Preferential binding of two compatible solutes to the glycan moieties of Peniophora lycii phytase
Preferential binding of two compatible solutes to the glycan moieties of Peniophora lycii phytase
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DOI：
10.1021/bi034693i
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发表时间：
2003-09-02
期刊：
影响因子：
2.9
通讯作者：
Westh, P
中科院分区：
文献类型：
--
作者：
Bagger, HL;Fuglsang, CC;Westh, P
水化行为的调节，以及对溶解度和其他性质的伴随影响，已被认为是蛋白质糖基化的主要功能。在这项工作中，我们研究了严重糖基化的隔孢霉植酸酶在两种相容溶质甘油和山梨醇的溶液（0.15-1.1 μ m）中的水合作用。渗透压测量显示甘油优先结合植酸酶（即，甘油-糖蛋白相互作用比水-糖蛋白相互作用更有利，导致甘油在蛋白质界面附近优先积累），而山梨糖醇优先被排除在水合球之外（水-糖蛋白相互作用更有利）。为了评估碳水化合物和肽部分的贡献，分别，我们比较了植酸酶（Phy）和修饰的，但酶活性的形式（dgPhy），其中90%的聚糖已被删除。这表明，两种多元醇均显示出明显且近似相等的与碳水化合物部分的优先结合程度。多元醇与聚糖的这种优先结合与本文（对于dgPhy）和许多先前关于非糖基化蛋白质的报告中观察到的肽界面的排除相反。尽管肽和碳水化合物基团之间存在明显差异，但糖基化对甘油和山梨醇提供的稳定作用没有影响。在此基础上，得出的结论是，Phy的碳水化合物地幔是同样可访问的天然和热变性状态，分别（最有可能完全访问在这两个），因此，它与相容的溶质的相互作用有很少或没有影响的糖蛋白的构象平衡。对于溶解度和聚集平衡，另一方面，结果表明多元醇诱导的单体形式的稳定。
Regulation of hydration behavior, and the concomitant effects on solubility and other properties, has been suggested as a main function of protein glycosylation. In this work, we have studied the hydration of the heavily glycosylated Peniophora lycii phytase in solutions (0.15-1.1 m) of the two compatible solutes glycerol and sorbitol. Osmometric measurements showed that glycerol preferentially binds to phytase (i.e., glycerol-glycoprotein interactions are more favorable than water-glycoprotein interactions resulting in a preferential accumulation of glycerol near the protein interface), while sorbitol is preferentially excluded from the hydration sphere (water-glycoprotein interactions are the more favorable). To assess contributions from carbohydrate and peptide moieties, respectively, we compared phytase (Phy) and a modified, yet enzymatically active form (dgPhy) in which 90% of the glycans had been removed. This revealed that both polyols showed a pronounced and approximately equal degree of preferential binding to the carbohydrate moiety. This preferential binding of polyols to glycans is in contrast to the exclusion from peptide interfaces observed here (for dgPhy) and in numerous previous reports on nonglycosylated proteins. Despite the distinct differences between peptide and carbohydrate groups, glycosylation had no effect on the stabilizing action provided by glycerol and sorbitol. On the basis of this, it was concluded that the carbohydrate mantle of Phy is equally accessible in the native and thermally denatured states, respectively (most likely fully accessible in both), and thus that its interactions with compatible solutes have little or no effect on conformational equilibria of the glycoprotein. For solubility and aggregation equilibria, on the other hand, the results suggest a polyol-induced stabilization of monomeric forms.