Complete assignment of H and C NMR signals of monoglucosylated high-mannose type glycan attached to asparagine.

GlcManGlcNAc (G1M9) glycan and other high mannose-type glycans play key roles in the quality control mechanisms of glycoprotein synthesis. The lectin-like proteins calnexin (CNX) and calreticulin (CRT) specifically recognize G1M9 glycan and assist newly synthesized glycoproteins to achieving correct folding. Nuclear magnetic resonance (NMR) spectroscopy is a unique method for analyzing the conformation, dynamics and interactions of glycans like G1M9 glycan and CNX/CRT. Accurate assignment of H and C signals is a prerequisite for such analyses. Here, we present the complete assignment of H and C signals for the Asn-linked G1M9 glycan, modified at its N-terminus with a 9-fluorenylmethyloxycarbonyl (Fmoc) group (Fmoc-Asn-G1M9). Using conventional two-dimensional NMR techniques including H-H COSY, H-H NOESY, H-C HSQC, H-C HMBC and H-C HSQC-TOCSY, we achieved a comprehensive spectral assignment. Our results are consistent with previously reported assignments of the partial pentasaccharide structure of G1M9 glycan. This complete assessment of G1M9 glycan signals provides a foundation for detailed studies of its interactions with CNX/CRT, which will advance our understanding of the molecular mechanisms underlying glycoprotein quality control.