Sequential Reorganization of Cornified Cell Keratin Filaments Involving Filaggrin-Mediated Compaction and Keratin 1 Deimination
2002; Elsevier BV; Volume: 118; Issue: 2 Linguagem: Inglês
10.1046/j.0022-202x.2001.01671.x
ISSN1523-1747
AutoresAkemi Ishida‐Yamamoto, Tatsuo Senshu, Robin A.J. Eady, Hidetoshi Takahashi, Hiroshi Shimizu, Masashi Akiyama, Hajime Iizuka,
Tópico(s)Hair Growth and Disorders
ResumoThe final step of keratinocyte differentiation, transition from the granular cells to the cornified cells, involves various post-translational modifications that include deimination of arginine residues. Major deiminated epidermal proteins are derived from K1. Two preferred deimination sites were identified in mouse K1, one in the V1 and the other in the V2 subdomains. An antibody against the deiminated peptide sequence in the V2 subdomain recognized not only deiminated mouse K1 but also deiminated human K1. In this study we analyzed distribution of deiminated K1 in normal human skin and in bullous congenital ichthyosiform erythroderma at light and electron microscopic levels. In normal skin the first few (1–3) cornified cell layers were positive for filaggrin and negative for the antibody against deiminated mouse K1 peptide, whereas the more superficial cells were negative for filaggrin and strongly positive for the antibody against deiminated mouse K1 peptide, indicating slightly delayed onset of K1 deimination at the initial stage of cornification. The clumped keratin in bullous congenital ichthyosiform erythroderma that was not properly compacted with filaggrin was poorly positive to the antibody against deiminated mouse K1 peptide. In addition, K1 derivatives in bullous congenital ichthyosiform erythroderma reacted poorly with the antibody against deiminated mouse K1 peptide compared with the normal control in immunoblot analyses. Our results suggest sequential reorganization of cornified cell keratin filaments involving filaggrin-mediated compaction and K1 deimination. Abnormal keratin aggregation in bullous congenital ichthyosiform erythroderma is likely to disturb the normal deimination of K1. The final step of keratinocyte differentiation, transition from the granular cells to the cornified cells, involves various post-translational modifications that include deimination of arginine residues. Major deiminated epidermal proteins are derived from K1. Two preferred deimination sites were identified in mouse K1, one in the V1 and the other in the V2 subdomains. An antibody against the deiminated peptide sequence in the V2 subdomain recognized not only deiminated mouse K1 but also deiminated human K1. In this study we analyzed distribution of deiminated K1 in normal human skin and in bullous congenital ichthyosiform erythroderma at light and electron microscopic levels. In normal skin the first few (1–3) cornified cell layers were positive for filaggrin and negative for the antibody against deiminated mouse K1 peptide, whereas the more superficial cells were negative for filaggrin and strongly positive for the antibody against deiminated mouse K1 peptide, indicating slightly delayed onset of K1 deimination at the initial stage of cornification. The clumped keratin in bullous congenital ichthyosiform erythroderma that was not properly compacted with filaggrin was poorly positive to the antibody against deiminated mouse K1 peptide. In addition, K1 derivatives in bullous congenital ichthyosiform erythroderma reacted poorly with the antibody against deiminated mouse K1 peptide compared with the normal control in immunoblot analyses. Our results suggest sequential reorganization of cornified cell keratin filaments involving filaggrin-mediated compaction and K1 deimination. Abnormal keratin aggregation in bullous congenital ichthyosiform erythroderma is likely to disturb the normal deimination of K1. antibody against deiminated mouse K1 peptide antibody against chemically modified citrulline bullous congenital ichthyosiform erythroderma peptidylarginine deiminase The process of epidermal differentiation or keratinization is characterized by a series of morphologic changes as keratinocytes leave the basal compartment and move upwards following the programmed cell death pathway (Ishida-Yamamoto et al., 1999Ishida-Yamamoto A. Tanaka H. Nakane H. Takahashi H. Hashimoto Y. Iizuka H. Programmed cell death in normal epidermis and loricrin keratoderma. Multiple functions of profilaggrin in keratinization.J Invest Dermatol Symp Proc. 1999; 4: 145-149Abstract Full Text PDF PubMed Scopus (35) Google Scholar). The cells finally differentiate from the granular cells through transitional cells to enucleated, dead cornified cells. This final step involves post-translational modifications of various proteins. Deimination is among such modifications and is catalyzed by a family of calcium-dependent enzyme, peptidylarginine deiminases (PAD) that convert protein arginine residues to citrulline residues (Rothnagel and Rogers, 1984Rothnagel J.A. Rogers G.E. Citrulline in proteins from the enzymatic deimination of arginine residues.in: Wald F. Moldave K. Methods in Enzymology. Vol. 107. Academic Press, Orlando1984: 624-631Google Scholar). Three types of PAD (types I, III, and IV) are thought to be expressed in the epidermis (Watanabe et al., 1988Watanabe K. Akiyama K. Hikichi K. Ohtsuka R. Okuyama A. Senshu T. Combined biochemical and immunochemical comparison of peptidylarginine deiminase present in various tissues.Biochim Biophys Acta. 1988; 966: 375-383Crossref PubMed Scopus (130) Google Scholar;Terakawa et al., 1991Terakawa H. Takehara H. Sugawara K. Three types of mouse peptidylarginine deiminase: characterization and tissue distribution.J Biochem. 1991; 110: 661-666PubMed Google Scholar;Nishijyo et al., 1997Nishijyo T. Kawada A. Kanno T. Shiraiwa M. Takahara H. Isolation and molecular cloning of epidermal- and hair follicle-specific peptidylarginine deiminase (type III) from rat.J Biochem. 1997; 121: 868-875Crossref PubMed Scopus (43) Google Scholar;Ishigami et al., 1998Ishigami A. Kuramoto M. Yamada M. Watanabe K. Senshu T. Molecular cloning of two novel types of peptidylarginine deiminase cDNAs from retinoic acid-treated culture of a newborn rat keratinocyte cell line.FEBS Lett. 1998; 433: 113-118Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar;Yamakoshi et al., 1998Yamakoshi A. Ono H. Nishijyo T. Shiraiwa M. Takahara H. Cloning of cDNA encoding a novel isoform (type IV) of peptidylarginine deiminase from rat epidermis.Biochim Biophys Acta. 1998; 227–232: 1998Google Scholar;Rus'd et al., 1999Rus'd A.A. Ikejiri Y. Ono H. Yonekawa T. Shiraiwa M. Kawada A. Takahara H. Molecular cloning of cDNAs of mouse peptidylarginine deiminase type I, type III and type IV, and the expression pattern of type I in mouse.Eur J Biochem. 1999; 259: 660-669Crossref PubMed Scopus (65) Google Scholar;Kanno et al., 2000Kanno T. Kawada A. Yamanouchi J. et al.Human peptidylarginine deiminase type III. Molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin.J Invest Dermatol. 2000; 115: 813-823Crossref PubMed Scopus (112) Google Scholar). It has previously been shown that major deiminated epidermal proteins are derived from keratin, K1, and minor deiminated proteins are from filaggrin and K10, using a monospecific antibody (AMC) that recognizes chemically modified citrulline residues (Senshu et al., 1995Senshu T. Akiyama K. Kan S. Asaga H. Ishigami A. Manabe M. Detection of deiminated proteins in rat skin: probing with a monospecific antibody after modification of citrulline residues.J Invest Dermatol. 1995; 105: 163-169Crossref PubMed Scopus (125) Google Scholar, Senshu et al., 1996Senshu T. Kan S. Ogawa H. Manabe M. Asaga H. Preferential deimination of keratin K1 and filaggrin during the terminal differentiation of human epidermis.Biochem Biophys Res Commun. 1996; 225: 712-719Crossref PubMed Scopus (128) Google Scholar). Two preferred deimination sites were identified in mouse K1, one in the V1 and the other in the V2 subdomains (Senshu et al., 1999bSenshu T. Akiyama K. Nomura K. Identification of citrulline residues in the V subdomains of keratin K1 derived from the cornified layer of newborn mouse epidermis.Exp Dermatol. 1999; 8: 392-401Crossref PubMed Scopus (31) Google Scholar). These subdomains in K1 are characterized by high glycine and serine contents with a few arginine residues as only charged amino acids (Steinert et al., 1985Steinert P.M. Parry D.A.D. Idler W.W. Johnson L.D. Steven A.C. Roop D.R. Amino acid sequences of mouse and human epidermal type II keratins of Mr67,000 provide a systematic basis for the structural and functional diversity of the end domains of keratin intermediate filament subunits.J Biol Chem. 1985; 260: 7142-7149Abstract Full Text PDF PubMed Google Scholar). We have raised an antibody against a deiminated undecapeptide containing the deimination site in the V2 subdomain of mouse K1 (ACP) (Senshu et al., 1999aSenshu T. Akiyama K. Ishigami A. Nomura K. Studies on specificity of peptidylarginine deiminase reactions using an immunochemical probe that recognizes an enzymatically deiminated partial sequence of mouse keratin K1.J Dermatol Sci. 1999; 21: 113-126Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Similar sequences are present in V subdomains of human K1. ACP reacted with deiminated mouse K1 and also cross-reacted with deiminated human K1 (Ishida-Yamamoto et al., 2000Ishida-Yamamoto A. Senshu T. Takahashi H. Akiyama K. Nomura K. Iizuka H. Decreased deiminated keratin K1 in psoriatic hyperproliferative epidermis.J Invest Dermatol. 2000; 114: 701-705https://doi.org/10.1046/j.1523-1747.2000.00936.xCrossref PubMed Scopus (90) Google Scholar), but did not react with unmodified K1; also, it did not react with deiminated filaggrin. Albeit these studies the biologic significance of deimination of V subdomains of K1 is still unknown. To gain some insights into this question, we analyzed ultrastructural localization of the deiminated K1 in normal human skin and in bullous congenital ichthyosiform erythroderma (BCIE), a genetic skin disease of K1/K10 (Fuchs, 1997Fuchs E. of mice and men: genetic disorders of the cytoskeleton.Mol Biol Cell. 1997; 8: 189-203Crossref PubMed Scopus (73) Google Scholar;Ishida-Yamamoto et al., 1998Ishida-Yamamoto A. Tanaka H. Nakane H. Takahashi H. Iizuka H. Inherited disorders of epidermal keratinization.J Dermatol Sci. 1998; 18: 139-154Abstract Full Text PDF PubMed Scopus (20) Google Scholar;Irvine and McLean, 1999Irvine A.D. McLean W.H.I. Human keratin diseases: the increasing spectrum of disease and subtlety of the phenotype-genotype correlation.Br J Dermatol. 1999; 140: 815-828https://doi.org/10.1046/j.1365-2133.1999.02810.xCrossref PubMed Scopus (322) Google Scholar). Our results suggest that there is a sequential reorganization of cornified cell keratin filaments involving deimination and compaction with filaggrin. In addition, we performed comparative immunoblot analyses of stratum corneum samples from normal and BCIE subjects using ACP and AMC. The results suggested that keratin aggregation in BCIE interferes with the domain specific K1 deimination and causes aberrant deimination of K1 and other epidermal proteins, thus providing insights into the mechanisms and biologic significance of K1 deimination. Normal human skin was obtained from the neck and the chest at the time of plastic surgery. Biopsy from lesional skin of two patients with BCIE (cases 1 and 2) was taken after obtaining informed consent. These patients correspond to cases 8 and 6 from previous reports, respectively (Ishida-Yamamoto et al., 1992Ishida-Yamamoto A. McGrath J.A. Judge M.R. Leigh I.M. Lane E.B. Eady R.A.J. Selective involvement of keratins K1 and K10 in the cytoskeletal abnormality of epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma).J Invest Dermatol. 1992; 99: 19-26Crossref PubMed Scopus (114) Google Scholar, Ishida-Yamamoto et al., 1994Ishida-Yamamoto A. Eady R.A.J. Underwood R.A. Dale B.A. Holbrook K.A. Filaggrin expression in epidermolytic ichthyosis (epidermolytic hyperkeratosis).Br J Dermatol. 1994; 131: 767-779Crossref PubMed Scopus (24) Google Scholar). Keratin mutations in these patients have not been determined. Scales of three other typical BCIE patients (cases 3–5; mutations in keratins in these patients are to be published elsewhere) were collected by scraping. The outermost cornified cells were collected from four healthy volunteers by tape-stripping. Preparation and affinity purification of the rabbit polyclonal antibody (ACP) to a deiminated undecapeptide corresponding to the identified deimination site in the V2 subdomain of mouse K1 (amino acid residues 545 GSSGGGRGGSS 555) were described previously (Senshu et al., 1999aSenshu T. Akiyama K. Ishigami A. Nomura K. Studies on specificity of peptidylarginine deiminase reactions using an immunochemical probe that recognizes an enzymatically deiminated partial sequence of mouse keratin K1.J Dermatol Sci. 1999; 21: 113-126Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Immunofluorescence studies on semithin sections of Lowicryl-embedded skin (see below) or cryostat sections of unfixed normal skin were incubated with either a mixture of ACP (4 µg per ml) and a mouse monoclonal antibody, 34βB4, that recognizes suprabasal keratins, including K1 (Gown and Vogel, 1984Gown A.M. Vogel A.M. Monoclonal antibodies to human intermediate filament proteins. II. Distribution of filament proteins in normal human tissues.Am J Pathol. 