Interrelationship of Steric Stabilization and Self-Crowding of a Glycosylated Protein
Interrelationship of Steric Stabilization and Self-Crowding of a Glycosylated Protein
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DOI：
10.1016/j.bpj.2009.05.045
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发表时间：
2009-09-02
影响因子：
3.4
通讯作者：
Arleth, L.
中科院分区：
文献类型：
--
作者：
Hoiberg-Nielsen, R.;Westh, P.;Arleth, L.
在真核细胞中，蛋白质糖基化发生在内质网拥挤的环境中。为了阐明高浓度对糖蛋白相互作用的影响，我们对重糖基化酶lycii phytase （Phy）和去糖基化酶dgPhy进行了一系列小角度x射线散射实验。小角度x射线散射数据分析使用单独的数值形式因子，分别结合两个结构因素，一个硬球和一个屏蔽库仑势结构因素，由从头算分析确定。基于这些数据分析，可以得出三个主要结论。首先，在相当的蛋白质浓度（mg/ml）下，Phy的相对排除体积比dgPhy高75%，表明聚糖显著增加了排除体积相互作用。第二，dgPhy的相对排除体积随浓度增加，与预期一致；然而，在Phy中观察到相反的效果，相对排除体积随着蛋白质浓度的增加而减少。第三，在两种乙二醇形式之间观察到盐度对排除体积相互作用的影响的明显差异。虽然dgPhy的相对排除体积随着离子强度的增加而减小，但Phy的相对排除体积对盐度的增加基本不敏感。我们认为来自聚糖的突出力有助于蛋白质的空间稳定，并且糖基化有助于在拥挤条件下维持排斥静电相互作用。结合起来，这有助于稳定高浓度的糖基化蛋白。
In the eukaryotic cell, protein glycosylation takes place in the crowded environment of the endoplasmatic reticulum. With the purpose of elucidating the impact of high concentration on the interactions of glycoproteins, we have conducted a series of small-angle x-ray scattering experiments on the heavily glycosylated enzyme Peniophora lycii phytase (Phy) and its deglycosylated counterpart (dgPhy). The small-angle x-ray scattering data were analyzed using an individual numerical form factor for each of the two glycoforms combined with two structure factors, a hard sphere and a screened coulomb potential structure factor, respectively, as determined by ab initio analysis. Based on this data analysis, three main conclusions could be drawn. First, at comparable protein concentrations (mg/ml), the relative excluded volume of Phy was similar to 75% higher than that of dgPhy, showing that the glycans significantly increase excluded-volume interactions. Second, the relative excluded volume of dgPhy increased with concentration, as expected; however, the opposite effect was observed for Phy, where the relative excluded volume decreased in response to increasing protein concentration. Third, a clear difference in the effect of salinity on the excluded-volume interactions was observed between the two glycol forms. Although the relative excluded volume of dgPhy decreased with increasing ionic strength, the relative excluded volume of Phy was basically insensitive to increased salinity. We suggest that protrusion forces from the glycans contribute to steric stabilization of the protein, and that glycosylation helps to sustain repulsive electrostatic interactions under crowded conditions. In combination, this aids in stabilizing high concentrations of glycosylated proteins.