Nuclear proteins of the bovine esophageal epithelium. I. Monoclonal antibody W2 specifically reacts with condensed nuclei of differentiated superficial cells

Cells from three layers of the bovine esophageal epithelium, representing different stages of differentiation, were dissociated and separated by Percoll gradient centrifugation into fractions of small, medium and large sizes. A majority of the large cells possessed condensed nuclei, a characteristic feature of terminal differentiation of the superficial epithelium. The small cells resembled the proliferate cells of the basal layer. In vitro culture of the esophageal epithelial cells resulted in proliferation of the small cells, colony formation, and, in some cases, differentiation into cells with condensed nuclei. Nuclei, or nuclear subfractions derived from cells of the different layers, were used as immunogens for the generation of hybridomas secreting monoclonal antibodies that bound specifically to different regions of the esophageal tissue. One such antibody, designated W2, labeled the condensed nuclei from the superficial laver of stratified esophageal and corneal epithelia in situ, as well as the large cells from esophageal culture in vitro. Thus, the expression of the W2 antigen may be associated with the process of nuclear condensation during epithelial differentiation. Immunoisolation of the target antigen of W2 from extracts of large cells of the bovine esophagus yielded a band of M(r) ~33,000 on nonreducing polyacrylamide gels. This band dissociated into two polypeptides, of M(r) ~22,000 and ~11,000, upon treatment with dithiothreitol. Amino acid sequence analysis of the larger polypeptide showed extensive homology to a group of small calcium-binding proteins, including two helix-turn-helix motifs designated as the EF-hand, characteristic of the configuration of the metal-ion coordinating ligands of the calcium-binding site. Similarly the sequence at the amino terminus of the polypeptide of ~11,000 indicated that it was the light chain counterpart of the same calcium-binding protein complex.