Xiong Yongao, Karuppanan Kalimuthu, Bernardi Austen, Li Qiongyu, Kommineni Vally, Dandekar Abhaya M, Lebrilla Carlito B, Faller Roland, McDonald Karen A, Nandi Somen
Department of Chemical Engineering, University of California, Davis, Davis, CA, United States.
Department of Chemistry, University of California, Davis, Davis, CA, United States.
Front Plant Sci. 2019 Jun 28;10:768. doi: 10.3389/fpls.2019.00768. eCollection 2019.
Protein N-glycosylation is an important post-translational modification and has influences on a variety of biological processes at the cellular and molecular level, making glycosylation a major study aspect for glycoprotein-based therapeutics. To achieve a comprehensive understanding on how N-glycosylation impacts protein properties, an Fc-fusion anthrax decoy protein, rCMG2-Fc, was expressed in plant with three types of N-glycosylation profiles. Three variants were produced by targeting protein to plant apoplast (APO), endoplasmic reticulum (ER) or removing the N-glycosylation site by a point mutation (Agly). Both the APO and ER variants had a complex-type N-glycan (GnGnXF) as their predominant glycans. In addition, ER variant had a higher concentration of mannose-type N-glycans (50%). The decoy protein binds to the protective antigen (PA) of anthrax through its CMG2 domain and inhibits toxin endocytosis. The protein expression, sequence, N-glycosylation profile, binding kinetics to PA, toxin neutralization efficiency, and thermostability were determined experimentally. In parallel, we performed molecular dynamics (MD) simulations of the predominant full-length rCMG2-Fc glycoform for each of the three N-glycosylation profiles to understand the effects of glycosylation at the molecular level. The MAN8 glycoform from the ER variant was additionally simulated to resolve differences between the APO and ER variants. Glycosylation showed strong stabilizing effects on rCMG2-Fc during accumulation, evidenced by the over 2-fold higher expression and less protein degradation observed for glycosylated variants compared to the Agly variant. Protein function was confirmed by toxin neutralization assay (TNA), with effective concentration (EC) rankings from low to high of 67.6 ng/ml (APO), 83.15 ng/ml (Agly), and 128.9 ng/ml (ER). The binding kinetics between rCMG2-Fc and PA were measured with bio-layer interferometry (BLI), giving sub-nanomolar affinities regardless of protein glycosylation and temperatures (25 and 37°C). The protein thermostability was examined utilizing the PA binding ELISA to provide information on EC differences. The fraction of functional ER variant decayed after overnight incubation at 37°C, and no significant change was observed for APO or Agly variants. In MD simulations, the MAN8 glycoform exhibits quantitatively higher distance between the CMG2 and Fc domains, as well as higher hydrophobic solvent accessible surface areas (SASA), indicating a possibly higher aggregation tendency of the ER variant. This study highlights the impacts of N-glycosylation on protein properties and provides insight into the effects of glycosylation on protein molecular dynamics.
蛋白质N-糖基化是一种重要的翻译后修饰，在细胞和分子水平上对多种生物学过程产生影响，这使得糖基化成为基于糖蛋白的治疗药物的一个主要研究方面。为了全面了解N-糖基化如何影响蛋白质特性，一种Fc融合炭疽诱饵蛋白rCMG2-Fc在植物中表达，具有三种类型的N-糖基化谱。通过将蛋白质靶向植物质外体（APO）、内质网（ER）或通过点突变去除N-糖基化位点（Agly）产生了三种变体。APO和ER变体都以复合型N-聚糖（GnGnXF）作为其主要聚糖。此外，ER变体具有更高浓度的甘露糖型N-聚糖（50%）。该诱饵蛋白通过其CMG2结构域与炭疽保护性抗原（PA）结合，并抑制毒素内吞作用。通过实验确定了蛋白质表达、序列、N-糖基化谱、与PA的结合动力学、毒素中和效率和热稳定性。同时，我们对三种N-糖基化谱中每种的主要全长rCMG2-Fc糖型进行了分子动力学（MD）模拟，以在分子水平上了解糖基化的影响。对ER变体的MAN8糖型进行了额外模拟，以解析APO和ER变体之间的差异。糖基化在rCMG2-Fc积累过程中显示出强烈的稳定作用，与Agly变体相比，糖基化变体的表达高出2倍以上且蛋白质降解更少，证明了这一点。通过毒素中和试验（TNA）证实了蛋白质功能，有效浓度（EC）从低到高的排名为67.6 ng/ml（APO）、83.15 ng/ml（Agly）和128.9 ng/ml（ER）。用生物层干涉术（BLI）测量了rCMG2-Fc与PA之间的结合动力学，无论蛋白质糖基化和温度（25和37°C）如何，亲和力均为亚纳摩尔级。利用PA结合ELISA检测了蛋白质热稳定性，以提供有关EC差异的信息。ER变体功能部分在37°C过夜孵育后衰减，而APO或Agly变体未观察到显著变化。在MD模拟中，MAN8糖型在CMG2和Fc结构域之间表现出定量更高的距离，以及更高的疏水溶剂可及表面积（SASA），表明ER变体可能具有更高的聚集倾向。这项研究突出了N-糖基化对蛋白质特性的影响，并深入了解了糖基化对蛋白质分子动力学的作用。
