Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial...

Treatment of bovine tongue mucosa with 1 M KCl induced a split in the lamina densa of the basement membrane zone (BMZ). The epithelium was then separated from the underlying connective tissue. Electron microscopic analysis of the stripped epithelium revealed that hemidesmosomes and their associated intermediate filaments (IF) remain along the basal surface of the epithelium. This surface was solubilized in an SDS/urea-containing buffer. Characterization of components of this protein mixture was undertaken using human autoantibodies from bullous pemphigoid (BP) patients that have been shown to recognize hemidesmosomal plaque elements (Mutasim, D. F., Y. Takahashi, R. S. Labib, G. J. Anhalt, H. P. Patel, and L. A. Diaz. 1985. J. Invest. Dermatol. 84:47-53) and by production of mAbs. Affinity-purified autoantibodies directed against 180- and 240-kD polypeptides present in the protein preparation generated strong immunofluorescence staining patterns along the BMZ of bovine tongue mucosa. Furthermore, immunogold localization revealed that these two polypeptides are associated with the hemidesmosomal plaque. A mAb preparation directed against a 125-kD polypeptide present in this same protein mixture lamina lucida side of the hemidesmosome. Autoantibodies in BP serum samples, affinity-purified 180-kD autoantibodies and the mAb preparation generated a punctate stain along the substratum attached surface of epithelial cells maintained on glass substrata for approximately 1 wk. The spots appeared to be associated with bundles of IF in cultured mouse keratinocytes. These monospecific antibody probes should prove invaluable for the study of hemidesmosome structure, assembly, and function.