Defining the enzymatic pathway for polymorphic O-glycosylation of the pneumococcal serine-rich repeat protein PsrP
Defining the enzymatic pathway for polymorphic O-glycosylation of the pneumococcal serine-rich repeat protein PsrP
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定义肺炎球菌富含丝氨酸重复蛋白 PsrP 多态性 O-糖基化的酶促途径
DOI：
10.1074/jbc.m116.770446
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发表时间：
2017-04-14
影响因子：
4.8
通讯作者：
Zhou, Cong-Zhao
中科院分区：
文献类型：
--
作者：
Jiang, Yong-Liang;Jin, Hua;Zhou, Cong-Zhao
蛋白质o -糖基化是所有生物体中重要的翻译后修饰，但由于其结构的复杂性，破译这些聚糖的特定功能是困难的。了解粘蛋白样蛋白的糖基化是一个特别的挑战，因为它们经过多次修饰，所涉及的酶和糖基化模式都知之甚少。在这里，我们系统地探索了来自人肺炎链球菌TIGR4的粘蛋白样富含丝氨酸的重复蛋白PsrP的o糖基化途径。先前的研究发现，在被认为修饰PsrP的10种糖基转移酶中，有3种、GtfA/B和Gtf3催化了前两个反应，形成了统一的双糖核心结构。我们现在使用体内和体外糖基化分析结合水解活性分析来鉴定能够在糖基化的第三和第四步修饰该核心结构的糖基转移酶。具体来说，全长GlyE和GlyG蛋白以及GlyD DUF1792结构域参与了这两个步骤，而全长GlyA和GlyD GT8结构域只催化了第四步。在富含丝氨酸的重复序列的多个位点将不同的糖结合到双糖核心结构中，会产生高度多态性的产物。此外，载脂蛋白和udp络合GlyE的晶体结构与结构分析相结合，揭示了一个新的罗斯曼折叠“附加”结构域，我们推测该结构域作为GlyD， GlyE和GlyA共享的通用模块，将肽受体从一种酶传递到另一种酶。这些发现定义了细菌糖蛋白的完整糖基化途径，并提供了糖基转移酶协调如何促进聚糖组装的可测试假设。
Protein O-glycosylation is an important post-translational modification in all organisms, but deciphering the specific functions of these glycans is difficult due to their structural complexity. Understanding the glycosylation of mucin-like proteins presents a particular challenge as they are modified numerous times with both the enzymes involved and the glycosylation patterns being poorly understood. Here we systematically explored the O-glycosylation pathway of a mucin-like serine-rich repeat protein PsrP from the human pathogen Streptococcus pneumoniae TIGR4. Previous works have assigned the function of 3 of the 10 glycosyltransferases thought to modify PsrP, GtfA/B, and Gtf3 as catalyzing the first two reactions to form a unified disaccharide core structure. We now use in vivo and in vitro glycosylation assays combined with hydrolytic activity assays to identify the glycosyltransferases capable of decorating this core structure in the third and fourth steps of glycosylation. Specifically, the full-length GlyE and GlyG proteins and the GlyD DUF1792 domain participate in both steps, whereas full-length GlyA and the GlyD GT8 domain catalyze only the fourth step. Incorporation of different sugars to the disaccharide core structure at multiple sites along the serine-rich repeats results in a highly polymorphic product. Furthermore, crystal structures of apo- and UDP-complexed GlyE combined with structural analyses reveal a novel Rossmann-fold “add-on” domain that we speculate to function as a universal module shared by GlyD, GlyE, and GlyA to forward the peptide acceptor from one enzyme to another. These findings define the complete glycosylation pathway of a bacterial glycoprotein and offer a testable hypothesis of how glycosyltransferase coordination facilitates glycan assembly.