Lipids Are Covalently Attached to Rigid Corneocyte Protein Envelopes Existing Predominantly as β-Sheets: A Solid-State Nuclear Magnetic Resonance Study

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 ...

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“…This supports the prediction that involucrin may serve as a scaffold for the covalent attachment of CE lipids (6,75).…”

Section: Unknown Variants 46 Timessupporting

confidence: 87%

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

Steinert

Marekov

1997

Journal of Biological Chemistry

195

194

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“…However, rather less is known about how the lipid envelope becomes attached to the protein envelope. Specifically, although a model has been suggested (2,21,24), the nature of the protein substrate for lipid addition is not yet known. In this study, we explored this question.…”

mentioning

confidence: 99%

Ceramides Are Bound to Structural Proteins of the Human Foreskin Epidermal Cornified Cell Envelope

Marekov

Steinert

1998

Journal of Biological Chemistry

202

193

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An important component of barrier function in human epidermis is contributed by ceramides that are bound by ester linkages to undefined proteins of the cornified cell envelope (CE). In this paper, we have examined the protein targets for the ceramide attachment. By partial saponification of isolated foreskin epidermal CEs followed by limited proteolysis, we have recovered several lipopeptides. Biochemical and mass spectroscopic characterization revealed that all contained near stoichiometric amounts of ceramides of masses ranging from about 690 to 890 atomic mass units, of which six quantitatively major species were common. The array of ceramides was similar to that obtained from pig skin, the composition of which is known, thereby providing strong indirect data for their fatty acid and sphingosine compositions. The recovered peptides accounted for about 20% of the total foreskin CE ceramides. By amino acid sequencing, about 35% of the peptides were derived from ancestral glutamine-glutamate-rich regions of involucrin, an important CE structural protein. Another 18% derived from rod domain sequences of periplakin and envoplakin, which are also known or suspected CE proteins. Other peptides were too short for unequivocal identification. Together, these data indicate that involucrin, envoplakin, periplakin, and possibly other structural proteins serve as substrates for the attachment of ceramides by ester linkages to the CE for barrier function in human epidermis.Mammalian epidermis lies at the interface with the environment, where it plays an essential role in providing a physical, chemical, and water barrier for the organism (1-4). Cornified keratinocytes, which constitute the major cell type of the epidermis, have evolved an elaborate barrier system. Part of this is contributed by the cornified cell envelope (CE), 1 which is a 15-20-nm thick layer on the periphery of the corneocyte, and consists of two components. An Ϸ15-nm-thick layer of several defined structural proteins is deposited on the intracellular surface of the cell membrane in the upper spinous and granular cells of the living epidermis (5-8). These proteins become crosslinked together by disulfide bonds and N ⑀ (␥-glutamyl)lysine or N 1 ,N 8 -bis(␥-glutamyl)spermidine isopeptide bonds formed by the action of transglutaminases (5-9). This process appears to begin with the cross-linking of certain early protein components, such as involucrin and envoplakins, at or near to the site of desmosomes (10 -13), which together form a scaffold (12,14) for the subsequent stages of addition of elafin, small prolinerich proteins, and much larger amounts of loricrin (10 -12, 15-17). At a late stage of protein envelope assembly, perhaps in upper granular cells, the lipid envelope component is assembled (reviewed in Refs. 2-4). The phospholipid-rich cellular plasma membrane is rebuilt as a 5-nm-thick layer of ceramide lipids, which subsequently become covalently attached to the protein envelope on the extracellular surface (18 -21). The lipids are synthesized, pac...

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“…A) Milder scarring (1 point each) Macular erythematous pigmented Mildly atrophic dish-like used concentration for acne scars is 30% in multiple sessions, 3-5 times, every 3-4 weeks [23,24]. Possible side effects of salicylic acid peeling are mild and temporary; they include mainly erythema and dryness.…”

Section: Number Of Lesions 3(>20)mentioning

confidence: 99%

The Management of Atrophic Acne Scars: Overview and New Tools

Fabbrocini¹

2013

J Clin Exp Dermatol Res

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Lipids Are Covalently Attached to Rigid Corneocyte Protein Envelopes Existing Predominantly as β-Sheets: A Solid-State Nuclear Magnetic Resonance Study

Cited by 67 publications

References 24 publications

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

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

Ceramides Are Bound to Structural Proteins of the Human Foreskin Epidermal Cornified Cell Envelope

The Management of Atrophic Acne Scars: Overview and New Tools

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