Structural and functional characterization of the interactions of platelet derived chemokines CCL5, CXCL4 and CXCL4L1
Year: 2011
Type: dissertation
Abstract: Chemokines play an important role in the development of inflammation and in the recruitment of leukocytes from the vessel into the inflamed tissue. Activated platelets serve as a rich source of chemokines, e.g. CCL5, CXCL4 and its closely related variant CXCL4L1. The pro-inflammatory CCL5 is a known potent chemoattractant for monocytes and also responsible for its arrest on inflamed endothelium. Since this arrest is enhanced by the interaction of CCL5 with CXCL4, we aimed to determine the structural interaction of this CCL5-CXCL4 heterodimer in the first part of this thesis. For this purpose, CCL5, CXCL4 and CXCL4L1 were expressed recombinantly in E. coli by different techniques, CCL5 as inclusion bodies in the cytoplasm, CXCL4 and CXCL4L1 as soluble proteins in the periplasmic space. For structural analysis of the heterodimerization of the CCL5-CXCL4 complex, 15N-enriched CCL5 was expressed. Additionally, CXCL4L1 was chemically synthesized by the solid-phase peptide synthesis method. All generated proteins were purified by sequential steps with chromatographic methods using FPLC and HPLC. Chemical shift mapping of the 15N-CCL5 by the 15N-1H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) and 3D modeling revealed that the N-termini were responsible for the formation of the CCL5-CXCL4 heterodimer. In the second part of this dissertation, a functional comparison between CXCL4 and CXCL4L1 was carried out to evaluate the differences of these closely related chemokines. This comparison revealed that the angiogenesis, as assessed by endothelial tube formation, was inhibited by CXCL4 as well as CXCL4L1, but only CXCL4L1 triggered undirected locomotion of endothelial cells (chemokinesis). The chemotactic response towards the growth factors VEGF and bFGF was attenuated by both variants but only CXCL4L1-induced chemokinesis was blocked by bFGF or VEGF. Endothelial cell proliferation was inhibited by CXCL4 but not by CXCL4L1, while both chemokines bound directly to VEGF and bFGF. Moreover, CXCL4 enhanced CCL5-induced monocyte arrest in vitro and as well in vivo, whereas CXCL4L1 had no effect. CXCL4L1 revealed lower affinity for CCL5 than CXCL4 as quantified by isothermal fluorescence titration. As evidenced by the reduction of the activated partial thromboplastin time, CXCL4L1 exhibited less heparin-neutralizing activity than CXCL4. In addition, activation of protein C by thrombin/thrombomodulin was less effectively accelerated by CXCL4L1 compared with CXCL4. It was also discovered that the C-terminal natural changes in CXCL4L1 compared to CXCL4 determine a different quaternary structure since CXCL4 predominantly exists as tetramer and CXCL4L1 as dimer under physiological conditions despite a comparable general positive charge. These structural differences are closely related to the different biological effects most prominently exemplified by the distinct effects in monocyte recruitment and angiogenesis. Summing up, CXCL4L1 may act angiostatically by causing random endothelial cell locomotion, disturbing directed migration towards angiogenic chemokines, serving as a homeostatic chemokine with a moderate structural distinction yet different functional profile from CXCL4.
Source: RWTH Publications (RWTH Aachen)
Authors: Alisina Sarabi, Christian Weber
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W628597830