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J Biol Chem, Vol. 275, Issue 5, 3231-3238, February 4, 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^*

Jonas Ångström, Malin Bäckström^§, Anna Berntsson, Niclas Karlsson, Jan Holmgren^§, Karl-Anders Karlsson, Michael Lebens^§, and Susann Teneberg^¶

From the  Institute of Medical Biochemistry, Göteborg University, P. O. Box 440, SE 405 30 Göteborg, Sweden, and the ^§ Department of Medical Microbiology and Immunology, Göteborg University, Guldhedsgatan 10, SE 413 46 Göteborg, Sweden

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) are structurally and functionally related. However, the carbohydrate binding specificities of the two proteins differ. While both CTB and LTB bind to the GM1 ganglioside, LTB also binds to N-acetyllactosamine-terminated glycoconjugates. The structural basis of the differences in carbohydrate recognition has been investigated by a systematic exchange of amino acids between LTB and CTB. Thereby, a CTB/LTB hybrid with a gain-of-function mutation resulting in recognition of blood group A and B determinants was obtained. Glycosphingolipid binding assays showed a specific binding of this hybrid B-subunit, but not CTB or LTB, to slowly migrating non-acid glycosphingolipids of human and animal small intestinal epithelium. A binding-active glycosphingolipid isolated from cat intestinal epithelium was characterized by mass spectrometry and proton NMR as GalNAc3(Fuc2)Gal4(Fuc3)GlcNAc3Gal4Glc NAc3Gal4Glc1Cer. Comparison with reference glycosphingolipids showed that the minimum binding epitope recognized by the CTB/LTB hybrid was Gal3(Fuc2)Gal4(Fuc3)GlcNAc. The blood group A and B determinants bind to a novel carbohydrate binding site located at the top of the B-subunit interfaces, distinct from the GM1 binding site, as found by docking and molecular dynamics simulations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Enterotoxins produced by Vibrio cholerae and enterotoxigenic Escherichia coli are causative agents of diarrheal diseases leading to millions of deaths annually (1). Both cholera toxin (CT)¹ and E. coli type I heat-labile enterotoxin (LT) are oligomeric proteins with one A-subunit and five B-subunits (2). The A-subunits have ADP-ribosyltransferase activity, while the B-subunits mediate binding to receptors on the eukaryotic cell surface. LT type I B-subunits originating from porcine and human isolates of enterotoxigenic E. coli (pLTB and hLTB, respectively) share 96% sequence identity with each other, but only approximately 80% with CTB (3).

Despite great similarity between the carbohydrate binding sites of the B-subunits, evidenced by recent crystal complexes (4-6), the carbohydrate binding specificities of CTB and LTB differ. Both B-subunits bind with high affinity to GM1 (7), whereas only LTB interacts with N-acetyllactosamine-terminated glycoconjugates (8-10). Binding of LTB, but not CTB, to gangliotetraosylceramide and the GD1b ganglioside has also been reported (8, 10, 11). The structural basis of the differences in carbohydrate binding between CTB and hLTB have been investigated by construction of a number of CTB/hLTB hybrids, having CTB amino acids substituted with heterologous amino acids of hLTB (12). By introducing hLTB residues in the 1-25 region and at positions 94 and 95 of CTB, a hybrid B-subunit (designated LCTBH) was created, with N-acetyllactosamine-binding properties almost indistinguishable from hLTB.

These studies have now been extended by re-substitution of single amino acids of LCTBH back to the original CTB residues. By re-substituting from Ser⁴ to Asn, a daughter hybrid with reduced N-acetyllactosamine-binding capacity was obtained. However, this daughter hybrid had a novel carbohydrate binding specificity, as described in the present paper.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmids and DNA Manipulations-- All recombinant B-subunits were produced from plasmids derived from pML-CTBtac, essentially as described (13). pML-LCTBtacK was obtained by introduction of synthetic oligonucleotides between the unique SacI and PstI sites in pML-LCTBtacH. The mutated plasmid was electroporated into the classical 01 V. cholerae strain JS1569 (DctxA). The structure of the hybrid gene was confirmed by DNA sequencing using Sequenase 2.0 from USB (Amersham Pharmacia Biotech, United Kingdom). Oligonucleotides were from KEBO Lab, Spånga, Sweden, and restriction enzymes were from Roche Molecular Biochemicals or New England Biolabs, and used according to the manufacturer's instructions.

Production, Purification, and Characterization of B-subunits-- Recombinant B-subunits were produced and purified as described (12). Purified B-subunits were analyzed by SDS-polyacrylamide gel electrophoresis. Protein concentrations were determined using Bradford's protein assay (14) (Bio-Rad) with bovine serum albumin as standard. Sequential Edman degradation was performed on a Procise 492 protein sequencer (Perkin Elmer).

Glycosphingolipid Binding Assays-- Glycosphingolipids were isolated and characterized by mass spectrometry, ¹H NMR, and degradation studies, as outlined in (8, 10).

Mixtures of glycosphingolipids (20-40 µg/lane) or pure compounds (0.1-4 µg/lane) were separated on aluminum-backed silica gel 60 high-performance thin-layer chromatography plates (Merck), using chloroform/methanol/water (60:35:8, by volume) as solvent. Chemical detection was done with anisaldehyde (15).

Binding of B-subunits to glycosphingolipids on thin-layer chromatograms or adsorbed in microtiter wells were performed as described (8, 10), using ¹²⁵I-labeled B-subunits diluted in phosphate-buffered saline, pH 7.2, containing 2% (w/v) bovine serum albumin and 0.1% (w/v) NaN_3, to approximately 5 × 10⁶ cpm/ml.

Chromatogram binding assays with monoclonal antibodies directed against blood group A, B, and H determinants (Dakopatts a/s, Glostrup, Denmark) were done as described (16) using ¹²⁵I-labeled anti-mouse antibodies for detection.

Isolation of an LCTBK-binding Non-acid Glycosphingolipid from Epithelial Cells of Cat Small Intestine-- A total non-acid glycosphingolipid fraction (64 mg) was obtained from pooled epithelial cell scrapings from the small intestines of 12 cats by standard methods (17). The non-acid glycosphingolipids (50 mg) were first separated on a silicic acid column, stepwise eluted with increasing amounts of methanol in chloroform. The fractions containing triglycosylceramides and larger glycosphingolipids were pooled, giving 45 mg, and further separated by HPLC on a Kromasil 5 Silica column (2.12 × 25 cm, inner diameter; particle size, 5 mm; Skandinaviska Genetec, Kungsbacka, Sweden) eluted with a linear gradient of chloroform/methanol/water 80:20:1 to 40:40:12 (by volume) during 180 min with a flow rate of 4 ml/min. Each 4-ml fraction was analyzed by thin-layer chromatography using anisaldehyde for detection. The fractions containing tetraglycosylceramides and larger glycosphingolipids were tested for binding of LCTBK using the chromatogram binding assay. The binding-active compound eluted in tubes 151-158, and after pooling of these fractions 0.4 mg was obtained.

Mass Spectrometry-- For electron ionization mass spectrometry, aliquots of the isolated glycosphingolipid were permethylated (18), or permethylated and reduced with LiAlH₄ (19). The samples were analyzed on a JEOL SX-102A mass spectrometer (JEOL, Tokyo, Japan) using the in-beam technique (20). The analyses of both derivatives were performed with an electron energy of 70 eV, trap current of 300 µA, and acceleration voltage of 10 kV. The temperature was raised from 150 °C to 410 °C, by increases of 10 °C/min.

Proton NMR Spectroscopy-- ¹H NMR spectra were obtained on a Varian 500-MHz spectrometer at 30 °C. Samples were dissolved in dimethyl sulfoxide-d₆/D₂O (98/2, by volume) after deuterium exchange.

Molecular Modeling and Dynamics Simulations-- Docking and molecular dynamics simulations were conducted on a Silicon Graphics Indigo²Extreme workstation using the Quanta97/CHARMm22 software package (Molecular Simulations Inc., Waltham, MA), whereas the A pentasaccharide (GalNAc3(Fuc2)Gal4(Fuc3)GlcNAc) initially was constructed using Biograf software package (Molecular Simulations Inc.) before transferring the structure to the aforementioned program. The glycosidic dihedral angles of the CHARMm-refined structure deviated insignificantly from literature values (21). All protein structures were constructed from the crystal structure of the pLT-lactose complex (Ref. 4; Protein Data Bank entry 1LTT): two whole B-subunits (G and H) were thus used in the construction of corresponding subunits of hLTB and the hybrid mutants listed in Table I following procedures outlined earlier (22). The root-mean-square deviations for the main chain atoms of energy-refined hLT, LCTBK, and LCTBK in complex with the A pentasaccharide (snapshot at 180 ps) relative to the pLT crystal structure were 0.55, 0.86, and 0.94 Å, respectively. A comparison between LCTBK and its complex with the A pentasaccharide yielded root-mean-square values of 0.38 Å for the backbone atoms and 0.62 Å when all atoms were considered. Of the several starting structures for subsequent dynamics runs that were generated through manual docking of the A pentasaccharide into the putative LCTBK binding site, only a few very similar orientations of the pentasaccharide gave a satisfactory surface complementarity as well as involving the amino acid side chains implicated in the binding studies. Vacuum dynamics simulations at 300 K using a distance-dependent dielectric constant ( = 6r) and a 2-fs time step were performed as described (22). The final 1-ns run lasted for 380 CPU h. In these runs, 70 residues surrounding the binding site were allowed freedom of movement, whereas in energy minimization the whole dimer was movable.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation and Characterization of Hybrid CT/hLT B-subunits-- The amino acid sequences of the B-subunits utilized in this study are summarized in Table I. The daughter hybrid designated LCTBK was obtained by re-substituting from Ser (found in hLTB) to Asn (found in CTB) at position 4. SDS-polyacrylamide gel electrophoresis analysis of LCTBK indicated that it formed stable pentamers. It also appeared identical to LCTBH in terms of reactivity with CTB- and LTB-specific monoclonal antibodies (23). However, identification of the 14 N-terminal amino acids of LCTBK by sequential Edman degradation confirmed the presence of Asn at position 4. 

Binding to GM1 Ganglioside and N-Acetyllactosamine-terminated Glycosphingolipids in Microtiter Wells-- The microtiter well assay showed that all B-pentamers bound to GM1 with similar affinities (Fig. 1, A), demonstrating that the mutations introduced had not affected the ability to interact with the GM1 ganglioside. Half-maximal binding of all B-subunit proteins occurred at approximately 5 pmol/well.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Intact GM1 ganglioside binding capacity, but reduced binding of N-acetyllactosamine-terminated glycosphingolipids by the hybrid B-subunit LCTBK, demonstrated by binding of 125I-labeled B-subunits to serial dilutions of glycosphingolipids in microtiter wells. Data are expressed as mean values of triplicate determinations.

As reported previously hLTB and LCTBH, but not CTB, bound to the N-acetyllactosamine-terminated glycosphingolipids neolactotetraosylceramide (Gal4GlcNAc3Gal4Glc1 Cer; Fig. 1B) (12). Linear neolactohexaosylceramide (Gal 4GlcNAc3Gal4GlcNAc3Gal4Glc1Cer; data not shown) and branched neolactohexaosylceramide (Gal4GlcNAc6 (Gal4GlcNAc3)Gal4Glc1Cer; Fig. 1C) were also bound by hLTB and LCTBH. However, the level of binding of LCTBK to N-acetyllactosamine-terminated compounds was reduced virtually to the level of CTB. Furthermore, while hLTB and LCTBH bound to gangliotetraosylceramide (Gal3Gal NAc4Gal4Glc1Cer), no binding of CTB or LCTBK to this glycosphingolipid was observed (data not reproduced).

Binding to Glycosphingolipids on Thin-layer Chromatograms-- In accordance with the results from the microtiter well assay, the hybrid B-subunits bound to the GM1 ganglioside on thin-layer chromatograms (Fig. 2, lane 9, and present in lane 8). By binding of B-pentamers to glycosphingolipids from small intestinal epithelium of single human individuals on thin-layer chromatograms, an aberrant behavior of LCTBK was detected. Unlike the other B-pentamers, LCTBK bound selectively to slowly migrating non-acid glycosphingolipids present in some individuals (Fig. 2C, lanes 3 and 4). LCTBK-specific binding to slowly migrating glycosphingolipids was also detected in the non-acid fractions of rabbit (Fig. 2C, lane 1), rat, dog, pig, and cat intestine and human meconium (data not shown).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Selective binding of the hybrid B-subunit LCTBK to slowly migrating non-acid glycosphingolipids of human and rabbit small intestinal epithelium. Glycosphingolipids were chromatographed on aluminum-backed silica gel plates using chloroform/methanol/water (60:35:8, by volume) as solvent system, and visualized with anisaldehyde (A). Duplicate chromatograms were incubated with 125I-labeled LCTBH (B) and LCTBK (C), followed by autoradiography for 12 h, as described under "Experimental Procedures." The lanes were: non-acid glycosphingolipids of rabbit small intestinal epithelium, 40 µg (lane 1); non-acid glycosphingolipids of human small intestinal epithelium of three separate individuals, 40 µg/lane (lanes 2-4); acid glycosphingolipids of human small intestinal epithelium, 40 µg (lane 5); GM1 ganglioside, 0.4 µg (lane 6). The arrow marks the migration of residual GM1 ganglioside in the samples.

