Nuclear Localization of Peptidylarginine Deiminase V and Histone Deimination in Granulocytes
2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês
10.1074/jbc.m208795200
ISSN1083-351X
AutoresKatsuhiko Nakashima, Teruki Hagiwara, Michiyuki Yamada,
Tópico(s)Signaling Pathways in Disease
ResumoPeptidylarginine deiminase (PAD) deiminates arginine residues in proteins to citrulline residues Ca2+ dependently. There are four types of PADs, I, II, III, and V, in humans. We studied the subcellular distribution of PAD V in HL-60 granulocytes and peripheral blood granulocytes. Expression of green fluorescent protein-tagged PADs in HeLa cells revealed that PAD V is localized in the nucleus, whereas PAD I, II, and III are localized in the cytoplasm. PAD V deletion mutants indicated that the sequence residues 45–74 have a nuclear localization signal (NLS). A sequence feature of this NLS is a three-lysine residue cluster preceded by a proline residue and is not found in the three other PADs. Substitution of the lysine cluster by an alanine cluster abrogated the nuclear import activity. These results suggested that the NLS is a classical monopartite NLS. HL-60 granulocytes, neutrophils, and eosinophils stained with antibody specific for PAD V exhibited distinct positive signals in the nucleus. Subcellular fractionation of HL-60 granulocytes also showed the nuclear localization of the enzyme. When neutrophils were stimulated with calcium ionophore A23187, protein deimination occurred in the nucleus. The major deiminated proteins were identified as histones H2A, H3, and H4. The implication of PAD V in histone modifications is discussed. Peptidylarginine deiminase (PAD) deiminates arginine residues in proteins to citrulline residues Ca2+ dependently. There are four types of PADs, I, II, III, and V, in humans. We studied the subcellular distribution of PAD V in HL-60 granulocytes and peripheral blood granulocytes. Expression of green fluorescent protein-tagged PADs in HeLa cells revealed that PAD V is localized in the nucleus, whereas PAD I, II, and III are localized in the cytoplasm. PAD V deletion mutants indicated that the sequence residues 45–74 have a nuclear localization signal (NLS). A sequence feature of this NLS is a three-lysine residue cluster preceded by a proline residue and is not found in the three other PADs. Substitution of the lysine cluster by an alanine cluster abrogated the nuclear import activity. These results suggested that the NLS is a classical monopartite NLS. HL-60 granulocytes, neutrophils, and eosinophils stained with antibody specific for PAD V exhibited distinct positive signals in the nucleus. Subcellular fractionation of HL-60 granulocytes also showed the nuclear localization of the enzyme. When neutrophils were stimulated with calcium ionophore A23187, protein deimination occurred in the nucleus. The major deiminated proteins were identified as histones H2A, H3, and H4. The implication of PAD V in histone modifications is discussed. A family of peptidylarginine deiminases (PAD) 1The abbreviations used are: PAD, peptidylarginine deiminase; anti-MC, anti-modified citrulline IgG; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; GST, glutathioneS-transferase; MBP, eosinophil major basic protein; MPO, myeloperoxidase; NLS, nuclear localization signal; PBS(−), Mg2+- and Ca2+-free phosphate-buffered saline; RA, all-trans-retinoic acid; TRITC, tetramethylrhodamine isothiocyanate (EC 3.5.3.15) catalyzes the conversion of arginine residues in proteins into citrulline residues in the presence of calcium ion. Four types of rodent PADs, I, II, III, and IV, and of human PADs, I, II, III, and V, are known (1Watanabe K. Senshu T. J. Biol. Chem. 1989; 264: 15255-15260Abstract Full Text PDF PubMed Google Scholar, 2Tsuchida M. Takahara H. Minami N. Arai T. Kobayashi Y. Tsujimoto H. Fukazawa C. Sugawara K. 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Chem. 1999; 274: 27786-27792Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). PAD V was also found in peripheral blood granulocytes (22Asaga H. Nakashima K. Senshu T. Ishigami A. Yamada M. J. Leukocyte Biol. 2001; 70: 46-51PubMed Google Scholar). PAD V in HL-60 cells can be activated to deiminate nuclear proteins of nucleophosmin/B23 and histones by stimulation with calcium ionophore (23Hagiwara T. Nakashima K. Hirano H. Senshu T. Yamada M. Biochem. Biophys. Res. Commun. 