1984; 114: 309-321PubMed Google Scholar) (Enzo Diagnostics, New York, NY, 1:20 dilution in phosphate-buffered saline) or a mixture of ACP and a mouse monoclonal anti-filaggrin antibody (BT576, Biomedical Technologies, Stoughton, MA, 1:1000–4000 dilution) for 30 min at 37°C. This was followed by incubation with a mixture of fluorescein isothiocyanate-conjugated swine anti-rabbit immunoglobulins (1:20 dilution, DAKO, Glostrup, Denmark) and Texas red-conjugated sheep anti-mouse immunoglobulins (1:10 dilution, Amersham Life Science, Buckinghamshire, U.K.) or R-phycoerythrin-conjugated F(ab′)2 fragment of goat anti-mouse immunoglobulins (1:10 dilution, DAKO). Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (Nacalai Tesque, Kyoto, Japan). Fluorescence immunolabeling was observed using an Olympus BX-FLA-1 system (Tokyo, Japan). Digital images were captured using an electric-cooled CCD camera (SenSys, Photometrics, Tucson, AZ). The camera and image processing were controlled using IP Laboratory Spectrum software (Signal Analytics, Vienna, VA). Skin tissue samples were cryofixed, cryosubstituted, and embedded in Lowicryl K11M resin (Chemische Werke Lowi, Waldkraiburg, Germany) according to the methods previously described (Shimizu et al., 1989Shimizu H. McDonald J.N. Kennedy A.R. Eady R.A.J. Demonstration of intra- and extracellular localization of bullous pemphigoid antigen using cryofixation and freeze substitution for postembedding immunoelectron microscopy.Arch Dermatol Res. 1989; 281: 443-448Crossref PubMed Scopus (113) Google Scholar;Ishida-Yamamoto et al., 1996Ishida-Yamamoto A. Eady R.A.J. Watt F.M. Roop D.R. Hohl D. Iizuka H. Immunoelectron microscopic analysis of cornified cell envelope formation in normal and psoriatic epidermis.J Histochem Cytochem. 1996; 44: 167-175Crossref PubMed Scopus (74) Google Scholar). Ultrathin sections were cut, collected on formvar-coated nickel grids, and immunostained as described previously (Ishida-Yamamoto et al., 1996Ishida-Yamamoto A. Eady R.A.J. Watt F.M. Roop D.R. Hohl D. Iizuka H. Immunoelectron microscopic analysis of cornified cell envelope formation in normal and psoriatic epidermis.J Histochem Cytochem. 1996; 44: 167-175Crossref PubMed Scopus (74) Google Scholar). As the primary antibodies ACP (4 µg per ml), 34βB4 (1:20 dilution) and anti-filaggrin antibody (BT576, 1:4000 dilution) were used. As labels, 5 or 10 nm gold-conjugated goat anti-rabbit or mouse IgG (Amersham, 1:10 dilution) were used. In some experiments, the immunogold particles were enhanced using a silver staining kit (Amersham). For all immunohistochemistry, negative controls included incubation in the presence of a secondary antibody alone, and incubation with unrelated primary antibodies. For quantitative evaluations, numbers of gold particles per μm2 were counted over cytoplasmic areas filled with filamentous keratin and that of amorphous keratin clumps in the stratum corneum of a BCIE patient (case 1). For each compartment 30 areas were randomly chosen on photomicrographs of immunoelectron microscopy. Differences in labeling densities were tested for significance using the Student's t test. An epidermal extract enriched with undegraded keratins derived from the viable epidermal cells of normal skin was prepared as described previously (Senshu et al., 1996Senshu T. Kan S. Ogawa H. Manabe M. Asaga H. Preferential deimination of keratin K1 and filaggrin during the terminal differentiation of human epidermis.Biochem Biophys Res Commun. 1996; 225: 712-719Crossref PubMed Scopus (128) Google Scholar;Ishida-Yamamoto et al., 2000Ishida-Yamamoto A. Senshu T. Takahashi H. Akiyama K. Nomura K. Iizuka H. Decreased deiminated keratin K1 in psoriatic hyperproliferative epidermis.J Invest Dermatol. 2000; 114: 701-705https://doi.org/10.1046/j.1523-1747.2000.00936.xCrossref PubMed Scopus (90) Google Scholar). Scale samples of three BCIE patients (cases 3–5) were homogenized with 62.5 mM Tris–HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, and 2% 2-mercaptoethanol (Laemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (202763) Google Scholar) by sonication. The homogenate was centrifuged at 10,000 × g for 5 min The supernatant containing keratins was used for the analyses. Tape-stripped cornified cells were extracted with the same buffer. Protein concentrations were estimated by the method ofLowry et al., 1951Lowry O.H. Rosebrough N.J. .farr A.L. Randall R.J. Protein measurement with the Folin phenol_reagent.J Biol_Chem_. 1951; 193: 265-275PubMed Google Scholar. Proteins in the extract were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis for western blotting as described previously (Senshu et al., 1996Senshu T. Kan S. Ogawa H. Manabe M. Asaga H. Preferential deimination of keratin K1 and filaggrin during the terminal differentiation of human epidermis.Biochem Biophys Res Commun. 1996; 225: 712-719Crossref PubMed Scopus (128) Google Scholar;Ishida-Yamamoto et al., 2000Ishida-Yamamoto A. Senshu T. Takahashi H. Akiyama K. Nomura K. Iizuka H. Decreased deiminated keratin K1 in psoriatic hyperproliferative epidermis.J Invest Dermatol. 2000; 114: 701-705https://doi.org/10.1046/j.1523-1747.2000.00936.xCrossref PubMed Scopus (90) Google Scholar). The blot was first incubated with ACP, that reacts preferentially with deiminated peptide sequences in V subdomains of K1, and then horseradish peroxidase-labeled goat anti-rabbit IgG (Bio-Rad, Hercules, CA) for the detection by the enhanced luminol reaction using Renaissance (Dupont NEN, Boston, MA). The chemiluminescence image was recorded using a Bio-Rad Fluor S Max Chemiluminescence Analyzer (Bio-Rad). In order to make all citrulline residues in the separated proteins detectable, the blot was next incubated in 0.0125% FeCl3, 2.3 M H2SO4, 1.5 M H3PO4, 0.25% diacetyl monoxime, and 0.125% anti-pyrine (modification medium) at 37°C for 3 h to modify citrulline residues chemically. This treatment also abolished the reactivities of the antibodies bound in the preceding step. The blot was then incubated with a monospecific antibody to chemically modified citrulline (AMC) (Senshu et al., 1992Senshu T. Sato T. Inoue T. Akiyama K. Asaga H. Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane.Anal Biochem. 1992; 203: 94-100Crossref PubMed Scopus (143) Google Scholar) and the labeled second antibody for chemi luminescence detection in a similar manner. The blot was finally stained with Amido Black 10B to visualize proteins. The data were presented together with superimposed color images, that were prepared using an Atto Spot Screener (Atto Corporation, Tokyo, Japan). The ACP antibody raised against the deiminated mouse K1 peptide strongly stained epidermal cornified cells of the normal human skin as reported previously (Ishida-Yamamoto et al., 2000Ishida-Yamamoto A. Senshu T. Takahashi H. Akiyama K. Nomura K. Iizuka H. Decreased deiminated keratin K1 in psoriatic hyperproliferative epidermis.J Invest Dermatol. 2000; 114: 701-705https://doi.org/10.1046/j.1523-1747.2000.00936.xCrossref PubMed Scopus (90) Google Scholar). Some ACP-negative, 34βB4-positive, and filaggrin-positive cells were detected at the lower cornified layers (!Figure 1). As detailed nature of these thin layers of epidermal cells was difficult to assess by immunofluorescence microscopy, we performed immunoelectron microscopy. We confirmed that the first few cell layers of stratum corneum were ACP-negative (Figure 2a). A difference in the texture of cytoplasm between the ACP-positive cells and ACP-negative cells was also appreciated. Namely, positive cells had homogeneous cytoplasm, whereas negative cells showed some fine reticular patterns. In double staining with 34βB4, all cornified cells were 34βB4-positive, including ACP-negative lower cornified cells (Figure 2b). In double staining with filaggrin, the first few layers were filaggrin positive, but ACP negative (Figure 2c). The next few layers were double positive, and the more superficial cell layers were filaggrin negative/ACP positive (Figure 2d).Figure 2Immunoelectron microscopy shows sequential reorganization of cornified cell keratin filaments. (a) The first (1) and second (2) cornified cell layers