Reconstruction of the immunogenic peptide RNase(43-56) by identification and transfer of the critical residues into an unrelated peptide backbone.
The involvement of each of the amino acid residues of the I-Ak-restricted T cell determinant RNase(43-56) was examined in detail using a series of peptides containing single amino acid substitutions. Four positions were identified as being essential for the formation of the determinant, Phe-46, Val-47, His-48, and Leu-51. When these four residues were substituted into the backbone of the unrelated peptide HA(130-144), a nonstimulatory peptide was obtained. The inclusion of an additional residue, Val-54, resulted in a chimeric peptide, RN/HA2, which was nearly as active as the native molecule. The peptide RN/HA2 was able to prime in vivo for RNase reactivity, confirming that these five residues contained all of the specificity of the RNase(43-56) determinant. The role of three of these critical residues was examined using both a functional competition assay and an in vivo priming assay. It was ascertained that the Phe-46 was directly involved in contacting the TCR, while the His-48 and Leu-51 were either involved in binding to the I-Ak molecule or in determining the conformation of the peptide. Thus, by critically evaluating the contribution of each of the amino acid residues in a T cell determinant, we were able to generate a chimeric peptide only containing 5 of 15 residues from the RNase(43-56) sequence that was functionally identical to the native RNase(43-56) molecule both in vitro and in vivo.
Lorenz
R G
RG
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Tyler
A N
AN
Allen
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D002678:Chimera; D004261:DNA Replication; D000939:Epitopes / Q000235:genetics; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D006825:Hybridomas; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation; D008285:Major Histocompatibility Complex; D051379:Mice; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D012260:Ribonucleases / Q000235:genetics / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*
D000939:Epitopes; D000949:Histocompatibility Antigens Class II; D007376:Interleukin-2; D012260:Ribonucleases