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

被引:18
作者
De Marzi, MC
Fernández, MM
Sundberg, EJ
Molinero, L
Zwirner, NW
Llera, AS
Mariuzza, RA
Malchiodi, EL
机构
[1] Univ Buenos Aires, Fac Farm & Bioquim, CONICET, Catedra Inmunol, RA-1053 Buenos Aires, DF, Argentina
[2] Univ Buenos Aires, Fac Farm & Bioquim, CONICET, IDEHU, RA-1053 Buenos Aires, DF, Argentina
[3] Univ Maryland, Inst Biotechnol, WM Keck Lab Struct Biol, Ctr Adv Res Biotechnol, Rockville, MD USA
[4] Univ Buenos Aires, Fac Med, Hosp Clin Jose de San Martin, Lab Inmunogenet, RA-1053 Buenos Aires, DF, Argentina
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2004年 / 271卷 / 20期
关键词
affinity constant; biosensor; SSA; Streptococcus pyogenes; T-cell receptor;
D O I
10.1111/j.1432-1033.2004.04345.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 K-d of 9-11 mum with a fast k(ass) and a moderately fast k(diss). In spite of the reported stimulation of Vbeta2.1 bearing T-cells by SSA, we observed no measurable interaction.
引用
收藏
页码:4075 / 4083
页数:9
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