Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, India.
Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville 7530, South Africa.
Int J Biol Macromol. 2018 Jun;112:490-498. doi: 10.1016/j.ijbiomac.2018.01.201. Epub 2018 Feb 1.
In this study, the effect of N-glycosylation on the conformational and functional stability of Putranjiva roxburghii family 1 β-glucosidase (PRGH1) enzyme was investigated. The deglycosylation of PRGH1 was carried out by using PNGase F enzyme and confirmed by SDS-PAGE and carbohydrate estimation. Comparative analysis with respect to enzyme activity, stability and aggregation behaviour was carried out for the glycosylated and deglycosylated PRGH1. The deglycosylation of PRGH1 affected enzyme activity to a certain extent only where K was not affected but a slight reduction in V for various substrates was observed. Circular dichroism, fluorescence studies and differential scanning calorimetry (DSC) analysis demonstrated the possible effect of glycosylation on local and/or global conformational dynamics of protein and its effect on the thermostability of PRGH1. DSC results showed deglycosylated form had lower T as compared to the glycosylated form of PRGH1. The PRGH1 was found to be more sensitive to proteolysis after deglycosylation suggesting that the glycosylated PRGH1 was quite compact and rigid. Mutagenesis studies revealed that out of seven potential N-linked glycosylation sites, only three were glycosylated. The results demonstrated that N-linked glycosylation played an important role in conformational stability of PRGH1; however, it did not affect the enzyme function drastically.
在这项研究中，研究了 N-糖基化对猪屎豆族 1 型β-葡萄糖苷酶（PRGH1）酶构象和功能稳定性的影响。使用 PNGase F 酶对 PRGH1 进行去糖基化，并通过 SDS-PAGE 和碳水化合物估计进行确认。对糖基化和去糖基化的 PRGH1 进行了酶活性、稳定性和聚集行为的比较分析。PRGH1 的去糖基化在一定程度上仅影响酶活性，其中 K 不受影响，但观察到各种底物的 V 略有降低。圆二色性、荧光研究和差示扫描量热法（DSC）分析表明，糖基化可能影响蛋白质的局部和/或整体构象动力学及其对 PRGH1 热稳定性的影响。DSC 结果表明，去糖基化形式的 T 比 PRGH1 的糖基化形式低。PRGH1 在去糖基化后更容易受到蛋白水解的影响，这表明糖基化的 PRGH1 相当紧凑和坚硬。突变研究表明，在七个潜在的 N-连接糖基化位点中，只有三个被糖基化。结果表明，N-糖基化对 PRGH1 的构象稳定性起着重要作用；然而，它并没有极大地影响酶的功能。
