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 Vbeta domain of T-cell receptors (TCRs). In this study, recombinant TCR beta chains were constructed with human variable domains Vbeta5.2, Vbeta1 and Vbeta2.1, 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 Vbeta5.2 and Vbeta1 with Kd of 9-11 microm with a fast kass and a moderately fast kdiss. In spite of the reported stimulation of Vbeta2.1 bearing T-cells by SSA, we observed no measurable interaction.
Cátedra de Inmunología and Instituto de Estudios de la Inmunidad Humoral (IDEHU), CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Mauricio C
MC
De Marzí
Marisa M
MM
Fernández
Eric J
EJ
Sundberg
Luciana
L
Molinero
Norberto W
NW
Zwirner
Andrea S
AS
Llera
Roy A
RA
Mariuzza
Emilio L
EL
Malchiodi
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D004306:Dose-Response Relationship, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D008213:Lymphocyte Activation; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000276:immunology*; D018089:Superantigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism
D000942:Antigens, Bacterial; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins; D018089:Superantigens