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• W2035052662 abstract "Three types of peptidylarginine deiminase (PAD), which converts a protein arginine residue to a citrulline residue, are widely distributed in animal tissues. Little is known about PAD of hemopoietic cells. We found that PAD activity in human myeloid leukemia HL-60 cells was induced with the granulocyte-inducing agents retinoic acid and dimethyl sulfoxide and with the monocyte-inducing agent 1α,25-dihydroxyvitamin D3. We cloned and characterized a PAD cDNA from retinoic acid-induced cells. The cDNA was 2,238 base pairs long and encoded a 663-amino acid polypeptide. The HL-60 PAD had 50–55% amino acid sequence identities with the three known enzymes and 73% identity with the recently cloned keratinocyte PAD. The recombinant enzyme differs in kinetic properties from the known enzymes. Immunoblotting and Northern blotting with an antiserum against the enzyme and the cDNA, respectively, showed that a protein of approximately 67 kDa increased concomitantly with increase of mRNA of approximately 2.6 kilobases during granulocyte differentiation. During monocyte differentiation the same mRNA and protein increased as in granulocyte differentiation. Neither the enzyme activity nor the protein was found in macrophage-induced cells. These results suggested that expression of the PAD gene is tightly linked to myeloid differentiation. Three types of peptidylarginine deiminase (PAD), which converts a protein arginine residue to a citrulline residue, are widely distributed in animal tissues. Little is known about PAD of hemopoietic cells. We found that PAD activity in human myeloid leukemia HL-60 cells was induced with the granulocyte-inducing agents retinoic acid and dimethyl sulfoxide and with the monocyte-inducing agent 1α,25-dihydroxyvitamin D3. We cloned and characterized a PAD cDNA from retinoic acid-induced cells. The cDNA was 2,238 base pairs long and encoded a 663-amino acid polypeptide. The HL-60 PAD had 50–55% amino acid sequence identities with the three known enzymes and 73% identity with the recently cloned keratinocyte PAD. The recombinant enzyme differs in kinetic properties from the known enzymes. Immunoblotting and Northern blotting with an antiserum against the enzyme and the cDNA, respectively, showed that a protein of approximately 67 kDa increased concomitantly with increase of mRNA of approximately 2.6 kilobases during granulocyte differentiation. During monocyte differentiation the same mRNA and protein increased as in granulocyte differentiation. Neither the enzyme activity nor the protein was found in macrophage-induced cells. These results suggested that expression of the PAD gene is tightly linked to myeloid differentiation. peptidylarginine deiminase N α-benzoyl-l-arginine ethyl ester N α-benzoyl-l-arginine dithiothreitol glutathione S-transferase myeloperoxidase polyacrylamide gel electrophoresis Mg2+- and Ca2+-free phosphate-buffered saline polymerase chain reaction all-trans-retinoic acid rapid amplification of cDNA ends 12-O-tetradecanoylphorbol-13-acetate 1α,25-dihydroxyvitamin D3 base pairs kilobases dimethyl sulfoxide nucleotides polyacrylamide gel electrophoresis Peptidylarginine deiminases (PADs)1 (protein-arginine deiminase, protein l-arginine iminohydrolase, EC 3.5.3.15) are a family of post-translational modification enzymes which convert arginine residues to citrulline residues in the presence of calcium ion. Enzymatic deimination in vitro changes the functional properties of various proteins and alters their secondary and tertiary structures (1Takahara H. Okamoto H. Sugawara K. J. Biol. Chem. 1985; 260: 8378-8383Abstract Full Text PDF PubMed Google Scholar, 2Imparl J.M. Senshu T. Graves D.J. Arch. Biochem. Biophys. 1995; 318: 370-377Crossref PubMed Scopus (30) Google Scholar, 3Lamensa J.W. Moscarello M.A. J. Neurochem. 1993; 61: 987-996Crossref PubMed Scopus (96) Google Scholar, 4Tarcsa E. Marekov L.N. Mei G. Melino G. Lee S.-C. Steinert P.M. J. Biol. Chem. 1996; 271: 30709-30716Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Deimination of keratins, filaggrin, and trichohyalin is involved in the process of keratinization of skin and hair (4Tarcsa E. Marekov L.N. Mei G. Melino G. Lee S.-C. Steinert P.M. J. Biol. Chem. 1996; 271: 30709-30716Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 5Steinert P.M. Harding H.W. Rogers G.E. Biochim. Biophys. Acta. 1969; 175: 1-9Crossref PubMed Scopus (42) Google Scholar, 6Rogers G.E. Harding H.W. Llewellyn-Smith I.J. Biochim. Biophys. Acta. 1977; 495: 159-175Crossref PubMed Scopus (117) Google Scholar, 7Senshu T. Akiyama K. Kan S. Asaga H. Ishigami A. Manabe M. J. Invest. Dermatol. 1995; 105: 163-169Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 8Senshu T. Kan S. Ogawa H. Manabe M. Asaga H. Biochem. Biophys. Res. Commun. 1996; 225: 712-719Crossref PubMed Scopus (135) Google Scholar, 9Rogers G. Winter B. McLaughlan C. Powell B. Nesci T. J. Invest. Dermatol. 1997; 108: 700-707Abstract Full Text PDF PubMed Scopus (73) Google Scholar). Deiminated keratins and filaggrin are found in the cornified layer of the epidermis and deiminated trichohyalin is localized in the medulla of hair and the inner root sheath of hair follicles and these modifications are tightly linked to cell-specific stages of epidermis differentiation and hair follicle development (5Steinert P.M. Harding H.W. Rogers G.E. Biochim. Biophys. Acta. 1969; 175: 1-9Crossref PubMed Scopus (42) Google Scholar, 6Rogers G.E. Harding H.W. Llewellyn-Smith I.J. Biochim. Biophys. Acta. 1977; 495: 159-175Crossref PubMed Scopus (117) Google Scholar, 7Senshu T. Akiyama K. Kan S. Asaga H. Ishigami A. Manabe M. J. Invest. Dermatol. 1995; 105: 163-169Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 8Senshu T. Kan S. Ogawa H. Manabe M. Asaga H. Biochem. Biophys. Res. Commun. 1996; 225: 712-719Crossref PubMed Scopus (135) Google Scholar, 9Rogers G. Winter B. McLaughlan C. Powell B. Nesci T. J. Invest. Dermatol. 1997; 108: 700-707Abstract Full Text PDF PubMed Scopus (73) Google Scholar). Extensively deiminated forms of myelin basic protein are also found in normal infant brain and in demyelinated areas of brain with multiple sclerosis, and this deimination is thought to be associated with immature myelination (10McLaurin J. Hashim G. Moscarello M.A. J. Neurochem. 1992; 59: 1414-1420Crossref PubMed Scopus (14) Google Scholar, 11Wood D.D. Bilbao J.M. O'Connors P. Moscarello M.A. Ann. Neurol. 1996; 40: 18-24Crossref PubMed Scopus (225) Google Scholar). We reported a correlation between deimination of vimentin in mouse peritoneal macrophages and ionomycin-induced apoptosis (12Asaga H. Yamada M. Senshu T. Biochem. Biophys. Res. Commun. 1998; 243: 641-646Crossref PubMed Scopus (179) Google Scholar). Deimination of a 70-kDa nuclear protein in cultured keratinocytes associated with apoptosis was also reported recently (13Mizoguchi M. Manabe M. Kawamura Y. Kondo Y. Ishidoh K. Kominami E. Watanabe K. Asaga H. Senshu T. Ogawa H. J. Histochem. Cytochem. 1998; 46: 1303-1309Crossref PubMed Scopus (50) Google Scholar). All these findings suggest involvements of PAD in biological as well as pathological processes. There are at least three types of PAD in various rodent tissues which seem to be cell type specific (3Lamensa J.W. Moscarello M.A. J. Neurochem. 1993; 61: 987-996Crossref PubMed Scopus (96) Google Scholar, 14Kubilus J. Baden H.P. Biochim. Biophys. Acta. 1983; 745: 285-291Crossref PubMed Scopus (71) Google Scholar, 15Watanabe K. Akiyama K. Hikichi K. Ohtsuka R. Okuyama A. Senshu T. Biochim. Biophys. Acta. 1988; 966: 375-383Crossref PubMed Scopus (131) Google Scholar, 16Terakawa H. Takahara H. Sugawara K. J. Biochem. ( Tokyo ). 