A chimeric class I glycoprotein was created to investigate the functional contribution of the alpha helices and the beta-pleated sheets in forming the antigen recognition site (ARS) of antigen-presenting molecules. This novel molecule was generated by replacing the DNA sequences encoding the alpha helices of the Ld gene with the corresponding sequences from the Kb gene. Serologic analysis of transfected L cells that expressed the chimeric molecule (Kb alpha Ld beta) revealed that the engineered class I glycoprotein retains two conformational epitopes associated with the alpha helices of Kb, as defined by monoclonal antibodies K10.56 and 28-13-3. These results demonstrate that the alpha helices of Kb can associate with the beta-pleated sheets of Ld to form a stable structure, which is expressed on the cell surface. To address the role of the alpha helices of the ARS in determining T cell crossreactivity, alloreactive cytotoxic T lymphocytes (CTL) were used to analyze L cells expressing Kb alpha Ld beta. CTL raised against Kb or Ld as alloantigens showed little, if any, ability to lyse L cells expressing Kb alpha Ld beta. Thus, alloreactive CTL did not recognize structures determined by the alpha helices alone or by the beta sheets of the ARS alone. However, bulk and cloned alloreactive CTL that were generated against the mutant Kb glycoprotein Kbm8 reacted strongly with Kb alpha Ld beta. In addition to the Kb alpha helices, the Kbm8 ARS shares a single polymorphic amino acid at position 24 with Kb alpha Ld beta. Amino acid 24 is located on the beta 2 strand that forms part of the floor of the ARS and has been identified as a component of pocket B in the HLA class I ARS. The substitution of Glu to Ser at this position was shown previously to be the central determinant of the Kbm8 mutant alloantigenicity. The functional significance of this position in determining crossreactivity between bm8 and Kb alpha Ld beta identifies pocket B as a strong anchor for allogenic self-peptides. These findings demonstrate that determinants recognized by CTL on class I alloantigens are formed by interactions involving both the alpha helices and beta sheets of the ARS. These interactions are best explained by the influence of the alpha helices and beta sheets on the peptide-binding properties of these antigen-presenting molecules.

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