Isolation and Characterization of an LCTBK-binding Glycosphingolipid from Epithelial Cells of Cat Small Intestine-- The non-acid glycosphingolipid fraction from epithelial cells of cat small intestine was separated by chromatography on a silicic acid column, followed by HPLC on straight-phase silica gel. The fractions containing the LCTBK-binding compound were pooled, giving 0.4 mg, which was used for structural characterization by electron ionization mass spectrometry and proton NMR spectroscopy.

The mass spectrum of the permethylated LCTBK-binding glycosphingolipid isolated from cat intestinal epithelial cells (Fig. 3A) has ions characteristic of a terminal blood group A determinant: terminal HexNAc (m/z 260), terminal fucose (m/z 189), and A trisaccharide (HexNAc(Fuc)Hex; m/z 638 and 606). The next ions that can be attributed to the carbohydrate chain are seen at m/z 1056 and 1024, indicating a HexNAc(Fuc)Hex(Fuc)HexNAc pentasaccharide. The next carbohydrate units toward the reducing end is a Hex (hexasaccharide ions at m/z 1261 and 1229), followed by a HexNAc (heptasaccharide ions at m/z 1506 and 1474), a Hex (octasaccharide ions at m/z 1710 and 1678), and a Hex (nonasaccharide ions at m/z 1914 and 1882). The concluded sequence was supported by the ion at m/z 1988, containing the whole carbohydrate chain and part of the fatty acid, and by molecular ions at m/z 2596, 2624 and 2652 (nonasaccharide with phytosphingosine and hydroxy 20:0, 22:0 and 24:0 fatty acids, respectively).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Electron ionization mass spectra of the permethylated (A), and permethylated and LiAlH4-reduced (B), LCTBK-binding glycosphingolipid isolated from the epithelial cells of cat small intestine. Above the spectra are simplified interpretation formulae representing the species with phytosphingosine and hydroxy 22:0 fatty acid. The analytical conditions were: electron energy, 70 eV; trap current, 300 µA; and acceleration voltage, 10 kV. The temperature was raised from 150 °C to 410 °C, by increases of 10 °C/min. Both spectra were recorded at 380 °C.

The major long chain base is phytosphingosine (m/z 396), and the ceramide composition is given by the ions at m/z 694 and 722, indicating phytosphingosine combined with hydroxy 22:0 and 24:0 fatty acids, respectively.

The mass spectrum of the permethylated and reduced glycosphingolipid (Fig. 3B) has a series of prominent immonium ions (F-fragments), formed by loss of part of the long chain base, at m/z 2200-2312. These ions give information about the number and type of sugars, and the fatty acid composition, and in the present case demonstrate the presence of a saccharide composed of two fucoses, three N-acetylhexosamines, and four hexoses, combined with hydroxy 16:0 to 24:0 fatty acids. Carbohydrate sequence ions are found at m/z 189 (terminal fucose), m/z 246 (terminal HexNAc), m/z 624 (terminal A trisaccharide), m/z 1028 (HexNAc(Fuc)Hex(Fuc)HexNAc pentasaccharide), m/z 1233 and 1201 (hexasaccharide), and m/z 1668 (octasaccharide).

