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

Abstract

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...

Accumulation of protein‐bound epidermal glucosylceramides in β‐glucocerebrosidase deficient type 2 Gaucher mice

“…ester linked) g-hydroxylated Cers (Cer(OS)) and fatty acids [2,3,11]. Although little is known about the attachment of lipids to proteins of the epidermal corni¢ed cell envelope, it was assumed that free Cer(EOS) are the direct precursors of bound Cer(OS).…”

Harlequin ichthyosis and other autosomal recessive congenital ichthyoses: The underlying genetic defects and pathomechanisms

“…Similarly, transglutaminase 1 is an enzyme that was reported to crosslink hydroxyceramide to the cornified cell envelope [23,24]. Cornified cell envelope formation is essential as the scaffold upon which normal intercellular lipid layer formation in the stratum corneum can then take place [25].…”

Bricks and mortar of the epidermal barrier

“…One minor but important component of the extruded lipids are the ω-hydroxyceramides, which become covalently attached to the outer surface of the protein envelope of the CE forming an ~5 nm thick lipid envelope coat surrounding each corneocyte (Swartzendruber et al, 1987;Wertz and Downing, 1987a;Wertz et al, 1989b). The ceramides are attached by way of ester bonds to glutamic acid and glutamine residues of several CE proteins, including involucrin, envoplakin and periplakin ( Wertz et al, 1989b;Marekov and Steinert, 1998). The protein-bound ω-hydroxyceramides are built from sphingosine coupled to highly saturated, uniquely long ( C 3 0 -3 6 ) chain fatty acids having a chain terminal ("ω" ) hydroxyl group (Wertz and Downing , 1991).…”