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• W2010670539 abstract "•F241 and F243 of IgG1 Fc both restrict N-glycan motion•Disrupting μs motion of the Fc N-glycan reduces affinity for FcγRIIIa•IgG1 Fc K246F stabilizes the N-glycan and enhances FcγRIIIa affinity•IgD, IgE, IgG, and IgM share similar N-glycan restricting features; IgA does not Immunoglobulin G1 (IgG1)-based therapies are widespread, and many function through interactions with low-affinity Fc γ receptors (FcγR). N-glycosylation of the IgG1 Fc domain is required for FcγR binding, though it is unclear why. Structures of the FcγR:Fc complex fail to explain this because the FcγR polypeptide does not bind the N-glycan. Here we identify a link between motion of the N-glycan and Fc:FcγRIIIa affinity that explains the N-glycan requirement. Fc F241 and F243 mutations decreased the N-glycan/polypeptide interaction and increased N-glycan mobility. The affinity of the Fc mutants for FcγRIIIa was directly proportional to the degree of glycan restriction (R2 = 0.82). The IgG1 Fc K246F mutation stabilized the N-glycan and enhanced affinity for FcγRIIIa. Allosteric modulation of a protein/protein interaction represents a previously undescribed role for N-glycans in biology. Conserved features suggesting a similar N-glycan/aromatic interaction were also found in IgD, IgE, and IgM, but not IgA. Immunoglobulin G1 (IgG1)-based therapies are widespread, and many function through interactions with low-affinity Fc γ receptors (FcγR). N-glycosylation of the IgG1 Fc domain is required for FcγR binding, though it is unclear why. Structures of the FcγR:Fc complex fail to explain this because the FcγR polypeptide does not bind the N-glycan. Here we identify a link between motion of the N-glycan and Fc:FcγRIIIa affinity that explains the N-glycan requirement. Fc F241 and F243 mutations decreased the N-glycan/polypeptide interaction and increased N-glycan mobility. The affinity of the Fc mutants for FcγRIIIa was directly proportional to the degree of glycan restriction (R2 = 0.82). The IgG1 Fc K246F mutation stabilized the N-glycan and enhanced affinity for FcγRIIIa. Allosteric modulation of a protein/protein interaction represents a previously undescribed role for N-glycans in biology. Conserved features suggesting a similar N-glycan/aromatic interaction were also found in IgD, IgE, and IgM, but not IgA. The fragment crystallizable (Fc) of human immunoglobulin G1 (IgG1) engages Fc γ receptors (FcγRs) displayed on the surface of immune cells. In an adaptive immune response, Fc links the target-specific recognition of antigen binding fragments (Fabs) to a proinflammatory cascade, resulting in destruction of the invading pathogen (Janeway et al., 2008Janeway C. Murphy K.P. Travers P. Walport M. Janeway’s Immunobiology.Seventh Edition. Garland Science, New York2008Google Scholar). IgG1 Fc contains a conserved asparagine-linked carbohydrate (N-glycan) that is required for productive engagement of the low-affinity FcγRs (Jefferis, 2009Jefferis R. Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action.Trends Pharmacol. Sci. 2009; 30: 356-362Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, Lux et al., 2013Lux A. Yu X. Scanlan C.N. Nimmerjahn F. Impact of immune complex size and glycosylation on IgG binding to human FcγRs.J. Immunol. 2013; 190: 4315-4323Crossref PubMed Scopus (189) Google Scholar). The IgG1 Fc N-glycan is heterogeneous in nature as a result of the template-independent synthesis of carbohydrates in the Golgi (reviewed in Varki, 2009Varki A. Essentials of Glycobiology.Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor2009Google Scholar). Despite this source of compositional variability, a relatively small number of Fc glycoforms are observed and are predominantly of a core fucosylated, biantennary complex type with low levels of terminal sialic acid modification (Arnold et al., 2007Arnold J.N. Wormald M.R. Sim R.B. Rudd P.M. Dwek R.A. The impact of glycosylation on the biological function and structure of human immunoglobulins.Annu. Rev. Immunol. 2007; 25: 21-50Crossref PubMed Scopus (993) Google Scholar). The Fc N-glycan composition correlates strongly with rheumatoid arthritis (RA) disease state and is dominated by ungalactosylated forms in patients with advanced disease (Parekh et al., 1985Parekh R.B. Dwek R.A. Sutton B.J. Fernandes D.L. Leung A. Stanworth D. Rademacher T.W. Mizuochi T. Taniguchi T. Matsuta K. et al.Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG.Nature. 1985; 316: 452-457Crossref PubMed Scopus (989) Google Scholar). Furthermore, changes in glycan distribution can be observed years before RA symptoms arise (Ercan et al., 2010Ercan A. Cui J. Chatterton D.E. Deane K.D. Hazen M.M. Brintnell W. O’Donnell C.I. Derber L.A. Weinblatt M.E. Shadick N.A. et al.Aberrant IgG galactosylation precedes disease onset, correlates with disease activity, and is prevalent in autoantibodies in rheumatoid arthritis.Arthritis Rheum. 2010; 62: 2239-2248Crossref PubMed Scopus (172) Google Scholar), and glycan anomalies return to normal during pregnancy-induced remission (Alavi et al., 2000Alavi A. Arden N. Spector T.D. Axford J.S. Immunoglobulin G glycosylation and clinical outcome in rheumatoid arthritis during pregnancy.J. Rheumatol. 2000; 27: 1379-1385PubMed Google Scholar, Bondt et al., 2013Bondt A. Selman M.H. Deelder A.M. Hazes J.M. Willemsen S.P. Wuhrer M. Dolhain R.J. Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation.J. Proteome Res. 2013; 12: 4522-4531Crossref PubMed Scopus (117) Google Scholar). The Fc N-glycan is predominantly of a biantennary complex type with a high level of core fucosylation (Figure 1). It was suggested that native sialic acid modification, which converts proinflammatory Fc to a potently anti-inflammatory form, is prevented when a galactose (Gal) residue at the nonreducing termini of the glycan is absent (Anthony et al., 2008Anthony R.M. Nimmerjahn F. Ashline D.J. Reinhold V.N. Paulson J.C. Ravetch J.V. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc.Science. 2008; 320: 373-376Crossref PubMed Scopus (644) Google Scholar, Kaneko et al., 2006Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1379) Google Scholar). RA is a multifactorial disease, and though it is not known if IgG N-glycan anomalies cause RA, it is known that compositional changes to the Fc N-glycan alter FcγRIIIa affinity (Okazaki et al., 2004Okazaki A. Shoji-Hosaka E. Nakamura K. Wakitani M. Uchida K. Kakita S. Tsumoto K. Kumagai I. Shitara K. Fucose depletion from human IgG1 oligosaccharide enhances binding enthalpy and association rate between IgG1 and FcgammaRIIIa.J. Mol. Biol. 2004; 336: 1239-1249Crossref PubMed Scopus (283) Google Scholar, Yamaguchi et al., 2006Yamaguchi Y. Nishimura M. Nagano M. Yagi H. Sasakawa H. Uchida K. Shitara K. Kato K. Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy.Biochim. Biophys. Acta. 2006; 1760: 693-700Crossref PubMed Scopus (175) Google Scholar). Structural models of IgG1 Fc show the N-glycan interacting with the Fc polypeptide surface between the Cγ2 domains (Figure 1A; Deisenhofer, 1981Deisenhofer J. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution.Biochemistry. 1981; 20: 2361-2370Crossref PubMed Scopus (1357) Google Scholar, Huber et al., 1976Huber R. Deisenhofer J. Colman P.M. Matsushima M. Palm W. Crystallographic structure studies of an IgG molecule and an Fc fragment.Nature. 1976; 264: 415-420Crossref PubMed Scopus (312) Google Scholar). Surprisingly, the glycan termini were distal to the site of FcγRIIIa binding (Figure 1B; Mizushima et al., 2011Mizushima T. Yagi H. Takemoto E. Shibata-Koyama M. Isoda Y. Iida S. Masuda K. Satoh M. Kato K. Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans.Genes Cells. 2011; 16: 1071-1080Crossref PubMed Scopus (187) Google Scholar, Sondermann et al., 2000Sondermann P. Huber R. Oosthuizen V. Jacob U. The 3.2-A crystal structure of the human IgG1 Fc fragment-Fc gammaRIII complex.Nature. 2000; 406: 267-273Crossref PubMed Scopus (600) Google Scholar), and it seems unlikely that direct interactions between the branch termini of the Fc N-glycan and the proinflammatory FcγRIIIa explain how composition differences at the Fc N-glycan termini affect Fc:FcγRIIIa affinity (Yamaguchi et al., 2006Yamaguchi Y. Nishimura M. Nagano M. Yagi H. Sasakawa H. Uchida K. Shitara K. Kato K. Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy.Biochim. Biophys. Acta. 2006; 1760: 693-700Crossref PubMed Scopus (175) Google Scholar). A different model must be used to explain this phenomenon. Solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations revealed significant motions of the Fc N-glycan (Barb et al., 2012Barb A.W. Meng L. Gao Z. Johnson R.W. Moremen K.W. Prestegard J.H. NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation.Biochemistry. 2012; 51: 4618-4626Crossref PubMed Scopus (96) Google Scholar, Barb and Prestegard, 2011Barb A.W. Prestegard J.H. NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic.Nat. Chem. Biol. 2011; 7: 147-153Crossref PubMed Scopus (148) Google Scholar, Frank et al., 2014Frank M. Walker R.C. Lanzilotta W.N. Prestegard J.H. Barb A.W. Immunoglobulin G1 Fc domain motions: implications for Fc engineering.J. Mol. Biol. 2014; 426: 1799-1811Crossref PubMed Scopus (61) Google Scholar), which was unexpected considering the fixed N-glycan position observed by X-ray crystallography (Huber et al., 1976Huber R. Deisenhofer J. Colman P.M. Matsushima M. Palm W. Crystallographic structure studies of an IgG molecule and an Fc fragment.Nature. 1976; 264: 415-420Crossref PubMed Scopus (312) Google Scholar). These opposing observations agreed in one key aspect: the α1–6 branch of the N-glycan interacts with amino acid residues on the Fc polypeptide surface. Motions of the Fc Cγ2 domain motion also occur (Frank et al., 2014Frank M. Walker R.C. Lanzilotta W.N. Prestegard J.H. Barb A.W. Immunoglobulin G1 Fc domain motions: implications for Fc engineering.J. Mol. Biol. 2014; 426: 1799-1811Crossref PubMed Scopus (61) Google Scholar, Krapp et al., 2003Krapp S. Mimura Y. Jefferis R. Huber R. Sondermann P. Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity.J. Mol. Biol. 2003; 325: 979-989Crossref PubMed Scopus (550) Google Scholar, Saphire et al., 2002Saphire E.O. Stanfield R.L. Crispin M.D. Parren P.W. Rudd P.M. Dwek R.A. Burton D.R. Wilson I.A. Contrasting IgG structures reveal extreme asymmetry and flexibility.J. Mol. Biol. 2002; 319: 9-18Crossref PubMed Scopus (199) Google Scholar) and may be related to N-glycan motion. NMR spectroscopy provides a direct measurement of molecular motion with atom-level resolution. Though a single peak, corresponding to a resonance frequency, for each of the two Gal 13C2 nuclei was observed, further NMR analysis revealed that each peak represented the population-weighted average of two distinct states (Barb and Prestegard, 2011Barb A.W. Prestegard J.H. NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic.Nat. Chem. Biol. 2011; 7: 147-153Crossref PubMed Scopus (148) Google Scholar). The (α1–6 branch)Gal residue showed the greatest effects of this interconversion and was found to exchange between a polypeptide-bound state and an unrestricted, mobile state on a μs timescale. Each resonance in each state is characterized by a distinct resonance frequency that is largely determined by covalent bonds in the Gal moiety and the immediate nonbonded chemical environment (within 5 Å). Both the rate of exchange and the difference in the resonance frequencies for each state contributed to line broadening relaxation (R2) of the glycan resonances (Barb and Prestegard, 2011Barb A.W. Prestegard J.H. NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic.Nat. Chem. Biol. 2011; 7: 147-153Crossref PubMed Scopus (148) Google Scholar, Cavanagh, 2007Cavanagh J. Protein NMR Spectroscopy: Principles and Practice.Second Edition. Academic Press, Amsterdam, Boston2007Google Scholar). Two Fc amino acid residues likely restrict Fc N-glycan mobility (Lund et al., 1996Lund J. Takahashi N. Pound J.D. Goodall M. Jefferis R. Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fc gamma receptor I and influence the synthesis of its oligosaccharide chains.J. Immunol. 1996; 157: 4963-4969PubMed Google Scholar, Voynov et al., 2009Voynov V. Chennamsetty N. Kayser V. Helk B. Forrer K. Zhang H. Fritsch C. Heine H. Trout B.L. Dynamic fluctuations of protein-carbohydrate interactions promote protein aggregation.PLoS ONE. 2009; 4: e8425Crossref PubMed Scopus (38) Google Scholar, Yu et al., 2013Yu X. Baruah K. Harvey D.J. Vasiljevic S. Alonzi D.S. Song B.D. Higgins M.K. Bowden T.A. Scanlan C.N. Crispin M. Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.J. Am. Chem. Soc. 2013; 135: 9723-9732Crossref PubMed Scopus (72) Google Scholar) and contribute to stabilizing the polypeptide-bound state of the N-glycan (Barb and Prestegard, 2011Barb A.W. Prestegard J.H. NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic.Nat. Chem. Biol. 2011; 7: 147-153Crossref PubMed Scopus (148) Google Scholar). F241 and F243 are found at the IgG1 Fc N-glycan/polypeptide interface and >7 Å from the nearest FcγRIIIa residue (Figure 1B). F241 is positioned beneath the branch point mannose residue, and F243 interacts only with the glycan’s α1–6 branch (Figures 1B and 1C). Mutations at these positions increased the level of Gal and sialic acid incorporation at these positions (Lund et al., 1996Lund J. Takahashi N. Pound J.D. Goodall M. Jefferis R. Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fc gamma receptor I and influence the synthesis of its oligosaccharide chains.J. Immunol. 1996; 157: 4963-4969PubMed Google Scholar, Voynov et al., 2009Voynov V. Chennamsetty N. Kayser V. Helk B. Forrer K. Zhang H. Fritsch C. Heine H. Trout B.L. Dynamic fluctuations of protein-carbohydrate interactions promote protein aggregation.PLoS ONE. 2009; 4: e8425Crossref PubMed Scopus (38) Google Scholar, Yu et al., 2013Yu X. Baruah K. Harvey D.J. Vasiljevic S. Alonzi D.S. Song B.D. Higgins M.K. Bowden T.A. Scanlan C.N. Crispin M. Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.J. Am. Chem. Soc. 2013; 135: 9723-9732Crossref PubMed Scopus (72) Google Scholar). Kelly and coworkers determined that aromatic residues formed higher affinity interactions with a glycan than other amino acids types (Chen et al., 2013Chen W. Enck S. Price J.L. Powers D.L. Powers E.T. Wong C.H. Dyson H.J. Kelly J.W. Structural and energetic basis of carbohydrate-aromatic packing interactions in proteins.J. Am. Chem. Soc. 2013; 135: 9877-9884Crossref PubMed Scopus (75) Google Scholar). Though hydrophobic effects were measurable, the dispersion component of CH-π interactions contributed a greater proportion of free energy to the interaction. This observation highlights F241 and F243 as targets for mutation to disrupt the interface. Based on recent reports (Barb et al., 2012Barb A.W. Meng L. Gao Z. Johnson R.W. Moremen K.W. Prestegard J.H. NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation.Biochemistry. 2012; 51: 4618-4626Crossref PubMed Scopus (96) Google Scholar, Barb and Prestegard, 2011Barb A.W. Prestegard J.H. NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic.Nat. Chem. Biol. 2011; 7: 147-153Crossref PubMed Scopus (148) Google Scholar, Frank et al., 2014Frank M. Walker R.C. Lanzilotta W.N. Prestegard J.H. Barb A.W. Immunoglobulin G1 Fc domain motions: implications for Fc engineering.J. Mol. Biol. 2014; 426: 1799-1811Crossref PubMed Scopus (61) Google Scholar, Krapp et al., 2003Krapp S. Mimura Y. Jefferis R. Huber R. Sondermann P. Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity.J. Mol. Biol. 2003; 325: 979-989Crossref PubMed Scopus (550) Google Scholar, Malhotra et al., 1995Malhotra R. Wormald M.R. Rudd P.M. Fischer P.B. Dwek R.A. Sim R.B. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein.Nat. Med. 1995; 1: 237-243Crossref PubMed Scopus (677) Google Scholar, Yamaguchi et al., 1998Yamaguchi Y. Kato K. Shindo M. Aoki S. Furusho K. Koga K. Takahashi N. Arata Y. Shimada I. Dynamics of the carbohydrate chains attached to the Fc portion of immunoglobulin G as studied by NMR spectroscopy assisted by selective 13C labeling of the glycans.J. Biomol. NMR. 1998; 12: 385-394Crossref PubMed Scopus (56) Google Scholar), we propose that conformational oscillations of the Fc Cγ2 domains and N-glycans are integral to FcγR binding and vary between naturally occurring glycoforms. Such a dynamic system endows IgG-expressing B cells with the capacity to tune FcγR affinity by changing glycan composition and would explain why serum IgG N-glycan composition changes rapidly in response to injury or disease (Lauc et al., 2013Lauc G. Huffman J.E. Pučić M. Zgaga L. Adamczyk B. Mužinić A. Novokmet M. Polašek O. Gornik O. Krištić J. et al.Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers.PLoS Genet. 2013; 9: e1003225Crossref PubMed Scopus (257) Google Scholar, Novokmet et al., 2014Novokmet M. Lukic E. Vuckovic F. Ethuric Z. Keser T. Rajsl K. Remondini D. Castellani G. Gasparovic H. Gornik O. Lauc G. Changes in IgG and total plasma protein glycomes in acute systemic inflammation.Sci. Rep. 2014; 4: 4347Crossref PubMed Scopus (97) Google Scholar). If this model of Fc motion is accurate, one expects an Fc N-glycan with Gal on the α1–6 branch to experience less motion than an N-glycan lacking this Gal, and an Fc N-glycan with an N-acetylglucosamine residue (GlcNAc) on the α1–6 branch to experience less motion than an N-glycan lacking this GlcNAc. The restriction in both cases is due to extended glycan/polypeptide interactions. It is unclear how α1–3 branch modification might affect glycan/polypeptide interactions. This supposition is supported by the observation that Fc with longer glycans binds FcγRIIIa with higher affinity (Yamaguchi et al., 2006Yamaguchi Y. Nishimura M. Nagano M. Yagi H. Sasakawa H. Uchida K. Shitara K. Kato K. Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy.Biochim. Biophys. Acta. 2006; 1760: 693-700Crossref PubMed Scopus (175) Google Scholar); the effect of sialylation, an extending modification, on receptor affinity is unresolved (Kaneko et al., 2006Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1379) Google Scholar, Yu et al., 2013Yu X. Baruah K. Harvey D.J. Vasiljevic S. Alonzi D.S. Song B.D. Higgins M.K. Bowden T.A. Scanlan C.N. Crispin M. Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.J. Am. Chem. Soc. 2013; 135: 9723-9732Crossref PubMed Scopus (72) Google Scholar), though recent evidence suggests that sialylation does not alter either N-glycan motion or the interface character to a large extent (Barb et al., 2012Barb A.W. Meng L. Gao Z. Johnson R.W. Moremen K.W. Prestegard J.H. NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation.Biochemistry. 2012; 51: 4618-4626Crossref PubMed Scopus (96) Google Scholar, Crispin et al., 2013Crispin M. Yu X. Bowden T.A. Crystal structure of sialylated IgG Fc: implications for the mechanism of intravenous immunoglobulin therapy.Proc. Natl. Acad. Sci. USA. 2013; 110: E3544-E3546Crossref PubMed Scopus (73) Google Scholar). To simplify analysis of the relationship between dynamics and affinity, we generated a series of Fc mutants with alterations at the N-glycan/polypeptide interface and remodeled the N-glycans in vitro to near homogeneity. Here we report the N-glycan mobility and receptor binding affinity for these Fc mutants. We prepared F241I, F243I, and F241I/F243I Fc mutants to disrupt CH-π interactions but preserve hydrophobic contacts and F241S, F243S, and F241S/F243S Fc mutants to disrupt both CH-π and hydrophobic contacts. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis of N-glycans from purified Fc mutants showed a broader distribution of glycoforms and a greater degree of modification than those from wild-type (WT) Fc (Figure 2 and Figure S1 available online). Though complex-type, core fucosylated, biantennary types were the predominant glycans from all proteins, the double mutants contained triantennary glycans, extensive sialylation, as well as increased fucosylation. Glycan distributions from F241S/F243S and single F241 and F243 mutants shared similarities with published reports (Lund et al., 1996Lund J. Takahashi N. Pound J.D. Goodall M. Jefferis R. Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fc gamma receptor I and influence the synthesis of its oligosaccharide chains.J. Immunol. 1996; 157: 4963-4969PubMed Google Scholar, Voynov et al., 2009Voynov V. Chennamsetty N. Kayser V. Helk B. Forrer K. Zhang H. Fritsch C. Heine H. Trout B.L. Dynamic fluctuations of protein-carbohydrate interactions promote protein aggregation.PLoS ONE. 2009; 4: e8425Crossref PubMed Scopus (38) Google Scholar, Yu et al., 2013Yu X. Baruah K. Harvey D.J. Vasiljevic S. Alonzi D.S. Song B.D. Higgins M.K. Bowden T.A. Scanlan C.N. Crispin M. Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.J. Am. Chem. Soc. 2013; 135: 9723-9732Crossref PubMed Scopus (72) Google Scholar). These triantennary and branch-fucosylated glycoforms are not found on IgG1 Fc from human serum (Arnold et al., 2007Arnold J.N. Wormald M.R. Sim R.B. Rudd P.M. Dwek R.A. The impact of glycosylation on the biological function and structure of human immunoglobulins.Annu. Rev. Immunol. 2007; 25: 21-50Crossref PubMed Scopus (993) Google Scholar). The single mutant proteins showed degrees of modification that were intermediate between WT and the two Fc double mutants. One explanation for the increased diversity of glycans on mutant Fc offered by Lund et al., 1996Lund J. Takahashi N. Pound J.D. Goodall M. Jefferis R. Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fc gamma receptor I and influence the synthesis of its oligosaccharide chains.J. Immunol. 1996; 157: 4963-4969PubMed Google Scholar is that removing aromatic side chains reduced glycan/polypeptide interactions and decreased steric occlusion of the glycan. This would lead to greater glycan exposure and greater modification by glycosyltransferases in the Golgi during protein expression. Alternative explanations include increased retention in the Golgi or protein misfolding. The increased diversity of Fc mutant glycoforms introduces a significant challenge because N-glycan composition changes FcγRIIIa affinity and potentially Fc quaternary structure (Kaneko et al., 2006Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1379) Google Scholar, Krapp et al., 2003Krapp S. Mimura Y. Jefferis R. Huber R. Sondermann P. Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity.J. Mol. Biol. 2003; 325: 979-989Crossref PubMed Scopus (550) Google Scholar, Yamaguchi et al., 2006Yamaguchi Y. Nishimura M. Nagano M. Yagi H. Sasakawa H. Uchida K. Shitara K. Kato K. Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy.Biochim. Biophys. Acta. 2006; 1760: 693-700Crossref PubMed Scopus (175) Google Scholar, Yu et al., 2013Yu X. Baruah K. Harvey D.J. Vasiljevic S. Alonzi D.S. Song B.D. Higgins M.K. Bowden T.A. Scanlan C.N. Crispin M. Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.J. Am. Chem. Soc. 2013; 135: 9723-9732Crossref PubMed Scopus (72) Google Scholar). To reduce the effect of heterogeneity, N-glycans from WT and mutant Fc were enzymatically remodeled in vitro to contain 60%–95% of the G2F glycoform (Figures 1C, S2, and S3). All Fc proteins were judged using 1H NMR spectroscopy to be well folded following in vitro glycan remodeling (data not shown). Furthermore, each protein is bound to a Protein A affinity resin, which binds Fc through a protein-protein interaction and requires appropriately folded Fc (data not shown). Disulfide bonds that link the hinge region and stabilize the Fc dimer also formed (Figure S4). Heteronuclear single quantum coherence (HSQC) spectra provide high-resolution information regarding protein tertiary structure. We found that the 2d 1H-15N HSQC spectrum of [15N-tyrosine(Y)]-Fc F241S/F243S was nearly identical to that of [15N-Y]-Fc WT (Figure 3), indicating that the Cγ2 and Cγ3 domains of both proteins are similarly folded. One difference between the spectra was the disappearance of the Y300 peak in the Fc F241S/F243S spectrum. Y300 occupies the same loop as the glycan tether residue, asparagine(N)-297, and the Y300 resonance is sensitive to changes in the structure and motion of the loop (Figure 1B; Matsumiya et al., 2007Matsumiya S. Yamaguchi Y. Saito J. Nagano M. Sasakawa H. Otaki S. Satoh M. Shitara K. Kato K. Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1.J. Mol. Biol. 2007; 368: 767-779Crossref PubMed Scopus (234) Google Scholar, Yamaguchi et al., 2006Yamaguchi Y. Nishimura M. Nagano M. Yagi H. Sasakawa H. Uchida K. Shitara K. Kato K. Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy.Biochim. Biophys. Acta. 2006; 1760: 693-700Crossref PubMed Scopus (175) Google Scholar). Folded Fc WT and Fc mutants, remodeled to contain a high proportion of G2F N-glycans, bound to a soluble fragment of glycosylated human FcγRIIIa. We found the Fc WT:FcγRIIIa affinity, as measured by surface plasmon resonance (SPR; 0.55 ± 0.05 μM), isothermal titration calorimetry (ITC; 1.2 ± 0.1 μM), and a protein A resin-based pull down assay (0.5 μM), was similar to reported values (0.1–2 μM; Kaneko et al., 2006Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1379) Google Scholar, Yamaguchi et al., 2006Yamaguchi Y. Nishimura M. Nagano M. Yagi H. Sasakawa H. Uchida K. Shitara K. Kato K. Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy.Biochim. Biophys. Acta. 2006; 1760: 693-700Crossref PubMed Scopus (175) Google Scholar, Yu et al., 2013Yu X. Baruah K. Harvey D.J. Vasiljevic S. Alonzi D.S. Song B.D. Higgins M.K. Bowden T.A. Scanlan C.N. Crispin M. Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.J. Am. Chem. Soc. 2013; 135: 9723-9732Crossref PubMed Scopus (72) Google Scholar)). Single mutant proteins showed 3- to 4-fold reduced affinity by SPR when compared with Fc WT and the double mutant proteins bound with a 20- to 60-fold less affinity than WT (Table 1). A previous study found a similar binding effect with single F241A and F243A mutants (Yu et al., 2013Yu X. Baruah K. Harvey D.J. Vasiljevic S. Alonzi D.S. Song B.D. Higgins M.K. Bowden T.A. Scanlan C.N. Crispin M. Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.J. Am. Chem. Soc. 2013; 135: 9723-9732Crossref PubMed Scopus (72) Google Scholar). These data are noteworthy because the effect of glycan heterogeneity has been removed by efficient in vitro glycan remodeling to a physiologically relevant form. The trend of FcγRIIIa binding by Fc mutants also mirrored the MALDI-MS data that showed greater N-glycan modification during expression for Fc double mutants and intermediate degrees observed for the single mutants (Figure 2).Table 1Equilibrium FcγRIIIa Dissociation Constants for Fc WT and Mutants from SPR ExperimentsIgG1 FcFcγRIIIa KD (μM)± ErrorG2F glycanWT0.550.05F241I2.10.2F241S1.50.5F243S1.90.3F241I F243I101F241S F243S293K246F0.340.06GlcNAc glycanWT2.40.2F241I F243I6.30.6F241S F243S3.50.9No glycan T299Anb–Each observation represents the average of three independent measurements ± SD of the mean. n.b., no binding detected. Open table in a new tab Each observation represents the average of three independent measurements ± SD of the mean. n.b., no binding detected. An enzymatically truncated Fc glycoform was prepared that provided a mechanism to test the role of the F241 and F243 mutations. It was reported that deglycosylated Fc and Fuc-GlcNAc-Fc (Figure 1C) bound FcγRIIIa below the levels of detectability (Yamag" @default.
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• W2010670539 title "Restricted Motion of the Conserved Immunoglobulin G1 N-Glycan Is Essential for Efficient FcγRIIIa Binding" @default.
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