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The Journal of Immunology, 1999, 163: 6686-6693.
Copyright © 1999 by The American Association of Immunologists

Staphylococcal Enterotoxin H Displays Unique MHC Class II-Binding Properties

Helen Nilsson1,*, Per Björk*, Mikael Dohlsten{dagger} and Per Antonsson*

* Active Biotech Research AB and {dagger} Department of Cell and Molecular Biology, Section for Tumor Immunology, Lund University, Lund, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Staphylococcal enterotoxin H (SEH) has been described as a superantigen by sequence homology with the SEA subfamily and briefly characterized for its in vivo activity. In this study, we demonstrate that SEH is a potent T cell mitogen and inducer of T cell cytotoxicity that possesses unique MHC class II-binding properties. The apparent affinity of SEH for MHC class II molecules is the highest affinity ever measured for a staphylococcal enterotoxin (Bmax1/2 ~ 0.5 nM for MHC class II expressed on Raji cells). An excess of SEA or SEAF47A, which has reduced binding to the MHC class II {alpha}-chain, is able to compete for binding of SEH to MHC class II, indicating an overlap in the binding sites at the MHC class II ß-chain. The binding of SEH to MHC class II is like SEA, SED, and SEE dependent on the presence of zinc ions. However, SEH, in contrast to SEA, binds to the alanine-substituted DR1 molecule, ßH81A, believed to have impaired zinc-bridging capacity. Furthermore, alanine substitution of residues D167, D203, and D208 in SEH decreases the affinity for MHC class II as well as its in vitro potency. Together, this indicates an MHC class II binding site on SEH with a different topology as compared with SEA. These unique binding properties will be beneficial for SEH to overcome MHC class II isotype variability and polymorphism as well as to allow an effective presentation on APCs also at low MHC class II surface expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Superantigens (SAgs)2 are a group of highly potent immunostimulatory proteins of bacterial and viral origin. In contrast to conventional Ags, SAgs interact as unprocessed proteins with MHC class II molecules outside the peptide-binding groove (1) on APCs and activate a high frequency of T lymphocytes. The SAg binds and activates all T lymphocytes expressing particular TCR Vß chains (2). Activation results in vigorous T cell proliferation and production of cytokines as well as induction of T cell cytotoxic activity.

Staphylococcal enterotoxins (SEs) are a group of structurally and functionally homologous SAgs and the major cause of food poisoning in humans (3, 4). SEs are proteins with a molecular mass of about 25 kDa, and to date 10 enterotoxins have been characterized: staphylococcal enterotoxin A, B, C, D, E, G, H, I, and J (SEA, SEB, etc.) and toxic shock syndrome toxin-1 (TSST-1). SEA and SEB are, together with TSST-1, among the most well-characterized SAgs, whereas SEH, SEG, SEI, and SEJ have only recently been identified (5, 6, 7, 8). Crystallographic studies of SEA (9, 10), SEB (11, 12), SEC2 (13), SED (14), and TSST-1 (15, 16) show a remarkably conserved topology, indicating a strong evolutionary pressure on SE structural integrity. Based on sequence similarities, SEs can be divided into two subfamilies of which one includes SEB, SEC1–3, and SEG. The second subfamily consists of SEA, SED, SEE, SEH, SEI, and SEJ, which all share a zinc-binding motif in their C-terminal domain. It has been shown that coordination of Zn2+ is a major requirement for SEA, SED, and SEE to be efficiently presented on MHC class II molecules (17, 18, 19, 20, 21).

Despite their high degree of structural homology, SEs interact with MHC class II molecules in different manners and with different affinities. The co-crystal structures of SEB/HLA-DR1 (22) and TSST-1/HLA-DR1 (23) have been resolved and show marked differences in their interaction with MHC class II. SEB interacts exclusively with residues of the MHC class II {alpha}-chain, whereas TSST-1 contacts the bound peptide as well as both the {alpha}- and ß-chain of MHC class II. The crystal structure of SEA in complex with a MHC class II molecule has not yet been determined. However, inhibition studies demonstrate that SEA competes with both SEB and TSST-1 (24, 25), indicating that the binding of these toxins to MHC class II is partly overlapping. On the other hand, SEB and TSST-1 are not able to inhibit binding of SEA to MHC class II (24, 25). These findings strongly suggest that a common binding region exists for these SEs as well as an additional MHC class II binding in SEA. Site-directed mutagenesis reveals two distinct regions in SEA that influence its affinity for MHC class II molecules (17, 18, 19, 26). The binding site residing in the N-terminal domain of SEA, interacting with the {alpha}-chain of MHC class II, is structurally homologous to the same region in SEB. The binding surface includes the residues F47 and D70 in SEA (17, 18, 19, 26), which are equivalent to F44 and E67 in SEB. These residues are important contact residues in the interface between SEB and HLA-DR1 (22). Furthermore, a zinc-dependent binding site resides in the C-terminal domain of SEA, binding to the MHC class II ß-chain through zinc coordination by H187, H225, and D227 in SEA (9, 10, 18, 19). Together, these two binding sites allow SEA to bind to MHC class II with an affinity of about 10 nM (18).

