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

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 Ca 2− 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.