Epitope diversity of N-glycans from bovine peripheral myelin glycoprotein P0 revealed by mass spectrometry and nano probe magic angle spinning H-1 NMR spectroscopy

The carbohydrate structures present on the glycoproteins in the central and peripheral nerve systems are essential in many cell adhesion processes. Th e PO glycoprotein, expressed by myelinating Schwann cells, plays an importa nt role during the formation and maintenance of myelin, and it is the most abundant constituent of myelin. Using monoclonal antibodies, the homophilic binding of the PO glycoprotein was shown to be mediated via the human natu ral keller cell (HNK)-1 epitope (3-O-SO3H-GlcUA(beta1-3)Gal(beta1-4)GlcNAc) present on the N-glycans. We recently described the structure of the N-gly can carrying the HNK-1 epitope, present on bovine peripheral myelin PO (Vos hol, H., van Zuylen, C. W. E. M., Orberger, G., Vliegenthart, J. F. G., and Schachner, M. (1996) J. Biol Chem. 271, 22957-22960). In this study, we re port on the structural characterization of the detectable glycoforms, prese nt on the single N-glycosylation site, using state-of-the-art NMR and mass spectrometry techniques. Even though all structures belong to the hybrid- o r biantennary complex-type structures, the variety of epitopes is remarkabl e. In addition to the 3-O-sulfate present on the HNK-1-carrying structures, most of the glycans contain a 6-O-sulfated N-acetylglucosamine residue. Th is indicates the activity of a 6-O-sulfo-GlcNAc-transferase, which has not been described before in peripheral nervous tissue. The presence of the dis ialo-, galactosyl-, and 6-O-sulfosialyl-Lewis X epitopes provides evidence for glycosyltransferase activities not detected until now. The finding of s uch an epitope diversity triggers questions related to their function and w hether events, previously attributed merely to the HNK-1 epitope, could be mediated by the structures described here.