The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.

The cornified cell envelope (CE) is a 15-nm thick layer of insoluble protein deposited on the intracellular side of the cell membrane of terminally differentiated stratified squamous epithelia. The CE is thought to consist of a complex amalgam of proteins cross-linked by isodipeptide bonds formed by the action of transglutaminases, but little is known about how or in which order the several putative proteins are cross-linked together. In this paper, CEs purified from human foreskin epidermis were digested in two steps by proteinase K, which released as soluble peptides about 30% and then another 35% of CE protein mass, corresponding to approximately the outer third (cytoplasmic surface) and middle third, respectively. Following fractionation, 145 unique peptides containing two or more sequences cross-linked by isodipeptide bond(s) were sequenced. Based on these data, most (94% molar mass) of the outer third of CE structure consists of intra- and interchain cross-linked loricrin, admixed with SPR1 and SPR2 proteins as bridging cross-links between loricrin. Likewise, the middle third of CE structure consists largely of cross-linked loricrin and SPR proteins, but is mixed with the novel protein elafin which also forms cross-bridges between loricrin. In addition, cross-links involving loricrin and keratins 1, 2e, and 10 or filaggrin were recovered in both levels. The data establish for the first time that these several proteins are indeed cross-linked protein components of the CE structure. In addition, the data support a model for the intermediate to final stages of CE assembly: the proteins elafin, SPR1 and SPR2, and loricrin begin to be deposited on a preformed scaffold; later, elafin deposition decreases as loricrin and SPR accumulation continues to effect final assembly. The recovery of cross-links involving keratins further suggests that the subjacent cytoplasmic keratin intermediate filament-filaggrin network is anchored to the developing CE during these events.