A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation.
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.
Hang
Howard C
HC
Center for New Directions in Organic Synthesis and Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.
Yu
Chong
C
Kato
Darryl L
DL
Bertozzi
Carolyn R
CR
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D037102:Lectins / Q000737:chemistry; D051379:Mice; D008954:Models, Biological; D008956:Models, Chemical; D009005:Monosaccharides / Q000737:chemistry; D009077:Mucins / Q000737:chemistry*; D041681:NIH 3T3 Cells; D020543:Proteome; D011994:Recombinant Proteins / Q000737:chemistry
D006023:Glycoproteins; D037102:Lectins; D009005:Monosaccharides; D009077:Mucins; D020543:Proteome; D011994:Recombinant Proteins