N-Linked Glycan Structures of the Human FcÎ³ ... - ACS Publications

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N‑Linked Glycan Structures of the Human Fcγ Receptors Produced in NS0 Cells Eoin F. J. Cosgrave,†,‡ Weston B. Struwe,†,§ Jerrard M. Hayes,† David J. Harvey,∥ Mark R. Wormald,∥ and Pauline M. Rudd*,† †

NIBRT Glycobiology Group, National Institute for Bioprocessing Research and Training, Foster’s Avenue, Mount Merrion, Blackrock, County Dublin, Ireland ‡ Pharmaceutical Life Sciences Group, Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, United States § Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom ∥ Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom ABSTRACT: Immune recognition of nonself is coordinated through complex mechanisms involving both innate and adaptive responses. Circulating antibodies communicate with effector cells of the innate immune system through surface receptors known as Fcγ receptors (FcγRs). The FcγRs are single-pass transmembrane glycoproteins responsible for regulating innate effector responses toward antigenic material. Although immunoglobulin G (IgG) antibodies bind to a range of receptors, including complement receptors and C-type lectins, we have focused on the Fcγ receptors. A total of five functional FcγRs are broadly classified into three families (FcγRI, FcγRII, and FcγRIII) and together aid in controlling both inflammatory and anti-inflammatory responses of the innate immune system. Due to the continued success of monoclonal antibodies in treating cancer and autoimmune disorders, research is typically directed toward improving the interaction of antibodies with the FcγRs through manipulation of IgG properties such as N-linked glycosylation. Biochemical studies using recombinant forms of the FcγRs are often used to quantitate changes in binding affinity, a key indicator of a likely biological outcome. However, analysis of the FcγRs themselves is imperative as recombinant FcγRs differ greatly from those observed in humans. In particular, the N-linked glycan composition is significantly important due to its function in the IgG−FcγR interaction. Here, we present data for the N-linked glycans present on FcγRs produced in NS0 cells, namely, FcγRIa, FcγRIIa, FcγRIIB, FcγRIIIa, and FcγRIIIb. Importantly, these FcγRs demonstrate typical murine glycosylation in the form of α-galactose epitopes, N-glycolylneuraminic acid, and other key glycan properties that are generally expressed in murine cell lines and therefore are not typically observed in humans. KEYWORDS: glycosylation, antibody, IgG, Fcγ receptors, N-linked glycan, α-galactosylation, N-glycolylneuraminic acid, HPLC, mass spectrometry, N-acetyllactosamine, CD16, CD32, CD64

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INTRODUCTION Clearance of foreign material from the host system is achieved in part by the activity of immunoglobulins (Igs), an important class of glycoproteins responsible for the recognition of nonself epitopes and the subsequent broadcasting of this identification to the innate and adaptive arms of the immune system. However, recognition of potentially harmful foreign antigens followed by opsonization is insufficient for their removal, and immunoglobulins rely on activation of innate effector cells that carry out the elimination process. Coupling of soluble immunoglobulins to innate host cell membranes is facilitated through binding to membrane-bound receptors (Fc receptors) specific for the Fc region of Igs, which then trigger immune activation. This leads to clustering of receptors, immune complex formation, and triggering of an intracellular signaling cascade involving phosphorylation/dephosphorylation of © 2013 American Chemical Society

ITAMs (immuno-tyrosine-like activatory motifs) and ITIMs (immuno-tyrosine-like inhibitory motifs). Key to the ability of immunoglobulin G (IgG) in coordinating immune responses is glycosylation, a nontemplate-driven post-translational modification that when absent adversely affects the structural integrity of the antibody molecule, therein eliminating or significantly reducing binding to its Fc receptors C1q and MBL.1−5 It is now widely appreciated that this binding and activation event is mediated by glycosylation. Carbohydrates attached to IgG, the predominant class of Ig found in human serum, play a critical role in its function of linking antigen recognition to immune effector functions.6 Antibody-mediated immune responses Received: April 14, 2013 Published: June 18, 2013 3721

dx.doi.org/10.1021/pr400344h | J. Proteome Res. 2013, 12, 3721−3737

Journal of Proteome Research

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titration calorimetry (ITC), each of which typically uses recombinant forms of the individual Fcγ receptors. Production of recombinant receptors is performed in similar cell expression systems used for antibody generation such as NS0, CHO, BHK, or HEK cells largely due to the fact that the Fcγ receptors are heavily glycosylated proteins and glycosylation is required for their biological activity. Typically these cell lines perform all the necessary post-translational modifications required for production of complex glycoproteins. While glycosylation is performed by these various cell lines, it is done so in a manner divergent to normal human glycosylation. This raises obvious concern in light of compounding evidence for the role of FcγR glycosylation in IgG biological activity. Nonhuman glycosylation of Fcγ receptors used in these assessments could potentially generate data which are not biologically relevant. It is therefore necessary to characterize the glycosylation of recombinant Fcγ receptors to determine where possible how the heterologous forms compare to their natural counterparts. Several groups have employed recombinant Fcγ receptors from NS0 cells for use in affinity studies without detailed knowledge of the carbohydrate content of the receptors or how glycosylation affects antibody binding and kinetics. 26−31 In this study we therefore set out to comprehensively characterize the N-glycans present on commercially available and industrially relevant Fcγ receptors produced in the murine cell line NS0. Specifically, N-glycans from FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb were investigated. The information provided here is of vital importance to the biopharmaceutical industry where NS0expressed receptors are used to assess the activity of candidate monoclonal antibodies. Using both HPLC- and MS-based approaches, we have demonstrated that NS0-derived recombinant soluble human FcγRs are substantially heterogeneous with multiantennary structures containing extensive immunogenic α(1,3)-galactosylation, outer arm fucosylation, poly(N-acetyllactosamine), and N-glycolylneuraminic acid. These findings show that significant differences are likely to exist in the glycosylation of Fcγ receptors which are recombinantly produced and those which are found in humans. These receptors could potentially produce misleading IgG binding interaction data, and the information provided here could lead to a more physiologically relevant assessment of monoclonal antibody function when using murine-derived receptors which could potentially have differences in protein structure as well as carbohydrate epitopes. This also raises the question of whether human-derived receptors should be exclusively used for the analysis of therapeutic monoclonal antibodies.