SEH is a bacterial SAg with only a few functional properties known (5, 6). SEH shares about 35% amino acid identity with SEA, SED, and SEE. Sequence alignment of SEH and SEA shows interesting similarities as well as differences in the regions contributing to MHC class II interaction in SEA (Fig. 1Go). The crystal structure of SEH has recently been resolved (Maria Håkansson et al., manuscript in preparation), and a superimposition of SEH with the other available crystal structures shows that the alignment of these regions is correct. The C-terminal zinc-binding motif of SEA, which contributes to the interaction with the ß-chain of MHC class II, also seems to be present in SEH. The zinc-binding motif in SEA, established by x-ray diffraction structure determinations (9, 10), is composed by H225 and D227 in the outermost C-terminal part of SEA together with H187. Single alanine substitution of all three zinc-coordinating residues in SEA results in reduced MHC class II-binding affinity accompanied by a decreased in vitro potency of the SAg (18). The amino acid residues D167, H206, and D208 in SEH, all potential residues for coordinating zinc, align with H187, H225, and D227 in SEA. The coordination of zinc in SEA is almost identical to that of the related SED, as shown by the x-ray-determined structure (14). In the SED crystal, however, the fourth position of the tetrahedral coordination is occupied by H218 from the neighboring SED, resulting in a SED homodimer. The presence of SED homodimers in solution was confirmed by covalent cross-linking experiments as well as by gel filtration (14). Furthermore, single alanine substitution of all zinc-coordinating residues as well as absence of zinc was demonstrated to abrogate the ability of SED to form homodimers in solution.



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  		FIGURE 1. Sequence alignment of SEH, SED, SEE, and SEA created by the pileup program, Genetic Computer Group. Amino acid identities between SEH and any of the other SEs are boxed. Amino acids in SED, SEA, and SEE, important for MHC class II {alpha}-chain binding (SEDF42, SEAF47, SEEF47, SEAD70, and SEED70), MHC class II ß-chain binding (SEDD182, SEAH187, SEEH187, SEDH220, SEAH225, SEEH225, SEDD222, SEAD227, and SEED227), and homodimerization of SED (SEDH218) are highlighted. Amino acid residue numbering is based on the SEA sequence.

 
Homodimerization opens new possibilities of SED to interact with MHC class II, and may be of biological importance, as indicated by functional cross-linking of MHC class II on monocytes (21). In SEH, the aspartate in position 203 aligns with H218 in SED, indicating that this residue could participate in a similar zinc-dependent homodimerizaton. In the N-terminal region, SEH differs more markedly compared with other SEs. The phenylalanine, F44 in SEB and F47 in SEA, which is important for binding to the MHC class II {alpha}-chain, aligns with a serine residue in SEH (S36). Moreover, E67 in SEB interacting with K39 in the MHC class II {alpha}-chain and equivalent D70 in SEA, probably involved in a similar interaction (17, 22), aligns with a lysine residue in SEH (K56). The differences in these important positions indicate an incapability of SEH to interact with the MHC class II {alpha}-chain with the same binding topology as all SEs characterized up to now.

In this study, we have investigated the binding properties of SEH to MHC class II, using alanine-substituted SEH and MHC class II molecules in a sensitive scintillation proximity assay, and characterized the effects of MHC class II binding on in vitro potency. We evaluate the potential zinc-binding motif found in the C-terminal domain of SEH and show that SEH displays a unique binding to MHC class II.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning and construction of SEH expression vectors

The amino acid sequence from the published SEH (5) was back-translated using Escherichia coli codon preferences for highly expressed genes. The dsDNA, encoding the mature SEH, was produced using PCR-based sequence overlap extension (27). To facilitate future point substitutions of this sequence, several unique restriction enzyme sites, silent in respect of amino acid coding, were introduced. The resulting gene, which produces mature SEH protein, shares a 72% nucleotide identity with the published sequence (5). Single amino acid substitutions of the SEH gene were introduced using sequence overlap extension (27). PCRs were performed with AmpliTaq (Perkin-Elmer, Foster City, CA), according to recommendations of the manufacturer. PCR-generated fragments were cloned in PCR-script (Stratagene, La Jolla, CA) or PCR-TA (Invitrogen, Leek, The Netherlands). After sequence analysis with ABI prism 310, using a recommended standard protocol (Perkin-Elmer), the SEH variants were cloned in the expression vector pLR16. This vector contains a PBR322 origin of replication, the staphylococcal protein A promoter, a synthetic signal peptide (L. Abrahmsén, unpublished results), and a kanamycin-resistance sequence.

Protein expression and purification

The E. coli K12 strain UL635, harboring the vectors encoding the different SEH variants, was cultured overnight at 25°C in 2x YT (Difco Laboratories, Detroit, MI) supplemented with kanamycin (100 mg/L). The cell pellets were collected after centrifugation at 5 x 103 g and subsequently frozen. The periplasmic content was released by thawing the cell pellets in 5 mM Tris-HCl (ICN Biomedicals, Aurora, OH), pH 7.5, on ice under agitation. The periplasmic extracts were clarified by centrifugation at 1 x 104 g and were subjected to purification.

