Conformational changes in the rod domain of human keratin 8 following heterotypic association with keratin 18 and its implication for filament stability.
Keratin intermediate filaments are heteropolymers of type I and type II polypeptides that constitute the bulk of the epithelial cytoskeleton. We microinjected seven keratin monoclonal antibodies into human epithelial cells, and two of them, only A45-B/B3 and LP3K, caused the formation of keratin aggregates. The keratin filaments in human epithelial cells were also disrupted by a monovalent A45-B/B3 Fab fragment, suggesting that the binding of the antibody, rather than cross-linking, collapses the filaments. Immunoblotting and ELISA experiments suggested that the antibody reacted weakly with recombinant K8 but did not react with recombinant K18 at all. However, the antibody reactivity increased substantially when a mixture of the two keratin polypeptides, either recombinant or derived from MCF-7, was used. The epitopes of 15 monoclonal antibodies recognizing human K8 were characterized by their reactivity with recombinant fragments of K8. Reactivity of antibody A45-B/B3 with fragments of K8 in the presence of K18 revealed that the antibody recognizes an epitope in the rod domain of K8, between residues 313 and 332, on the amino-terminal side of the stutter in helix 2B, which is involved in heterotypic association. The data suggest that this region of K8 undergoes a conformational change following interaction with the complementary K18 either to expose the epitope or to increase its affinity for the antibody. Taken together, the data highlight the role of this epitope in heterotypic association and in filament stabilization.
Waseem
Ahmad
A
Programme in Oral Oncology, Oral Diseases Research Centre, Bart's and The London, Queen Mary's School of Medicine and Dentistry, London, UK. a.waseem@qmul.ac.uk
Karsten
Uwe
U
Leigh
Irene M
IM
Purkis
Patricia
P
Waseem
Naushin H
NH
Lane
E Birgitte
EB
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D000939:Epitopes / Q000032:analysis / Q000235:genetics / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007382:Intermediate Filaments / Q000737:chemistry* / Q000378:metabolism*; D053533:Keratin-8; D007633:Keratins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D008845:Microinjections; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D017384:Sequence Deletion; D013997:Time Factors
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; C426203:KRT8 protein, human; D053533:Keratin-8; D010446:Peptide Fragments; D020033:Protein Isoforms; D011994:Recombinant Proteins; D007633:Keratins