Direct Evidence That Involucrin Is a Major Early Isopeptide Cross-linked Component of the Keratinocyte Cornified Cell Envelope

Abstract

Involucrin was the first protein to be identified as a likely constituent of the insoluble cornified cell envelope (CE) of stratified squamous epithelia. However, to date, direct isolation from CEs of involucrin crosslinked by way of the transglutaminase-induced isopeptide bond has not been reported. We have treated human foreskin CEs with methanol/KOH (saponification) to hydrolyze off much of the lipids. By immunogold electron microscopy, this exposed large amounts of involucrin epitopes as well as of desmoplakin, a desmosomal structural protein. About 20% of the total CE protein could be solubilized by proteolytic digestion after saponification, of which involucrin was the most abundant. Subsequent amino acid sequencing revealed many peptides involving involucrin cross-linked either to itself or to a variety of other known CE protein components, including cystatin ␣, desmoplakin, elafin, keratins, members of the small proline-rich superfamily, loricrin, and unknown proteins related to the desmoplakin family. Specific glutamines or lysines of involucrin were used to cross-link the different proteins, such as glutamines 495 and 496 to desmoplakin, glutamine 288 to keratins, and lysines 468, 485, and 508 and glutamines 465 and 489 for interchain involucrin cross-links. Many identical peptides were obtained from immature CEs isolated from the inner living cell layers of foreskin epidermis. The multiple crosslinked partners of involucrin provide experimental confirmation that involucrin is an important early scaffold protein in the CE. Further, these data suggest that there is significant redundancy in the structural organization of the CE. The cornified cell envelope (CE)1 is a specialized structure formed during terminal differentiation of stratified squamous epithelia and serves as a vital barrier for the tissue. Foreskin epidermal CEs, for example, consist of an ϳ15-nm-thick layer of insoluble protein (about 90% of CE mass) on the intracellular or cytoplasmic surface, overlaid by ϳ5 nm of lipid envelope (10% of mass) located on the extracellular or outer surface (1-6). A similar CE structure of about 5 nm is formed in hair cuticle cells (7,8). Most other internal "wet" epithelia commonly assemble a 5-10-nm protein but not a lipid component of a CE (3).The insolubility of the protein portion of the CE is due to extensive cross-linking of several constituent proteins by both disulfide bonds and the N ⑀ -(␥-glutamyl)lysine isopeptide crosslink introduced by the action of transglutaminases (1-5). Analysis of the protein composition of the CE has been hampered by the simple fact that the cross-link cannot be cleaved by reagents that do not also cleave peptide bonds. Nevertheless, many studies using biochemical and immunological techniques have identified several protein components of CEs of epidermal or other epithelia, including cystatin ␣ (9, 10), formerly named keratolinin (11), elafin (12-15), involucrin (4, 16 -21, 23, 24), loricrin (25-30), members of the small proline-rich superfamily (Spr) (Spr1 and Spr2 in ...

Bricks and mortar of the epidermal barrier

“…Mature CEs cover and incorporate desmosomal attachment plaques and utilize proteins of those for CE assembly Robinson et al, 1997;Steinert and Marekov, 1997) forming a morphologically uniform layer about 15 nm thick in the terminally differentiated dead cells (Jarnik et al, 1998).…”

“…In epidermis it is expressed in the spinous layer (Jarvinen et al, 1987). Cystatin A is a substrate for epidermal TGases and has been identified as a minor cross-linked component of CEs (Zettergren et a l., 1984;Takahashi et al, 1992;Steinert and Marekov, 1997). The protein is a known cysteine protease inhibitor (Jarvinen et al ., 1987;Takahashi et al, 1994;Shibuya et al, 1995) and it has been suggested that this feature might be relevant for the bacteriostatic properties of the skin (Takahashi e t al., 1994).…”

“…The fate of proelafin after synthesis is somewhat obscure, as the protein is stored in secretory granules and is later extruded from the cells (Pfundt et al, 1996). However, TGases utilize glutamine residues in the cementoin sequence as sub-strates (Molhuizen et al, 1993), and proelafin has been identified as a crosslinked component of epidermal CEs (Steinert and M a r e k o v, 1995;Steinert and Marekov, 1997).…”

“…Keratin intermediate filaments (KIF) are of course the major protein of the corneocyte, and some keratin p r o t e i n s become cross-linked to the peripheral CE during terminal differentiation Steinert and Marekov, 1997;Candi et al, 1998a). KIF and their roles in health and disease have been reviewed in detail elsewhere (Parry and Steinert, 1995;Steinert, 1996).…”

“…A large body of evidence indicates that the KIF meet at the site of the desmosome where they may interact directly or indirectly through various other intermediary proteins, with the terminal domains of desmoplakin (Green et al, 1990;Virata et al, 1992;Kouklis et al, 1994;Garrod et a l., 1996;Green and Jones, 1996;Fuchs et al, 1997). There is direct evidence from protein sequencing that desmo-plakin becomes crosslinked to a variety of other CE proteins, although interestingly, the crosslink connection with the type II keratins seems to be indirectly through other proteins Steinert and Marekov, 1997;Candi et al, 1998a). Several other desmosomal proteins including desmoglein 3, desmo-collins 3A/3B, plakoglobin, and plakophilin were found among proteolyzed CE fragments .…”