coordinated by IgG are defined by interaction with receptors specific for the Fc region of the IgG molecule (Fcγ receptors, FcγRs). The Fcγ receptors are widely distributed across the hematopoietic system and are instrumental in the activation of innate effector cells following interaction with immune complexes. They are categorized into three main classes: (i) the high-affinity (kD ≈ 10−9 M) FcγRI (CD64) class consisting of FcγRIa, (ii) the low-affinity (kD ≈ 10−7 M) FcγRII (CD32) class consisting of the activatory FcγRIIa and inhibitory FcγRIIb, and (iii) the low-affinity (kD ≈ 10−6 M) FcγRIII (CD16) class consisting of FcγRIIIa and the GPI-anchored FcγRIIIb. It has been shown that the glycan composition of IgG significantly affects the ability of FcγRIIIa to bind IgG. Most notably, the loss of core α(1−6) fucose on the Fc glycans at Asn-297 on each heavy chain significantly increases not only the binding affinity to FcγRIIIa7−9 but also the associated cellular cytotoxicity through NK cell activation.10−13 The precise mechanism for this phenomenon is well-defined and involves glycosylation of the Fcγ receptor itself. The carbohydrate at Asn-162 on FcγRIIIa is instrumental to the activity of afucosylated IgG, and elimination of the glycan at this position significantly reduced the binding affinity of IgG by up to an order of magnitude.14 Further studies of FcγRIIIa glycosylation have demonstrated that N-linked glycosylation at position Asn-45 reduces the binding affinity of IgG while a point mutation of Asn-45 positively influences affinity.15 In light of such findings, it is becoming increasingly evident that glycosylation of the Fcγ receptors as well as the IgG Fc domain plays a significant role in the ultimate binding event between these two molecules and subsequent downstream activation. This has critical implications for the glycoengineering of therapeutic monoclonal antibodies (mAbs) and the potential approach the biopharmaceutical industry takes toward development of new candidate mAb therapies. There are multiple levels of complexity in FcγR biology, including cellular distribution, regulation, activation, and function; however, these topics have been discussed elsewhere,16−21 and in the present study we will focus on cellular FcγR epitopes. Fc−FcγR interactions represent an area of intense focus in the generation of monoclonal antibody therapeutics destined for use in cancer immunotherapy. Targeted elimination of tumorigenic cells relies on the identification of tumor-associated antigens (TAAs) and the recruitment of immune cells capable of destroying aberrant cancer cells. Antibody-mediated tumor eradication depends on two principal mechanisms targeted for activation, complementdependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). ADCC is primarily faciliated by the activity of NK cells through interaction of IgG immune complexes with FcγRIIIa, although other innate effector cells such as neutrophils22 and eosinophils23 are also capable of performing this function. Enhancing the cytotoxic activity of NK cells through engineering of monoclonal antibodies has been of paramount interest to the pharmaceutical industry as it presents opportunites to generate therapies with enhanced biological activity and therefore increased potency and efficacy in tumor eradication. Guidelines governing the biological activity of candidate mAbs specify that precise information on the binding affinity and specificity of the proposed mAb with FcγRs is required.24,25 Determination of binding kinetics is largely achieved through measurement of protein−protein interactions using techniques such as surface plasmon resonance (SPR) or isothermal

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EXPERIMENTAL SECTION

N-Linked Glycan Release and 2-Aminobenzamide (2AB) Labeling

Three separate lots of Fcγ receptors were purchased from R&D Systems (Minneapolis, MN) and delivered as lyophilized products. Each receptor was reconstituted in phosphatebuffered saline (PBS) or PBS containing 0.1% bovine serum albumin (BSA) to 1 mg mL −1, on the basis of the manufacturer’s recommendation. N-Linked glycans of each receptor were released using a 0.1 U mL−1 concentration of the peptide N-glycanase PNGase F (ProZyme, Hayward, CA). Glycans were subsequently isolated, labeled with 2AB (Ludger, United Kingdom), and reconstituted in deionized water as previously described.32 3722



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Table 1. Summary of Properties for Commercially Acquired Fc Receptorsa

a

Fc receptors were purchased from R&D Systems. For each Fc receptor, a structure is presented with putative N-linked glycan sites illustrated in red. The mass recorded is based on the amino acid sequence.

Hydrophilic Interaction High-Performance Liquid Chromatography (HI-HPLC)

Exoglycosidase Arrays

Full carbohydrate characterization in terms of sequence, composition, and linkage specificities was facilitated by use of exoglycosidase digestion arrays. All digestion reactions were performed with enzymes purchased from ProZyme. Fluorescently labeled glycans were digested in 50 mM sodium acetate, pH 5.5, at 37 °C overnight using a preselected panel of enzymes, with each digestion reaction brought to a final volume of 10 μL using ddH2O. Digested glycans were then separated from the enzyme mixtures using Nanosep 10 kDa MWCO centrifugal filters (Pall Corp., Port Washington, NY). Digested 2AB-labeled glycans were then prepared for separation on hydrophilic interaction liquid chromatography (HILIC)− HPLC using the 180 min gradient method as previously described.32 Specific monosaccharides were removed as follows: terminal sialic acid in all linkages was removed with 1 mU μL−1 Arthrobacter ureafaciens sialidase (ABS), terminal galactose monosaccharides were removed using 0.5 mU μL−1 bovine testes β-galactosidase (BTG), which releases both β(1,3)- and β(1,4)-linked galactose, terminal galactose-α(1,3)-galactose was released using 40 mU μL−1 coffee bean α-galactosidase (CBG), terminal N-acetylglucosamine (GlcNAc) monosaccharides were released with 40 mU μL−1 Streptococcus pneumoniae hexosaminidase (GUH), capable of cleaving β-linked GlcNAc moieties, core α(1,6)-fucose was selectively removed using 1 mU μL−1 bovine kidney α-fucosidase (BKF), terminal outer arm α(1,2)linked fucose was investigated using 0.8 mU μL−1 Xanthomonas manihotis α-fucosidase, and α-linked mannose was removed using 150 mU μL−1 jack bean α-mannosidase (JBM).

