Brown Aaron J, Sepuru Krishna Mohan, Sawant Kirti V, Rajarathnam Krishna
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, United States.
Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, United States.
Front Immunol. 2017 Oct 2;8:1248. doi: 10.3389/fimmu.2017.01248. eCollection 2017.
Platelet-derived chemokine CXCL7 (also known as NAP-2) plays a crucial role in orchestrating neutrophil recruitment in response to vascular injury. CXCL7 exerts its function by activating the CXC chemokine receptor 2 (CXCR2) receptor and binding sulfated glycosaminoglycans (GAGs) that regulate receptor activity. CXCL7 exists as monomers, dimers, and tetramers, and previous studies have shown that the monomer dominates at lower and the tetramer at higher concentrations. These observations then raise the question: what, if any, is the role of the dimer? In this study, we make a compelling observation that the dimer is actually the favored form in the GAG-bound state. Further, we successfully characterized the structural basis of dimer binding to GAG heparin using solution nuclear magnetic resonance (NMR) spectroscopy. The chemical shift assignments were obtained by exploiting heparin binding-induced NMR spectral changes in the WT monomer and dimer and also using a disulfide-linked obligate dimer. We observe that the receptor interactions of the dimer are similar to the monomer and that heparin-bound dimer is occluded from receptor interactions. Cellular assays also show that the heparin-bound CXCL7 is impaired for CXCR2 activity. We conclude that the dimer-GAG interactions play an important role in neutrophil-platelet crosstalk, and that these interactions regulate gradient formation and the availability of the free monomer for CXCR2 activation and intrathrombus neutrophil migration to the injury site.
血小板衍生的趋化因子CXCL7（也称为NAP-2）在协调中性粒细胞对血管损伤的募集反应中起关键作用。CXCL7通过激活CXC趋化因子受体2（CXCR2）并结合调节受体活性的硫酸化糖胺聚糖（GAG）发挥其功能。CXCL7以单体、二聚体和四聚体形式存在，先前的研究表明，单体在较低浓度下占主导，而四聚体在较高浓度下占主导。这些观察结果进而引发了一个问题：二聚体的作用是什么（如果有作用的话）？在本研究中，我们有一个引人注目的发现，即二聚体实际上是GAG结合状态下的优势形式。此外，我们利用溶液核磁共振（NMR）光谱成功表征了二聚体与GAG肝素结合的结构基础。通过利用野生型单体和二聚体中肝素结合诱导的NMR光谱变化以及使用二硫键连接的专一性二聚体，获得了化学位移归属。我们观察到二聚体与受体的相互作用与单体相似，并且肝素结合的二聚体被阻断与受体相互作用。细胞实验还表明，肝素结合的CXCL7的CXCR2活性受损。我们得出结论，二聚体与GAG的相互作用在中性粒细胞与血小板的串扰中起重要作用，并且这些相互作用调节梯度形成以及游离单体用于CXCR2激活和血栓内中性粒细胞向损伤部位迁移的可用性。
