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The Journal of Immunology, 2008, 181, 6664 -6669
Copyright © 2008 by The American Association of Immunologists, Inc.
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Crucial Role of Aspartic Acid at Position 265 in the CH2 Domain for Murine IgG2a and IgG2b Fc-Associated Effector Functions1

Lucie Baudino*, Yasuro Shinohara{dagger}, Falk Nimmerjahn{ddagger}, Jun-Ichi Furukawa{dagger}, Munehiro Nakata§, Eduardo Martínez-Soria*, Franz Petry, Jeffery V. Ravetch||, Shin-Ichiro Nishimura{dagger} and Shozo Izui2,*

* Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland; {dagger} Laboratory of Advanced Chemical Biology, Graduate School of Advanced Life Science, Hokkaido University, Sapporo, Japan; {ddagger} Laboratory for Experimental Immunology and Immunotherapy, Nikolaus-Fiebiger-Center for Molecular Medicine, University of Erlangen-Nuremberg, Erlangen, Germany; § Department of Applied Biochemistry, Tokai University, Hiratsuka, Kanagawa, Japan; Institute of Medical Microbiology and Hygiene, Johannes Gutenberg-University, Mainz, Germany; and || The Rockefeller University, New York, NY 10065


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Replacement of aspartic acid by alanine at position 265 (D265A) in mouse IgG1 results in a complete loss of interaction between this isotype and low-affinity IgG Fc receptors (Fc{gamma}RIIB and Fc{gamma}RIII). However, it has not yet been defined whether the D265A substitution could exhibit similar effects on the interaction with two other Fc{gamma}R (Fc{gamma}RI and Fc{gamma}RIV) and on the activation of complement. To address this question, 34-3C anti-RBC IgG2a and IgG2b switch variants bearing the D265A mutation were generated, and their effector functions and in vivo pathogenicity were compared with those of the respective wild-type Abs. The introduction of the D265A mutation almost completely abolished the binding of 34-3C IgG2a and IgG2b to all four classes of Fc{gamma}R and the activation of complement. Consequently, these mutants were hardly pathogenic. Although oligosaccharide side chains of these mutants were found to contain higher levels of sialic acids than those of wild-type Abs, the analysis of enzymatically desialylated D265A variants ruled out the possibility that very poor Fc-associated effector functions of the D265A mutants were due to an increased level of the mutant Fc sialylation. Thus, our results demonstrate that aspartic acid at position 265 is a residue critically implicated in triggering the Fc-associated effector functions of IgG, probably by defining a crucial three-dimensional structure of the Fc region.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Fc region of IgG Abs interacts with IgG Fc{gamma}R and complement, thereby mediating Ab-dependent effector functions. Three classes of activating Fc{gamma}R (Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV) and one inhibitory receptor (Fc{gamma}RIIB) are expressed on murine immune effector cells (1). They mediate various cellular responses, such as phagocytosis by macrophages, Ab-dependent cell-mediated cytotoxicity by NK cells, and degranulation of mast cells (2). Each Fc{gamma}R displays different binding affinities and IgG subclass specificities. Fc{gamma}RI is the only receptor capable of binding monomeric IgG2a with high affinity; low-affinity Fc{gamma}RIIB and Fc{gamma}RIII are specific for IgG1, IgG2a and IgG2b; and Fc{gamma}RIV binds IgG2a and IgG2b with intermediate affinity. Notably, the relative balance of engagement of activating and inhibitory Fc{gamma}R is critical for the overall effector functions of individual IgG subclasses in vivo (1). Furthermore, the classical pathway of complement activation is also dependent on IgG subclass characteristics because only IgG2a, IgG2b, and IgG3, but not IgG1, efficiently activate complement (3, 4).

