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

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 Nglycans that contact polypeptide residues and apply the method to profile the five N-glycans attached to the human antibody receptor CD16A (Fc gamma receptor Human embryonic kidney 293S cells expressed CD16A with C-13(U)-labeled N-glycans using standard protein expression techniques and medium supplemented with 3 g/L [Cu-13]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 Nglycan. Correlations between anomeric (1)H1 and (13)C1 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 its all-atom computational simulations to identify unexpected contacts between the N45 N-glycan and CD16A polypeptide residues.