Immunological and Biochemical Characterization of Streptococcal Pyrogenic Exotoxins I and J (SPE-I and SPE-J) from Streptococcus pyogenes 1 Thomas Proft,* Vickery L. Arcus, ² Vanessa Handley,* Edward N. Baker, ² and John D. Fraser 2 * Recently, we described the identification of novel streptococcal superantigens (SAgs) by mining the Streptococcus pyogenes M1 genome database at Oklahoma University. Here, we report the cloning, expression, and functional analysis of streptococcal pyrogenic exotoxin (SPE)-J and another novel SAg (SPE-I). SPE-I is most closely related to SPE-H and staphylococcal enterotoxin I, whereas SPE-J is most closely related to SPE-C. Recombinant forms of SPE-I and SPE-J were mitogenic for PBL, both reaching half maximum responses at 0.1 pg/ml. Evidence from binding studies and cell aggregation assays using a human B-lymphoblastoid cell line (LG-2) suggests that both toxins exclusively bind to the polymorphic MHC class II -chain in a zinc-dependent mode but not to the generic MHC class II -chain. The results from analysis by light scattering indicate that SPE-J exists as a dimer in solution above concentrations of 4.0 mg/ml. Moreover, SPE-J induced a rapid homotypic aggregation of LG-2 cells, suggesting that this toxin might cross-link MHC class II molecules on the cell surface by building tetramers of the type HLA-DR–SPE-J–SPE- J–HLA-DR. SPE-I preferably stimulates T cells bearing the V18.1 TCR, which is not targeted by any other known SAg. SPE-J almost exclusively stimulates V2.1 T cells, a V that is targeted by several other streptococcal SAgs, suggesting a specific role for this T cell subpopulation in immune defense. Despite a primary sequence diversity of 51%, SPE-J is functionally indistin- guishable from SPE-C and might play a role in streptococcal disease, which has previously been addressed to SPE-C. The Journal of Immunology, 2001, 166: 6711– 6719. S treptococcus pyogenes is a major human pathogenic bac- terium that causes a wide range of diseases including acute tonsillitis, streptococcal toxic shock syndrome, scarlet fe- ver, necrotizing fasciitis, cellulitis, and bacteremia (1– 4). This Gram-positive bacterium produces a variety of exotoxins, known as streptococcal pyrogenic exotoxins (SPEs), 3 which are believed to be involved in pathogenicity or virulence. Together with the staphylococcal enterotoxins (SEs) and the toxic shock syndrome toxin (TSST) produced by Staphylococcus aureus, they build a larger family of structurally related proteins (5–7). These proteins are also known as superantigens (SAgs), due to their ability to stimulate large populations of T cells (8, 9). In contrast to conventional Ags, SAgs are not processed inside APCs, but instead directly bind to the MHC class II protein outside the Ag binding groove (10 –14). Simultaneously, they bind to all TCRs bearing particular V regions (15, 16). This trimolecular complex subsequently cross-links a large number of APCs and T cells resulting in the production of high systemic levels of the cytokines TNF- and IL-1 and of T cell mediators, such as IL-2 and IFN- (8, 17–19). Thus far, four streptococcal SAgs have been identified after pu- rification from cell culture supernatants. These are SPE-A (20), SPE-C (21), streptococcal mitogenic exotoxin Z (SMEZ) (22), and streptococcal SAg (SSA) (23). Recently, two novel sag genes (spe-g and spe-h) and one incomplete sag gene (spe-j) have been identified by screening the incomplete S. pyogenes M1 genome database at Oklahoma University (24, 25). The predicted superan- tigenic properties of SPE-G and SPE-H have been confirmed by biochemical and immunological analysis of the corresponding re- combinant proteins (24). SPE-B and SPE-F were originally added to the list of strepto- coccal SAgs, but this has been controversial. Both proteins are genetically unrelated to the streptococcal and staphylococcal SAgs, and the superantigenic properties of SPE-B (streptococcal cysteine protease) were shown to be due to contamination (15). There are now crystal structures for 11 SAgs: SPE-A (26), SPE-C (27), SPE-H (28), SMEZ-2 (28), and SSA (29), and the staphylococcal toxins SEA (30), SEB (31), SEC2 (32), SED (33), SEH (34), and TSST (35). Despite the limited primary sequence homology (sometimes 25%), all structures show a conserved folding pattern, comprising a NH 2 -terminal -barrel globular do- main and a COOH-terminal globular domain based on a -grasp motif. All examined staphylococcal SAgs, as well as the streptococcal SSA and SPE-A, have a generic binding site for the invariant -chain of MHC class II located in the NH 2 -terminal domain (10, 19). In contrast, SPE-C, SPE-G, SPE-H, and all SMEZ variants bind the polymorphic MHC class II -chain, probably mediated by a zinc coordination complex between three SAg residues and the highly conserved His81 of the HLA-DR1 -chain (24, 27, 36). SEA and SEE combine both binding modes to cross-link MHC *Division of Molecular Medicine and ² School of Biological Sciences, University of Auckland, Auckland, New Zealand Received for publication January 10, 2001. Accepted for publication March 27, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Health Research Council of New Zealand. 2 Address correspondence and reprint requests to Prof. John D. Fraser, Division of Molecular Medicine, School of Medicine, University of Auckland, Private Bag 92019, Auckland, New Zealand. E-mail address: jd.fraser@auckland.ac.nz 3 Abbreviations used in this paper: SPE, streptococcal pyrogenic exotoxin; SE, staph- ylococcal enterotoxin; TSST, toxic shock syndrome toxin; SAg, superantigen; SMEZ, streptococcal mitogenic exotoxin Z; SSA, streptococcal SAg; RPMI-10, RPMI 1640 with 10% FCS; SCRs, structurally conserved regions; pI, isoelectric point. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00