Structure-function analysis of human IFN-alpha. Mapping of a conformational epitope by homologue scanning.
The purpose of this study was to map a conformational epitope of a mAb that binds the IFN subtype alpha 4a. Binding of this mAb, designated I-4-A, to IFN-alpha 4a 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-alpha 4a subtype to locate the epitope, and the remainder of the nonreactive IFN-alpha 14 subtype to provide the overall conformation of an IFN-alpha 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-alpha (hybrid) molecule was formed, immunoreactivity and biologic activity were lost, indicating the importance of the C-terminus for correct folding of IFN-alpha molecules. The epitope for I-4-A was localized to the N-terminal 23 residues of IFN-alpha 4a. Furthermore, the immunoreactivity of IFN-alpha 4a analogues, with alterations in the putative receptor-binding region of IFN-alpha 4a 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-alpha 4a, 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.
Wang
L
L
Centre for Molecular Biology and Medicine, Monash University, Melbourne, Australia.
Hertzog
P J
PJ
Galanis
M
M
Overall
M L
ML
Waine
G J
GJ
Linnane
A W
AW
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000998:Antiviral Agents / Q000737:chemistry; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D000939:Epitopes; D006801:Humans; D016898:Interferon-alpha / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000998:Antiviral Agents; D017931:DNA Primers; D000939:Epitopes; D016898:Interferon-alpha; D011994:Recombinant Proteins