2002; 290: 979-983Crossref PubMed Scopus (152) Google Scholar). This has suggested the location of PAD V in the nucleus. The locations of PADs in cells are important for understanding a role of PADs in cellular functions, but have not yet been studied comprehensively (15Moscarello M.A. Pritzker L. Mastronardi F.G. Wood D.D. J. Neurochem. 2002; 81: 335-343Crossref PubMed Scopus (111) Google Scholar, 22Asaga H. Nakashima K. Senshu T. Ishigami A. Yamada M. J. Leukocyte Biol. 2001; 70: 46-51PubMed Google Scholar, 24Takahara H. Tsuchida M. Kusubata M. Akutsu K. Tagami S. Sugawara K. J. Biol. Chem. 1989; 264: 13361-13368Abstract Full Text PDF PubMed Google Scholar). Here we show the nuclear localization of PAD V in granulocytes compared with three other PADs and discuss a role of PAD V in histone modification. HL-60 and HeLa cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) (25Kawamura H. Tomozoe Y. Akagi T. Kamei D. Ochiai M. Yamada M. J. Biol. Chem. 2002; 277: 2732-2739Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). HL-60 granulocytes were produced by culturing HL-60 cells (3 × 105 cells/ml) with 1 μm all-trans-retinoic acid for 3 days (7Nakashima K. Hagiwara T. Ishigami A. Nagata S. Asaga H. Kuramoto M. Senshu T. Yamada M. J. Biol. Chem. 1999; 274: 27786-27792Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Human granulocytes were prepared from heparinized blood of healthy donors with PolymorphprepTM (AXIS-SHIELD) according to the manufacturer's instructions. Neutrophils and eosinophils were separated in a Percoll density reagent (Amersham Biosciences) (22Asaga H. Nakashima K. Senshu T. Ishigami A. Yamada M. J. Leukocyte Biol. 2001; 70: 46-51PubMed Google Scholar). A whole PAD V encoding region in pGEX-PAD V was subcloned into a pEGFP vector (Clontech) (7Nakashima K. Hagiwara T. Ishigami A. Nagata S. Asaga H. Kuramoto M. Senshu T. Yamada M. J. Biol. Chem. 1999; 274: 27786-27792Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). A human PAD I cDNA (KAT12008) was purchased from Takara Bio. A human PAD II cDNA (KIAA 0994) was from Kazusa DNA Research Institute (26Nagase T. Kikuno R. Ishikawa K. Hirosawa M. Ohara O. DNA Res. 2000; 7: 143-150Crossref PubMed Scopus (109) Google Scholar). A whole coding region of PAD II was amplified with PCR using a pair of primers attached with anEcoRI linker. PAD I was amplified with PCR using a pair of primers attached with a SalI linker. PAD III was amplified with PCR using pKKhPAD3 as a template and a pair of 5′ and 3′ primers attached with an EcoRI linker and a SalI linker, respectively. The amplified cDNAs were subcloned into a pEGFP vector. PAD V deletion mutants 1–262 and 262–663 were prepared by digestion of a PAD V cDNA with SmaI, and 1–394 was prepared by digestion with XhoI. Deletion mutants 58–663, 182–663, 1–104, 35–104, and 45–74 were prepared with PCR using a pGEX-PAD V as a template and a pair of 5′ and 3′ primers attached with an EcoRI linker. Alanine substitution mutant 1–104 (K59A/K60A/K61A) for three lysine residues (59–61) in a wild type was constructed by overlap extension (27Sambrook J. Russel D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001: 13.36-13.39Google Scholar). Sequences in constructs prepared with PCR were verified by sequencing. Growing HeLa cells were collected by trypsinization, washed with RPMI 1640, and resuspended in RPMI 1640 medium without fetal bovine serum and antibiotics. The cells (4 × 106 cells) were mixed with 30 μg of the CsCl-purified plasmid in 0.5 ml of medium and subjected to electroporation at 200 V, 1180 μF, and low ohm with a Cell-Porator (Invitrogen). The cells were incubated on ice for 10 min and then cultured in the complete medium for 24 h. Escherichia coliBL-21 carrying PAD I, II, and V cDNAs in a pGEX-6P vector were cultured with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside at 25 °C for 16 h (7Nakashima K. Hagiwara T. Ishigami A. Nagata S. Asaga H. Kuramoto M. Senshu T. Yamada M. J. Biol. Chem. 1999; 274: 27786-27792Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). From their extracts GST-fused PADs were affinity purified by glutathione-Sepharose 4B chromatography and digested with Prescission protease (Amersham Biosciences), and excised PADs were recovered in a flow-through fraction through a glutathione-Sepharose 4B column. Recombinant PAD III was a gift from Hidenari Takahara, Ibaraki University (8Kanno T. Kawada A. Yamanouchi J. Yosida-Noro C. Yoshiki A. Siraiwa M. Kusakabe M. Manabe M. Tezuka T. Takahara H. J. Invest. Dermatol. 