Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies

We report that N-linked oligosaccharide structures can be present on an asparagine residue not adhering to the consensus site motif NX(S/T), where X is not proline, described in the literature. We have observed oligosaccharides on a non-consensus asparaginyl residue in the C_H1 constant domain of IgG1 and IgG2 antibodies. The initial findings were obtained from characterization of charge variant populations evident in a recombinant human antibody of the IgG2 subclass. HPLC-MS results indicated that cation-exchange chromatography acidic variant populations were enriched in antibody with a second glycosylation site, in addition to the well documented canonical glycosylation site located in the C_H2 domain. Subsequent tryptic and chymotryptic peptide map data indicated that the second glycosylation site was associated with the amino acid sequence TVSWN¹⁶²SGAL in the C_H1 domain of the antibody. This highly atypical modification is present at levels of 0.5–2.0% on most of the recombinant antibodies that have been tested and has also been observed in IgG1 antibodies derived from human donors. Site-directed mutagenesis of the C_H1 domain sequence in a recombinant-human IgG1 antibody resulted in an increase in non-consensus glycosylation to 3.15%, a greater than 4-fold increase over the level observed in the wild type, by changing the ?1 and +1 amino acids relative to the asparagine residue at position 162. We believe that further understanding of the phenomenon of non-consensus glycosylation can be used to gain fundamental insights into the fidelity of the cellular glycosylation machinery.