Staphylococcal enterotoxins (SEs) are a family of structurally related exotoxin molecules produced by certain Gram-positive Staphylococcus aureus bacterial strains. SEs are a major cause of food poisoning and are involved in bacterial Gram-positive shock in humans. SEs bind to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) and subsequently activate a large fraction, 5–20%, of T lymphocytes (1). This property has led to their classification of superantigens (SAg). The T cells are activated by SAg to proliferate and produce cytokines such as interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor-α and β (TNF-α and β) (2,3). Depending on origin, superantigens can be divided in two groups, viral and bacterial. For reviews see Refs. 4–8.

Tec family protein tyrosine kinases (TFKs) play a central role in hematopoietic cellular signaling. Initial activation takes place through specific tyrosine phosphorylation situated in the activation loop. Further activation occurs within the SH3 domain via a transphosphorylation mechanism, which for Bruton's tyrosine kinase (Btk) affects tyrosine 223. We found that TFKs phosphorylate preferentially their own SH3 domains, but differentially phosphorylate other member family SH3 domains, whereas non-related SH3 domains are not phosphorylated. We demonstrate that SH3 domains are good and reliable substrates. We observe that transphosphorylation is selective not only for SH3 domains, but also for dual SH3SH2 domains. However, the dual domain is phosphorylated more effectively. The major phosphorylation sites were identified as conserved tyrosines, for Itk Y180 and for Bmx Y215, both sites being homologous to the Y223 site in Btk. There is, however, one exception because the Tec-SH3 domain is phosphorylated at a non-homologous site, nevertheless a conserved tyrosine, Y206. Consistent with these findings, the 3D structures for SH3 domains point out that these phosphorylated tyrosines are located on the ligand-binding surface. Because a number of Tec family kinases are coexpressed in cells, it is possible that they could regulate the activity of each other through transphosphorylation.

Monoclonal antibodies have a potential for cancer therapy that may be further improved by linking them to effector molecules such as superantigens. Tumor targeting of a superantigen leads to a powerful T cell attack against the tumour tissue. Encouraging results have been observed preclinically and in patients using the superantigen staphylococcal enterotoxin A, SEA. To further improve the concept, we have reduced the reactivity to antibodies against superantigens, which is found in all individuals. Using epitope mapping, antibody binding sites in SEA and SEE were found around their MHC class II binding sites. These epitopes were removed genetically and a large number of synthetic superantigens were produced in an iterative engineering procedure. Properties such as decreased binding to anti-SEA as well as higher selectivity to induce killing of tumour cells compared to MHC class II expressing cells, were sequentially improved. The lysine residues 79, 81, 83 and 84 are all part of major antigenic epitopes, Gln204, Lys74, Asp75 and Asn78 are important for optimal killing of tumour cells while Asp45 affects binding to MHC class II. The production properties were optimised by further engineering and a novel synthetic superantigen, SEA/E-120, was designed. It is recognised by approximately 15% of human anti-SEA antibodies and have more potent tumour cell killing properties than SEA. SEA/E-120 is likely to have a low toxicity due to its reduced capacity to mediate killing of MHC class II expressing cells. It is produced as a Fab fusion protein at approximately 35 mg/l in Escherichia coli.

Psoriasis is a T-cell-mediated immune dermatosis probably triggered by bacterial superantigens. This pathomechanism has been experimentally reproduced in a SCID-hu xenogeneic transplantation model. We analyzed the effects of different bacterial superantigens on the induction of psoriasis in this model. Staphylococcal enterotoxin B and exfoliative toxin triggered the onset of psoriasis when administered repetitively intracutaneously over a period of 2 wk, whereas staphylococcal enterotoxin A representing a distinct subfamily of staphylococcal enterotoxins only mimicked certain aspects of psoriasis. The biologic effects of staphylococcal enterotoxin A were more pronounced when a mutated form, SEA(H187A), of this superantigen with reduced affinity to major histocompatibility complex class II was coinjected. Another mutated variant, SEA(F47A/D227A), exhibiting no measurable major histocompatibility complex class II affinity blocked the effects triggered by wild-type staphylococcal enterotoxin A when injected in a 10-fold higher dose. Inhibition was specific as induction of psoriasiform epidermal changes by staphylococcal enterotoxin B could not be blocked. As staphylococcal enterotoxin A, in contrast to the other superantigens tested, is capable of inducing epidermal thickening but not the typical appearance of psoriasis, we conclude that bacterial superantigens may differ with regard to their effects on human nonlesional psoriatic skin. Staphylococcal-enterotoxin-A-mediated effects were blocked by a genetically engineered superantigen highlighting the potential therapeutic use of mutated superantigens.

The structure of a mutant form of staphylococcal enterotoxin A (SEA) has been determined to 2.1 Å resolution. The studied SEA substitution H187→A187 (SEAH187A) leads to an almost 10-fold reduction of the binding to major histocompatibility complex (MHC) class II. H187 is important for this interaction since it coordinates Zn2+. The zinc ion is thought to hold MHC class II and SEA together in a complex. Interestingly, only one of two molecules in the asymmetric unit binds Zn2+. H225, D227, a water molecule, and H44 from a symmetry-related molecule ligate Zn2+. The symmetry-related histidine is necessary for this substituted Zn2+ site to bind to Zn2+ at low zinc concentration (no Zn2+ added). Since a water molecule replaces the missing H187, H44 binds Zn2+ at the position where βH81 from MHC class II probably will bind. Dynamic light scattering analysis reveals that in solution as well as in the crystal lattice the SEAH187A mutant forms aggregates. The substitution per se does not cause aggregation since wild-type SEA also forms aggregates. Addition of EDTA reduces the size of the aggregates, indicating a cross-linking function of Zn2+. In agreement with the biological function, the aggregation is weak (i.e. not revealed by gel filtration) and non-specific.

The superantigens staphylococcal enterotoxin A and E (SEA and SEE) can activate a large number of T-cells. SEA and SEE have approximately 80% sequence identity but show some differences in their biological function. Here, the two superantigens and analogues were characterized biophysically. SEE was shown to have a substantially higher thermal stability than SEA. Both SEA and SEE were thermally stabilized by 0.1 mM Zn(2+) compared with Zn(2+)-reduced conditions achieved using 1 mM EDTA or specific replacements that affect Zn(2+) coordination. The higher stability of SEE was only partly caused by the T-cell receptor (TCR) binding regions, whereas regions in the vicinity of the major histocompatibility complex class II binding sites affected the stability to a greater extent. SEE exhibited a biphasic denaturation between pH 5.0-6.5, influenced by residues in the TCR binding regions. Interestingly, enzyme-linked immunosorbent assay, isoelectric focusing, and circular dichroism analysis indicated that conformational changes had occurred in the SEA/E chimerical constructs relative to SEA and SEE. Thus, it is proposed that the Zn(2+) binding site is very important for the stability and potency of SEA and SEE, whereas residues in the TCR binding site have a substantial influence on the molecular conformation to control specificity and function.

The superantigens staphylococcal enterotoxin A and E (SEA and SEE) can activate a large number of T-cells. SEA and SEE have approximately 80% sequence identity but show some differences in their biological function. Here, the two superantigens and analogues were characterized biophysically. SEE was shown to have a substantially higher thermal stability than SEA. Both SEA and SEE were thermally stabilized by 0.1 mM Zn compared with Zn-reduced conditions achieved using 1 mM EDTA or specific replacements that affect Zn coordination. The higher stability of SEE was only partly caused by the T-cell receptor (TCR) binding regions, whereas regions in the vicinity of the major histocompatibility complex class II binding sites affected the stability to a greater extent. SEE exhibited a biphasic denaturation between pH 5.0–6.5, influenced by residues in the TCR binding regions. Interestingly, enzymelinked immunosorbent assay, isoelectric focusing, and circular dichroism analysis indicated that conformational changes had occurred in the SEA/E chimerical constructs relative to SEA and SEE. Thus, it is proposed that the Zn binding site is very important for the stability and potency of SEA and SEE, whereas residues in the TCR binding site have a substantial influence on the molecular conformation to control specificity and function.

The X-ray structure of the superantigen staphylococcal enterotoxin H (SEH) has been determined at 1.69 A resolution. In this paper we present two structures of zinc-free SEH (apoSEH) and one zinc-loaded form of SEH (ZnSEH). SEH exhibits the conventional superantigen (SAg) fold with two characteristic domains. In ZnSEH one zinc ion per SEH molecule is bound to the C-terminal beta-sheet in the region implicated for major histocompatibility complex class II (MHC class II) binding in SEA, SED and SEE. Surprisingly, the zinc ion has only two ligating amino acid residues His206 and Asp208. The other ligands to the zinc ion are two water molecules. An extensive packing interaction between two symmetry-related molecules in the crystal, 834 A(2)/molecule, forms a cavity that buries the zinc ions of the molecules. This dimer-like interaction is found in two crystal forms. Nevertheless, zinc-dependent dimerisation is not observed in solution, as seen in the case of SED. A unique feature of SEH as compared to other staphylococcal enterotoxins is a large negatively charged surface close to the Zn(2+) site. The interaction of SEH with MHC class II is the strongest known among the staphylococcal enterotoxins. However, SEH seems to lack a SEB-like MHC class II binding site, since the side-chain properties of structurally equivalent amino acid residues in SEH and those in SEB-binding MHC class II differ dramatically. There is also a structural flexibility between the domains of SEH. The domains of two apoSEH structures are related by a 5 degrees rotation leading to at most 3 A difference in C(alpha) positions. Since the T-cell receptor probably interacts with both domains, SEH by this rotation may modulate its binding to different TcR Vbeta-chains.

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 approximately 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 beta-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, betaH81A, 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.