Martin L, Blanpain C, Garnier P, Wittamer V, Parmentier M, Vita C
CEA (Commissariat à l'Energie Atomique) Saclay, Département d'Ingénierie et d'Etudes des Protéines, F-91191 Gif-sur-Yvette, France.
Biochemistry. 2001 May 29;40(21):6303-18. doi: 10.1021/bi002670n.
Chemokines mediate their biological activity through activation of G protein coupled receptors, but most chemokines, including RANTES, are also able to bind glycosaminoglycans (GAGs). Here, we have investigated, by site-directed mutagenesis and chemical acetylation, the role of RANTES basic residues in the interaction with GAGs using surface plasmon resonance kinetic analysis. Our results indicate that (i) RANTES exhibited selectivity in GAGs binding with highest affinity (K(d) = 32.1 nM) for heparin, (ii) RANTES uses the side chains of residues R44, K45, and R47 for heparin binding, and blocking these residues in combination abolished heparin binding. The biological relevance of RANTES-GAGs interaction was investigated in CHO-K1 cells expressing CCR5, CCR1, or CCR3 and the various GAGs that bind RANTES. Our results indicate that the heparin binding site, defined as the 40s loop, is only marginally involved in CCR5 binding and activation, but largely overlaps the CCR1 and CCR3 binding and activation domain in RANTES. In addition, enzymatic removal of cell surface GAGs by glycosidases did not affect CCR5 binding and Ca(2+) response. Furthermore, addition of soluble GAGs inhibited both CCR5 binding and functional response, with a rank of potency similar to that found in surface plasmon resonance experiments. Thus, cell surface GAGs is not a prerequisite for receptor binding or signaling, but soluble GAGs can inhibit the binding and the functional response of RANTES to CCR5 expressing cells. However, the marked selectivity of RANTES for different GAGs may serve, in vivo, to control the concentration of specific chemokines in inflammatory situations and locations.
趋化因子通过激活G蛋白偶联受体来介导其生物学活性，但大多数趋化因子，包括RANTES，也能够结合糖胺聚糖（GAGs）。在此，我们通过定点诱变和化学乙酰化，利用表面等离子体共振动力学分析研究了RANTES碱性残基在与GAGs相互作用中的作用。我们的结果表明：（i）RANTES在与GAGs结合时表现出选择性，对肝素具有最高亲和力（K(d)=32.1 nM）；（ii）RANTES利用残基R44、K45和R47的侧链与肝素结合，同时阻断这些残基会消除肝素结合。在表达CCR5、CCR1或CCR3以及各种结合RANTES的GAGs的CHO-K1细胞中研究了RANTES-GAGs相互作用的生物学相关性。我们的结果表明，被定义为40s环的肝素结合位点仅在一定程度上参与CCR5的结合和激活，但在很大程度上与RANTES中的CCR1和CCR3结合及激活结构域重叠。此外，糖苷酶对细胞表面GAGs的酶促去除并不影响CCR5的结合和Ca(2+)反应。此外，添加可溶性GAGs会抑制CCR5的结合和功能反应，其效力排序与表面等离子体共振实验中发现的相似。因此，细胞表面GAGs不是受体结合或信号传导的先决条件，但可溶性GAGs可以抑制RANTES与表达CCR5的细胞的结合和功能反应。然而，RANTES对不同GAGs的显著选择性可能在体内用于控制炎症情况下和炎症部位特定趋化因子的浓度。
