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(beta1-3)Gal(beta1-4)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-O-sulfate 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-O-sulfo-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.
Highlights
Domain in its extracellular part, a transmembranous domain, and a cytoplasmic tail
We recently described the structure of the N-glycan carrying the human natural keller cell (HNK)-1 epitope, present on bovine peripheral myelin P0
Due to variations in the pulsed amperometric detection response, no estimation of the percentages for the individual fractions could be made. These combined fractions accounted for Ͼ90% of all glycans on P0. At this stage of purification, only three fractions were homogeneous according to one-dimensional 1H NMR spectroscopy and mass spectrometry; no further purification was carried out in order to prevent any loss of material
Summary
Isolation of P0 and Release and Isolation of the Carbohydrate Chains—The P0 isolation procedure and release of the carbohydrate chains were described previously by Voshol et al [17]. Two-dimensional TOCSY spectra in a 5-mm probe were recorded using a “clean” MLEV-17 spin-lock pulse [21,22,23] of 90 –110 ms as described by Voshol et al [17]. Twodimensional rotating frame NOESY spectra in a 5-mm probe [24] were recorded at spin-lock times of 150 –225 ms as described by Voshol et al [17]. A two-dimensional NOESY spectrum [25] in a nano probe was acquired with a mixing time of 200 ms and pre-saturation delay of 1.2 s. Negative-ion mode mass spectrometric analyses of all other fractions were performed on a Reflex III MALDI-TOF spectrometer (Bruker Daltonik GmbH) equipped with a SCOUT ion source, a N2 laser (337 nm), and a 2-GHz digitizer. Data recorded were processed using Bruker Daltonik XMASS/NT Version 5 software
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