NMR Characterization of Immunoglobulin G Fc Glycan Motion on Enzymatic Sialylation

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 antiinflammatory upon addition of alpha 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) Science 313, 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 alpha 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 alpha 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.