title: 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 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 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 α2-6 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 α1-6Man-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.
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, United States.
authors.forename: Adam W
authors.initials: AW
authors.lastname: Barb
authors.forename: Lu
authors.initials: L
authors.lastname: Meng
authors.forename: Zhongwei
authors.initials: Z
authors.lastname: Gao
authors.forename: Roy W
authors.initials: RW
authors.lastname: Johnson
authors.forename: Kelley W
authors.initials: KW
authors.lastname: Moremen
authors.forename: James H
authors.initials: JH
authors.lastname: Prestegard
mesh_terms: D000818:Animals; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D012799:Sialyltransferases / Q000378:metabolism*
chemical_list: D007141:Immunoglobulin Fc Fragments; D011134:Polysaccharides; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid