title: Methylation of arginine residues interferes with citrullination by peptidylarginine deiminases in vitro.
abstract: Peptidylarginine deiminase (PAD) enzymes catalyze the conversion of arginine residues in proteins to citrulline residues. Citrulline is a non-standard amino acid that is not incorporated in proteins during translation, but can be generated post-translationally by the PAD enzymes. Although the existence of citrulline residues in proteins has been known for a long time, only a few proteins have been reported to contain this amino acid under normal conditions. These include the nuclear histones, which also contain a wide variety of other post-translational modifications, as for instance methylation of arginine residues. It has been suggested that citrullination and methylation of arginine residues are competing processes and that PAD enzymes might "reverse" the methylation of arginine residues by converting monomethylated arginine into citrulline. However, conflicting data have been reported on the capacity of PADs to citrullinate monomethylated peptidylarginine. Using synthetic peptides that contain either arginine or methylated arginine residues, we show that the human PAD2, PAD3 and PAD4 enzymes and PAD enzyme present in several mouse tissues in vitro can only convert non-methylated peptidylarginine into peptidylcitrulline and that hPAD6 does not show any deiminating activity at all. A comparison of bovine histones either treated or untreated with PAD by amino acid analysis also supported the interference of deimination by arginine methylation. Taken together, these data indicate that it is unlikely that methyl groups at the guanidino position of peptidylarginine can be removed by peptidylarginine deiminases, which has important implications for the recently reported role of these enzymes in gene regulation.
authors.affiliation: Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands. R.Raijmakers@ncmls.ru.nl
authors.forename: Reinout
authors.initials: R
authors.lastname: Raijmakers
authors.forename: Albert J W
authors.initials: AJ
authors.lastname: Zendman
authors.forename: Wilma Vree
authors.initials: WV
authors.lastname: Egberts
authors.forename: Erik R
authors.initials: ER
authors.lastname: Vossenaar
authors.forename: Jos
authors.initials: J
authors.lastname: Raats
authors.forename: Claudia
authors.initials: C
authors.lastname: Soede-Huijbregts
authors.forename: Floris P J T
authors.initials: FP
authors.lastname: Rutjes
authors.forename: Peter A
authors.initials: PA
authors.lastname: van Veelen
authors.forename: Jan W
authors.initials: JW
authors.lastname: Drijfhout
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Pruijn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002478:Cells, Cultured; D002956:Citrulline / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D008745:Methylation; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D006657:Histones; D002956:Citrulline; D001120:Arginine; D011484:Protein-Arginine N-Methyltransferases; D006867:Hydrolases; C000620352:PADI2 protein, human; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases