Selective ¹³C‐Labels on Repeating Glycan Oligomers to Reveal Protein Binding Epitopes through NMR: Polylactosamine Binding to Galectins

Abstract

A combined chemo‐enzymatic synthesis/NMR‐based methodology is presented to identify, in unambiguous manner, the distinctive binding epitope within repeating sugar oligomers when binding to protein receptors. The concept is based on the incorporation of 13C‐labels at specific monosaccharide units, selected within a repeating glycan oligomeric structure. No new chemical tags are added, and thus the chemical entity remains the same, while the presence of the 13C‐labeled monosaccharide breaks the NMR chemical shift degeneracy that occurs in the non‐labeled compound and allows the unique identification of the different components of the oligomer. The approach is demonstrated by a proof‐of‐concept study dealing with the interaction of a polylactosamine hexasaccharide with five different galectins that display distinct preferences for these entities.

Pre-B cell receptor acts as a selectivity switch for Galectin-1 at the pre-B cell surface

“…Thus, λ5-UR interaction modifies Gal-1 binding to α2,3 sialylated glycan by allowing additional contacts with the pentasaccharide including with the internal galactose unit. While recent studies have elegantly rubber-stamped the selectivity of Gal-1 towards terminal galactose 21 , here we establish that λ5-UR binding to Gal-1 induces a selectivity switch converting Gal-1 from an exclusive exo-type lectin to an endo-type lectin interacting with both terminal and internal galactose units (Fig. 5i).…”

“…To the best of our knowledge, this result is the first example of such conversion dictating the glycan preference of a Galectin. Gal-1 bound to λ5-UR is able to interact with terminal and internal galactose like the tandem-repeat Gal-8 21 . Tandem-repeat Galectins (Galectins with two CRDs linked by a flexible linker) are known to be more potent in triggering cellular responses 46 , therefore λ5-UR induced conversion might increase Gal-1 efficiency at the pre-B cell surface through the formation of higher-order multimers as seen for tandem-repeat Galectins 46 .…”

“…Gal-1 is an exo-type lectin i.e. it interacts specifically with terminal units of polysaccharides 20,21 . During B cell differentiation in the bone marrow, pre-BCRs are expressed at the surface of pre-B cells, which are present in a specialized cellular niche consisting of stromal cells secreting Gal-1.…”

Exploring multivalent carbohydrate–protein interactions by NMR

“…For instance, the synthesis of 13 C labelled carbohydrates or carbohydrates with fluorine tags has enabled performing STD-HSQC and STD-TOCSY experiments. 61,62 Diffusion ordered spectroscopy Diffusion Ordered Spectroscopy (DOSY) experiments can also be used to monitor protein-carbohydrate interactions. 63 DOSY experiments are pseudo-2D experiments, which relate the diffusion coefficient, in the Y axis, to the regular 1 H NMR spectrum (chemical shifts) in the X axis.…”

Atom-economical synthesis of 1,2-bis(phosphine oxide)ethanes from calcium carbide with straightforward access to deuterium- and13C-labeled bidentate phosphorus ligands and metal complexes

“…[1][2][3] Labels, namely, deuterium and carbon-13, provide essential data for tuning the absorption, distribution, metabolism, and excretion of drugs. 4,5 The identification of products, [6][7][8][9][10][11][12] reaction mechanisms, [13][14][15][16][17][18][19][20][21] spectroscopy, 22 and many other applications [23][24][25][26][27][28][29][30][31][32][33] require the incorporation of a deuterium label at a specific position.…”