Glutamine-linked and non-consensus asparagine-linked oligosaccharides present in human recombinant antibodies define novel protein glycosylation motifs.
We report the presence of oligosaccharide structures on a glutamine residue present in the V(L) domain sequence of a recombinant human IgG2 molecule. Residue Gln-106, present in the QGT sequence following the rule of an asparagine-linked consensus motif, was modified with biantennary fucosylated oligosaccharide structures. In addition to the glycosylated glutamine, analysis of a lectin-enriched antibody population showed that 4 asparagine residues: heavy chain Asn-162, Asn-360, and light chain Asn-164, both of which are present in the IgG1 and IgG2 constant domain sequences, and Asn-35, which was present in CDR(L)1, were also modified with oligosaccharide structures at low levels. The primary sequences around these modified residues do not adhere to the N-linked consensus sequon, NX(S/T). Modeling of these residues from known antibody crystal structures and sequence homology comparison indicates that non-consensus glycosylation occurs on Asn residues in the context of a reverse consensus motif (S/T)XN located on highly flexile turns within 3 residues of a conformational change. Taken together our results indicate that protein glycosylation is governed by more diversified requirements than previously appreciated.
Valliere-Douglass
John F
JF
Department of Analytical and Formulation Sciences, Amgen Inc, Seattle, Washington 98119, USA.
Eakin
Catherine M
CM
Wallace
Alison
A
Ketchem
Randal R
RR
Wang
Wesley
W
Treuheit
Michael J
MJ
Balland
Alain
A
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001216:Asparagine / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D009844:Oligosaccharides / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D046188:Protein Modification, Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011994:Recombinant Proteins; D018698:Glutamic Acid; D001216:Asparagine