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NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation.

ABSTRACT: The terminal carbohydrate residues of the N-glycan on the immunoglobulin G (IgG) fragment crystallizable (Fc) determine whether IgG activates pro- or anti-inflammatory receptors. The IgG Fc alone becomes potently anti-inflammatory upon addition of ?2-6-linked N-acetylneuraminic acid residues to the N-glycan, stimulating interest in use of this entity in novel therapies for autoimmune disease [Kaneko et al. (2006) Science313, 670-3]. Complete Fc sialylation has, however, been deemed challenging due to a combination of branch specificity and perceived protection by glycan-protein interactions. Here we report the preparation of high levels of disialylated Fc by using sufficient amounts of a highly active ?2-6 sialyltransferase (ST6Gal1) preparation expressed in a transiently transformed human cell culture. Surprisingly, ST6Gal1 sialylated the two termini of the complex-type binantennary glycan in a manner remarkably similar to that observed for the free N-glycan, suggesting the Fc polypeptide does not greatly influence ST6Gal1 specificity. In addition, sialylation of either branch terminus does not appear to dramatically alter the motional behavior of the N-glycan as judged by solution NMR spectroscopy. Together these, data suggest the N-glycan occupies two distinct states: one with both glycan termini sequestered from enzymatic modification by an ?1-6Man-branch interaction with the polypeptide surface and the other with both glycan termini exposed to the bulk solvent and free from glycan-polypeptide interactions. The results suggest new modes by which disialylated Fc can act as an anti-inflammatory effector.

SUBMITTER: Barb AW

PROVIDER: S-EPMC3447994 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation.

Barb Adam W AW Meng Lu L Gao Zhongwei Z Johnson Roy W RW Moremen Kelley W KW Prestegard James H JH

Biochemistry 20120522 22

The terminal carbohydrate residues of the N-glycan on the immunoglobulin G (IgG) fragment crystallizable (Fc) determine whether IgG activates pro- or anti-inflammatory receptors. The IgG Fc alone becomes potently anti-inflammatory upon addition of α2-6-linked N-acetylneuraminic acid residues to the N-glycan, stimulating interest in use of this entity in novel therapies for autoimmune disease [Kaneko et al. (2006) Science313, 670-3]. Complete Fc sialylation has, however, been deemed challenging d ...[more]

PMID: 22574931

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Project description:Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (Fc?Rs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for Fc?R and complement activity. Bisection had no effect on Fc?R or C1q-binding, and sialylation had no- or little effect on Fc?R binding. We confirmed that hypo-fucosylation of IgG1 increased binding to Fc?RIIIa and Fc?RIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for Fc?RIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for Fc?R- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for Fc?RIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.

Dataset Information

NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation.

Publications

NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation.

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