Portal de Periódicos da CAPES

Human Peptidylarginine Deiminase Type III: Molecular Cloning and Nucleotide Sequence of the cDNA, Properties of the Recombinant Enzyme, and Immunohistochemical Localization in Human Skin

2000; Elsevier BV; Volume: 115; Issue: 5 Linguagem: Inglês

10.1046/j.1523-1747.2000.00131.x

1523-1747

Takuya Kanno, Masakazu Shiraiwa, Hidenari Takahara, Akira Kawada, Tadashi Tezuka, Jun Yamanouchi, Chikako Yoshida, Atsushi Yoshiki, Moriaki Kusakabe, Motomu Manabe,

Virus-based gene therapy research

Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions. In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization. Of these isoforms, only type III was detected in epidermis and hair follicles. Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase. In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription–polymerase chain reaction and by rapid amplification of cDNA ends methods. This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770). To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria. The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III. The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively. An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles. Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone. The enzyme was also expressed in the cuticle layer of hair. On the other hand, expression of the enzyme in the epidermis was very low. These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation. Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions. In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization. Of these isoforms, only type III was detected in epidermis and hair follicles. Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase. In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription–polymerase chain reaction and by rapid amplification of cDNA ends methods. This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770). To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria. The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III. The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively. An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles. Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone. The enzyme was also expressed in the cuticle layer of hair. On the other hand, expression of the enzyme in the epidermis was very low. These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation. acetyl anti-human peptidylarginine deiminase type III benzoyl -amide O-ethylester O-methylester peptidylarginine deiminase polyacrylamide gel electrophoresis rapid amplification of cDNA ends human trichohyalin domain 8 tosyl Peptidylarginine deiminase (PAD; EC 3.5.3.15) is a post-translational modification enzyme that converts the guanidino group of arginine in proteins to the ureido group of citrulline in a Ca2+-dependent manner. To date, PAD has been identified in various tissues of vertebrates (Rogers and Taylor, 1977Rogers G.E. Taylor L.D. The enzymic derivation of citrulline residues from arginine residues in situ during the biosynthesis of hair proteins that are cross-linked by isopeptide bonds.Adv Exp Med Biol. 1977; 86A: 283-294Crossref PubMed Scopus (51) Google Scholar;Kubilus et al., 1980Kubilus J. Waitkus R.F. Baden H.P. Partial purification and specificity of an arginine-converting enzyme from bovine epidermis.Biochim Biophys Acta. 1980; 615: 246-251Crossref PubMed Scopus (68) Google Scholar;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;Kubilus and Baden, 1983Kubilus J. Baden H.P. Purification and properties of a brain enzyme which deiminates proteins.Biochim Biophys Acta. 1983; 745: 285-291Crossref PubMed Scopus (69) Google Scholar,Kubilus and Baden, 1985Kubilus J. Baden H.P. The occurrence and immunology of peptidylarginine deiminase and its preparation from bovine epidermis by an improved method.J Invest Dermatol. 1985; 85: 232-234Crossref PubMed Scopus (25) Google Scholar;Takahara et al., 1983Takahara H. Oikawa Y. Sugawara K. Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle.J Biochem (Tokyo). 1983; 94: 1945-1953Crossref PubMed Scopus (65) Google Scholar,Takahara et al., 1986bTakahara H. Okamoto H. Sugawara K. Affinity chromatography of peptidylarginine deiminase from rabbit skeletal muscle on a column of soybean trypsin inhibitor (Kunitz) -Sepharose.J Biochem (Tokyo). 