The CH2 domain of the Fc portion carries N-linked biantennary complex-type oligosaccharide side chains. These oligosaccharide structures are highly heterogeneous in terms of galactosylation and sialylation: most of them are core fucosylated, and end with two galactose residues, one galactose and one N-acetylglucosamine, or two N-acetylglucosamines (5, 6). However, a small but significant fraction of galactosylated oligosaccharide chains bear one or two terminal sialic acids. It has been believed that glycosylation is required for IgG integrity and for the optimal activation of effector mechanisms through complement and Fc{gamma}R. Notably, the interaction with activating Fc{gamma}R was substantially down-modulated by the presence of terminal sialic acid residues in IgG oligosaccharide side chains (7, 8).

The binding sites for C1q and Fc{gamma}R are located in the CH2 domain of IgG. Mutagenesis analysis of an IgG2b mAb identified the glutamic acid, lysine, and lysine residues at positions 318, 320, and 322, respectively, as a key binding motif for C1q (9). In addition, amino acid residues at positions 234–238 are involved in the high-affinity interaction of murine IgG2a with Fc{gamma}RI (10, 11). Furthermore, replacement of aspartic acid by alanine at position 265 (D265A) has been shown to abrogate the interaction of murine IgG1 with the low-affinity Fc{gamma}RIIB and Fc{gamma}RIII (12, 13). It has also been reported that the D265A substitution in human chimeric IgG3 resulted in a loss of complement activation and interaction with human Fc{gamma}RI in vitro, in association with a substantial increase in the content of sialylated glycoforms (14). However, it remains to be determined whether the D265A substitution could also affect the interaction of IgG2a and IgG2b with Fc{gamma}R and whether increased Fc sialylation is causally implicated in the reduction of Fc-associated effector functions of this mutant.

We have previously demonstrated that the development of anemia induced by different IgG subclasses of the 34-3C anti-RBC mAb is due to Fc{gamma}R- and complement receptor-mediated phagocytosis and that the respective contributions of Fc{gamma}R and complement are markedly different among the four IgG subclasses (4, 11). Using this experimental model of autoimmune hemolytic anemia, we now assessed the effect of replacement of aspartic acid by alanine at position 265 on the effector function of IgG2a and IgG2b subclasses in relation to the extent of their Fc sialylation. Our results demonstrate that 34-3C IgG2a and IgG2b D265A mutants are hardly pathogenic due to their poor Fc-associated effector functions, which were unrelated to levels of oligosaccharide side chain sialylation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 (B6) mice were purchased from The Jackson Laboratory. B6 mice deficient in Fc receptor common {gamma} chain (FcR{gamma}–/–), lacking the functional expression of the activating Fc{gamma}RI, Fc{gamma}RIII, or Fc{gamma}RIV, were generated with B6-derived embryonic stem cells (15). Mice were provided by Dr. T. Saito (RIKEN Research Center for Allergy and Immunology, Yokohama, Japan). C3-deficient mice, provided by Dr. M. Carroll (Harvard Medical School, Boston, MA), were generated by gene targeting in 129-derived embryonic stem cells (16), backcrossed for six generations on a B6 background.

Monoclonal Abs

The hybridoma secreting the 34-3C IgG2a anti-RBC monoclonal autoantibody was derived from unmanipulated New Zealand Black mice (17). The generation of its IgG2b subclass switch variant was previously described (4). 34-3C IgG2a and IgG2b D265A mutants at position 265 (aspartic acid to alanine) and IgG2a F243A mutant at position 243 (phenylalanine to alanine) were generated by transfecting a 34-3C H chain-loss cell line with VDJ34-3C-C{gamma}2a(D265A), VDJ34-3C-C{gamma}2b(D265A) or VDJ34-3C-C{gamma}2a(F243A) mutant plasmid, which was generated by oligonucleotide-directed mutagenesis, as described (18). IgG mAb were purified from culture supernatants by protein A column chromatography. The purity of IgG was >95% as documented by SDS-PAGE. Mouse RBC-binding activity of 34-3C mAb was assessed in vitro by a flow cytometric analysis using a biotinylated rat anti-mouse {kappa}-chain mAb (H139.52.1.5), followed by PE-conjugated streptavidin, as previously described (19).

