Complete assignment of 1H and 13C NMR signals of monoglucosylated high-mannose type glycan attached to asparagine

Glc1Man9GlcNAc2 (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 1H and 13C signals is a prerequisite for such analyses. Here, we present the complete assignment of 1H and 13C 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 1H-1H COSY, 1H-1H NOESY, 1H-13C HSQC, 1H-13C HMBC and 1H-13C 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.