Avenues to Characterize the Interactions of Extended N‐Glycans with Proteins by NMR Spectroscopy: The Influenza Hemagglutinin Case

Abstract

Long‐chain multiantenna N‐glycans are extremely complex molecules. Their inherent flexibility and the presence of repetitions of monosaccharide units in similar chemical environments hamper their full characterization by X‐ray diffraction or standard NMR methods. Herein, the successful conformational and interaction analysis of a sialylated tetradecasaccharide N‐glycan presenting two LacNAc repetitions at each arm is presented. This glycan has been identified as the receptor of the hemagglutinin protein of pathogenic influenza viruses. To accomplish this study, a N‐glycan conjugated with a lanthanide binding tag has been synthesized, enabling analysis of the system by paramagnetic NMR. Under paramagnetic conditions, the NMR signals of each sugar unit in the glycan have been determined. Furthermore, a detailed binding epitope of the tetradecasaccharide N‐glycan in the presence of HK/68 hemagglutinin is described.

Recent Advances in the Chemical Biology of N-Glycans

“…On the other hand, no differences in the strengths of STD signals of each branch were observed with Ricinus communis agglutinin (RCA120), which recognizes terminal galactose, indicating that RCA120 recognizes all branches without distinction. In a similar analysis between sialic acid containing biantennary N-glycans and HA, STD signals from both sialic acids were observed, suggesting the contribution of two sialic acids in a multivalent effect [45]. Furthermore, interesting results have been reported showing that the N-glycan conformation directly affects lectin recognition ( Figure 6) [87].…”

“…While conformation is an important factor for glycan recognition, the flexibility of glycans makes conformational analysis difficult. In addition to analysis based on coupling constants and the nuclear Overhauser effect (NOE), an analysis using pseudocontact shift (PCS) by paramagnetic metals has recently been developed, and its efficiency has been demonstrated [ 42 , 43 , 44 , 45 , 46 , 47 , 48 ].…”

“…Conformation analysis of glycans using NMR provides important insights into complex glycan–lectin interactions. The N -glycan conformation can be predicted by combining PCS-based NMR analysis and MD simulations ( Figure 5 ) [ 42 , 43 , 44 , 45 , 46 , 47 , 48 ]. Kato et al analyzed the conformation of high-mannose glycans by PCS-based NMR analysis using 13 C-labeled compounds and Tm 3+ as a paramagnetic metal ion tag [ 38 ].…”

Conformational Studies of Oligosaccharides

“…The use of paramagnetic NMR was also applied to the study of the conformation of multiantenna N‐glycans and their interaction with HK/68 hemagglutinin from influenza viruses. This study was enabled by chemoenzymatic synthesis of long‐chain N‐glycans containing poly‐LacNAc and Neu5Ac residues, followed by conjugation with a lanthanide binding tag …”

The Interaction of Fluorinated Glycomimetics with DC-SIGN: Multiple Binding Modes Disentangled by the Combination of NMR Methods and MD Simulations

“…The last years have enjoyed large advances in the Glycoscience field [6][7][8][9][10][11], providing new tools and perspectives to expand the understanding on the structure-function relationship of the events mediated by sugar-lectin interactions. These developments have also led to important progress in the use of glycomimetics [12,13], sugar analogues devised to bind lectins, antibodies, or enzymes [14][15][16].…”