title: Extracellular citrullination inhibits the function of matrix associated TGF-β.
abstract: In inflammatory arthritis peptidyl arginine deiminase (PAD) enzymes can citrullinate arginine residues in extracellular matrix (ECM) proteins, such as collagens and fibronectin. This may lead to the generation of anti-citrullinated protein antibodies, important diagnostic markers in rheumatoid arthritis. In addition, the citrullination may directly affect protein function. Based on structural analysis, we found that most ECM-associated growth factors (GFs) have arginine residues in their receptor recognition sites. Thus, they are potential functional targets of extracellular citrullination. To examine this further, we focused on the citrullination of transforming growth factor-βs (TGF-β), well-known ECM-associated GFs. PAD-treatment of CHO-LTBP1 cell derived matrix, rich with TGF-β, decreased the level of TGF-β activity as detected by HaCaT and MLEC-PAI-1/Lu reporter cells. Additional experiments indicated that PAD-treatment inhibits the integrin-mediated TGF-β activation since PAD-treatment decreased the binding of integrin αVβ6 ectodomain as well as integrin-mediated spreading of MG-63 and HaCaT cells to β1-latency associated peptide (TGF-β1 LAP). The citrullination of the RGD site, an important integrin recognition motif, was confirmed by mass spectrometry. Furthermore, the citrullination of active TGF-β1 inhibited its binding to recombinant TGF-β receptor II, and prevented its ability to activate TGF-β signaling. Thus, extracellular PAD activity can affect the function of ECM-associated growth factors by different mechanisms. Importantly, the citrullination of both latent and active TGF-β has the potency to regulate the inflammatory process.
authors.affiliation: University of Turku, Department of Biochemistry, FI-20014 Turku, Finland.
authors.forename: Kalle H
authors.initials: KH
authors.lastname: Sipilä
authors.affiliation: Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, FI-20520 Turku, Finland.
authors.forename: Vipin
authors.initials: V
authors.lastname: Ranga
authors.affiliation: University of Turku, Department of Biochemistry, FI-20014 Turku, Finland.
authors.forename: Pekka
authors.initials: P
authors.lastname: Rappu
authors.affiliation: University of Turku, Department of Biochemistry, FI-20014 Turku, Finland.
authors.forename: Annamari
authors.initials: A
authors.lastname: Torittu
authors.affiliation: Turku University Hospital, Division of Medicine, Department of Rheumatology and University of Turku, FI-20014, Turku, Finland.
authors.forename: Laura
authors.initials: L
authors.lastname: Pirilä
authors.affiliation: University of Turku, Department of Biochemistry, FI-20014 Turku, Finland.
authors.forename: Jarmo
authors.initials: J
authors.lastname: Käpylä
authors.affiliation: Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, FI-20520 Turku, Finland.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Johnson
authors.affiliation: University of British Columbia, Department of Oral Biological and Medical Sciences, Vancouver, British Columbia, Canada.
authors.forename: Hannu
authors.initials: H
authors.lastname: Larjava
authors.affiliation: University of Turku, Department of Biochemistry, FI-20014 Turku, Finland. Electronic address: jyheino@utu.fi.
authors.forename: Jyrki
authors.initials: J
authors.lastname: Heino
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000378:metabolism; D016466:CHO Cells; D045744:Cell Line, Tumor; D000076302:Citrullination; D006224:Cricetinae; D003412:Cricetulus; D005109:Extracellular Matrix / Q000201:enzymology*; D006801:Humans; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D000076342:Protein-Arginine Deiminases / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000502:physiology; D015398:Signal Transduction; D053773:Transforming Growth Factor beta1 / Q000502:physiology*
chemical_list: D018125:Receptors, Transforming Growth Factor beta; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D000076342:Protein-Arginine Deiminases
keywords: Citrullination; Integrin; PAD; TGF-β