A comprehensive assessment of selective amino acid 15N-labeling in human embryonic kidney 293 cells for NMR spectroscopy.

A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-[15N]- atoms from 10 amino acids experience minimal metabolic scrambling (C, F, H, K, M, N, R, T, W, Y). Two more interconvert to each other (G, S). Six others experience significant scrambling (A, D, E, I, L, V). We also demonstrate that tuning culture conditions suppressed V and I scrambling. These results define expectations for 15N-labeling in HEK293 cells.

Inhibiting N-glycan processing increases the antibody binding affinity and effector function of human natural killer cells.

Novel approaches are required to improve the efficacy of immunotherapies and increase the proportion of patients who experience a benefit. Antibody-dependent cell-mediated cytotoxicity (ADCC) contributes to the efficacy of many monoclonal antibodies therapies. Natural killer (NK) cells mediate ADCC, though responses are highly variable and depend on prior treatment as well as other factors. Thus, strategies to increase NK cell activity are expected to improve multiple therapies. Both cytokine treatment and NK cell receptor engineering are being explored to increase ADCC. Post-translational modifications, including glycosylation, are widely recognized as mediators of cellular processes but minimally explored as an alternative strategy to increase ADCC. We evaluated the impact of treatment with kifunensine, an inhibitor of asparagine-linked (N-)glycan processing, on ADCC using primary and cultured human NK cells. We also probed affinity using binding assays and CD16a structure with nuclear magnetic resonance spectroscopy. Treating primary human NK cells and cultured YTS-CD16a cells with kifunensine doubled ADCC in a CD16a-dependent manner. Kifunensine treatment also increased the antibody-binding affinity of CD16a on the NK cell surface. Structural interrogation identified a single CD16a region, proximal to the N162 glycan and the antibody-binding interface, perturbed by the N-glycan composition. The observed increase in NK cell activity following kifunensine treatment synergized with afucosylated antibodies, further increasing ADCC by an additional 33%. These results demonstrate native N-glycan processing is an important factor that limits NK cell ADCC. Furthermore, optimal antibody and CD16a glycoforms are defined that provide the greatest ADCC activity.

The Preparation and Solution NMR Spectroscopy of Human Glycoproteins Is Accessible and Rewarding.

The majority of proteins excreted by human cells and borne at the cell surface are modified with carbohydrates. Glycoproteins mediate a wide range of processes and adopt fundamental roles in many diseases. The carbohydrates covalently attached to proteins during maturation in the cell directly impact protein structure and function as integral and indispensable components. However, the ability to study the structure of glycoproteins to high resolution was historically limited by technical barriers including a limited availability of appropriate recombinant protein expression platforms, limited methods to generate compositional homogeneity, and difficulties analyzing glycoprotein composition. Furthermore, glycoproteins and in particular the glycan moieties themselves often exhibit a high degree of conformational heterogeneity. Solution NMR spectroscopy is a powerful tool to study biological macromolecules that is capable of characterizing mobile elements of molecules with atomic-level resolution. Methods to express glycoproteins, incorporate stable isotope labels, and analyze glycoproteins have recently opened new avenues to prepare and investigate glycoproteins. These methods are accessible to many laboratories with experience expressing and purifying proteins from prokaryotic expression hosts.

Intradomain Interactions in an NMDA Receptor Fragment Mediate N-Glycan Processing and Conformational Sampling.

The structural and functional roles of highly conserved asparagine-linked (N)-glycans on the extracellular ligand-binding domain (LBD) of the N-methyl-D-aspartate receptors are poorly understood. We applied solution- and computation-based methods that identified N-glycan-mediated intradomain and interglycan interactions. Nuclear magnetic resonance (NMR) spectra of the GluN1 LBD showed clear signals corresponding to each of the three N-glycans and indicated the reducing end of glycans at N440 and N771 potentially contacted nearby amino acids. Molecular dynamics simulations identified contacts between nearby amino acids and the N440- and N771-glycans that were consistent with the NMR spectra. The distal portions of the N771-glycan also contacted the core residues of the nearby N471-glycan. This result was consistent with mass spectrometry data indicating the limited N471-glycan core fucosylation and reduced branch processing of the N771-glycan could be explained by interglycan contacts. We discuss a potential role for the GluN1 LBD N-glycans in interdomain contacts formed in NMDA receptors.