are ACP negative, whereas the third layer (3) is positive. Note that the cytoplasm of negative cells shows a reticular pattern, whereas that of positive cells is amorphous in texture. Five nanometer gold labels have been enhanced using a silver staining method. The inset is to show these cells at lower magnification. G, granular cell. (b) Double staining with ACP (5 nm gold) and 34βB4 (10 nm gold). All the cornified cells are similarly positive to 34βB4. ACP staining is not detected in the first layer (1), only a few labels are seen in the second layer (2) and many labels in the third layer (3). These are shown in a lower magnification in the inset. G, granular cell. (c, d) Double staining with anti-filaggrin (5 nm gold) and ACP (10 nm gold). The first cornified cell (1) is filaggrin positive/ACP negative. The second cell (2) is double positive, and the third cell (3) is filaggrin negative/ACP positive. The inset in c is to show cells 1–3 in c and d at lower magnification. Scale bars: 0.1 μm; 1 μm in the inset.View Large Image Figure ViewerDownload (PPT) We next examined skin from patients with BCIE. This is a genetic skin disease caused by K1/K10 mutations. In BCIE skin, we have found that keratin filaments were abnormally clumped throughout the differentiated cell layers (Ishida-Yamamoto et al., 1992Ishida-Yamamoto A. McGrath J.A. Judge M.R. Leigh I.M. Lane E.B. Eady R.A.J. Selective involvement of keratins K1 and K10 in the cytoskeletal abnormality of epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma).J Invest Dermatol. 1992; 99: 19-26Crossref PubMed Scopus (114) Google Scholar) and some clumps in the stratum corneum were poorly associated with filaggrin (Ishida-Yamamoto et al., 1994Ishida-Yamamoto A. Eady R.A.J. Underwood R.A. Dale B.A. Holbrook K.A. Filaggrin expression in epidermolytic ichthyosis (epidermolytic hyperkeratosis).Br J Dermatol. 1994; 131: 767-779Crossref PubMed Scopus (24) Google Scholar). This led us to speculate that K1 deimination might be altered in BCIE. Immuno fluorescence showed that 34βB4 was positive in all the differentiated epidermal cells, whereas ACP labeling was noted only in a portion of stratum corneum (!Figure 3). Immunoelectron microscopy revealed that amorphous keratin clumps were less labeled with ACP (Figure 4a) and filaggrin antibodies than the filamentous keratin (Figure 4b;Ishida-Yamamoto et al., 1994Ishida-Yamamoto A. Eady R.A.J. Underwood R.A. Dale B.A. Holbrook K.A. Filaggrin expression in epidermolytic ichthyosis (epidermolytic hyperkeratosis).Br J Dermatol. 1994; 131: 767-779Crossref PubMed Scopus (24) Google Scholar). To clarify the difference between clumped and filamentous keratins, we compared the labeling over the keratin clumps and keratin filaments. The numbers of ACP-positive gold particles per μm2 were 17.5 ± 2.0 (SEM) and 79.2 ± 5.9, respectively (p <0.0001). Those of filaggrin labels were 94.3 ± 5.6 and 174.1 ± 13.4, respectively (p <0.0001).Figure 4Immunoelectron microscopy shows that deimination and association with filaggrin are heterogeneous in BCIE keratins. More labels for ACP (a) and for filaggrin (b) (silver enhanced 5 nm gold) are seen over filamentous keratin (*) than over round amorphous keratin clumps (arrows). Scale bar: 0.5 μm.View Large Image Figure ViewerDownload (PPT) The BCIE scale extracts were subjected to western blotting analyses in parallel with that of stripped normal cornified cells (!Figure 5). Keratins derived from viable epidermal cells were used as references. Amido Black staining visualized four major bands, that were estimated to be K1, K5, K10, and K14 from their mobilities (Figure 5a, lane 1). The cornified cells extracts from all normal subjects examined showed broad bands migrating slightly ahead of K1 and K10 (a representative example is shown in Figure 5a, lane 2), corresponding to partially degraded K1 and K10, respectively, as described (Senshu et al., 1996Senshu T. Kan S. Ogawa H. Manabe M. Asaga H. Preferential deimination of keratin K1 and filaggrin during the terminal differentiation of human epidermis.Biochem Biophys Res Commun. 1996; 225: 712-719Crossref PubMed Scopus (128) Google Scholar;Ishida-Yamamoto et al., 2000Ishida-Yamamoto A. Senshu T. Takahashi H. Akiyama K. Nomura K. Iizuka H. Decreased deiminated keratin K1 in psoriatic hyperproliferative epidermis.J Invest Dermatol. 2000; 114: 701-705https://doi.org/10.1046/j.1523-1747.2000.00936.xCrossref PubMed Scopus (90) Google Scholar). Those from three BCIE patients displayed more heterogeneous profiles containing a band comigrating with undegraded K1 and those migrating ahead of K10, suggesting aberrant expression and/or processing of keratins (Figure 5a, lanes 3–5). In the normal control ACP visualized a strong major signal comigrating with partially degraded K1 and a few more minor bands (45–51 kDa) (Figure 5b, lane 2), that were not detectable in the stained total profile. Similar bands were detected in the keratome biopsy samples (Ishida-Yamamoto et al., 2000Ishida-Yamamoto A. Senshu T. Takahashi H. Akiyama K. Nomura K. Iizuka H. Decreased deiminated keratin K1 in psoriatic hyperproliferative epidermis.J Invest Dermatol. 2000; 114: 701-705https://doi.org/10.1046/j.1523-1747.2000.00936.xCrossref PubMed Scopus (90) Google Scholar). The intensity of these ACP-positive bands was decreased greatly in all three BCIE scales examined, suggesting decreased deimination of specific arginine residues in K1 as well as in 45–51 kDa proteins (Figure 5b, lanes 3–5). The same blot was incubated in the modification medium followed by probing with AMC to detect all citrulline residues in the separated proteins. This treatments visualized diffuse bands comigrating with partially degraded K1 and K10 in the normal control (Figure 5d, lane 2) as described (Senshu et al., 1996Senshu T. Kan S. Ogawa H. Manabe M. Asaga H. Preferential deimination of keratin K1 and filaggrin during the terminal differentiation of human epidermis.Biochem Biophys Res Commun. 1996; 225: 712-719Crossref PubMed Scopus (128) Google Scholar;Ishida-Yamamoto et al., 2000Ishida-Yamamoto A. Senshu T. Takahashi H. Akiyama K. Nomura K. Iizuka H. Decreased deiminated keratin K1 in psoriatic hyperproliferative epidermis.J Invest Dermatol. 2000; 114: 701-705https://doi.org/10.1046/j.1523-1747.2000.00936.xCrossref PubMed Scopus (90) Google Scholar). An AMC-positive band comigrating with partially degraded K1 was also detected in the BCIE scales at intensities no less than that of the normal control (Figure 5d, lanes 3–5). It should be noted that all three BCIE scales showed heterogeneous profiles, and that the overall intensity of AMC-positive signals in these samples was comparable or even greater than that of the normal control. We have studied the possible role of V subdomain specific deimination of K1. To address these questions, we analyzed immunohistochemical and ultrastructural localization as well as immunoblotting profiles of the deiminated keratins in normal human skin and in BCIE using ACP and AMC. ACP reacts preferentially with deiminated peptide sequences in V subdomains of K1, whereas AMC detects all citrulline residues present in resolved proteins on the blot. The present immunohistochemical study showed that deimination of specific arginine residue(s) in the V subdomains of K1 remained below the limit of detection in the first few layers of the stratum corneum. This indicates that K1 deimination is not completed at the initial stage of cornification. We observed a marked ultrastructural change in the texture of corneocyte cytoplasm, namely from a reticular form in the ACP-negative lower cells to the homogeneous one in the ACP-positive upper layers. This raises an interesting possibility that deimination of the V subdomains of K1 accounts for the ultrastructural change, at least in part. Other types of keratin modifications such as cross-linking by transglutaminases or sulfhydryl oxidases and proteolysis might also be involved in the conformational alterations of K1. As the V1 and V2 subdomains of K1 contain a few arginine residues as the only positively charged amino acids (Johnson et al., 1985Johnson L.D. Idler W.W. Zhou X.-M. Roop D.R. Steinert P.M. Structure of a gene for the human epidermal 67-kDa keratin.Proc Natl Acad Sci USA. 1985; 82: 1896-1900Crossref PubMed Scopus (107) Google Scholar;Steinert et al., 1985Steinert P.M. Parry D.A.D. Idler W.W. Johnson L.D. Steven A.C. Roop D.R. Amino acid sequences of mouse and human epidermal type II keratins of Mr67,000 provide a systematic basis for the structural and functional diversity of the end domains of keratin intermediate filament subunits.J Biol Chem. 1985; 260: 7142-7149Abstract Full Text PDF PubMed Google Scholar), deimination of even a single arginine residue would markedly decrease the net positive charge of K1 as well as the hydrophilicity of these subdomains. Although very little is known about substrate configuration required for the susceptibility to PAD,Tarcsa et al., 1996Tarcsa E. Marekov L.N. Mei G. Melino G. Lee S.-C. Steinert P.M. Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin.J Biol Chem. 1996; 271: 30709-30716Crossref PubMed Scopus (280) Google Scholar found that the degree and rate of modification of arginine residues to citrulline residues directly correlated with the structural order of the substrate. Proteins such as filaggrin having only simple β-turn secondary structures were rapidly modified by PAD III in vitro, whereas those with high α-helical contents such as trichohyalin were modified less readily (Tarcsa et al., 1996Tarcsa E. Marekov L.N. Mei G. Melino G. Lee S.-C. Steinert P.M. Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin.J Biol Chem. 1996; 271: 30709-30716Crossref PubMed Scopus (280) Google Scholar). Arginine residues in the V subdomains of keratins are probably more susceptible to PAD type I, which is presumed to be a major epidermal PAD, than those in the rod domain. Filaggrin bundles keratin filaments into macrofibrils (Dale et al., 1990Dale B.A. Resing K.A. Haydock P.V. Filaggrins.in: Goldman R.D. Steinert P.M. Cellular and Molecular Biology of Intermediate Filaments. Plenum Press, New York1990: 393-412Crossref Google Scholar). In the normal human skin, aggregation of keratins with filaggrin was detected earlier than K1 deimination in the V subdomains (!Figure 2). In BCIE, deimination in the V subdomain occurred poorly (Figure 5), particularly in clumped keratins (Figure 4a) that had failed to interact properly with filaggrin (Figure 4b) (Ishida-Yamamoto et al., 1994Ishida-Yamamoto A. Eady R.A.J. Underwood R.A. Dale B.A. Holbrook K.A. Filaggrin expression in epidermolytic ichthyosis (epidermolytic hyperkeratosis).Br J Dermatol. 1994; 131: 767-779Crossref PubMed Scopus (24) Google Scholar). Interestingly, although the intensity of ACP-positive signals was markedly decreased in BCIE scales, the overall intensity of AMC-positive signals in these samples was comparable or even greater than that of the normal control. These data suggest that PAD are not deficient in BCIE epidermis, but cause aberrant deimination of K1 and possibly other epidermal proteins. Therefore, it might be that the deimination of specific arginine residues in the V subdomains occurs preferentially on keratin filaments compacted with filaggrin, and that the decreased ACP signals in BCIE may be due to decreased susceptibility of certain arginine residues to the action of PAD in abnormally clumped keratins. In addition, the decreased ACP staining in BCIE could be due to its hyperproliferative condition, as a similar decrease has been observed in psoriasis vulgaris epidermis (Ishida-Yamamoto et al., 2000Ishida-Yamamoto A. Senshu T. Takahashi H. Akiyama K. Nomura K. Iizuka H. Decreased deiminated keratin K1 in psoriatic hyperproliferative epidermis.J Invest Dermatol. 2000; 114: 701-705https://doi.org/10.1046/j.1523-1747.2000.00936.xCrossref PubMed Scopus (90) Google Scholar). The minor ACP-positive bands (45–51 kDa) found in normal cornified cells, which were thought to have a similar deiminated peptide sequence, remain to be identified. In summary we have presented evidence suggesting that cornification, the final step of keratinocyte differentiation, is not completed instantaneously. It proceeds as sequential steps of intermolecular interactions and modifications involving keratin, filaggrin, and probably many other proteins. Alteration of these events would hamper the normal keratinization process and might be responsible for some keratinization disorders. We thank Professor Michiyuki Yamada (Graduate School of Integrated Science, Yokohama City University) for the use of his facilities. This study was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to AI-Y (11877138) and a grant from the Ministry of Health and Welfare, Japan to HI. Electron microscopy was performed in the Electron Microscopy Unit, Central Laboratory for Research and Education, Asahikawa Medical College.
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