PDF(Pubmed)
Abstract：
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.
摘要：
N-糖基化是真核生物中分泌蛋白的常见翻译后修饰。该修饰靶向共有序列内的天冬酰胺残基,N-X-S/T.虽然该序列是糖基化所必需的，通过寡糖基转移酶A或B（OST-A或OST-B）的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还没有得到很好的理解，了解它们可以深入了解这些重要酶的功能。这里,我们使用质谱（MS）同时测量重组糖蛋白N端IgV结构域上三个N糖基化位点的相对占用率，HCEACAM1.我们证明了添加主要是通过OST-B酶，并提出了OST-BN-糖基化的动力学模型。将动力学模型拟合到MS数据在大多数位点处产生不同的聚糖添加速率,并且表明聚糖添加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，并且在中心或N-末端位点的糖基化导致死端产物,其很少导致所有三个位点的完全糖基化。只有一条逐渐糖基化的途径，一种是由C端位点的糖基化引发的，可以有效地导致所有三个站点的完全占用。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。
