Site-to-site cross-talk in OST-B glycosylation of hCEACAM1-IgV.
Site-to-site cross-talk in OST-B glycosylation of hCEACAM1-IgV.
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DOI：
10.1073/pnas.2202992119
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发表时间：
2022-10-25
影响因子：
11.1
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中科院分区：
文献类型：
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n -糖基化是一种常见的细胞外蛋白翻译后修饰。它在内质网的蛋白质折叠中起作用，随后参与重要的蛋白质-碳水化合物相互作用。特别是病毒，通常高度糖基化。了解特定位点的修饰决定因素对于更好地理解这些过程非常重要。获得这一信息的一个挑战是分离两种糖基转移酶OST-A和OST-B的作用。我们的研究重点是OST-B，并揭示了在OST-B糖基化的背景下，多个紧密间隔的糖基化位点如何相互影响。n -糖基化是真核生物中常见的分泌蛋白翻译后修饰。该修饰针对的是一致序列N-X-S /T内的天冬酰胺残基。虽然这个序列是糖基化所必需的，但低聚糖转移酶a或B （OST-A或OST-B）对高甘露糖聚糖的初始转移可能导致在给定位点的不完全占据。决定转移程度的因素尚不清楚，了解这些因素可能有助于深入了解这些重要酶的功能。在这里，我们使用质谱（MS）同时测量重组糖蛋白hCEACAM1的n端IgV结构域上三个n -糖基化位点的相对占有率。我们证明了加成主要是由OST-B酶进行的，并提出了OST-B n -糖基化的动力学模型。将动力学模型拟合到质谱数据中，在大多数位点上可以得到不同的聚糖加成速率，并表明聚糖加成的初始顺序在很大程度上是随机的。该模型还表明，一个位点的糖基化会影响其他位点后续修饰的效率，而中心或n端位点的糖基化会导致终端产物，很少导致所有三个位点的完全糖基化。只有一条进行性糖基化途径，即由c末端的糖基化启动的途径，才能有效地导致所有三个位点的完全占用。因此，hCEACAM1结构域提供了一个有效的模型系统来研究OST-B对糖基化序列的位点特异性识别，并表明翻译后糖基化的顺序和效率受到相邻受体位点之间的空间串扰的影响。
N-glycosylation is a common posttranslational modification of extracellular proteins. It plays a role in protein folding in the endoplasmic reticulum and later participates in important protein–carbohydrate interactions. Viruses, in particular, are often highly glycosylated. Knowledge of the determinants of modification at a particular site is important for better understanding these processes. One challenge in obtaining this information is separating the effect of the two glycosyltransferases, OST-A and OST-B. Our study focuses on OST-B and reveals how multiple, closely spaced glycosylation sites can influence one another in the context of OST-B glycosylation. N-glycosylation is a common posttranslational modification of secreted proteins in eukaryotes. This modification targets asparagine residues within the consensus sequence, N–X–S/T. While this sequence is required for glycosylation, the initial transfer of a high-mannose glycan by oligosaccharyl transferases A or B (OST-A or OST-B) can lead to incomplete occupancy at a given site. Factors that determine the extent of transfer are not well understood, and understanding them may provide insight into the function of these important enzymes. Here, we use mass spectrometry (MS) to simultaneously measure relative occupancies for three N-glycosylation sites on the N-terminal IgV domain of the recombinant glycoprotein, hCEACAM1. We demonstrate that addition is primarily by the OST-B enzyme and propose a kinetic model of OST-B N-glycosylation. Fitting the kinetic model to the MS data yields distinct rates for glycan addition at most sites and suggests a largely stochastic initial order of glycan addition. The model also suggests that glycosylation at one site influences the efficiency of subsequent modifications at the other sites, and glycosylation at the central or N-terminal site leads to dead-end products that seldom lead to full glycosylation of all three sites. Only one path of progressive glycosylation, one initiated by glycosylation at the C-terminal site, can efficiently lead to full occupancy for all three sites. Thus, the hCEACAM1 domain provides an effective model system to study site-specific recognition of glycosylation sequons by OST-B and suggests that the order and efficiency of posttranslational glycosylation is influenced by steric cross-talk between adjoining acceptor sites.
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影响因子：
5.7
作者：
Mohorko, Elisabeth;Owen, Robin L.;Glockshuber, Rudi
通讯作者：
Glockshuber, Rudi
影响因子：
4.8
作者：
Zhuo, You;Yang, Jeong-Yeh;Prestegard, James H.
通讯作者：
Prestegard, James H.
影响因子：
3
作者：
Pettersen, EF;Goddard, TD;Ferrin, TE
通讯作者：
Ferrin, TE
影响因子：
7.4
作者：
Zubarev, RA;Horn, DM;McLafferty, FW
通讯作者：
McLafferty, FW
影响因子：
56.9
作者：
Ramirez, Ana S.;Kowal, Julia;Locher, Kaspar P.
通讯作者：
Locher, Kaspar P.