Phosphorylated cystatin α is a natural substrate of epidermal transglutaminase for formation of skin cornified envelope

Abstract
Both keratohyalin granules (KHG) and cornified envelopes were stained histochemically in an indirect immunofluorescent study by antiphosphorylated cystatin α antibody, indicating that phosphorylated cystatin α is a component of the cornified envelope proteins. When phosphorylated cystatin α (P-cystatin α) was incubated with epidermal transglutaminase (TGase) and Ca2− ions, polymerized protein was produced by formation of ϵ-(γ-glutamyl)lysine cross-linking peptide bonds between lysine residues of cystatin α and glutamine residues of suitable protein(s) in the enzyme preparation. However, phosphorylated and non-phosphorylated cystatins were polymerized to similar extents by the TGase. Immunofluorescent and immunoelectron microscopic observations revealed that P-cystatin α could be detected in vivo in the KHG and cornified envelopes. Treatment of sphingosine, a specific inhibitor of protein kinase C, markedly suppressed the incorporation of cystatin α into KHG. Thus phosphorylation of cystatin α by protein kinase C may play an important role in targeting cystatin α into KHG