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

Abstract

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.

Filaggrin Genotype in Ichthyosis Vulgaris Predicts Abnormalities in Epidermal Structure and Function

“…Since FLG is a component of the CE, [37][38][39] it is not surprising that CEs are attenuated in IV (Figure 3). Yet, the CE alterations reported here do not appear to solely account for the extracellular alterations in lamellar bilayer morphology.…”

“…Alternatively, dominant-negative disruption of the cytoskeleton, as occurs with mutations in either keratin 1 or 10 in epidermolytic ichthyosis (epidermolytic hyperkeratosis), blocks lamellar body (LB) secretion, also resulting in a downstream, paracellular permeability barrier defect. 36 Since FLG peptides normally associate with both the CE [37][38][39] and keratin filaments, 40 FLG deficiency could compromise permeability barrier function by either mechanism, by both mechanisms, or by a mechanism that is unique to IV. Although in the pre-genotype era, barrier function was shown to be moderately abnormal in IV and AD patients, [41][42][43][44][45] this has not been assessed in genotyped, FLG-deficient IV patients, and there is little evidence for the mechanisms of a potential barrier defect.…”

Involvement of c-JUN in the regulation of terminal differentiation genes in normal and malignant keratinocytes

“…The small proline rich protein 2 (SPRR2), a corni®ed cell envelope precursor (Hohl et al, 1995;Steinert and Marekov, 1995), whose expression is restricted to terminally di erentiated keratinocytes, was used as a marker for terminal di erentiation. Immuno-histochemical analysis of strati®ed cultures of normal human keratinocytes revealed that SPRR2 proteins are located in the cytoplasm and cell periphery of suprabasal, differentiating keratinocytes whereas c-JUN is detected in the nuclei of cells in the basal, proliferating layer ( Figure 1a).…”

S100A11, S100A10, Annexin I, Desmosomal Proteins, Small Proline-rich Proteins, Plasminogen Activator Inhibitor-2, and Involucrin Are Components of the Cornified Envelope of Cultured Human Epidermal Keratinocytes

“…The proteins that have been identified as constituents of the CE include loricrin (8 -10), involucrin (11)(12)(13)(14), the small proline-rich (SPR) family of proteins (15)(16)(17)(18), cystatin A (19,20), elafin (21)(22)(23), filaggrin (24 -26), keratin (27,28), desmosomal components (27), annexin I (29 -31), plasminogen activator inhibitor-2 (PAI-2) (32), and the 195-kDa and 210-kDa proteins (33,34). Cross-links have been identified within loricrin, elafin, filaggrin, keratin, SPR1, SPR2, and desmoplakin (35). Loricrin is present as a partner in most of these cross-links.…”

“…Loricrin is present as a partner in most of these cross-links. It has been proposed that envelope formation is initiated with formation of an envelope scaffolding that consists of soluble precursors, and that other precursors (both soluble and insoluble) are later deposited (3,11,35). The scaffolding has been proposed to consist of involucrin, cystatin A, and possibly other proteins (25).…”