2AB-labeled glycans were prepared as 80% (v/v) acetonitrile solutions for injection onto a TSKgel amide-80, 5 μm (4.6 mm × 250 mm) column as previously described.32 All separations were performed over a 180 min period at 30 °C using 50 mM ammonium formate, pH 4.4, as solvent A and 100% (v/v) acetonitrile as solvent B. All separations were performed using Waters Alliance 2695 instruments coupled with a Waters 2475 fluorescence detector with glycans detected through fluorescence excitation at 420 nm and emission at 330 nm. Each 180 min separation was achieved by running a 20−58% gradient of solvent A over a 152 min time frame and a flow rate of 0.4 mL min−1. HPLC instrumentation was calibrated using 2AB-labeled dextran as an internal calibration standard. Retention times for peaks arising in the dextran hydrolysate separation were used to fit a fifth-order polynomial distribution curve using Waters Empower software (Milford, MA), which provided the means for converting chromatographic retention times into standardized glucose unit (GU) values as previously described.32 Weak Anion Exchange (WAX) HPLC

The extent of terminal sialylation and charged peak fractionation was determined using WAX-HPLC. A Glyko Glycosep C (7.5 mm × 75 mm) column (ProZyme) was used for separation of charged carbohydrate structures. Solvent A was prepared as 100 mM ammonium acetate, pH 7, in 20% (v/ v) methanol, while solvent B was 20% (v/v) acetonitrile. All WAX-HPLC separations were performed on a Waters Alliance 2695 instrument coupled with a Waters 474 fluorescence detector. Carbohydrate samples were separated over a 30 min gradient method. The relative number of terminal charged glycan structures was determined by comparison to released Nlinked glycans from fetuin. For WAX fractionation procedures, glycan pools were first separated by WAX-HPLC and peaks arising from this separation were manually collected. Excess solvent in each glycan collection was then evaporated, and the resulting isolated glycans were reconstituted in a predetermined volume of doubly distilled water (ddH2O). Each fraction was prepared for injection onto TSKgel amide-80 columns using the aforementioned 180 min gradient method.

MALDI MS

Released N-glycans from 50 μg of Fcγ receptors were released in a volume of 50 μL and subsequently diluted with 200 μL of H2O following reaction. N-Glycans were purified by retentive solid-phase extraction using porous graphitized carbon (PGC) as previously described33 with minor alterations. In brief, Nglycans were loaded to the top of the PGC bed, washed with HPLC-grade H2O, and eluted with 25% acetonitrile/0.05% trifluoroacetic acid (TFA). N-Glycans were dried, reconstituted in 100 μL of H2O, and aliquoted into 5 × 20 μL fractions for exoglycosidase digestions. N-Glycans were digested with ABS, 3723



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ABS + CBG, ABS + CBG + BTG, and ABS + CBG + BTG + BKF as described above. Exoglycosidases were removed via PGC solid-phase extraction (SPE), and N-glycans were reconstituted in 20 μL of H2O and 5 μL of MeOH. Glycan samples (3 μL) were spotted on 1 μL of 2,5-dihydroxylbenzoic acid matrix (10 mg mL−1 in 50% (v/v) acetonitrile aqueous solution) on a stainless steel MALDI target plate. Sample spots were recrystallized with 0.7 μL of ethanol. MALDI spectra were acquired using a Micromass MALDI-Micro MX mass spectrometer (Waters, Manchester, U.K.) operated in positive reflectron mode. Instrument control, spectral acquisition, and processing were performed with MassLynx software version 4.1 (Waters, Milford, MA). ESI−MS/MS

Samples (3 μL) of Fcγ receptor N-glycans following PGC cleanup were placed on the surface of a Nafion 117 membrane (Aldrich, Poole, Dorset, U.K.) which was atop water for further desalting. Spots were removed, and 1 μL of a 1:1 (v/v) mixture of water/methanol containing 0.2 M ammonium phosphate was added. The samples were transferred to a Proxeon borosilicate capillary (Proxeon Biosystems, Odense, Denmark) and injected into a Micromass QToF Ultima Global mass spectrometer (Waters, Manchester, U.K.). MS/MS spectra (collision-induced dissociation) were acquired in negative ion mode. Data acquisition and processing were conducted with Waters MassLynx version 4.1.

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RESULTS

The Carbohydrates of Fcγ Receptors Consist of High-Mannose and Complex Multiantennary Structures

Antennarity Is Varied between FcγRs. The principal aim of this study focused on the N-glycan composition of commercial Fcγ receptors produced in NS0 cells. Specifically, five Fcγ receptors (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) were investigated. Table 1 summarizes the key features of each Fcγ receptor. To ascertain the complexity of glycosylation present on each FcγR, N-linked glycans were released with PNGase F, fluorescently labeled with 2AB, and separated using HILIC with fluorescence detection (FLD). Assessment of the resulting chromatograms indicated extensive variability and complexity within and across each Fcγ receptor sample (Figure 1). Using GU values together with structural assignments via exoglycosidase arrays and subsequent confirmation by mass spectrometry, several structures were found to be common to all receptor subtypes. Numerous structures were also found to be exclusive to individual receptors. A total of 12 glycan structures were common to all Fcγ receptors, although the relative distribution of these common structures varied significantly between receptors (Table 2). Mono- and biantennary branched structures were found predominantly on FcγRIa and FcγRIIa, while tri- and tetraantennary structures constituted the majority of branched structures on the remaining FcγRs (Figure 2). FcγRs Demonstrate Lot-to-Lot Variance. To determine lot-to-lot variability, three separate lots of each FcγR were purchased from R&D Systems and analyzed for N-linked glycan content. The glycan pool composition was found to be consistent for each Fcγ receptor with the same peaks observed across three separately released batches (data not shown). However, significant differences were found in the distribution of the common structures within the glycan pool. As a result, the relative standard deviation observed for individual peaks in

Figure 1. N-Linked glycans released from Fcγ receptors demonstrate significant complexity and heterogeneity. Fcγ receptors containing murine-derived N-linked glycosylation were assessed for glycan content by enzymatically releasing the carbohydrate structures and analyzing using hydrophilic interaction HPLC. Each glycan pool was fluorescently labeled with 2-aminobenzamide and separated using TSKgel amide-80 columns. Peaks common to all FcγR glycan pools are identified by vertical dashed lines and numerical annotation. Key: (A) FcγRIa, (B) FcγRIIa, (C) FcγRIIb, (D) FcγRIIIa, and (E) FcγRIIIb.

each Fcγ receptor glycan pool was found to be significantly high, suggesting variability in the relative abundance of common structures across different batches of Fcγ receptors (Table 3). Sialylation of FcγRs Demonstrates Variability in Abundance and Heterogeneity. Analysis of sialylation was performed using either WAX-HPLC with fluorescence detection or HILIC-FLD. HILIC-based analysis of sialic acid content was determined through comparative assessment of the undigested Fcγ receptor glycan samples with equivalent ABStreated samples. Monitoring the effect of ABS on each glycan pool provided a means of identifying the peaks containing sialic acid as well as their relative percentages. For each sample, the total peak area change due to ABS treatment was measured and 3724