Surface plasmon resonance (SPR)3 analysis

A Biacore 3000 biosensor system was used to determine the interaction of soluble murine Fc{gamma}R classes (Fc{gamma}RI, Fc{gamma}RIIB, Fc{gamma}RIII, and Fc{gamma}RIV) with different 34-3C IgG anti-RBC mAb, as described (13). Briefly, soluble versions of murine Fc{gamma}R were injected through flow cells containing immobilized Abs at five different concentrations (0.25, 0.5, 1, 2, and 4 µg/ml). Background binding to a reference flow cell containing immobilized BSA was subtracted.

Detection of C1q and C3 deposits on RBC in vivo

The deposition of C1q and C3 on RBC 24 h after an i.v. injection of 34-3C anti-RBC mAb in BALB/c mice was detected by a flow cytometric assay, using biotinylated goat anti-mouse C1q (20) or goat anti-mouse C3 (Cappel Laboratories), followed by PE-conjugated streptavidin, as previously described (4).

Experimental autoimmune hemolytic anemia

Autoimmune hemolytic anemia was induced by a single i.v. injection of purified anti-RBC mAb into 2- to 3-mo-old mice. The injection of mAb was controlled by assessing the level of Ab opsonization of RBC by using biotinylated rat anti-mouse {kappa}-chain mAb. Blood samples were collected into heparinized microhematocrit tubes every 2 days after the injection, and hematocrit (Ht) values were directly determined after centrifugation. Livers, obtained 8 days after injection of mAb, were processed for histological examination, and the extent of in vivo RBC destruction by Kupffer cell-mediated phagocytosis was determined by Perls iron staining.

Analysis of oligosaccharide structures

Purification of oligosaccharides from 34-3C IgG2a and IgG2b mAb, and from polyclonal human IgG (Sigma-Aldrich) as a control, were performed based on chemoselective glycoblotting technique, as described (21, 22). Briefly, IgG samples were reductively alkylated under the presence of detergent, and then digested with trypsin and peptide N-glycosidase F (Roche Diagnostics) (23). The digested sample was mixed with a novel hydrazide-functionalized glycoblotting polymer (22) and following washing of the unbound substances (e.g., peptides, detergent, enzymes), sialic acids were methyl-esterified to render sialylated oligosaccharides chemically equivalent to neutral oligosaccharides, as described (24). The IgG oligosaccharides were finally recovered as derivatives of aoWR (N{alpha}-((aminooxy)acetyl)tryptophanylarginine methyl ester), an oligosaccharide labeling reagent that allows highly sensitive detection on mass spectrometry (MS) (25). Then, the recovered glycans were subjected to MALDI-TOF MS using an Ultraflex II Mass Spectrometer (Bruker Daltonik) controlled by the FlexControl 2.0 software package. Estimation of N-linked oligosaccharide structures was obtained by input of peak masses into the GlycoMod Tool (http://au.expasy.org/tools/glycomod/) or GlycoSuite Tool (https:// glycosuite.proteomesystems.com/glycosuite/glycodb).

Desialylation of IgG2a and IgG2b D265A mutants

The 2 mg of IgG2a or IgG2b D265A mutants in 1 ml of 0.05 M sodium citrate buffer (pH 6.0) were incubated with 400 U of recombinant {alpha}2,3/{alpha}2,6-neuraminidase cloned from Clostridium perfringens (New England Biolabs) at 37°C for 48 h. Then, neuraminidase-treated IgG samples were dialyzed against PBS and purified by protein A column chromatography for the analysis of oligosaccharide structures by MALDI-TOF MS. As a control, these mutant Abs were treated similarly in the absence of neuraminidase.

