C-terminal truncation of CXCL10 attenuates inflammatory activity but retains angiostatic properties

Abstract

Interferon-{gamma}-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. The truncated CXCL10 proteoform CXCL10(1-73), lacking the four endmost C-terminal amino acids, was previously identified in human cell culture supernatant. To further explore the functioning of CXCL10(1-73), we optimized its production in this study through Fmoc-based solid phase peptide synthesis (SPPS) and propose an SPPS strategy to efficiently generate human CXCL10 proteoforms. Compared to intact CXCL10(1-77), CXCL10(1-73) had diminished affinity for glycosaminoglycans 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 reduced primary human T lymphocyte chemotaxis in vitro and evoked less peritoneal ingress of CXCR3+ T lymphocytes in mice receiving intraperitoneal chemokine injections. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on spontaneous and/or FGF-2-induced migration, proliferation, wound healing, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. Thus, C-terminally truncated CXCL10 has attenuated inflammatory properties, but preserved anti-angiogenic capacity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/548382v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@10c1207org.highwire.dtl.DTLVardef@173bc5eorg.highwire.dtl.DTLVardef@153d6a2org.highwire.dtl.DTLVardef@1302a76_HPS_FORMAT_FIGEXP M_FIG C_FIG