Deimination of histone H2A and H4 at arginine 3 in HL-60 granulocytes.
Interplay of various covalent modifications of histone tails has an essential role in regulation of chromatin function. Peptidylarginine deiminase (PADI) 4 deiminates protein arginine to citrulline in a Ca(2+)-dependent manner and is present in the nucleus of granulocyte-differentiated HL-60 cells. When these cells are treated with the calcium ionophore A23187, core histone deimination occurs. To determine the deimination sites of histones, histone species were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) from the cells. Immunoblotting using antimodified citrulline antibody indicated that histones H2A, H3, and H4 but not H2B were deiminated. H2A and H4 were digested with Staphylococcus aureus V8 protease, and the digests were separated by RP-HPLC. Immuno dot-blotting and mass spectrometry showed that the deiminated residues were present in H2A (1-56) and H4 (1-52) regions but not in other regions. The H2A peptide (1-56) was digested with alpha-chymotrypsin, and the deiminated peptide was separated from the corresponding nondeiminated peptide by RP-HPLC. The deiminated residue was found to be limited to residues 1-23. Similarly, digestion of the H4 peptide (1-52) with endoproteinase Asp-N and separation of the deiminated peptide from the nondeiminated peptide indicated that the deiminated residue was limited to residues 1-23. Mass spectrometry of lysylendopeptidase digests of the H2A (1-23) and H4 (1-23) peptides showed that deimination occurred at arginine 3 of the N-terminal sequence Ac-SGRGK common to H2A and H4. These results suggest that PADI4 deiminates only a restricted site of target proteins in cells. Deimination of histones is discussed in relation to chromatin structure and function.
Hagiwara
Teruki
T
Graduate School of Integrated Science, Yokohama City University, 22-2, Seto, Kanazawa-ku, Yokohama 236-0027, Japan.
Hidaka
Yuji
Y
Yamada
Michiyuki
M
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D000001:Calcimycin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D018922:HL-60 Cells; D006657:Histones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D007097:Imines / Q000737:chemistry; D007476:Ionophores / Q000494:pharmacology; D008969:Molecular Sequence Data; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006657:Histones; D007097:Imines; D007476:Ionophores; D000001:Calcimycin; D001120:Arginine; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases