Abstract
The natural diversity in glycan structures found on glycoproteins appears to be due in large part to tissue-specific regulation of glycosyltransferase activities. In this review, we describe a method for examining tissue-specific patterns of O-glycosylation using aryl-α-D-GalNAC to prime O-glycan biosynthesis in vivo. The addition of p-nitrophenyl-, phenyl-, or benzyl-α-D-GalNAc to cells in tissue culture competitively inhibits extension of GalNAcα-Ser/Thr on endogenous glycoproteins, while priming aryl-oligosaccharide biosynthesis. The aryl-oligosaccharidesare produced in the Golgi and move by membrane bulk-flow into the culture medium. Absorbance of the aryl group at 303nm can be used to follow the purification of aryl-oligosaccharides, and sufficient glycan can be made for analysis by proton nuclear magnetic resonance and mass spectrometry. Structural analysis of oligosaccharides primed by aryl-α-D-GalNAc in vivo, combined with measurements of glycosyltransferase activity in vitro provide a means of characterizing O-glycosylation in diverse cell types. The limitations of this approach are also discussed.