1986; 99: 1417-1424Crossref PubMed Scopus (36) Google Scholar;Watanabe et al., 1988Watanabe K. Akiyama K. Hikichi K. Ohtsuka R. Okuyama A. Senshu T. Combined biochemical and immunochemical comparison of peptidylarginine deiminases present in various tissues.Biochim Biophys Acta. 1988; 966: 375-383Crossref PubMed Scopus (130) Google Scholar), and classified into four isoforms (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;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; 1386: 227-232Crossref PubMed Scopus (25) Google Scholar): (i) the type I enzyme is detected in the epidermis and uterus (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;Rus'd et al., 1999Rus'd A.A. Ikejiri Y. Ono H. Yonekawa T. Shiraiwa M. Kawada A. Takahara H. Molecular cloning of cDNA 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); (ii) the type II enzyme is widely expressed in various tissues, such as brain, pituitary, spinal cord, salivary gland, pancreas, skeletal muscle, uterus, spleen, stomach, and thymus (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;Kubilus and Baden, 1983Kubilus J. Baden H.P. Purification and properties of a brain enzyme which deiminates proteins.Biochim Biophys Acta. 1983; 745: 285-291Crossref PubMed Scopus (69) Google Scholar;Senshu et al., 1989Senshu T. Akiyama K. Nagata S. Watanabe K. Hikichi K. Peptidylarginine deiminase in rat pituitary: sex difference, estrous cycle-related changes, and estrogen dependence.Endocrinology. 1989; 124: 2666-2670Crossref PubMed Scopus (51) Google Scholar;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); (iii) the type III enzyme is expressed only in epidermis and hair follicles (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); and (iv) type IV is a newly classified PAD, which is a sort of ubiquitous enzyme, being expressed in the pancreas, spleen, ovary, liver, lung, stomach, kidney, uterus, dermis, brain, heart, and epidermis (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; 1386: 227-232Crossref PubMed Scopus (25) Google Scholar). In rodents, the cDNA sequences of these isoforms have been determined (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;Tsuchida et al., 1993Tsuchida M. Takahara H. Minami N. et al.cDNA nucleotide sequence and primary structure of mouse uterine peptidylarginine deiminase. Detection of a 3′-untranslated nucleotide sequence common to the mRNA of transiently expressed genes and rapid turnover of this enzyme's mRNA in the estrous cycle.Eur J Biochem. 1993; 215: 677-685Crossref PubMed Scopus (28) 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;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; 1386: 227-232Crossref PubMed Scopus (25) Google Scholar;Ishigami et al., 1998Ishigami A. Kuramoto M. Yamada M. Watanabe K. Senshu T. Molecular cloning of two novel types of peptidylarginine deiminase cDNA 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 cDNA 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), and PAD type III cDNA has also been cloned from sheep hair follicles (Rogers et al., 1997Rogers G.E. 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). Although little is known about the physiologic functions of PAD, it has been documented that PAD in brain plays an important part in the central nervous system (Kubilus and Baden, 1985Kubilus J. Baden H.P. The occurrence and immunology of peptidylarginine deiminase and its preparation from bovine epidermis by an improved method.J Invest Dermatol. 1985; 85: 232-234Crossref PubMed Scopus (25) Google Scholar;Takahara et al., 1986aTakahara H. Sueyoshi K. Sugawara K. Activities and properties of peptidylarginine deiminase of several vertebrates brains.Agric Biol Chem. 1986; 50: 1303-1306Crossref Scopus (4) Google Scholar;Wood and Moscarello, 1989Wood D.D. Moscarello M.A. The isolation, characterization, and lipid-aggregating properties of a citrulline containing myelin basic protein.J Biol Chem. 1989; 264: 5121-5127Abstract Full Text PDF PubMed Google Scholar;Lamensa and Moscarello, 1993Lamensa J.W. Moscarello M.A. Deimination of human myelin basic protein by a peptidylarginine deiminase from bovine brain.J Neurochem. 1993; 61: 987-996Crossref PubMed Scopus (94) Google Scholar) and a key role in the development of multiple sclerosis (Mastronardi et al., 1993Mastronardi F.G. Ackerley C.A. Arsenault L. Roots B.I. Moscarello M.A. Demyelination in a transgenic mouse: a model for multiple sclerosis.J Neurosci Res. 1993; 36: 315-324Crossref PubMed Scopus (81) Google Scholar,Mastronardi et al., 1996Mastronardi F.G. Mak B. Ackerley C.A. Roots B.I. Moscarello M.A. Modifications of myelin basic protein in DM20 transgenic mice are similar to those in myelin basic protein from multiple sclerosis.J Clin Invest. 1996; 97: 349-358Crossref PubMed Scopus (43) Google Scholar). On the other hand, the finding of citrulline residues in hair follicles (Rogers, 1962Rogers G.E. Occurrence of citrulline in proteins.Nature. 1962; 194: 1149-1151Crossref PubMed Scopus (48) Google Scholar) has focused the attention of dermatologic researchers on the role of PAD in mammal skin. PAD in the epidermis seems to be involved in the terminal processing of filaggrin, its disassembly with keratin intermediate filaments, and the degradation of filaggrin to free amino acids, which are thought to maintain moisture in the upper stratum corneum (Scott et al., 1982Scott I.R. Harding C.R. Barrett J.G. Histidine-rich protein of the keratohyalin granules. Source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum.Biochim Biophys Acta. 1982; 719: 110-117Crossref PubMed Scopus (219) Google Scholar;Harding and Scott, 1983Harding C.R. Scott I.R. Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation.J Mol Biol. 1983; 170: 651-673Crossref PubMed Scopus (190) Google Scholar;Manabe et al., 1991Manabe M. Sanchez M. Sun T.T. Dale B.A. Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in ichthyosis vulgaris.Differentiation. 1991; 48: 43-50Crossref PubMed Scopus (89) Google Scholar;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). Furthermore, it was reported that PAD in the inner root sheath (IRS) of hair follicles probably causes a change in the conformation of trichohyalin during hair growth (Rogers et al., 1997Rogers G.E. 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). Steinert's group proposed an in vivo model in which insoluble cytoplasmic droplets of trichohyalin that are located in the IRS of hair follicles and the medulla were initially modified by PAD, which converts the trichohyalin to a more soluble form, and subsequently the trichohyalin is cross-linked by transglutaminase (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.J Biol Chem. 1996; 271: 30709-30716Crossref PubMed Scopus (280) Google Scholar,Tarcsa et al., 1997Tarcsa E. Marekov L.N. Andreoli J. Idler W.W. Candi E. Chung S.I. Steinert P.M. The fate of trichohyalin. Sequential post-translational modifications by peptidyl-arginine deiminase and transglutaminases.J Biol Chem. 1997; 272: 27893-27901Crossref PubMed Scopus (128) Google Scholar). Recently, it was reported that there is an intimate relationship between epidermal PAD and skin disease, i.e., the deimination by PAD is involved in rheumatoid arthritis-associated anti-filaggrin autoantibodies (Girbal-Neuhauser et al., 1999Girbal-Neuhauser E. Durieux J.J. Arnaud M. et al.The epitopes targeted by the rheumatoid arthritis-associated anti-filaggrin autoantibodies are posttranslationally generated on various sites of (pro) filaggrin by deimination of arginine residues.J Immunol. 1999; 162: 585-594PubMed Google Scholar). These findings prompted us to look more carefully at the molecular biology of epidermal human PAD. In this study, we focused on human PAD type III. We cloned the full-length cDNA of human PAD type III from cultured human epidermal keratinocytes, constructed the recombinant enzyme in bacteria, and produced a monospecific anti-human PAD type III antibody for the immunohistochemical staining of human skin. Our immunohistochemical observations indicate that the type III isoform acts predominantly in hair follicles. Normal human epidermal keratinocytes from foreskin were purchased from Kurabo (Tokyo, Japan) and cultured in serum-free keratinocyte growth medium supplemented with 0.4% bovine pituitary extract, 0.1 ng epidermal growth factor per ml, 5 μg insulin per ml, 0.5 μg hydrocortisone per ml, 50 μg gentamycin per ml, and 0.05 μg amphotericin per ml in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The medium was changed every 2 d. After the cells were maintained in a proliferative phase in keratinocyte growth medium with 0.15 mM Ca2+, terminal differentiation was induced by exposure to keratinocyte growth medium with 1.2 mM Ca2+ (Hennings et al., 1980Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1449) Google Scholar). The differentiated cells after 96 h were used in this study. Total RNA was extracted using the acid guanidinium–thiocyanate–phenol–chloroform method (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62286) Google Scholar). Single-stranded cDNA was synthesized with 5 μg of total RNA, 100 μM random primers (GIBCO-BRL, Gaithersburg, MD), 50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT), 0.5 mM deoxynucleoside triphosphate (dNTP), 20 units SuperScriptII RNaseH– Reverse transcriptase (GIBCO-BRL). Reverse transcription was done for 60 min at 50°C, and then enzyme was inactivated by heating at 95°C for 3 min. (PCR) The PCR amplification was performed with 1–5 μg of the single-stranded cDNA, 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, 0.5 μM of sense and anti-sense primer, and 0.025 units TaKaRa EX Taq (Takara Shuzo, Shiga, Japan). Typically, the PCR conditions were 40 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 3 min, followed by a final elongation at 72°C for 7 min, using the Program Temp Control System PC-700 (Astec, Tokyo, Japan). The PCR products were subjected to 0.8–2.0% agarose gel electrophoresis, purified with a DNA PREP Kit (Dia-iatron, Tokyo, Japan), and cloned into dT-tailed vectors prepared from pBluescript II SK(+) (Stratagene, La Jolla, CA) (Marchuk et al., 1991Marchuk D. Drumm M. Saulino A. Collins F.S. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products.Nucleic Acids Res. 1991; 19: 1154Crossref PubMed Scopus (1105) Google Scholar). Plasmid cDNA was isolated from five different clones and sequenced by using the AutoRead Sequencing Kit on an ALF DNA sequencer II (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. Analysis of nucleotide and deduced amino acid sequence was performed with GENETYX-MAC, version 9.0 (Software Development, Tokyo, Japan). The strategy for human PAD type III cDNA cloning is shown in Figure 1. First, we designed the degenerate primers on the basis of the conserved amino acid sequences among rat and mouse PAD isoforms (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;Tsuchida et al., 1991Tsuchida M. Minami N. Tsujimoto H. Fukazawa C. Takahara H. Sugawara K. Molecular cloning of mouse peptidylarginine deiminase, and its possible isozyme cDNA.Agric Biol Chem. 1991; 55: 295-297Crossref PubMed Scopus (6) 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;Rus'd et al., 1999Rus'd A.A. Ikejiri Y. Ono H. Yonekawa T. Shiraiwa M. Kawada A. Takahara H. Molecular cloning of cDNA 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). The details of these primers are given below: P1 [sense, 5′-AT(C/T)CTGCTGGT(G/C)AA(C/T)TG(C/T)GA-3′], which corresponds to the conserved peptide motif of ILLVNCD; P2C [anti-sense, 5′-AGGGCCGAAGGG(C/T)TT(A/C/G/T)GG(A/G/T)AT-3′], which corresponds to IPKPFGP; P3 (sense, 5′-GACCGCTGGATCCAG GATGA-3′), which corresponds to DRWIQDE; and P4 [sense, 5′-GC(A/C/T)CC(A/C)TGGAT(C/T)ATGAC(A/C/G)CCC-3′], which corresponds to APWIMTP. The reverse transcription–PCR was carried out by using the single-stranded cDNA synthesized from cultured human epidermal keratinocytes and the degenerate primers. The PCR with a primer pair (P1/P2C) yielded a ≈ 1.4 kbp product (product A in Figure 1). As product A contained a mixture of cDNA of several PAD isoforms, we carried out a nested PCR using product A as a template and a degenerate primer pair of P3/P2C. By this amplification, we obtained a ≈ 0.8 kbp product (phPAD3–1 in Figure 1). This cDNA fragment was cloned into the dT-tailed vectors. Sequencing analysis of phPAD3–1 showed extremely high homology with rodent PAD type III cDNA (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;Rus'd et al., 1999Rus'd A.A. Ikejiri Y. Ono H. Yonekawa T. Shiraiwa M. Kawada A. Takahara H. Molecular cloning of cDNA 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) and sheep PAD type III cDNA (Rogers et al., 1997Rogers G.E. 