Nuclear Localization of Peptidylarginine Deiminase V and Histone Deimination in Granulocytes

Abstract

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. 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 with PAD I was amplified with PCR using a pair of with a PAD III was amplified with PCR using as a and a pair of and with an and a The amplified were subcloned into a pEGFP PAD V deletion mutants and were prepared by of a PAD V cDNA with and was prepared by with mutants and 45–74 were prepared with PCR using a pGEX-PAD V as a and a pair of and with an for three lysine residues in a was by J. A Scholar). in prepared with PCR were by HeLa cells were by with RPMI and in RPMI 1640 medium fetal bovine serum and The cells × were with of the in of medium and to V, and with a The cells were on for and in the medium for PAD I, II, and V in a vector were with for (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). PADs were by and with (Amersham and PADs were in a a PAD III was a from (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 PADs I, II, III, and V showed specific of and when the (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 V was on an N-terminal PAD V (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 was with and was a with and The was to of and the was with The was with with a of proteins were to by the of PubMed Scopus Google and the proteins were to a were with (Clontech) and was with a of using a reagent of deiminated proteins were with the medium for citrulline residues for 3 and citrulline residues were with a antibody to citrulline T. T. T. Akiyama K. Asaga H. Biochem. PubMed Scopus (152) Google Scholar). The citrulline medium of 1 of reagent A and and 1 of reagent and The signal was and in a and the signal were with HL-60 cells and blood granulocytes in were with of calcium and and were with in for The cells were in PBS(−), and on by for in a The cells were with for of HL-60 cells and blood eosinophils were to in a for and to J. 2001; PubMed Scopus Google Scholar). This was for The cells were with for with in for and with serum bovine serum in HL-60 cells were with a of and mouse antibody Yamada M. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar). The were with a a and a Neutrophils were with and and eosinophils with and mouse antibody The were using a and a tetramethylrhodamine isothiocyanate of and 3 of PAD V protein were for and × for The was used for the were stained for nuclear DNA with and in a and HL-60 granulocytes were in A 1 and and with a (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 was × for to into and The and were to granulocytes were × in stimulated with 1 μm calcium for and with for The were with medium for citrulline residues for 3 and were with and (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, T. Nakashima K. Hirano H. Senshu T. Yamada M. Biochem. Biophys. Res. Commun. 2002; 290: 979-983Crossref PubMed Scopus (152) Google Scholar). The medium of 1 of A and and 1 of and for were with as a substrate and the cells were stained with were from a nuclear with and with and the were to and as (23Hagiwara T. Nakashima K. Hirano H. Senshu T. Yamada M. Biochem. Biophys. Res. Commun. 2002; 290: 979-983Crossref PubMed Scopus (152) Google Scholar). the subcellular localization of PAD by we prepared PADs I, II, III, and V cDNA with a of the and used to HeLa cells for in were by using serum PADs with were in but not in The of these proteins were 1 of of cells with but a of deiminated proteins with a of were with The deiminated protein with 1 and of 1 of these cells in The was the a and PAD V was in the stained with whereas PADs I, II, and III were in the These results suggested a nuclear location of PAD V in the an NLS of PAD V, we prepared and C-terminal deletion mutants that were with the These were in HeLa cells for of deletion proteins were by using serum on the of not deletion mutants PAD not in the show of these deletion mutants in these The was the cells The N-terminal deletion mutants and were the more in the in the the C-terminal deletion mutants and and deletion were in the 45–74 as as was also in the the presence of an NLS. This has three lysine preceded by proline residue These are of a basic NLS that was not found in the three other PADs the of the three lysine residues to the NLS we prepared a and has three alanine of the three lysine residues and with the These were to HeLa cells for of and proteins in cells were by using not of 3 the of these The was in the nucleus, whereas the was in the and the was found the These results suggested that the in this be for the nuclear import of PAD V. The results the of PAD V to in the nucleus. We the locations of PAD V in HL-60 and blood granulocytes by We an antibody specific for PAD V from PAD V with N-terminal was The antibody was first with human PADs I, II, III, and V by with PAD V, but not with amounts of PADs I, II, and This antibody was also on of HL-60 cells HL-60 granulocytes PAD V were prepared by culturing HL-60 cells with These cells distinct of PAD on a whereas HL-60 cells signal immunocytochemical of HL-60 cells were first in a and with a of and was used as a specific for a This was for of PAD V of HL-60 granulocytes, the distinct signals for PAD V were in the nucleus, but not in the In HL-60 cells signal was in the nucleus, but a signal was in the for were found in the of HL-60 cells and HL-60 granulocytes a and These results indicated that HL-60 PAD V is localized in the nucleus. We PAD