Structure-function analysis of human IFN-alpha. Mapping of a conformational epitope by homologue scanning

J Immunol. 1994 Jan 15;152(2):705-15.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal / immunology
  • Antiviral Agents / chemistry
  • Base Sequence
  • DNA Primers / chemistry
  • Epitopes
  • Humans
  • Interferon-alpha / chemistry
  • Interferon-alpha / immunology*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Structure, Tertiary
  • Recombinant Proteins
  • Structure-Activity Relationship

Substances

  • Antibodies, Monoclonal
  • Antiviral Agents
  • DNA Primers
  • Epitopes
  • Interferon-alpha
  • Recombinant Proteins