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). We next synthesized an anti-sense primer P5C (5′-TTGACCTGCT-CATCATCA ACAAC-3′, at nucleotides 1590–1612 in Figure 2) based on the internal nucleotide sequences of phPAD3–1, and performed a nested PCR using product A as a template and a primer pair of P4/P5C. An approximately 0.7 kbp cDNA (phPAD3–2 in Figure 1) produced by this amplification overlapped with phPAD3–1. Furthermore, we synthesized an anti-sense primer of P6C (5′-ATCCACAAAACACGTGTTGTTCC-3′, at 982–1004) based on the internal nucleotide sequence of phPAD3–2 and performed a nested PCR using product A as a template and a primer pair of P1/P6C. From this amplification, we obtained a ≈ 0.5 kbp product (phPAD3–3 in Figure 1), which overlapped with phPAD3–2. The remaining sequences of the 5′ and 3′ terminal regions were determined by the method of 5′-rapid amplification of cDNA ends (RACE) and 3′-RACE (Frohman et al., 1988Frohman M.A. Dush M.K. Martin G.R. Rapid production of full-length cDNA from rare transcripts: amplification using a single gene-specific oligonucleotide primer.Proc Natl Acad Sci USA. 1988; 85: 8998-9002Crossref PubMed Scopus (4277) Google Scholar). For obtaining the 5′ terminal region of the cDNA, single-stranded cDNA was synthesized with the P6C primer and then a poly(dA) tail was added by using deoxyadenosine triphosphate and terminal deoxynucleotidyl transferase (GIBCO-BRL). We amplified a cDNA fragment (product B in Figure 1) using a primer pair of (dT)18-adapter primer [sense, 5′-(GA)10ACTGTCTCGAG(T)18-3′] and P7C (anti-sense, 5′-CTGTGCCCGTTTGGCATCATAGCT-3′, at 654–677 in Figure 2) based on the internal nucleotide sequence of phPAD3–3, and followed by a nested PCR with the (dT)5-adapter primer [5′-(GA)4ACTG-TCTCGAG(T)5-3′] as a sense primer and P8C (5′-GTTTGTGGTCA-TCAAAGAGGGCTG-3′, at 614–637 in Figure 2) as an anti-sense primer. The amplified cDNA fragment (phPAD3–4 in Figure 1) overlapped with phPAD3–3 and covered the 5′-terminal nucleotide sequence of human PAD type III cDNA containing the initiation Met codon. For obtaining the 3′ terminal region of cDNA, total RNA was reverse transcribed with the (dT)18-adapter primer. The single-stranded cDNA was subjected to PCR using P9 (5′-CATCAACTACAATAAGTTTGTG-3′, at 1652–1673) as a sense primer and the (dT)5-adapter primer as an anti-sense primer, and the resulting product (product C in Figure 1) was used for a template followed by a nested PCR using P10 [5′-ATCGACAT(A/C/T) CCCCAG(C/T)T(A/G/C/T)TT(C/T)AA-3′, at 1740–1762] as a sense primer and the (dT)5-adapter primer as an anti-sense primer. The amplified fragment (phPAD3–5 in Figure 1) overlapped with phPAD3–1 and contained a polyadenylation signal (AATAAA at 3124–3129 in Figure 2) and a poly(A) tail.Figure 2Nucleotide sequence and deduced amino acid sequence of human PAD type III. The DNA sequence is numbered from the first nucleotide of the cDNA. The numbers are on the right of each line. The deduced amino acid sequence is displayed below the nucleotide sequence in one letter code starting from the putative initiation methionine. A putative polyadenylation signal is underlined. The amino acid sequence indicated by wave line is that of a synthetic peptide for producing anti-hPAD3 antibody.View Large Image Figure ViewerDownload (PPT) We constructed an expression plasmid (pKKhPAD3) for producing the human PAD type III in E. coli cells as previously described (Ohsugi et al., 1995Ohsugi I. Takahara H. Shiraiwa M. Sugawara K. Expression of mouse uterine peptidylarginine deiminase in Escherichia coli: construction of expression plasmid and properties of the recombinant enzyme.Arch Biochem Biophys. 1995; 317: 62-68Crossref PubMed Scopus (5) Google Scholar). Briefly, two fragments of human PAD type III cDNA, which overlapped and covered the whole coding region, were amplified by PCR as follows. The 5′-upstream region was amplified with a primer pair of Ex1 (sense, 5′-ACTGAATTCaggaTATTACTATG-GaggaTTGATCTATGTCGCTGCAGAGAATCGTGCG-3′ as shown in Figure 1) and P6C (anti-sense). Ex1 contained an EcoRI site (indicated by a double underline), a pair of Shine-Dalgarno sequences (indicated by small letter), and a short preceding cistron (indicated by italics) inserted into the adjacent deduced amino terminal sequence (indicated by an underline). The 3′-downstream region was amplified with a primer pair of P1 (sense) and Ex2C (anti-sense, 5′-GGAAAGCTTCCAGGGGGACAGGATG- GTG-3′ in Figure 1). Ex2C contained a HindIII site (indicated by a double underline) and the complementary nucleotide sequence of 3′-untranslational region (indicated by an underline, corresponding to nucleotides 2050–2068 in Figure 2). These amplified DNA were digested with EcoRI/KpnI (the 5′-upstream region) or KpnI/HindIII (the 3′-downstream region), ligated to each other, and then inserted into the EcoRI/HindIII site of pKK223–3 vector (Amersham Pharmacia Biotech). The nucleotide sequence of the construct was confirmed by DNA sequence analysis. The pKKhPAD3 was transformed