| 1. |
Yogo R, Yamaguchi Y, Watanabe H, Yagi H, Satoh T, Nakanishi M, Onitsuka M, Omasa T, Shimada M, Maruno T, Torisu T, Watanabe S, Higo D, Uchihashi T, Yanaka S, Uchiyama S, Kato K, The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III, Scientific Reports, 10.1038/s41598-019-48323-w, 9, 1, 11957, 2019.12. |
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Saeko Yanaka, Rina Yogo, Koichi Kato, Biophysical characterization of dynamic structures of immunoglobulin G., Biophysical reviews, 10.1007/s12551-020-00698-1, 2020.05, Immunoglobulin G (IgG) is a major antibody and functions as a hub linking specific antigen binding and recruitment of effector molecules typified by Fcγ receptors (FcγRs). These activities are associated primarily with interactions involving its Fab and Fc sites, respectively. An IgG molecule is characterized by a multiple domain modular structure with conserved N-glycosylation in Fc. The molecule displays significant freedom in internal motion on various spatiotemporal scales. The consequent conformational flexibility and plasticity of IgG glycoproteins are functionally significant and potentially important factors for design and engineering of antibodies with enhanced functionality. In this article, experimental and computational approaches are outlined for characterizing the conformational dynamics of IgG molecules in solution. In particular, the importance of integration of these approaches is highlighted, as illustrated by dynamic intramolecular interactions between the pair of N-glycans and their proximal amino acid residues in Fc. These interactions can critically affect effector functions mediated by human IgG1 and FcγRIII. Further improvements in individual biophysical techniques and their integration will advance understanding of dynamic behaviors of antibodies in physiological and pathological conditions. Such understanding will provide opportunities for engineering antibodies through controlling allosteric networks in IgG molecules.. |
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Saeko Yanaka, Yoshiki Yamaguchi, Takeshi Takizawa, Yohei Miyanoiri, Rina Yogo, Ichio Shimada, Koichi Kato, NMR assignments of the N-glycans of the Fc fragment of mouse immunoglobulin G2b glycoprotein., Biomolecular NMR assignments, 10.1007/s12104-020-10004-5, 15, 1, 187-192, 2021.01, The Fc portion of immunoglobulin G (IgG) promotes defensive effector functions in the immune system by interacting with Fcγ receptors and complement component C1q. These interactions critically depend on N-glycosylation at Asn297 of each CH2 domain, where biantennary complex-type oligosaccharides contain microheterogeneities resulting primarily from the presence or absence of non-reducing terminal galactose residues. Crystal structures of Fc have shown that a pair of N-glycans is located between the two CH2 domains. Here we applied our metabolic isotope labeling technique using mammalian cells for in-solution structural characterization of mouse IgG2b-Fc glycoforms with a molecular mass of 54 kDa. Based on spectral assignments of the N-glycans as well as polypeptide backbones of Fc, we probed conformational perturbations of Fc induced by N-glycan trimming, especially enzymatic degalactosylation. The results indicated that degalactosylation structurally perturbed the Fc region through rearrangement of glycan-protein interactions. The spectral assignments of IgG2b-Fc glycoprotein will provide the basis for NMR investigation of its dynamic conformations and interactions with effector molecules in solution.. |
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Saeko Yanaka, Hirokazu Yagi, Rina Yogo, Masayoshi Onitsuka, Koichi Kato, Glutamine-free mammalian expression of recombinant glycoproteins with uniform isotope labeling: an application for NMR analysis of pharmaceutically relevant Fc glycoforms of human immunoglobulin G1, Journal of Biomolecular NMR, 10.1007/s10858-021-00387-5, 76, 1-2, 17-22, 2022.01, Mammalian cells are widely used for producing recombinant glycoproteins of pharmaceutical interest. However, a major drawback of using mammalian cells is the high production costs associated with uniformly isotope-labeled glycoproteins due to the large quantity of labeled L-glutamine required for their growth. To address this problem, we developed a cost-saving method for uniform isotope labeling by cultivating the mammalian cells under glutamine-free conditions, which was achieved by co-expression of glutamine synthase. We demonstrate the utility of this approach using fucosylated and non-fucosylated Fc glycoforms of human immunoglobulin G1.. |