1991; 110: 661-666Crossref PubMed Scopus (97) Google Scholar). Their substrate specificities for BAEE and Bz-l-Arg and their antigenic properties are different. PAD type II purified from rat muscle has been well characterized. It is also present in the brain, spinal cord, and some secretory tissues. PAD types I and III are mainly present in the epidermis and uterus and in hair follicles, respectively. PAD cDNAs for types I, II, and III have been isolated from rat, mouse, and sheep, but not from humans (9Rogers G. Winter B. McLaughlan C. Powell B. Nesci T. J. Invest. Dermatol. 1997; 108: 700-707Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 17Watanabe K. Senshu T. J. Biol. Chem. 1989; 264: 15255-15260Abstract Full Text PDF PubMed Google Scholar, 18Nishijyo T. Kawada A. Kanno T. Shiraiwa M. Takahara H. J. Biochem. ( Tokyo ). 1997; 121: 868-875Crossref PubMed Scopus (43) Google Scholar, 19Rus'd A.A. Ikejiri Y. Ono H. Yonekawa T. Shiraiwa M. Kawada A. Takahara H. Eur. J. Biochem. 1999; 259: 660-669Crossref PubMed Scopus (67) Google Scholar). Their amino acid sequences constituting 662 to 673 amino acid residues have been deduced. Recently, a novel PAD cDNA named type IV was isolated from a keratinocyte cell line from a newborn rat and rat epidermis, but the distribution of the enzyme in cells and tissues is not yet known (20Ishigami A. Kuramoto M. Yamada M. Watanabe K. Senshu T. FEBS Lett. 1998; 433: 113-118Crossref PubMed Scopus (42) Google Scholar, 21Yamakoshi A. Ono H. Nishijyo T. Shiraiwa M. Takahara H. Biochim. Biophys. Acta. 1998; 1386: 227-232Crossref PubMed Scopus (26) Google Scholar). PAD activities in rat granulocytes and mouse peritoneal macrophages have been reported, but nothing is known about the enzyme properties or structures of the enzymes (22Nagata S. Senshu T. Experientia ( Basel ). 1990; 46: 72-74Crossref PubMed Scopus (23) Google Scholar). We studied PAD in human myeloid leukemia HL-60 cells, which can be induced to differentiate into granulocytes by retinoic acid and into monocyte/macrophages by 1α,25-(OH)2D3 or TPA (23Collins S.J. Blood. 1987; 70: 1233-1244Crossref PubMed Google Scholar). We report here the molecular characterization of HL-60 cell PAD induced by retinoic acid and regulation of its expression in myeloid differentiation. Gigapack III Gold packaging extract and λZAP II/EcoRI/calf intestine alkaline phosphatase-treated vector were from Stratagene. Hybond-N nylon membranes, cyanogen bromide-activated Sepharose 4B, a GST expression system, and PreScissionTM protease of a 3C protease were from Amersham Pharmacia Biotech. pCRII and a Fast Track mRNA isolation kit were from Invitrogen. SequeThermTM and Long-ReadTMCycle Sequencing kits were from Epicentre Technologies. SuperScript II RT was from Life Technologies, Inc. Expand Taq DNA polymerase was from Roche Molecular Biochemicals. BAEE was from the Peptide Institute, Inc. Bz-l-Arg and RA were from Sigma. 1α,25-(OH)2D3 was from Wako Pure Chemicals Co. TPA was from Midland Corp. Rat muscle PAD type II (15Watanabe K. Akiyama K. Hikichi K. Ohtsuka R. Okuyama A. Senshu T. Biochim. Biophys. Acta. 1988; 966: 375-383Crossref PubMed Scopus (131) Google Scholar), rat recombinant PAD type IV (20Ishigami A. Kuramoto M. Yamada M. Watanabe K. Senshu T. FEBS Lett. 1998; 433: 113-118Crossref PubMed Scopus (42) Google Scholar), rat PAD type II cDNA (17Watanabe K. Senshu T. J. Biol. Chem. 1989; 264: 15255-15260Abstract Full Text PDF PubMed Google Scholar), rabbit anti-rat PAD type II serum (15Watanabe K. Akiyama K. Hikichi K. Ohtsuka R. Okuyama A. Senshu T. Biochim. Biophys. Acta. 1988; 966: 375-383Crossref PubMed Scopus (131) Google Scholar), rabbit anti-modified citrulline IgG (24Senshu T. Sato T. Inoue T. Akiyama K. Asaga H. Anal. Biochem. 1992; 203: 94-100Crossref PubMed Scopus (149) Google Scholar), rabbit-anti MPO serum (25Yamada M. Kurahashi K. J. Biol. Chem. 1984; 259: 3021-3025Abstract Full Text PDF PubMed Google Scholar), and MPO cDNA (26Tsuchiya N. Kamei D. Takano A. Matsui T. Yamada M. J. Biochem. ( Tokyo ). 1998; 123: 499-507Crossref PubMed Scopus (27) Google Scholar) were described previously. HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (General Scientific Laboratories) and 50 μg/ml kanamycin sulfate. For granulocyte differentiation, the cells were seeded at a density of 3 × 105 cells/ml and cultured in the presence of 1 μm RA or 1.25% Me2SO (25Yamada M. Kurahashi K. J. Biol. Chem. 1984; 259: 3021-3025Abstract Full Text PDF PubMed Google Scholar). For monocyte/macrophage differentiation, the cells were cultured in the presence of 0.1 μm1α,25-(OH)2D3 or 10 ng/ml TPA (26Tsuchiya N. Kamei D. Takano A. Matsui T. Yamada M. J. Biochem. ( Tokyo ). 1998; 123: 499-507Crossref PubMed Scopus (27) Google Scholar). For TPA treatment, the cells were seeded at a density of 9 × 105 cells/ml. PAD activity was determined using BAEE as a substrate as described previously (15Watanabe K. Akiyama K. Hikichi K. Ohtsuka R. Okuyama A. Senshu T. Biochim. Biophys. Acta. 1988; 966: 375-383Crossref PubMed Scopus (131) Google Scholar). Harvested HL-60 cells were resuspended at 2 × 108 cells/ml in a lysis buffer containing 20 mm Tris-HCl (pH 7.6), 1 mmEDTA, 1 mm phenylmethylsulfonyl fluoride, and 1% Triton X-100 and ruptured by freeze-thawing 3 times. The reaction mixture (50 μl) containing 0.1 m Tris-HCl (pH 7.6), 10 mmCaCl2, 5 mm dithiothreitol, 10 mmBAEE, and 25 μl of the cell lysate was incubated at 50 °C for 1 h. Then the reaction was stopped by adding 12.5 μl of 5m perchloric acid. The perchloric acid-soluble fraction was subjected to a colorimetric reaction with citrulline as a standard. The reaction was linear with time up to 3 h with cell lysates and recombinant enzyme under the assay conditions. One unit of the enzyme was defined as the amount of enzyme catalyzing the formation of 1 μmol of citrulline derivative in 1 h under the assay conditions. Kinetic parameters for BAEE and Bz-l-Arg were estimated from the activities assayed at 37 °C for 1 h from Lineweaver-Burk plots. Protein concentrations were determined by the method of Bradford with bovine serum albumin as a standard (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Poly(A)+ RNA was prepared from HL-60 cells treated with 1 μm RA for 3 days using a Fast Track mRNA isolation kit. cDNA was synthesized from 5 μg of poly(A)+ RNA with oligo(dT)25 (dA/C/G) as a primer using Moloney murine leukemia virus reverse transcriptase, followed by addition ofEcoRI-NotI-SalI adaptor and phosphorylation of the 5′ end using a Great Lengths cDNA synthesis kit (CLONTECH) according to the supplier's manual. The cDNA was ligated into an EcoRI site of λZAP II vector and then packaged at 22 °C for 2 h using Gigapack III GOLD phage extract. Approximately 5 × 105 plaques were screened by plaque hybridization with a32P-labeled rat PAD type II cDNA probe prepared by the random oligoprimer DNA labeling method (28Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16562) Google Scholar). Hybridization was carried out in a solution containing 5 × SSPE, 5 × Denhardt's solution, 50% formamide, 10% dextran sulfate, 1% SDS, and the probe (8 × 107 cpm/5 ng/ml) at 45 °C overnight. The membranes were washed twice with 2 × SSC, 0.1% SDS at room temperature and 1 × SSC, 0.1% SDS at 65 °C for 15 min. They were exposed to x-ray film at −80 °C (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Positive cDNA clones were characterized by restriction enzyme digestions. Two PAD cDNA clones, 7-2 and 13-2, were chosen, subcloned into plasmids and sequenced. Clones with cDNAs for the 5′-end of the PAD were isolated by the 5′-RACE method (30Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4314) Google Scholar). A sample of 1 μg of poly(A)+ RNA isolated from RA-treated HL-60 cells was reverse-transcribed with SuperScript IITM using an antisense primer (nt 360–342); 5′-CGGTGAGGTAGAGTAGAGC-3′. The first strand cDNA synthesized was polyguanylated with terminal deoxynucleotidyl transferase. The second strand cDNA was synthesized with Expand Taq DNA polymerase using the polyguanylated cDNA as a template and a C primer; 5′-GGCCCGACGTCGCATGAATTCGCCCCCCCCCCCC-3′ and then the cDNA was amplified by PCR using an ApaI primer; 5′-GGGCCCGACGTCGCATG-3′ and a nested antisense primer (nt 329–310); 5′-AGTCTTGGGTCCGTAGTATG-3′. The PCR product was subcloned into pCR II and sequenced. The cycle sequencing reaction was performed using an IRD41-labeled primer and SequeTherm DNA polymerase by the chain termination method (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Nucleotide sequences were determined with an Li-COR DNA sequencer, model 4000L. The current nucleotide sequence and protein sequence data bases were searched with a BLAST program (31Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (68368) Google Scholar). Total RNAs were isolated from HL-60 cells by the acid guanidine thiocyanate method (32Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar) and poly(A)+RNA was isolated using an oligo(dT)-cellulose column (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The poly(A)+ RNA was separated by electrophoresis in denaturing 0.8% agarose gel containing 2.2 m formaldehyde, transferred to a Hybond-N nylon membrane, and UV cross-linked (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The membrane was hybridized with a 32P-labeled full-length hPAD cDNA probe (8.5 × 106 cpm/6.3 ng/ml) in a solution of 50% formamide, 6 × SSPE, 0.1% SDS, 0.01% sonicated heat-denatured salmon sperm DNA, 5 × Denhart's solution, and 5% dextran sulfate at 42 °C for 24 h. The membranes were finally washed in 0.1 × SSC, 0.1% SDS at 65 °C, and autoradiographed as described above (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The entire coding sequence of PAD cDNA was constructed from a 5′-RACE cDNA and 7-2 cDNA by overhang extension by PCR. Briefly, a 5′-RACE cDNA was amplified using an M13 p8 primer (TOYOBO) and the antisense primer (nt 329–310) described above and then treated with T4 DNA polymerase to excise the 3′ extruded portion. The PCR product, whose 3′ end overlaps the 5′ end (nt 246–329) of the 7-2 cDNA sequence, was annealed with KpnI-cut 7-2 cDNA and elongated at 68 °C. The elongated product was amplified with a sense primer (hPAD-ex1: 27-mer) consisting of a 5′ EcoRI site (underlined) and a 19-nt sequence (nt 27–45): 5′-CCGAATTCATGGCCCAGGGGACATTGA-3′, an antisense primer (hPAD-ex2: 35-mer) consisting of a 5′ EcoRI-NotI site (underlined) and a 19-nt sequence (nt 2,093–2,075): 5′-CCGAATTCGCGGCCGCGAGCTCTTGCTTGCCACAC-3′ and Expand Taq DNA polymerase. The amplified cDNA was digested with EcoRI and subcloned into an EcoRI site of pGEX 4T-1 containing a thrombin site and named pGEX-hPAD. The hPAD cDNA was also subcloned into an EcoRI site of pGEX 6P-1 containing a 3C protease site. BL-21 cells transformed with pGEX-hPAD were grown in 2 × YT medium at 25 °C to a cell density of 1.0 at 600 nm and then after addition of 0.1 mmisopropyl-β-d-thiogalactopyranoside for a further 5 h. The cells were resuspended in a lysis buffer containing 20 mm Tris-HCl (pH 7.6), 1 mm EDTA, and 0.1% Triton X-100 and disrupted by 2–3 passages through a French press. The cell lysate was brought to a concentration of 1 m NaCl and centrifuged at 15,000 × g for 30 min. The supernatant was loaded on a glutathione-Sepharose 4B column and the column was thoroughly washed with lysis buffer containing 0.1 m NaCl. The recombinant fusion protein was eluted with a solution of 10 mm glutathione in 50 mm Tris-HCl (pH 8.0), 0.1m NaCl, and 0.1% Triton X-100. The yield of enzyme activity was about 26%. Purified GST-hPAD (360 μg) in complete Freund's adjuvant was injected into rabbits and then they were given a booster injection of the same antigen in incomplete Freund's adjuvant. Anti-PAD serum was applied to a GST-Sepharose column. The unabsorbed fraction contained anti-PAD activity. An aliquot was diluted 100-fold with PBS(−) and then incubated with 280 μg/ml recombinant GST at room temperature for 20 min before use for immunoblotting. This preincubation was necessary for bleaching a nonspecific band of about 70 kDa. Sample proteins were subjected to SDS-10% PAGE by the method of Laemmli (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) and then transferred to a nitrocellulose membrane. For immunostaining of deiminated proteins, the membrane was treated at 37 °C for 3 h with the medium for chemically modifying citrulline residues and then modified citrulline residues were detected by coupled immunoreactions with rabbit anti-modified citrulline IgG (0.125 μg/ml) for 1 h and horseradish peroxidase conjugate of goat anti-rabbit IgG (1:5,000) for 1 h by a reported method (7Senshu T. Akiyama K. Kan S. Asaga H. Ishigami A. Manabe M. J. Invest. Dermatol. 1995; 105: 163-169Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 24Senshu T. Sato T. Inoue T. Akiyama K. Asaga H. Anal. Biochem. 1992; 203: 94-100Crossref PubMed Scopus (149) Google Scholar) with slight modification. Immunoblotting of PAD was performed using anti-GST-hPAD serum (1:3,000) or anti-rat type II PAD and bound IgG was detected with a horseradish peroxidase conjugate of goat anti-rabbit IgG (1:5,000) (Bio-Rad) using a chemiluminescence reagent kit, Renaissance (NEN Life Science Products). The blot was reprobed with anti-MPO serum (1:3,000) after deprobing with a solution of 2% SDS, 62.5 mm Tris-HCl (pH 6.5), 0.1 m 2-mercaptoethanol as described (34Kaufmann S.H. Ewing C.M. Shaper J.H. Anal. Biochem. 1987; 161: 89-95Crossref PubMed Scopus (143) Google Scholar). When HL-60 cells were grown in the presence of RA, a granulocyte inducing agent, their PAD activity increased in the exponential phase of cell growth and reached a plateau in the stationary phase. No activity was detected in the absence of RA throughout the 3-day culture period (Fig.1, A and B). During cell growth in the presence of RA, the MPO activity of the cells rapidly decreased to about 10% of that of control cells, indicating differentiation of the HL-60 cells into granulocytes (Fig.1 C), as reported previously (25Yamada M. Kurahashi K. J. Biol. Chem. 1984; 259: 3021-3025Abstract Full Text PDF PubMed Google Scholar). Various compounds are known to induce differentiation of HL-60 cells into granulocytes, monocytes, or macrophages (23Collins S.J. Blood. 1987; 70: 1233-1244Crossref PubMed Google Scholar). After additions of these compounds, the cells were examined for expression of PAD. TableI summarizes the effects of various differentiation inducers on the expressions of PAD in cells cultured for 2 days. Like RA, another granulocyte inducing agent, Me2SO also caused increase in PAD activity. The monocyte inducing agent 1α,25-(OH)2D3 increased PAD activity of the cells, while cells cultured with the macrophage inducing agent TPA, like control cells, did not show induction of PAD activity. The PAD in cells cultured with RA showed a ratio of activities to Bz-l-Arg and BAEE of about 1.5. The PAD in cells cultured with 1α,25-(OH)2D3 also showed similar activities to Bz-l-Arg and BAEE (data not shown). The ratios of the two HL-60 PADs differed from those of four known rat enzymes (1.0, 0.2, 0.2, and 0.2 for type I, II, III, and IV, respectively) (15Watanabe K. Akiyama K. Hikichi K. Ohtsuka R. Okuyama A. Senshu T. Biochim. Biophys. Acta. 1988; 966: 375-383Crossref PubMed Scopus (131) Google Scholar, 16Terakawa H. Takahara H. Sugawara K. J. Biochem. ( Tokyo ). 1991; 110: 661-666Crossref PubMed Scopus (97) Google Scholar, 20Ishigami A. Kuramoto M. Yamada M. Watanabe K. Senshu T. FEBS Lett. 1998; 433: 113-118Crossref PubMed Scopus (42) Google Scholar).Table IPAD activities of HL-60 cells treated with various differentiation inducing agentsAdded inducersActivity1-aValues are mean ± SD for three separate cell cultures.units/mgNoneND1-bND, not detectable.RA0.091 ± 0.018Me2SO0.041 ± 0.0121α,25-(OH)2D30.052 ± 0.005TPANDHL-60 cells were grown for 2 days in the absence or presence of the granulocyte inducers 1 μm RA and 1.25% Me2SO and the monocyte/macrophage inducers 0.1 μm1α,25-(OH)2D3 and 10 ng/ml TPA. PAD activities of the cell lysates were determined using BAEE as a substrate as described under “Experimental Procedures.”a Values are mean ± SD for three separate cell cultures.b ND, not detectable. Open table in a new tab HL-60 cells were grown for 2 days in the absence or presence of the granulocyte inducers 1 μm RA and 1.25% Me2SO and the monocyte/macrophage inducers 0.1 μm1α,25-(OH)2D3 and 10 ng/ml TPA. PAD activities of the cell lysates were determined using BAEE as a substrate as described under “Experimental Procedures.” We then examined whether PADs produced in HL-60 cells also act on cellular proteins (Fig. 2 A). Lysates of cells cultured with RA or 1α,25-(OH)2D3 for 3 days were incubated at 37 °C for 1 h with or without 10 mmCaCl2 and then subjected to SDS-PAGE. Deiminated proteins in the protein blots were probed with anti-modified citrulline IgG. On incubation with Ca2+, both the cell lysates showed numerous deiminated proteins migrating in a wide molecular weight range (lanes 5 and 6), but on incubation without Ca2+ no deiminated proteins were detected (lanes 2 and 3). Untreated cell lysates did not show any deiminated proteins, regardless of the presence or absence of Ca2+ (Fig. 2 A, lanes 1 and 4). These results indicate that PADs in the RA cell lysates and 1α,25-(OH)2D3 cell lysates can deiminate various cellular proteins in the presence of Ca2+. In addition, the absence of detectable deiminated proteins in the intact cells suggested that a few proteins might be targeted slightly underin vivo conditions. Immunostaining of similar protein blots loaded with RA- and 1α,25-(OH)2D3 cell lysates containing 7 milliunits of PAD with anti-rat PAD type II IgG did not give any positive signals, although 2.8 and 14 milliunits of PAD of rat muscle PAD type II gave bands of about 72 kDa (Fig.2 C). This also suggested that the HL-60 PADs produced in cells cultured with RA or 1α,25-(OH)2D3differ from the type II enzyme. To isolate and characterize the HL-60 PAD, we used a cDNA cloning strategy. We constructed a cDNA library in λZAP II from HL-60 cells treated with RA for 3 days, and then screened the library by plaque hybridization with rat PAD type II cDNA as a probe. Two positive cDNA clones, 7-2 and 13-2, were selected and sequenced. Their sequences overlapped, but a sequence for a 5′ portion of PAD mRNA was missing. Thus, a 5′ portion of PAD cDNA was prepared by the 5′-RACE method. Several 5′-RACE cDNAs were obtained and sequenced. They had the same sequence. Three overlapping cDNAs were 5′-RACE cDNA (nt 1 to 329), 7-2 cDNA (nt 246 to 2, 286), and 13-2 cDNA (nt 1,374 to 2,286). Alignment of the 5′-RACE cDNA and the 7-2 and 13-2 cDNAs gave a full-length cDNA named human PAD V cDNA (hPAD V cDNA). The cDNA was 2,286 bp long, and consisted of a 5′-untranslated region of 26 bp, a coding region of 1,992 bp, a 3′-untranslated region of 268 bp including a polyadenylation signal, AATAAA (nt 2,236 to 2,241), and a poly(A) tail (nt 2,264 to 2,286). The coding sequence encoded a polypeptide of 663 amino acid residues with a calculated molecular mass of 74,100 Da. The