Structural analysis of the carbohydrate chains of mucin-type glycoproteins by high-resolution 1H-n.m.r. spectroscopy.

Abstract

Mucins are high-molecular-weight glycoproteins consisting of numerous carbohydrate chains that are attached via GalNAc to hydroxyl groups in the side chains of Serine and threonine in the polypeptide backbone. These O-glycosidically linked oligosaccharides may vary in size from dito 'megalo'-saccharides. Similar structures are encountered on soluble glycoproteins like fetuin and ic-casein, and also on membrane-bound glycoproteins, e.g. glycophorin and glycocalicin. The aforementioned compounds all together are referred to as mucin-type glycoproteins. The carbohydrate structures of mucin-type glycoproteins may be divided into three domains: the core, backbone and peripheral regions. In particular the two last-mentioned are of considerable importance since they may express a variety of antigenic activities. As such, they are involved as receptors in cellular interactions and reactions with microbial agents. Furthermore, these parts of the structures may be altered during cell differentiation, as well as in neoplastic conditions (Hounsell & Feizi, 1982). Structural analysis of mucin-type carbohydrate chains is greatly hampered by their heterogeneity, making it virtually impossible to study them at the level of the intact glycoprotein. The chains are most conveniently studied, after release from the peptide by alkaline borohydride treatment, as oligosaccharide alditols; a number of subsequent separation and purification steps are usually required to make them amenable for structural analysis. In the past few years, we have had the opportunity to demonstrate that high-resolution I H-n.m.r. spectroscopy, in conjunction with sugar analysis, is an extremely powerful method for primary-structural characterization of such oligosaccharide alditols. For reasons of resolution and sensitivity, if is desirable that the highest magnetic field strengths currently available, i.e. 10-14T, corresponding to radiofrequencies of 400-600MHz, are employed. Even then, at least 20nmol of oligosaccharide alditol, free from non-carbohydrate contaminants, are required; however, the n.m.r. method is nondestructive, thus leaving open the possibility of performing chemical, enzymic or immunological studies afterwards. Moreover, H-n.m.r. is able to handle adequately mixtures of structurally related and therefore hardly separable oligosaccharide alditols, in that it affords the complete structures of the constituents and the ratio in which they occur in the mixture. So, this approach can cope with (micro-)heterogeneity to a considerable extent. Finally, if 5-10mmol of a sufficiently pure oligosaccharide alditol can be made available, more advanced n.m.r. techniques allow insight to be gained into its solution conformation. The present article outlines briefly, how to arrive at core, backbone and peripheral portions of mucin-type oligosaccharide alditol structures, and eventually at their solution conformation, by employment of oneand twodimensional ' H-n.m.r. spectroscopy. The data compiled below stem from investigations of oligosaccharide alditols