After adjusting pH, the periplasmic extracts were applied to a SP Sepharose column. The column was washed and proteins were eluted with a linear gradient from 20 to 500 mM ammonium acetate (ICN Biomedicals), pH 4.65, containing 0.02% Tween 80. Fractions containing SEH were once again applied to a SP Sepharose column and eluted with reduced flow and increased volume of the gradient. The SEH protein preparations were >95% pure according to Coomassie-stained SDS-polyacrylamide gels as well as liquid chromatography-mass spectrometry (LCQ) combined with UV detection. Liquid chromatography-mass spectrometry was also used to determine the molecular mass of the produced SEH variants. The deviation from the theoretical molecular mass, calculated from the amino acid sequence, was below ±4 Da (0.02%) for all produced SEH variants.

Cells

The human B cell lymphoma cell line Raji, the human colon carcinoma Colo205, Ltk- cells, RJ 2.2.5 cells, CHO cells, and the murine fibroblast L cell line DAP-3 transfected with HLA-DR1 were cultured in complete R medium (RPMI 1640; BioWhittaker, Verviers, Belgium) supplemented with 10% FCS (HyCrone, Logan, UT), 5 x 10-5 M 2-ME (Merck, Darmstadt, Germany), and 0.1 mg/ml gentamicin (Biological Industries, Beit Haemek, Israel). DAP cells transfected with DR1ßH81A were cultured in complete R medium supplemented with 1 mg/ml Geniticin (Calbiochem-Novabiochem, La Jolla, CA). The DAP transfectants were kindly provided by R.-P. Sékaly (26). CHO transfectants expressing HLA-DR4 were cultured in complete R medium without L-glutamine supplemented with 1 mM MSX (Sigma, St. Louis, MO). PBMCs were prepared from heparinized blood from healthy donors. The cells were isolated by density centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden), according to recommendations of the manufacturer. The human SEH-reactive T cell line was generated according to a standard protocol (28).

Lymphocyte proliferation assay

Human PBMCs, 2 x 105 cells per 0.2 ml complete R medium, were incubated with varying amounts of SEs in flat-bottom microtiter plates at 37°C for 72 h. Each well was pulsed with 5 µCi [3H]thymidine (NEN Life Science Products, Boston, MA) for 4 h. Cells were harvested and proliferation was measured by incorporation of [3H]thymidine in a beta scintillation counter.

Cytotoxicity assays

Cytotoxicity was measured in a standard 4-h 51Cr release assay (28). 51Cr-labeled human Raji cells were used as target cells at 2500 cells per 0.2 ml complete medium in V-shaped microtiter wells. The effector cells, the SEH or SEA-reactive human T cell line, were used at an E:T ratio of 30:1 and with SEs added at varying concentrations. The percentage of specific cytotoxicity was calculated as 100 x [(cpm experimental release - cpm background release)/(cpm total release - cpm background release)].

Binding assays

Influence of zinc on the interaction between SEH and MHC class II was investigated with a combined ELISA and plasma membrane-bound assay (29). Isolation of plasma membrane fractions from human Raji and RJ 2.2.5 cells and preparation of microtiter plates coated with plasma membrane were performed, as previously described (29, 30). Biotinylated SEs diluted in TBS (10 mM Tris-HCl, 150 mM NaCl (Merck), pH 7.4) with 1% BSA (Hoffmann-LaRoche, Basel, Switzerland) were preincubated with either 1 µM ZnCl2 (Sigma) or 2 µM EDTA (ICN Biomedicals). The SE solution, 0.2 ml/well at a concentration giving rise to half-maximal binding, was added to wells coated with 0.165 µg plasma membrane preparation. Biotinylation of SEs with NHS-biotin (long arm) was performed according to the recommendations of the manufacturer (Vector Laboratories, Burlingame, CA). Interaction between the SEs and MHC class II was detected using Vectastain ABC kit (Vector Laboratories) and SIGMA FAST OPD peroxidase substrate tablet set (Sigma).

A cell-based scintillation proximity assay (SPA) was used to determine apparent affinity between SEs and MHC class II. Detached DAP cells transfected with HLA-DR1wt and HLA-DR1ßH81A, CHO cells transfected with HLA-DR4, and Raji cells were washed twice in leucine-deficient medium (RPMI 1640 medium without Arg, Cys, Leu, Met, inositol, glucose, and L-glutamine (Life Technologies, Middlesex, U.K.) supplemented with 2% FCS, 240 mg/L L-arginine (Life Technologies), 59.6 mg/L disodium cysteine (Life Technologies), 15 mg/L L-methionine (Life Technologies), 35 mg/L myo-inositol (Life Technologies), 2 g/L D-glucose (Life Technologies), 300 mg/L L-glutamine (Life Technologies), and 10 mg/L L-leucine (Life Technologies)). Suspended cells were radiolabeled overnight at 37°C at 3 x 106 cells per 6 ml in leucine-deficient medium supplemented with L-[4,5-3H]leucine (Movarek Biochemicals, Brea, CA). A sp. act. of 0.4, 0.5, 0.2, and 0.3 mCi [3H]leucine per 106 cells was used for DAP-DR1wt, DAP-DR1ßH81A, CHO-DR4, and Raji cells, respectively. Before use, the cells were detached and washed twice in assay buffer: HBSS (Life Technologies) supplemented with 25 mM HEPES (BioWhittaker) and 1% BSA. Saturation analysis was performed with 1.5 mg poly(vinyl toluene) SPA beads coated with anti-mouse IgG Ab (Amersham Pharmacia Biotech, Uppsala, Sweden), 15 x 103 radiolabeled cells, and varying concentrations of C215Fab-SE fusion proteins in a total volume of 0.15 ml assay buffer per well. SPA beads and C215Fab-SE were preincubated for at least 30 min before addition of the cells. The specific binding was calculated by subtraction of background signal achieved with C215Fab alone. The assay was performed in 96-well Isoplates (Wallac, Turko, Finland). After 8-h incubation in room temperature, the plates were counted in a beta scintillation counter. MHC class II-negative Ltk- cells, CHO cells, and RJ 2.2.5 cells were used as negative controls for DAP-DR1wt, CHO-DR4, and Raji cells, respectively, to assure that the achieved signal is specific for MHC class II binding. It was assumed that all C215Fab-SE bound to the cells until saturation was reached. Half-maximum of saturation (Bmax1/2), estimated from the saturation curves, was used as a measurement of binding affinity.

Competition of different SEs with C215Fab-SEHwt or C215Fab-SEHD208A for binding to radiolabeled MHC class II-expressing cells was performed with the same SPA system, as described above. However, SPA beads were preincubated with a concentration of C215Fab-SE, giving rise to half-maximal binding in a saturation curve, before addition of competitors. SPA beads, C215Fab-SE, and SEs were incubated at least 30 min before addition of the cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro activity of SEH

The ability of SEHwt to stimulate human PBMCs to proliferate was analyzed by measuring the amount of incorporated [3H]thymidine in the DNA. The effect of three different alanine substitutions in SEH, D167A, D203A, and D208A was also investigated to determine the contribution of these specific amino acid residues to biological function. It should be kept in mind that there is a risk that substitutions give rise to structural changes in the molecule. However, in Results and Discussion, we assume that the effects seen on the biological function are a consequence of the altered amino acid residue. SEH is a potent T cell mitogen showing a dose-dependent proliferative response with a half-maximum effect concentration (EC50) of about 0.2 pM (Fig. 2GoA). This proliferative potency is comparable with that seen with SEA (EC50 ~ 0.2 pM) on matched donors (Fig. 2GoB). Alanine substitution in the potential zinc-binding motif, D167A and D208A, respectively, results in a 10- and 300-fold decreased ability to induce proliferation as compared with SEHwt (Fig. 2GoA and Table IGo). The proliferative activity of SEHD167A is similarly reduced as alanine substitution of the analogous amino acid residue in SEA, SEAH187A (Fig. 2Go, A and B, and Table IGo). However, the effect of D208A in SEH is somewhat lower as compared with the equivalent D227A substitution in SEA (Fig. 2Go, A and B, and Table IGo), indicating that residue D208 is less significant for total activity. In addition, SEHD203A showed a decreased ability to induce proliferation, comparable with that of SEHD167A (Fig. 2GoA and Table IGo). This is in contrast to the equivalent substitution H218A in SED, showing wild-type activity in both in vitro proliferation and T cell cytotoxicity (14). In SED, substitution of H218 only influences the ability to form zinc-dependent homodimers (14, 21). The ability of SEH to homodimerize in the presence of zinc was investigated by the use of cross-linking agents and subsequent SDS-PAGE analysis of the product. In contrast to SED, SEH did not show any ability to form homodimeric products under these conditions (data not shown), indicating a somewhat different biological function for D203 in SEH.



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  		FIGURE 2. In vitro proliferation of human PBMCs. The cells were stimulated for 72 h, and proliferation was measured as incorporation of [3H]thymidine after a 4-h pulse. Data from one of three representative experiments are shown.

 

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  		Table I. Biological effects of alanine substitutions in SEH and SEA

 
The ability of SEHwt and substituted variants to induce SAg-dependent cellular cytotoxicity by targeting SEH-reactive human CTLs against MHC class II+ Raji cells was measured in a 4-h 51Cr release assay (Fig. 3GoA). SEH induces a strong cytotoxicity with an EC50 value of about 1 pM. The SEH variants, SEHD167A and SEHD203A, show a decreased capacity to induce cytotoxicity with EC50 values (Table IGo) of the same order of potency as did the results obtained with PBMC proliferation. SEHD208A, however, seems to influence the induction of cytotoxic effect to lesser extent. Cross-reactivity is observed between SEA- and SEH-reactive human CTLs, demonstrating that SEH and SEA share some TCR Vß specificity (Fig. 3Go, A and B).



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  		FIGURE 3. SAg-dependent cellular cytotoxicity against MHC class II+ Raji cells was analyzed in a 4-h 51Cr release assay using either an SEH-stimulated (A) or an SEA-stimulated (B) human T cell line as effector cells. Data from one of three representative experiments are shown.

 
High biological activity was also achieved with C215Fab-SEH fusion protein in both in vitro proliferation and T cell cytotoxicity (data not shown), which demonstrates that the SE, although fused with a Fab-tag, retains its biological function. This is of significance for the experiments described below.

SEH shows a zinc-dependent binding to MHC class II

SEA and the related SED and SEE have the ability to bind zinc (10, 14, 19, 20), and coordination of zinc is crucial for efficient binding of these SEs to MHC class II (17, 18, 19, 20, 21). Sequence alignments of SEH with SEA, SED, and SEE reveal a conserved zinc-binding motif within the C-terminal domain of SEH composed by D167, H206, and D208 (Fig. 1Go). Whether SEH also shows a zinc-dependent interaction to MHC class II was investigated using a modified ELISA assay (29). Plates coated with plasma membranes from MHC class II-expressing Raji cells were incubated with SEs in the presence or absence of zinc (1 µM Zn2+ and 2 µM EDTA, respectively). In conformity with SEA and SED, SEH shows a dependency on zinc for its MHC class II binding (Fig. 4Go). SEB does not show any requirement for zinc for MHC class II binding, a result expected due to the lack of zinc-binding motif in SEB.



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  		FIGURE 4. Influence of zinc on the interaction between SEs and MHC class II. The SEs were incubated in the presence or absence of zinc in plates coated with plasma membranes from Raji cells. Binding of biotinylated SEs to MHC class II was detected by a streptavidin-conjugated enzyme. Data from one of three representative experiments are shown.

 
No binding of the SEs was observed using plasma membranes prepared from RJ 2.2.5, an MHC class II-negative cell line derived from Raji cells (data not shown).

SEH is a high affinity binder to MHC class II

To examine the interaction between SEH and MHC class II, a homogeneous and real time SPA was used. C215Fab in fusion with SEHwt, SEAwt, or SEHD208A was attached through the Fab moiety to scintillation beads coated with anti-mouse Ig Abs. These beads were mixed with MHC class II-expressing cells metabolically labeled with [3H]leucine. Upon interaction between the SEs and MHC class II, the beads and the labeled cells come in close proximity, which allows transfer of the energy from the radioactive decay on the cells into a light emission signal. Saturation curves reveal that SEH binds to MHC class II with at least 4 times higher affinity compared with SEAwt when using either DR1- and DR4-transfected cell lines or Raji cells as MHC class II-expressing cells (Fig. 5Go, A–C). The competition experiments described below, which include SEs without the C215Fab-tag, reveal a stoichiometry close to 1:1, thus excluding contribution of the Fab moiety. The highest affinity between SEH and MHC class II, Bmax1/2 ~ 0.5 nM, was obtained with Raji cells, expressing DR10 and DR17 among other MHC class II molecules. The affinity of SEA for Raji cells was calculated to 1.9 nM. Also, when using cell lines transfected with specific HLA-DR molecules, SEH exhibited a higher MHC class II affinity than SEA. The affinity of SEH and SEA for DAP cells transfected with DR1 was 1.1 and 9 nM, respectively. When using DR4-transfected CHO cells, SEH and SEA bind to these cells with an affinity of 2.7 and 15 nM, respectively.



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  		FIGURE 5. Binding characteristics for interaction of C215Fab-SE fusion proteins with MHC class II using SPA. SPA beads coated with anti-mouse Ig Abs bind to the Fab portion of the C215Fab-SE fusion protein, which in turn interacts with MHC class II on the surface of [3H]leucine-labeled cells. Raji cells (A), DAP transfectants expressing HLA-DR1 (B), CHO transfectants expressing HLA-DR4 (C), and DAP transfectants expressing HLA-DR1ßH81A (D) were used as MHC class II-expressing cells. Data from one of three representative experiments are shown.

 
To investigate the direct effects of an amino acid substitution in the putative zinc-binding motif, C215Fab-SEHD208A was also included in the binding experiments. With all MHC class II-expressing cell lines, C215Fab-SEHD208A shows about a 75-fold decrease in affinity as compared with C215Fab-SEHwt.

No binding was observed when analogous cells lacking expression of MHC class II (parental CHO cells together with Ltk- and RJ 2.2.5 cells) were used (data not shown), demonstrating that the binding is specific for MHC class II.

SEH binds to the ß-chain of MHC class II

To characterize the binding of SEH to MHC class II, we investigated the ability of SEBwt, SEAwt, and alanine-substituted variants of SEA to inhibit the binding of C215Fab-SEHwt to DR1-expressing cells. The same binding assay, based on scintillation proximity as previously described, was used. Varying concentrations of SEs, without the C215Fab-tag, were allowed to compete with 2 nM of C215Fab-SEHwt to determine the concentrations giving rise to 50% inhibition (IC50). SEHwt itself was found to be by far the most efficient competitor (IC50 ~ 2.1 nM) (Fig. 6GoA). As much as a 275-fold excess of SEAwt was needed to inhibit binding of SEH to MHC class II to the same extent (IC50 ~ 570 nM). In addition, SEH proved to be about 6 times a better competitor than SEA itself when investigating the ability to inhibit MHC class II binding of C215Fab-SEA in the same assay (data not shown). Again, this demonstrates that SEH binds to MHC class II with a higher affinity than SEA. The fact that SEH and SEA compete for binding clearly demonstrates that SEH and SEA share closely related binding sites on MHC class II. The great excess of SEA needed to inhibit MHC class II binding of SEH (275-fold), as compared with the modest difference in affinity (4-fold), indicates that the binding sites are only partially overlapping.



