Natural nitration of CXCL12 reduces its signaling capacity and chemotactic activity <i>in vitro</i> and abrogates intra-articular lymphocyte recruitment <i>in vivo</i>

Abstract

// Rik Janssens 1, 2 , Anneleen Mortier 1 , Daiane Boff 1, 2 , Vincent Vanheule 1 , Mieke Gouwy 1 , Charlotte Franck 1, 3 , Olav Larsen 3 , Mette M. Rosenkilde 3 , Jo Van Damme 1 , Fl&aacute;vio A. Amaral 2 , Mauro M. Teixeira 2 , Sofie Struyf 1 , Paul Proost 1 1 Laboratory of Molecular Immunology, Department of Microbiology and Immunology, Rega Institute, KU Leuven, B-3000 Leuven, Belgium 2 Departamento de Bioqu&iacute;mica e Imunologia, Instituto de Ci&ecirc;ncias Biol&oacute;gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil 3 Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark Correspondence to: Paul Proost, email: paul.proost@rega.kuleuven.be Keywords: chemokine, posttranslational modification, lymphocyte migration, extravasation, chemotaxis Received: November 16, 2015&emsp;&emsp;&emsp;&emsp; Accepted: August 13, 2016&emsp;&emsp;&emsp;&emsp; Published: August 23, 2016 ABSTRACT The chemokine CXCL12/stromal cell-derived factor-1 is important for leukocyte migration to lymphoid organs and inflamed tissues and stimulates tumor development. In vitro, CXCL12 activity through CXCR4 is abolished by proteolytic processing. However, limited information is available on in vivo effects of posttranslationally modified CXCL12. Natural CXCL12 was purified from the coculture supernatant of stromal cells stimulated with leukocytes and inflammatory agents. In this conditioned medium, CXCL12 with a nitration on Tyr 7 , designated [3-NT 7 ]CXCL12, was discovered via Edman degradation. CXCL12 and [3-NT 7 ]CXCL12 were chemically synthesized to evaluate the biological effects of this modification. [3-NT 7 ]CXCL12 recruited &beta;-arrestin 2 and phosphorylated the Akt kinase similar to CXCL12 in receptor-transfected cells. Also the affinity of CXCL12 and [3-NT 7 ]CXCL12 for glycosaminoglycans, the G protein-coupled chemokine receptor CXCR4 and the atypical chemokine receptor ACKR3 were comparable. However, [3-NT 7 ]CXCL12 showed a reduced ability to enhance intracellular calcium concentrations, to generate inositol triphosphate, to phosphorylate ERK1/2 and to induce monocyte and lymphocyte chemotaxis in vitro . Moreover, nitrated CXCL12 failed to induce in vivo extravasation of lymphocytes to the joint. In summary, nitration on Tyr 7 under inflammatory conditions is a novel natural posttranslational regulatory mechanism of CXCL12 which may downregulate the CXCR4-mediated inflammatory and tumor-promoting activities of CXCL12.