Site-specific glycosylation of an aglycosylated human IgG1-Fc antibody protein generates neoglycoproteins with enhanced function

Gregory M. Watt, John Lund, Michaela Levens, V. S.Kumar Kolli, Royston Jefferis, Geert Jan Boons*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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 (Δh-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 FcγRI by inhibiting superoxide production by γ -interferon-stimulated U937 cells. The neoglycoproteins displayed enhanced superoxide inhibition relative to aglycosylated Δh-Fc N297C, where increased glycan size correlated positively with increased inhibition.

Original languageEnglish
Pages (from-to)807-814
Number of pages8
JournalChemistry and Biology
Volume10
Issue number9
DOIs
Publication statusPublished - 1 Sept 2003
Externally publishedYes

Funding

The authors are grateful for financial support for this work from the University of Georgia (G.M.W., G.-J.B., and V.S.K.K.), the Leverhulme Trust UK (grant number F/00094/R to J.L.), and the BBSRC UK (grant number 6/B15663 to R.J.).

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