Evidence for conformationally different states of interleukin-10: binding of a neutralizing antibody enhances accessibility of a hidden epitope.
We present the mapping of two anti-human interleukin-10 (hIL-10) antibodies (CB/RS/2 and CB/RS/11) which have been described as binding their antigen cooperatively. The epitopes were identified using hIL-10-derived overlapping peptide scans prepared by spot synthesis. To identify residues essential for binding within the two epitopes, each position was replaced by all other L-amino acids. The epitope-derived peptides were further characterized with respect to antibody affinity and their inhibition of the antibody-hIL-10 interaction. One antibody (CB/RS/11) binds to residues which are completely buried in the X-ray structure of IL-10. Accessibility of this hidden epitope is enhanced upon binding of the antibody CB/RS/2, which recognizes a discontinuous epitope located nearby. The recognition of the hidden CB/RS/11 epitope, as well as the cooperative binding behaviour of the two antibodies, provides evidence that IL-10 can adopt a conformational state other than that observed in the crystal structure.
Reineke
U
U
Institut für Medizinische Immunologie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Campus Mitte, D-10098 Berlin, Germany.
Schneider-Mergener
J
J
Glaser
R W
RW
Stigler
R D
RD
Seifert
M
M
Volk
H D
HD
Sabat
R
R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000378:metabolism* / Q000494:pharmacology; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D016753:Interleukin-10 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016753:Interleukin-10