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

Abstract

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 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.The 10 nm intermediate filaments (IF) 1 provide resilience in response to mechanical stress by forming a stable network attached to specific desmosome cell-cell contacts (1). IF can be classified into groups expressed in higher eukaryotes in a tissue-specific and cell type-restricted pattern (2, 3). In epithelial cells, IFs consist of stoichiometrically equal amounts of type I (smaller and acidic) and type II (larger, neutral, or basic) cytokeratin polypeptides, which form strongly interacting heterodimers (4 -6). Cytokeratin 8 (K8) and cytokeratin 18 (K18), type I and II, respectively, are the major components of IFs of simple or single layer epithelia, such as those of the intestine, the liver, and the breast ducts, although cytokeratin 19 (K19) is also present and forms heterodimers with K8 (2, 5). The assembly of IF involves several association steps where the ␣-helical rod domain of the cytokeratin molecules plays the central role (7-9). The head and the tail domains are not thought to be part of the filamentous backbone but to protrude laterally and contribute to profilame...

Cleavage of Host Keratin 8 by a Chlamydia-Secreted Protease

“…Although increased expression of keratin 8 has been found at the invasive front of some tumors (11,21), the degree of keratinization was inversely correlated with the metastasis potential of tumors (18), suggesting that the increased expression of keratins may lead to generation of soluble keratins and keratin fragments that are associated with cellular malignancy. This hypothesis is supported by the observation that both proteolytic fragments of keratin 8 and increased levels of keratins have been detected in cancer cells but not in normal epithelial cells (5). In addition, keratin 8 was detected on the external surfaces of some tumor cells (10), and carcinoma cell-released keratin 8 can act as a plasminogen receptor (8,9).…”

“…Various viral proteases have been shown to cleave host cell keratins (4,23), which may cause cytoskeleton collapse and facilitate viral release (27). On the other hand, proteolytic fragments of keratin 8 have been detected in carcinoma cells but not in normal epithelial cells (5,14), suggesting that there is a correlation between keratin cleavage and cellular malignancy. We hypothesize that proteolytic modification of keratin 8 may benefit chlamydial intracellular growth and survival by increasing the fluidity of the host cell cytoskeleton and promoting the survival of the infected cells.…”

Surface-Affinity Profiling To Identify Host-Pathogen Interactions

“…Our experiments suggest that CK8 has a surface-exposed component in colon adenocarcinoma cells that allows its interaction with intestinal pathogens. This theory is supported by several reports on CK8/CK18 membrane localization in colon tumor cells (10,13,17,20,24). Furthermore, Pankov et al found that this surface localization was present only in tumor cells and not in healthy tissue (29).…”

Increased IgG Antibody-Induced Cytotoxicity Against Airway Epithelial Cells in Patients with Nonallergic Asthma

“…The recombinant human cytokeratin 8, cytokeratin 18, and alpha-enolase proteins were produced and purified from E. coli using commercially available full open reading frame cDNA clones for each protein (RZPD, Heidelberg, Germany) as previously described [16,17]. …”