Involucrin is a covalently crosslinked constituent of highly purified epidermal corneocytes: Evidence for a common pattern of involucrin crosslinking in vivo and in vitro

Abstract

Involucrin (hINV) is an important structural component of the keratinocyte cornified envelope that is expressed early in the keratinocyte differentiation process and is thought to be a component of the initial envelope scaffolding. We have previously shown that cyanogen bromide (CNBr) cleavage of cornified envelopes isolated from cultured foreskin keratinocytes releases several discrete insalucrin-immunoreactive peptides. In this study, we compare the pattern of release of immunoreactive hINV fragments from envelopes prepared from human breast skin and foreskin, and from spontaneous and induced envelopes prepared from cultured keratinocytes. We also identify one of the released products. Envelopes prepared from human breast skin or foreskin, or spontaneous or induced envelopes prepared from cultured cells differ significantly in structure. The envelopes isolated from epidermis appear to be structurally complete, whereas spontaneous envelopes appear less complete and the induced envelopes appear to be the least complete. In spite of these structural differences, CNBr cleavage releases an identical quartet of hINV-immunoreactive peptides migrating between 68 and 81 kDa from each preparation. Immunoblots indicate that the quantity of hINV-immunoreactive material released per μg of envelope protein is as follows: induced > spontaneous > foreskin > breast skin. The fastest migrating peptide (68 kDa) comigrates with a peptide that is released after CNBr cleavage of bacterially produced recombinant hINV. Amino-terminal amino acid sequencing of this peptide from recombinant hINV and from the cornified envelopes yields the sequence G-Q-L-K-H-L-E-Q-Q-E-G-Q-P-K-H. These results suggest that this fragment is the 275-amino acid segment of hINV beginning at G311 and extending to K5855 and that this peptide is not crosslinked to another protein. These results indicate that a population of the envelope associated hINV present in cultured and in vivo keratinocytes is crosslinked in the amino-terminal half. It is possible that this species represents an early intermediate in the involucrin crosslinking process.