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Carbohydrate-Polypeptide Contacts in the Antibody Receptor CD16A Identified through Solution NMR Spectroscopy.

ABSTRACT: Asparagine-linked carbohydrates (N-glycans) are common modifications of eukaryotic proteins that confer multiple properties, including the essential stabilization of therapeutic monoclonal antibodies. Here we present a rapid and efficient strategy for identifying N-glycans that contact polypeptide residues and apply the method to profile the five N-glycans attached to the human antibody receptor CD16A (Fc ? receptor IIIA). Human embryonic kidney 293S cells expressed CD16A with 13CU-labeled N-glycans using standard protein expression techniques and medium supplemented with 3 g/L [13CU]glucose. Anomeric resonances on the protein-linked N-acetylglucosamine residue at the reducing end of the glycan are particularly well suited to studies of multiply glycosylated N-glycoproteins because only one reducing end and nitrogen-linked residue is present in each N-glycan. Correlations between anomeric 1H1 and 13C1 nuclei on the reducing end residue generate crosspeaks in a conventional two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance (NMR) experiment that appear in a region of the spectrum devoid of other carbohydrate peaks or background protein signals. Two N-glycan peaks corresponding to the N45 and N162 N-glycans were dispersed from the rapidly averaged peaks corresponding to the N38, N74, and N169 N-glycans. We used a combination of NMR and 1 ?s all-atom computational simulations to identify unexpected contacts between the N45 N-glycan and CD16A polypeptide residues.

SUBMITTER: Subedi GP

PROVIDER: S-EPMC5925738 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Carbohydrate-Polypeptide Contacts in the Antibody Receptor CD16A Identified through Solution NMR Spectroscopy.

Subedi Ganesh P GP Falconer Daniel J DJ Barb Adam W AW

Biochemistry 20170616 25

Asparagine-linked carbohydrates (N-glycans) are common modifications of eukaryotic proteins that confer multiple properties, including the essential stabilization of therapeutic monoclonal antibodies. Here we present a rapid and efficient strategy for identifying N-glycans that contact polypeptide residues and apply the method to profile the five N-glycans attached to the human antibody receptor CD16A (Fc γ receptor IIIA). Human embryonic kidney 293S cells expressed CD16A with <sup>13</sup>C<sub ...[more]

PMID: 28613884

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Project description:BACKGROUND:Caldicellulosiruptor saccharolyticus is a thermophilic, Gram-positive, non-spore forming, strictly anaerobic bacterium of interest in potential industrial applications, including the production of biofuels such as hydrogen or ethanol from lignocellulosic biomass through fermentation. High-resolution, solution-state nuclear magnetic resonance (NMR) spectroscopy is a useful method for the identification and quantification of metabolites that result from growth on different substrates. NMR allows facile resolution of isomeric (identical mass) constituents and does not destroy the sample. RESULTS:Profiles of metabolites produced by the thermophilic cellulose-degrading bacterium Caldicellulosiruptor saccharolyticus DSM 8903 strain following growth on different monosaccharides (D-glucose, D-mannose, L-arabinose, D-arabinose, D-xylose, L-fucose, and D-fucose) as carbon sources revealed several unexpected fermentation products, suggesting novel metabolic capacities and unexplored metabolic pathways in this organism. Both 1H and 13C nuclear magnetic resonance (NMR) spectroscopy were used to determine intracellular and extracellular metabolite profiles. One dimensional 1H NMR spectral analysis was performed by curve fitting against spectral libraries provided in the Chenomx software; 2-D homonuclear and heteronuclear NMR experiments were conducted to further reduce uncertainties due to unassigned, overlapping, or poorly-resolved peaks. In addition to expected metabolites such as acetate, lactate, glycerol, and ethanol, several novel fermentation products were identified: ethylene glycol (from growth on D-arabinose), acetoin and 2,3-butanediol (from growth on D-glucose, L-arabinose, and D-xylose), and hydroxyacetone (from growth on D-mannose, L-arabinose, and D-xylose). Production of ethylene glycol from D-arabinose was particularly notable, with around 10% of the substrate carbon converted into this uncommon fermentation product. CONCLUSIONS:The present research shows that C. saccharolyticus, already of substantial interest due to its capability for biological ethanol and hydrogen production, has further metabolic potential for production of higher molecular weight compounds, such as acetoin and 2,3-butanediol, as well as hydroxyacetone and the uncommon fermentation product ethylene glycol. In addition, application of nuclear magnetic resonance (NMR) spectroscopy facilitates identification of novel metabolites, which is instrumental for production of desirable bioproducts from biomass through microbial fermentation.

Dataset Information

Carbohydrate-Polypeptide Contacts in the Antibody Receptor CD16A Identified through Solution NMR Spectroscopy.

Publications

Carbohydrate-Polypeptide Contacts in the Antibody Receptor CD16A Identified through Solution NMR Spectroscopy.

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