Author:
Dillemans Luna,Yu Karen,De Zutter Alexandra,Noppen Sam,Gouwy Mieke,Berghmans Nele,Verhallen Lisa,De Bondt Mirre,Vanbrabant Lotte,Brusselmans Stef,Martens Erik,Schols Dominique,Verschueren Patrick,Rosenkilde Mette M.,Marques Pedro Elias,Struyf Sofie,Proost Paul
Abstract
Abstract
Background
Interferon-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. We studied CXCL10(1–73), lacking the four endmost C-terminal amino acids, which was previously identified in supernatant of cultured human fibroblasts and keratinocytes.
Methods
Relative levels of CXCL10(1–73) and intact CXCL10(1–77) were determined in synovial fluids of patients with rheumatoid arthritis (RA) through tandem mass spectrometry. The production of CXCL10(1–73) was optimized through Fmoc-based solid phase peptide synthesis (SPPS) and a strategy to efficiently generate human CXCL10 proteoforms was introduced. CXCL10(1–73) was compared to intact CXCL10(1–77) using surface plasmon resonance for glycosaminoglycan (GAG) binding affinity, assays for cell migration, second messenger signaling downstream of CXCR3, and flow cytometry of CHO cells and primary human T lymphocytes and endothelial cells. Leukocyte recruitment in vivo upon intraperitoneal injection of CXCL10(1–73) was also evaluated.
Results
Natural CXCL10(1–73) was more abundantly present compared to intact CXCL10(1–77) in synovial fluids of patients with RA. CXCL10(1–73) had diminished affinity for GAG including heparin, heparan sulfate and chondroitin sulfate A. Moreover, CXCL10(1–73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1–73) incited significantly less primary human T lymphocyte chemotaxis in vitro and peritoneal ingress of CXCR3+ T lymphocytes in mice. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on migration, proliferation, wound closure, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells.
Conclusion
Our study shows that the C-terminal residues Lys74-Pro77 of CXCL10 are important for GAG binding, signaling through CXCR3A, T lymphocyte chemotaxis, but dispensable for angiostasis.
Graphical Abstract
Funder
Fonds Wetenschappelijk Onderzoek
Onderzoeksraad, KU Leuven
Publisher
Springer Science and Business Media LLC
Subject
Cell Biology,Molecular Biology,Biochemistry