Sequence analysis of the gene for a novel superantigen produced by Yersinia pseudotuberculosis and expression of the recombinant protein.
We previously reported that the Gram-negative bacterium Yersinia pseudotuberculosis produces a superantigen (YPM, Y. pseudotuberculosis-derived mitogen) that expands T cells bearing V betas 3, 9, 13.1, and 13.2 in an MHC class II-dependent manner. Based on the previously determined N-terminal 23 amino acids of YPM (T-D-Y-D-N-T-L-N-S-I-P-S-L-R-I-P-N-I-A-T-Y-T-G- (one-letter code)), we cloned the ypm gene and analyzed the nucleotide sequence. The gene encodes a 151-amino acid protein with a 20-amino acid signal peptide at its N terminus. The recombinant YPM expressed by the cloned gene exerted a mitogenic activity on human PBMC at a concentration of approximately 1 pg/ml. T cells bearing V beta 13.3 were preferentially expanded as well as T cells bearing the same V beta repertoires stimulated by native YPM. T cells were stimulated by the recombinant YPM in the presence of either fixed or unfixed HLA class II-transfected mouse fibroblasts. Furthermore, sequence diversity in the junctional region of the TCR beta-chain containing the V beta 3 element could be observed after stimulation by the recombinant YPM. These results indicate that YPM belongs to the category of superantigens and should be included as a novel member. The amino acid sequence of the mature protein showed no significant homology to other superantigens derived from Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pyogenes. This observation, together with the substantially smaller m.w. suggest that ypm must have evolved from a different ancestral gene.
Ito
Y
Y
Department of Allergy and Immunology, National Children's Medical Research Center, Tokyo, Japan.
Abe
J
J
Yoshino
K
K
Takeda
T
T
Kohsaka
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D018089:Superantigens / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection; D015011:Yersinia pseudotuberculosis / Q000276:immunology*
D001426:Bacterial Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins; D018089:Superantigens; C093313:Yersinia pseudotuberculosis-derived mitogen