Monoclonal cytokeratin antibody recognizing a heterotypic complex: immunological probing of conformational states of cytoskeletal proteins in filaments and in solution.
A novel type of monoclonal murine antibody (Ks18.18) directed against an epitope depending on human cytokeratin (CK) 18, a member of the acidic (type I) CK subfamily, is described. We show by SDS-PAGE immunoblots and dot-blot assays that this antibody is unreactive with both the denatured and the renatured individual polypeptides but binds strongly to heterotypic coiled-coil complexes of CK 18 with several members of the complementary basic (type II) CK subfamily, notably with CK 8; i.e., its most frequent natural partner. We also show that specific interactions between complementary CK polypeptides take place during the incubation steps of immunoblotting procedures as polypeptides, or fragments thereof, that detach from the substrate can bind to complementary polypeptides attached to the substratum, which may result in false assignments of antibody reactivities. The conformation-specific, CK 18-dependent epitope of Ks18.18 was detected in intermediate filaments (IFs) of cultured cells, simple epithelia, and many carcinomas and, surprisingly, also in the basal cells of some stratified epithelia. Ks18.18 also reacts with altered CK configurations as present in the spheroidal bodies of mitotic cells and in the Mallory bodies of hepatocytes intoxicated with certain drugs, thus indicating that the heterotypic CK complexes are maintained in these structures. We have also used antibody Ks18.18 to demonstrate the existence of heterotypic CK 8 and 18 complexes in a distinct soluble form among supernatant proteins from cell homogenates which is indistinguishable from the heterotypic tetramer obtained after experimental disintegration of IFs. The potential value of such IF conformation-specific antibodies in cell biological research and pathology is discussed.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003599:Cytoskeleton / Q000276:immunology*; D004848:Epithelium / Q000276:immunology; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D006801:Humans; D007163:Immunosorbent Techniques; D007382:Intermediate Filaments / Q000276:immunology*; D007633:Keratins / Q000276:immunology*; D046911:Macromolecular Substances; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D007633:Keratins