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Functional analysis of the disulphide loop mutant of staphylococcal enterotoxin C2

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The superantigen staphylococcal enterotoxin C2 (SEC2) tremendously activate T lymphocytes bearing certain T-cell receptor Vβ domains when binding to MHC II molecules, which launches a powerful response of tumour inhibition in vitro as well as in vivo. However, the toxicity of SEC2 performed in clinic limited its broad application for immunotherapy. The previous studies suggested that the disulphide loop may be important for the toxicity of some SEs, which prompted us to investigate the potential roles of the disulphide loop in biological activity of SEC2. Site-directed mutagenesis was used to disturb the formation of the disulphide bond by substituting Ala or Ser for Cys-93 and Cys-110. The expressed mutants in Escherichia coli were used to determine their superantigen activity and toxicity. Results showed that all of the mutated proteins exhibited reduced abilities to induce T-cell proliferation and cytotoxic effects on tumour cells L929 and Hepa1-6, suggesting that the disulphide loop plays functional role in maintaining the maximal superantigen activity of SEC2. Furthermore, the toxicity assays in vivo showed that all of the mutants induced a reduced emetic and pyrogenic responses compared with native SEC2, which might be important for further construction of lowly toxic superantigen agent.

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  1. Acharya KR, Passalacqua EF, Jones EY, Harlos K, Stuart DI, Brehm RD, Tranter HS (1994) Structural basis of superantigen action inferred from crystal structure of toxic-shock syndrome toxin-1. Nature 367:94–97

  2. Anonymous (2002) The United States Pharmacopeia. U.S. Pharmacopeial Convention, Rockville

  3. Bohach GA, Fast DJ, Nelson RD, Schlievert PM (1990) Staphylococcal and streptococcal pyrogenic toxins involved in toxic shock syndrome and related illnesses. Crit Rev Microbiol 17:251–272

  4. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochemistry 72:248–254

  5. Chen TZ (2001) The exploitation of HAS and its application in tumor therapy. Prog Microbiol Immunol China 29:63–69

  6. Clark WG, Page JS (1968) Pyrogenic responses to staphylococcal enterotoxins A and B in cats. J Bacteriol 96:1940–1946

  7. Creighton TE (1988) Disulphide bonds and protein stability. Bioessays 8:57–63

  8. Dellabonna P, Peccoud J, Kappler J, Marrack P, Benoist C, Mathis D (1990) Superantigens interact with MHC class II molecules outside of the antigen groove. Cell 62:1115–1121

  9. Dinges MM, Orwin PM, Schlievert PM (2000) Exotoxins of Staphylococcus aureus. Clin Microbiol Rev 13:16–34

  10. Fraser JD (1989) High-affinity binding of staphylococcal enterotoxins A and B to HLA-DR. Nature 339:221–223

  11. Grossman D, Cook RD, Sparrow JT, Mollick JA, Rich RR (1990) Dissociation of the stimulatory activities of staphylococcal enterotoxins for T cells and monocytes. J Exp Med 172:1831–1841

  12. Grossman D, Van M, Mollick JA, Highlander SK, Rich RR (1991) Mutation of the disulfide loop in staphylococcal enterotoxin A. Consequences for T cell recognition. J Immunol 147:3274–3281

  13. Gupta A, Van Vlijmen HWT, Singh J (2004) A classification of disulfide patterns and its relationship to protein structure and function. Protein Sci 13:2045–2058

  14. Hansson J, Ohlsson L, Persson R, Andersson G, Ilbäck NG, Litton MJ, Kalland T, Dohlsten M (1997) Genetically engineered superantigens as tolerable antitumor agents. Proc Natl Acad Sci U S A 94:2489–2494

  15. Harris TO, Betley MJ (1995) Biological activities of staphylococcal enterotoxin type A mutants with N-terminal substitutions. Infect Immun 63:2133–2140

  16. Harris TO, Grossman D, Kappler JW, Marrack P, Rich RR, Betley MJ (1993) Lack of complete correlation between emetic and T-cell-stimulatory activities of staphylococcal enterotoxins. Infect Immun 61:3175–3183

  17. Hedlund G, Dohlsten M, Petersson C, Kalland T (1993) Superantigen-based tumor therapy: in vivo activation of cytotoxic T cells. Cancer Immunol Immunother 36:89–93

  18. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59

  19. Hoffman M, Tremaine M, Mansfield J, Betley M (1996) Biochemical and mutational analysis of the histidine residues of staphylococcal enterotoxin A. Infect Immun 64:885–890

