Mutational analysis of the binding of staphylococcal enterotoxin D to the T cell receptor Vbeta chain and major histocompatibility complex class II.
In an attempt to define the binding of staphylococcal enterotoxin D (SED) to the T cell receptor Vbeta (TCR Vbeta) 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 Vbeta 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 Vbeta5 were significantly reduced. When stimulated with SED-F45A, T cells bearing TCR Vbeta5, TCR Vbeta8 and TCR Vbeta12.1 were all significantly reduced. These results suggest Asn23 is an important residue involved SED interacting with TCR Vbeta; Phe45 is required for effective interaction with MHC class II molecules; and the ability of SED stimulating certain TCR Vbeta+ T cells is dependent on Phe45 binding to MHC class II molecules.
Li
Yafei
Y
Department of Epidemiology, Third Military Medical University, Chongqing 400038, PR China. yafeilye@yahoo.com.cn
Zhu
Xihua
X
Huang
Yunhui
Y
Xiong
Hongyan
H
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008213:Lymphocyte Activation / Q000187:drug effects; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000378:metabolism*; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology
D004269:DNA, Bacterial; D004768:Enterotoxins; D000949:Histocompatibility Antigens Class II; D016693:Receptors, Antigen, T-Cell, alpha-beta; C019368:enterotoxin D, Staphylococcal