Hang Howard C, Yu Chong, Kato Darryl L, Bertozzi Carolyn R
Center for New Directions in Organic Synthesis and Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.
Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):14846-51. doi: 10.1073/pnas.2335201100. Epub 2003 Dec 1.
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.
粘蛋白型O-连接糖蛋白参与高等真核生物中的多种生物相互作用。这些糖蛋白的生物合成由一族多肽N-乙酰-α-半乳糖胺基转移酶（ppGalNAcTs）起始，它们在分泌途径中修饰蛋白质。由于ppGalNAcTs缺乏明确的共有序列，仅基于一级序列很难预测粘蛋白型O-连接糖基化。在此，我们提出一种用独特化学标签叠氮化物标记粘蛋白型O-连接糖蛋白的方法，该方法允许从复杂的细胞裂解物中对其进行选择性共价修饰。从一组合成衍生物中，我们鉴定出一种叠氮基GalNAc类似物（N-叠氮乙酰半乳糖胺，GalNAz），它可被多种细胞类型代谢，并由ppGalNAcTs安装在粘蛋白型O-连接糖蛋白上。叠氮化物作为一种生物正交化学手柄，可用于使用施陶丁格连接反应与生化或生物物理探针进行选择性修饰。通过标记一种已知带有O-连接聚糖的重组糖蛋白验证了该方法。此外，GalNAz能有效地标记内源性表达的粘蛋白型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。
