Use of synthetic peptides to identify an N-terminal epitope on mouse γ interferon that may be involved in function

The authors previously have assigned N-terminal specificity to three hamster monoclonal antibodies (mAbs I, II, and III) produced to mouse recombinant γ interferon (IFN-γ), based on the ability of the N-terminal peptide IFN-γ-(1-39) to block binding of 125I-labeled IFN-γ (125I-IFN-γ) and on the ability of these antibodies to bind 125I-IFN-γ-(1-39). Only mAb I blocked function and binding to the IFN-γ receptor, suggesting that it may bind to a region of the molecule involved in interaction with the receptor. To further define the epitope specificities of the antibodies, a series of N-terminal peptides were synthesized and tested for their ability to block antibody binding of 125I-IFN-γ. All three antibodies had similar K/sub d/ values for 125I-IFN-γ. A prediction of the secondary structure of the molecule and the peptide inhibition data suggest that the epitope for mAb I involves a loop in the area containing residues 12-20, with sequences N-terminal to these residues possibly stabilizing the loop conformation. Direct evidence that the N-terminal 1-39 region of IFN-γ is important in receptor binding was the observation that IFN-γ-(1-39), but not the C-terminal IFN-γ-(95-133), competed with 125I-IFN-γ for the receptor on mouse L cells. The fact that IFN-γ-(1-39) was the only peptide that blocked both IFN-γ binding to receptor and function is consistent with the antibody competition data