationship with ABH blood group determinants. Mol. Cell. Biochem. 194, 53–62. 32. Ångström, J., Bäckström, M., Berntsson, A., Karlsson, N., Holmgren, J., Karlsson, K.-A. et al. (2000). 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. J. Biol. Chem. 275, 3231–3238. 33. Holmner, Å., Lebens, M., Teneberg, S., Ångström, J., Ökvist, M. & Krengel, U. (2004). Novel binding site identified in a hybrid between cholera toxin and heatlabile enterotoxin: 1.9 Å crystal structure reveals the details. Structure, 12, 1655–1667 (Erratum : Structure (2007). 15, 253.). 34. Balanzino, L. E., Barra, J. L., Monferran, C. G. & Cumar, F. A. (1994). Differential interaction of Escherichia coli heat-labile toxin and cholera toxin with pig intestinal brush border glycoproteins depending on their ABH and related blood group antigenic determinants. Infect. Immun. 62, 1460–1464. 35. Mekalanos, J., Swartz, D. J., Pearson, G. D. N., Harford, N., Groyne, F. & de Wilde, M. (1983). Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature, 306, 551–557. 36. Clemens, J. D., Sack, D. A., Harris, J. R., Chakraborty, J., Khan, M. R., Huda, S. et al. (1989). ABO blood groups and cholera: new observations on specificity of risk and modification of vaccine efficacy. J. Infect. Dis. 159, 770–773. 37. Faruque, A. S. G., Mahalanabis, D., Hoque, S. S. & Albert, M. J. (1994). The Relationship between ABO blood groups and susceptibility to diarrhea due to Vibrio cholerae 0139. Clin. Infect. Dis. 18, 827–828. 38. Lebens, M. & Holmgren, J. (1994). Structure and arrangement of the cholera toxin genes in Vibrio cholerae O139. FEMS Microbiol. Letters, 117, 197–202. 39. Greenwell, P. (1997). Blood group antigens: molecules seeking a function? Glycoconj. J. 14, 159–173. 40. Horstman, A. L. & Kuehn, M. J. (2000). Enterotoxi-
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Edited by I. Wilson (Received 29 November 2006; received in revised form 16 May 2007; accepted 18 May 2007) Available online 31 May 2007
J. Mol. Biol. (2007) 371, 754–764
Blood Group Antigen Recognition by Escherichia coli Heat-labile Enterotoxin Åsa Holmner 1,2 ⁎, Glareh Askarieh 2 , Mats Ökvist 2 and Ute Krengel 1,2 ⁎ 1
Department of Chemistry and Bioscience, Chalmers University of Technology, PO box 462, SE-40530 Göteborg, Sweden 2
Department of Chemistry, University of Oslo, PO box 1033 Blindern, NO-0315 Oslo, Norway
In a number of bacterial infections, such as Helicobacter pylori, Campylobacter jejuni and Vibrio cholerae infections, a correlation between the severity of disease and blood group phenotype of infected individuals has been observed. In the present investigation, we have studied the molecular basis of this effect for enterotoxigenic Escherichia coli (ETEC) infections. ETEC are non-invasive bacteria, which act through second messenger pathways to cause diarrhea. It has been suggested that the major virulence factor of ETEC from human isolates, i.e. the human heat-labile enterotoxin (hLT), recognizes certain blood group epitopes, although the molecular basis of blood group antigen recognition is unknown. The 2.5 Å crystal structure of the receptor-binding B-subunit of hLT in complex with the blood group A antigen analog GalNAcα3(Fucα2)Galβ4(Fucα3)Glcβ provides evidence of a previously unknown binding site in the native toxin. The structure reveals the molecular interactions underlying blood group antigen recognition and suggests how this protein can discriminate between different blood group epitopes. These results support the previously debated role of hLT in the blood group dependence of ETEC infections. Similar observations regarding the closely related cholera toxin in V. cholera infections are also discussed. © 2007 Elsevier Ltd. All rights reserved.
*Corresponding authors
Keywords: blood-group recognition; bacterial toxins; clinical/epidemiological data; protein-carbohydrate interactions; X-ray crystal structure
Introduction Bacterial infections have historically killed more people than any other disease and the annual burden of diarrheal diseases alone is estimated to three million deaths, according to the World Health Present address: Å. Holmner, Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden. Abbreviations used: CCP4, Collaborative Computational Project 4; CT, cholera toxin; CTB, cholera toxin B-pentamer; ETEC, enterotoxigenic Escherichia coli; Fuc, L-fucose; Gal, D-galactose; GalNAc, 2′-N-acetyl galactosamine; Glc, D-glucose; LT, heat-labile enterotoxin; LTB, heat-labile enterotoxin B-pentamer; hLTB, B-pentamer of human isolates of heat-labile enterotoxin; PBS, phosphate-buffered saline; PDB, Protein Data Bank; r.m.s.d., root-mean-square difference/deviation; WHO, world health organization; LPS, lipopolysaccharide(s). E-mail addresses of the corresponding authors: [email protected]; [email protected]
Organization (WHO). Cholera, which is caused by the human pathogen Vibrio cholerae, gives rise to the most severe form of secretory diarrhea, whereas the most frequently isolated enteropathogen is enterotoxigenic Escherichia coli (ETEC), accounting for 210 million diarrhea episodes annually. When infected with certain V. cholerae strains, people of blood group O phenotype are more prone to develop severe symptoms than people of blood group A, B or AB phenotype.1–4 A similar correlation has been found for ETEC infections, although the epidemiological evidence in this case is contradictory. For reasons not yet understood, one ETEC study shows a clear blood group correlation,5 whereas two others find no such association.1,6 The blood group ABO antigens are fucosylated carbohydrate structures carried on both glycolipids and glycoproteins. They are widely distributed throughout the body and are found also in the small intestine, which is the site of cholera and ETEC infections. In this tissue, blood group antigens are found on the intestinal epithelium as glycoproteins and glycolipids.7–9 In addition, people belonging to
0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
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the secretor phenotype express blood group antigens also in secretions, such as saliva and gastric mucosa.10–12 The shortest ABO blood group antigen, termed H, is characterized by the addition of Fucα2 to the terminal β-galactosyl residue of tissue specific glycan precursors. People with blood group O only carry the H antigen in its unmodified form, while for people of blood group A and B phenotype, the H antigen is further modified by the addition of either GalNAcα3 or Galα3 to generate the A and B antigens, respectively. It is widely accepted that the dense glycan layer lining the respiratory, gastric and urinary tracts serves to protect the body from invasion of foreign pathogens13 and it has been suggested that the human ABO polymorphism has evolved for the same purpose.12,14 For cholera and ETEC infections several attempts have been made to explain the molecular basis of blood group dependence and the role of ABH antigens in disease development. The risk of infection has earlier been related to the primary steps of infection, such as colonization and mucosal adherence.15,16 However, it has later become increasingly clear that only the severity of disease and not the risk of infection is correlated with the observed blood group dependence.1,2,4 This points to the significance of later steps in pathogenesis, such as the elaboration of the major virulence factors, i.e. cholera toxin (CT) and heatlabile enterotoxin (LT). CT and LT are structurally and functionally related and consist of one enzymatically active A-subunit anchored in the center of a homo B-pentamer.17–19 The A-subunit displays ADP-ribosyl transferase activity whereas the B-pentamers, CTB and LTB, anchor the toxin to carbohydrate receptors present on the target cells.19–21 In addition to significant homology in sequence and structure, the two toxins share the primary cellular receptor in the small intestine, i.e. the GM1 ganglioside.21 GM1 is believed to induce CT and LT endocytosis and is found to be critical for targeting the toxins to the intracellular compartment, which is required to elicit a cellular response.22–24 However, at least for LT there seem to be GM1-independent routes of intoxication, since blocking of this receptor does not completely inhibit the cellular response typical for the enzymatic activity of the toxin.25,26 In differentiated HT29 cells, for example, the secretory response caused by LT can only be inhibited by simultaneously blocking both GM1 and blood group A active glycoconjugates, which points to a possible functional role of blood group antigens also in ETEC infections.27 Recombinantly expressed CTB and LTB from human isolates, i.e. hLTB, have indeed been shown to bind blood group antigens, with preference for A and B-epitopes, and it has been hypothesized that these antigens might disturb the action of the toxin by interfering with binding to GM1 in the small intestine.28–31 However, the ability of CTB to recognize these receptors is controversial. Furthermore, the well-conserved GM1 binding site is believed to be ganglioside-specific and cannot accommodate
the fucosylated blood group antigens according to computer modeling studies.32 Consequently, the molecular basis for blood group antigen recognition in CTB and hLTB has remained unclear. In a recent investigation of a CTB/ hLTB toxin hybrid in complex with a blood group A type 2 antigen analog, we identified a potential binding site for blood group ligands, which is clearly distinct from the primary GM1 binding site.33 This hybrid showed an enhanced binding affinity for blood group A and B antigens compared to the parent toxins,32 an affinity that has later been re-created by introducing the CTB residue Asn in LTB at position 4 (Ser in hLTB).33 Here, we have solved the structure of hLTB in complex with the same ligand, confirming the presence of a second binding site in the native toxin. The crystal structure provides an explanation to why the toxin binds to blood group A and B, rather than H antigens and brings further insight into the role of blood group antigens in ETEC and cholera infections.
Results Final crystallographic model Recombinant hLTB in complex with the pentasaccharide GalNAcα3(Fucα2)Galβ4(Fucα3)Glcβ crystallized in space group P21212 and there are two homo-pentamers, i.e. two copies of the biological unit, in the asymmetric unit. Each of the protomers is composed of a five-stranded antiparallel β-sheet as well as two helices, which together form the center of the doughnut-shaped pentamer. Despite the modest resolution, the data are of high quality and the two B-pentamers are characterized by clear electron density. Only a few solvent exposed regions, mainly loop regions 53–60, are poorly ordered. This is also observed in other reported CTB and LTB structures crystallized in the absence of ligands of the primary GM1 receptor site. The blood group binding site, however, is well-defined and the carbohydrate ligand could be unambiguously modeled into all ten crystallographically independent sites (Figure 1), with the exception of subunit I, for which Fucα3 and Glcβ are characterized by weak electron density. The final crystallographic model (R/Rfree = 18.2/23.9; Table 1) contains 1012 amino acid residues, 280 water molecules and ten pentasaccharides. Root-mean-square deviations (r.m.s.d.) from ideal geometry are 0.011 Å and 1.34° for bond lengths and bond angles, respectively (see Table 1). Details of blood group antigen recognition The ligand used in the present study is the pentasaccharide GalNAcα3(Fucα2)Galβ4(Fucα3) Glcβ, which is closely related to the blood group A type 2 pentasaccharide GalNAcα3(Fucα2)Galβ4 (Fucα3)GlcNAcβ; the only difference being the substitution of the GlcNAc group at the reducing
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Figure 1. (a) Surface representation of the hLT B-pentamer. The blood group A antigen analog GalNAcα3(Fucα2) Galβ4(Fucα3)Glcβ is shown in red stick representation. A model of the GM1 pentasaccharide (black) is depicted for comparison. (b) Well-defined electron density (2Fo−Fc map, at 1σ) of the pentasaccharide, at the interface of two B-subunits (in green and blue, respectively) The Figure was generated with PyMOL: DeLano, W.L. [http://www.pymol.org].
end of the blood group determinant by an unmodified Glc residue. The blood group A analog was found to bind to a new binding pocket, which is clearly distinct from the primary GM1 binding site. Whereas GM1 binding occurs at what is considered the bottom of the B-pentamer, i.e. the side facing the target cell upon ligand binding and endocytosis, the blood group antigens bind at the periphery of the ringshaped structure (Figure 1(a)). All ten ligand binding sites of the final model are crystallographically unique and display subtle differences with respect to the topology of the ligand and the amino acid residues constituting the binding pocket. However, the essential interaction partners and most of the contacts are well conserved and have served as a basis for the structural analysis. In subunits D and L, there are some additional contacts caused by the crystal packing, but these are not discussed further.
Similar to the GM1 binding site, the blood group recognition site is located at the interface of two Bsubunits, with one of the two B-subunits providing the majority of the contacts to the ligand (Figure 1). The branched pentasaccharide is positioned almost parallel to the protein surface, with Fucα2 entering a shallow extended depression close to the B-subunit interface. It is primarily the terminal trisaccharide, i.e. GalNAcα3(Fucα2)Galβ4, which is bound by direct contacts to the shallow cavity. The central amino acid residues involved in direct hydrogen bonding are Gly45, Thr47, Asn94 and Gln#3 (the hash indicates that the residue belongs to a neighboring B-subunit) (Figure 2(a)). Three other residues, i.e. Tyr18, Ala46 and Phe48, provide extensive hydrophobic contacts and in this way contribute significantly to binding (Table 2). These three residues, together, form a hydrophobic pocket that accommodates the methyl groups of the two
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Blood Group Recognition in ETEC Infections Table 1. Data collection and refinement statistics Space group Unit cell parameters (Å) Resolution (Å) Completeness (%) I/σ Rmerge (%) Redundancy Number of observed reflections Number of unique reflections Reflections in test set (%) Rcryst (%) Rfree (%) r.m.s.d. bond lengths (Å) r.m.s.d. bond angles (°) B-factors (Å2) Ramachandran profile (%)a
Real-space correlation coefficientb Real-space R-factorb PDB id code
a b c
Protein Ligand Water Most favorable Additionally allowed Generously allowed Disallowed
P21212 117.8 167.0 57.5 96.2–2.5 99.0 (94.2) 24.9 (12.4) 3.3 (8.0) 6.1 (3.7) 233,311 38,366 5 18.2 23.9 0.011 1.34 15.3 26.3 24.5 92.2 7.6 0.2 0.0 0.89 0.11 202L
Values in parentheses correspond to the data in the highest resolution shell (2.5–2.6 Å). a According to the program PROCHECK.60 b From the program CNS.52
fucose residues. Tyr18 further plays an important role for the stabilization of the reducing end of the pentasaccharide, as major coordinator of a water network at this site and through strong contacts with the ring structure of Glcβ. Otherwise, there are only few interactions observed at the reducing end of the pentasaccharide (apart from internal carbohydrate– carbohydrate interactions). Consequently, Glcβ as well as Fucα3 are characterized by higher B-factors and somewhat weaker electron density as compared to the terminal trisaccharide. It should be noted that Glcβ is positioned at a water-filled cavity. In particular its 2-OH group, which is modified by an N-acetyl group in pure blood group structures, is highly solvent-accessible and an N-acetyl group could easily be accomodated in the binding site, with no steric interference. In comparison to the reducing end of the pentasaccharide, the terminal trisaccharide and in particular Fucα2 is characterized by exemplary welldefined electron density. Fucα2 forms, as judged from the short distances to its hydrogen bond donors/acceptors, strong hydrogen bonds to three of the four central amino acid residues, i.e. the backbone of Thr47 and Asn94 and the side chain of Gln#3 (Table 2). In addition, there are significant hydrophobic contacts to Phe48 and, to a lesser extent, to Tyr18 and Ala46. This ligand residue alone contributes to 40% of the solvent accessible surface buried upon ligand binding. The monosaccharide determining the blood group A activity, i.e. GalNAcα3, engages in direct hydrogen bonds to Gly45
and Thr47, while Galβ4 forms two hydrogen bonds to Gly45. In addition to the direct interactions, which are well conserved in all or a significant number of the ten individual binding sites, there are a number of water molecules mediating contacts to the ligand both at the reducing and at the non-reducing end of the sugar (Figure 2(a) and Table 2). The water molecules at the reducing end are highly conserved in most high-resolution LTB structures in the Protein Data Bank (PDB) and can therefore be considered as an extension of the protein surface. These watermediated interactions are also preserved in the crystal structure of a CTB/hLTB hybrid, in complex with the same pentasaccharide, which was solved to a higher resolution (1.9 Å).33 Due to the moderate resolution of the hLTB structure presented here, clear and well-ordered electron density is, however, not observed in all of the subunits and water molecules have only been introduced in some of the ten individual sites (Table 2).
