Glycosylation of erythropoietin affects receptor binding kinetics: Role of electrostatic interactions
Glycosylation of erythropoietin affects receptor binding kinetics: Role of electrostatic interactions
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DOI：
10.1021/bi0265022
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发表时间：
2002-12-10
期刊：
影响因子：
2.9
通讯作者：
Beals, JM
中科院分区：
文献类型：
--
作者：
Darling, RJ;Kuchibhotla, U;Beals, JM
促红细胞生成素（EPO）是一种由肾脏产生的细胞因子，其功能是刺激骨髓中的红细胞生成。先前，已显示EPO对其受体EPOR的亲和力与EPO碳水化合物的唾液酸化成反比。为了更好地理解EPO调节其受体亲和力的性质，使用表面等离子体共振分析了各种糖型。所使用的系统已经被很好地表征，并且是基于使用在蛋白A表面上捕获的EPOR-Fc嵌合体的先前报道。使用含有不同水平唾液酸化的EPO的三种变体，我们确定唾液酸使缔合速率常数（k（on））降低约3倍。此外，糖基化EPO的k（on）比非糖基化EPO慢20倍，表明核心碳水化合物也对k（on）产生负面影响。通过测量不同NaCl浓度下的结合动力学来研究静电力对EPO结合的影响。增加NaCl浓度导致较慢的k（on），而对k（off）几乎没有影响，这表明长程静电相互作用在确定EPO和EPOR之间的缔合速率中是主要重要的。此外，糖基化含量（即，非糖基化vs糖基化，唾液酸化vs去唾液酸化）影响了k（on）对[NaCl]的总体敏感性，表明唾液酸和聚糖本身各自影响这些静电力的总体效应。
Erythropoietin (EPO) is a cytokine produced by the kidney whose function is to stimulate red blood cell production in the bone marrow. Previously, it was shown that the affinity of EPO for its receptor, EPOR, is inversely related to the sialylation of EPO carbohydrate. To better understand the properties of EPO that modulate its receptor affinity, various glycoforms were analyzed using surface plasmon resonance. The system used has been well characterized and is based on previous reports employing an EPOR-Fc chimera captured on a Protein A surface. Using three variants of EPO containing different levels of sialylation, we determined that sialic acid decreased the association rate constant (k(on)) about 3-fold. Furthermore, glycosylated EPO had a 20-fold slower k(on) than nonglycosylated EPO, indicating that the core carbohydrate also negatively impacted k(on). The effect of electrostatic forces on EPO binding was studied by measuring binding kinetics in varying NaCl concentrations. Increasing NaCl concentration resulted in a slower k(on) while having little impact on k(off), suggesting that long-range electrostatic interactions are primarily important in determining the rate of association between EPO and EPOR. Furthermore, the glycosylation content (i.e., nonglycosylated vs glycosylated, sialylated vs desialylated) affected the overall sensitivities of k(on) to [NaCl], indicating that sialic acid and the glycan itself each impact the overall effect of these electrostatic forces.