TAILIEUCHUNG - Báo cáo khoa học: Cloning, expression and interaction of human T-cell receptors with the bacterial superantigen SSA

Superantigens (SAgs) are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate a large number of T-cells through interaction with the Vbdomain of T-cell receptors (TCRs). In this study, recombinant TCRbchains were constructed with human variable domains , Vb1and , expressed as inclusion bodies, refolded and purified. The Streptococcus pyogenesSAg SSA-1 was cloned and expressed as a soluble periplasmicprotein. SSA-1wasobtainedbothas amonomer and a dimer that has an intermolecular disulfide bond. . | Eur. J. Biochem. 271 4075-4083 2004 FEBS 2004 doi Cloning expression and interaction of human T-cell receptors with the bacterial superantigen SSA Mauricio C. De Marzi1 Marisa M. Fernandez1 Eric J. Sundberg2 Luciana Molinero3 Norberto W. Zwirner3 Andrea S. Llera1 i Roy A. Mariuzza2 and Emilio L. Malchiodi1 lCátedra de Inmunologia and Instituto de Estudios de la Inmunidad Humoral IDEHU CONICET Facultad de Farmacia y Bioqulmica Universidad de Buenos Aires Argentina 2Center for Advanced Research in Biotechnology W. M. Keck Laboratory for Structural Biology University of Maryland Biotechnology Institute Rockville MD USA 3Laboratorio de Inmunogenetica Hospital de Clinicas Jose de San Martin Facultad de Medicina Universidad de Buenos Aires Argentina Superantigens SAgs are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate a large number of T-cells through interaction with the Vb domain of T-cell receptors TCRs . In this study recombinant TCR b chains were constructed with human variable domains Vb1 and expressed as inclusion bodies refolded and purified. The Streptococcus pyogenes SAg SSA-1 was cloned and expressed as a soluble periplasmic protein. SSA-1 was obtained both as a monomer and a dimer that has an intermolecular disulfide bond. We analyzed the biological activity of the recombinant SAgs by proliferation assays. The results suggest that SSA dimerization occludes the TCR interaction site. Naturally occurring SSA dimerization was also observed in supernatants of S. pyogenes isolates. An SSA mutant SSA C26S was produced to eliminate the Cys responsible for dimerization. Affinity assays using a resonant biosensor showed that both the mutant and monomeric wild type SSA have affinity for human and Vb1 with Kd of 9-11 M with a fast kass and a moderately fast kdiss. In spite of the reported stimulation of bearing T-cells by SSA we observed no measurable .

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