Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy

The Fe portion of immunoglobulin G (IgG) expresses the biantennary complex type oligosaccharides at Asn297 of the C(H)2 domain of each heavy chain with microheterogeneities depending oil physiological and pathological states. These N-glycans are known to be essential for promotion of proper effector functions of IgG such as complement activation and Fc gamma receptor (Fc gamma R)-mediated activities. To gain a better understanding of the role of Fc glycosylation, we prepared a series of truncated glycoforms of human IgGl-Fe and analyzed their interactions with human soluble Fc gamma RIIIa (sFc gamma RIIIa) and with staphylococcal protein A by surface plasmon resonance and nuclear magnetic resonance (NMR) methods. Progressive but less pronounced reductions in the affinity for sFc gamma RIIIa were observed as a result of the galactosidase and subsequent N-acetylhexosaminidase treatments of IgGI-Fc. The following endoglycosidase D treatment, giving rise to a disaccharide structure composed of a fucosylated GlcNAc, abrogated the affinity of IgGI-Fc for sFc gamma RIIIa. Oil the other hand, those glycosidase treatments did not significantly affect the affinity of IgGI-Fc for protein A. Inspection of stable-isotope-assisted NMR data of a series of Fc glycoforms indicates that the stepwise trimming Out of the carbohydrate residues results in concomitant increase in the number of amino acid residues perturbed thereby in the CH2 domains. Furthermore, the cleavage at the GlcNAc beta 1-4GlcNAc glycosidic linkage induced the conformational alterations of part of the lower hinge region, which makes no direct contact with the carbohydrate moieties and forms the major Fc gamma R-binding site, while the conformation of the C(H)2/C(H)3 interface was barely perturbed that is the protein A-binding site. These results indicate that the carbohydrate moieties are required for maintaining the structural integrity of the Fc gamma R-binding site. (c) 2005 Elsevier B.V. All rights reserved.