Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, Karl-Franzens-University Graz, Schubertstrasse 1, 8010 Graz, Austria.
Antagonis Biotherapeutics GmbH, Strasserhofweg 77a, 8045 Graz, Austria.
Int J Mol Sci. 2022 Jun 10;23(12):6519. doi: 10.3390/ijms23126519.
Proinflammatory chemokine ligand 26 (CCL26, eotaxin-3) mediates transendothelial cell migration of eosinophils by binding and activating the G-protein-coupled (GPC) chemokine receptor 3 on the surface of eosinophilic cells. Here we have investigated the role of glycosaminoglycans (GAGs) as potential co-receptors in the process of CCL26-induced eosinophil chemotaxis. For this purpose, we have first identified the GAG-binding site of CCL26 by a site-directed mutagenesis approach in the form of an alanine screening. A panel of GAG-binding-deficient mutants has been designed, generated, and analyzed with respect to their binding affinities to heparan sulphate (HS) by isothermal fluorescence titration studies. This showed that basic amino acids in the α-helical part of CCL26 are strongly involved in GAG-binding. In chemotaxis experiments, we found that decreased GAG-binding affinity correlated with decreased chemotactic activity, which indicates an involvement of GAGs in eosinophil migration. This was further proven by the negative impact of heparinase III treatment and, independently, by the incubation of eosinophils with an anti heparan sulfate antibody. We finally investigated eosinophils' proteoglycan (PG) expression patterns by real-time PCR, which revealed the highest expression level for serglycin. Including an anti-serglycin antibody in CCL26-induced eosinophil migration experiments reduced the chemotaxis of these immune cells, thereby proving the dependence of eosinophil mobilization on the proteoglycan serglycin.
趋化因子配体 26（CCL26，嗜酸性粒细胞趋化因子-3）通过结合并激活嗜酸性粒细胞表面的 G 蛋白偶联（GPC）趋化因子受体 3，介导嗜酸性粒细胞穿过内皮细胞的迁移。在这里，我们研究了糖胺聚糖（GAG）作为 CCL26 诱导嗜酸性粒细胞趋化过程中潜在共受体的作用。为此，我们首先通过定点诱变方法以丙氨酸筛选的形式确定了 CCL26 的 GAG 结合位点。设计、生成了一组 GAG 结合缺陷突变体，并通过等温荧光滴定研究分析了它们与硫酸乙酰肝素（HS）的结合亲和力。这表明 CCL26 的α螺旋部分中的碱性氨基酸强烈参与 GAG 结合。在趋化实验中，我们发现 GAG 结合亲和力降低与趋化活性降低相关，这表明 GAGs 参与了嗜酸性粒细胞的迁移。肝素酶 III 处理的负面影响以及嗜酸性粒细胞与抗硫酸乙酰肝素抗体孵育进一步证明了这一点。最后，我们通过实时 PCR 研究了嗜酸性粒细胞的蛋白聚糖（PG）表达模式，发现硫酸乙酰肝素蛋白聚糖的表达水平最高。在 CCL26 诱导的嗜酸性粒细胞迁移实验中加入抗硫酸乙酰肝素蛋白聚糖抗体，减少了这些免疫细胞的趋化性，从而证明了嗜酸性粒细胞动员依赖于蛋白聚糖硫酸乙酰肝素蛋白聚糖。
