Evidence that Involucrin Is a Covalently Linked Constituent of Highly Purified Cultured Keratinocyte Cornified Envelopes

The cornified envelop, the terminal product of keratinocyte differentiation, is composed of a variety of covalently cross-linked proteins that form a rigid three-dimensional structure. Our present studies show that preparations of intact envelopes prepared from cultured human keratinocytes contain soluble involucrin, keratin, and filaggrin. Sequential extraction with sodium dodecyl sulfate (SDS) and urea followed by sonication produces envelop fragments that are largely free of soluble proteins, including involucrin. Digestion of these highly purified envelope fragments with cyanogen bromide (CNBr) releases a smear of anti-involucrin immunoreactive material (40–180 kDa) with two enriched clusters of bands at approximately 52 and 70 kDa. The 52-kDa band cluster co-migrates with products released by CNBr digestion of purified involucrin. The CNBr-mediated release of discrete (52- and 70-kDa) involucrin-immunoreactive bands suggests that many involucrin molecules may be cross-linked at relatively few glutamyl residues/molecule in envelopes prepared from cultured keratinocytes. Moreover, what appears to be cross-linked involucrin can be localized on highly purified sonicated envelope fragments using colloidal gold electron microscopy. These results provide evidence that involucrin is a cross-linked component of the keratinocyte marginal band.