Cloning, expression and interaction of human T-cell receptors with the bacterial superantigen SSA

MAURICIO DE MARZI; MARISA FERNÁNDEZ; ERIC SUNDBERG; LUCIANA MOLINERO; NORBERTO ZWIRNER; ANDREA LLERA; ROY MARIUZZA; EMILIO MALCHIODI

EUROPEAN JOURNAL OF BIOCHEMISTRY

FEBS

Año: 2004 vol. 271 p. 4075 - 4083

0014-2956

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 Vb5.2, Vb1 and Vb2.1, expressed as inclusion bodies, refolded and puri.ed. 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 disul.de 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 ofb domain of T-cell receptors (TCRs). In this study, recombinant TCR b chains were constructed with human variable domains Vb5.2, Vb1 and Vb2.1, expressed as inclusion bodies, refolded and puri.ed. 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 disul.de 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 ofb chains were constructed with human variable domains Vb5.2, Vb1 and Vb2.1, expressed as inclusion bodies, refolded and puri.ed. 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 disul.de 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 ofb5.2, Vb1 and Vb2.1, expressed as inclusion bodies, refolded and puri.ed. 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 disul.de 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 ofStreptococcus 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 disul.de 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 ofSAg 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 disul.de 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. A.nity assays using a resonant biosensor showed that both the mutant and monomeric wild type SSA have a.nity for human Vb5.2 and Vb1 with Kd of 9–11 lM with a fast kassisolates. An SSA mutant [SSA(C26S)] was produced to eliminate the Cys responsible for dimerization. A.nity assays using a resonant biosensor showed that both the mutant and monomeric wild type SSA have a.nity for human Vb5.2 and Vb1 with Kd of 9–11 lM with a fast kassb5.2 and Vb1 with Kd of 9–11 lM with a fast kass and a moderately fast kdiss. In spite of the reported stimulation of Vb2.1 bearing T-cells by SSA, we observed no measurable interaction.kdiss. In spite of the reported stimulation of Vb2.1 bearing T-cells by SSA, we observed no measurable interaction.b2.1 bearing T-cells by SSA, we observed no measurable interaction.