A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation
A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation
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DOI：
10.1073/pnas.2335201100
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发表时间：
2003-12-09
影响因子：
11.1
通讯作者：
Bertozzi, CR
中科院分区：
文献类型：
--
作者：
Hang, HC;Yu, C;Bertozzi, CR
粘蛋白型O-连接糖蛋白在高等真核生物中参与多种生物相互作用。这些糖蛋白的生物合成由修饰分泌途径中的蛋白质的多肽N-乙酰基-α-半乳糖胺转移酶（ppGalNAcTs）家族启动。ppGalNAcTs缺乏确定的共有序列使得仅基于一级序列难以预测粘蛋白型0-连接的糖基化。在这里，我们提出了一种方法，用于标记粘蛋白型O-连接的糖蛋白与一个独特的化学标签，叠氮化物，这使得他们的选择性共价修饰复杂的细胞裂解物。从一组合成衍生物中，我们鉴定了叠氮基GalNAc类似物（N-叠氮基乙酰半乳糖胺，GalNAz），其被许多细胞类型代谢并通过ppGalNAcTs安装在粘蛋白型O-连接的糖蛋白上。叠氮化物用作生物正交化学手柄，用于使用Staudinger连接用生物化学或生物物理探针进行选择性修饰。通过用GalNAz标记已知具有O-连接聚糖的重组糖蛋白来验证该方法。此外，GalNAz有效地标记了内源性水平表达的粘蛋白型O-连接糖蛋白。用化学标签标记粘蛋白型O-连接糖蛋白的能力应有助于通过蛋白质组学策略对其进行鉴定。
Mucin-type O-linked glycoproteins are involved in a variety of biological interactions in higher eukaryotes. The biosynthesis of these glycoproteins is initiated by a family of polypeptide N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs) that modify proteins in the secretory pathway. The lack of a defined consensus sequence for the ppGalNAcTs makes the prediction of mucin-type O-linked glycosylation difficult based on primary sequence alone. Herein we present a method for labeling mucin-type O-linked glycoproteins with a unique chemical tag, the azide, which permits their selective covalent modification from complex cell lysates. From a panel of synthetic derivatives, we identified an azido GalNAc analog (N-azidoacetylgalactosamine, GalNAz) that is metabolized by numerous cell types and installed on mucin-type O-linked glycoproteins by the ppGalNAcTs. The azide serves as a bioorthogonal chemical handle for selective modification with biochemical or biophysical probes using the Staudinger ligation. The approach was validated by labeling a recombinant glycoprotein that is known to possess O-linked glycans with GalNAz. In addition, GalNAz efficiently labeled mucin-type O-linked glycoproteins expressed at endogenous levels. The ability to label mucin-type O-linked glycoproteins with chemical tags should facilitate their identification by proteomic strategies.