title: Site-specific glycosylation of an aglycosylated human IgG1-Fc antibody protein generates neoglycoproteins with enhanced function.
abstract: A range of well-defined IgG glycoforms was prepared by employing a combination of synthetic carbohydrate chemistry and genetic engineering. The key aspect of this methodology is the coupling of thioaldoses with cysteine-containing proteins to give disulfide-linked neoglycoproteins. This technology was applied to the synthesis of a series of synthetic N-glycan thioaldoses which were coupled to an aglycosylated IgG1-Fc fragment, engineered to have Cys-297 in place of glycan-linked Asn (Deltah-Fc N297C). Analysis of the resulting Fc neoglycoproteins by mass spectrometry and trypsin digestion showed that the saccharides were site-selectively incorporated at Cys-297 to full occupancy without affecting other Fc protein disulfides. The neoglycoproteins were tested for their ability to interact with human FcgammaRI by inhibiting superoxide production by gamma-interferon-stimulated U937 cells. The neoglycoproteins displayed enhanced superoxide inhibition relative to aglycosylated Deltah-Fc N297C, where increased glycan size correlated positively with increased inhibition.
authors.affiliation: Complex Carbohydrate Research Center, The University of Georgia, 220 Riverbend Road, Athens, GA 30602, USA. gjboons@ccrc.uga.edu
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Watt
authors.forename: John
authors.initials: J
authors.lastname: Lund
authors.forename: Michaela
authors.initials: M
authors.lastname: Levens
authors.forename: V S Kumar
authors.initials: VS
authors.lastname: Kolli
authors.forename: Royston
authors.initials: R
authors.lastname: Jefferis
authors.forename: Geert Jan
authors.initials: GJ
authors.lastname: Boons
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D003545:Cysteine; D006023:Glycoproteins / Q000138:chemical synthesis* / Q000378:metabolism / Q000502:physiology; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D007371:Interferon-gamma / Q000494:pharmacology; D011134:Polysaccharides / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D013481:Superoxides
chemical_list: D006023:Glycoproteins; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011134:Polysaccharides; D013481:Superoxides; D007371:Interferon-gamma; D003545:Cysteine