Quaternary structure-dependent idiotope and antigen binding of a monoclonal antibody specific for conformational epitope on type II collagen.
We previously generated a monoclonal antibody (mAb) against a putative pathogenic epitope on native type II collagen (CII) for the induction of collagen-induced arthritis in mice (mAb1), and an anti-idiotypic mAb which appears to possess the internal image of the CII epitope (mAb2). In the present study, the structural basis of the antigen/mAb1 and mAb1/mAb2 interactions was examined. When partially SH-reduced mAb1 was analysed on Western blots, only fragments containing both heavy (H) and light (L) chains were recognized by mAb2. When mAb2 was partially SH-reduced, only fragments containing both H and L chains were recognized by mAb1. H and L chains were separated from mAb1 in a reduced, denatured condition, and each chain and a mixture of the two were refolded. mAb2 reacted specifically to the renatured whole IgG molecule of mAb1, but not to the refolded L or to H chains. Recombinant single chain Fv (scFv) generated from mAb1 and mAb2 had properties of the original mAbs, whereas genetically constructed chimeric scFvs, consisting of VH from mAb1 and an irrelevant VL. or VL of mAb1 and an irrelevant VH. did not react either to CII or to mAb2. Thus, interactions among CII, mAb1 and mAb2 appear to depend on quaternary structures containing different protein subunits. These observations support the internal image property of the mAb2. In addition, this dependency on quaternary structure for recognition of proteins may also be relevant to other protein-protein interactions.
Ito
H O
HO
Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka, Japan. hitoded@mbox.nc.kyushu-u.ac.jp
Ueda
T
T
Hashimoto
Y
Y
Imoto
T
T
Koga
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D015153:Blotting, Western; D003094:Collagen / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D013329:Structure-Activity Relationship
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D003094:Collagen