Measuring N and C Enrichment Levels in Sparsely Labeled Proteins Using High-Resolution and Tandem Mass Spectrometry.

Isotope labeling of both N and C in selected amino acids in a protein, known as sparse labeling, is an alternative to uniform labeling and is particularly useful for proteins that must be expressed using mammalian cells, including glycoproteins. High levels of enrichment in the selected amino acids enable multidimensional heteronuclear NMR measurements of glycoprotein three-dimensional structure. Mass spectrometry provides a means to quantify the degree of enrichment.

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa/CD16a structure.

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa/CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity.

Automated Assignment of N And C Enrichment Levels in Doubly-Labeled Proteins.

Uniform enrichment of N and C in proteins is commonly employed for 2D heteronuclear NMR measurements of the three-dimensional protein structure. Achieving a high degree of enrichment of both elements is important for obtaining high quality data. Uniform labeling of proteins and glycoproteins expressed in higher organisms (yeast or mammalian cell lines) is more challenging than expression in , a prokaryote that grows on simple, chemically defined media but does not provide appropriate eukaryotic modifications.

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa / CD16a structure.

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa / CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity.

Distinct CD16a features on human NK cells observed by flow cytometry correlate with increased ADCC.

Natural killer (NK) cells destroy tissue that have been opsonized with antibodies. Strategies to generate or identify cells with increased potency are expected to enhance NK cell-based immunotherapies. We previously generated NK cells with increased antibody-dependent cell mediated cytotoxicity (ADCC) following treatment with kifunensine, an inhibitor targeting mannosidases early in the N-glycan processing pathway.

A comprehensive assessment of selective amino acid N-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.

Uniform [C,N]-labeled and glycosylated IgG1 Fc expressed in Saccharomyces cerevisiae.

Despite the prevalence and importance of glycoproteins in human biology, methods for isotope labeling suffer significant limitations. Common prokaryotic platforms do not produce mammalian post-translation modifications that are essential to the function of many human glycoproteins, including immunoglobulin G1 (IgG1). Mammalian expression systems require complex media and thus introduce significant costs to achieve uniform labeling.

Macrophage N-glycan processing inhibits antibody-dependent cellular phagocytosis.

Factors regulating macrophage effector function represent potential targets to optimize the efficacy of antibody-mediated therapies. Macrophages are myeloid cells capable of engulfing and destroying diseased or damaged target cells. Antibodies binding to the target cell surface can engage macrophage Fc gamma receptors (FcγRs) to elicit antibody-dependent cellular phagocytosis (ADCP), a process that contributes to treatments mediated by anti-tumor antibodies.

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.

Decoding human-macaque interspecies differences in Fc-effector functions: The structural basis for CD16-dependent effector function in Rhesus macaques.

Fc mediated effector functions of antibodies play important roles in immunotherapies and vaccine efficacy but assessing those functions in animal models can be challenging due to species differences. Rhesus macaques, (Mm) share approximately 93% sequence identity with humans but display important differences in their adaptive immune system that complicates their use in validating therapeutics and vaccines that rely on Fc effector functions. In contrast to humans, macaques only have one low affinity FcγRIII receptor, CD16, which shares a polymorphism at position 158 with human FcγRIIIa with Ile and Val variants.

The weaker-binding Fc γ receptor IIIa F158 allotype retains sensitivity to N-glycan composition and exhibits a destabilized antibody-binding interface.

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.

MetabolicN labeling of the N-glycosylated immunoglobulin G1 Fc with an engineered Saccharomyces cerevisiae strain.

The predominant protein expression host for NMR spectroscopy is Escherichia coli, however, it does not synthesize appropriate post-translation modifications required for mammalian protein function and is not ideal for expressing naturally secreted proteins that occupy an oxidative environment. Mammalian expression platforms can address these limitations; however, these are not amenable to cost-effective uniform  N labeling resulting from highly complex growth media requirements. Yeast expression platforms combine the simplicity of bacterial expression with the capabilities of mammalian platforms, however yeasts require optimization prior to isotope labeling.

The antibody-binding Fc gamma receptor IIIa / CD16a is N-glycosylated with high occupancy at all five sites.

The antibody-binding Fc γ receptors (FcγRs) trigger life-saving immune responses and many therapeutic monoclonal antibodies require FcγR engagement for full effect. One proven strategy to improve the efficacy of antibody therapies is to increase receptor binding affinity, in particular binding to FcγRIIIa/CD16a. Currently, affinities are measured using recombinantly-expressed soluble extracellular FcγR domains and CD16a-mediated antibody-dependent immune responses are characterized using cultured cells.

Method for Identifying Galectin Ligands on Lymphocyte Membrane Glycoproteins.

Glycosylation is one of the most common protein posttranslational modifications. Most human lymphocyte membrane receptors are modified by diverse glycan structures, and functional studies have indicated that a family of glycan-binding proteins, galectins, can significantly modulate lymphocyte development and function by interacting with these glycans. Several galectins have a varying degree of affinity for the N-acetyllactosamine (LacNAc) disaccharide, and some critical lymphocyte receptors can be modified by glycan structures carrying this motif.

Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain.

Interactions with cell surface receptors enhance the therapeutic properties of many important antibodies, including the low-affinity Fc γ Receptors (FcγRs). These interactions require proper processing of the immunoglobulin G Fc -glycan, and eliminating the -glycan abolishes binding, restricting antibody production to mammalian expression platforms. Yeasts, for example, generate extensively mannosylated -glycans that are unsuitable for therapeutics.

Recent Advances Toward Engineering Glycoproteins Using Modified Yeast Display Platforms.

Yeast are capable recombinant protein expression hosts that provide eukaryotic posttranslational modifications such as disulfide bond formation and N-glycosylation. This property has been used to create surface display libraries for protein engineering; however, yeast surface display (YSD) with common laboratory strains has limitations in terms of diversifying glycoproteins due to the incorporation of high levels of mannose residues which often obscure important epitopes and are immunogenic in humans. Developing new strains for efficient and appropriate display will require combining existing technologies to permit efficient glycoprotein engineering.

Fc γ receptor IIIa/CD16a processing correlates with the expression of glycan-related genes in human natural killer cells.

Many therapeutic monoclonal antibodies require binding to Fc γ receptors (FcγRs) for full effect and increasing the binding affinity increases efficacy. Preeminent among the five activating human FcγRs is FcγRIIIa/CD16a expressed by natural killer (NK) cells. CD16a is heavily processed, and recent reports indicate that the composition of the five CD16a asparagine(N)-linked carbohydrates (glycans) impacts affinity.

Fc γ receptor compositional heterogeneity: Considerations for immunotherapy development.

The antibody-binding crystallizable fragment (Fc) γ receptors (FcγRs) are expressed by leukocytes and activate or suppress a cellular response once engaged with an antibody-coated target. Therapeutic mAbs that require FcγR binding for therapeutic efficacy are now frontline treatments for multiple diseases. However, substantially fewer development efforts are focused on the FcγRs, despite accounting for half of the antibody-receptor complex.

Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.

Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E.coli.

Allotype-specific processing of the CD16a N45-glycan from primary human natural killer cells and monocytes.

Fc γ receptor IIIa/CD16a is an activating cell surface receptor with a well-defined role in natural killer (NK) cell and monocyte effector function. The extracellular domain is decorated with five asparagine (N)-linked glycans; N-glycans at N162 and N45 directly contribute to high-affinity antibody binding and protein stability. N-glycan structures at N162 showed significant donor-dependent variation in a recent study of CD16a isolated from primary human NK cells, but structures at N45 were relatively homogeneous.

Site-specific N-glycan Analysis of Antibody-binding Fc γ Receptors from Primary Human Monocytes.

FcγRIIIa (CD16a) and FcγRIIa (CD32a) on monocytes are essential for proper effector functions including antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Indeed, therapeutic monoclonal antibodies (mAbs) that bind FcγRs with greater affinity exhibit greater efficacy. Furthermore, post-translational modification impacts antibody binding affinity, most notably the composition of the asparagine(N)-linked glycan at N162 of CD16a.

A synopsis of recent developments defining how N-glycosylation impacts immunoglobulin G structure and function.

Therapeutic monoclonal antibodies (mAbs) are the fastest growing group of drugs with 11 new antibodies or antibody-drug conjugates approved by the Food and Drug Administration in 2018. Many mAbs require effector function for efficacy, including antibody-dependent cell-mediated cytotoxicity triggered following contact of an immunoglobulin G (IgG)-coated particle with activating crystallizable fragment (Fc) γ receptors (FcγRs) expressed by leukocytes. Interactions between IgG1 and the FcγRs require post-translational modification of the Fc with an asparagine-linked carbohydrate (N-glycan).

Primary Human Natural Killer Cells Retain Proinflammatory IgG1 at the Cell Surface and Express CD16a Glycoforms with Donor-dependent Variability.

Post-translational modification confers diverse functional properties to immune system proteins. The composition of serum proteins such as immunoglobulin G (IgG) strongly associates with disease including forms lacking a fucose modification of the crystallizable fragment (Fc) asparagine(N)-linked glycan that show increased effector function, however, virtually nothing is known about the composition of cell surface receptors or their bound ligands because of low abundance in the circulating blood. We isolated primary NK cells from apheresis filters following plasma or platelet donation to characterize the compositional variability of Fc γ receptor IIIa/CD16a and its bound ligand, IgG1.

From Rhesus macaque to human: structural evolutionary pathways for immunoglobulin G subclasses.

The Old World monkey, Rhesus macaque (Macaca mulatta, Mm), is frequently used as a primate model organism in the study of human disease and to test new vaccines/antibody treatments despite diverging before chimpanzees and orangutans. Mm and humans share 93% genome identity with substantial differences in the genes of the adaptive immune system that lead to different functional IgG subclass characteristics, Fcγ receptors expressed on innate immune cells, and biological interactions. These differences put limitations on Mm use as a primary animal model in the study of human disease and to test new vaccines/antibody treatments.

Multiple Variables at the Leukocyte Cell Surface Impact Fc γ Receptor-Dependent Mechanisms.

Fc γ receptors (FcγR) expressed on the surface of human leukocytes bind clusters of immunoglobulin G (IgG) to induce a variety of responses. Many therapeutic antibodies and vaccine-elicited antibodies prevent or treat infectious diseases, cancers and autoimmune disorders by binding FcγRs, thus there is a need to fully define the variables that impact antibody-induced mechanisms to properly evaluate candidate therapies and design new intervention strategies. A multitude of factors influence the IgG-FcγR interaction; one well-described factor is the differential affinity of the six distinct FcγRs for the four human IgG subclasses.