Ganesh P. Subedi

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

ABSTRACT 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.

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 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 glycoproteins 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.

Antibody Fucosylation Lowers the Fc gamma RIIIa/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)β-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.

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.

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.