The weaker-binding Fc γ receptor IIIa F158 allotype retains sensitivity to N-glycan composition and exhibits a destabilized antibody-binding interface
2022; Elsevier BV; Volume: 298; Issue: 9 Linguagem: Inglês
10.1016/j.jbc.2022.102329
ISSN1083-351X
Autores Tópico(s)T-cell and B-cell Immunology
ResumoAntibodies engage Fc γ receptors (FcγRs) to elicit healing cellular immune responses following binding to a target antigen. Fc γ receptor IIIa/CD16a triggers natural killer cells to destroy target tissues with cytotoxic proteins and enhances phagocytosis mediated by macrophages. Multiple variables affect CD16a antibody-binding strength and the resulting immune response, including a genetic polymorphism. The predominant CD16a F158 allotype binds antibodies with less affinity than the less common V158 allotype. This polymorphism likewise affects cellular immune responses and clinical efficacy of antibodies relying on CD16a engagement, though it remains unclear how V/F158 affects CD16a structure. Another relevant variable shown to affect affinity is composition of the CD16a asparagine-linked (N)-glycans. It is currently not known how N-glycan composition affects CD16a F158 affinity. Here, we determined N-glycan composition affects the V158 and F158 allotypes similarly, and N-glycan composition does not explain differences in V158 and F158 binding affinity. Our analysis of binding kinetics indicated the N162 glycan slows the binding event, and shortening the N-glycans or removing the N162 glycan increased the speed of binding. F158 displayed a slower binding rate than V158. Surprisingly, we found N-glycan composition had a smaller effect on the dissociation rate. We also identified conformational heterogeneity of CD16a F158 backbone amide and N162 glycan resonances using NMR spectroscopy. Residues exhibiting chemical shift perturbations between V158 and F158 mapped to the antibody-binding interface. These data support a model for CD16a F158 with increased interface conformational heterogeneity, reducing the population of binding-competent forms available and decreasing affinity. Antibodies engage Fc γ receptors (FcγRs) to elicit healing cellular immune responses following binding to a target antigen. Fc γ receptor IIIa/CD16a triggers natural killer cells to destroy target tissues with cytotoxic proteins and enhances phagocytosis mediated by macrophages. Multiple variables affect CD16a antibody-binding strength and the resulting immune response, including a genetic polymorphism. The predominant CD16a F158 allotype binds antibodies with less affinity than the less common V158 allotype. This polymorphism likewise affects cellular immune responses and clinical efficacy of antibodies relying on CD16a engagement, though it remains unclear how V/F158 affects CD16a structure. Another relevant variable shown to affect affinity is composition of the CD16a asparagine-linked (N)-glycans. It is currently not known how N-glycan composition affects CD16a F158 affinity. Here, we determined N-glycan composition affects the V158 and F158 allotypes similarly, and N-glycan composition does not explain differences in V158 and F158 binding affinity. Our analysis of binding kinetics indicated the N162 glycan slows the binding event, and shortening the N-glycans or removing the N162 glycan increased the speed of binding. F158 displayed a slower binding rate than V158. Surprisingly, we found N-glycan composition had a smaller effect on the dissociation rate. We also identified conformational heterogeneity of CD16a F158 backbone amide and N162 glycan resonances using NMR spectroscopy. Residues exhibiting chemical shift perturbations between V158 and F158 mapped to the antibody-binding interface. These data support a model for CD16a F158 with increased interface conformational heterogeneity, reducing the population of binding-competent forms available and decreasing affinity. Fc γ receptor (FcγR) IIIa/CD16a binds to the crystallizable fragment (Fc) of immunoglobulin G (IgG) antibodies (Fig. 1). CD16a triggers cell activation upon encountering antibodies clustered on a surface, which may include a damaged tissue, foreign pathogen, or particle. Possible cell-mediated immune responses are phagocytic and cytotoxic, leading to destruction of the antibody-coated target (1Chiesa S. Tomasello E. Vivier E. Vely F. Coordination of activating and inhibitory signals in natural killer cells.Mol. Immunol. 