Statistical analysis

Statistical analysis was performed with the Wilcoxon two-sample test. Probability values <5% were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lack of binding to soluble forms of all four classes of Fc{gamma}R by 34-3C IgG2a and IgG2b D265A mutants

To determine the effect of the substitution of aspartic acid with alanine at position 265 in the CH2 domain of IgG2a and IgG2b on the binding to different classes of Fc{gamma}R, D265A mutants of the 34-3C anti-RBC mAb were generated for these two subclasses. By using SPR with soluble forms of Fc{gamma}R, we assessed the ability of the IgG2a and IgG2b D265A mutant to bind four different classes of Fc{gamma}R (Fc{gamma}RI, Fc{gamma}RIIB, Fc{gamma}RIII, and Fc{gamma}RIV). As expected, 34-3C IgG2a wild-type (WT) mAb displayed binding to all four types of Fc{gamma}R, and its IgG2b variant bound to all Fc{gamma}R except one, Fc{gamma}RI (Table I). However, the replacement of aspartic acid by alanine at position 265 completely abolished the interaction of both IgG2a and IgG2b with all four classes of Fc{gamma}R.


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  		Table I. Binding affinity of Fc{gamma}RI, Fc{gamma}RIIB, Fc{gamma}RIII, and Fc{gamma}RIV for 34-3C IgG2a D265A, IgG2a F243A, IgG2b D265A, and their respective WT counterpartsa

 
Lack of complement activation by 34-3C IgG2a and IgG2b D265A mutants

To investigate the possible implication of aspartic acid at position 265 in the activation of complement by the IgG2a and IgG2b subclass, we analyzed by flow cytometry the extent of C1q and C3 deposition on circulating RBC in B6 mice 24 h after a single i.v. injection of 200 µg of 34-3C IgG2a and IgG2b D265A mutants, in comparison with their respective WT counterparts. As expected, the injection of WT IgG2a and IgG2b mAb induced substantial C1q and C3 deposition on RBC (Fig. 1). In contrast, the extents of C1q deposition were markedly and moderately diminished in mice injected with IgG2a and IgG2b D265A mutants, respectively. In addition, none of them induced detectable levels of C3 deposition. Notably, D265A mutants and WT Abs displayed comparable mouse RBC-binding activity in vivo (Fig. 1). These results indicated that the substitution of aspartic acid with alanine at position 265 completely abrogated the activation of complement by IgG2a and IgG2b in vivo.


Figure 1
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  		FIGURE 1. Flow cytometric analysis of complement activation in vivo by 34-3C IgG2a and IgG2b D265A mutants, and their respective WT counterparts. At 24 h after an i.v. injection of 200 µg of 34-3C anti-RBC IgG2a or IgG2b into B6 mice, RBC were stained with biotinylated goat anti-mouse C1q, goat anti-C3, or rat anti-mouse {kappa}-chain Abs, followed by PE-conjugated streptavidin. Mutant Abs (thick line histogram) and WT Abs (dotted line histogram) are shown. Shaded histogram indicates the background staining obtained with untreated B6 mice.

 
Very poor pathogenicity of 34-3C IgG2a and IgG2b D265A mutants

Because the results we described suggested an incapacity of 34-3C IgG2a and IgG2b D265A mutants to induce Fc-dependent effector functions, their pathogenic activity was assessed in B6 mice, in comparison with their respective WT counterparts. As shown previously (4), a single injection of 34-3C IgG2a and IgG2b WT mAb at a dose of 200 µg induced very severe anemia with maximal drops of Ht values peaking at day 4 (IgG2a WT: 14.8 ± 2.3% and IgG2b WT: 17.1 ± 1.5% (Fig. 2)). In contrast, 200 µg of the IgG2a D265A mutant provoked only very mild anemia (mean Ht values at day 4: 36.8 ± 1.7%, p < 0.0005), and IgG2b D265A mutant was unable to induce any appreciable anemia (44.4 ± 1.5%, p < 0.001). Because histological analysis revealed the presence of a minimal, but still significant erythrophagocytosis by Kupffer cells in mice injected with 200 µg of IgG2a D265A mutant (data not shown), we determined the pathogenic effect of IgG2a D265A mutant in B6 mice deficient either in C3 or FcR{gamma}. Although C3–/– B6 mice still developed mild anemia (mean Ht values at day 4: 36.8 ± 1.9%) to an extent comparable to WT B6 mice, FcR{gamma}–/– B6 mice were completely resistant to the pathogenic effect of the IgG2a D265A mutant (44.0 ± 1.0%, p < 0.01 (Fig. 2)). Histological analysis confirmed the complete absence of iron deposits in Kupffer cells in FcR{gamma}–/– B6 mice (data not shown). These results indicate that despite the lack of binding to soluble forms of Fc{gamma}R in SPR analysis in vitro, the 34-3C IgG2a D265A mutant was still able to interact, though weakly, with Fc{gamma}R expressed on the surface of Kupffer cells to trigger erythrophagocytosis.


Figure 2
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  		FIGURE 2. Development of anemia in B6 mice following the injection of 34-3C IgG2a and IgG2b D265A mutants, and their respective WT counterparts. The 200 µg of 34-3C IgG2a WT ({circ}) or D265A mutant (•) were injected i.v. into WT, C3–/–, or FcR{gamma}–/– ({gamma}–/–) B6 mice (left), and 200 µg of 34-3C IgG2b WT ({circ}) or D265A mutant (•) were injected into WT B6 mice (right). Ht values of individual mice measured 4 days after injection of 34-3C mAb are shown. Mean Ht value is indicated by horizontal bar. The normal range of Ht (mean ± 3SD) of 2- to 3-mo-old B6 mice is represented at shaded area.

 
Increased sialylation of 34-3C IgG2a and IgG2b D265A mutants

It has previously been shown that the D265A mutant of a human chimeric IgG3 displayed a marked increase in sialylation when this mutant mAb was expressed in Chinese hamster ovary cells (14). In view of anti-inflammatory properties of IgG Abs enriched with sialic acids (7, 8), we determined whether the modulation of Fc effector functions observed with 34-3C IgG2a and IgG2b D265A mutants could be attributed to a possible increase in sialic acid contents of their carbohydrate side chains. To this end, the oligosaccharide side chains liberated from 34-3C D265A mutants and WT counterparts were subjected to MALDI-TOF MS analysis, and the content of sialylated (monosialylated A1 and disialylated A2) and nonsialylated (agalactosylated G0, monogalactosylated G1 and digalactosylated G2) glycoforms (Fig. 3) was estimated. Both IgG2a and IgG2b WT mAb were poorly sialylated, with less than 1% of total oligosaccharides containing terminal sialic acid residues (Table II). In contrast, contents of sialylated oligosaccharides were increased in both IgG2a and IgG2b D265A mutants to levels of 6.6% and 31.7% of total oligosaccharides, respectively.


Figure 3
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  		FIGURE 3. Biantennary complex-type oligosaccharide structures released from 34-3C anti-RBC IgG2a and IgG2b Abs. Structures of different sialylated (A1 and A2) and nonsialylated glycoforms (G0, G1, and G2) are summarized. F, fucose; G, galactose; GN, N-acetylglucosamine; M, mannose; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid.

 

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  		Table II. Structural analysis of N-linked oligosaccharide chains purified from 34-3C IgG2a D265A, IgG2a F243A, IgG2b D265A, and their respective WT counterpartsa

 
No increases in pathogenicity of desialylated 34-3C IgG2b D265A mutant

To determine whether the increased content of sialic acids could be responsible for the poor effector functions of 34-3C IgG2a and IgG2b D265A mutants, we attempted to generate desialylated variants of D265A mutants by treating them with {alpha}2,3/{alpha}2,6-neuraminidase in vitro. When the extent of desialylation was controlled by MALDI-TOF MS analysis on oligosaccharides released from these two neuraminidase-treated mutant Abs, we observed that nearly 90% of sialic acids were successfully removed from the IgG2b D265A mutant following treatment with {alpha}2,3/{alpha}2,6-neuraminidase (Table III). In contrast, this treatment was hardly efficient for the IgG2a D265A mutant (data not shown), which may in part be related to the fact that the content of sialic acids in this mutant was already very limited before neuraminidase treatment as compared with that of IgG2b mutant. Therefore, we only determined whether neuraminidase-treated IgG2b D265A mutant became more pathogenic than its nondesialylated counterpart. We found that an injection of 200 µg of neuraminidase-treated IgG2b D265A mutant in B6 mice still did not induce anemia (mean Ht values of n = 4 mice at day 4: 43.0 ± 2.0%), as was the case with D265A control, which was treated similarly but in the absence of neuraminidase (44.2 ± 0.3%). Notably, neuraminidase-treated and untreated mutant Abs both displayed comparable mouse RBC-binding activities, and failed to induce measurable C3 deposition on RBC in vivo (data not shown).


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  		Table III. Structural analysis of N-linked oligosaccharide chains purified from neuraminidase-treated 34-3C IgG2b D265A mutanta

 
Markedly increased sialylation without modulation of Fc-associated effector functions and pathogenicity of 34-3C IgG2a F243A mutant

To further assess the effect of IgG sialylation with respect to the pathogenic potential of IgG subclasses, we generated another 34-3C IgG2a F243A mutant with a replacement of phenylalanine by alanine at position 243. This replacement has been shown to result in a remarkable increase in Fc sialylation of a human chimeric IgG3, in which more than 50% of oligosaccharides were terminally sialylated (14, 26). Structural analysis of oligosaccharides released from IgG2a F243A mutant confirmed a marked increase in sialylated glycoforms, which accounted for 25% of total oligosaccharides (Table II). Notably, the observed sialic acid species in the IgG2a F243A mutant was mostly (>85%) NeuGc (N-glycolylneuraminic acid) rather than NeuAc (N-acetylneuraminic acid), as was the case with IgG2a and IgG2b D265A mutants (data not shown). In vitro SPR measurements revealed that the substitution of phenylalanine with alanine at position 243 led to only moderate (3- to 4-fold) decreases in the ability of IgG2a to bind Fc{gamma}RIIB and Fc{gamma}RIII, but did not affect its interaction with Fc{gamma}RI and Fc{gamma}RIV (Table I). The injection of 200 µg of 34-3C IgG2a F243A mutant in B6 mice induced C1q and C3 depositions on RBC at levels essentially identical with those obtained with IgG2a WT Ab (Fig. 4A). Moreover, the pathogenic effect of the F243A mutant assessed at two different doses (50 and 200 µg) was indistinguishable from that of the WT Ab (Fig. 4B).


Figure 4
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  		FIGURE 4. Efficient complement activation and induction of anemia by 34-3C IgG2a F243A mutant. A, At 24 h after an i.v. injection of 200 µg of 34-3C anti-RBC IgG2a WT or F243A mutant into B6 mice, RBC were stained with biotinylated goat anti-mouse C1q, goat anti-C3, or rat anti-mouse {kappa}-chain Abs, followed by PE-conjugated streptavidin. F243A mutant (black line histogram) and WT Ab (gray line histogram) are shown. Shaded histogram indicates the background staining obtained with untreated B6 mice. B, Either 50 or 200 µg of 34-3C IgG2a WT ({circ}) or F243A mutant (•) were injected i.v. into B6 mice. Ht values of individual mice measured 4 days after injection of 34-3C mAb are shown. Mean Ht value is indicated by horizontal bar. The normal range of Ht (mean ± 3SD) of 2- to 3-mo-old B6 mice is represented by shaded area.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study was designed to define the implication of aspartic acid at position 265 of the IgG2a and IgG2b subclass in the interaction with four different classes of Fc{gamma}R and in complement activation, in relation to the levels of sialylation of the IgG oligosaccharide side chains. Comparative in vitro and in vivo analyses of WT and D265A mutants of 34-3C IgG2a and IgG2b anti-RBC mAb revealed that the replacement of aspartic acid by alanine at position 265 resulted in an almost complete abrogation of the capacity of IgG2a and IgG2b subclasses to interact with all four classes of Fc{gamma}R and activate complement, hence dramatically reducing their pathogenic potential. Analysis of desialylated D265A variants excluded a role for increased sialylation in the poor Fc-mediated effector functions observed with D265A mutants, thus indicating that aspartic acid at position 265 is a critical residue for triggering Fc effector functions of IgG.

The lack of any detectable binding by 34-3C IgG2a and IgG2b D265A mutants to soluble forms of four different Fc{gamma}R in vitro confirms and extends the previous finding that an IgG1 D265A mutant fails to bind to Fc{gamma}RIIB and Fc{gamma}RIII (12, 13), and further underscores the critical role of aspartic acid at position 265 in the interaction of IgG with all four classes of Fc{gamma}R. Moreover, the remarkably decreased pathogenicity of D265A mutants confirmed the results obtained by the in vitro binding analysis. Our present results also revealed that aspartic acid at position 265 is involved in the interaction with C1q and hence complement activation. In this regard, it is worth mentioning that C1q deposits on RBC in mice injected with D265A mutants of 34-3C IgG2a and IgG2b were still detectable, although markedly decreased, but this was not the case for C3 deposits. The lack of C3 activation despite detectable C1q deposits is likely due to the activity of several complement inhibitory proteins present on the RBC membranes. Indeed, it has been shown that RBC opsonized with 34-3C IgG2a were more rapidly eliminated by complement than by the Fc{gamma}R pathway in mice deficient in membrane C3 regulatory proteins (27); this observation contrasts with the fact that Fc{gamma}R-mediated erythrophagocytosis plays a major role in the development of anemia induced by 34-3C IgG2a in WT animals (4).

The remarkable effects of the D265A substitution on IgG effector functions might be attributed either to a modification in the overall Fc structure or to a change in glycosylation patterns, such as an increase in the content of terminal sialic acid residues in the oligosaccharide side chains attached to the CH2 domain of IgG. Notably, IgG2a and IgG2b WT Abs were very poorly sialylated, whereas D265A mutants, in particular IgG2b D265A, showed markedly increased levels of sialylation. However, a higher binding to C1q by the more sialylated IgG2b D265A mutant compared with the less sialylated IgG2a D265A mutant, and no increases in pathogenicity after desialylation of the IgG2b D265A mutant clearly indicated that the very poor Fc effector functions of the D265A mutants were unrelated to increased levels of their Fc sialylation. This indication was also confirmed by the analysis of a desialylated IgG1 D265A mutant of the 6A6 antiplatelet mAb (our unpublished data). As previous studies identified the distinct sequence motifs implicated in the interaction with C1q and Fc{gamma}RI (9, 11), it is difficult to imagine that aspartic acid at position 265 is part of the sequence motif involved in the binding to C1q as well as to the four different classes of Fc{gamma}R. Instead, it is more likely that the D265A substitution leads to a conformational change in the Fc region, thereby interfering with the efficient binding of IgG to C1q and Fc{gamma}R.

It is interesting to note that the pathogenic activity of 34-3C IgG2a F243A mutant, which contained 25% sialylated glycovariants, was comparable to that of its barely sialylated WT counterpart. SPR analysis revealed an only limited (3- to 4-fold) decrease in the affinity of Fc{gamma}RIIB and Fc{gamma}RIII to the 34-3C F243A mutant, without any decrease in the affinity of Fc{gamma}RI and Fc{gamma}RIV. In contrast, the affinity of Fc{gamma}RIIB, Fc{gamma}RIII, and Fc{gamma}RIV to very highly sialylated 6A6 IgG1 and IgG2b variants containing 60% of terminal sialic acid residues was reduced by 7–20 times, as compared with their WT counterparts. Thus, the relatively unaltered Fc-mediated effector functions of the 34-3C IgG2a F243A mutant could be due to its lesser content of sialylated glycoforms than those of 6A6 IgG1 and IgG2b sialylated variants prepared through lectin affinity column chromatography.

Using human IgG1, however, it was recently reported that the ability to interact with Fc{gamma}RIIIA was not necessarily affected by an overall increase in Fc sialylation, but was rather dependent on how the sialylated variants were derived (i.e., from different Ab production processes or from lectin-based column fractionation) (8). Notably, higher sialylated variants (containing 67% sialylated glycoforms) obtained through lectin column chromatography displayed binding to Fc{gamma}RIIIA comparable to that of lesser sialylated variants (containing 5% sialylated glycoforms). In contrast, naturally selected sialylated variants exhibited a significantly reduced binding to Fc{gamma}RIIIA despite an only modest increase in sialic acid contents (29% sialylated glycoforms in the selected variant vs 20% in WT Ab). Moreover, this study also reported an increased binding by a highly sialylated variant of human IgG1 mAb to high-affinity Fc{gamma}RI, but a reduced binding to low-affinity Fc{gamma}RIIIA. The Fc{gamma}R-specific sensitivity to Fc sialylation may be compatible with our finding obtained with the IgG2a F243A mutant, which displayed reduced bindings to Fc{gamma}RIIB and Fc{gamma}RIII, but comparable bindings to Fc{gamma}RI and Fc{gamma}RIV. Because sialic acids are present on the {alpha}1,6- or {alpha}1,3-linked mannose arms of the biantennary glycan structure, relative amounts of sialic acid residues on the {alpha}1,6 arm vs the {alpha}1,3 arm may differ depending on how the sialylated variants were derived, and such differences in sialic acid distribution may critically influence the interaction of IgG with different classes of Fc{gamma}R. In addition, it has been shown that inhibitory effects of sialylated IgG on Fc-associated effector functions were apparently dependent on the sialic acid-galactose linkage specificity because a human chimeric IgG3 mAb containing only {alpha}2,3-sialylated glycoforms was less efficient in interaction with human Fc{gamma}R and C1q than its variant containing comparable amounts of both {alpha}2,3- and {alpha}2,6-sialylated glycoforms, despite the fact that the overall contents of sialylated glycoforms were not different between these two variants (26). In contrast, it has recently been shown that terminal {alpha}2,6-sialylated IgG, but not {alpha}2,3-sialylated IgG, was responsible for anti-inflammatory activity of sialylated i.v. Ig (IVIG) (28). Notably, anti-inflammatory properties of sialylated IVIG were primarily mediated by an up-regulation of inhibitory Fc{gamma}RIIB expression, and did not depend on a direct interaction of IVIG with canonical Fc{gamma}R (7, 29, 30). Collectively, these data suggest that the qualitative features of IgG sialylation may play a more crucial role in IgG Fc-associated effector functions than the purely quantitative aspects of sialylation. In this respect, it is worth mentioning that Ag-binding activities could also be affected in highly sialylated IgG variants, possibly as a result of reduced flexibility of the hinge region (8).

In conclusion, our present study revealed the loss of Fc-associated effector functions of IgG when aspartic acid at position 265 was substituted with alanine. Because this substitution did not affect the Ag-binding ability of IgG, the D265A variant may present an advantage for therapeutic applications in which Ab is required to bind to Ag, but must not trigger Fc-mediated effector activities. Clearly, further understanding of the interplay between structure and function of sialylated glycoforms of IgG would provide useful guiding principles for the engineering of mAb for in vivo applications.


   Acknowledgments
 
We thank Dr. T. Moll for critical reading of the manuscript and G. Celetta, G. Brighouse, G. Sealy, and T. Le Minh for excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Swiss National Foundation for Scientific Research, by Special Coordination Funds for Promoting Science and Technology of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government and by a grant from the Roche Research Foundation. F.N. was supported by grants from the German Research Foundation (DFG) and from the Bavarian Genome Research Network (BayGene). Back

2 Address correspondence and reprint requests to Dr. Shozo Izui, Department of Pathology and Immunology, Centre Médicale Universitaire, 1211 Geneva 4, Switzerland. E-mail address: Shozo.Izui{at}medecine.unige.ch Back

3 Abbreviations used in this paper: SPR, surface plasmon resonance; Ht, hematocrit; MS, mass spectrometry; IVIG, i.v. Ig; WT, wild type. Back

Received for publication June 11, 2008. Accepted for publication August 26, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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