Epitope Diversity of N-Glycans from Bovine Peripheral Myelin Glycoprotein P0 Revealed by Mass Spectrometry and Nano Probe Magic Angle Spinning 1H NMR Spectroscopy

Abstract

The carbohydrate structures present on the glycoproteins in the central and peripheral nerve systems are essential in many cell adhesion processes. The P0 glycoprotein, expressed by myelinating Schwann cells, plays an important 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 P0 glycoprotein was shown to be mediated via the human natural keller cell (HNK)-1 epitope (3-O-SO 3 H-GlcUA(␤1-3)Gal(␤1-4)GlcNAc) present on the N-glycans. We recently described the structure of the N-glycan carrying the HNK-1 epitope, present on bovine peripheral myelin P0 (Voshol, 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 report on the structural characterization of the detectable glycoforms, present on the single N-glycosylation site, using state-of-the-art NMR and mass spectrometry techniques. Even though all structures belong to the hybrid-or biantennary complex-type structures, the variety of epitopes is remarkable. In addition to the 3-Osulfate present on the HNK-1-carrying structures, most of the glycans contain a 6-O-sulfated N-acetylglucosamine residue. This indicates the activity of a 6-Osulfo-GlcNAc-transferase, which has not been described before in peripheral nervous tissue. The presence of the disialo-, galactosyl-, and 6-O-sulfosialyl-Lewis X epitopes provides evidence for glycosyltransferase activities not detected until now. The finding of such an epitope diversity triggers questions related to their function and whether events, previously attributed merely to the HNK-1 epitope, could be mediated by the structures described here.The P0 glycoprotein consists of a single immunoglobulin-like domain in its extracellular part, a transmembranous domain, and a cytoplasmic tail. It is the most abundant protein constituent of peripheral myelin. P0 contains a single N-glycosylation site and heterogeneity in its glycosylation pattern that originates from variable contents of fucose, galactose, and sialic acid residues; sulfate; and the HNK-1 carbohydrate epitope (1-4). P0 appears at the initial stage of myelination and contributes to the formation and maintenance of myelin compaction as an adhesion molecule (5). The essential functional role of P0 in the processes of myelination has been demonstrated by creating P0 knockout mice, which show severe hypomyelination and myelin degeneration (5, 6). In humans, several neurological disorders such as Charcot-Marie-Tooth disease, Dejerine-Sottas disease, and congenital hypomyelination have been associated with mutations in the P0 gene (7).It has been reported that the glycan moiety of P0 plays an important role in cell-cell adhesion via homophilic binding. This homophilic binding has been mapped to the SDNGT sequence composing amino acids 91-95, which harbors the single N-glycosylation site on P0 (8). Thus, it was observed that this glycopeptide fragment inhibits cell adhesion t...

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A Non-sulfated Form of the HNK-1 Carbohydrate Is Expressed in Mouse Kidney

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