Breaking the Limits in Analyzing Carbohydrate Recognition by NMR Spectroscopy: Resolving Branch‐Selective Interaction of a Tetra‐Antennary N‐Glycan with Lectins

Abstract

The biological recognition of complex-type Nglycans is part of many key physiological and pathological events.Despite their importance,the structural characterization of these events remains unsolved. The inherent flexibility of Nglycans hampers crystallization and the chemical equivalence of individual branches precludes their NMR characterization. By using achemoenzymatically synthesized tetra-antennary Nglycan conjugated to al anthanide binding tag,t he NMR signals under paramagnetic conditions discriminated all four N-acetyl lactosamine antennae with unprecedented resolution. The NMR data revealed the conformation of the N-glycan and permitted for the first time the direct identification of individual branches involved in the recognition by two N-acetyllactosamine-binding lectins,D atura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA120).N-glycans are ubiquitous in nature and functionalize glycoproteins.[1] Protein glycosylation is required for proper biological and biophysical function and often, alterations in glycosylation are related to diseases. [2] Complex glycosylation patterns containing multi-antennary N-glycans are typically found in mature glycoproteins. However,t he structural characterization of these glycans is rather challenging.U sually,N MR spectroscopy and X-ray diffraction techniques fail to provide specific answers on the structure and molecular recognition features owing to the intrinsic attributes of the glycan. Thep roperties of the glycosidic bond and especially the presence of 1-6 linkages endow al arge flexibility to the molecule.T his feature precludes crystallization or hampers the detection of enough electron density for most of the glycan part in the X-ray analysis of glycoproteins.Moreover,the standard use of the corresponding fitting programs to deduce three-dimensional structures frequently give rise to incorrect structures of the glycans.[3] Thus,a ny advance in this area is of high value. As ap romising approach, carbohydrates conjugated to lanthanide-binding tags have shown high potential toward this aim. [4,5] In its vicinity,acomplexed paramagnetic ion induces significant chemical shift changes of the NMR signals of the glycan as ar esult of dipolar interactions involving the unpaired electron of the metal. These pseudocontact shifts (PCS) depend on the distance between each proton and the metal (proportional to 1/r 3 ).[6] This methodology has first been applied to the study of small oligosaccharides (di-, tri-, and tetrasaccharides), [7][8][9][10][11] then to N-glycans.T he conformational properties of complex-type bi-antennary and high-mannosetype N-glycans were elucidated and could be resolved in each case down to the level of individual branches. [4,5] Proceeding from this experimental basis,weherein 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 ag alactosylated complex-type

Strategies for the highly efficient synthesis of erythropoietin N‐glycopeptide hydrazides

“…After coupling, deprotection and purification the isolated yield was further improved (71%), indicating that impurities contained in the selectively deprotected peptide 5 that are removable by HPLC are the cause of low yields in pseudoproline‐assisted Lansbury aspartylations. Because the N‐glycans of EPO are mainly of the tetraantennary complex type, a synthetic tetraantennary glycosylamine 10 36 (1 eq.) was coupled to purified 5 (1.3 eq.).…”

Recent Advances in the Chemical Biology of N-Glycans

“…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]. "Off resonance" experiment gives a reference spectrum.…”

“…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].…”

“…They elucidated the conformational change caused by mannose trimming during the N-glycan biosynthetic process. Unverzagt and Barbero et al distinguished each branch of tetraantennary N-glycan based on the PCS method and analyzed the differences in the recognition of each branch by lectins [44]. Datura stramonium seed lectin (DSL), which recognizes the lactosamine structure, interacts more strongly with the lactosamine on the α1,6-branched chain than with that on the α1,3-branched chain.…”

Theoretical DFT interpretation of infrared spectra of biologically active arabinogalactan sulphated derivatives

“…Recently, interest in the study of polysaccharides by theoretical methods was increasing (Profant V et al 2019, Akman F et al 2020. These methods are based on calculations of spectroscopic characteristics of electron circular dichroism (ECD) (Rudd TR et al 2009, Matsuo K. et al 2009), infrared absorption with Fourier transform (FTIR) (Mainreck N. et al 2009, Akman F et al 2020, Garnjanagoonchorn W. et al 2007), Raman spectroscopy (Profant V et al 2019, Rüther, A et al 2017, Yaffe NR et al 2010, Ellis R et al 2009, spectroscopy nuclear magnetic resonance (Canales A et al 2017, Klepach T et al 2015, Gerbst AG et al 2017, Shklyaev OE et al 2014, Akman F et al 2016. The methods allow one to obtain the key properties of a number of polymers (Profant V et al 2019).…”