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Table 2. Summary of Common N-Glycan Structures Identified in All Fcγ Receptorsa FcR relative peak area (%) peak

GU

m/z

composition

structure

IA

IIA

IIB

IIIA

IIIB

x̅

σ

1 2 3 4 5 6 7 8 9 10 11 12

6.21 6.84 7.88 8.07 8.48 8.90 9.61 9.99 10.45 10.96 11.24 12.38

1257.42

Hex5HexNAc2 Fuc1Hex3HexNAc6 Hex5HexNAc2 Fuc1Hex5HexNAc5 Fuc1Hex5HexNAc6 Fuc1Hex6HexNAc5 Fuc1Hex7HexNAc5 Fuc1Hex7HexNAc5 Fuc1Hex8HexNAc5 Fuc1Hex7HexNAc5NeuAc1 Fuc1Hex9HexNAc5 Fuc1Hex11HexNAc6

M5 F(6)A4 F(6)A3G1Gal1 F(6)A3G2 F(6)A4G1Gal1 F(6)A3G3 F(6)A3G3Gal1 F(6)A3G2Gal2 F(6)A3G3Gal2 F(6)A3G3Gal1S1 F(6)A3G3Gal3 F(6)A4G4Gal4

14.41 0.59 0.79 5.02 6.83 5.67 2.80 3.16 5.23 3.24 15.93 1.22

0.27 0.29 1.68 1.89 2.71 4.54 7.36 4.68 11.70 17.69 16.05 1.88

0.16 0.13 1.66 2.87 1.54 4.37 4.58 6.82 7.53 3.36 20.12 4.27

0.38 1.14 0.54 3.64 4.66 4.84 4.66 4.01 5.08 4.47 24.08 2.21

2.65 0.49 1.48 4.97 3.11 6.17 4.87 4.46 13.89 16.02 13.41 2.15

3.57 0.53 1.23 3.68 3.77 5.12 4.85 4.63 8.69 8.96 17.92 2.35

6.15 0.39 0.53 1.35 2.04 0.77 1.63 1.36 3.95 7.25 4.20 1.14

1581.53 2012.72

2336.83 2498.88 2660.93

a Following characterization of each Fcγ receptor, common structures were identified and tabulated. GU values together with m/z values and exoglycosidase digestions confirmed the presence of each structure. The relative peak area for each common structure is presented with the average peak area (x)̅ reported with the standard deviation (σ). Glycan naming is performed as outlined in the Oxford notation.

composition of charged glycan structures present in each Fcγ receptor glycan pool and assisted in categorizing the sialic acid content into mono-, di-, tri-, and tetrasialylated species (data not shown). A representative result of sialic acid content determination is presented in Figure 3. FcγRIa contained approximately 24% sialylation, present primarily as monosialylated structures and distributed across five separate structures (see Figure 3 and Table 2). FcγRIIa contained an equivalent amount of approximately 24%, although sialylation was observed as mono-, di-, and triantennary structures distributed over 11 peaks. In contrast, FcγRIIb contained less overall sialylation of only approximately 18% predominantly as two monosialylated structures. FcγRIIIa contained four monosialylated structures totaling approximately 27% of the relative peak area, while FcγRIIIb contained nearly 35% sialylation with a total of seven structures ranging from monosialylated to trisialylated. Generally speaking, the FcγRs displayed heterogeneity in both the amount of sialylation and the extent of terminal sialic acids present. It is particularly interesting to compare the different FcγR classes, which contain extensive homology in their extracellular domains. FcγRIIa contained more sialylation and greater diversity of sialylated structures in general than FcγRIIb. Similarly, FcγRIIIb contained a greater extent and diversity of sialylation compared to FcγRIIIa. Reasons for this are unclear, but it is interesting to observe the different molecular functions of each receptor and what impact glycosylation may have on their cellular role. High-Mannose Structures Vary in Abundance and Distribution across FcγR. Early processing of N-linked glycans generates a number of high-mannose structures in the endoplasmic reticulum, typically producing structures from M9 down to M4, which are usually processed to complex glycans. To measure the oligomannose content, N-linked glycans were sequentially digested with exoglycosidase arrays that exposed terminal mannose residues. Exoglycosidase treatment and subsequent separation of the digested glycans by HILIC-FLD allowed for the quantitation of the various high-mannose structures. All tentative high-mannose structures were confirmed by further digestion of the glycan pool with JBM, which exhibits enzymatic activity only on structures containing terminal mannose. In the present study high-mannose structures refer only to structures bearing four terminal mannose (M4) to nine

Figure 2. Fcγ receptor N-glycans contain varying amounts of branched structures. The abundance of antennary structures within glycan pools obtained from each Fcγ receptor was determined by enzymatically reducing each glycan population to branched, hybrid, and highmannose structures. Symbols denoted on the right of each chromatogram indicate the individual monosaccharides removed through exoglycosidase digestion. Peaks are identified with structures reported in Oxford notation. Key: (A) FcγRIa, (B) FcγRIIa, (C) FcγRIIb, (D) FcγRIIIa, and (E) FcγRIIIb. Peaks denoted by an asterisk refer to a structure with an unconfirmed structural composition.

used to approximate the amount of sialylation in each sample. WAX-HPLC was also used to study the sialic acid content of the receptors. This technique was used to analyze the 3725



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Table 3. Total N-Glycan Structural Composition Determined across All Fcγ Receptorsa

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Table 3. continued

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Table 3. continued

a

Structures identified within each Fcγ receptor sample are tabulated. All N-glycans are reported in GU values with structure and structure name using the Oxford notation. For each GU value, the relative peak area (RPA) is reported with the standard deviation (±σ). Standard deviation values are based on the analysis of three separate lots of each Fcγ receptor. The presence of values within a cell position indicates the identification of the given structure within the NS0 FcγR sample. The relative standard deviation (RSD, %) is reported to give a reflection of the variability observed between different lots of the same NS0 Fcγ receptor. All peaks with a relative peak area greater than 0.5% are reported.

terminal mannose (M9) residues. FcγRs expressed in NS0 cells were selectively found to contain M4 to M9. The total proportions of oligomannose structures ranged from less than 2% to as high as 16% of the total glycan population for individual Fcγ receptors (Figure 4). FcγRIa demonstrated the largest content of high-mannose structures (16%), with over 13% of the total glycan pool dominated by M5 alone, suggesting that it is at one site protected from further processing by subsequent glycosyltransferases. The remaining 3% was comprised of M4, M6, and M7. The increased level of high mannosylation did not appear to affect the ability for full glycan processing of the remaining glycan pool as the level of sialylation was still comparable to that of the other FcγRs. FcγRIIa contained the least amount of high-mannose structures, with just under 2% of the total glycan population represented by M5 and M6. FcγRIIb contained nearly 4% high mannose structure in the form of M6, M7, and M8, while both FcγRIIIa and FcγRIIIb contained nearly 6% of each highmannose structure. Despite similar peak area percentages, FcγRIIIa and FcγRIIIb were markedly different in the diversity

of high-mannose structures. FcγRIIIa contained M4, M5, and M6, while FcγRIIIb contained all structures from M4 to M9. Reasons for the differences in high-mannose diversity are not clear at this point, but represent a unique and defining feature between the various FcγRs. Fcγ Receptors Contain Extensive Gal-α(1,3)-Gal Epitopes

Murine-derived recombinant proteins incorporate monosaccharide linkages not typically found in humans, such as galactose linked to galactose in an α(1−3) orientation.34 This terminal disaccharide is known to be immunogenic in humans,35,36 and therefore, it is important to identify any glycans bearing this epitope. To determine if such moieties existed in the NS0-expressed Fcγ receptors, two approaches were utilized, including HILIC-FLD with exoglycosidase arrays and negative mode ESI−MS/MS. To identify structures containing galactose-α(1−3)-galactose, CBG causes a signature GU shift on N-glycans containing the Gal-α-Gal epitope, where glycan peaks shift by 0.8−0.9 GU per α-galactose monomer when assessed by HILIC-FLD. Glycans released from each receptor were used in targeted digestions using enzyme 3728



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reactions containing CBG. A representative chromatographic comparison is illustrated in Figure 5. Many of the dominant peaks in each chromatogram demonstrated a shift corresponding to a loss of at least one and at most four galactose monomers linked in an α(1−3) position. The overall change in peak area due to CBG activity was subsequently used to determine the total amount of the glycan pool containing α-galactose. Of the five Fcγ receptors analyzed, FcγRIIb contained the highest amount of α-galactosylation with an approximate amount of 71% of the total glycan pool. FcγRIa contained the lowest amount of α-galactose with an approximate amount of 35%. The lower amount of α-galactose present on FcγRIa is possibly due to the elevated levels of highmannose structures observed with FcγRIa, although the exact relationship between mannose content and α-galactosylation as it pertains to Fcγ receptor biology remains to be determined. FcγRIIa was found to contain approximately 56% αgalactosylation, with FcγRIIIa and FcγRIIIb determined to have 61% and 44%, respectively. It is evident from these numbers that α-galactose is a prominent monosaccharide on NS0-derived FcγRs. FcγRs Contain N-Glycolylneuraminic Acid

With evidence supporting the presence of sialic acids on each Fcγ receptor and reports indicating the prevalence of Nglycolylneuraminic acid (Neu5Gc) in NS0 cells, 37 we determined if any of the receptor-associated sialic acids were present as Neu5Gc. ESI−MS/MS confirmed the presence of Neu5Gc within the NS0 Fcγ receptor N-glycan pool. Neu5Gc structures were identified by tandem ESI−MS of [M − H + H2PO4] precursor ions (Figure 6). The fragmentation spectra of sialic acid containing negative ions results predominantly in the detection of the sialic acid monosaccharide peaks. The Btype fragment of Neu5Gc is detected at 307 m/z (Nacetylneuraminic acid (Neu5Ac) B-type ions are detected at 290 m/z). This and the isolated precursor mass distinguished Neu5Gc-containing structures. Glycans Contain poly(N-acetyllactosamine)

Core branched structures are identified and quantified using a series of exoglycosidases that truncate the glycan pool to structures bearing terminal GlcNAc residues. In most cases, the largest branched structure identified is the tetraantennary structure A4 with a GU value of approximately 6.4. In situations where structures with GU values greater than 6.4 persist, other glycan modifications are possible that can be identified with selected exoglycosidase digestions. Glycans that are likely to appear with a larger mass than the core antennary structures after an ABS/BTG/CBG/BKF digestion typically include either high-mannose structures or branched structures containing poly(N-acetyllactosamine) (LacNAc) repeats. Glycan peaks containing repeating units of 1.3 GU values in combination with the core antennary structures indicate the presence of LacNAcs. This is based on the composition of a LacNAc consisting of a GlcNAc (0.5 GU) and a galactose (0.8 GU). The presence of higher mass structures suggested either multiple high-mannose structures (M5 to M9) or terminal glycan structures that prevented complete digestion of the glycan pool with the exoglycosidases ABS, BTG, BKF, and CBG used in each reaction (Figure 7). Reducing end glycan termination by motifs such as outer arm fucosylation or LacNAc repeats could potentially explain the persistence of higher order glycan structures. To confirm the presence of LacNAcs, glycans were digested using ABS, CBG,

Figure 3. FcγR sialylation is identified by enzymatic digestion. Sialidase digestion of each Fcγ receptor glycan pool was performed to determine the abundance of sialylation. FcγRIIIb N-glycans were used as a representative for each Fcγ receptor glycan pool. (A) WAXHPLC-FLD was used to separate the FcγRIIIb glycan pool on the basis of charge, where 2AB-labeled N-glycans from fetuin were used to identify retention positions of neutral glycans and glycans containing one (mono), two (di), three (tri), and four (tetra) terminal sialic acids. Each sialylated peak was confirmed by separating ABS-digested glycans from the FcRIIIb glycan pool and identifying missing peaks. Additional charges not arising from sialylation were also observed and are denoted by an asterisk. (B) HILIC-FLD chromatograms for both the undigested and ABS-digested N-glycans from FcγRIIIb were used to identify individual peaks containing sialic acid. Peak migrations from sialylated to neutral structures are denoted by dashed arrows between each chromatogram. Sialylated peaks are annotated using the Oxford linear notation.

compositions with and without CBG. All digestion reactions were separated by HILIC, and shifts corresponding to losses of α-linked galactose monomers were observed following 3729



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Figure 4. Extent of high-mannose structures in various NS0 FcγRs. High-mannose structures were identified for each FcγR through exoglycosidase array analysis using ABS, CBG, BTG, BKF, and GUH. Total high-mannose structure abundance was determined by quantitation of each highmannose peak present in HILIC-FLD chromatograms. The relative abundance of high-mannose glycans for each FcγR is illustrated as a bar graph, and the distribution (M4 to M9) for each FcγR is depicted with pie charts.

corresponding to a Fuc-Hex C- and B-type fragment, respectively. Furthermore, the E-type m/z 1139 ion from the 3-arm located the fucose on either GlcNAc present on this arm; however, the linkage position of fucose was not identified. Mass spectrometric analysis of FcγRIIIb was able to demonstrate the presence of multiple structures with outer arm fucosylation and, in some instances, multiple fucose monosaccharide residues per N-linked glycan. To determine the linkage of outer arm fucose in each Fcγ receptor, α-fucosidases specific for terminal fucose in various linkage positions were used. AMF, which cleaves fucose linked to N-acetylglucosamine in either an α(1−3) or α(1−4) position was used with no effect on any of the glycan samples (data not shown). To ensure full access for the enzyme to its substrate, terminal monosaccharides that potentially blocked access of AMF were first removed before digestion with AMF. This had no impact on the activity of AMF. The second fucosidase used was XMF, which specifically removes α(1−2)fucose linked to galactose. Similar to AMF, no obvious effect was observed (data not shown) despite similar efforts to remove any interfering terminal monosaccharides. Interestingly and somewhat unexpectedly, BKF (1 mU μL−1) was found to be more capable of removing outer arm fucose when compared to AMF, where the latter demonstrated no activity on any FcγR glycan pool (data not shown). BKF releases α(1−2)- and α(1−6)-linked nonreducing terminal fucose residues more efficiently than α(1−3)- and α(1−4)-

BTG, BKF, and GUH, and the resulting chromatogram was analyzed and compared to the same digest without GUH. Following digestion with GUH, all higher mass structures digested to the M3 core, indicating that all structures above GU values of 6.4 most likely correlated to structures containing LacNAc repeats. Using the known incremental shifts in GU values due to exoglycosidase activity, the composition of the various LacNAc-containing structures was determined. This HILIC and exoglycosidase approach identified structures containing both one and two terminal LacNAcs. The relative proportions of LacNAc structures was low in all cases, contributing from as low as 1% to no greater than 6% of the total glycan pool in all the Fcγ receptors studied. FcγRs Contain Outer Arm Fucosylation

The possibility of glycan modification by outer arm fucosylation was investigated due to the appearance of undigested glycan during routine exoglycosidase array analysis. To avoid issues with enzyme specificity and/or activity, mass spectrometry was used to search for structures containing more than one fucose. Outer arm fucosylation was initially confirmed by ESI−MS/MS of negative ions. The MS/MS spectrum of the Hex8HexNAc 5Fuc2 [M + H2PO 4]− ion from FcγRIIIb identified fucose linked to the reducing end GlcNAc residue from the 2,4AR fragment as well as terminally from the 510 and 570 m/z ions (Figure 8C). The outer arm fucose was linked to a GlcNAc residue from the absence of 370 and 325 m/z ions, 3730



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Figure 5. NS0 FcγRs contain extensive α-galactosylation. Released and 2AB-labeled glycans from each FcγR were first digested with the sialidase ABS to remove terminal sialylation (A). The asialylated glycan pool was then digested with coffee bean α-galactosidase (B) followed by bovine testes β-galactosidase (C) to determine which peaks contained the differentially linked galactose structures. The chromatograms presented are based on data obtained from FcγRIIa and are representative of a common outcome for all FcγRs. Dashed lines represent the migration of various structures due to enzyme activity. All peaks are identified and reported using the Oxford linear notation.

Figure 6. N-Glycolylneuraminic acid residues are expressed on Fcγ receptors. The nano-ESI−MS/MS spectrum of the m/z 1430.4 [M − H + H2PO4]2− ion from FcRγIIIb demonstrated the presence of NGNA residues. The 306.1 m/z peak corresponds to the B-fragment of the NGNA monosaccharide.

The migration of the F(6)A3F1 peak to the A3 peak following BKF digestion provided evidence that BKF was able to remove both the core α(1−6) fucose and the outer arm fucose. Due to the ability of BKF to remove both α(1−3) and α(1−4) fucose, the exact linkage of the outer arm fucose could not be

linked fucose under the reaction conditions used. Outer arm fucosylation was identified in the Fcγ receptor samples through observation of an unidentifiable peak during routine exoglycosidase digestion. Subsequent analysis revealed a bifucosylated triantennary structure, F(6)A3F1 (Figure 8A). 3731



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Figure 7. N-Acetyllactosamine is identified in NS0 FcγRs. To identify the presence of N-acetyllactosamine (LacNAc) structures, each 2AB-labeled FcγR glycan pool was first digested with ABS, CBG, BTG, and BKF to reduce the glycan pool to structures containing terminal N-acetylglucosamine (GlcNAc), hybrids, and high-mannose structures (A). A hexosaminidase (GUH) was used to identify peaks containing terminal GlcNAc (B). Comparison of the two resulting chromatograms allowed for identification of LacNAc structures.

However, because this carbohydrate structure is not found in humans, any positive or negative effect on IgG binding will not be representative of a natural IgG interaction. It is also worth noting that potential therapeutic use of these receptors could elicit an immune response due to circulating anti-α-Gal antibodies, resulting in an undesirable reaction. This has been found previously with therapeutic proteins containing α-Gal epitopes.35 The presence of terminal α-galactosylation also causes a reduction in the extent of sialylation, which may have negative connotations for FcγR interactions. Sialyltransferase competes for the same acceptor substrate as α(1−3) galactosyltransferase, meaning that the presence of an α-Gal residue on a terminal galactose will prevent addition of a sialic acid. This could potentially affect the net charge of the receptor-associated glycans and could also interfere with IgG binding.

determined. Despite this, evidence is presented that demonstrates Fcγ receptors expressed in NS0 cells contain both core fucose and outer arm fucose linked to N-acetylglucosamine.