2000; 115: 813-823Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The purified recombinant PADs I, II, III, and V showed specific activities of 165, 37, 120, and 224, respectively, when determined withN α-benzoyl-l-arginine ethyl ester under the conditions described previously (7Nakashima K. Hagiwara T. Ishigami A. Nagata S. Asaga H. Kuramoto M. Senshu T. Yamada M. J. Biol. Chem. 1999; 274: 27786-27792Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The previously described rabbit antiserum against GST-PAD V was affinity chromatographed on an N-terminal PAD V fragment-(1–262) bound column (7Nakashima K. Hagiwara T. Ishigami A. Nagata S. Asaga H. Kuramoto M. Senshu T. Yamada M. J. Biol. Chem. 1999; 274: 27786-27792Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The serum (2 ml) was fractionated with 17% sodium sulfate, and precipitated IgG was dialyzed, passed through a DEAE-Sephacel column equilibrated with 17.5 mm phosphate buffer, pH 6.3, and then passed through GST-bound Sepharose. The unabsorbed fraction was bound to GST-PAD V-(1–262)-bound Sepharose (a 0.6-ml column containing 2.5 mg of protein) and the column was washed with 20 mm Tris-HCl, pH 7.4, 0.5 m NaCl. The bound IgG was eluted with 0.1m glycine-HCl, pH 3.0, with a yield of about 200 μg. Sample proteins were subjected to SDS-10% PAGE by the method of Laemmli (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207497) Google Scholar) and the resolved proteins were electrotransferred to a nitrocellulose membrane. Blots were probed with anti-PAD V(N) (0.15 μg/ml) or rabbit anti-GFP antiserum (Clontech) (1:5,000), and then 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 (PerkinElmer Life Sciences). Blots of deiminated proteins were treated with the medium for chemically modifying citrulline residues at 37 °C for 3 h and then modified citrulline residues were detected with a rabbit monospecific antibody to modified citrulline (anti-MC, 0.125 μg/ml) (29Senshu T. Sato T. Inoue T. Akiyama K. Asaga H. Anal. Biochem. 1992; 203: 94-100Crossref PubMed Scopus (152) Google Scholar). The citrulline modifying medium consisted of 1 part of reagent solution A containing 0.0416% FeCl3-6H2O, 4.6 mH2SO4, and 3.0 mH3PO4 and 1 part of reagent solution B containing 0.5% diacetylmonoxime and 0.25% antipyrine. The chemiluminescent signal was captured and recorded in a Fluor-S MAX MultiImager and the signal intensities were determined with Quantity One software (Bio-Rad). HL-60 cells and blood granulocytes in suspension were washed with Hank's balanced salt solution free of calcium chloride and magnesium chloride and were fixed with ice-cold 4% paraformaldehyde in PBS(−) for 30 min. The fixed cells were washed, suspended in ice-cold PBS(−), and spread on glass slides by cytospinning at 1,600 rpm for 6 min in a CytofugeTM2 (StatSpin Inc). The cells were post-fixed with 4% paraformaldehyde for 15 min at room temperature. Cytospin preparations of HL-60 cells and blood eosinophils were heated to boiling in 10 mm citrate buffer, pH 7.0, under a microwave at 550 W for 10 min and cooled to room temperature (30Shi S.R. Cote R.J. Taylor C.R. J. Histochem. Cytochem. 2001; 49: 931-937Crossref PubMed Scopus (323) Google Scholar). This antigen retrieval procedure was omitted for neutrophils. The fixed cells were incubated with 2 m Tris-HCl, pH 7.4, for 15 min, permeabilized with 0.1% Triton X-100 in PBS(−) for 10 min, and then blocked with 2% normal goat serum containing 2% bovine serum albumin in PBS(−). HL-60 cells were double-stained with a mixture of rabbit anti-PAD V(N) (1.5 μg/ml) and mouse anti-MPO monoclonal antibody 3-2H3 (0.5 μg/ml) (31Hur S.J. Toda H.K. Yamada M. J. Biol. Chem. 1989; 264: 8542-8548Abstract Full Text PDF PubMed Google Scholar). The bound IgGs were detected with a biotin-labeled goat anti-rabbit IgG (Wako) (1:200), a streptavidin-Cy3 and a fluorescein isothiocyanate-labeled goat anti-mouse IgG (Jackson ImmunoResearch Inc.) (1:200). Neutrophils were double-stained with anti-PAD V(N) and anti-MPO, and eosinophils with anti-PAD V(N) and mouse anti-MBP monoclonal antibody BMK-13 (Monosan) (1:40 dilution). The bound IgGs were detected using a fluorescein isothiocyanate-labeled goat anti-rabbit IgG (Wako) (1:200) and a tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-mouse IgG (Sigma) (1:200). For preabsorption, 0.15 μg of either anti-PAD V(N) or nonimmune IgG and 3 μg of recombinant PAD V protein were mixed, incubated for 30 min at room temperature, and centrifuged at 15,000 × g for 20 min. The supernatant was used for the immunoreaction. Cells were stained for nuclear DNA with DAPI and mounted in a solution containing 0.1 m Tris-HCl, pH 9.0, 50% glycerol, 0.1% NaN3, and 2.5% 1,4-diazabicyclo[2,2,2]octane. HL-60 granulocytes were suspended in buffer A (20 mm Tris-HCl, pH 7.6, 5 mmMgCl2, 1.5 mm KCl, 1 mmphenylmethanesulfonyl fluoride, 2 mm dithiothreitol, and 0.1% Nonidet P-40) and disrupted with a Dounce homogenizer (25Kawamura H. Tomozoe Y. Akagi T. Kamei D. Ochiai M. Yamada M. J. Biol. Chem. 2002; 277: 2732-2739Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The homogenate was centrifuged at 760 × g at 4 °C for 10 min to separate it into supernatant (cytoplasmic) and pellet (nuclear) fractions. The supernatant and pellet fractions were subjected to immunoblotting. Blood granulocytes were suspended at 2 × 106 cells/ml in Locke's solution, stimulated with 1 μm calcium ionophoreA23187 for 5 min at 37 °C, and fixed with ice-cold 4% paraformaldehyde for 30 min. The cytospin preparations were incubated with medium for chemically modifying citrulline residues for 3 h at 37 °C and then were incubated successively with anti-MC (0.625 μg/ml), goat anti-rabbit IgG-biotin conjugate, and streptavidin-peroxidase conjugate (11Senshu T. Akiyama K. Kan S. Asaga H. Ishigami A. Manabe M. J. Invest. Dermatol. 1995; 105: 163-169Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 23Hagiwara T. Nakashima K. Hirano H. Senshu T. Yamada M. Biochem. Biophys. Res. Commun. 2002; 290: 979-983Crossref PubMed Scopus (152) Google Scholar). The medium consisted of 1 part of solution A (0.0416% FeCl3-6H2O, 4.6m H2SO4, and 3.0 mH3PO4) and 1 part of solution B (1% diacetylmonoxime, 0.5% antipyrine, and 0.5 m acetic acid). Signals for bound peroxidase were developed with 3,3′-diaminobenzidine as a substrate and the cells were stained with Giemsa. Histones were extracted from a nuclear fraction with 0.4 n H2SO4 and precipitated with acetone, and the precipitates were subjected to SDS-15% PAGE and immunoblotting as described previously (23Hagiwara T. Nakashima K. Hirano H. Senshu T. Yamada M. Biochem. Biophys. Res. Commun. 2002; 290: 979-983Crossref PubMed Scopus (152) Google Scholar). To investigate the subcellular localization of PAD by fluorescence microscopy, we prepared plasmid constructs containing PADs I, II, III, and V cDNA fused with a 3′ end of the EGFP gene and used them to transfect HeLa cells for their expression. After 24 h in culture, their expressions were confirmed by immunoblotting using anti-GFP serum (Fig.1 A). GFP-tagged PADs with about 98 kDa were detected in GFP-PAD gene-transfected cells, but not in control cells. The enzymatic activities of these GFP-PAD fusion proteins were confirmed (Fig. 1 B). On incubation of lysates of GFP-PAD gene-transfected cells with Ca2+, but without Ca2+, a number of deiminated proteins with a wide range of molecular weights were detected with anti-MC (lanes 3–10). The control cell lysate formed no deiminated protein with or without CaCl2 (lanes 1 and 2). Panel C of Fig. 1 shows fluorescence micrographs of these cells in parallel cultures. The control EGFP was located throughout the cell (panels a and f). GFP-tagged PAD V was located exclusively in the nucleus