Natural carboxyterminal truncation of human CXCL10 attenuates glycosaminoglycan binding, CXCR3A signaling and lymphocyte chemotaxis, while retaining angiostatic activity

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: (Figure presented.).