Effects of Anchor Structure and Glycosylation of FcÎ 3 Receptor III on Ligand Binding Affinity Ning

Isoforms of the Fcγ receptor III (FcγRIII or CD16) are cell surface receptors for the Fc portion of IgG and important regulators of humoral immune responses. Different ligand binding kinetics of FcγRIII isoforms are obtained in three dimensions by surface plasmon resonance and in two dimensions by a micropipette adhesion frequency assay. We show that the anchor structure of CD16 isoforms isolated from the cell membrane affects their binding affinities in a ligand-specific manner. Changing the receptor anchor structure from full to partial to none decreases the ligand binding affinity for human IgG1 (hIgG1) but increases it for murine IgG2a (mIgG2a). Removing N-glycosylation from the CD16 protein core by tunicamycin also increases the ligand binding affinity. Molecular dynamics simulations indicate that deglycosylation at Asn-163 of CD16 removes the steric hindrance for the CD16-hIgG1 Fc binding and thus increases the binding affinity. These results highlight an unexpected sensitivity of ligand binding to the receptor anchor structure and glycosylation and suggest their respective roles in controlling allosterically the conformation of the ligand binding pocket of CD16. INTRODUCTION In humans, type III cell surface receptors for the Fc portion of immunoglobulin (Ig) G (Fcγ receptor III or CD16) are encoded by two genes, A and B, which give two protein products, CD16a and CD16b, respectively (Kimberly et al., 2002; Nimmerjahn and Ravetch, 2008). CD16a is expressed on macrophages, mast cells, and natural killer cells as a transmembrane receptor (Ravetch and Perussia, 1989; Selvaraj et al., 1989). Expressed exclusively on neutrophils, CD16b is the only Fc receptor (FcR) anchored by a glycosyl-phosphatidylinositol (GPI) linker to the plasma membrane (Selvaraj et al., 1988). CD16b is polymorphic, with two alleles—neutrophil alloantigen 1 (NA1) and 2 (NA2). Despite the lack of a signaling component, CD16b plays an active role in triggering Ca2+ mobilization and neutrophil degranulation (Kimberly et al., 1990; Unkeless et al., 1995). Furthermore, in conjunction with FcγRIIa (CD32a), CD16b activates phagocytosis, degranulation, and oxidative burst, which lead to clearance of opsonized pathogens by neutrophils. Moreover, soluble forms of CD16 containing the Ig-like extracellular domains of the receptor circulate in plasma (Teillaud et al., 1994). CD16 are highly glycosylated molecules, with five (CD16a), four (CD16bNA1), or six (CD16bNA2) N-glycosylation sites on their extracellular domains (Figure 1). CD16a on NK cells has a higher affinity for ligands than on monocytes due to differential cell type–specific glycosylation (high mannose– and complex-type oligosaccharides for NK cell CD16a but not monocyte CD16a), despite their identical protein cores (Edberg and Kimberly, 1997). Single-residue replacements of the four N-glycosylation sites (Asn-39, Asn-75, Asn163, and Asn-170) of CD16bNA1 show that the N163Q mutant binds monomeric IgG with higher affinity than the other mutants (Drescher et al., 2003). In addition, preincubation of wild-type Monitoring Editor Jennifer Lippincott-Schwartz Howard Hughes Medical Institute Received: Jun 29, 2016 Revised: Aug 25, 2016 Accepted: Aug 26, 2016 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E16-06-0470) on August 31, 2016. †These authors contributed equally. Present addresses: ‡Department of Biomedical Engineering, University of Texas at Austin, Austin, TX 78712; §Schrodinger, Inc., 120 West 45th Street, New York, NY 10036; ∥Department of Pharmaceutical Sciences, School of Pharmacy, Philadelphia College of Osteopathic Medicine, Suwannee, GA 30024. *Address correspondence to: Cheng Zhu (cheng.zhu@bme.gatech.edu). © 2016 Jiang, Chen, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by -nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. Abbreviations used: BSA, bovine serum albumin; 2D, two dimensional; 3D, three dimensional; FcγR, Fcγ receptor; GPI, glycosyl-phosphatidylinositol; hFc1, Fc fragment of hIgG1; hIgG1, human IgG1; Ig, immunoglobulin; mIgG2a, mouse IgG2a; NA1, neutrophil alloantigen 1; NA2, neutrophil alloantigen 2; PIPLC, phosphatidylinositol-specific phospholipase C; pMHC, peptide-major histocompatibility complex; RBC, red blood cell; SPR, surface plasma resonance; TCR, T-cell receptor; TM, transmembrane. Ning Jianga,†,‡, Wei Chenb,†,§, Prithiviraj Jothikumara, Jaina M. Patelc, Rangaiah Shashidharamurthyc,∥, Periasamy Selvarajc, and Cheng Zhua,b,* aCoulter Department of Biomedical Engineering and bWoodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332; cDepartment of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322 http://www.molbiolcell.org/content/suppl/2016/08/29/mbc.E16-06-0470v1.DC1. html Supplemental Material can be found at: