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

• Published in: Biochimica et biophysica acta Vol. 1760; no. 4; pp. 693 - 700
• Main Authors: Yamaguchi, Yoshiki, Nishimura, Mamiko, Nagano, Mayumi, Yagi, Hirokazu, Sasakawa, Hiroaki, Uchida, Kazuhisa, Shitara, Kenya, Kato, Koichi
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2006
• Subjects: Binding Sites; Fcγ receptor; Glycoform; Glycoprotein; Glycoside Hydrolases - metabolism; Glycosylation; Humans; IgG; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin Fc Fragments - metabolism; Immunoglobulin G - chemistry; NMR; Nuclear Magnetic Resonance, Biomolecular; Oligosaccharides - analysis; Protein Binding; Protein Conformation; Receptors, IgG - metabolism; Staphylococcal Protein A - metabolism; C1q; Endo D; RU; Glycoform; Glycoprotein; PNGase F; IgG; Fcγ receptor; CHO; NMR; SPR; HSQC; ADCC; FcγR; Fc
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2005.10.002

The Fc portion of immunoglobulin G (IgG) expresses the biantennary complex type oligosaccharides at Asn297 of the C H2 domain of each heavy chain with microheterogeneities depending on 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 Fcγ receptor (FcγR)-mediated activities. To gain a better understanding of the role of Fc glycosylation, we prepared a series of truncated glycoforms of human IgG1-Fc and analyzed their interactions with human soluble FcγRIIIa (sFcγRIIIa) and with staphylococcal protein A by surface plasmon resonance and nuclear magnetic resonance (NMR) methods. Progressive but less pronounced reductions in the affinity for sFcγRIIIa were observed as a result of the galactosidase and subsequent N-acetylhexosaminidase treatments of IgG1-Fc. The following endoglycosidase D treatment, giving rise to a disaccharide structure composed of a fucosylated GlcNAc, abrogated the affinity of IgG1-Fc for sFcγRIIIa. On the other hand, those glycosidase treatments did not significantly affect the affinity of IgG1-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 C H2 domains. Furthermore, the cleavage at the GlcNAcβ1-4GlcNAc 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 FcγR-binding site, while the conformation of the C H2/C H3 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 FcγR-binding site.