Conformational Changes in the Rod Domain of Human Keratin 8 following Heterotypic Association with Keratin 18 and Its Implication for Filament Stability

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• 作者：Ahmad Waseem ; Uwe Karsten ; Irene M. Leigh ; Patricia Purkis ; Naushin H. Waseem ; and E. Birgitte Lane
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：February 10, 2004
• 年：2004
• 卷：43
• 期：5
• 页码：1283 - 1295
• 全文大小：478K
• 年卷期：v.43,no.5(February 10, 2004)
• ISSN：1520-4995

文摘

Keratin intermediate filaments are heteropolymers of type I and type II polypeptides thatconstitute the bulk of the epithelial cytoskeleton. We microinjected seven keratin monoclonal antibodiesinto human epithelial cells, and two of them, only A45-B/B3 and LP3K, caused the formation of keratinaggregates. 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 thefilaments. Immunoblotting and ELISA experiments suggested that the antibody reacted weakly withrecombinant K8 but did not react with recombinant K18 at all. However, the antibody reactivity increasedsubstantially 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 theirreactivity with recombinant fragments of K8. Reactivity of antibody A45-B/B3 with fragments of K8 inthe presence of K18 revealed that the antibody recognizes an epitope in the rod domain of K8, betweenresidues 313 and 332, on the amino-terminal side of the stutter in helix 2B, which is involved in heterotypicassociation. The data suggest that this region of K8 undergoes a conformational change following interactionwith 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 filamentstabilization.