The purpose of this study was to map a conformational epitope of a mAb that binds the IFN subtype α4a. Binding of this mAb, designated I-4-A, to IFN-α4a does not block receptor binding, but does neutralize biologic activity by inhibition of signal transduction. A novel strategy was developed, termed homologue scanning, which uses template-coupled polymerase chain reaction to generate hybrid molecules consisting of part (N-terminus) of the reactive IFN-α4a subtype to locate the epitope, and the remainder of the nonreactive IFN-α14 subtype to provide the overall conformation of an IFN-α molecule. Hybrid molecules were expressed as 35S-methionine-labeled proteins and tested for immunoreactivity by Western blotting and antiviral activity by cytopathic effect reduction bioassays. Unless an entire IFN-α (hybrid) molecule was formed, immunoreactivity and biologic activity were lost, indicating the importance of the C-terminus for correct folding of IFN- α molecules. The epitope for 1-4-A was localized to the N-terminal 23 residues of IFN-α4a. Furthermore, the immunoreactivity of IFN-α4a analogues, with alterations in the putative receptor-binding region of IFN- α4a residues 30 to 40 was unaffected, in contrast to the biologic activity that was reduced by several orders of magnitude. Thus, the N-terminal 23 residues of IFN-α4a, which probably are not involved in receptor binding, may be important for other interactions of the receptor-bound ligand. In general terms, the novel approach of homologue scanning, using template- coupled PCR to facilitate the generation of hybrid proteins, will have broad application in the mapping of conformational epitopes of proteins that are members of a homologous family. The ability to identify conformational epitopes will increase our understanding important interactions of proteins with antibodies, receptors, and other macromolecules.
|Number of pages||11|
|Journal||Journal of Immunology|
|Publication status||Published - 15 Jan 1994|