Abstract
We previously have assigned N-terminal specificity to three hamster monoclonal antibodies (mAbs I, II, and III) produced to mouse recombinant gamma interferon (IFN-gamma), based on the ability of the N-terminal peptide IFN-gamma-(1-39) to block binding of 125I-labeled IFN-gamma (125I-IFN-gamma) and on the ability of these antibodies to bind 125I-IFN-gamma-(1-39). Only mAb I blocked function and binding to the IFN-gamma 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-gamma. Peptides IFN-gamma-(1-39), IFN-gamma-(1-20), IFN-gamma-(3-20), and IFN-gamma-(5-20) inhibited binding of 125I-IFN-gamma by mAb I in order of decreasing effectiveness, while peptide IFN-gamma-(7-20) was without effect. Peptides IFN-gamma-(1-39), IFN-gamma-(1-20), and IFN-gamma-(3-20) also inhibited binding of 125I-IFN-gamma by mAb II but were less effective when compared with their inhibition of mAb I. IFN-gamma-(5-20) and IFN-gamma-(7-20) did not inhibit binding by mAb II. Peptides IFN-gamma-(1-10), IFN-gamma-(10-30), and IFN-gamma-(21-44) did not inhibit either mAb I or mAb II. While IFN-gamma-(1-39) and IFN-gamma-(10-30) inhibited binding by mAb III, neither IFN-gamma-(1-20) nor any of its truncated forms were inhibitory. All three antibodies had similar Kd values for 125I-IFN-gamma. A prediction of the secondary structure of the molecule and the peptide inhibition data suggest that the epitope (possible receptor binding region) 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-gamma is important in receptor binding was the observation that IFN-gamma-(1-39), but not the C-terminal IFN-gamma-(95-133), competed with 125I-IFN-gamma for the receptor on mouse L cells. IFN-gamma-(1-39) also specifically blocked IFN-gamma antiviral activity at concentrations that blocked binding to the receptor. The fact that IFN-gamma-(1-39) was the only peptide that blocked both IFN-gamma binding to receptor and function is consistent with the antibody competition data, where it was the most effective peptide in blocking binding of 125I-IFN-gamma by the N-terminal-specific mAbs. The combination of peptide mapping of epitope specificities and receptor competition should further help define the structural basis for IFN-gamma action.