Proc. Nati. Acad. Sci. USA Vol. 88, pp. 125-128, January 1991 Medical Sciences

Structural basis for differential binding of staphylococcal enterotoxin A and toxic shock syndrome toxin 1 to class II major histocompatibility molecules (microbial superantlgens/synthetic peptides)

CAROL H. PONTZER, JEFFRY K. RUSSELL, AND HOWARD M. JOHNSON Department of Microbiology and Cell Science, University of Florida, 1059 McCarty Hall, Gainesville, FL 32611

Communicated by George K. Davis, September 17, 1990

ABSTRACT The related staphylococcal toxins staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) are microbial superantigens. They require interaction with class II major histocompatibility complex (MHC) molecules to activate T cells. We have previously identified a binding site on SEA, the N-terminal 45 amino acids, as well as its corresponding receptor on the MHC antigen, residues 65-85 of the fi chain. To further characterize the structural basis for SEA binding to class II MHC molecules we have examined its relationship to TSST-1 binding. Both toxins bound similarly to murine A20 cells, but blockage of binding was observed only with the homologous toxin, which suggests that the binding sites for the two toxins on A20 cells are distinct. In contrast, specific binding of SEA was greater than that of TSST-1 on human Raji cells. Further, SEA was a better inhibitor of TSST-1 binding than was TSST-1 itself at low concentrations, but TSST-1 only minimally inhibited SEA binding. The data suggest that TSST-1 interacts with Raji cells at an SEA binding site, but with a lower affinity. The peptides SEA-(1-45) and I-AI&-(6585) were capable of blocking SEA binding on both A20 and Raji cells, but blockage was more effective on A20 cells. Neither peptide was capable of blocking TSST-1 binding on either cell line. The data are compatible with a model in which SEA has a binding site on A20 cells involving SEA-(1-45) and I-A$Ib(65-85) which is distinct from that which binds TSST-1, while at least two binding sites are present on Raji cells. One site involves predominantly the residue 1-45 region on SEA and the 65-85 region of the MHC .3 chain, while the other site involves both a different region on the SEA molecule and a different site on the class II MHC molecule to which it binds. This latter site also binds TSST-1.

Staphylococcal enterotoxin A (SEA) is the most potent T-cell mitogen known, stimulating DNA synthesis, interferon-y production, and interleukin 2 production at concentrations as low as 10-16 M (1-3). It has been described as a microbial superantigen because of its ability to stimulate all T cells bearing particular T-cell antigen receptor VB regions (4, 5). Antigen-presenting cells are required for SEA activity (6, 7), and recently it has been shown that class II major histocompatibility complex (MHC) molecules are the specific receptors on the antigen-presenting cell for SEA (8-10). Unlike classically presented antigens SEA is not processed prior to binding (6, 7, 11), nor is its presentation restricted by polymorphic portions of class II molecules (7). The N-terminal region of SEA has been identified previously as a site on SEA that is responsible for its interaction with MHC class II antigens (12). Moreover, the same region of SEA is capable of binding to class II antigens from different species-i.e., human leukocyte antigens (HLA) and murine

immune-associated antigens (Ia) (12). SEA and staphylococcal enterotoxin B (SEB) are reported to compete for binding to HLA-DR, implying that they bind to the same region on the MHC molecule (10). In contrast, SEB and toxic shock syndrome toxin 1 (TSST-1) have been shown to interact at different sites (13). We have further examined the complex binding ofmicrobial superantigens to class II MHC molecules by using synthetic peptides, and we have detected multiple binding sites for SEA on human Raji cells, one of which overlaps with that for TSST-1.

