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• W2012207611 abstract "Post-translational conversion of arginine to citrulline residues is catalyzed by peptidylarginine deiminases (PAD). Although the existence of five isoforms of PAD has been reported in rodents and humans, their tissue distribution, substrate specificity, and physiological function have yet to be explored. In the epidermis, deimination of filaggrin and keratins is involved in maintaining hydration of the stratum corneum (SC), and hence the cutaneous barrier function. Here, RT-PCR, western blotting, and confocal microscopy analyses with anti-peptide antibodies highly specific for each of the PAD1–4 demonstrated that only PAD1–3 are expressed in mouse and human epidermis. PAD1 was detected in all layers, including the SC, and PAD2 in all the living layers, whereas PAD3 expression was shown to be restricted to the granular layer and lower SC. Moreover, PAD1 and 3 were observed to co-localize with (pro)filaggrin, and PAD2 to be located at the keratinocyte periphery in the stratum granulosum. We also detected PAD1 in extracts of superficial SC, where K1 is deiminated. Moreover, we showed that PAD1 and 3 are able to modify filaggrin in vitro. These data strongly suggest that each enzyme exerts a specific role in the course of epidermis differentiation. Post-translational conversion of arginine to citrulline residues is catalyzed by peptidylarginine deiminases (PAD). Although the existence of five isoforms of PAD has been reported in rodents and humans, their tissue distribution, substrate specificity, and physiological function have yet to be explored. In the epidermis, deimination of filaggrin and keratins is involved in maintaining hydration of the stratum corneum (SC), and hence the cutaneous barrier function. Here, RT-PCR, western blotting, and confocal microscopy analyses with anti-peptide antibodies highly specific for each of the PAD1–4 demonstrated that only PAD1–3 are expressed in mouse and human epidermis. PAD1 was detected in all layers, including the SC, and PAD2 in all the living layers, whereas PAD3 expression was shown to be restricted to the granular layer and lower SC. Moreover, PAD1 and 3 were observed to co-localize with (pro)filaggrin, and PAD2 to be located at the keratinocyte periphery in the stratum granulosum. We also detected PAD1 in extracts of superficial SC, where K1 is deiminated. Moreover, we showed that PAD1 and 3 are able to modify filaggrin in vitro. These data strongly suggest that each enzyme exerts a specific role in the course of epidermis differentiation. desmoglein immunofluorescence monoclonal antibody peptidylarginine deiminase polyacrylamide gel electrophoresis stratum corneum sodium dodecyl sulfate soybean trypsin inhibitor Peptidylarginine deiminases (PAD) (protein–arginine deiminase, protein–L-arginine iminohydrolase, EC 3.5.3.15) are post-translational modification enzymes that convert the guanidino group of arginine in proteins to the ureido group of citrulline, in a Ca2+-dependent manner (Fujisaki and Sugawara, 1981Fujisaki M. Sugawara K. Properties of peptidylarginine deiminase from the epidermis of newborn rats.J Biochem (Tokyo). 1981; 89: 257-263Crossref PubMed Scopus (100) Google Scholar;Sugawara et al., 1982Sugawara K. Oikawa Y. Ouchi T. Identification and properties of peptidylarginine deiminase from rabbit skeletal muscle.J Biochem (Tokyo). 1982; 91: 1065-1071Crossref PubMed Scopus (52) Google Scholar;Vossenaar et al., 2003Vossenaar E.R. Zendman A.J. van Venrooij W.J. Pruijn G.J. PAD, a growing family of citrullinating enzymes: Genes, features and involvement in disease.Bioessays. 2003; 25: 1106-1118Crossref PubMed Scopus (685) Google Scholar). A family of five conserved PAD isoforms (PAD1–6) has been identified in various tissues of vertebrates and are encoded by five genes, clustered on chromosome 1p35–36 in humans. PAD1 has mainly been detected in the epidermis and uterus, although mRNA expression data suggest a broader tissue distribution (Terakawa et al., 1991Terakawa H. Takahara H. Sugawara K. Three types of mouse peptidylarginine deiminase: Characterization and tissue distribution.J Biochem (Tokyo). 1991; 110: 661-666Crossref PubMed Scopus (96) 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;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;Guerrin et al., 2003Guerrin M. Ishigami A. Méchin M.C. et al.cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I.Biochem J. 2003; 370: 167-174Crossref PubMed Scopus (102) Google Scholar). PAD2 is widely expressed and is particularly abundant in the skeletal muscle, brain, uterus, salivary glands, and pancreas (Takahara et al., 1989Takahara H. Tsuchida M. Kusubata M. Akutsu K. Tagami S. Sugawara K. Peptidylarginine deiminase of the mouse. Distribution, properties, and immunocytochemical localization.J Biol Chem. 1989; 264: 13361-13368Abstract Full Text PDF PubMed Google Scholar;Watanabe and Senshu, 1989Watanabe K. Senshu T. Isolation and characterization of cDNA clones encoding rat skeletal muscle peptidylarginine deiminase.J Biol Chem. 1989; 264: 15255-15260Abstract Full Text PDF PubMed Google Scholar;Akiyama et al., 1990Akiyama K. Inoue K. Senshu T. Immunocytochemical demonstration of skeletal muscle type peptidylarginine deiminase in various rat tissues.Cell Biol Int Rep. 1990; 14: 267-273Crossref PubMed Scopus (20) Google Scholar;Terakawa et al., 1991Terakawa H. Takahara H. Sugawara K. Three types of mouse peptidylarginine deiminase: Characterization and tissue distribution.J Biochem (Tokyo). 1991; 110: 661-666Crossref PubMed Scopus (96) Google Scholar;Ishigami et al., 2002Ishigami A. Ohsawa T. Asaga H. Akiyama K. Kuramoto M. Maruyama N. Human peptidylarginine deiminase type II: Molecular cloning, gene organization, and expression in human skin.Arch Biochem Biophys. 2002; 407: 25-31Crossref PubMed Scopus (84) Google Scholar). PAD3 is known to be expressed in hair follicles and in rodent epidermis (Terakawa et al., 1991Terakawa H. Takahara H. Sugawara K. Three types of mouse peptidylarginine deiminase: Characterization and tissue distribution.J Biochem (Tokyo). 1991; 110: 661-666Crossref PubMed Scopus (96) 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 (Tokyo). 1997; 121: 868-875Crossref PubMed Scopus (43) Google Scholar;Rogers et al., 1997Rogers G. Winter B. McLaughlan C. Powell B. Nesci T. Peptidylarginine deiminase of the hair follicle: Characterization, localization, and function in keratinizing tissues.J Invest Dermatol. 1997; 108: 700-707Crossref PubMed Scopus (67) Google 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). PAD4 (formerly known as PAD5) is found primarily in hematopoietic cells (Nakashima et al., 1999Nakashima K. Hagiwara T. Ishigami A. et al.Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1alpha, 25-dihydroxyvitamin D(3).J Biol Chem. 1999; 274: 27786-27792Crossref PubMed Scopus (158) Google Scholar;Asaga et al., 2001Asaga H. Nakashima K. Senshu T. Ishigami A. Yamada M. Immunocytochemical localization of peptidylarginine deiminase in human eosinophils and neutrophils.J Leukoc Biol. 2001; 70: 46-51PubMed Google Scholar;Vossenaar et al., 2004Vossenaar E.R. Radstake T.R. Van Der Heijden A. et al.Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages.Ann Rheum Dis. 2004; 63: 373-381Crossref PubMed Scopus (308) Google Scholar). Human PAD6 mRNA were detected by RT-PCR in the ovary, peripheral blood leukocytes, and testis, and, at minor levels, in the small intestine, spleen, lung, liver, and skeletal muscle (Chavanas et al., 2004Chavanas S. Méchin M.C. Takahara H. Kawada A. Nachat R. Serre G. Simon M. Comparative analysis of the mouse and human peptidylarginine deiminase gene clusters reveals highly conserved non-coding segments and a new human gene, PADI6.Gene. 2004; 330: 19-27Crossref PubMed Scopus (138) Google Scholar). The latest isoform was also identified from mouse eggs by a proteomic approach and named ePAD (Wright et al., 2003Wright P.W. Bolling L.C. Calvert M.E. et al.ePAD, an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets.Dev Biol. 2003; 256: 73-88Crossref PubMed Scopus (110) Google Scholar). Although their exact physiological functions are not known, PAD have been suggested to play important roles in several human diseases. Indeed, an increased deimination of myelin basic protein and glial fibrillary acidic protein (Kim et al., 2003Kim J.K. Mastronardi F.G. Wood D.D. Lubman D.M. Zand R. Moscarello M.A. Multiple sclerosis: An important role for post-translational modifications of myelin basic protein in pathogenesis.Mol Cell Proteomics. 2003; 2: 453-462PubMed Scopus (152) Google Scholar;Nicholas et al., 2004Nicholas A.P. Sambandam T. Echols J.D. Tourtellotte W.W. Increased citrullinated glial fibrillary acidic protein in secondary progressive multiple sclerosis.J Comp Neurol. 2004; 473: 128-136Crossref PubMed Scopus (72) Google Scholar) is involved in the pathogenesis of multiple sclerosis. In rheumatoid arthritis, disease-associated circulating autoantibodies to citrullinated proteins recognize deiminated fibrin extracted from patient synovial membranes, and are suspected to contribute to the joint inflammation (Girbal-Neuhauser et al., 1999Girbal-Neuhauser E. Durieux J.J. Arnaud M. et al.The epitopes targeted by the rheumatoid arthritis-associated antifilaggrin autoantibodies are posttranslationally generated on various sites of (pro)filaggrin by deimination of arginine residues.J Immunol. 1999; 162: 585-594PubMed Google Scholar;Masson-Bessière et al., 2001Masson-Bessière C. Sebbag M. Girbal-Neuhauser E. Nogueira L. Vincent C. Senshu T. Serre G. The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the alpha- and beta-chains of fibrin.J Immunol. 2001; 166: 4177-4184Crossref PubMed Scopus (551) Google Scholar). Furthermore, a recent study revealed the association in a Japanese population between functional haplotypes of the PADI4 gene and rheumatoid arthritis (Suzuki et al., 2003Suzuki K. Sawada T. Murakami A. et al.High diagnostic performance of ELISA detection of antibodies to citrullinated antigens in rheumatoid arthritis.Scand J Rheumatol. 2003; 32: 197-204Crossref PubMed Scopus (138) Google Scholar). Alterations in the pattern of deiminated epidermal proteins have been reported in some skin disorders, namely psoriasis and bullous congenital ichthyosiform erythroderma (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: 701-705Crossref PubMed Scopus (90) Google Scholar,Ishida-Yamamoto et al., 2002Ishida-Yamamoto A. Senshu T. Eady R.A. Takahashi H. Shimizu H. Akiyama M. Iizuka H. Sequential reorganization of cornified cell keratin filaments involving filaggrin-mediated compaction and keratin 1 deimination.J Invest Dermatol. 2002; 118: 282-287Crossref PubMed Scopus (65) Google Scholar), suggesting a crucial role of PAD during the late stages of epidermal differentiation. Lastly, deimination of histones was observed in granulocytes, suggesting a role in chromatin remodelling (Hagiwara et al., 2002Hagiwara T. Nakashima K. Hirano H. Senshu T. Yamada M. Deimination of arginine residues in nucleophosmin/B23 and histones in HL-60 granulocytes.Biochem Biophys Res Commun. 2002; 290: 979-983Crossref PubMed Scopus (141) Google Scholar;Nakashima et al., 2002Nakashima K. Hagiwara T. Yamada M. Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes.J Biol Chem. 2002; 277: 49562-49568Crossref PubMed Scopus (266) Google Scholar). Using an antiserum that detects chemically modified citrulline residues independent of the surrounding amino acid sequences, deiminated proteins were detected in the stratum corneum (SC) (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). Keratin K1 was identified as the major deiminated epidermal protein, K10 and the intermediate filament-associated protein filaggrin as the minor ones (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). Deimination of the three proteins leads to a drastic charge loss inducing the disassembly of filaggrin from the keratin intermediate filaments (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). Then, breakdown of filaggrin to free amino acids occurs. These amino acids would be involved in maintaining a proper hydration of the SC necessary for the barrier function of the skin (Rawlings and Harding, 2004Rawlings A.V. Harding C.R. Moisturization and skin barrier function.Dermatol Ther. 2004; 17: 43-48Crossref PubMed Google Scholar). In human adult epidermis, we detected PAD1 in the cytoplasm of all the nucleated keratinocytes with a higher level in the upper spinous and granular layers (Guerrin et al., 2003Guerrin M. Ishigami A. Méchin M.C. et al.cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I.Biochem J. 2003; 370: 167-174Crossref PubMed Scopus (102) Google Scholar), and we demonstrated that PAD6 is not expressed (Chavanas et al., 2004Chavanas S. Méchin M.C. Takahara H. Kawada A. Nachat R. Serre G. Simon M. Comparative analysis of the mouse and human peptidylarginine deiminase gene clusters reveals highly conserved non-coding segments and a new human gene, PADI6.Gene. 2004; 330: 19-27Crossref PubMed Scopus (138) Google Scholar). No clear data, however, are available about the expression pattern of the other PAD. Moreover, although PAD1 has been proposed to be responsible for K1 deimination (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), which PAD are involved in the modification of filaggrin and K10 is not known. To gain more information on the physiological role of PAD in the epidermis, we describe in this paper their expression pattern using affinity-purified anti-peptide antibodies highly specific for each isoform in western blotting and confocal laser microscopy analysis. In our previous papers, PADI1 tissue distribution was characterized by RT-PCR as major in the placenta, prostate, testis, thymus, and epidermis, and PADI6 in the ovary (Guerrin et al., 2003Guerrin M. Ishigami A. Méchin M.C. et al.cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I.Biochem J. 2003; 370: 167-174Crossref PubMed Scopus (102) Google Scholar;Chavanas et al., 2004Chavanas S. Méchin M.C. Takahara H. Kawada A. Nachat R. Serre G. Simon M. Comparative analysis of the mouse and human peptidylarginine deiminase gene clusters reveals highly conserved non-coding segments and a new human gene, PADI6.Gene. 2004; 330: 19-27Crossref PubMed Scopus (138) Google Scholar). In this work, the expression of PADI2–4 in human adult tissues was also investigated by RT-PCR (Figure 1). PADI2 was found to be transcribed in all samples tested, including the skin, epidermis, and HL-60 cells. PADI3 showed a more restricted expression profile, in the colon, prostate, small intestine, testis, thymus, lung, kidney, pancreas, skin, epidermis, and HL-60 cells. PADI4 mRNA also seems to be expressed in a large panel of tissues with higher levels in peripheral blood leukocytes and spleen, intermediate levels in the prostate, small intestine, testis, heart, placenta, lung, and liver and lower levels in the thymus, kidney, and pancreas. By contrast, PADI4 cDNA was not amplified from human adult epidermis. With respect to skin, PADI4 cDNA was found to be transcribed in only one of three different samples. In HL-60 cells, besides PADI2–4, we were also able to detect the expression of PADI6 but not of PADI1 (data not shown). To explore the expression in the epidermis of PAD at the protein level, anti-peptide antibodies were developed against PAD1–4, only these four isoforms being known when we started work. To design isoform-specific potentially immunogenic peptides, multi-alignment of the human, mouse, and rat PAD sequences was performed. For each PAD, three peptides identified in the most differential regions, but which were also orthologous conserved regions, were synthesized and used for immunizing rabbits (Table I and Table III). The anti-PAD1 serum has been described previously (Guerrin et al., 2003Guerrin M. Ishigami A. Méchin M.C. et al.cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I.Biochem J. 2003; 370: 167-174Crossref PubMed Scopus (102) Google Scholar). The specificity of the anti-PAD2 and anti-PAD3 antisera was extensively checked. The antisera were first tested by ELISA on their corresponding synthetic peptides (data not shown). The anti-PAD2 serum reacted strongly with the peptide corresponding to residues 514–527 (peptide C2). The anti-PAD3 serum predominantly reacted with the peptide corresponding to residues 49–66 (peptide B3). The anti-PAD4 serum presented a high reactivity against the three PAD4-derived peptides. The antisera were then purified by affinity chromatography on a mixture of their corresponding three peptides. Competition immunoblotting experiments showed a dramatic or complete loss of immunoreactivity of the affinity-purified anti-PAD2, anti-PAD3, and anti-PAD4 antibodies after adsorption with peptides C2, B3, and B4, respectively (Figure 2a). Moreover, the proteins were not detected when non-immune sera were used (data not shown). Based on these results, the anti-PAD2 and anti-PAD3 sera were affinity-purified on peptides C2 and B3, respectively. The anti-peptide antibodies obtained were named anti-PAD2 (C2) and anti-PAD3 (B3).Table IAntibodies used for immunoblotting and/or immunohistologyAntibodiesSpecificity (peptides used in purification)Source, referenceAffinity-purified anti-peptide antibodies Anti-PAD1Human PAD1 (peptides A1, B1, C1)Guerrin et al., 2003Guerrin M. Ishigami A. Méchin M.C. et al.cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I.Biochem J. 2003; 370: 167-174Crossref PubMed Scopus (102) Google Scholar Anti-PAD2Human PAD2 (peptides A2, B2, C2)This work Anti-PAD2 (C2)Human PAD2 (peptide C2) Anti-PAD3Human PAD3 (peptides A3, B3, C3)This work Anti-PAD3 (B3)Human PAD3 (peptide B3) Anti-PAD4Human PAD4 (peptides A4, B4, C4)This workMonoclonal antibodies AHF3Human (pro)filaggrinSimon et al., 1995Simon M. Sebbag M. Haftek M. et al.Monoclonal antibodies to human epidermal filaggrin, some not recognizing profilaggrin.J Invest Dermatol. 1995; 105: 432-437Crossref PubMed Scopus (28) Google Scholar AHF5Human (pro)filaggrinSimon et al., 1995Simon M. Sebbag M. Haftek M. et al.Monoclonal antibodies to human epidermal filaggrin, some not recognizing profilaggrin.J Invest Dermatol. 1995; 105: 432-437Crossref PubMed Scopus (28) Google Scholar DG3.10Human DSG1 and DSG2Progen Biotechnik GmbHPAD, peptidylarginine deiminase isoforms; DSG, desmoglein. Open table in a new tab Table IIPrimers used for RT-PCR or full cDNA cloningSequenceProduct length (bp)Primers for PCRPADI2 Sense5′-ATGCACCTTCATCGACGACATTT-3′332 Antisense5′-TTTCAGCAGGGACAGAGTCGAG-3′PADI3 Sense5′-CAGAGACAGGCCCTGAACGATAA-3′477 Antisense5′-AAGATGGTTCCGCCCTGATCTAA-3′PADI4 Sense5′-TCTTGTGAATATTGTGGCTCCCT-3′133 Antisense5′-AGAGCAGAACTGAGTGTGCAGTG-3′G3PDH Sense5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′983 Antisense5′-CATGTGGGCCATGAGGTCCACCAC-3′Primers for full cDNA cloningPADI4 Sense5′-CCGACGATGGCCCAGGGGACATTGAT-3′2000 Antisense5′-GCTCAGGGCACCATGTTCCACCACTT-3′G3PDH, glyceraldehyde-3-phosphate dehydrogenase; PAD, peptidylarginine deiminase isoforms. Open table in a new tab Table IIIPeptides used for antibody productionPeptidesSequenceAmino acid positionGenBank accession numberAnti-PAD1 Peptide A1CMAPKRVVSQLSLKM1–13 Peptide B1CNHRSAEPDLTHSWLM158–172AB033768 Peptide C1CARGGNSLSDYKQ215–227Anti-PAD2 Peptide A2MLRERTVRLQYGSC1–13 Peptide B2CTPWLPKEDMRDEK159–171AB030176 Peptide C2MFKGLGGMSSKRITC514–527Anti-PAD3 Peptide A3MSLQRIVRVSLEHC1–13 Peptide B3DIYISPNMERGRERADTRC49–66AB026831 Peptide C3CEAYRHVLGQDKV223–235Anti-PAD4 Peptide A4MAQGTLIRVTPEQC1–13 Peptide B4VVDIAHSPPAKKKSTC49–63AB017919 Peptide C4FQATRGKLSSKC214–225PAD, peptidylarginine deiminase isoforms. Open table in a new tab PAD, peptidylarginine deiminase isoforms; DSG, desmoglein. G3PDH, glyceraldehyde-3-phosphate dehydrogenase; PAD, peptidylarginine deiminase isoforms. PAD, peptidylarginine deiminase isoforms. We then investigated the cross-reactivity of each affinity-purified anti-PAD antibody to the other PAD isoforms, including PAD6. Recombinant GST-PAD1, PAD3, PAD4, and PAD6, and purified rabbit muscle PAD2 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by western blotting (Figure 2b). The anti-PAD1, anti-PAD2 (C2), anti-PAD3 (B3), and anti-PAD4 antibodies were shown to be highly specific for their respective PAD isoform. A recombinant human PAD2 was also only detected by the anti-PAD2 (C2) antibodies (data not shown). When an extract of human epidermis homogenized in a detergent-containing buffer (TE-NP40 buffer extract) was immunoblotted with the anti-PAD1, anti-PAD2 (C2), and anti-PAD3 (B3) antibodies, immunoreactive bands corresponding to human PAD1–3 were observed as 70, 76, and 70 kDa proteins, respectively (Figure 3a). The reactivities disappeared after adsorption of the antibodies on the corresponding peptides. Confirming the RT-PCR results, PAD4 was not detected in the human epidermis extract and was observed as a 70 kDa protein in an HL-60 cell extract. To enrich the epidermal extract in PAD, it was applied to a column of soybean trypsin inhibitor (STI), a substrate of PAD, immobilized on agarose. Each fraction of the affinity chromatography was then analyzed by immunoblotting with the anti-PAD antibodies (Figure 3b). PAD1 and 2 were shown to be eluted from the affinity column in high amounts (lanes 8 and 9). PAD3 was only detected in the unbound protein fractions (lanes 2–4), whereas PAD4 was not detected (data not shown). To analyze the solubility properties of PAD1–3 and to confirm the absence of expression of PAD4, the human epidermis was sequentially extracted first in TE-NP40 buffer, then in the presence of urea and finally in the presence of a reducing agent. Extracted proteins were separated by SDS-PAGE and analyzed by immunoblotting with the anti-PAD antibodies. PAD1–3 were only detected in the TE-NP40 buffer extract. Once again, PAD4 was not detected (data not shown). When total extracts of superficial SC from six normal volunteers were immunoblotted using the anti-PAD antibodies (Figure 4), an immunoreactive band of 70 kDa, corresponding to PAD1, was detected with a variable intensity between samples. By contrast, PAD2 and 3 were not detected. Indirect immunofluorescence (IF) on unfixed cryosections of normal human skin allowed us to localize PAD1–3 in the human epidermis. We performed double-label IF stainings and confocal analysis, using the anti-PAD1, anti-PAD2 (C2), and anti-PAD3 (B3) antibodies, and two monoclonal antibody (MoAb) directed to either the desmosomal cadherins desmoglein 1 and 2 (DSG1 and DSG2) (DG3.10) or (pro)filaggrin (AHF3). On control sections incubated in the absence of primary antibody, no significant immunoreactivity was observed (data not shown). DG3.10 produced pericellular labelling in basal, spinous, and granular layers (Figure 5a, Figure 6a, and Figure 7a), as expected. AHF3 showed strong cytoplasmic staining of the stratum granulosum and diffuse staining of the lower SC (Figure 5e, Figure 6e, and Figure 7e) as described previously (Simon et al., 1995Simon M. Sebbag M. Haftek M. et al.Monoclonal antibodies to human epidermal filaggrin, some not recognizing profilaggrin.J Invest Dermatol. 1995; 105: 432-437Crossref PubMed Scopus (28) Google Scholar). The cytoplasmic location of PAD1 in keratinocytes of all living epidermis layers, and the higher staining intensity in the granular layer, previously observed using an epifluorescence microscope (Guerrin et al., 2003Guerrin M. Ishigami A. Méchin M.C. et al.cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I.Biochem J. 2003; 370: 167-174Crossref PubMed Scopus (102) Google Scholar), was confirmed by confocal microscopy (Figure 5b–d and f–h). Moreover, a granular pattern was clearly observed in the cytoplasm of the granular layer keratinocytes (Figure 5d and h). The double-label IF staining combining anti-PAD1 and AHF3 antibodies indicates the co-location of PAD1 and (pro)filaggrin in the granular layer (Figure 5g and h).Figure 6Immunofluorescence staining of peptidylarginine deiminase isoforms (PAD)2 in human epidermis. Normal human skin cryosections were analyzed by confocal microscopy using the anti-PAD2 (C2) antibodies, the anti-desmoglein 1 and 2 (DSG1–2) monoclonal antibody, DG3.10, and the anti-(pro)filaggrin monoclonal antibody, AHF3, as indicated. Image superimpositions of DSG1–2/PAD2 and pro(filaggrin)/PAD2 are shown. Scale bar: 40 μm (E–G), 20 μm (A–C), 10 μm (H), 5 μm (D).View Large Image Figure ViewerDownload (PPT)Figure 7Immunofluorescence staining of peptidylarginine deiminase isoforms (PAD)3 in human epidermis. Normal human skin cryosections were analyzed by confocal microscopy using the anti-PAD3 (B3) antibodies, the anti-desmoglein 1 and 2 (DSG1—2) monoclonal antibody, DG3.10, and the anti-(pro)filaggrin monoclonal antibody, AHF3, as indicated. Image superimpositions of DSG1–2/PAD3 and pro(filaggrin)/PAD3 are shown. Scale bar: 40 μm (E–G), 20 μm (A–C), 10 μm (D), 5 μm (H).View Large Image Figure ViewerDownload (PPT) PAD2 was localized in the cytoplasm of keratinocytes of all living epidermis layers, with a gradient of expression increasing from the basal (very low level of detection) to the granular layer (Figure 6b). Furthermore, intense pericellular labelling was observed in the granular layer (Figure 6b–d and h). Double staining, performed with anti-PAD2 (C2) and AHF3 antibodies, confirmed the apparent accumulation of PAD2 at the keratinocyte periphery and showed a rare co-location of the two proteins (Figure 6g and h). PAD3 was only immunodetected in the granular layer and lower SC (Figure 7b–d and f–h), and cytoplasmic location was observed at a high magnification (Figure 7h). Double staining using anti-PAD3 (B3) and AHF3 antibodies revealed a convincing co-location of PAD3 with (pro)filaggrin in the granular layer and in the lower SC (Figure 7g and h). The anti-PAD antibodies were used to analyze PAD expression in epidermis extracts of adult B6/CBA mice. In a TE-NP40 buffer extract, immunoreactive bands of 76 kDa, corresponding to mouse PAD1 and 2, and of 80 kDa, corresponding to the mouse PAD3 (Figure 8a), were observed. In agreement with the results obtained with human epidermis, PAD4 was not detected in the mouse epidermis extracts. To determine the PAD location in mouse epidermis, immunohistochemical studies were performed on unfixed cryosections of newborn mouse skin (Figure 8b). Anti-PAD1 antibodies stained the strat" @default.
• W2012207611 created "2016-06-24" @default.
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