Systems analysis of N-glycan processing in mammalian cells.
Systems analysis of N-glycan processing in mammalian cells.
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DOI：
10.1371/journal.pone.0000713
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发表时间：
2007-08-08
期刊：
影响因子：
3.7
通讯作者：
Hu WS
中科院分区：
文献类型：
--
作者：
Hossler P;Mulukutla BC;Hu WS
N-糖基化在许多治疗性糖蛋白生物制剂的质量中起关键作用。这些寡糖的生物合成反应是一种网络，其中相对少量的酶产生大量的N-聚糖作为反应中间体和末端产物。多个聚糖出现在糖蛋白分子上，并产生异质产物。控制聚糖分布对于产品的质量控制至关重要。了解N-聚糖的生物合成和微观异质性的病因学将提供生理学的见解，并促进细胞工程，以提高糖蛋白的质量。我们开发了一个数学模型，在高尔基体的聚糖生物合成和分析的各种反应变量上得到的聚糖分布。高尔基体模型被建模为串联的四个隔室。蛋白质通过高尔基体的转运机制仍有争议。从其保持时间分布特性的角度来看，两个主要的假设机制，囊泡运输和高尔基体成熟模型，类似于四个连续混合罐（4CSTR）和四个活塞流反应器（4PFR）串联，分别。这两个假设相应地建模和比较。本征反应动力学首先使用分批（或单个PFR）反应器进行评价。需要足够的放置时间来生产最终加工的聚糖。改变酶浓度对最终聚糖分布具有复杂的影响，因为这些变化通常会影响网络中的许多反应步骤。通过比较4CSTR和4PFR模型预测的聚糖谱，指出4PFR系统更可能是真正的机制。为了评估在生物治疗剂的生物合成中是否可以消除聚糖异质性，进一步使用4PFR模型来评估是否可以通过代谢工程产生同质聚糖谱。我们证明了酶的空间定位到特定的隔间，所有终端加工的N-聚糖可以合成为均匀的产品与足够的保持时间在高尔基室。开发的模型可以作为指导未来工程的糖蛋白。
N-glycosylation plays a key role in the quality of many therapeutic glycoprotein biologics. The biosynthesis reactions of these oligosaccharides are a type of network in which a relatively small number of enzymes give rise to a large number of N-glycans as the reaction intermediates and terminal products. Multiple glycans appear on the glycoprotein molecules and give rise to a heterogeneous product. Controlling the glycan distribution is critical to the quality control of the product. Understanding N-glycan biosynthesis and the etiology of microheterogeneity would provide physiological insights, and facilitate cellular engineering to enhance glycoprotein quality. We developed a mathematical model of glycan biosynthesis in the Golgi and analyzed the various reaction variables on the resulting glycan distribution. The Golgi model was modeled as four compartments in series. The mechanism of protein transport across the Golgi is still controversial. From the viewpoint of their holding time distribution characteristics, the two main hypothesized mechanisms, vesicular transport and Golgi maturation models, resemble four continuous mixing-tanks (4CSTR) and four plug-flow reactors (4PFR) in series, respectively. The two hypotheses were modeled accordingly and compared. The intrinsic reaction kinetics were first evaluated using a batch (or single PFR) reactor. A sufficient holding time is needed to produce terminally-processed glycans. Altering enzyme concentrations has a complex effect on the final glycan distribution, as the changes often affect many reaction steps in the network. Comparison of the glycan profiles predicted by the 4CSTR and 4PFR models points to the 4PFR system as more likely to be the true mechanism. To assess whether glycan heterogeneity can be eliminated in the biosynthesis of biotherapeutics the 4PFR model was further used to assess whether a homogeneous glycan profile can be created through metabolic engineering. We demonstrate by the spatial localization of enzymes to specific compartments all terminally processed N-glycans can be synthesized as homogeneous products with a sufficient holding time in the Golgi compartments. The model developed may serve as a guide to future engineering of glycoproteins.
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