Glycosaminoglycans mediate cell surface oligomerization of chemokines
Glycosaminoglycans mediate cell surface oligomerization of chemokines
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DOI：
10.1021/bi971125s
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发表时间：
1997-11-04
期刊：
影响因子：
2.9
通讯作者：
Wells, TNC
中科院分区：
文献类型：
--
作者：
Hoogewerf, AJ;Kuschert, GSV;Wells, TNC
趋化因子是8 - 10 kDa的蛋白质，参与白细胞运输和激活的调控。在自由溶液中，在生理浓度下趋化因子是单体，尽管在较高浓度下许多会形成多聚体。细胞表面的硫酸乙酰肝素可能会隔离趋化因子，提高它们的局部浓度并促进它们与白细胞上表达的受体结合。在使用固定化肝素的竞争性结合实验中，随着纳摩尔范围内未标记趋化因子浓度的增加，观察到结合的放射性标记趋化因子增加了2 - 3倍。需要0.25到50 μM之间的未标记趋化因子浓度来竞争结合的放射性。对于N - 甲基 - L25 IL - 8（一种不能二聚化的IL - 8变体），没有观察到这种双相竞争曲线。此外，从凝胶过滤柱洗脱的趋化因子和肝素复合物的表观分子量为33 - 60 kDa，这表明趋化因子发生了多聚化。从使用人内皮细胞的研究中可以看出这种多聚化过程的生理相关性。由于竞争实验显示出在固定化肝素研究中看到的双相曲线和微摩尔IC50值，并且未检测到已知趋化因子受体的mRNA，因此推断IL - 8、RANTES和MCP - 1的内皮细胞结合位点是糖胺聚糖。此外，用糖胺聚糖酶消化内皮细胞单层使趋化因子结合减少多达80%。糖胺聚糖可作为携带趋化因子受体细胞的配体结合亲和力的调节剂。从表达趋化因子受体CXCR1、CCR1或CCR2的CHO细胞中去除糖胺聚糖导致RANTES、MCP - 1、IL - 8和MIP - 1α的结合减少40 - 70%。我们的数据表明，细胞表面糖胺聚糖诱导趋化因子聚合，提高它们的局部浓度，从而增强它们在局部微环境中对高亲和力受体的作用。
Chemokines are 8-10 kDa proteins involved in the control of leukocyte trafficking and activation. In free solution, chemokines are monomers at physiologic concentrations, although many multimerize at higher concentrations. Cell surface heparan sulfate may sequester chemokines, increasing their local concentrations and facilitating their binding to receptors expressed on leukocytes. In competitive binding assays using immobilized heparin, a 2-3-fold increase in the bound radiolabeled chemokine was seen with increasing concentrations of unlabeled chemokine in the nanomolar range. Unlabeled chemokine concentrations between 0.25 and 50 mu M were needed to compete the bound radioactivity. This biphasic competition curve was not seen for N-methyl-L25 IL-8, a variant of IL-8 which is unable to dimerize. In addition, complexes of chemokine and heparin eluted from gel filtration columns with apparent molecular masses of 33-60 kDa, suggesting that chemokine multimerization had occurred. The physiological relevance of this multimerization process was seen from studies using human endothelial cells. The endothelial cell binding sites for IL-8, RANTES, and MCP-1 were deduced to be glycosaminoglycans since competition assays showed the biphasic curves and micromolar IC50 values seen in studies with immobilized heparin, and mRNA for known chemokine receptors was not detected. Furthermore, digestion of endothelial cell monolayers with glycosaminidases decreased chemokine binding by up to 80%. Glycosaminoglycans can act as modulators of the ligand binding affinity of chemokine receptor-bearing cells. Removal of glycosaminoglycans from CHO cells expressing chemokine receptors CXCR1, CCR1, or CCR2 resulted in 40-70% decreases in the binding of RANTES, MCP-1, IL-8, and MIP-1 alpha. Our data show that cell surface glycosaminoglycans induce polymerization of chemokines, increasing their local concentration and therefore enhancing their effects on high-affinity receptors within the local microenvironment.