calculated pI of the protein was 6.12. The deduced amino acid sequence showed 55, 50, and 55% identities with those of rat PAD types I, II, and III, respectively, and 73% identity with rat keratinocyte PAD type IV, whose distribution in cells and tissues is not yet known. The carboxyl two-thirds of the sequences were relatively conserved, while the sequences of their amino-terminal one-thirds were more divergent (data not shown). To express the above cloned PAD cDNA as a GST fusion protein inE. coli, we constructed the entire coding sequence (nt 27 to 2,093) of PAD from a 5′-RACE cDNA and a 7-2 cDNA by overhang extension and PCR and inserted it into the pGEX 4T-1 vector. An isolated construct of pGEX-hPAD contained one base substitution of G for A at nt 1,367, which did not result in any change in the amino acid sequence encoded by the original PAD cDNA. Cells transformed with pGEX-hPAD showed high PAD activity (specific activity 18.3 with BAEE as a substrate), but cells transformed with pGEX-hPADα containing the PAD cDNA in the reverse direction had no activity (data not shown). Most of the enzyme activity in cell extracts was recovered in a soluble fraction and then was affinity purified on a GSH-Sepharose column with a yield of 26%. The preparation gave a single major band of approximately 97 kDa on SDS-PAGE (Fig.3). Its specific activity (units/mg) was about 399, which was close to that of a homogeneous preparation of PAD type II purified from rat muscle. A GST-hPAD fusion protein was digested with PreScission 3C protease and then the recombinant enzyme was isolated. The activities of this enzyme on the synthetic substrates BAEE and Bz-l-Arg were studied at 37 °C. The kinetic parameters V max, K m,K cat, andK cat/K m for these substrates were estimated from Lineweaver-Burk plots (TableII). The K catvalue for BAEE was the same as that for Bz-l-Arg. TheK m for BAEE was larger than that for Bz-l-Arg. TheK cat/K m ratio for Bz-l-Arg was 1.5 times that for BAEE." @default.
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• W2035052662 title "Molecular Characterization of Peptidylarginine Deiminase in HL-60 Cells Induced by Retinoic Acid and 1α,25-Dihydroxyvitamin D3" @default.
• W2035052662 cites W1492219703 @default.
• W2035052662 cites W1500939789 @default.
• W2035052662 cites W1507990006 @default.
• W2035052662 cites W1514133613 @default.
• W2035052662 cites W1516626843 @default.
• W2035052662 cites W1773256867 @default.
• W2035052662 cites W1918768783 @default.
• W2035052662 cites W1964310248 @default.
• W2035052662 cites W1966572145 @default.
• W2035052662 cites W1967034105 @default.
• W2035052662 cites W1977114497 @default.
• W2035052662 cites W1980325059 @default.
• W2035052662 cites W1995734706 @default.
• W2035052662 cites W1996985500 @default.
• W2035052662 cites W1998528712 @default.
• W2035052662 cites W2002832567 @default.
• W2035052662 cites W2010694257 @default.
• W2035052662 cites W2014669590 @default.
• W2035052662 cites W2015339616 @default.
• W2035052662 cites W2017212166 @default.
• W2035052662 cites W2023300261 @default.
• W2035052662 cites W2030366638 @default.
• W2035052662 cites W2033573660 @default.
• W2035052662 cites W2034228147 @default.
• W2035052662 cites W2038011107 @default.
• W2035052662 cites W2041015449 @default.
• W2035052662 cites W2051252146 @default.
• W2035052662 cites W2055043387 @default.
• W2035052662 cites W2062871507 @default.
• W2035052662 cites W2100164281 @default.
• W2035052662 cites W2100837269 @default.
• W2035052662 cites W2108388593 @default.
• W2035052662 cites W2109578241 @default.
• W2035052662 cites W2117263637 @default.
• W2035052662 cites W2157378804 @default.
• W2035052662 cites W2161766888 @default.
• W2035052662 cites W4293247451 @default.
• W2035052662 cites W4294216491 @default.
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