MATERIALS AND METHODS Toxins. SEA and TSST-1 were obtained from Toxin Tech-

nology (Madison, WI). SEA was homogenous by SDS gel electrophoresis (1). For biotinylation, 1 mg ofSEA or TSST-1 was dissolved in 50 mM sodium bicarbonate buffer (pH = 9.6) with 0.02 mg of sulfosuccinimidyl 6-(biotinamide) hexanoate (NHS-LC-biotin; Pierce) and incubated on ice for 2 hr. Unreacted biotin was removed by dialysis. Synthetic Peptides. Peptides corresponding to the N-terminal 45 amino acids of SEA [SEA-(1-45)] and the a-helical region of the ( chain of I-Ab [I-A,-b-(65-85)] were synthesized in collaboration with PepTech (Alachua, FL) using a Biosearch 9500 AT automated peptide synthesizer and fluorenylmethyloxycarbonyl (Fmoc) chemistry (14). Peptides were cleaved from the resins by using trifluoroacetic acid/ ethanedithiol/thioanisole/anisole at a ratio of 90:3:5:2 (vol/ vol). The cleaved peptides were then extracted in diethyl ether and ethyl acetate and subsequently dissolved in water and lyophilized. Reverse-phase HPLC analysis of crude peptides indicated one major peak in each profile. Hence, further purification was not warranted. Amino acid analysis of these peptides showed that the amino acid composition corresponded closely to theoretical. Toxin Binding Assays. Cells (1 x 106) of the human Burkitt lymphoma line Raji or the murine Iad B-cell line A20 were washed with phosphate-buffered saline (PBS; Sigma) and incubated with 50 jgl of FACS buffer (PBS containing 10 mM sodium azide and 0.5% bovine serum albumin) or a competitor at 37°C for 40 min. Competitors included unlabeled SEA, unlabeled TSST-1, or synthetic peptides at various concentrations. After incubation, each tube received 50 yd of200 nM biotinylated-SEA or biotinylated-TSST-1 and was incubated for an additional 40 min. The cells were washed twice with FACS buffer and further incubated with 50 ,u of streptavidinphycoerythrin (Becton Dickinson) at 1 pg/ml for 15 min at 4°C, washed two more times, and analyzed. Fluorescence Abbreviations: SEA, staphylococcal enterotoxin A; TSST-1, toxic shock syndrome toxin 1; SEB, staphylococcal enterotoxin B; Ia, immune-associated antigens; HLA, human leukocyte antigens; MHC, major histocompatibility complex; I-Ap", Ia , chain molecule of the A isotype, b haplotype.

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was analyzed on a flow cytometer (FACStar; Becton Dickinson) using logarithmic amplifiers. Fluorescence is expressed as relative intensity. Reverse cross-inhibition studies were performed in a similar manner, with the incubation with biotinylated toxin preceding the addition of competitor.

RESULTS AND DISCUSSION We have examined binding of SEA and TSST-1 to both human and murine cells. Both toxins bound to human Raji cells as well as murine A20 cells (Fig. 1). Specific binding of biotinylated toxin was defined as that which could be inhibited by a 50-fold molar excess of the identical unbiotinylated toxin. SEA and TSST-1 exhibited essentially the same level of specific binding to the murine cell line, A20, as seen by displacement with the homologous toxin (Fig. 1 A and B). Cross-inhibition studies were performed by using TSST-1 to inhibit SEA, and SEA to inhibit TSST-1, on A20 cells. TSST-1 could not inhibit SEA binding, and similarly SEA could not inhibit the binding of TSST-1 (Fig. 1 A and B). These data indicate that the binding sites for the two toxins on mouse lad molecules are distinct. In contrast, a different pattern of toxin binding was observed on the human cell line, Raji. In cross-inhibition studies performed with Raji cells, TSST-1 could be displaced by both excess TSST-1 and excess SEA, while SEA was only minimally displaced by excess TSST-1 (Fig. 1 C and D). This suggests that the binding of the toxins is different between mouse and human class II MHC molecules, and that an SEA

Proc. Natl. Acad. Sci. USA 88 (1991)

binding site is also a binding site for TSST-1 on the human line.

Specific binding of SEA to Raji cells was much greater than to A20 cells, while the level of TSST-1 binding was relatively equivalent on the two cell lines. The increased specific binding of SEA to the human cell line may reflect a higher MHC antigen expression on the Raji cells, binding to more than one site, or a higher-affinity interaction between SEA and HLA, relative to mouse Ta. Raji cells express DR3 and DRw1O as well as DQwl and DQw2 (15), while A20 cells express I-Ad and I-Ed (16). The broad peaks observed in histograms of Raji cells labeled with either toxin alone may reflect variable affinity of SEA and TSST-1 for different HLA class II haplotypes and/or for different sites on a given class II molecule on Raji cells. Data suggestive of a higher affinity of SEA relative to TSST-1 for a common binding site were obtained in doseresponse studies. Fig. 2 presents data on the relative ability of SEA and TSST-1 to displace biotinylated TSST-1 binding. At low concentrations SEA was a better competitor than the homologous toxin. At high concentrations the two were equally effective competitors of TSST-1 binding, but even at the high concentrations used in cross-inhibition studies, TSST-1 exhibited minimal ability to compete with SEA. Thus, only a small proportion of the SEA specific binding appears to be shared with TSST-1. While separate sites are involved in the interaction of these superantigens with mouse Ta, overlapping binding regions are apparent on HLA, where the affinity of SEA for the same site on HLA class II molecules may be greater than that for TSST-1.