A prominent ion at m/z 182 is present in the mass spectra of both derivatives. This ion is due to rearrangement of GlcNAc, and is only found in spectra of glycosphingolipids with a type 2 core (Hex4HexNAc) (24, 25). The proposed carbohydrate sequence has HexHexNAc at two positions, either of which could have a type 2 core. However, further information may be obtained from the spectrum of the permethylated and reduced derivative. In glycosphingolipids having a Hex in 1-3 linkage to a HexNAc (type 1 core), a series of rearrangement ions derived from the immonium ions are found (26). These ions are obtained by fragmentation of the Hex1-3HexNAc linkage and rearrangement of the GlcNAc with loss of the acetamido group of 58 mass units. A type 1 linkage at the non-reducing HexHexNAc would give rise to a series of ions at m/z 1502-1614 (F  640  58), while a type 1 linkage of the HexHexNAc close to the reducing end would produce a series at m/z 893-1005 (F  1249  58). The absence of both ion series thus suggests that the LCTBK-binding glycosphingolipid has type 2 core chains at both positions.

Thus, by mass spectrometry, the LCTBK-binding glycosphingolipid was tentatively identified as a nonaglycosylceramide with a terminal A trisaccharide and a HexNAc(Fuc)Hex(Fuc)HexNAcHexHexNAcHexHex sequence, with two type 2 linkages (Hex4HexNAc).

The ¹H NMR spectrum at 30 °C of the fraction containing the nonaglycosylceramide with a terminal A trisaccharide (not shown) displayed anomeric proton signals, which could easily be assigned on the basis of earlier published spectra. Thus, the signals at 5.10 ppm (Fuc2), 5.08 ppm (GalNAc3), 4.86 ppm (Fuc3), 4.66 ppm (GlcNAc3), and 4.45 ppm (Gal4) confirm the presence of an ALe^y determinant as in the A7 type 2 glycosphingolipid (27) whereas the signals at 4.26, 4.72, 4.28, and 4.22 ppm are consistent with the internal sequence Gal4GlcNAc3Gal4Glc1 (28, 29). Combined with the data from mass spectrometry the identity of the LCTBK-binding glycosphingolipid can thus be established as GalNAc3 (Fuc2)Gal4(Fuc3)GlcNAc3Gal4GlcNAc3Gal4Glc1Cer, i.e. the A9 type 2 glycosphingolipid.

Binding of B-subunits to Blood Group-active Glycosphingolipids in Microtiter Wells-- Binding to the A9 type 2 glycosphingolipid from cat small intestine in microtiter wells (Fig. 4A) confirmed that this glycosphingolipid was preferentially recognized by LCTBK, with a half-maximal binding at approximately 20 pmol/well.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Selective binding of LCTBK to glycosphingolipids with terminal GalNAc3(Fuc2)Gal4(Fuc3)GlcNAc sequences. A, binding of LCTBK, but not CTB, hLTB or LCTBH, to the A9 type 2 glycosphingolipid (GalNAc3(Fuc2)Gal4(Fuc3)GlcNAc3Gal 4GlcNAc3Gal4Glc1Cer) adsorbed in microtiter wells. B, binding LCTBK to the A7 type 2 glycosphingolipid, but not to glycosphingolipids with related structures in microtiter wells. Open circles, GalNAc3(Fuc2)Gal4(Fuc3)GlcNAc3Gal4Glc1Cer (A7-2); open squares, GalNAc3(Fuc2)Gal3(Fuc4)GlcNAc3Gal4Glc1Cer (A7-1); filled circles, GalNAc3(Fuc2)Gal4GlcNAc3Gal4Glc1Cer (A6-2); filled squares, Fuc2Gal4(Fuc3)GlcNAc3Gal4Glc1Cer (Y-6). Data are expressed as mean values of triplicate determinations.

Next, the binding of LCTBK to a number of glycosphingolipids with structures related to the A9 type 2 glycosphingolipid was tested in microtiter wells (summarized in Table II). LCTBK, but not the other B-subunits, bound to the A7 type 2 glycosphingolipid (GalNAc3(Fuc2)Gal4(Fuc3)GlcNAc3 Gal4Glc1Cer) with a half-maximal binding at approximately 40 pmol/well (Fig. 4B). No binding of LCTBK to the A7 type 1 glycosphingolipid (GalNAc3(Fuc2)Gal3(Fuc4)GlcNAc3 Gal4Glc1Cer), the A6 type 2 glycosphingolipid (Gal NAc3(Fuc2)Gal4GlcNAc3Gal4Glc1Cer), or the Y6 glycosphingolipid (Fuc2Gal4(Fuc3)GlcNAc3Gal4Glc1Cer) was obtained. Furthermore, the binding of LCTBK to the B7 type 2 glycosphingolipid (Gal3(Fuc2)Gal4(Fuc3)GlcNAc 3Gal4Glc1Cer of human erythrocytes (Fig. 5, lane 2) indicated that the acetamido group of the terminal GalNAc was not essential for the interaction, and thus the minimal structural element involved in the recognition process was Gal3 (Fuc2)Gal4(Fuc3)GlcNAc.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Recognition of blood group A- and B-active heptaglycosylceramides and larger glycosphingolipids by LCTBK. Glycosphingolipids were chromatographed on aluminum-backed silica gel plates using chloroform/methanol/water (60:35:8, by volume) as solvent system, and visualized with anisaldehyde (A). Duplicate chromatograms were incubated with 125I-labeled LCTBK (B), hLTB (C), and monoclonal antibodies directed against the blood group A determinant (D) and the blood group B determinant (E), followed by autoradiography for 12 h, as described under "Experimental Procedures." The lanes were: non-acid glycosphingolipids of human blood group A erythrocytes, 40 µg (lane 1); non-acid glycosphingolipids of human blood group B erythrocytes, 40 µg (lane 2); non-acid glycosphingolipids of human blood group O erythrocytes, 40 µg (lane 3); neolactotetraosylceramide (Gal4GlcNAc3Gal4Glc1Cer), 1 µg (lane 4); A7 type 2 glycosphingolipid (GalNAc3(Fuc2)Gal4(Fuc3)GlcNAc3Gal4Glc1Cer), 1 µg (lane 5); GM1 ganglioside (Gal3GalNAc4(NeuAc3)Gal4Glc1 Cer),0.5 µg (lane 6). The numbers to the left of A indicate the approximate number of the carbohydrate units in the bands.

Molecular Modeling and Dynamics Simulations-- Inspection of Table I and the pLT/CT crystal structures (4-6) show that the non-conserved amino acids at positions 7, 25, 83, and 102 are in close proximity to Asn⁴ of LCTBK, strongly suggesting a binding site location within this perimeter. Docking a blood group A pentasaccharide (GalNAc3(Fuc2)Gal4(Fuc3) GlcNAc) into the LCTBK hybrid and ensuing molecular dynamics simulations (1 ns) of the complex yields the picture shown in Figs. 6-9. The pentasaccharide lies in a shallow depression of the protein surface at the subunit interfaces with the fucoses exposed to the solvent. In addition to an excellent protein-saccharide surface complementarity, critical hydrogen bond interactions with the side chains of Gln³, Asn⁴, Ser²⁶, Thr²⁸, Thr⁴¹, Thr⁴⁷, Glu⁸³, and Lys⁸⁴ as well as with the peptide backbone are found involving all five sugars (Figs. 7 and 9). The electrostatic potential energy surfaces for the LCTBK binding site (Fig. 7), generated by a water probe, and the corresponding surface for the pentasaccharide (partially shown in Fig. 8), reveal several regions of advantageous complementary potentials of opposite sign. This is particularly evident for the interactions of the GalNAc3 and Fuc3 residues, which are essential for binding to occur (see Table II). Due to its rigid nature (21), conformational changes of the pentasaccharide are very minor. Protein conformational changes are small as well, being restricted to re-orientation of the Leu²⁵ and Glu⁸³ side chains in order to accommodate the pentasaccharide. Other changes include movement of Ser²⁶ approximately 1.2 Å toward Gal4 and an average downward movement of the 42-46 loop by 2 Å compared with the pLT crystal structure (4).

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Ribbon representation of two subunits of the LCTBK B-pentamer showing the location of the blood group A pentasaccharide binding site at the subunit interface (top center) relative to the classic GM1 pentasaccharide binding site (bottom right). The B-dimer is shown with the top slightly tilted toward the viewer and with the -helices lining the central pore of the B-pentamer facing away from the viewer. The terminal GalNAc residue of the A pentasaccharide is pointing to the right, whereas the GlcNAc at the reducing end is seen to point to the left. The terminal Gal residue of the GM1 oligosaccharide is seen at the top whereas the sialic acid is seen pointing to the left.

