Functional Analysis of the Antigen Binding Site

نویسندگان

  • Eric A. Nalefski
  • Shailaja Kasibhatla
  • Anjana Rao
چکیده

We have identified residues on a T ce]l receptor (TCR) ot chain that are important for interaction with antigen/major histocompatibility complex (MHC). Using site-directed mutagenesis, we modified DNA encoding the postulated antigen/MHC binding loops on the TCR r chain expressed by the T cell done D5, which recognizes p-azobenzenearsonate--conjugated antigens presented by ceUs bearing I-A a. These variant TCR a chains were expressed in conjunction with the wildtype D5 TCR 13 chain on the surface of hybridoma cells, and were tested for the ability to recognize hapten-conjugated antigens presented by I-A a. Individual amino acid substitutions in each of the three antigen binding loops (or1, cx2, c~3) of the D5 TCR ot chain affected antigen recognition, demonstrating that all three loops are important in recognition of antigen/MHC. A subset of the single amino acid substitutions completely eliminated antigen recognition, thus identifying the residues that are particularly important in the recognition of antigenic peptide/MHC by the D5 TCR. Because the wild-type D5 TCR recognizes arsonate and certain structural analogues of arsonate conjugated to a variety of protein antigens, we were able to test whether the TCR substitutions affected the specificity of the D5 TCR for hapten or carrier antigen. One substitution introduced into antigen binding loop or3 markedly altered the pattern of carrier recognition. Together, these results verify the Ig model for the TCR and are consistent with the proposition that residues forming the first and second antigen binding loops of the TCR contact the MHC, while those forming the third loop contact mainly antigenic peptides.

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تاریخ انتشار 2003