Detection of Deiminated Proteins in Rat Skin: Probing with a Monospecific Antibody After Modification of Citrulline Residues

We performed a systematic study on deiminated proteins present in rat epidermis. Proteins extracted from various epidermal samples were resolved by either one- or two-dimensional gel electrophoresis and Western blotted to nitrocellulose membranes. Deiminated proteins were detected by modification of citrulline residues followed by probing with an anti-modified citrulline monospecific antibody. The cornified layer of adult plantar skin gave multiple series of isoelectric variants, most of which were found to be differentially deiminated type II keratins (60 kDa, and 67 kDa or above). The whole epidermis of 5-day-old rat back skin showed isoelectric variants of 60-kDa keratin as major deiminated components, and deiminated 55-kDa keratin and deiminated filaggrin as minor spots. In addition, we found highly deiminated proteins (200–220 kDa) thought to be derived from trichohyalin. The immunoreactivity of deiminated proteins was mainly localized in the granular and cornified layers of epidermis. Co-localization of deiminated filaggrin and keratins in the granular layer suggests the possible role of protein deimination during the terminal stage of epidermal differentiation.