Backbone 1H, 13C, and 15N resonance assignments of the Fc fragment of human immunoglobulin G glycoprotein

The Fc portion of immunoglobulin G (IgG) recruits complements and its cognate receptors, thereby promoting defensive mechanisms in the humoral immune system. These effector functions critically depend on N-glycosylation at the Fc region, which is therefore regarded as a crucial factor in the design and production of therapeutic antibodies. NMR spectroscopy plays a unique role in the characterization of conformational dynamics and intermolecular interactions of IgG-Fc in solutions. Here, we report NMR assignments of the glycosylated Fc fragment (Mr 53 kDa), cleaved from a chimeric antibody with human IgG1 constant regions, which was produced in Chinese hamster ovary cells with uniform 13C- and 15N-labeling. Electronic supplementary material The online version of this article (doi:10.1007/s12104-014-9586-7) contains supplementary material, which is available to authorized users.


Biological context
Immunoglobulin G (IgG) is a multifunctional glycoprotein composed of an Fc region and two Fab regions, which are connected through the hinge region ). The Fab regions recognize and capture specific antigens, while the Fc region recruits complements and its cognate receptors, Fcc receptors (FccRs), and offers acceptor sites for bacterial proteins including protein A and protein G. The Fc region has a homodimeric structure comprising the C-terminal halves of the heavy chains, each composed of the C H 2 and C H 3 domains. The C H 2 domain possesses a conserved N-glycosylation site, Asn297, at which a biantennary complex-type oligosaccharide is expressed with microheterogenieties characterized by the presence and absence of the non-reducing terminal galactose, fucose, sialic acid, and bisecting N-acetylglucosamine residues.
The effector function of IgG critically depends on Nglycosylation in the Fc region. The outer carbohydrate moieties govern the structural integrity of the FccR-binding site of IgG, while the core fucosylation impairs antibody-dependent cellular cytotoxicity because of its negative steric effect against IgG interaction with FccRIII (Ferrara et al. 2011;Krapp et al. 2003;Mizushima et al. 2011;Yamaguchi et al. 2006). Hence, the Fc glycoforms are now considered as a crucial factor in the design and Hirokazu Yagi and Ying Zhang have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s12104-014-9586-7) contains supplementary material, which is available to authorized users. production of therapeutic antibodies in biopharmaceutical fields (Berkowitz et al. 2012;Jiang et al. 2011).
NMR spectroscopy offers unique tools for characterizing the conformational dynamics and intermolecular interactions of IgG-Fc in solution (Kato et al. 1991a(Kato et al. , 1993aKim et al. 1994a;Latypov et al. 2012). We developed protocols for uniform and amino acid-selective stable isotope labeling of an IgG glycoprotein and its functional fragments, using mammalian expression systems Yamaguchi and Kato 2010). Based on partially (approximately 66 %) achieved spectral assignments (Yamaguchi et al. 2006), we previously reported NMR analytical results to characterize the Nglycosylation-dependent conformational changes of human IgG1-Fc and its interaction with a specific RNA aptamer (Matsumiya et al. 2007;Miyakawa et al. 2008;Yamaguchi et al. 2006).
In an extension of these studies, we herein report NMR assignments of the glycosylated version of Fc fragment (Mr 53 kDa) cleaved from a chimeric antibody with human IgG1 constant regions that was expressed by Chinese hamster ovary (CHO) cells with uniform 13 C-and 15 Nlabeling.
Backbone resonance assignments were made on the basis of 2D 1 H-15 N HSQC spectral data of uniformly or selectively 13 C/ 15 N-labeled IgG1-Fc, and 3D spectral data obtained with the following experiments: HNCA, HNCO, HN(CA)CO, CBCA(CO)NH, and HNCACB. All NMR data were processed using NMRPipe software (Delaglio et al. 1995), and analyzed with SPARKY (Goddard and Kneller 1993) and CcpNmr (Vranken et al. 2005) software. Figure 1 shows the 1 H-15 N HSQC spectrum of human IgG1-Fc. Although the use of a mammalian expression system is mandatory for preparing antibodies with physiological glycosylation, uniform deuteration of the glycoprotein is not facile in such a system (Liu et al. 2007). Hence, we established spectral assignments based on the triple resonance spectral dataset recorded at a higher temperature, i.e. 52°C, complemented with HSQC spectral data obtained by amino acid-selective 13 C/ 15 N-labeling. Chemical shift assignments were made for protein backbone resonances: Ca (99 %), Cb (84 %), CO (80 %), HN (99 %), and N (99 %) (except for N of prolines). The spectral assignments at lower temperatures could be extrapolated by observing progressive spectral changes, depending on temperature, as exemplified by the spectrum at 42°C (Supplemental Fig.1). The present spectral assignments indicate that a cluster of amino acid residues in the vicinity of the N-glycans, i.e. Gln295-Thr299 exhibit significant chemical shift differences in comparison with the previously reported assignments of human Fc produced in Escherichia coli (Liu et al. 2007