  20. Hovde CJ, Marr JC, Hoffmann ML, Hackett SP, Chi YI, Crum KK, Stevens DL, Stauffacher CV, Bohach GA (1994) Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1. Mol Microbiol 13:897–909

  21. Hufnagle WO, Tremaine MT, Betley MJ (1991) The carboxylterminal region of staphylococcal enterotoxin A is required for a fully active molecule. Infect Immun 59:2126–2134

  22. Hui J, Cao Y, Xiao F, Zhang J, Li H, Hu FQ (2008) Staphylococcus aureus enterotoxin C2 mutants: biological activity assay in vitro. J Ind Microbial Biotechnol 35:975–980

  23. Lamphear JG, Bohach GA, Rich RR (1998) Structural dichotomy of Staphylococcal enterotoxin C superantigens leading to MHC Class II-independent activation of T lymphocytes. J Immunol 160:2107–2114

  24. Letertre C, Perelle S, Dilasser F, Fach P (2003) Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus. J Appl Microbiol 95:38–43

  25. Martin WJ, Marcus S (1964) Relation of pyrogenic and emetic properties of enterobacteriaceal endotoxin and of staphylococcal enterotoxin. J Bacteriol 87:1019–1026

  26. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

  27. Noskova VP, Ezepchuk YV, Noskov AN (1984) Topology of the functions in molecule of staphylococcal enterotoxin type A. Int J Biochem 16:201–206

  28. Ochi A, Migita K, Xu J, Siminovitch K (1993) In vivo tumor immunotherapy by a bacterial superantigen. J Immunol 151:3180–3186

  29. Omoe K, Imanishi K, Hu DL, Kato H, Fugane Y, Abe Y, Hamaoka S, Watanabe Y, Nakane A, Uchiyama T, Shinagawa K (2005) Characterization of novel staphylococcal enterotoxin-like toxin type p. Infect Immun 73:5540–5546

  30. Orwin PM, Leung DY, Donahue HL, Novick RP, Schlievert PM (2001) Biochemical and biological properties of staphylococcal enterotoxin K. Infect Immun 69:360–366

  31. Papageorgiou AC, Acharya KR, Shapiro R, Passalacqua EF, Brehm R, Tranter HS (1995) Crystal structure of the superantigen enterotoxin C2 from Staphylococcus aureus reveals a zinc-binding site. Structure 3:769–779

  32. Pardoll DM (1995) Parakrine cytokine adjuvants in cancer immunotherapy. Annu Rev Immunol 13:399

  33. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci 12:5436–5467

  34. Schad EM, Zaitseva I, Zaitseva VN, Dohlsten M, Kalland T, Schlievert PM, Ohlendorf DH, Svensson LA (1995) Crystal structure of the superantigen staphylococcal enterotoxin type A. EMBO J 14:3292–3301

  35. Schad EM, Papageorgiou AC, Svensson LA, Acharya KR (1997) A structural and functional comparison of staphylococcal enterotoxins A and C2 reveals remarkable similarity and dissimilarity. J Mol Biol 269:270–280

  36. Schlievert PM, Jablonski LM, Roggiani M, Sadler I, Callantine S, Mitchell DT, Ohlendorf DH, Bohach GA (2000) Pyrogenic toxin superantigen site specificity in toxic shock syndrome and food poisoning in animals. Infect Immun 68:3630–3634

  37. Swaminathan S, Furey W, Pletcher J, Sax M (1992) Crystal structure of staphylococcal enterotoxin B, a superantigen. Nature 359:801–806

  38. Xu MK, Zhang CG (2006) Gene expression and function study of fusion immunotoxin anti-Her-2-scFv-SEC2 in Escherichia coli. Appl Microbiol Biotechnol 70:78–84

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This work was supported by a grant from the Ph.D. programs initializing foundation of the Institute of Applied Ecology of the CAS (08SBS111S3) and Shenyang Xiehe Bio-pharmaceutical Co. Ltd.

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Correspondence to Mingkai Xu.

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Wang, X., Xu, M., Cai, Y. et al. Functional analysis of the disulphide loop mutant of staphylococcal enterotoxin C2. Appl Microbiol Biotechnol 82, 861–871 (2009).

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  • Superantigen
  • Staphylococcal enterotoxin C2
  • Immunotherapy
  • Toxicity
  • The disulphide loop
  • Site-directed mutagenesis