title: Primary sequence, together with other factors, influence peptide deimination by peptidylarginine deiminase-4.
abstract: Enzymes of the peptidylarginine deiminase (PAD) family catalyze the posttranslational deimination of polypeptide-bound arginine residues. Here, we report the selection of peptide substrates by PAD-4, an isoform thought to be involved in the pathogenesis of rheumatoid arthritis. First, we investigated whether PAD-4-mediated deimination is influenced by the nature of amino acid residues flanking the targeted arginine. Using two peptide substrates, residues in positions -2, -1, +1, and +2 relative to the central arginine targeted by PAD-4 were systematically replaced by all natural L-amino acids except cysteine. Each peptide was treated with recombinant human PAD-4 and deimination was analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In all four flanking positions, amino acids which positively or negatively influenced deimination were identified. We next designed peptides with expected high or low deimination rates and determined their Km and kcat values. These peptides showed PAD-4 substrate behavior as predicted, demonstrating that residues flanking the targeted arginine are important for deimination. Further truncation of peptide substrates suggested additional effects on deimination by residues outside the -2 to +2 region. Finally, we observed that a methylated lysine residue flanking the targeted arginine influences PAD-4-mediated deimination, also suggesting that posttranslational modifications can affect substrate efficiency.
authors.affiliation: Centre for Immune Regulation, Institute of Immunology, University of Oslo, Department of Rheumatology, Rikshospitalet University Hospital, N-0027 Oslo, Norway. maria.stensland@rr-research.no
authors.forename: Maria E
authors.initials: ME
authors.lastname: Stensland
authors.forename: Sylvie
authors.initials: S
authors.lastname: Pollmann
authors.forename: Øyvind
authors.initials: Ø
authors.lastname: Molberg
authors.forename: Ludvig M
authors.initials: LM
authors.lastname: Sollid
authors.forename: Burkhard
authors.initials: B
authors.lastname: Fleckenstein
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D002956:Citrulline / Q000737:chemistry; D003500:Cyclization; D006801:Humans; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010455:Peptides; D002956:Citrulline; D001120:Arginine; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases