Mutational analysis of the binding of staphylococcal enterotoxin D to the T cell receptor Vβ chain and major histocompatibility complex class II

In an attempt to define the binding of staphylococcal enterotoxin D (SED) to the T cell receptor Vβ (TCR Vβ) and major histocompatibility complex (MHC) class II molecules, site-directed mutagenesis has been used to introduce alanine substitutions at Asn23, Phe45, Leu59, Asn61, Ile92 and Phe203 in SED. SED-N23A (SEA with Asn23 replaced with alanine) and SED-F45A mutants exhibit a significantly reduced ability to induce T cell proliferation. However, SED-L59A, SED-N61A, SED-I92A and SED-F203A mutants exhibit the normal mitogenic activity. The ability binding to MHC class II and the TCR Vβ specificity of SED-N23A and SED-F45A were then detected. SED-N23A, but not SED-F45A, was able to compete effectively with FITC-conjugated SED for binding to Raji cells. This finding indicates that the mitogenic activity defection of SED-N23A is not due to poor binding to SED-MHC class II molecules. When stimulated with SED-N23A, T cells bearing TCR Vβ5 were significantly reduced. When stimulated with SED-F45A, T cells bearing TCR Vβ5, TCR Vβ8 and TCR Vβ12.1 were all significantly reduced. These results suggest Asn23 is an important residue involved SED interacting with TCR Vβ; Phe45 is required for effective interaction with MHC class II molecules; and the ability of SED stimulating certain TCR Vβ+ T cells is dependent on Phe45 binding to MHC class II molecules.