stained with DAPI (panels e andj), whereas GFP-tagged PADs I, II, and III were located only in the cytoplasm (panels b–d andg–i). These results suggested a unique nuclear location of PAD V in the cells. To identify an NLS of PAD V, we prepared N- and C-terminal deletion mutants that were fused with the EGFP gene (Fig.2 A). These constructs were expressed in HeLa cells for 24 h. Expressions of all GFP-tagged deletion mutant proteins were confirmed by immunoblotting using anti-GFP serum on the basis of their expected sizes (data not shown). No isolated deletion mutants had PAD activity (data not shown). As shown in Fig. 2 B, the fluorescence micrographs show localizations of these deletion mutants in these cells. The control GFP was distributed throughout the cells (panel a). The N-terminal deletion mutants 58–663, 182–663, and 261–663 were distributed throughout the cells, although more densely in the cytoplasm than in the nucleus (panels b–d). However, the C-terminal deletion mutants 1–394, 1–262, and 1–104 and deletion mutant 35–104 were located in the nucleus (panels e–h). Deletion mutant 45–74 as short as 30 amino acids was also located in the nucleus (panel i), indicating the presence of an NLS. This fragment has three consecutive lysine residues, 59–61, preceded by proline residue 57 (Fig. 2 C). These are features of a basic type NLS motif (Fig. 2 D). But that was not found in the three other PADs (Fig. 2 C). To determine the contribution of the three lysine residues to the NLS activity, we prepared a wild type 1–104 and its mutant 1–104 (K59A/K60A/K61A) which has three alanine (59–61) instead of the three lysine residues and fused them with the GFP-GST gene (Fig.3 A). These constructs were transfected to HeLa cells for expression. Expressions of GFP-GST-tagged wild type and mutant proteins in 24-h culture cells were confirmed by immunoblotting using anti-GFP antiserum (data not shown). Panel B of Fig. 3 shows the fluorescence micrographs of these cells. The wild type GFP-GST-PAD-(1–104) was located in the nucleus, whereas the mutant GFP-GST-PAD-(1–104) was located in the cytoplasm, and the control GFP-GST was found throughout the cell. These results suggested that the KKK motif in this position might be essential for the nuclear import of PAD V. The above results indicate the ability of recombinant PAD V to become located in the nucleus. We next determined the locations of native PAD V in HL-60 and blood granulocytes by immunocytochemistry. We affinity purified an antibody specific for PAD V from antiserum against PAD V with its N-terminal segment 1–261-conjugated Sepharose beads. It was termed anti-PAD V(N). The antibody was first tested with recombinant human PADs I, II, III, and V by immunoblotting (Fig. 4 A). It reacted with PAD V, but not with similar amounts of PADs I, II, and III. This antibody was also tested on lysates of HL-60 cells (Fig.4 B). HL-60 granulocytes expressing PAD V were prepared by culturing HL-60 cells with RA. These cells gave one distinct band of 67 kDa PAD on a blot (lane 2), whereas control HL-60 cells gave no signal (lane 1). For immunocytochemical staining, cytospin preparations of paraformaldehyde-fixed HL-60 cells were first immersed in a boiling citrate buffer and then double-stained with a mixture of anti-PAD V(N) and anti-MPO. MPO was used as a marker specific for a neutrophil cytoplasmic granule. This antigen retrieval procedure was essential for visualization of PAD V staining. On staining of HL-60 granulocytes, the distinct signals for PAD V were detected in the nucleus, but not in the cytoplasm (Fig. 4 C, panel e). In control HL-60 cells no signal was detected in the nucleus, but a faint signal was detected in the cytoplasm (panel b). Signals for MPO were consistently found in the cytoplasm of control HL-60 cells and HL-60 granulocytes (panels a and d). These results indicated that HL-60 PAD V is localized in the nucleus. We next examined PAD V in peripheral blood granulocytes. Granulocytes gave only a single band with 67 kDa on blots and its signal intensity increased with increase in the cell number from 1 × 104 to 4 × 104 (Fig.5 A, lanes 5–7). The cellular content of PAD V was estimated to be ∼1.9 × 106 molecules per cell using recombinant PAD V as a standard (lanes 1–4). Neutrophils were double-stained with a mixture of anti-PAD V(N) and anti-MPO (Fig. 5 B) without boiling in citrate buffer. Distinctive signals of anti-PAD V were confined to narrow diffuse DAPI-staining regions located along segmented forms of the nucleus (panels b and cand e and f). This is more clearly shown at higher magnification (panel d–f). The PAD-positive cells were identified as neutrophils by their nuclear morphology and by the cytoplasmic location of the MPO signal (panels a, d, g, and j). When nonimmune IgG was used as a control, no signals were detected in the nucleus, whereas the cytoplasm was stained faintly (panel h). Preabsorption of the antibody with recombinant PAD V completely abolished the signal in the nucleus, although the faint cytoplasmic signals remained (panel k). Preabsorption of nonimmune IgG also showed only the faint cytoplasmic signals (data not shown). These results indicated that neutrophil PAD V is localized in the nucleus. An eosinophil fraction prepared in a Percoll density gradient was >50% in purity. Eosinophil cytospin preparations were treated by microwave to boiling and double stained with a mixture of anti-PAD V(N) and anti-eosinophil MBP, as a marker specific for a cytoplasmic granule. Panel C of Fig. 3 shows the fluorescence micrographs. The major signal of PAD was found overlapping with the nucleus stained with DAPI, whereas a weaker signal was found in the cytoplasm overlapped with the MBP signal (panel a–c). When cells were stained with nonimmune IgG, no nuclear signal was detected, but the cytoplasmic signal was observed again (d–f). These cytoplasmic signals could be accounted for by nonspecific IgG binding to the cytopolasm. These results indicate that eosinophil PAD is localized in the nucleus. To substantiate the PAD V nuclear location shown above, we examined PAD V of HL-60 granulocytes by fractionation of their lysates into nuclear and cytoplasmic fractions. Immunoblotting of these fractions indicated that PAD V was present only in the nuclear fraction, and α-tubulin was mostly in the cytoplasmic fraction (Fig.6 A, upper panel). Protein staining of a gel showed that all core histones were recovered in the nuclear fraction (lower panel). The virtual absence of α-tubulin and histones in the nuclear and cytoplasmic fractions, respectively, showed negligible cross-contamination between these fractions. The amounts of lactate dehydrogenase activity, PAD activity, and DNA determined were 99% of the total in the cytoplasmic fraction, and 97 and 100% in the nuclear fraction, respectively. These results also indicated the nuclear localization of PAD V. To examine the solubilization of PAD V from the nucleus, we extracted the nuclear fraction with increasing concentrations of NaCl with or without 0.5% CHAPS (Fig. 6 B). PAD was almost fully solubilized at 0.2m NaCl without CHAPS and addition of 0.5% CHAPS solubilized PAD at 0.1 m NaCl (upper panel). Core histones remained insoluble under all the conditions employed (lower panel). To investigate where protein deimination occurs in blood granulocytes, we suspended cells in medium containing 2 mm CaCl2 and gave a 5-min stimulus with 0.5 μm calcium ionophore A23187. These cells were stained for deiminated proteins with anti-MC. Panel A of Fig.7 shows the micrographs of these cells. The positive signals were confined to segmented forms of the nucleus in neutrophils with the stimulus (panel b), but not without the stimulus (panel a). The number of positive cells amounted to about 50% of the total cells within 5 min and reached almost all cells by 15 min. Next, we identified deiminated proteins in cells by immunoblotting using anti-MC (Fig. 7 B). The unstimulated cells gave no bands on blots (lanes 1 and 5), whereas the stimulated cells gave three major distinct bands with ∼14, 17, and 18 kDa and several other weak bands with higher molecular weight (lanes 2–4 and 5–8). The signal intensities of these bands (14–18 kDa) increased with increase in the A23187 concentrat
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