Development and Application of High Performance Liquid Chromatography Map of Glucuronyl N-glycans

Although the multi-dimensional HPLC maps of neutral, sialyl, and sulfated N-glycans have been reported and widely used for glycosylation profiling, those of glucuronyl oligosaccharides have not yet been available. In the present study, by in vitro enzymatic reactions, we prepared 55 different glucuronyl PA-oligosaccharides that include 6 kinds of HNK-1-containing N-glycans, and established their HPLC map. Furthermore, we applied this map to the characterization of branch specificity in glucuronylation reaction catalyzed by human GlcAT-S, revealing that this enzyme transfers the glucuronyl residues preferentially onto the Galβ1→4GlcNAcβ1→4Manα1→3 and Galβ1→4GlcNAcβ1→2Manα1→3 branches of a galactose-terminated tri-antennary oligosaccharide. The HPLC map developed in the present study will be a useful glycomics tool for identification and profiling of glucuronyl N-glycans expressed in the neural and other biological systems.