V in peripheral blood granulocytes. a with on and signal increased with in the from 1 × to × The cellular of PAD V was to be × using PAD V as a Neutrophils were with a of and in signals of V were to of the and and This is more The cells were identified as neutrophils by nuclear and by the location of the signal and When was used as a signals were in the nucleus, whereas the was stained of the antibody with PAD V the signal in the nucleus, the signals of also showed the signals not These results indicated that PAD V is localized in the nucleus. eosinophil prepared in a Percoll density was in were by to and stained with a of and MBP, as a specific for a of 3 the The major signal of PAD was found with the stained with DAPI, whereas a signal was found in the with the signal When cells were stained with nuclear signal was but the signal was These signals be for by to the These results that eosinophil PAD is localized in the nucleus. the PAD V nuclear location we PAD V of HL-60 granulocytes by fractionation of into nuclear and of these indicated that PAD V was in the nuclear and was in the of a showed that histones were in the nuclear The of and histones in the nuclear and showed these The amounts of PAD and DNA were of the in the and and in the nuclear These results also indicated the nuclear localization of PAD V. the of PAD V from the nucleus, we the nuclear with of with PAD was and of PAD histones the protein deimination in blood granulocytes, we cells in medium and a with μm calcium ionophore These cells were stained for deiminated proteins with A of the of these The positive signals were to of the in neutrophils with the but not the The of positive cells to of the cells and cells by we identified deiminated proteins in cells by using The cells on 1 and whereas the stimulated cells three major distinct with and and other with and The signal of these increased with in the from to 1 and a not and a a with 1 These deiminated proteins were in the nuclear These and proteins were identified as histones H2A, and H3, by of nuclear proteins with and by for histones not as found in HL-60 granulocytes (23Hagiwara T. Nakashima K. Hirano H. Senshu T. Yamada M. Biochem. Biophys. Res. Commun. 2002; 290: 979-983Crossref PubMed Scopus (152) Google Scholar). These results indicated that protein deimination in the nucleus. This of for the nuclear localization of PAD V in HL-60 granulocytes and peripheral blood granulocytes. immunocytochemical of HL-60 granulocytes, neutrophils, and eosinophils using revealed distinct signals of PAD V in the nucleus, but signals in the cytoplasm. These nuclear signals in neutrophils were by of the antibody with an of PAD V. These results the nuclear localization of most of PAD V in these in of neutrophils and eosinophils using an V, we the positive signals in the but signals in the and these to the V in the (22Asaga H. Nakashima K. Senshu T. Ishigami A. Yamada M. J. Leukocyte Biol. 2001; 70: 46-51PubMed Google Scholar). of the in the and we found that in neutrophils the the nuclear signals were to an The of the of and the This to and in of nuclear In 3 of neutrophils the nuclear but of this the distinct positive nuclear not In of of of the the signals to the nuclear in the not This the signal of this the of the nuclear positive signal as in this of to the in eosinophils is also by S. P. B. R. J. Immunol. 1998; PubMed Scopus Google Scholar). in neutrophils and eosinophils and of in eosinophils to a in signals and in the of is that the nuclear PAD signal in neutrophils was to a region that is from a region by and a distribution of PAD V is with the of and with nuclear of HeLa cells PADs more revealed the nuclear location of PAD V the and We identified an NLS of PAD V as a acid This has an NLS that is to monopartite basic of and M. R. B. 2000; PubMed Scopus Google Scholar, J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. M. Biol. 1999; PubMed Google Scholar). This sequence feature is also found in mouse and PAD but not in human and rodent PADs I, II, and III The locations of these PAD IV in cells to be import of this of NLS is by an and a There are types of with E. M. L. G. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, The T. B. J. Biol. 2000; PubMed Scopus Google Scholar, D. Biol. 1999; PubMed Scopus Google Scholar). types of and the nuclear import of PAD V be immunocytochemical of deiminated proteins revealed protein deimination in the when blood granulocytes were stimulated with the calcium ionophore We identified these deiminated proteins as histones H3, H2A, and H4. The of protein deimination in the is also found in HL-60 granulocytes (23Hagiwara T. Nakashima K. Hirano H. Senshu T. Yamada M. Biochem. Biophys. Res. Commun. 2002; 290: 979-983Crossref PubMed Scopus (152) Google Scholar). 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T. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is that arginine deimination also by positive in the histone of the arginine deimination on histones is in The of of histones and other nuclear proteins in is known a role of the PAD in differentiation and Neutrophils are activated by of the cells with and and of the Google Scholar). neutrophils are cells a with of D. The and of Scholar, C. FEBS Lett. 2001; PubMed Scopus Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). studies on not PAD V is with a and and on of calcium is to nuclear PAD V are We of for a of anti-MC, and a of the for of human PAD III cDNA and and Takahashi for We also of this for of a