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J. Biol. Chem., Vol. 277, Issue 24, 21712-21722, June 14, 2002

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Cancer-associated Cleavage of Cytokeratin 8/18 Heterotypic Complexes Exposes a Neoepitope in Human Adenocarcinomas^*

Henrik J. Ditzel^§, Merel C. M. Strik, Morten K. Larsen^¶, Antony C. Willis, Ahmad Waseem^**, Karin Kejling^¶, and Jens C. Jensenius

From the  Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, ^¶ Biomedical Laboratory, University of Southern Denmark, 5000 Odense, Denmark,  MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, OX1 3QU Oxford, United Kingdom, ^** Head and Neck Cancer Research Program, Guys, King's, and St. Thomas' Dental Institute, King's College London, London SE1 9RT, United Kingdom, and the  Department of Medical Microbiology and Immunology, University of Aarhus, 8000 Aarhus, Denmark

The intermediate filament network in simple glandular epithelial cells predominantly consists of heterotypic complexes of cytokeratin 8 (K8) and cytokeratin 18 (K18). In contrast to other cytokeratins, K8 and K18 are persistently expressed during malignant transformation, but changes in cell morphology are accompanied by alterations in the intermediate filament network. To study molecular changes, K8 and K18 were purified from surgically removed colon cancer and normal epithelia tissues. Western blotting and amino acid sequencing revealed the presence of abundant K8 and K18 fragments, truncated at the N terminus, from cancerous, but not normal, epithelial cells. The fragmentation pattern indicates proteolysis mediated by several enzymes, including trypsin-like enzymes. The cancer-associated forms of K8 and K18 are specifically recognized by the human antibody, COU-1, cloned from the B cells of a cancer patient. We demonstrate that COU-1 recognizes a unique conformational epitope presented only by a complex between K8 and K18. The epitope is revealed after proteolytic removal of the head domain of either K8 or K18. A large panel of recombinant K8 and K18 fragments, deleted N- or C-terminally, allowed for the localization of the COU-1 epitope to the N-terminal part of the rod domains. Using surface plasmon resonance, the affinity of COU-1 for this epitope was determined to be 10⁹ M¹, i.e. more than 2 orders of magnitude higher than for intact heterotypic K8/K18 complexes. The cellular distribution of truncated K8/K18 heterotypic complexes in viable adenocarcinomas cells was probed using COU-1 showing small fibrillar structures distinct from those of intact K8/K18 complexes. Previously we demonstrated the binding and subsequent internalization of recombinant Fab COU-1 to live cancer cells. We have thus characterized a cancer neoepitope recognized by the humoral immune system. The results have biological as well as clinical implications.

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