2005; 42: 477-484Crossref PubMed Scopus (41) Google Scholar, 2Weiskopf K. Weissman I.L. Macrophages are critical effectors of antibody therapies for cancer.mAbs. 2015; 7: 303-310Crossref PubMed Scopus (166) Google Scholar). The strength of the cellular response depends on the antibody-binding affinity of CD16a.Two major CD16a forms in humans result from a genetic polymorphism affecting the amino acid residue at position 158 on the extracellular antibody-binding domain (3Mahaweni N.M. Olieslagers T.I. Rivas I.O. Molenbroeck S.J.J. Groeneweg M. Bos G.M.J. et al.A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism.Sci. Rep. 2018; 815983Crossref PubMed Scopus (29) Google Scholar). The allele frequency varies by population but several studies collectively suggest the V158-encoding allele is present at 30 to 39% frequency, with the remainder encoding F158 (3Mahaweni N.M. Olieslagers T.I. Rivas I.O. Molenbroeck S.J.J. Groeneweg M. Bos G.M.J. et al.A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism.Sci. Rep. 2018; 815983Crossref PubMed Scopus (29) Google Scholar, 4Lehrnbecher T. Foster C.B. Zhu S. Leitman S.F. Goldin L.R. Huppi K. et al.Variant genotypes of the low-affinity Fcgamma receptors in two control populations and a review of low-affinity Fcgamma receptor polymorphisms in control and disease populations.Blood. 1999; 94: 4220-4232Crossref PubMed Google Scholar, 5Kastbom A. Coster L. Arlestig L. Chatzidionysiou A. van Vollenhoven R.F. Padyukov L. et al.Influence of FCGR3A genotype on the therapeutic response to rituximab in rheumatoid arthritis: an observational cohort study.BMJ Open. 2012; 2e001524Crossref PubMed Scopus (33) Google Scholar). In binding affinity assays, the CD16a F158 allotype binds IgG1 4 to 5 fold weaker than V158 (6Bruhns P. Iannascoli B. England P. Mancardi D.A. Fernandez N. Jorieux S. et al.Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses.Blood. 2009; 113: 3716-3725Crossref PubMed Scopus (959) Google Scholar, 7Dekkers G. Treffers L. Plomp R. Bentlage A.E.H. de Boer M. Koeleman C.A.M. et al.Decoding the human immunoglobulin G-glycan repertoire reveals a spectrum of Fc-receptor- and complement-mediated-effector activities.Front. Immunol. 2017; 8: 877Crossref PubMed Scopus (170) Google Scholar).Natural Killer (NK) cells function in the innate immune system but respond to antibody-coated targets through CD16a as the primary FcγR. NK cells expressing F158 showed less cytotoxic activity following antibody treatment than V158 (8Hatjiharissi E. Xu L. Santos D.D. Hunter Z.R. Ciccarelli B.T. Verselis S. et al.Increased natural killer cell expression of CD16, augmented binding and ADCC activity to rituximab among individuals expressing the Fc{gamma}RIIIa-158 V/V and V/F polymorphism.Blood. 2007; 110: 2561-2564Crossref PubMed Scopus (207) Google Scholar). Similarly, Binyamin et al. (9Binyamin L. Alpaugh R.K. Hughes T.L. Lutz C.T. Campbell K.S. Weiner L.M. Blocking NK cell inhibitory self-recognition promotes antibody-dependent cellular cytotoxicity in a model of anti-lymphoma therapy.J. Immunol. 2008; 180: 6392-6401Crossref PubMed Scopus (131) Google Scholar) showed increased antibody-dependent cell-mediated cytotoxicity for V158 with NK92 cells. Data from primary human NK cells also supports V158 promoting antibody-dependent cell-mediated cytotoxicity compared to F158 (7Dekkers G. Treffers L. Plomp R. Bentlage A.E.H. de Boer M. Koeleman C.A.M. et al.Decoding the human immunoglobulin G-glycan repertoire reveals a spectrum of Fc-receptor- and complement-mediated-effector activities.Front. Immunol. 2017; 8: 877Crossref PubMed Scopus (170) Google Scholar). Clinical studies link the F158 allotype to increased susceptibility to a wide assortment of diseases (10Wu J. Edberg J.C. Redecha P.B. Bansal V. Guyre P.M. Coleman K. et al.A novel polymorphism of FcgammaRIIIa (CD16) alters receptor function and predisposes to autoimmune disease.J. Clin. Invest. 1997; 100: 1059-1070Crossref PubMed Scopus (590) Google Scholar, 11Karassa F.B. Trikalinos T.A. Ioannidis J.P. Fc gamma RIIIA-SLE Meta-Analysis InvestigatorsThe Fc gamma RIIIA-F158 allele is a risk factor for the development of lupus nephritis: a meta-analysis.Kidney Int. 2003; 63: 1475-1482Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 12Zhu X.W. Wang Y. Wei Y.H. Zhao P.P. Wang X.B. 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Perez-Guillermo M. et al.Association between the FCGR3A V158F polymorphism and the clinical response to infliximab in rheumatoid arthritis and spondyloarthritis patients.Scand. J. Rheumatol. 2010; 39: 518-520Crossref PubMed Scopus (26) Google Scholar, 16Shimizu C. Mogushi K. Morioka M.S. Yamamoto H. Tamura K. Fujiwara Y. et al.Fc-gamma receptor polymorphism and gene expression of peripheral blood mononuclear cells in patients with HER2-positive metastatic breast cancer receiving single-agent trastuzumab.Breast Cancer. 2016; 23: 624-632Crossref PubMed Scopus (11) Google Scholar, 17Weng W.K. Levy R. Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma.J. Clin. Oncol. 2003; 21: 3940-3947Crossref PubMed Scopus (1100) Google Scholar). Though these correlations are known, it is not known how a single amino acid substitution in this receptor affects the ligand-binding affinity.In addition to the CD16a residue at 158, glycosylation was recently shown to affect antibody-binding affinity. CD16a is a transmembrane receptor modified with five asparagine(N)-linked carbohydrates (glycans). N-glycans on secreted proteins exhibit a high degree of compositional heterogeneity due to the template-independent glycan processing in the ER and Golgi (18Moremen K.W. Tiemeyer M. Nairn A.V. Vertebrate protein glycosylation: diversity, synthesis and function.Nat. Rev. Mol. Cell Biol. 2012; 13: 448-462Crossref PubMed Scopus (1065) Google Scholar). CD16a exhibits a high degree of heterogeneity. Among the five N-glycosylation sites, N162 appears the most heterogeneous on CD16a isolated from primary human cells (19Patel K.R. Nott J.D. Barb A.W. Primary human natural killer cells retain proinflammatory IgG1 at the cell surface and express CD16a glycoforms with donor-dependent variability.Mol. Cell. Proteomics. 2019; 18: 2178-2190Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 20Roberts J.T. Patel K.R. Barb A.W. Site-specific N-glycan analysis of antibody-binding Fc gamma receptors from primary human monocytes.Mol. Cell. Proteomics. 2020; 19: 362-374Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Furthermore, CD16a V158 N162 glycan heterogeneity impacts Fc-binding affinity. Minimally processed oligomannose glycans, present on CD16a isolated from human donors, provide greater affinity than highly processed complex-type glycans on CD16a (21Patel K.R. Roberts J.T. Subedi G.P. Barb A.W. Restricted processing of CD16a/Fc gamma receptor IIIa N-glycans from primary human NK cells impacts structure and function.J. Biol. Chem. 2018; 293: 3477-3489Abstract Full Text Full Text PDF PubMed Google Scholar, 22Subedi G.P. Barb A.W. CD16a with oligomannose-type N-glycans is the only "low-affinity" Fc gamma receptor that binds the IgG crystallizable fragment with high affinity in vitro.J. Biol. Chem. 2018; 293: 16842-16850Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Hayes et al. (23Hayes J.M. Frostell A. Karlsson R. Muller S. Martin S.M. Pauers M. et al.Identification of Fc gamma receptor glycoforms that produce differential binding kinetics for rituximab.Mol. Cell. Proteomics. 