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.
Department of Analytical and Formulation Sciences, Amgen Inc, Seattle, Washington 98119, USA.
John F
JF
Valliere-Douglass
Catherine M
CM
Eakin
Alison
A
Wallace
Randal R
RR
Ketchem
Wesley
W
Wang
Michael J
MJ
Treuheit
Alain
A
Balland
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