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Published April 2016 | Accepted Version + Supplemental Material
Journal Article Open

Structural characterization of GASDALIE Fc bound to the activating Fc receptor FcγRIIIa

Abstract

The Fc region of Immunoglobulin G (IgG) initiates inflammatory responses such as antibody-dependent cell-mediated cytotoxicity (ADCC) through binding to activating Fc receptors (FcγRI, FcγRIIa, FcγRIIIa). These receptors are expressed on the surface of immune cells including macrophages, dendritic cells, and natural killer cells. An inhibitory receptor, FcγRIIb, is expressed on macrophages and other myeloid leukocytes simultaneously with the activating receptor FcγRIIa, thereby setting a threshold for cell activation. The affinity of IgG Fc for binding activating Fc receptors depends on IgG subclass and the composition of N-linked glycans attached to a conserved asparagine in the Fc C_H2 domain. For example, Fc regions with afucosylated glycans bind more tightly to FcγRIIIa than fucosylated Fc, and afucosylated Fcs exhibit enhanced ADCC activity in vivo and in vitro. Enhanced pro-inflammatory responses have also been seen for Fc regions with amino acid substitutions. GASDALIE Fc is an Fc mutant (G236A/S239D/A330L/I332E) that exhibits a higher affinity for FcγRIIIa and increased effector functions in vivo compared to wild-type Fc. To explore its altered functions, we compared the affinities of GASDALIE and wild-type Fc for activating and inhibitory FcγRs. We also determined the crystal structure of GASDALIE Fc alone and bound to FcγRIIIa. The overall structure of GASDALIE Fc alone was similar to wild-type Fc structures, however, increased electrostatic interactions in the GASDALIE Fc:FcγRIIIa interface compared with other Fc:FcγR structures suggest a mechanism for the increased affinity of GASDALIE Fc for FcγRIIIa.

Additional Information

© 2016 Elsevier Inc. Received 18 October 2015; Received in revised form 28 January 2016; Accepted 1 February 2016; Available online 2 February 2016. We thank Stylianos Bournazos, John Desjarlais, and Jeffrey Ravetch for helpful discussions, the Caltech Protein Expression Center for protein production, members of the Bjorkman lab for critical reading of the manuscript, Marta Murphy for help making figures, and Jens Kaiser and members of the staff at the Advanced Light Source (ALS) for help with data collection and processing. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. This research was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health Grant HIVRAD P01 AI100148 (P.J.B.); (the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health) and the Molecular Observatory at Caltech supported by the Gordon and Betty Moore Foundation.

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Accepted Version - nihms758390.pdf

Supplemental Material - mmc1.pdf

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Created:
August 22, 2023
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October 17, 2023