2017; 16: 1770-1788Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) also found binding differences related to CD16a glycosylation and the 158 allotype. It is not currently known how the N162 N-glycan composition affects the antibody-binding affinity of the CD16a F158 allotype.We investigated the structural and functional consequences of a phenylalanine residue at CD16a position 158 relative to a valine. We first characterized the impact of N-glycans on antibody-binding affinity by CD16a F158. Next, we identified backbone and N-glycan atoms that were differentially affected by V158 and F158 using NMR spectroscopy. These data provide insight into the unique structural and functional characteristics of the predominant CD16a F158 allotype with atomic-level resolution.ResultsStructural similarity of V158 and F158Multiple examples of the CD16a V158 structure from X-ray crystallography are reported and are highly similar (24Ferrara C. Grau S. Jager C. Sondermann P. Brunker P. Waldhauer I. et al.Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 12669-12674Crossref PubMed Scopus (530) Google Scholar, 25Mizushima T. Yagi H. Takemoto E. Shibata-Koyama M. Isoda Y. Iida S. et al.Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans.Genes Cells. 2011; 16: 1071-1080Crossref PubMed Scopus (184) Google Scholar, 26Falconer D.J. Subedi G.P. Marcella A.M. Barb A.W. Antibody fucosylation lowers the FcgammaRIIIa/CD16a affinity by limiting the conformations sampled by the N162-glycan.ACS Chem. Biol. 2018; 13: 2179-2189Crossref PubMed Scopus (46) Google Scholar). As of this writing, no models of the F158 variant determined by X-ray crystallography or cryo-EM are reported despite the single amino acid difference and greater prevalence in the human population. AlphaFold, however, provided a computational model for comparison (27Varadi M. Anyango S. Deshpande M. Nair S. Natassia C. Yordanova G. et al.AlphaFold protein structure database: massively expanding the structural coverage of protein-sequence space with high-accuracy models.Nucleic Acids Res. 2022; 50: D439-D444Crossref PubMed Scopus (404) Google Scholar). As expected, the structures of the extracellular antibody-binding domains are highly similar (Fig. 1, C and D). Subtle differences in the packing around position 158 are notable, stemming from steric contacts that prevent the F158 sidechain from occupying the same position as V158. This comparison does not clearly indicate why F158 binds weaker than V158. However, relatively small changes in binding affinity, like those noted for F158 and V158, may not result from large structural rearrangements and may be instead due to differences in conformational sampling or glycosylation that are not always identifiable in models from X-ray crystallography or AlphaFold.The effect of N-glycan composition on CD16a F158 affinityWe examined whether glycosylation explained the reported differences in affinity. Our laboratory previously determined that the composition of the N162 glycan affects CD16a V158 antibody-binding affinity (22Subedi G.P. Barb A.W. CD16a with oligomannose-type N-glycans is the only "low-affinity" Fc gamma receptor that binds the IgG crystallizable fragment with high affinity in vitro.J. Biol. Chem. 2018; 293: 16842-16850Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Thus, it is possible that F158 affects N-glycan processing, weakening affinity. WT HEK293F cells expressed both CD16a V158 and F158 and are capable of performing extensive N-glycan remodeling reactions and generating complex-type N-glycoforms. SDS-PAGE showed substantial processing as both proteins migrated much higher than expected based on the predicted molecular weight of the unmodified polypeptide (50.5 kDa; Fig. S1). Mass spectrometry analysis demonstrated that both allotypes contain similar N162-glycans, predominantly complex-type, core fucosylated glycoforms (Fig. S2 and Table S1). A small amount of oligomannose (6%) appeared in the V158 allotype. Furthermore, a recent analysis demonstrated a high degree of N-glycan occupancy for both allotypes (28Lampros E.A. Kremer P.G. Aguilar Diaz de Leon J.S. Roberts E.T. Rodriguez Benavente M.C. Amster I.J. et al.The antibody-binding Fc gamma receptor IIIa/CD16a is N-glycosylated with high occupancy at all five sites.Curr. Res. Immunol. 2022; 3: 128-135Crossref PubMed Scopus (1) Google Scholar).The CD16a F158 allotype bound IgG1 Fc with a six-fold reduction in affinity compared to V158 (Figs. 2 and 3 and Table 1), consistent with previous reports (6Bruhns P. Iannascoli B. England P. Mancardi D.A. Fernandez N. Jorieux S. et al.Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses.Blood. 2009; 113: 3716-3725Crossref PubMed Scopus (959) Google Scholar, 21Patel K.R. Roberts J.T. Subedi G.P. Barb A.W. Restricted processing of CD16a/Fc gamma receptor IIIa N-glycans from primary human NK cells impacts structure and function.J. Biol. Chem. 2018; 293: 3477-3489Abstract Full Text Full Text PDF PubMed Google Scholar, 29Cambay F. Forest-Nault C. Dumoulin L. Seguin A. Henry O. Durocher Y. et al.Glycosylation of Fcgamma receptors influences their interaction with various IgG1 glycoforms.Mol. Immunol. 2020; 121: 144-158Crossref PubMed Scopus (15) Google Scholar). We next expressed both CD16a allotypes in a Gnt1-cell line that expresses glycoproteins with predominantly oligomannose (Man5GlcNAc2) N-glycans (30Reeves P.J. Callewaert N. Contreras R. Khorana H.G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13419-13424Crossref PubMed Scopus (501) Google Scholar). The mobility of these proteins in SDS-PAGE increased from the presence of smaller N-glycans (Fig. S1). Both proteins bound IgG1 Fc with increased affinity compared to the same protein expressed in the WT cell line; however, the F158 allotype bound with a 13-fold weaker affinity than V158 (Fig. S3). While both allotypes gain increased affinity with oligomannose glycans, the gain in affinity by V158 is more substantial than F158 (3.1× versus 1.6×; Table S2). This result indicates that the factor causing weaker F158 binding is also present in the oligomannose N-glycoform.Figure 2Representative surface plasmon resonance (SPR) sensorgrams of CD16a allotypes. Glycoforms are complex-types (A and E) with two variants missing the N162 glycan (C and G). Fits of the dissociation constants and associated errors from equilibrium binding measurements are also shown (B, D, F, and H). Cartoons show the expected N-glycan at the N162 site. The S164A mutation disrupts the N162 N-glycan sequon and thus lacks an N-glycan at that site.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Affinity of each CD16a allotype glycoform. Each mean (horizontal red bar) represents a minimum of five individual measurements (gray circles). Glycan representations reflect the modification at the N162 site. A) V158, B) F158.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The F158-encoding polymorphism introduces an aromatic residue at a position not found in V158. Aromatic residues form strong interactions through CH-π dispersive forces with carbohydrate residues (31Chen W. Enck S. Price J.L. Powers D.L. Powers E.T. Wong C.H. et al.Structural and energetic basis of carbohydrate-aromatic packing interactions in proteins.J. Am. Chem. Soc. 2013; 135: 9877-9884Crossref PubMed Scopus (74) Google Scholar). Thus, it is possible that F158 introduces an inhibitory intramolecular contact. We enzymatically truncated the N-glycans to a single GlcNAc residue and again found that F158 bound with six-fold weaker affinity than V158. Lastly, we prevented N162 glycosylation through the S164A mutation to disrupt the NVS glycosylation sequon. Again, the F158 allotype bound IgG1 Fc with six-fold weaker affinity than V158. When comparing to the native form of the receptor, both allotypes possess nearly identical relative affinity of the single GlcNAc and S164A forms (Table S2). These data demonstrate that the weaker F158 binding affinity is independent of N-glycan composition and the presence of the N162 glycan.CD16a F158 exhibits slower binding kineticsA detailed analysis of the binding curves revealed differences in binding rates. We fitted a two-state binding model to the sensorgrams that revealed major (ka1, kd1) and minor (ka2, kd2) events, with the major event dominating the observed data (Fig. 4, Table 2). The fitting errors were less than 2% in all instances. Dissociation constants resulting from the kinetic fits were lower than those found with fitting the equilibrium responses; however, the relative differences between forms were preserved. Fitting a 1:1 binding model revealed rates highly comparable to the major event in the two-state model, however with slightly increased residuals due to an inability of this model to accommodate slight nonlinearity at the later timepoints of the association curves. This nonlinearity may be due to reversible nonspecific interactions with the chip surface or a slow conformational rearrangement once bound.Figure 4Binding kinetics show different rates for the glycoform and allotype variants. IgG1 Fc binding (A) CD16a V158 with complex-type N-glycans, (B) CD16a V158 S164A with complex-type N-glycans, (C) CD16a V158 with truncated (1)GlcNAc N-glycans, or (D) with Man5 glycans. E, CD16a F158 with (1)GlcNAc N-glycans. Sensorgrams were fitted with a two-state kinetic model (black line). Cartoons show the expected N-glycan at the N162 site. IgG1, immunoglobulin G1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1CD16a-binding affinities from at least five individual measurements (Ave ± StDev)AllotypeN-GlycoformKD (nM)Error (nM)V158Complex27040V158Man58613V158(1)GlcNAc223V158S164A23040F158Complex1660150F158Man51070120F158(1)GlcNAc12616F158S164A1470100 Open table in a new tab Table 2Kinetic parameters from a two-state binding modelProteinN-glycoformka1 (1/Ms)ka2 (1/s)kd1 (1/s)kd2 (1/s)KD (nM)Errorka1 (1/Ms)ka2 (1/s)kd1 (1/s)kd2 (1/s)V158Complex-type1.7E+056.1E-032.5E-026.6E-03760.65%0.67%0.84%0.25%V158Man57.1E+054.9E-037.1E-026.0E-03550.72%0.33%0.78%0.17%V158 S164AComplex-type1.4E+061.4E-031.8E-017.8E-031090.82%0.64%0.85%0.51%V158(1)GlcNAc2.5E+063.2E-034.0E-024.7E-039.20.81%0.90%1.01%0.52%F158(1)GlcNAc1.1E+061.8E-037.9E-025.0E-03521.81%0.91%1.94%0.72% Open table in a new tab We observed differences between CD16a V158 binding and binding of the V158 S164A variant, both with complex-type N-glycans but the latter lacking the N162 glycan. The V158 S164A variant bound with an eight-fold increased on rate and a seven-fold increased off rate. These data indicate that the N162 glycan slows complex formation, though the resulting complex exhibits a slower dissociation rate. Shortening the V158 N-glycan with the Man5 glycoforms increased the binding rate by 4.2-fold and also increased the dissociation rate by 2.8-fold. The F158 association proved too weak and too fast for reproducible measurements of the binding kinetics.The CD16a V158 variant with the truncated (1)GlcNAc N-glycans revealed a 15-fold faster association rate than the complex-type glycoform; however, the dissociation rates were similar (1.6-fold difference). These results support the evidence from the S164A example above that the extended N-glycan slowed the association rate. In contrast to S164A, however, the truncated (1)GlcNAc proved sufficient to stabilize the complex.The increased binding affinity for the CD16a F158 allotype with truncated (1)GlcNAc N-glycans provided the opportunity to measure binding kinetics and compare V158 and F158 directly. The CD16a F158 (1)GlcNAc protein exhibited an on rate that was 2.3-fold slower than the CD16a V158 (1)GlcNAc glycoform and a dissociation rate two-fold faster. Thus, the presence of F158 slowed the binding and increased the rate of complex dissociation. These differences are consistent with a greater conformational heterogeneity of the F158 allotype.NMR shows CD16a structural differencesWe evaluated CD16a V158 and F158 using solution NMR spectroscopy to identify possible structural differences. Glycosylated proteins represent a substantial challenge for solution NMR spectroscopy, largely because the common expression host for NMR, Escherichia coli, does not N-glycosylate. We expressed CD16a in the same human cell line used for the binding affinity measurements (HEK293F). These cells glycosylate appropriately but do not allow uniform amino acid labeling from metabolic precursors. Instead, we first supplemented the expression medium with [15N]-phenylalanine, an essential amino acid that does not scramble under the conditions used for expression.An HSQC-TROSY spectrum of [15N-phenylalanine]-CD16a V158 showed seven clear peaks corresponding to seven phenylalanine residues (Fig. 5A). A similar spectrum of [15N-F]-CD16a F158 showed nine peaks, seven of these similar to V158 peaks. The two remaining peaks are likely due to F158. It is curious to note that the F158 N-H correlation revealed two peaks, indicating the presence of two different conformations exchanging slowly on the NMR timescale. It is not known if the V158 N-H correlation likewise samples two conformations; the nitrogen atom from [15N]-valine is metabolically scrambled to other amino acids during expression and a V158 peak was not identified (data not shown). It is also notable that the F133 and F153 peaks do not perfectly overlay in spectra of the [15N-phenylalanine]-CD16a allotypes, instead showing chemical shift perturbations.Figure 5NMR spectroscopy of the CD16a allotypes. A, overlay of HSQC-TROSY spectra for [15N-phenylalanine]-labeled CD16a collected at 30 °C and 21.1 T. B, overlay of HSQC-TROSY spectra for [15N-phenylalanine/lysine/tyrosine]-labeled CD16a. C, overlay of 1H-(13C direct observe) spectra of [13C-glucose]-labeled CD16a showing the region corresponding to the (1)GlcNAc 1H1-13C1 correlation. D, mapping residues with different positions (red spheres) and similar positions (gray spheres) from panel (B) onto a structure of CD16a. The IgG1 Fc–binding interface is shown with dashed lines. IgG1, immunoglobulin G1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next probed chemical shift perturbations in other amino acid residues by adding [15N]-lysine, [15N]-phenylalanine, and [15N]-tyrosine to the expression medium. Lysine and tyrosine likewise did not scramble. We were able to match each peak to a residue using the assignment of a related receptor by Kato et al. (32Yogo R. Yanaka S. Kato K. Backbone (1)H, (13)C, and (15)N assignments of the extracellular region of human Fcgamma receptor IIIb.Biomol. NMR Assign. 2018; 12: 201-204Crossref PubMed Scopus (1) Google Scholar). In total, these spectra showed ten residues with chemical shift perturbations, notably at F133, K131, F153, and K161 (Fig. 5B).The N162 glycan is also in the general vicinity of these perturbed residues. N-glycans can be labeled by supplementing the expression medium with [13C]-glucose, which is then incorporated into each N-glycan sugar and alanine methyls (33Yamaguchi Y. Kato K. Shindo M. Aoki S. Furusho K. Koga K. et al.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, 34Rogals M.J. Yang J.Y. Williams R.V. Moremen K.W. Amster I.J. Prestegard J.H. Sparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose.Glycobiology. 2021; 31: 425-435Crossref PubMed Scopus (4) Google Scholar). One unique N-glycan proton-carbon correlation provides unique insight into each individual N-glycan at the point of attachment: the 1H1-13C1 correlation on the (1)GlcNAc residue that is covalently bonded to the N-glycosylated asparagine residue (35
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