CD16a with oligomannose-type N-glycans is the only "low-affinity" Fc γ receptor that binds the IgG crystallizable fragment with high affinity in vitro.

Fc γ receptors (FcγRs) bind circulating IgG (IgG1) at the surface of leukocytes. Antibodies clustered at the surface of a targeted particle trigger a protective immune response through activating FcγRs. Three recent reports indicate that the composition of the asparagine-linked carbohydrate chains (N-glycans) of FcγRIIIa/CD16a impacted IgG1-binding affinity. Here we determined how N-glycan composition affected the affinity of the "low-affinity" FcγRs for six homogeneous IgG1 Fc N-glycoforms (G0, G0F, G2, G2F, A2G2, and A2G2F). Surprisingly, CD16a with oligomannose N-glycans bound to IgG1 Fc (A2G2) with a KD = 1.0 ± 0.1 nm This affinity represents a 51-fold increase over the affinity measured for CD16a with complex-type N-glycans (51 ± 8 nm) and is comparable with the affinity of FcγRI/CD64, the sole "high-affinity" FcγR. CD16a N-glycan composition accounted for increases in binding affinity for the other IgG1 Fc glycoforms tested (10-50-fold). This remarkable sensitivity could only be eliminated by preventing glycosylation at Asn162 with an Asn-to-Gln mutation; mutations at the four other N-glycosylation sites preserved tighter binding in the Man5 glycoform. None of the other low-affinity FcγRs showed more than a 3.1-fold increase upon modifying the receptor N-glycan composition, including CD16b, which differs from CD16a by only four amino acid residues. This result indicates that CD16a is unique among the low-affinity FcγRs, and modifying only the glycan composition of both the IgG1 Fc ligand and receptor provides a 400-fold range in affinities.

Antibody Fucosylation Lowers the FcγRIIIa/CD16a Affinity by Limiting the Conformations Sampled by the N162-Glycan.

Therapeutic monoclonal antibodies (mAbs) are largely based on the immunoglobulin G1 (IgG1) scaffold, and many elicit a cytotoxic cell-mediated response by binding Fc γ receptors. Core fucosylation, a prevalent modification to the asparagine (N)-linked carbohydrate on the IgG1 crystallizable fragment (Fc), decreases the Fc γ receptor IIIa (CD16a) binding affinity and mAb efficacy. We determined IgG1 Fc fucosylation reduced the CD16a affinity by 1.7 ± 0.1 kcal/mol when compared to that of afucosylated IgG1 Fc; however, CD16a N-glycan truncation decreased this penalty by 1.2 ± 0.1 kcal/mol or 70%. Fc fucosylation restricted the manifold of conformations sampled by displacing the CD16a Asn162-glycan that impinges upon the linkage between the α-mannose(1-6)ß-mannose residues and promoted contacts with the IgG Tyr296 residue. Fucosylation also impacted the IgG1 Fc structure as indicated by changes in resonance frequencies and nuclear spin relaxation observed by solution nuclear magnetic resonance spectroscopy. The effects of fucosylation on IgG1 Fc may account for the remaining 0.5 ± 0.1 kcal/mol penalty of fucosylated IgG1 Fc binding CD16a when compared to that of afucosylated IgG1 Fc. Our results indicated the CD16a Asn162-glycan modulates the antibody affinity indirectly by reducing the volume sampled, as opposed to a direct mechanism with intermolecular glycan-glycan contacts previously proposed to stabilize this system. Thus, antibody engineering to enhance intermolecular glycan-glycan contacts will likely provide limited improvement, and future designs should maximize the affinity by maintaining the CD16a Asn162-glycan conformational heterogeneity.

Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function.

CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody-CD16a interactions, the asparagine-linked carbohydrates (N-glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N-glycans (23%). These proportions indicated restricted N-glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N-glycans (82%). Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via coexpression with the Fc ϵ receptor γ-chain in HEK293F cells was expected to produce N-glycoforms similar to NK cell-derived CD16a but yielded N-glycoforms different from NK cell-derived CD16a and recombinant soluble CD16a. Of note, these differences in CD16a N-glycan composition affected antibody binding: CD16a with oligomannose N-glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N-glycans. The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein's structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines CD16a N-glycan composition and antibody-binding affinity.

Carbohydrate-Polypeptide Contacts in the Antibody Receptor CD16A Identified through Solution NMR Spectroscopy.

Asparagine-linked carbohydrates (N-glycans) are common modifications of eukaryotic proteins that confer multiple properties, including the essential stabilization of therapeutic monoclonal antibodies. Here we present a rapid and efficient strategy for identifying N-glycans that contact polypeptide residues and apply the method to profile the five N-glycans attached to the human antibody receptor CD16A (Fc γ receptor IIIA). Human embryonic kidney 293S cells expressed CD16A with 13CU-labeled N-glycans using standard protein expression techniques and medium supplemented with 3 g/L [13CU]glucose. Anomeric resonances on the protein-linked N-acetylglucosamine residue at the reducing end of the glycan are particularly well suited to studies of multiply glycosylated N-glycoproteins because only one reducing end and nitrogen-linked residue is present in each N-glycan. Correlations between anomeric 1H1 and 13C1 nuclei on the reducing end residue generate crosspeaks in a conventional two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance (NMR) experiment that appear in a region of the spectrum devoid of other carbohydrate peaks or background protein signals. Two N-glycan peaks corresponding to the N45 and N162 N-glycans were dispersed from the rapidly averaged peaks corresponding to the N38, N74, and N169 N-glycans. We used a combination of NMR and 1 µs all-atom computational simulations to identify unexpected contacts between the N45 N-glycan and CD16A polypeptide residues.

The immunoglobulin G1 N-glycan composition affects binding to each low affinity Fc γ receptor.

Immunoglobulin G1 (IgG1) is the most abundant circulating human antibody and also the scaffold for many therapeutic monoclonal antibodies (mAbs). The destruction of IgG-coated targets by cell-mediated pathways begins with an interaction between the IgG Fc region and multiple varieties of membrane-bound Fc γ receptors (FcγRs) on the surface of leukocytes. This interaction requires the presence of an asparagine-linked (N-)glycan on the Fc, and variations in the N-glycan composition can affect the affinity of CD16A binding (an FcγR). Contemporary efforts to glycoengineer mAbs focus on increasing CD16A affinity, and thus treatment efficacy, but it is unclear how these changes affect affinity for the other FcγRs. Here, we measure binding of the extracellular Fc-binding domains for human CD16A and B, CD32A, B and C, and CD64 to 6 well-defined IgG1 Fc glycoforms that cover ∼85% of the pool of human IgG1 Fc glycoforms. Core α1-6 fucosylation showed the greatest changes with CD16B (8.5-fold decrease), CD16A (3.9-fold decrease) and CD32B/C (1.8-fold decrease), but did not affect binding to CD32A. Adding galactose to the non-reducing termini of the complex-type, biantennary glycan increased affinity for all CD16s and 32s tested by 1.7-fold. Sialylation did not change the affinity of core-fucosylated Fc, but increased the affinity of afucosylated Fc slightly by an average of 1.16-fold for all CD16s and CD32s tested. The effects of fucose and galactose modification are additive, suggesting the contributions of these residues to Fc γ receptor affinity are independent.

An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins.

Metal ions serve important roles in structural biology applications from long-range perturbations seen in magnetic resonance experiments to electron-dense signatures in X-ray crystallography data; however, the metal ion must be secured in a molecular framework to achieve the maximum benefit. Polypeptide-based lanthanide-binding tags (LBTs) represent one option that can be directly encoded within a recombinant protein expression construct. However, LBTs often exhibit significant mobility relative to the target molecule. Here we report the characterization of improved LBTs sequences for insertion into a protein loop. These LBTs were inserted to connect two parallel alpha helices of an immunoglobulin G (IgG)-binding Z domain platform. Variants A and B bound Tb(3+) with high affinity (0.70 and 0.13 µM, respectively) and displayed restricted LBT motion. Compared to the parent construct, the metal-bound A experienced a 2.5-fold reduction in tag motion as measured by magnetic field-induced residual dipolar couplings and was further studied in a 72.2 kDa complex with the human IgG1 fragment crystallizable (IgG1 Fc) glycoprotein. The appearance of both pseudo-contact shifts (-0.221 to 0.081 ppm) and residual dipolar couplings (-7.6 to 14.3 Hz) of IgG1 Fc resonances in the IgG1 Fc:(variant A:Tb(3+))2 complex indicated structural restriction of the LBT with respect to the Fc. These studies highlight the applicability of improved LBT sequences with reduced mobility to probe the structure of macromolecular systems.