View larger version (56K): [in this window] [in a new window]   Fig. 7.   Stereo views of the blood group A pentasaccharide and its interactions with surrounding LCTBK amino acid side chains. Both panels were generated from an energy-refined snapshot 180 ps into a 1-ns molecular dynamics run. In the upper panel the backbones of the two different subunits are colored red and blue, respectively, whereas the side chains are in light blue. The  carbon atoms of several amino acids are numbered as they appear in the sequence and can be identified from Fig. 9. The blood group A pentasaccharide is shown in yellow with the terminal GalNAc residue at the top and the GlcNAc at the reducing end at the bottom. Hydrogen bonds are shown as white dashed lines. The glycosidic dihedral angles (, ) of the pentasaccharide differed insignificantly from those of the isolated pentasaccharide. The lower panel shows the electrostatic potential energy surface of LCTBK in the blood group A pentasaccharide binding site, generated by a water probe with a 1.4-Å radius, where blue represents the most negative potential and red the most positive one (±30 kcal/mol). The complementary surface generated for the pentasaccharide is almost identical in shape but reveals potentials of opposite sign in several significant parts of the surface.

View larger version (52K): [in this window] [in a new window]   Fig. 8.   Close-up view of the interactions of the Asn4 residue of LCTBK with the blood group A pentasaccharide. The electrostatic potential energy surface of the pentasaccharide, generated by a 1.4-Å water probe, is color-coded corresponding to the LCTBK surface shown in Fig. 7, whereas the van der Waals surface of the amide moiety of Asn4 is colored using standard atom colors. Note the groove, formed by the GalNAc3 acetamido moiety, the Gal4 4-CH, 4-OH, and 6-CH2 groups and the Fuc3 3-OH (the two latter groups are located just below the border of the figure), into which the amide moiety of Asn4 fits precisely. For clarity, the contribution of the Asn4 CH2 group to the van der Waals surface was omitted but makes the side chain fit even tighter.

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Schematic view of the blood group A pentasaccharide and its interactions with surrounding amino acid side chains of LCTBK as found from molecular dynamics simulations. Hydrogen bonds are indicated by dashed arrows.

The significance of the Ser⁴  Asn mutation in LCTBK lies partly in the additional hydrogen bonds formed between the Asn⁴ side chain and the Gal4 4-OH and the Fuc3 3-OH, and partly in the very snug fit of this side chain against the carbohydrate surface (Fig. 8). The orientation of the amide of the Asn⁴ side chain differs by 180° from that observed in CTB (5, 6). This is due to the nearby Glu⁷ in LCTBK, as opposed to the shorter Asp⁷ in CTB, which favors the opposite configuration. This also results in a preference of the hydroxyl group of Thr⁶ side chain to point downward to hydrogen-bond to Asn⁴ (Figs. 7 and 8).

Another significant difference between LCTBK and CTB is Leu²⁵, which in CTB is a phenylalanine. In CTB Phe²⁵ forms a hydrophobic patch with the methyl group of Thr⁴¹ as does Leu²⁵ of pLT (4). However, in LCTBK Leu²⁵ is too far from Thr⁴¹ due to a re-orientation of the Leu²⁵ side chain that appears necessary when the pentasaccharide complex is formed. In CTB, Phe²⁵ is locked in the crystal conformation, suggesting that its side chain would partially block access to the binding site by sterically interfering with the Fuc2 residue and also unfavorably interact with the 6-OH of the GlcNAc residue. The combination of these factors and the unfavorable orientation of Asn⁴ probably account for the absence of binding of CTB to the blood group A/B determinant.

The model also accounts for the relatively weak binding of A9-2 by hLTB and LCTBH since the Ser⁴ side chain present in these molecules is unable to provide the favorable carbohydrate interactions observed for the Asn⁴ side chain of LCTBK.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The binding of the B-subunits of CT and LT to receptor glycoconjugates on the small intestinal epithelial cells is a prerequisite for the following steps in toxin action leading to diarrhea. In addition, both CT and LT elicit strong immune responses, and are among the most potent mucosal adjuvants yet identified (30, 31). The immunogenicity, and to some extent the adjuvant activity, are also dependent on receptor binding, as shown by recent studies using the Gly³³ [arrow] Asp mutant of LT, which is devoid of GM1 binding capacity (32-34).

