Campus Units

Biochemistry, Biophysics and Molecular Biology, Roy J. Carver Department of

Document Type

Article

Publication Version

Accepted Manuscript

Publication Date

6-27-2017

Journal or Book Title

Biochemistry

Volume

56

Issue

25

First Page

3174

Last Page

3177

DOI

10.1021/acs.biochem.7b00392

Abstract

Asparagine-linked carbohydrates (N-glycans) are a common modification of eukaryotic proteins that confer multiple properties including the essential stabilization of therapeutic monoclonal antibodies. Here we present a rapid and efficient strategy to identify 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 [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 1H1-13C1 nuclei on the reducing end residue generate crosspeaks in a conventional 2d heteronuclear single quantum coherence 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 μs all-atom computational simulations to identify unexpected contacts between the N45 N-glycan and CD16A polypeptide residues.

Comments

This is a manuscript of an article published as Subedi, Ganesh P., Daniel J. Falconer, and Adam W. Barb. "Carbohydrate–Polypeptide Contacts in the Antibody Receptor CD16A Identified through Solution NMR Spectroscopy." Biochemistry 56, no. 25 (2017): 3174-3177. doi: 10.1021/acs.biochem.7b00392.

Copyright Owner

American Chemical Society

Language

en

File Format

application/pdf

Published Version

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