A TSST-1 on A20

B

SEA on A20

D

SEA on Raji

350

W

100

4-

101

0 0

C TSST-1 on Raji

.C

z 350

0

250

0

100 Fluorescence

1ol

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FIG. 1. Cross-inhibition of SEA and TSST-1 binding to A20 and Raji cells. Cells (1 x 106) were preincubated with either 50 ALI of FACS buffer or 5 gM each TSST-1 or SEA for 40 min at 37TC. Biotinylated TSST-1 (A and C) or SEA (B and D) was added at a final concentration of 100 nM and the mixture was incubated for an additional 40 min. Cells were washed and labeled with streptavidin-phycoerythrin at 1 ;Lg/ml for 15 min at 4TC. Binding was assessed by flow cytometry, and the results of one representative experiment of five replicates are presented as

single-parameter fluorescence histograms. Binding of biotinylated toxins in the absence of competitor is represented by widely spaced dots; binding in competition with the homologous toxin, by solid lines; and binding in competition with the heterologous toxin, by closely spaced dots.

Medical Sciences: Pontzer et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

a

cU

C:

0.1

0.2

1.0

Competitor, 4M

2.0

5.0

FIG. 2. Dose-response study of toxin inhibition of biotinylated TSST-1 binding to Raji cells. Raji cells (1 x 106) were preincubated for 40 min at 370C with FACS buffer or various concentrations of unlabeled SEA or TSST-1 ranging from 0.1 to 5 MM. Biotinylated TSST-1 was added at a final concentration of 100 nM, and the cells were incubated for an additional 40 min. Labeling with streptavidinphycoerythrin and assessment of fluorescence were performed as described for Fig. 1. Data were collected using logarithmic amplifiers, and representative results from one of three replicates are presented and expressed as mean fluorescence. TSST-1 binding in the absence of competition produced a mean fluorescence of 21.58 ± 0.58. Each determination was performed in triplicate, and the results are expressed as mean ± SEM. Competition by SEA is significantly different (P < 0.05) from that by TSST-1 at 0.1 MM as assessed by Student's t test. At lower doses of competitor (down to 0.01 AM), this difference stays the same but is not enhanced.

An alternative explanation for SEA inhibition of TSST-1 binding to Raji cells is that binding of SEA to a site in close proximity to that for TSST-1 could sterically hinder interaction of TSST-1 with its binding site. Incubation of cells with biotinylated toxins prior to addition of unlabeled toxin produced the same pattern of toxin binding as before: excess SEA was still capable of displacing biotinylated TSST-1 on Raji cells, while TSST-1 only minimally displaced SEA (Table 1). The data suggest that SEA blocks TSST-1 binding Table 1. SEA inhibition of TSST-1 binding is not a function of steric hindrance Mean fluorescence A20 Raji TSST-1 Competitor SEA TSST-1 SEA A. Preincubation with competitor

None TSST-1 SEA None TSST-1

30.53 8.22 29.12 B. Preincubation 28.10 11.30

24.26 21.86 11.85

50.23 11.98 12.94

534.75 457.79 74.44

with biotinylated toxin

18.41 18.07

33.63 10.41

393.12 375.63

SEA 26.09 11.95 11.98 240.32 Data from Fig. 1 are presented in part A, and the cross-inhibition studies presented in part B were performed by reversing the order of addition of competitor and biotinylated toxin. The two cell types, A20 and Raji, were initially incubated with either biotinylated TSST-1 or biotinylated SEA (100 nM final concentration) for 40 min at 37°C. FACS buffer or a 50-fold molar excess of competitor was subsequently added and the mixture was incubated for an additional 40 min. The cells were washed twice with FACS buffer and incubated with 50 ,l of streptavidin-phycoerythrin at 1 ,g/ml for 15 min at 4°C, washed twice again, and analyzed by flow cytometry. The data were collected on logarithmic amplifiers and are reported as mean fluorescence of 10,000 cells. Data from two experiments showed the same pattern of reactivity.