The binding of the B-subunits of cholera toxin to the GM1 ganglioside is a paradigm for protein-carbohydrate interactions. However, in the CTB/LTB hybrid LCTBK, with blood group A- and B-binding capacity, the blood group determinants are accommodated in a novel carbohydrate binding site, distinct from the GM1 binding site. This novel binding site is located at the top of the B-subunit interfaces as found by docking and molecular dynamics simulations. Changes of carbohydrate binding specificities by substitutions of specific amino acids within the carbohydrate binding sites have previously been reported for E-selectin, P-selectin, and mannose-binding protein (35-37), but this is the first report of the creation of a novel binding site in a carbohydrate-binding protein. The reason for the lost ability of LCTBK to bind to N-acetyllactosamine-terminated glycosphingolipids and gangliotetraosylceramide is, however, at present not apparent.

An additional observation is that also CTB exhibits a weak binding to branched neolactohexaosylceramide, but does not bind to linear to N-acetyllactosamine-terminated glycosphingolipids (Fig. 1). Docking studies and molecular dynamics simulations suggest that this is due to additional interactions between the the 6-linked branch and amino acid residues outside the GM1 binding site, i.e. van der Waals interactions between the -CH₂ group of His¹³ and the terminal galactose, and between Asn¹⁴ and the 3-OH of this galactose, in addition to the interactions described earlier for neolactotetraosylceramide (10).² The Gal4GlcNAc6(Gal3GlcNAc3)Gal element of branched neolactohexaosylceramide is found also in the carbohydrate chains of glycoproteins. However, CTB does not bind to human small intestinal glycoproteins on blotting membranes, and hLTB binds only after de-sialylation (38), indicating that certain substitutions on the Gal4GlcNAc6 (Gal3GlcNAc3)Gal core obliterates the binding.

An important further step will be to analyze whether the novel mode of binding of LCTBK, with recognition of carbohydrate receptors different from the GM1 ganglioside, will allow the toxin to exert its biological effects. The location of the binding site and the direction of the carbohydrate residues at the reducing end suggest that LCTBK may bind "upside down" with the GM1 binding sites directed away from the surface. The next step will thus be to produce an LCTBK holotoxin, where the presence of the A-subunit will prevent the upside down binding. This holotoxin should not bind to the relatively short glycosphingolipids tested here, but may bind to longer glycosphingolipids or A/B determinants on glycoproteins, and will be a novel tool for dissection of the relation of the binding event to the biological functions of the toxins. Thereafter, a holotoxin with an LCTBK/G33D mutation in the B-subunits will be constructed, giving a toxin that binds only to blood group A and B determinants. This hybrid toxin will allow further insights into how the biological activities of the toxins are related to recognition of different carbohydrate receptors.

In addition, efforts to crystallize LCTBK, alone and in complex with A pentasaccharide, are currently under way.

	ACKNOWLEDGEMENT

We gratefully acknowledge the use of the Varian 500-MHz machine at the Swedish NMR Center, Hasselblad Laboratory, Göteborg University.

	FOOTNOTES

^* This work was supported by Swedish Medical Research Council Grants 12628, 3967, and 10435; Swedish Technical Research Council Grant 97-296; the Swedish Cancer Foundation; and the Wallenberg Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Inst. of Medical Biochemistry, Göteborg University, P. O. Box 440, SE 405 30 Göteborg, Sweden. Tel.: 46-31-773-34-92; Fax: 46-31-413-190; E-mail: Susann. Teneberg@medkem.gu.se.

² S. Teneberg, A. Berntsson, and J. Ångström, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: CT, cholera toxin; CTB, cholera toxin B-subunit; Fuc, fucose; Hex, hexose; HexNAc, N-acetylhexosamine; LT, E. coli heat-labile enterotoxin; LTB, E. coli heat-labile enterotoxin B-subunit; HPLC, high performance liquid chromatography. The glycosphingolipid nomenclature follows the recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN) (CBN for Lipids (1977) Eur. J. Biochem. 79, 11-21; CBN for Lipids (1982) J. Biol. Chem. 257, 3347-3351; CBN for Lipids (1987) J. Biol. Chem. 262, 13-18). It is assumed that Gal, Glc, GlcNAc, GalNAc, NeuAc, and NeuGc are of the D-configuration, Fuc of the L-configuration, and all sugars present in the pyranose form..

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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