Immunochemical mapping of domains in human interleukin 4 recognized by neutralizing monoclonal antibodies.
Human interleukin 4 is a highly pleiotropic cytokine secreted by activated T cells that exerts multiple biological effects on B and T lymphocytes and other cell types. Elucidation of structure-function relations was accomplished by epitope mapping of a panel of monoclonal antibodies and by mutagenesis of selected amino acid residues. Epitope mapping of these monoclonal antibodies was achieved through binding studies with recombinant human interleukin 4 (rhuIL-4), proteolytic fragments produced by digestion with Staphylococcus aureus V8 protease and synthetic peptides derived from the sequence of the parent molecule. Monoclonal antibodies 25D2, 35F2, and 11B4 neutralized the in vitro T-cell proliferation activity of rhuIL-4 and also prevented binding of rhuIL4 to its cell surface receptor. These antibodies recognized sequences 104-129, 70-92, and 61-82, respectively. These regions comprise the BC loop/helix C (residues 61-92) and helix D (residues 104-129). A nonneutralizing monoclonal antibody (1A2) recognized a nonoverlapping region (residues 43-59) comprising almost entirely helix B. Mutagenesis of a cluster of residues within helix C showed that at least three residues (K84, R88, and N89) were potentially involved in receptor recognition. The existence of two distinct nonneighboring binding domains in the three-dimensional structure of rhuIL-4 provided preliminary evidence for a model of receptor interaction involving the formation of a ternary complex consisting of two molecules of the extracellular portion of the receptor and one molecule of rhuIL-4.
Ramanathan
L
L
Schering-Plough Research Institute, Kenilworth, New Jersey 07033.
Ingram
R
R
Sullivan
L
L
Greenberg
R
R
Reim
R
R
Trotta
P P
PP
Le
H V
HV
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D015847:Interleukin-4 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D015394:Molecular Structure; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013329:Structure-Activity Relationship; D014961:X-Ray Diffraction
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015847:Interleukin-4; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase