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.

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UL25 and UL17 are two essential minor capsid proteins of HSV-1, implicated in DNA packaging and capsid maturation. We used cryo-electron microscopy to examine their binding to capsids, whose architecture observes T = 16 icosahedral geometry. C-capsids (mature DNA-filled capsids) have an elongated two-domain molecule present at a unique, vertex-adjacent site that is not seen at other quasiequivalent sites or on unfilled capsids. Using SDS-PAGE and mass spectrometry to analyze wild-type capsids, UL25 null capsids, and denaturant-extracted capsids, we conclude that (1) the C-capsid-specific component is a heterodimer of UL25 and UL17, and (2) capsids have additional populations of UL25 and UL17 that are invisible in reconstructions because of sparsity and/or disorder. We infer that binding of the ordered population reflects structural changes induced on the outer surface as pressure builds up inside the capsid during DNA packaging. Its binding may signal that the C-capsid is ready to exit the nucleus.

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 ...

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...

Transglutaminases (TGases) are defined as enzymes capable of forming isopeptide bonds by transfer of an amine onto glutaminyl residues of a protein. Here we show that the membrane-bound form of the TGase 1 enzyme can also form ester bonds between specific glutaminyl residues of human involucrin and a synthetic analog of epidermal specific -hydroxyceramides. The formation of a Ϸ5-nm-thick lipid envelope on the surface of epidermal keratinocytes is an important component of normal barrier function. The lipid envelope consists of -hydroxyceramides covalently linked by ester bonds to cornified envelope proteins, most abundantly to involucrin. We synthesized an analog of natural -hydroxyceramides N-[16-(16-hydroxyhexadecyl)oxypalmitoyl]-sphingosine (lipid Z). When recombinant human TGase 1 and involucrin were reacted on the surface of synthetic lipid vesicles containing lipid Z, lipid Z was attached to involucrin and formed saponifiable protein-lipid adducts. By mass spectroscopy and sequencing of tryptic lipopeptides, the ester linkage formation used involucrin glutamine residues 107, 118, 122, 133, and 496 by converting the ␥-carboxamido groups to lipid esters. Several of these residues have been found previously to be attached to ceramides in vivo. Mass spectrometric analysis after acetonide derivatization also revealed that ester formation involved primarily the -hydroxyl group of lipid Z. Our data reveal a dual role for TGase 1 in epidermal barrier formation and provide insights into the pathophysiology of lamellar ichthyosis resulting from defects of TGase 1 enzyme.Terrestrial vertebrates protect themselves from chemical and physical damage and uncontrolled water loss by maintaining a water-impermeable barrier function of their epidermis. In mammals, this function is essentially accomplished by forming a highly insoluble protein structure on the surface of the corneocytes termed the cornified envelope (CE) and by impeding water diffusion across the stratum corneum by mortaring the corneocytes together by layers of skin-specific lipids (1, 2). These lipids differ in composition from other bilayerforming lipids found in living cells. Notably, their phospholipid content is lost, and instead they contain increased amounts of free fatty acids, cholesterol and its acyl and sulfate esters, and several classes of ceramides, including epidermal-specific longchain -hydroxy-and -hydroxyacylceramides (3). Synthesis of these lipids is initiated in the spinous layer, and they are temporarily stored in lamellar bodies of stratum granulosum, wherein they are arranged as stacks of tetralaminar sheets. Preceding or paralleling the formation of the protein envelope, the contents of the lamellar bodies are extruded into the intercellular space. One component of these lipids is epidermal-specific long-chain -hydroxyceramides that become covalently attached onto the outer surface of the CE as a Ϸ5-nm monomolecular layer. These protein-linked ceramides interdigitate with the intercellular lipid in a comb-like fashion, presuma...

Sjögren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder characterized by mental retardation, spasticity and ichthyosis. SLS patients have a profound deficiency in fatty aldehyde dehydrogenase (FALDH) activity. We have now cloned the human FALDH cDNA and show that it maps to the SLS locus on chromosome 17p11.2. Sequence analysis of FALDH amplified from fibroblast mRNA and genomic DNA from 3 unrelated SLS patients reveals distinct mutations, including deletions, an insertion and a point mutation. The cloning of FALDH and the identification of mutations in SLS patients opens up possibilities for developing therapeutic approaches to ameliorate the neurologic and cutaneous symptoms of the disease.

Loricrin is the major protein of the cornified cell envelope of terminally differentiated epidermal keratinocytes which functions as a physical barrier. In order to understand its properties and role in cornified cell envelope, we have expressed human loricrin from a fulllength cDNA clone in bacteria and purified it to homogeneity. We have also isolated loricrin from newborn mouse epidermis. By circular dichroism and fluorescence spectroscopy, the in vivo mouse and bacterially expressed human loricrins possess no ␣ or ␤ structure but have some organized structure in solution associated with their multiple tyrosines and can be reversibly denatured by either guanidine hydrochloride or temperature. The transglutaminase (TGase) 1, 2, and 3 enzymes expressed during epidermal differentiation utilized loricrin in vitro as a complete substrate, but the types of cross-linking were different. The TGase 3 reaction favored certain lysines and glutamines by forming mostly intrachain cross-links, whereas TGase 1 formed mostly large oligomeric complexes by interchain crosslinks involving different lysines and glutamines. Together, the glutamines and lysines used in vitro are almost identical to those seen in vivo. The data support a hypothesis for the essential and complementary roles of both TGase 1 and TGase 3 in cross-linking of loricrin in vivo. Failure to cross-link loricrin by TGase 1 may explain the phenotype of lamellar ichthyosis, a disease caused by mutations in the TGase 1 gene.Terminal differentiation in the epidermis involves the expression of a number of specific proteins that ultimately fulfill different structural roles in the cornified, dead stratum corneum cell. One set of proteins is the keratin intermediate filaments and the interfilamentous matrix protein filaggrin (1-3). A second set of proteins is used to construct the cornified cell envelope (CE), 1 a 15-nm-thick layer of protein deposited on the inner surface of the cell periphery, which serves as a physical barrier for the epidermis (4, 5). The CE proteins are rendered insoluble by cross-linking by both disulfide bonds and the N ⑀ -(␥-glutamyl)lysine isopeptide bond formed by the action of one or more of the three known epidermal transglutaminases (TGases) (4 -6). Several proteins have now been documented as CE constituents by direct sequencing analyses of cross-linked peptides (7), including loricrin, small proline-rich proteins 1 and 2 (SPR1 and SPR2), elafin, keratins, filaggrin, and desmoplakin. The proteins involucrin and cystatin ␣ are also likely constituents, but direct sequencing of cross-linked peptides involving these proteins has not yet been reported (reviewed in Ref. 8).In particular, a variety of data have suggested that loricrin comprises about 75% of the total CE protein mass (reviewed in Ref. 9), or 85-95% of the cytoplasmic two-thirds of the CE. In fact, amino acid sequencing of many peptides recovered by the proteolysis has now provided rigorous support for this idea (7). About 90% of the molar mass of peptides from the cytoplasmic...