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  		FIGURE 6. Competition of nonfused SEs with C215Fab-SEH wild-type or D208A for interaction with MHC class II-expressing cells. SPA was used by immobilizing C215Fab-SE fusion proteins on SPA beads coated with anti-mouse Ig Abs in the presence of [3H]leucine-labeled cells and competing SEs. Inhibition of SEHwt, SEAwt, and SEBwt together with alanine-substituted variants of SEH and SEA for binding of 2 nM C215Fab-SEHwt to HLA-DR1wt (A), 20 nM C215Fab-SEHD208A to HLA-DR1ßH81A (B), and 2 nM C215Fab-SEHwt to HLA-DR1wt (C). B and B0 indicate the obtained binding of the C215Fab-SE fusion protein in the presence and absence of competitors, respectively. Data from one of three representative experiments are shown.

 
The interaction between the zinc-dependent site seen in the C-terminal domain of SEA and the MHC class II ß-chain is of great importance for the overall high affinity binding of this SE to MHC class II (26, 31, 32). SEAF47A is alanine-substituted in a key residue of the second MHC class II binding site in SEA, located in the N-terminal domain, and found to interact with the {alpha}-chain of MHC class II (18, 19, 26). Hence, the remaining MHC class II affinity of SEAF47A is believed to mainly depend on interaction with the MHC class II ß-chain. Competition experiments reveal that SEAF47A inhibits binding of SEH to DR1 with an IC50 value of 1700 nM (Fig. 6GoA and Table IIGo) and clearly demonstrate that SEH also interacts with the ß-chain of MHC class II. The 3-fold decreased capacity to inhibit binding of SEH, as compared with SEAwt, may indicate the presence of a binding site to the MHC class II {alpha}-chain in SEH. However, it can also be the result of the lower overall affinity of SEAF47A for MHC class II.


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  		Table II. MHC class II binding of alanine substituted SEH and SEA1

 
An alanine substitution in SEA position D227 abolishes the ability of SEA to coordinate Zn2+ and to interact with the ß-chain of MHC class II (18, 19). Hence, both SEAD227A and SEBwt bind only to the {alpha}-chain of MHC class II. At high concentrations, SEB starts to compete with the binding of SEH to MHC class II, indicating a possible weak interaction between SEH and the MHC class II {alpha}-chain (Fig. 6GoA and Table IIGo). Because the affinity of SEA to the {alpha}-chain of MHC class II is lower than the binding of SEB to the same site, no inhibition is achieved with SEAD227A (Fig. 6GoA). The same experiment was performed with DR4-transfected CHO cells revealing almost identical binding patterns (data not shown).

His at position 81 of the ß-chain of MHC class II is critical for binding of SEA, SED, and SEE (21, 26, 31, 32). This residue probably functions as the fourth zinc-coordinating residue upon SE-MHC class II interaction. To examine the binding of SEH to the ß-chain of MHC class II, a binding study was performed with DAP cell transfectants expressing an alanine-substituted DR1 molecule carrying an alanine in position 81 of the ß-chain (DR1ßH81A). The binding of C215Fab-SEHwt, -SEAwt, and -SEHD208A to DR1ßH81A was investigated, and the apparent affinity of each SE was calculated. As shown previously for SEAwt (26), C215Fab-SEAwt displayed no detectable binding to DR1ßH81A (Fig. 5GoD). The affinity of C215Fab-SEHwt to DR1ßH81A was about 20-fold decreased (Bmax1/2 ~ 23 nM) as compared with DR1wt. The remaining interaction clearly indicates an additional binding of SEH to MHC class II. Interestingly, C215Fab-SEHD208A reveals as good binding as C215Fab-SEHwt to DR1ßH81A, indicating a mutual dependency between the residues D208 in SEH and H81 in the MHC class II ß-chain upon interaction. The small difference in affinity between C215Fab-SEHwt and C215Fab-SEHD208A is probably not significant (difference in Bmax1/2 is less than a factor 2).

To further elucidate the existence of additional MHC class II binding in SEH besides its interaction through ßH81, a competition assay using DR1ßH81A-transfected DAP cells and C215Fab-SEHD208A was performed. At a fixed concentration of C215Fab-SEHD208A (20 nM), SEHwt and SEHD208A compete with the same capacity, again demonstrating that these two SEs are equally good binders to DR1ßbH81A (Fig. 6GoB). Neither wild-type protein nor alanine-substituted variants of SEA were able to inhibit binding of C215Fab-SEHD208A, although the affinity of this SE for DR1ßH81A is decreased 20 times as compared with that of SEHwt for DR1wt. In analogy with the experiment using DR1wt, some inhibition is seen with SEBwt at high concentrations (Fig. 6GoB), further indicating the presence of SEH binding to the MHC class II {alpha}-chain.