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Table 2. SEA-(1-45) inhibits SEA but not TSST-1 binding to Raji cells Mean fluorescence Exp. 1 Exp. 2 SEA TSST-1 SEA TSST-1 Competitor None 109.62 9.60 182.71 16.29 SEA 6.83 5.00 11.65 4.59 TSST-1 92.51 4.87 78.93 8.56 120.77 SEA-(1-45) 19.81 Biotinylated toxins were employed at a final concentration of 100 nM, competitor toxins at 5 ,uM, and SEA-(1-45) at 1.5 mM. Competitors were preincubated with 1 x 106 Raji cells for 40 min at 370C. The data are presented as mean fluorescence of 10,000 cells, and the dash indicates experiments not done. as a result of interaction with a SEA binding site rather than as a result of steric hindrance. Using synthetic peptides, we have previously shown that a region of the SEA molecule, the N-terminal 45 amino acids,

is involved in its interaction with Ta and that the corresponding binding site on the class II MHC molecule involves the a-helix residues IaIb_(65-85) (refs. 12 and 17 and unpublished observations). Data in Table 2 show that the synthetic peptide SEA-(1-45) inhibited binding of SEA to Raji cells but failed to inhibit TSST-1 binding. Further, the I-Apb_(65-85) synthetic peptide, which represents a region of the I-Ab haplotype A chain corresponding to one of the two a-helical regions extending along the hypothetical antigen binding groove, blocked binding of SEA to both A20 and Raji cells, but it did not affect TSST-1 binding to either cell (Table 3). Peptide competition is known to require a large molar excess of peptide relative to native molecule, potentially because of conformational restraints (12, 17). On A20 cells the ratio of inhibition of SEA binding by the peptide I-A Bb_(65-85) relative to maximal inhibition by SEA itself was 0.82, while on Raji cells the ratio was 0.11 (derived from Table 3, experiment 1), which suggests that the binding sites for SEA on A20 and Raji cells are not identical but may involve common regions on SEA and la. The fact that SEA-(1-45) can block SEA binding to A20 cells (12, 17) and to a lesser extent to Raji cells demonstrates that this region of the SEA molecule interacts with both cell types. Similarly, SEA binding involves Ia, b(65-85) on A20 cells and to a lesser extent a region corresponding to its HLA counterpart on Raji cells. Therefore, the data suggest that there are at least two binding sites for SEA on Raji cells, one of which involves SEA-(1-45) and the counterpart of laf3-(65-85) which can displace SEA but not TSST-1 on Raji cells, and a second site which is also a binding site for TSST-1, since SEA can inhibit

I-Afpb_(65-85) inhibits SEA but not TSST-1 binding to

Table 3. A20 and Raji cells

Mean fluorescence A20 Raji Toxin Inhibitor Exp. 1 Exp. 2 Exp. 1 Exp. 2 TSST-1 None 18.62 10.68 22.19 8.28 TSST-1 TSST-1 6.53 4.61 5.65 3.59 TSST-1 I-Af8b_(65-85) 24.60 12.63 30.67 9.85 SEA None 9.50 12.95 231.48 99.05 SEA SEA 6.40 2.76 15.24 7.68 SEA I-AIBb_(65-85) 7.83 4.50 138.40 76.70 The data are from two of three replicate experiments. The synthetic peptide (1 mM) was incubated with biotinylated toxin (100 nM) for 40 min at 37°C followed by a similar incubation in the presence of washed cells. Excess (5 ,uM) unlabeled toxin was incubated with cells for 40 min at 37°C prior to similar incubation in the presence of biotinylated toxin.

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TSST-1 binding. The second binding site on Raji cells for SEA and the region on SEA involved is not known. Data suggest that this region also represents the binding site for TSST-1. This latter binding site may be located on the a rather than the 8 chain of HLA as suggested by experiments showing TSST-1 binding to chimeric HLA-DR molecules (18). The inability of the I-A P chain peptide to block TSST-1 binding is consistent with this observation. Thus, presentation of a given microbial superantigen can involve more than one site on the toxin and more than one site on class II MHC molecules. We therefore propose a model which is consistent with both cross-inhibition studies and data obtained from peptide competition. A single binding site for TSST-1 is postulated to be present on both murine and human class II MHC molecules, but this does not preclude interaction with more than one haplotype. On A20 cells, this TSST-1 site is separate from that which binds SEA. The previously described SEA binding region on class II MHC molecules of A20 and Raji cells is distinct from that which binds TSST-1, since neither SEA-(1-45) nor I-Apb_(65-85) blocked TSST-1 binding to A20 or Raji cells. However, the data presented here suggest an additional SEA binding region present on Raji cells at which TSST-1 binds with lower affinity. It could potentially be on a different haplotype or on a different portion of the same HLA molecule. This additional site is a common region which in this case overlaps the site where TSST-1 binds as seen by the ability of SEA to displace TSST-1. Thus, examination of binding of two microbial superantigens SEA and TSST-1 to class II MHC molecules on A20 and Raji cells reveals that rather than being a simple phenomenon, the interaction of superantigen and MHC may involve different sites on both the superantigen and the MHC molecule. The synthetic peptide approach should help to further delineate the sites of interaction of microbial superantigens and their MHC receptor molecules.

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