Identification of a novel keratin epitope: evidence for association between non-helical sub-domains L12 during filament assembly.
Keratin filaments in simple epithelial cells are heteropolymers of keratin 8 (K8) and keratin 18 (K18) polypeptides. The assembly of these polypeptides into intermediate filaments is a complex multi-stage phenomenon that involves several levels of associations. These molecular associations are not very well characterized. Monoclonal antibodies (MAbs) with defined specificities can be used to probe these associations and to isolate various intermediates in the assembly pathway. Here we describe the specificity of a MAb LE65 that has been widely used in keratin expression studies. We report that the MAb LE65 does not recognize individual keratin polypeptides but it instead reacts with a complex of K8 with K18. The MAb also did not react with complexes of K8 or K18 with other keratins. By allowing the antibody to react with complexes reconstituted from keratin fragments plus the complementary keratin, we have mapped the MAb LE65 epitope on the L12 sub-domains of K18, residues 214-231, and K8, residues 234-265, which must associate together to achieve antibody binding. These results suggest that the non-helical linkers, L12, of complementary keratins associate directly during filament assembly. This would explain why microinjection of MAb LE65 has been shown to disrupt keratin filaments. Furthermore, it may also help to explain the mechanism of filament disruption in some skin blistering syndromes induced by spontaneous mutations in the L12 region.
Waseem
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A
Department of Craniofacial Development, UMDS, Guy's Hospital, London, U.K.
White
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K
Waseem
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism*; D007633:Keratins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D007633:Keratins