Dataset Information

Flow Chemistry System for Carbohydrate Analysis by Rapid Labeling of Saccharides after Glycan Hydrolysis.

ABSTRACT: This study demonstrates the utilization of a flow chemistry system for continuous glycan hydrolysis and saccharide labeling to assist with the existing methods in glycan structural analysis. Acidic hydrolysis of glycans could be accelerated in a flow system. Aldoses and α-ketoacid-type saccharides were effectively labeled with naphthalene-2,3-diamine (NADA) at 60 °C for 10 min to form the fluorescent naphthimidazole (NAIM) and quinoxalinone (QXO) derivatives, respectively. The NADA-labeled derivatives improved the structural determination and composition analysis for their parent saccharides by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), liquid chromatography mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR). Furthermore, this protocol was applied to determine the SA-Gal-Glc sequence of GM3-sugar out of six possible permutations.

SUBMITTER: Hung WT

PROVIDER: S-EPMC7372588 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Flow Chemistry System for Carbohydrate Analysis by Rapid Labeling of Saccharides after Glycan Hydrolysis.

Hung Wei-Ting WT Chen Yi-Ting YT Chen Chung-Hsuan CH Lee Yuan Chuan YC Fang Jim-Min JM Yang Wen-Bin WB

SLAS technology 20200619 4

This study demonstrates the utilization of a flow chemistry system for continuous glycan hydrolysis and saccharide labeling to assist with the existing methods in glycan structural analysis. Acidic hydrolysis of glycans could be accelerated in a flow system. Aldoses and α-ketoacid-type saccharides were effectively labeled with naphthalene-2,3-diamine (NADA) at 60 °C for 10 min to form the fluorescent naphthimidazole (NAIM) and quinoxalinone (QXO) derivatives, respectively. The NADA-labeled deriv ...[more]

PMID: 32560600

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Project description:gal-1 (galectin-1) mediates cell-cell and cell-extracellular matrix adhesion, essentially by interacting with beta-galactoside-containing glycans of cell-surface glycoconjugates. Although most structural studies with gal-1 have investigated its binding to simple carbohydrates, in particular lactose and N-acetyl-lactosamine, this view is limited, because gal-1 functions at the cell surface by interacting with more complex glycans that are heterogeneous in size and composition. In the present study we used NMR spectroscopy to investigate the interaction of human gal-1 with a large (120 kDa) complex glycan, GRG (galactorhamnogalacturonate glycan), that contains non-randomly distributed mostly terminal beta(1-->4)-linked galactose side chains. We used 15N-1H-HSQC (heteronuclear single quantum coherence) NMR experiments with 15N-enriched gal-1 to identify the GRG-binding region on gal-1 and found that this region covers a large surface area on gal-1 that includes the quintessential lactose-binding site and runs from that site through a broad valley or cleft towards the dimer interface. HSQC and pulsed-field-gradient NMR diffusion experiments also show that gal-1 binds GRG with a gal-1:GRG stoichiometry of about 5:1 (or 6:1) and with average macroscopic and microscopic equilibrium dissociation constants (Kd) of 8 x 10(-6) M and 40 x 10(-6) M (or 48 x 10(-6) M) respectively, indicating stronger binding than to lactose (Kd=520 x 10(-6) M). Although gal-1 may bind GRG in various ways, the glycan can be competed for by lactose, suggesting that there is one major mode of interaction. Furthermore, even though terminal motifs on GRG are Gal-beta(1-->4)-Gal rather than the traditional Gal-beta(1-->4)-Glc/GlcNAc (where GlcNAc is N-acetylglucosamine), we show that the disaccharide Gal-beta(1-->4)-Gal can bind gal-1 at the lactose-binding domain. In addition, gal-1 binding to GRG disrupts inter-glycan interactions and decreases glycan-mediated solution viscosity, a glycan decongestion effect that may help explain why gal-1 promotes membrane fluidity and lateral diffusion of glycoconjugates within cell membranes. Overall, our results provide an insight into the function of galectin in situ and have potential significant biological consequences.

Dataset Information

Flow Chemistry System for Carbohydrate Analysis by Rapid Labeling of Saccharides after Glycan Hydrolysis.

Publications

Flow Chemistry System for Carbohydrate Analysis by Rapid Labeling of Saccharides after Glycan Hydrolysis.

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