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DISCUSSION

α-Galactosylation

Analysis of Fcγ receptors expressed in NS0 cells revealed several interesting attributes. Perhaps the most significant attribute identified was the presence of extensive galactoseα(1−3)-galactose epitopes throughout all Fcγ receptor subtypes. Glycan analysis revealed that, in all cases, the most abundant carbohydrates contained α-galactosylation present on bi-, tri-, and tetraantennary structures (Figure 5). In comparison to previously published Fcγ receptor glycan analyses from BHK, 38,39 HEK293,40 and CHO, 40 the carbohydrate structures presented here differ significantly and further reinforce the assertion that Fcγ receptors exhibit different glycoprofiles depending on the expression system used. FcγRIIIa, for example, when expressed in the human cell line HEK293, will contain mainly biantennary and hybrid glycans with terminal GalNAc residues, while in CHO cells it will predominantly present core fucosylated bi- and triantennary glycans.40 Detailed IgG interaction analysis with NS0-expressed FcγRs is required to determine the potential binding effects of α-Gal epitopes. To our knowledge this information is not available.

Fcγ Receptor Outer Arm Fucosylation

It has been previously established that BKF is capable of removing fucose from GlcNAc in a number of linkage positions, including α(1−3), α(1−4), and α(1−6). The possibility that BKF was removing fucose in a position inaccessible by either XMF or AMF was considered. The activity of BKF on outer arm fucose and its ability to remove fucose in either an α(1−3) or α(1−4) linkage to Nacetylglucosamine was an unexpected finding observed in the present study. Somewhat surprisingly, both AMF and XMF, 3732



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Figure 8. Fcγ receptors contain outer arm fucose. HILIC-FLD and ESI−MS/MS were used to confirm the presence of outer arm fucose on FcRIIIb. For HILIC-FLR analysis, N-linked glycans released from FcγRIIIb were digested with (A) ABS, BTG, and CBG or (B) ABS, BTG, CBG, and BKF to identify the presence of outer arm fucose. Chromatograms of the two digestion reactions indicated the movement of key peaks due to enzyme activity. The undigested glycan pool was subjected to static nanoinfusion ESI−MS/MS analysis (C) where diagnostic fragment ions were detected and confirmed the observations in the HILIC-FLD data.

two exoglycosidases capable of removing α(1−3)- or α(1−4)linked nonreducing terminal fucose, were unable to remove this residue. While BKF demonstrated activity where AMF and XMF could not, terminal fucose structures continued to persist that were unable to be modified by any of the available αfucosidases. N-Glycans digested with ABS, CBG, BTG, and BKF followed by analysis with HILIC-FLD revealed the

persistence of a peak with an approximate GU value of 5.6 (Figure 8A). The composition of this peak was determined to be M3A1F1, indicating an inability of any of our fucosidases for removing the fucose. Outer arm fucose was found predominantly on triantennary structures, but its exact arm specificity could not be determined. It is possible that one of the terminal GlcNAcs prevented access of BKF to the outer arm fucose, consequently generating a glycan structure in the form of A3F1. 3733



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Glycosylation of Human Fcγ Receptors Depends on the Cellular Expression System

If this is the case, it is interesting to observe that BKF is able to access outer arm fucose in a manner different from that observed for AMF. It is also possible that BKF is able to remove terminal fucose in a new linkage position, namely, a fucoseα(1−2)-GlcNAc, possibly an α(1−6)-linked fucose to a nonreducing GlcNAc, or indeed a fucose in an arm-specific nature. However, these suggestions are speculative and require further investigation.

To our knowledge three previous glycan analysis studies have been performed for recombinant human Fcγ receptors. These include both FcγRIIa38 and FcγRIIIb39 expressed in BHK cells and FcγRIIIa expressed in either HEK293 cells or CHO cells.40 As expected FcγRIIa and FcγRIIIb produced in BHK cells exhibit hamster kidney specific glycosylation patterns with α(2−3)-linked sialic acids only, no bisecting GlcNAc, and the presence of GalNAc residues. Eighteen and thirty-nine individual structures were identified for FcγRIIa and FcγRIIIb, respectively. Multiantennary structures containing up to four GlcNAcs with or without sialic acid capping were also described. While similarities exist between these analyses and the data presented here for the equivalent receptors from NS0 cells, such as the presence of oligomannose and multiantennary glycans, significant differences also exist, such as the presence of Neu5Gc and α-Gal and the absence of GalNac epitopes. More recently, a detailed site-specific analysis of the glycans present at each position of FcγRIIIa expressed in both CHO and HEK293 cells was reported. This illustrated stark differences in the glycans observed at individual sites on the Fcγ receptor.40 Specifically, an abundance of glycan structures containing outer arm fucose and GalNAcs linked to GlcNAcs were found on HEK-expressed FcγRIIIa, while CHO cells expressed FcγRIIIa with mainly complex sialylated biantennary structures. Interestingly, different glycoforms were observed at different sites on FcγRIIIa, providing evidence that glycoform subsets occur in a site-specific manner. In both HEK and CHO, there were no reports of α-galactosylation; however, outer arm fucose was described. Of additional interest was the observation of the effect of outer arm fucosylation on sialylation. Sialylation was not observed on antennae containing outer arm fucose as it is competitive. It is worth noting that depending on the expression system used human Fcγ receptors will have significantly different glycan contents, with or without immunogenic epitopes, and these glycosylation differences are likely to affect the interaction with IgG.

High-Mannose Glycans and Antennarity

The Fcγ receptors differ dramatically in their relative abundance and diversity of high-mannose structures as well as their distribution of antennary structures. The exact function of this diversity remains unclear, but future work is aimed at defining the glycovariants that exist at individual N-linked sites on these receptors as site occupancy of the Fcγ receptors is likely to be important. An interesting example of this is evident with FcγRIIIa, where one glycosylation site at Asn-162 has been shown to be important for positive antibody binding and a different site at Asn-45 has been shown to have inhibitory effects on afucosylated IgG binding.15 Zeck et al.40 have performed an extensive site occupancy study of Asn-162 of FcγRIIIa expressed in both HEK and CHO cells and show that different glycosylation patterns produced influence binding of IgG. Unfortunately, no significant structural information is available with regard to the glycosylation site occupancy based on the solved crystal structures of FcγRIIIb41 FcγRIIIa,42 FcγRIIa,43 and FcγRIa.44 In the case of FcγRIa and FcγRIIa, which are expressed in CHO and insect cells, respectively, the only glycans visible in the electron density map are away from the Fc−Fcγ receptor interface. Noting that the presence of carbohydrates is usually refractory to crystallization, it is likely that identified structures were the only crystallizable complexes and therefore not representative of the complete interaction. This is evident in the glycosylated FcγRIIIa structure complex with or without afucosylated antibody which shows carbohydrates in the binding interface play a pivotal role in the interaction and explain the higher affinity for afucosylated IgG to FcγRIIIa.45

