Howard C. Hang

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.

The glycosylation of serine and threonine residues with a single GlcNAc moiety is a dynamic posttranslational modification of many nuclear and cytoplasmic proteins. We describe a chemical strategy directed toward identifying O-GlcNAc-modified proteins from living cells or proteins modified in vitro. We demonstrate, in vitro, that each enzyme in the hexosamine salvage pathway, and the enzymes that affect this dynamic modification (UDP-GlcNAc:polypeptidtyltransferase and O-GlcNAcase), tolerate analogues of their natural substrates in which the N-acyl side chain has been modified to bear a bio-orthogonal azide moiety. Accordingly, treatment of cells with N-azidoacetylglucosamine results in the metabolic incorporation of the azido sugar into nuclear and cytoplasmic proteins. These O-azidoacetylglucosamine-modified proteins can be covalently derivatized with various biochemical probes at the site of protein glycosylation by using the Staudinger ligation. The approach was validated by metabolic labeling of nuclear pore protein p62, which is known to be posttranslationally modified with O-GlcNAc. This strategy will prove useful for both the identification of O-GlcNAc-modified proteins and the elucidation of the specific residues that bear this saccharide.