The Structural Role of Antibody N-Glycosylation in Receptor Interactions.

Asparagine(N)297-linked glycosylation of immunoglobulin G (IgG) Fc is required for binding to FcγRIIa, IIb, and IIIa, although it is unclear how it contributes. We found the quaternary structure of glycosylated Fc was indistinguishable from aglycosylated Fc, indicating that N-glycosylation does not maintain relative Fc Cγ2/Cγ3 domain orientation. However, the conformation of the C'E loop, which contains N297, was significantly perturbed in the aglycosylated Fc variant. The conformation of the C'E loop as measured with a range of Fc variants shows a strong correlation with FcγRIIIa affinity. These results indicate that the primary role of the IgG1 Fc N-glycan is to stabilize the C'E loop through intramolecular interactions between carbohydrate and amino acid residues, and preorganize the FcγRIIIa interface for optimal binding affinity. The features that contribute to the capacity of the IgG1 Fc N-glycan to restrict protein conformation and tune binding affinity are conserved in other antibodies including IgG2-IgG4, IgD, IgE, and IgM.

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Restricted motion of the conserved immunoglobulin G1 N-glycan is essential for efficient FcγRIIIa binding.

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 (R(2) = 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.

Expression and structural characterization of anti-T-antigen single-chain antibodies (scFvs) and analysis of their binding to T-antigen by surface plasmon resonance and NMR spectroscopy.

T-antigen (Galß1-3GalNAcα-1-Ser/Thr), also known as Thomsen-Friedenreich antigen (TF antigen), is an oncofetal antigen commonly found in cancerous tissues. Availability of anti-T-antigen human antibodies could lead to the development of cancer diagnostics and therapeutics. Four groups of single-chain variable fragment (scFv) genes were previously isolated from a phage library (Matsumoto-Takasaki et al. (2009) Isolation and characterization of anti-T-antigen single chain antibodies from a phage library. BioSci Trends 3:87-95.). Here, four anti-T-antigen scFv genes belonging to Group 1-4 were expressed and produced in a Drosophila S2 cell expression system. ELISA and surface plasmon resonance (SPR) analyses confirmed the binding activity of 1E8 scFv protein to various T-antigen presenting conjugates. NMR experiments provided evidence of the folded nature of the 1E8 scFv protein. ScFv-ligand contact was identified by STD NMR, indicating that the galactose unit of T-antigen at the non-reducing end was primarily recognized by 1E8 scFv. This thus provides direct evidence of T-antigen specificity.

Overproduction of anti-Tn antibody MLS128 single-chain Fv fragment in Escherichia coli cytoplasm using a novel pCold-PDI vector.

Overproduction of recombinant proteins in Escherichia coli is often hampered by their failure to fold correctly, leading to their accumulation within inclusion bodies. To overcome the problem, a variety of techniques aimed at soluble expression have been developed including low temperature expression and/or fusion of soluble tags and chaperones. However, a general protocol for bacterial expression of disulfide bond-containing proteins has hitherto not been established. Single chain Fv fragments (scFvs) are disulfide bond-containing proteins often difficult to express in soluble forms in E. coli. We here examine in detail the E. coli expression of a scFv originating from an anti-carbohydrate MLS128 antibody as a model system. We combine three techniques: (1) tagging scFv with thioredoxin, DsbC and protein disulfide isomerase (PDI), (2) expressing the proteins at low temperature using the pCold vector system, and (3) using Origami E. coli strains with mutations in the thioredoxin reductase and glutathione reductase genes. We observed a high expression level of soluble MLS128-scFv in the Origami strain only when PDI is used as a tag. The recombinant protein retains full binding activity towards synthetic carbohydrate antigens. The developed "pCold-PDI" vector has potential for overproduction of other scFvs and disulfide-containing proteins in the Origami strains.