Ruytinx Pieter, Janssens Rik, Berghmans Nele, Gouwy Mieke, Ronsse Isabelle, Liekens Sandra, Proost Paul, Van Damme Jo, Struyf Sofie
KU Leuven, University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Molecular Immunology, B-3000 Leuven, Belgium.
KU Leuven, University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium.
Biochem Pharmacol. 2017 Dec 1;145:123-131. doi: 10.1016/j.bcp.2017.08.020. Epub 2017 Aug 30.
CXCL4L1, a platelet-derived ELR-negative CXC chemokine, is a powerful angiostatic and anti-tumoral chemokine. We developed a mass spectrometric assay for the detection of different natural CXCL4L1 isoforms. Using this assay, we identified 4 different CXCL4L1 isoforms in the supernatant of thrombin-stimulated platelets from healthy volunteers: the classical isoform CXCL4L1(1-70), CXCL4L1(-4-70), which probably arises through alternative signal peptide removal and two COOH-terminally truncated isoforms CXCL4L1(1-69) and CXCL4L1(-4-69). CXCL4L1(1-70) was the most abundant isoform, whereas CXCL4L1(-4-70) was detected in 50% of the platelet preparations. Since alterations to the NH-terminus of chemokines can have severe biological consequences, we investigated the impact of the extension with 4 NH-terminal amino acids on the biological activity of CXCL4L1. In vitro, CXCL4L1(-4-70) was as potent as CXCL4L1(1-70) in inhibiting signal transduction and migration of human microvascular endothelial cells towards vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). In a FITC-conjugated dextran cell permeability assay, both splice variants showed a strong but comparable anti-permeable effect upon VEGF stimulation of the endothelial cell monolayer. In vivo angiogenesis induced by FGF-2 was equally reduced by CXCL4L1(1-70) and CXCL4L1(-4-70). In chemotaxis assays with CXCR3A-transfected cells the CXCL4L1 isoforms both induced migration from 125ng/ml onward. Finally, CXCL4L1(1-70) and CXCL4L1(-4-70) showed the same affinity for heparin. In conclusion, the investigated biological activities of CXCL4L1 are not influenced by the four extra NH-terminal residues present in the alternatively spliced isoform CXCL4L1(-4-70). Therefore, our results suggest that both isoforms equally interact with the CXCR3A and CXCR3B receptor.
CXCL4L1是一种血小板衍生的ELR阴性CXC趋化因子，是一种强大的血管生成抑制和抗肿瘤趋化因子。我们开发了一种质谱分析法来检测不同的天然CXCL4L1异构体。使用该分析法，我们在健康志愿者凝血酶刺激血小板的上清液中鉴定出4种不同的CXCL4L1异构体：经典异构体CXCL4L1(1-70)、CXCL4L1(-4-70)（可能通过选择性信号肽去除产生）以及两种COOH末端截短的异构体CXCL4L1(1-69)和CXCL4L1(-4-69)。CXCL4L1(1-70)是最丰富的异构体，而CXCL4L1(-4-70)在50%的血小板制剂中被检测到。由于趋化因子NH末端的改变可能会产生严重的生物学后果，我们研究了在NH末端添加4个氨基酸对CXCL4L1生物学活性的影响。在体外，CXCL4L1(-4-70)在抑制人微血管内皮细胞向血管内皮生长因子（VEGF）和成纤维细胞生长因子-2（FGF-2）的信号转导和迁移方面与CXCL4L1(1-70)一样有效。在FITC偶联葡聚糖细胞通透性试验中，两种剪接变体在VEGF刺激内皮细胞单层时均显示出强烈但相当的抗通透作用。FGF-2诱导的体内血管生成同样被CXCL4L1(1-70)和CXCL4L1(-4-70)降低。在用CXCR3A转染细胞进行的趋化试验中，两种CXCL4L1异构体从125ng/ml起均诱导迁移。最后，CXCL4L1(1-70)和CXCL4L1(-4-70)对肝素显示出相同的亲和力。总之，CXCL4L1的所研究生物学活性不受选择性剪接异构体CXCL4L1(-4-70)中存在的4个额外NH末端残基的影响。因此，我们的结果表明这两种异构体与CXCR3A和CXCR3B受体的相互作用相同。