Amino acid residues D167, D203, and D208 participate in binding of SEH to MHC class II

The ability of SEHD167A, SEHD203A, and SEHD208A to compete for binding of C215Fab-SEHwt to DAP-DR1 cells was investigated to characterize the C-terminal binding of SEH to MHC class II. All three variants of SEH showed a clearly reduced binding for MHC class II as compared with SEHwt (Fig. 6GoC and Table IIGo). The effect of the D208A substitution was most pronounced with a 5000-fold reduction in MHC class II binding, whereas a more moderate effect was obtained for D167A and D203A (35- and 22-fold reduction in binding, respectively). Consequently, residues D167, D203, and D208 are all involved in the interaction with MHC class II. The residues D167 and D208 are equivalent to two of the Zn2+-coordinating residues in SEA, SED, and SEE, which are important for the ability of these SEs to form an MHC class II ß-chain binding site (18, 19, 20, 21). The reduced binding activity of SEHD167A and SEHD208A indicates a similar function for these amino acid residues in SEH. In contrast, residue D203 in SEH, analogous to H218 in SED involved in homodimerization, appears to be directly involved in MHC class II binding.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
SEs are a group of SAgs displaying multiple binding modes to MHC class II that will enable T cell activation under different conditions. To understand the biological functions of the SEs, their interactions with MHC class II and the biological effects of these interactions need to be characterized. Data presented in this work show that SEH binds to HLA-DR with high affinity. In our set of experiments, SEH binds to both DR1- and DR4-transfected cells and to Raji cells with an affinity in the low nanomolar range (Bmax1/2 <= 3 nM). This is the strongest binding ever measured between an SE and MHC class II and is a suggestion that SEH shares the preference of SEA, SEB, SED, SEE, and TSST-1 for the human MHC class II isotype HLA-DR (33, 34). The higher affinity of SEH to MHC class II may also allow SEH to interact with a broader range of different MHC class II molecules. The biological relevance of varying binding affinities of SEs for MHC class II should be regarded in the context of an interplay with the TCR-SE interaction. An increased affinity of the SE for MHC class II can be compensatory for its weak affinity for TCR (35). Furthermore, the MHC class II-SE-TCR ternary complex may also be stabilized by a direct TCR-MHC class II interaction (36, 37). However, the significance of this interaction may differ depending on the affinity between SE and TCR (35, 36, 37). The complexity of this interplay is illustrated by SEH, which, despite the fact that its affinity for MHC class II is higher, induces T cell responses comparable with SEA.

The interaction of SEA, SED, and SEE with the ß-chain of MHC class II confers an ability for these SEs to bind MHC class II with a higher affinity as compared with the members of the SEB subfamily. In this study, we clearly demonstrate that SEH shares the ability of SEA, SED, and SEE to interact with the ß-chain of MHC class II. The binding of SEHwt to DR1wt is inhibited by SEAF47A, suggesting that SEH and SEA, at least partly, overlap in their binding site to the MHC class II ß-chain. In addition, SEH shows a decreased affinity for DR1ßH81A as compared with DR1wt, strongly indicating the importance of this ß-chain residue for the SEH-MHC class II interaction. In SEH, however, the effect of the ßH81A substitution in DR1 is by far not as pronounced as in SEA, in which binding to DR1 is abolished. This indicates an additional interaction between SEH and MHC class II and that the role of ßH81 as a contact residue in MHC class II is of lesser importance. It is also demonstrated that residue D208 in SEH depends directly on ßH81, because SEHD208A binds with the same affinity as SEHwt to DR1ßH81A. The interaction between SEHD208A and DR1wt shows a 75-fold decrease in affinity in comparison with SEHwt. These data correlate to the effect of SEHD208A on the in vitro potency, also demonstrating a less impaired function as compared with the equivalently substituted SEA. It is therefore reasonable to assume that the binding site in SEH involves other amino acid residues, which partly retain the binding to DR1ßH81A.

Interaction with zinc is of great importance for the SEs in the SEA subfamily. There is evidence that Zn2+ is sandwiched between SEA and DR1 (19) and that absence of Zn2+ significantly decreases the binding of SEA, SED, and SEE to MHC class II (Fig. 4Go) (17, 19, 20) as well as their ability to cluster MHC class II on the cell surface (21, 38). In addition, Zn2+ mediates homodimerization of SED, which may allow a different stoichiometry between SED and MHC class II molecules. SEH also demonstrates a zinc-dependent interaction to MHC class II. For SEA, it has been shown that all three residues in the zinc-binding motif (H187, H225, and D227) directly coordinate Zn2+ (19). Loss of zinc binding in SEA is directly correlated to decreased MHC class II affinity and reduced proliferative activity (17, 19, 20). Amino acid sequence alignment of SEH with its related SEs reveals the presence of a zinc-binding motif (Fig. 1Go). The importance of zinc ions for SEH-MHC class II interaction was demonstrated in a binding assay using cell membranes from Raji cells fixed to an ELISA plate. The addition of zinc increased the SEH binding, while the addition of a chelate complex binder (i.e., EDTA) markedly impaired binding in the assay. Both amino acids D167 and D208 are potential Zn2+-binding residues in SEH. Alanine substitution of these residues reduces the binding of SEH to MHC class II, as well as impairing the biological activity in vitro. It is reasonable to believe that D167 and D208 in SEH, together with H81 in the MHC class II ß-chain, contribute to the coordination of one zinc ion, and thereby to the binding of SEH for MHC class II. By using a Zn2+ ion for binding to the widely conserved ßH81 in MHC class II, SEH would efficiently utilize the otherwise polymorphic ß-chain for binding. However, coordination of the Zn2+ ion through ßH81 does not explain why chelation of zinc impairs SEH binding to such a great extent while the ßH81A substitution only has a modest effect on the affinity of SEH for MHC class II. It is possible that there exist other zinc-coordinating residues beyond ßH81 in the MHC class II molecule. Neither is it known whether the peptide bound to MHC class II is able to coordinate zinc or not. In addition, it cannot be excluded that an interaction between SEH and a Zn2+ ion gives rise to an allosteric change in the structure of the SEH molecule, which in turn may influence the ability of SEH to interact with MHC class II.

Hence, binding through ßH81 is not the only interaction between SEH and the MHC class II ß-chain. Alanine substitution of D203 in SEH reduces the binding to MHC class II as well as induction of PBMC proliferation and T cell cytotoxicity to almost the same extent as D167A in SEH. However, the equivalent residue in SED, H218, only influences the ability of SED to homodimerize. In contrast to SED, covalent cross-linking does not show homodimerization of SEH (data not shown). This demonstrates a unique biological role of this residue in SEH, different from that of H218 in SED. Residue D203 seems to be involved in MHC class II binding in a hitherto unknown manner, and may contribute to the additional binding seen for SEH to MHC class II. It is tempting to speculate that the additional binding of SEH to DR1ßH81A is dependent on a direct interaction between this residue in SEH and the MHC class II ß-chain. The extension of the SEH binding sites to MHC class II can be seen as an important ongoing evolution of the SEs to diverse their ability to cross-link APCs and T cells under various conditions. The SEs in the SEB subfamily have a monovalent binding to the MHC class II {alpha}-chain, which is common for all DR molecules. The interaction of SEA, SED, and SEE with MHC class II is extended to multivalent binding, by utilizing the polymorphic ß-chain. The additional unique binding of SEH is a further extension of MHC class II interaction and may represent a further adaptation of SEH to overcome host MHC class II variability. Moreover, there is evidence that MHC class II-associated peptides influence the presentation of SAgs to T cells (24, 39) and the different binding of SEH may result in lesser influence from the peptide. An increased interaction may also be a way to overcome differences in MHC class II between species.

All SEs investigated share the ability to interact with the {alpha}-chain of MHC class II. Data presented in this study suggest that such interaction also is present between SEH and MHC class II, despite unfavorable amino acid differences in key residues. The lysine residue at position 39 in the MHC class II {alpha}-chain is an important residue for the interaction with SEs (24, 26) and forms a saltbridge with E67 in SEB as well as probably also interacting with the analogous D70 in SEA (17, 22). When using DAP cells transfected with DR1{alpha}K39E in SPA, the affinity of C215Fab-SEHwt is decreased about 2-fold compared with DR1wt (data not shown), which further strengthens the indication of an interaction between SEH and the MHC class II {alpha}-chain. E67 in SEB and D70 in SEA align with K56 in SEH, an amino acid that does not favor a direct interaction with K39 in MHC class II {alpha}-chain. Hence, the interaction must involve other regions in SEH. The evolution of SEs with different binding sites to MHC class II can be seen as a strategy for the SE-producing bacteria to meet host variability and thereby increase their survival capabilities. The contribution of this interaction to the total affinity for MHC class II must, however, be of minor importance. As much as a 1000-fold excess of SEB is needed to achieve any inhibition at all of SEH binding, indicating that the N-terminal site in SEH has a weak affinity for MHC class II similar to that of the analogous site in SEA. Nevertheless, a second binding site, although of low affinity, would still be of significance for the biological function of SEH. Multivalent binding makes it possible for SEH to interact with MHC class II in different modes. For instance, it has been shown that two SEA molecules can form a trimeric complex with one DR molecule in solution (40). Moreover, SEA, SED, and SEE are able to cross-link MHC class II with an activation of the APCs as a result (21, 38). Hence, multivalent binding of SEH will increase its potency and facilitate SEH to interact with a greater range of different MHC class molecules. The existence of multiple binding sites of different affinities on both SEH and MHC class II also suggests that the stoichiometry of the complex may vary depending on the concentrations. Different binding modes of the SEH to MHC class II will influence the kind of complex that will interact with the TCR. This may broaden the TCR Vß specificity as well as increase the potency of SEH. Furthermore, due to its higher MHC class II-binding affinity, SEH may be able to bind MHC class II molecules also on cells expressing only low levels of the receptor. This will allow SEH to interact with and activate a broader range of APCs at different stages of activation.

Further investigations would be needed to explain the complex interaction of SEH with MHC class II. Structural information from the recently resolved crystal structure of SEH (Maria Håkansson et al., manuscript in preparation) will greatly facilitate the understanding and interpretation of possible contact areas in SEH to MHC class II.


   Acknowledgments
 
We thank E. Erlandsson, I. Andersson, H. Arozenius, and A. Cavallin for skillful technical assistance, and Dr. R.-P. Sékaly for providing the DAP transfectants. We are grateful to Drs. G. Forsberg and V. Avery for helpful discussions.


   Footnotes
 
1 Address correspondence and reprint requests to Helen Nilsson, Active Biotech Research AB, Box 724, S-220 07 Lund, Sweden. E-mail address: Back

2 Abbreviations used in this paper: SAg, superantigen; Bmax1/2, half-maximum of saturation; CHO, Chinese hamster ovary; SE, staphylococcal enterotoxin; SEA/B/C/D/E/G/H/I/J, staphylococcal enterotoxin A/B/C/D/E/G/H/I/J; SPA, scintillation proximity assay; TSST-1, toxic shock syndrome toxin-1; wt, wild type. Back

Received for publication May 5, 1999. Accepted for publication September 23, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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