Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Aug 16;9(1):11957.
doi: 10.1038/s41598-019-48323-w.

The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III

Rina Yogo  1   2 , Yuki Yamaguchi  3 , Hiroki Watanabe  1 , Hirokazu Yagi  2 , Tadashi Satoh  2 , Mahito Nakanishi  4 , Masayoshi Onitsuka  5 , Takeshi Omasa  3 , Mari Shimada  3 , Takahiro Maruno  3 , Tetsuo Torisu  3 , Shio Watanabe  6 , Daisuke Higo  6 , Takayuki Uchihashi  1   7 , Saeko Yanaka  1   2 , Susumu Uchiyama  8   9 , Koichi Kato  10   11
Affiliations

The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III

Rina Yogo et al. Sci Rep. .

Abstract

Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, FcγRIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with FcγRIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
HS-AFM observations of interactions between IgG1 antibodies and their Fc fragments with sFcγRIIIa. (a) We indicate full-length rituximab by a red arrow (Supplementary Videos 1–3). (b) We indicate rituximab Fc by a magenta arrow (Supplementary Videos 4–6). In (a,b), we show an immobilized sFcγRIIIa molecule having two extracellular domains by a white arrow.
Figure 2
Figure 2
Dwell times of three kinds of IgG1 and their Fc fragments on sFcγRIIIa.
Figure 3
Figure 3
HDX-MS analysis of the light chain of PMF37. (a) Differential plots of deuterium uptake degrees of peptides, showing time courses; 30 s (purple), 180 s (blue), 600 s (green), 3,600 s (yellow), and 14,400 s (red), along with their summational results (gray bar). We considered that amide proton environments were significantly different between free and bound states, with 99% confidence by independent t-test, when the summational result of a corresponding peptide showed difference more than 17% (gray dashed lines) and the result at a specific exposure time of the peptide showed difference more than 4% (brown dashed lines). (b) Deuterium uptake curves for the representative peptides showing significant differences between free (red) and bound (blue) states.
Figure 4
Figure 4
HDX-MS analysis of the heavy chain of PMF37. (a) Differential plots of deuterium uptake degrees of peptides, showing time courses; 30 s (purple), 180 s (blue), 600 s (green), 3,600 s (yellow), and 14,400 s (red), along with their summational results (gray bar). The criterion of significance of deuterium uptake difference between free and bound states is the same as that in Fig. 3. (b) Deuterium uptake curves for the representative peptides showing significant differences between free (red) and bound (blue) states.
Figure 5
Figure 5
HDX-MS analysis of sFcγRIIIa. (a) Differential plots of deuterium uptake degrees of peptides, showing time courses; 30 s (purple), 60 s (blue), 600 s (green), 3,600 s (yellow), and 14,400 s (red), along with their summational results (gray bar). The criterion of significance of deuterium uptake difference between free and bound states is the same as that in Fig. 3. (b) Deuterium uptake curves for the representative peptides showing significant differences between free (red) and bound (blue) states.
Figure 6
Figure 6
Mapping of HDX-MS data on 3D-structural models of IgG1 and sFcγRIIIa. We show the peptide segments exhibiting decreases in deuterium uptake rate upon interaction in red, magenta, or pink, and the peptide showing increased deuterium uptake in cyan, on a crystal structure of sFcγRIIIa extracted from Fc-sFcγRIIIa complex (PDB code:5XJE), and a homology model of PMF37 (shown as a half molecule composed of one light chain and one heavy chain). In the protected segments of PMF37, we classify residues into three types: the residues constituting the canonical sFcγRIIIa-binding sites in Fc (magenta), the remaining conserved residues among PMF37, trastuzumab, and rituximab (red), and the unconserved residues (pink). We classify the protected segments of sFcγRIIIa into two types; residues constituting the canonical Fc-binding sites (magenta) and the remaining conserved residues between FcγRIIIa and FcγRIIIb (red). The canonical binding sites are based on the crystal structure of Fc-sFcγRIIIa complex. We built the homology model based on crystal structures of the human anti-human immunodeficiency virus-1 gp120 IgG1 (PDB code: IHZH) using SWISS-MODEL Workspace. We prepared the molecular graphics using PyMOL (http://www.pymol.org/).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dorrington KJ, Klein MH. Binding sites for Fcγ receptors on immunoglobulin G and factors influencing their expression. Mol Immunol. 1982;19:1215–1221. doi: 10.1016/0161-5890(82)90286-3. - DOI - PubMed
    1. Burton DR. Immunoglobulin G: functional sites. Mol Immunol. 1985;22:161–206. doi: 10.1016/0161-5890(85)90151-8. - DOI - PubMed
    1. Jefferis R, Lund J, Pound JD. IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation. Immunol Rev. 1998;163:59–76. doi: 10.1111/j.1600-065X.1998.tb01188.x. - DOI - PubMed
    1. Yang D, Kroe-Barrett R, Singh S, Roberts CJ, Laue TM. IgG cooperativity - Is there allostery? Implications for antibody functions and therapeutic antibody development. MAbs. 2017;9:1231–1252. doi: 10.1080/19420862.2017.1367074. - DOI - PMC - PubMed
    1. Jay Jacob, Bray Brinkley, Qi Yaozhi, Igbinigie Eseosaserea, Wu Hao, Li Jinping, Ren Gang. IgG Antibody 3D Structures and Dynamics. Antibodies. 2018;7(2):18. doi: 10.3390/antib7020018. - DOI - PMC - PubMed

Publication types