Preferential Deimination of Keratin K1 and Filaggrin during the Terminal Differentiation of Human Epidermis (original) (raw)
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Experimental Dermatology, 1999
Citrulline residues are detected in keratins and filaggrin in the cornified layers of mammalian epidermis. Such citrulline residues are formed by the enzymatic deimination of arginine residues by peptidylarginine deiminase (EC 3.5.3.15). Major deiminated keratins are thought to be partially degraded/disulfide-cross-linked keratin K1 based on the immunoblotting profiles. In order to obtain more definitive evidence of the deimination of keratin K1 and also to investigate its functional significance, we attempted to identify its preferred acting sites of peptidylarginine deiminase. A partially degraded keratin K1 fraction obtained from the cornified layer of newborn mouse epidermis was subjected to limited proteolytic cleavages, and the resulting deiminated peptides were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or re-Key words: posttranslational modificationverse-phase high-performance liquid chromatography for N-terminal sepeptidylarginine deiminase-epidermal quencing and/or amino acid analysis. At least two sites were identified, one differentiation in the V1 and the other in the V2 subdomains of keratin K1. An undeca-Dr Tatsuo Senshu, Department of Bioactivity peptide sequence covering the latter shows about 70% homology with an Regulation, Tokyo Metropolitan Institute of undecapeptide sequence in the V2 subdomain of human K1, a presump-Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173, Japan tive site of deimination. We speculated that the deimination of arginine
European Journal of Biochemistry, 1984
Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two ofeach keratin subfamily; type I1 basic (PI 6.5 -8.5 ; human 68 kDa, 60.5 kDa and mouse 67 kDa, 60 kDa) and type I acidic (PI 4.7 -5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type 11 neutralacidic (PI 5.7 -6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (PI 4.9 -5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one-dimensional and two-dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type I1 basic and the type I acidic keratin subfamilies. A strong homology also existed between type I1 SC keratins and the larger basic (type 11) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor-product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation-related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.
Dynamic aspects of protein deimination in developing mouse epidermis
Experimental Dermatology, 1999
The cornified layer of mammalian epidermis contains deiminated keratins and filaggrin whose arginine residues are partly converted to citrulline residues by peptidylarginine deiminase (EC 3.5.3.15). We have attempted to study dynamic aspects of protein deimination using late embryonic to early postnatal mouse skin. The epidermis was separated from the dermis by brief immersion of skin into a weakly alkaline ammonium chloride solution. The total homogenate of the epidermis was subjected to western blotting analyses for quantitative densitometry of major keratins, deiminated proteins and immunoreactive filaggrin. We found marked increases in both deiminated keratins and deiminated filag-Key words: filaggrin-keratin-peptidylarginine grin from the 18th day of gestation to 2 h after birth followed by rapid deiminase-mouse development-epidermal decreases to minimum levels at 6 h and subsequent gradual increases surdifferentiation passing the earlier levels by 72 h after birth. Such variations were associ-Kyoichi Akiyama, Department of Cell Chemistry, ated with consistent changes of the intensity of deiminated proteins Tokyo Metropolitan Institute of Gerontology, stained immunocytochemically. These results suggest that the protein de-35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, imination might play a role in dealing with the drastic environmental
Journal of Anatomy, 2009
Historically, the term 'keratin' stood for all of the proteins extracted from skin modifications, such as horns, claws and hooves. Subsequently, it was realized that this keratin is actually a mixture of keratins, keratin filament-associated proteins and other proteins, such as enzymes. Keratins were then defined as certain filament-forming proteins with specific physicochemical properties and extracted from the cornified layer of the epidermis, whereas those filamentforming proteins that were extracted from the living layers of the epidermis were grouped as 'prekeratins' or 'cytokeratins'. Currently, the term 'keratin' covers all intermediate filament-forming proteins with specific physicochemical properties and produced in any vertebrate epithelia. Similarly, the nomenclature of epithelia as cornified, keratinized or non-keratinized is based historically on the notion that only the epidermis of skin modifications such as horns, claws and hooves is cornified, that the non-modified epidermis is a keratinized stratified epithelium, and that all other stratified and non-stratified epithelia are non-keratinized epithelia. At this point in time, the concepts of keratins and of keratinized or cornified epithelia need clarification and revision concerning the structure and function of keratin and keratin filaments in various epithelia of different species, as well as of keratin genes and their modifications, in view of recent research, such as the sequencing of keratin proteins and their genes, cell culture, transfection of epithelial cells, immunohistochemistry and immunoblotting. Recently, new functions of keratins and keratin filaments in cell signaling and intracellular vesicle transport have been discovered. It is currently understood that all stratified epithelia are keratinized and that some of these keratinized stratified epithelia cornify by forming a Stratum corneum . The processes of keratinization and cornification in skin modifications are different especially with respect to the keratins that are produced. Future research in keratins will provide a better understanding of the processes of keratinization and cornification of stratified epithelia, including those of skin modifications, of the adaptability of epithelia in general, of skin diseases, and of the changes in structure and function of epithelia in the course of evolution. This review focuses on keratins and keratin filaments in mammalian tissue but keratins in the tissues of some other vertebrates are also considered.
Journal of Dermatological Science, 1999
We devised a simple method to maintain an immortalized newbown rat keratinocyte cell line at the air-liquid interface using a tissue culture insert fitted with a microporous membrane. The cells formed stratified layers of flattened and anucleated cells resembling stratum corneum of the epidermis. Deiminated proteins, which are localized in the cornified layer of epidermis as the reaction products of peptidylarginine deiminase were detected immunohistochemically in the differentiated cells. Western blot analyses revealed that major deiminated proteins were type I keratins K10 and K14. Deiminated products of type II keratin K5 were found as minor components. Our observations show that deimination of keratins might be correlated with terminal differentiation of the immortalized keratinocyte cell line.
Keratin 15 Expression in Stratified Epithelia: Downregulation in Activated Keratinocytes
Journal of Investigative Dermatology, 1999
Keratin 15 (K15) is a type I keratin without a defined type II partner whose expression in epidermal diseases has not been investigated. In this study we have used LHK15, a monoclonal antibody raised against the last 17 amino acids of the K15 polypeptide, to show that K15 is expressed primarily in the basal keratinocytes of stratified tissues, including the fetal epidermis and fetal nail. Although K15 in normal hair follicles was virtually absent from hair bulbs, it was expressed by a subset of keratinocytes in the outer root sheath. By comparison, K14 expression was found throughout the outer root sheath of hair follicles; however, when both K14 alleles were naturally ablated, the expression of K15 was also observed throughout the outer root sheath of the follicles. Expression of K15 mRNA was assessed by in situ hybridization and corroborated the K eratins belong to the family of intermediate filament proteins that are specifically expressed in epithelia. They have a remarkable ability to polymerize into 10 nm filaments without the participation of auxiliary proteins (Coulombe and Fuchs, 1990; Hatzfeld and Weber, 1990; Steinert, 1990). They comprise a total of about 30 genes (including those of hair and nails, the trichocytic keratins) grouped into two types; type I are smaller (40-56.5 kDa) and acidic (pI Ͻ 7.0), whereas type II are larger (53-67 kDa) and basic/ neutral (pI ജ 7.0) (reviewed in Fuchs and Weber, 1994). The type I keratins include K9-K20 and the type II include K1-K8. The amino acid sequence of keratins is highly conserved in the central rod domain of the polypeptides and forms an α-helical structure (for a review see Albers and Fuchs, 1992). During filament assembly, two keratin polypeptides, one of each type, first form a parallel heterodimer, in which the rod domains assemble into coiled-coil (Hatzfeld and Weber, 1990; Steinert, 1990), which then undergoes further associations with other dimers to produce tetramers (Coulombe, 1993; Stewart, 1993). The association of tetramers produces protofilaments and finally mature filaments. Although any type I Manuscript
The Use of cDNA Clones and Monospecific Antibodies as Probes to Monitor Keratin Gene Expression
Annals of the New York Academy of Sciences, 1985
Approximately 20 different keratin proteins form the subunits of the intermediate filaments found in keratinocytes and other epithelial cells.'*2 Individual epithelial cells do not express all of these subunits at any given time but instead express limited subsets. The subunits expressed vary widely depending on cell type,) period of embryonic development: degree of differentiation:-' and growth environment of the The mechanism by which expression of this family of genes is regulated is not known. In addition, nothing is known about the structure or organization of these genes at the level of the chromosome. In order to address these questions, we have constructed cDNA libraries to messenger RNA isolated from terminally differentiating mouse epidermis'* and undifferentiated mouse epidermal cells grown in c~1ture.l~ The keratin cDNA clones isolated from these libraries consist of two subsets, those expressed predominantly in proliferating basal cells (the 50, 55, and 60 kD keratins) and those expressed predominantly in terminally differentiating cells (the 59 and 67 kD keratins). The DNA sequence of these cDNA clones has been determined to deduce their amino acid sequence."" All of these subunits contain a central domain of about 300 residues that forms a coiled-coil a-helical structure. Variations in both size and sequence are observed in the non-a-helical amino and carboxy terminal domains of these subunits. The 50, 55, and 60 kD keratin subunits expressed in undifferentiated cells contain conserved sequences enriched in serines, whereas subunits expressed in terminally differentiating cells contain numerous repeat sequences enriched in glycines. Variations in these sequences in keratins that are expressed at different states of differentiation implicate their involvement in changes in keratin filament properties
Posttranslational regulation of keratins: degradation of mouse and human keratins 18 and 8
Molecular and cellular biology, 1989
Human keratin 18 (K18) and keratin 8 (K8) and their mouse homologs, Endo B and Endo A, respectively, are expressed in adult mice primarily in a variety of simple epithelial cell types in which they are normally found in equal amounts within the intermediate filament cytoskeleton. Expression of K18 alone in mouse L cells or NIH 3T3 fibroblasts from either the gene or a cDNA expression vector results in K18 protein which is degraded relatively rapidly without the formation of filaments. A K8 cDNA containing all coding sequences was isolated and expressed in mouse fibroblasts either singly or in combination with K18. Immunoprecipitation of stably transfected L cells revealed that when K8 was expressed alone, it was degraded in a fashion similar to that seen previously for K18. However, expression of K8 in fibroblasts that also expressed K18 resulted in stabilization of both K18 and K8. Immunofluorescent staining revealed typical keratin filament organization in such cells. Thus, expres...