Breaking the limits in analyzing carbohydrate recognition by NMR: Resolving Branch‐ Selective Interaction of a Tetraantennary N‐Glycan with lectins

Abstract

N‐glycans are ubiquitous in nature and functionalize glycoproteins. Protein glycosylation is required for proper biological and biophysical function and often, alterations in glycosylation are related to diseases.[1]Complex glycosylation patterns containing multiantennary N‐glycans are typically found in mature glycoproteins. However, the structural characterization of these glycans is rather challenging. Usually, NMR and X‐ray diffraction techniques fail to provide specific answers on the structure and molecular recognition features due to the intrinsic attributes of the glycan. The properties of the glycosidic bond and especially the presence of 1–6 linkages endow a large flexibility to the molecule. This feature often precludes the crystallization of complex carbohydrates and/or hampers the detection of enough electron density for most of the glycan part in the X‐ray diffraction analysis of glycoproteins. Moreover, the standard use of the corresponding fitting programs to deduce the three‐dimensional structures frequently give rise to incorrect structures of the corresponding saccharide moieties.[2] Thus, any advance in this area is of fundamental value.As a promising approach, carbohydrates conjugated to lanthanide‐binding tags have recently revealed high potential toward this aim. [3,4] In its vicinity, a complexed paramagnetic ion induces significant chemical shift changes of the NMR signals of the glycan due to dipolar interactions involving the unpaired electron of the metal. These pseudocontact shifts (PCS) depend on the distance between each proton and the metal (PCS are proportional to 1/r3). [5] This methodology has first been applied to the study of small oligosaccharides (di‐, tri‐and tetrasaccharides) [6–10], then to N‐glycans. The conformational properties of complex‐type biantennary and high‐mannose‐type N‐glycans were elucidated and could be resolved in each case down to the level of individual branches.[3,4] Proceeding from this experimental basis, we herein extend this concept to the level of high‐degree branching, and show that it is possible to experimentally characterize the conformational behavior and recognition properties of a galactosylated complex‐type tetraantennary N‐glycan, which is not possible by standard NMR methodologies due to chemical shift degeneracy of the glycan. We have then tested the hypothesis of branch selectivity for two N‐acetyllactosamine‐binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA 120). [11]Support or Funding InformationWe thank Agencia Estatal de Investigacion and MINECO of Spain (Grants CTQ2016‐76263‐P, CTQ2015‐64597‐C2‐1P, CTQ2015‐64597‐C2‐2P, CTQ2015‐64624‐R and the SEV‐2016‐0644 Severo Ochoa Excellence Acreditation) and the Deutsche Forschungsgemeinschaft for financial support.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.