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 10(9) x m(-1), 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.