Discussion There is evidence that E. coli heat-labile enterotoxin, a close relative to cholera toxin, can bind to certain blood group ABO determinants,26,27,30,31,34 although the molecular basis for this specificity has remained unknown. This is particularly interesting, since the severity of cholera, and probably also ETEC infections, is correlated with the blood group ABO phenotype of the infected individual. The crystal structure of hLTB in complex with a blood group A antigen analog presented here (PDB id code 2O2L) proves that the toxin can in fact bind blood group antigens, and that these epitopes bind to a previously unknown receptor binding site. Discrimination of blood group antigens Based on earlier binding data, hLT binds both A and B antigens, which differ only in the terminal sugar residue.26,30,31 An analysis of the blood group antigen binding site reveals that the GalNAcα3 residue, which defines the blood group A activity, engages in hydrogen bonds mainly with the hydroxyl group on the 3-position and the nitrogen atom of the acetamido group. The terminal sugar residue of the B-antigen, i.e. Galα3, differs from the A-antigen only at the 2-position where the acetamido group is replaced by a hydroxyl group. This hydroxyl will most likely preserve the hydrogen bonds provided by the acetamido nitrogen, which explains why the toxin does not discriminate notably between the A and B epitopes.26,30,31 In contrast, the toxin does not bind the unmodified H antigen at all, or only with negligible affinity. 26,30,31,34 An earlier mutation study has shown that the loss of one single watermediated hydrogen bond to the terminal GalNAcα3 results in a reduced affinity for blood group A antigens.33 Hence, the H-antigen, which lacks the entire terminal sugar residue and all of its interac-
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Blood Group Recognition in ETEC Infections
Figure 2. (a) Detailed stereo-picture of the blood group antigen binding site. Hydrogen bonds are indicated by dashed lines for interactions with optimal geometry. The water molecules (green spheres) are well conserved also in the toxin hybrid structure33 (PDB id code 2NZG). In this structure, the CTB residue Asn4# is in addition engaged in an important water-mediated hydrogen bond to GalNAcα3 of the blood group A-determinant (water molecule not shown). (b) Stereosuperimposition of hLTB (red) with the CTB/hLTB toxin hybrid (green), CTB with El Tor characteristics (blue; featuring Tyr18) and CTB of classical O1 origin (yellow). The pentasaccharide belongs to the hLTB structure. Nearly all amino acid residues important for binding are shared by hLTB, the CTB/hLTB hybrid, and the El Tor model, including Tyr18, which provides important contacts at the reducing end of the ligand (the Figure was generated with PyMOL).
