Open AccessCloning, expression and interaction of human T‐cell receptors with the bacterial superantigen SSA
Author(s) -
De Marzi Mauricio C.,
Fernández Marisa M.,
Sundberg Eric J.,
Molinero Luciana,
Zwirner Norberto W.,
Llera Andrea S.,
Mariuzza Roy A.,
Malchiodi Emilio L.
Publication year - 2004
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2004.04345.x
Subject(s) - superantigen , t cell receptor , streptococcus pyogenes , recombinant dna , periplasmic space , microbiology and biotechnology , mutant , biology , receptor , t cell , chemistry , biochemistry , escherichia coli , gene , staphylococcus aureus , bacteria , genetics , immune system
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 Vβ domain of T‐cell receptors (TCRs). In this study, recombinant TCR β chains were constructed with human variable domains Vβ5.2, Vβ1 and Vβ2.1, 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 Vβ5.2 and Vβ1 with K d of 9–11 µ m with a fast k ass and a moderately fast k diss . In spite of the reported stimulation of Vβ2.1 bearing T‐cells by SSA, we observed no measurable interaction.