Human Hematoxylin-Stainable Protein of Keratohyalin Granules Origin. I. Extraction and Purification

Abstract

A human hematoxylin-stainable protein (HSP) was extracted from the massive stratum corneum of epidermal cysts. This protein was purified in two steps; first, through preparative isoelectric focusing, and, second, by affinity column chromatography bound with the specific, monoclonal antibody to keratohyalin granules (Ted-H-1). In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified HSP consisted of two proteins (70 and 62 kilodaltons [kd]), but only the 62-kd protein was detected in the 15,000 g supernatant fraction of the extract using the immunoblotting technique. The amino acid composition of the purified HSP included 30% glycine, 15% serine, 12% glutamic acid, and 4% ornithine, but only 2.3% histidine. Using the indirect immunofluorescent technique, observation showed that the monoclonal antibody, Ted-H-1, to the HSP, formed as a result of the partially purified antigen, was located in three places: 1) the keratohyalin granules; 2) in the cell membrane region of the lower part of the stratum corneum of the skin samples (forearm, cheek, and back); and 3) the keratohyalin granules in the follicular epithelium and on the trichohyalin granules. Reaction product was not seen in either the acrosyringium or in the plantar epidermis. As two positively reacted proteins with the Ted-H-1 were detected in the Tris-HCl extract from the plantar stratum corneum by the immunoblotting assay, however, the above negative result in the indirect immunofluorescent studies may be due to the masking of the antigenic site of keratohyalin granules in vivo. This hematoxylin-stainable protein was synthesized as one component of the keratohyalin granules in the stratum granulosum and may have been transferred to the stratum corneum cell membrane region.