Use of Fcγ Receptors in IgG Binding and Therapeutic mAb Evaluation

Receptor-Associated Neu5Gc

Recombinant soluble Fcγ receptors are commonly used in industry to assess the binding of potential therapeutic monoclonal antibodies and academia to investigate the binding of IgG and other nonantibody ligands. Through bioanalytical techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), the binding affinity of IgGs to Fcγ receptors and association/dissociation rates can be determined and used as a measure of performance and effectiveness of the antibody. In fact, regulatory agencies such as the FDA and EMA insist on such measurements being performed. Several publications have referenced the use of NS0-derived Fcγ receptors for use in SPR studies of antibody binding affinity.26,29,30,49−52 It is worth noting that in many cases these analyses are performed without a detailed knowledge of the glycosylation of the recombinant receptors used in the assays, and this is one of the main reasons why the work described here was performed. Interestingly, recent studies evaluating the biological activity of afucosylated antiCD20 antibody with FcγRIIIa has shown that specific glycan sites on the receptor significantly affect the binding of antibodies. Crystallization of glycosylated FcγRIIIa with an afucosylated antibody revealed a unique interface in the

Terminal Neu5Gc is a known feature of murine glycosylation but is considered a nonhuman sialic acid. It is present in many mammals, including chimpanzees and mice due to activity of the enzyme cytidine monophosphate N-acetylneuraminic acid hydroxylase (CMAH), which catalyzes the conversion of Neu5Ac to Neu5Gc.46 Negative mode ESI−MS/MS of released NS0 Fcγ receptor glycans clearly demonstrated the presence of this carbohydrate moiety. However, the relative abundance of this structure could not be determined in this study. Humans primarily synthesize the sialic acid Neu5Ac for incorporation into glycans but due to an inactivating mutation in the gene coding for the enzyme CMAH are unable to hyroxylate Neu5Ac to Neu5Gc.47 Similar to murine α-Gal epitopes, Neu5Gc is capable of acting as an antigen to stimulate the production of anti-Neu5Gc antibodies, and circulating antiNeu5Gc IgG has been found in healthy humans.48 This mammalian nonhuman sialic acid could therefore induce an immune response or result in neutralization and removal of biopharmaceuticals, which reinforces the need for vigilence toward glycan analysis of glycoprotein therapeutics expressed in murine cell lines. 3734



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structures reported here are not typically found in humans. It is widely accepted that key glycan epitopes such as αgalactosylation and N-glycolylneuraminic acid are a concern for regulatory agencies due to their known immunogenicity in humans, but what impact these particular properties have on in vitro binding studies remains to be seen. Neu5Gc and Neu5Ac differ by one oxygen atom at the C5 position, but is this unlikely to affect the affinity for IgG or kinetic rates of association and dissociation? Similarly, the replacement of terminal sialylation with terminal α-galactosylation significantly augments the charged glycan composition of Fcγ receptors, but understanding the overall impact on FcγR biology is currently unclear. Further complicating the matter is the fact that the local heterogeneity of glycosylation at individual sites throughout each Fcγ receptor is not yet known. Precise glycan distribution at individual sites remains paramount to fully elucidating the function of glycans in Fcγ receptor biology. Finally, knowledge of glycosylation of recombinant Fcγ receptors will only bear importance when the glycosylation status of their naturally occurring counterparts is elucidated, in both dormant and active states.

antibody−receptor complex consisting of carbohydrate− carbohydrate interactions.45 Significantly, these interactions are weakened or nonexistent in the complex of receptor and core-fucosylated antibody. On the basis of these important observations, it now seems increasingly evident that a role for glycosylation on FcγRs exists and that binding between IgGs and FcγRs is likely mediated in part by direct carbohydrate− carbohydrate interactions. Glycosylation of Fcγ Receptors in Their Natural Environment: The Key Challenge

Significant efforts have been made to determine the extent and nature of glycosylation of Fcγ receptors expressed in NS0 cells, principally as it is abundantly clear that the glycosylation state of Fcγ receptors affects IgG binding.14,15 In situations where glycosylation of a receptor or a ligand plays a prominent role in the binding interaction, it is far from ideal to lack detailed knowledge of how each binding partner is glycosylated, particulary in the case of in vitro versus in vivo and in vivo veritas situations. This can lead to spurious results, misleading data, and potentially dangerous situations if immunogenic carbohydrates are used in biotherapeutic applications. However, the key objective for Fcγ receptor glycobiology should not be focused around characterizing the glycosylation of recombinant glycoproteins expressed in cell lines and instead should be centered around understanding the glycosylation status of Fcγ receptors as they appear on innate effector cells such as macrophages and natural killer cells. Several important papers by Kimberly et al. used traditional glycoprotein analysis techniques to describe the glycosylation status of Fcγ receptors present on a variety of immune effector cells.53,54 Many critical features were described, such as the cell-type-specific glycosylation of Fcγ receptors and differential ligand binding to these receptors. Edberg and Kimberly demonstrated that FcγRIIIa expressed by monocytes differed in terms of its glycosylation when compared to FcγRIIIa expressed by NK cells.55 When expressed by NK cells, this receptor was shown to contain high-mannose- and complex-type oligosaccharides; however, when found on monocytes, it possessed no high-mannose-type glycans. This intriguing result suggests that natural glycoforms of Fcγ receptors are present as cell-type-specific glycoforms which bind IgG differentially. This has implications both for activation and for modulation of the immune system. Edberg et al. have also proposed that the presence of high-mannose structures is important for opsonin-independent receptor ligation, which is a key feature for the phagocytic uptake of bacterial pathogens displaying lectin-type adhesins (fimbriated Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium). This is fascinating, as it suggests that Fcγ receptors also have a function in nonclassical receptor ligation, although this has yet to be demonstrated experimentally. This potentially explains the presence of high-mannose structures in all of the Fcγ receptors described here. It remains a challenge, however, to determine the cell-type-specific glycosylation of Fcγ receptors and how it dictates ligand binding and downstream effector functions. Until this can be adequately described, the role of Fcγ receptors in immune function cannot be fully understood.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +353 (0)1 215 8100. Fax: +353 (0)1 215 8166. Notes

The authors declare no competing financial interest

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REFERENCES

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Closing Comments

The work presented here highlights the distinct glycan structural properties observed through recombinant expression of Fcγ receptors in NS0 cells. Importantly, many of the glycan 3735



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