Identification and partial characterization of pemphigoid antigen extracted from normal human skin

Abstract

Antibodies in the sera of patients with the disease bullous pemphigoid define a normal component of the basement membrane of stratified squamous epithelia. Pemphigoid antigen has been shown to be synthesized by mouse and human epidermal cells in culture as an approximately 220 kd protein when reduced. The purpose of this study was to characterize pemphigoid antigen extracted directly from normal human skin and to determine its relationship to the high molecular weight protein found in culture. Suction blister-derived epidermis was extracted with 2% sodium dodecyl sulfate (SDS) and the solubilized proteins were separated, after reduction, by SDS-polyacrylamide gel electrophoresis (PAGE). The separated proteins were electrophoretically transferred to nitrocellulose sheets. Pemphigoid antigen was then specifically identified by immunoperoxidase staining using pemphigoid sera. IgG from 5 different bullous pemphigoid patients bound a band of apparent molecular weight 225 kd. Antibodies from 6 normal sera and 4 pemphigus sera did not bind this molecule. On a lower percentage (4%) polyacrylamide gel the pemphigoid antigen could be resolved as a doublet (two closely spaced bands) in the range of 220-240 kd. When unreduced, the pemphigoid antigen extracted from skin was also detected as a doublet in the 220-240 kd range. This suggests that the two chains are not necessarily disulfide-linked to each other in skin. Newly synthesized pemphigoid antigen immunoprecipitated from extracts of cultured human epidermal cells could also be identified on SDS-PAGE, when reduced, as a doublet in the 220-240 kd range. Taken together these data demonstrate that the pemphigoid antigen can be extracted directly from normal human skin and is a molecule similar in molecular weight to the antigen synthesized in human epidermal cell culture.