title: The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities.
abstract: Human peptidylarginine deiminases (hPADs) have been implicated in several diseases, particularly in rheumatoid arthritis. Since hPAD2 and hPAD4 are the isotypes expressed in the inflamed joints of RA patients and protein citrullination by PADs has been proposed to play a pathophysiological role, they represent unique therapeutic targets. To facilitate the development of substrate-based PAD inhibitors the substrate specificity of hPAD2 and hPAD4 was determined. Recombinant hPADs were expressed in bacteria or mammalian cell lines and allowed to citrullinate proteins in cell lysates, as well as a series of synthetic peptides. The citrullinated residues in proteins and the efficiency of peptide citrullination were determined by mass spectrometry. In total 320 hPAD2 and 178 hPAD4 citrullination sites were characterized. Amino acid residues most commonly found in citrullination sites for both isotypes are Gly at +1 and Tyr at +3 relative to the target arginine. For hPAD4 several additional amino acids were observed to be preferred at various positions from -4 to +4. The substrate motifs determined by amino acid substitution analysis partially confirmed these preferences, although peptide context dependent differences were also observed. Taken together, our data show that the enzyme specificity for cellular substrates and synthetic peptides differs for hPAD2 and hPAD4. hPAD4 shows more restrictive substrate specificity compared to hPAD2. Consensus sequences, which can be used as the basis for the development of PAD inhibitors, were derived for the citrullination sites of both hPAD2 and hPAD4.
authors.affiliation: Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands. Electronic address: constance.assohou@gmail.com.
authors.forename: Constance
authors.initials: C
authors.lastname: Assohou-Luty
authors.affiliation: Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands. Electronic address: R.Raijmakers@uu.nl.
authors.forename: Reinout
authors.initials: R
authors.lastname: Raijmakers
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands. Electronic address: W.E.Benckhuijsen@lumc.nl.
authors.forename: Willemien E
authors.initials: WE
authors.lastname: Benckhuijsen
authors.affiliation: Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands. Electronic address: J.Vogelzangs@ncmls.ru.nl.
authors.forename: Judith
authors.initials: J
authors.lastname: Stammen-Vogelzangs
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands. Electronic address: A.H.de_Ru@lumc.nl.
authors.forename: Arnoud
authors.initials: A
authors.lastname: de Ru
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands. Electronic address: P.A.van_Veelen@lumc.nl.
authors.forename: Peter A
authors.initials: PA
authors.lastname: van Veelen
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands. Electronic address: C.L.M.C.Franken@lumc.nl.
authors.forename: Kees L M C
authors.initials: KL
authors.lastname: Franken
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands. Electronic address: j.w.drijfhout@lumc.nl.
authors.forename: Jan W
authors.initials: JW
authors.lastname: Drijfhout
authors.affiliation: Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands. Electronic address: G.Pruijn@ncmls.ru.nl.
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Pruijn
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D002956:Citrulline / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D002956:Citrulline; D014443:Tyrosine; D001120:Arginine; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D005998:Glycine
keywords: Citrullination; PAD; Peptidylarginine deiminase; Rheumatoid arthritis; Substrate specificity