tions, should have a significantly reduced ability to bind to the proposed binding pocket, if binding occurs at all. This is in agreement with previous binding data.30–32 Comparison of LTB and CTB When comparing the Cα trace of the LTB structure with other LTB and CTB structures in the PDB, the molecules superimpose with low r.m.s. deviations (0.4±0.1 Å). The deviation in the blood group binding pocket is even lower (0.2±0.1 Å) reflecting the high sequence conservation in this region (Figure 2(b)). Still, many reports claim that CT fails to recognize blood group determinants, despite a significant sequence and structure homology with hLTB.31,32,34 There are, however, small differences in the sequence of CT from different V. cholerae strains35 (Figure 3). Most studies have reported the use of toxin originating from the classical biotype of serogroup O1. Cholera outbreaks caused by this biotype show a weaker blood group correlation than those caused by serogroup O1 of the El Tor biotype and the more recently discovered serogroup O139 (Bengal).36,37 The toxins produced by the two latter V. cholerae subtypes are identical in amino acid sequence,38 and sequence and structure analyses suggest that these toxin variants are more likely to
bind blood group A antigens than classical CT O1 (Figures 2(b) and 3). First of all, CTB from the El Tor biotype shares with LTB a tyrosine residue at position 18, which has an important role in maintaining a well-conserved water network anchoring the reducing end of the ligand to the binding pocket (Figure 2(a) and (b)). This water network is well defined in the high-resolution CTB/hLTB hybrid structure in complex with the same pentasaccharide ligand and is also present in most high-resolution LTB structures in the PDB.33 Classical CTB of serogroup O1 usually features a histidine residue at this position (Figure 3) and cannot establish the same type of interactions. This is supported by the observation that high-resolution CTB structures from the classical strains are characterized by a different water shell in this area of the binding site. Tyr18 further provides strong contacts to Glcβ at the reducing end of the blood group ligand, which the shorter histidine side-chain most likely cannot match. The other non-conserved residue that is characteristic for El Tor and O139 V. cholerae (Figure 3), Ile47, has different chemical properties compared to Thr47 of the classical Ogawa and Inaba serotypes. Instead of the hydrogen bonds provided by the threonine residue, the isoleucine side-chain may, however, be able to contribute to binding with a number of strong hydrophobic interactions to
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Blood Group Recognition in ETEC Infections Table 2. Carbohydrate-protein interactions Ligand residue
Direct contacts (Å)
Interaction partner
GalNAc C4
3.1 ± 0.1 (5)a,b 3.1 ± 0.3 (5)a,b 3.2 ± 0.2 (5)b 3.1 ± 0.1 (5)b 3.1 ± 0.2 (10) 3.1 ± 0.2 (10) 3.8 ± 0.2 (10)
Thr47 Oγ1 W1c Thr47 Oγ1 Gly45 O Thr47 Oγ1 Gly45 O Gln#3 Cδ
Gal O3 Gal O4
3.1 ± 0.3 (9)b 2.8 ± 0.3 (10)a
Gly45 O Gly45 O
Fucα2 O2 Fucα2 O3
2.6 ± 0.2 (10) 3.3 ± 0.1 (2)b 3.1 (1) 3.0 ± 0.3 (10) 3.3 ± 0.1 (3)b 2.6 ± 0.2 (10) 3.1 ± 0.2 (10) 3.6 ± 0.1 (10) 3.9 ± 0.2 (10) 3.7 ± 0.2 (10) 3.5 ± 0.2 (10) 3.6 ± 0.2 (10) 3.9 ± 0.1 (5)
Gln#3 Oε1/Nε2 Asn94 N Thr92 Oγ1 Ans94 N Asn94 Oδ1/Nδ2 Thr47 O Thr47 N Asn94 Cγ Tyr18 Cε1/Cε2 Tyr18 Cζ Phe48 Cε1/Cε2 Phe48 Cζ Ala46 Cβ
Glc O2 Glc O6
3.2 ± 0.1 (3)a,b 3.0 ± 0.1 (2)b
Tyr18 Oη W3c
Glc Glc Glc Glc
C1 C2 C3 C5
3.3 ± 0.3 (10) 3.7 ± 0.3 (8)b 3.8 ± 0.3 (9)b 3.9 ± 0.1 (3)b
Tyr18 Oη Tyr18 Oη Tyr18 Oη Tyr18 Oη
Fucα3 C4 Fucα3 C6
3.1 ± 0.1 (3)b 3.8 ± 0.2 (7)b 3.6 ± 0.4 (9)b 3.6 ± 0.3 (9) 3.5 ± 0.2 (9)
Ser44 Oη Gly45 C Ser44 Oγ Ala46 Cα Ala46 Cβ
GalNAc O3 GalNAc O4 GalNAc O7 GalNAc N2
Fucα2 O4 Fucα2 O5 Fucα2 C4 Fucα2 C6
Indirect contacts (Å)
Interaction partner
3.1 ± 0.3 (2)b
Glu#7 Oε1/Oε2
3.2 ± 0.2 (3)b 3.2 ± 0.1 (4)b
Asn89 O Asn94 Oδ1/Nδ2
Listed are hydrogen bonds and van der Waals contacts with distances b 3.4 Å and 4.0 Å, respectively. For water-mediated interactions, only hydrogen bonds (and not van der Waals interactions) are listed, with the following further restrictions: the water molecules are conserved also in the CTB/hLTB hybrid structure and other known LTB structures and the contacts are present in the least 4 of the binding sites (from a total of ten independent sites in the asymmetric unit). (/) Orientation of functional group not known. (#) Residues from neighboring subunit in pentamer. () Number of binding sites, in which the particular interaction is present (). a Unfavorable angle for hydrogen bond (in one or more sites). b Interaction also present in other sites, although with greater distance than the stated cut-off. c Water molecules conserved in the CTB/hLTB toxin hybrid in complex with the same blood group A pentasaccharide (PDB id code 2NGZ).
Fucα2 and the terminal GalNAc N-acetyl group, as deduced from molecular modeling (unpublished data). Apart from the variation between different V. cholerae strains, there are a few notable sequence differences between LTB and CTB. Most importantly, as previously established, the Asn4 residue of CTB (Ser in hLTB) can interact with GalNAcα3 via a water molecule conserved in a significant number of CTB structures and should thus contribute significantly to blood group A antigen binding.33 His94 on the other hand (Asn in LTB) is less likely to affect binding affinity, since the side-chain of histidine should be able to preserve the interaction with the water molecule (W2 in Figure 2(a)) in the water cluster at the reducing end of the ligand (Figure 2). Arg94 of the classical Ogawa serotype may, however, be less compatible with binding blood group antigens.
In summary, as predicted from the sequence of the toxins, we expect variations in binding affinity for different V. cholerae strains. Specifically, we suspect that the toxins belonging to the El Tor biotype and to serogroup O139, which are strongly associated with a blood group dependence of disease outcome, are likely to show enhanced binding to blood group antigens compared to the classical V. cholera O1. The quantitative effect of these substitutions on binding affinity is, however, difficult to judge and this hypothesis remains to be tested. Biological significance of blood group antigen recognition in cholera and ETEC infections The interaction between hLT, presumably also CT, and the blood group A antigen discussed here may help to shed light on the epidemiological relationship pointing to a less severe infection of individuals
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Blood Group Recognition in ETEC Infections
Figure 3. Multiple sequence alignment of hLTB with different CTB strains (residues 1–100). Non-conserved amino acid residues are shown in white. Residues participating in blood group antigen binding are marked with red arrowheads (the Figure was generated with ESPript64).
carrying these blood group epitopes. However, despite the identification and delineation of this previously unknown molecular interaction, the mechanism by which these antigens influence the disease process is largely unknown. The small intestine, which is the site for cholera and ETEC infections, has a rich glycoconjugate flora, and blood group ABH antigens are found on both glycolipids and glycoproteins in the gastric epithelium.7–9 In addition, people of the secretor phenotype carry blood group epitopes on mucins constituting the protective mucosa.10–12 It is thus plausible to believe that people expressing blood group A and/or B epitopes in the target tissue, and in particular in the protective mucus layer,13,39 would be somewhat protected from severe intoxication and experience milder symptoms than individuals lacking these, i.e. people of the blood group O phenotype. However, several issues are still in apparent contradiction to this hypothesis and need to be further evaluated. First of all, recombinant LT binds to blood group antigens with higher affinity than CT, whereas cholera shows significantly stronger blood group dependence than ETEC infections, according to epidemiological data. In this context it is important to take into account that in vitro studies are traditionally performed using recombinantly expressed LT(B), which is not a biologically relevant form of the toxin. In vivo, CT is secreted solubly whereas LT was recently shown to be transported to the host cells in association with vesicles40,41 by binding surface lipopolysaccharides (LPS).42 This difference is not due to CTs inability to bind LPS in general as CT is able to bind to E. coli LPS, but rather due to the different chemical characteristics of ETEC and Vibrio LPS, which in contrast to the former are phosphorylated at the Kdo core.43 LT has the ability to simultaneously bind to LPS and host GM1-receptors,42 but it is still unclear to what extent this holds true for the blood group antigens. Interestingly, it has been proposed that the LPS binding site is located in the same general region as the blood group site in LT.43 If this is indeed the case, this could explain why CT is more susceptible to competition by blood-group
ligands (as inferred from clearer epidemiological evidence), since there is no additional competition with LPS. Another potentially critical issue concerns the possible competition of blood group ligands with GM1. The affinity of the toxins for GM1 is roughly in the nanomolar range44–46 and the affinity for blood group antigens seems to be significantly lower, as judged from qualitative binding studies.33 For this reason and on the basis of the location of the two distinct binding sites, the two receptors are unlikely to compete on equal terms. Blood group antigens are on the other hand more abundant than GM1 in the small intestine and could still slow down the process of GM1-directed endocytosis, especially if the competing epitopes are situated in the protective mucus layer, which represents the first line of defense against pathogens or injury. This hypothesis is in agreement with the observation that the prevalence of nonsecretors among people suffering from severe cholera is significantly higher than expected for the general population.47 Finally, A-antigens in the epithelial cells of the human small intestine were recently suggested to act as functional receptors for LT,27 which is in apparent contradiction to the epidemiological reports suggesting low disease prevalence in blood group A individuals. However, the distribution of functional blood group A receptors in this tissue, i.e. epitopes allowing internalization and activation of the enzymatic toxin A-subunit, is still unknown. Blood group O individuals might still attract a higher toxin dose due to the total lack of binding epitopes competing with GM1. It is also possible that non-secretors of blood group A phenotype (i.e. individuals, who do not express blood group A antigens on the secreted glycoproteins constituting the protecting and constantly shedding mucus layer) are at slightly higher risk of developing severe symptoms. This remains to be tested, since there are no epidemiological studies performed, which document both the blood group phenotype and secretor status of the affected individuals simultaneously.
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Blood Group Recognition in ETEC Infections
Concluding remarks Based on the crystal structure of hLTB in complex with a blood group A antigen analog presented here, the molecular basis for blood group antigen recognition of heat-labile enterotoxin is finally revealed. The structure explains how the toxin can discriminate between different ABH antigens and strengthens the hypothesis that hLT, and possibly CT, are responsible for the blood group dependence observed for cholera and ETEC infections. However, despite the identification and characterization of this previously unknown molecular interaction, the mechanism by which these antigens affect the disease process remains unclear and several issues need further clarification. In particular, there is an obvious need to assess epidemiological investigations with respect to secretor status, since it is currently unknown if the observed correlation between blood group phenotype and severity of disease also holds for nonsecretors. In this context, it is of considerable interest to investigate if blood group antigen binding influences toxin interaction with the primary cellular receptor GM1 and, in the case of hLT, if (as we suspect) blood group antigen binding is conflicted by LPS-binding during vesicle driven toxin transport. Second, the distribution of blood group antigens and in particular functional glycoconjugates in the human small intestinal epithelium should be further investigated in order to decide if blood group antigens can indeed act as functional receptors in this tissue. The above issues clearly need further clarification; however, the present evaluation points to a possible explanation for the previously observed correlation between blood group phenotype and severity of disease. All reviewed data are consistent with the hypothesis that toxin binding is prevented or slowed down by binding to A or B antigens on the protecting mucus layer, while the shorter H antigen of blood group O individuals, which lacks the terminal Gal or GalNAc residue, is unable to perform the same task and, hence, predisposes blood group O individuals to severe disease.
Materials and Methods Expression and purification Protein for crystallization purposes was obtained from M. Lebens and prepared in variation of a protocol described.33 Crystallization The protein, supplied in PBS buffer, was dialyzed against 10 mM Tris–HCl (pH 7.5) and concentrated to a final concentration of 3.5 mg/ml using a VivaSpin concentrator with 10kD cut-off (Viva Science). Several hours prior to the crystallization set-ups, protein and sugar ligand (Sigma-Aldrich, Sweden) were mixed to a
final molar ratio of 1:10 (the blood group A analog GalNAcα3(Fucα2)Galβ4(Fucα3)Glcβ was used rather than the pure blood group A pentasaccharide GalNAcα3 (Fucα2)Galβ4(Fucα3)GlcNAcβ, since only the former compound was available commercially). Crystals were grown at room temperature using the hanging drop vapor diffusion technique. Successful crystal growth was achieved in 18% (w/v) PEG 1000, 0.1–0.2 M sodium acetate, 5–7% (v/v) glycerol and 0.1 M bis-Tris propane (pH 9.0). The crystals belong to space group P21212, with two homo-pentamers in the asymmetric unit and cell axes of a = 117.8 Å, b = 167.0 Å and c = 57.5 Å. The Matthews coefficient is 2.4 Å3/Da, which corresponds to a solvent content of 48%. Data collection and processing Diffraction data to 2.5 Å data were collected at cryogenic temperature (100 K) at BL911-5, MaxII, Lund, Sweden on a MARCCD detector (600 frames, 0.3° oscillation). The data were processed and scaled using XDS and XSCALE.48–50 Scaling statistics are given in Table 1. Structure determination and refinement The structure was solved by molecular replacement using one LTB pentamer as a search model (PDB id code 1DJR). After rigid body minimization, followed by restrained refinement using Refmac5 in the CCP4 program suite51 and simulated annealing using CNS,52 clear electron density was observed for the ligand in all ten copies of the B-subunits. At this stage, the coordinates of the pentasaccharide were introduced into the structure (taken from the previously determined structure of a CTB/ hLTB hybrid; PDB id code 2NZG). Manual rebuilding using the programs O53 and Coot54 and restrained refinement using Refmac555 followed in an iterative fashion. Ten-fold non-crystallographic symmetry restraints were applied in all refinement steps. In later stages, water molecules were introduced using ARP/wARP.56 TLS refinement was performed with Refmac5 using TLS input parameters calculated by TLSMD.57,58 Each of the five individual subunits of the two pentamers was treated as rigid TLS groups in the following refinement steps. The occupancies of the carbohydrate ligands were refined to match the average B-factors of the coordinating protein residues. In a final step, water molecules were removed/ inserted manually using Coot. The structure was verified by composite simulated annealing OMIT maps calculated with CNS52 and with the validation programs SfCheck59 and Procheck60 of the CCP4 program suite. Refinement statistics are given in Table 1. Sequence alignment and structure comparison The ENTREZ protein sequence database interface was used in combination with BLAST61 and ClustalW62 to obtain and align toxin sequences of various V. cholerae and ETEC origins. Accordingly, proteins are stated with accession codes obtained from the ENTREZ entries; P13811, human LTB; 3CHBH, CTB of Classical O1 Ogawa; AAC34728, CTB of Classical O1 Inaba; CAA53976, CTB of Classical O139; P01556, CTB of El Tor O1 Inaba. Further, representative toxin structures with PBD accession numbers 1XTC (used as model for the El Tor toxin, based on identical sequences in the
762 blood group antigen binding site), 1CHQ (classical CTB O1), 2NZG (CTB/hLTB hybrid) and 2O2L (hLTB) were superimposed using the program Superpose of the CCP4 program suite.63 For a detailed analysis of the blood group binding site, only residues 3–4, 7, 16, 18, 45–47, 92 and 94 were used for the superimposition. R.m.s.d. values were calculated using O53 and the details were analyzed using the program Coot.54
Acknowledgements We thank S. Teneberg, J. Ångström, M. Lebens and R. Friemann for valuable discussions; M. Lebens for providing the protein for crystallization and Y. Cerenius for support at the MAXII synchrotron in Lund. This study has been supported by grants from the Glycoconjugates in Biological Systems program from the Swedish National Foundation for Strategic Research (research position of UK) and the Swedish (621-2003-4057) and Norwegian (171631/V40) Research Councils.
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9. Björk, S., Breimer, M. E., Hansson, G. C., Karlsson, K.-A. & Leffler, H. (1987). Structures of blood group glycosphingolipids of human small intestine. A relation between the expression of fucolipids of epithelial cells and the AbO, Le and Se phenotype of the donor. J. Biol. Chem. 262, 6758–6765. 10. D'Adamo, P. J. & Kelly, G. S. (2001). Metabolic and immunologic consequences of ABH secretor and Lewis subtype status. Altern. Med. Rev. 6, 390–405. 11. Henry, S., Oriol, R. & Samuelsson, B. (1995). Lewis histo-blood group system and associated secretory phenotypes. Vox Sang. 69, 166–182. 12. Henry, S. M. (2001). Molecular diversity in the biosynthesis of GI tract glycoconjugates. A blood group related chart of microorganism receptors. Transfus. Clin. Biol. 8, 226–230. 13. Moncada, D. M., Kammanadiminti, S. J. & Chadee, K. (2003). Mucin and Toll-like receptors in host defense against intestinal parasites. Trends Parasitol. 19, 305–311. 14. Seymour, R. M., Allan, M. J., Pomiankowski, A. & Gustafsson, K. (2004). Evolution of the human ABO polymorphism by two complementary selective pressures. Proc. Biol. Sci. 271, 1065–1072. 15. Barua, D. & Paguio, A. S. (1977). ABO blood groups and cholera. Ann. Hum. Biol. 4, 489–492. 16. Chaudhuri, A. & De, S. (1977). Cholera and bloodgroups. Lancet, 2, 404. 17. Zhang, R.-G., Scott, D. L., Westbrook, M. L., Nance, S., Spangler, B. D., Shipley, G. G. & Westbrook, E. M. (1995). The three-dimensional crystal structure of cholera toxin. J. Mol. Biol. 251, 563–573. 18. Sixma, T. K., Pronk, S. E., Kalk, K. H., Wartna, E. S., van Zanten, B. A. M., Witholt, B. & Hol, W. G. J. (1991). Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature, 351, 371–377. 19. Merritt, E. A. & Hol, W. G. J. (1995). AB5 toxins. Curr. Opin. Struct. Biol. 5, 165–171. 20. Cassel, D. & Pfeuffer, T. (1978). Mechanism of cholera toxin action: covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system. Proc. Natl Acad. Sci. USA, 75, 2669–2673. 21. Holmgren, J. (1973). Comparison of the tissue receptors for Vibrio cholerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid. Infect. Immun. 8, 851–859. 22. Wolf, A. A., Jobling, M. G., Wimer-Mackin, S., Ferguson-Maltzman, M., Madara, J. L., Holmes, R. K. & Lencer, W. I. (1998). Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia. J. Cell Biol. 141, 917–927. 23. Badizadegan, K., Wolf, A. A., Rodighiero, C., Jobling, M., Hirst, T. R., Holmes, R. K. & Lencer, W. I. (2000). Floating cholera toxin into epithelial cells: functional association with caveolae-like detergent-insoluble membrane microdomains. Int. J. Med. Microbiol. 290, 403–408. 24. Fujinaga, Y., Wolf, A. A., Rodighiero, C., Wheeler, H., Tsai, B., Allen, L. et al. (2003). Gangliosides that associate with lipid rafts mediate transport of cholera and related toxins from the plasma membrane to endoplasmic reticulm. Mol. Biol. Cell, 14, 4783–4793. 25. Holmgren, J., Fredman, P., Lindblad, M., Svennerholm, A.-M. & Svennerholm, L. (1982). Rabbit intestinal glycoprotein receptor for Escherichia coli heatlabile enterotoxin lacking affinity for cholera toxin. Infect. Immun. 38, 424–433.
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Edited by I. Wilson (Received 29 November 2006; received in revised form 16 May 2007; accepted 18 May 2007) Available online 31 May 2007