A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation

• DOI: 10.1073/pnas.2335201100
• Publication Date: 2003-12-09T18:17:48Z
• Authors: Howard C. Hang; Chong Yu; Darryl L. Kato; Carolyn R. Bertozzi
• Keywords: 0301 basic medicine; Glycosylation; Dose-Response Relationship, Drug; Proteome; Blotting, Western; Monosaccharides; Mucins; CHO Cells; Flow Cytometry; Models, Biological; Jurkat Cells; Mice; 03 medical and health sciences; Models, Chemical; Cricetinae; Lectins; COS Cells; NIH 3T3 Cells; Animals; Humans; Glycoproteins; HeLa Cells

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-α-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.