Mapping of neutralizing epitopes and the receptor binding site of human interleukin 1 beta.
Antibodies to synthetic peptides of human interleukin 1 beta (IL-1 beta) and to recombinant human IL-1 beta were used to identify epitopes of IL-1 beta associated with the neutralization of its biological activity. Analysis of antisera raised to 17 synthetic peptides derived from the mature IL-1 beta sequence showed that five regions (residues 6-15, 49-80, 58-80, 92-101, and 120-133) were both immunoprecipitating and neutralizing. Using a hexamer epitope mapping method, comparison of the regions recognized by four neutralizing rabbit antisera with those recognized by a rabbit antiserum raised to denatured IL-1 beta suggested two further neutralizing epitopes, residues 39-48 and 83-95. Finally, a neutralizing monoclonal antibody was shown to bind to the peptides 6-11 and 87-95 by peptide binding and mutagenesis. All of these regions appear predominantly on one face of IL-1 beta. The effect of mutations in residues 4-11 and 88-97, which lie within this face, on receptor binding and biological activity was determined. Most of the mutations tested affected both receptor binding and activity, whereas mutations in another face of IL-1 beta (residues 74-80) had no effect. Purification of two of the mutants with reduced bioactivity and receptor binding and analysis by two-dimensional NMR indicated no gross changes in tertiary structure. A third mutant had reduced bioactivity in two different bioassays but no change in receptor binding. Although two-dimensional NMR revealed no gross changes in conformation, small changes did occur at a site distal from that mutated. The data are consistent with other epitope mapping and receptor binding mutagenesis data and suggest that the neutralizing antibodies and receptor recognize different but overlapping regions of IL-1 beta.
Simon
P L
PL
Department of Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939.
Kumar
V
V
Lillquist
J S
JS
Bhatnagar
P
P
Einstein
R
R
Lee
J
J
Porter
T
T
Green
D
D
Sathe
G
G
Young
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D000939:Epitopes / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008223:Lymphoma; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011487:Protein Conformation; D017472:Receptors, Interleukin-1 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007375:Interleukin-1; D010455:Peptides; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins