Human IgG/FcγR Interactions Are Modulated by Streptococcal IgG Glycan Hydrolysis

Background The human pathogen Streptococcus pyogenes produces an endoglycosidase, EndoS that hydrolyzes the chitobiose core of the asparagine-linked glycan on the heavy chain of human IgG. IgG-binding to Fc gamma receptors (FcγR) on leukocytes triggers effector functions including phagocytosis, oxidative burst and the release of inflammatory mediators. The interactions between FcγR and the Fc domain of IgG depend on the IgG glycosylation state. Methodology/Principal Findings Here we show for the first time that EndoS hydrolyzes the heavy chain glycan of all four human IgG subclasses (IgG1-4), in purified form and in a plasma environment. An inactive form of EndoS, obtained by site-directed mutagenesis, binds IgG with high affinity, in contrast to wild type EndoS that only transiently interacts with IgG, as shown by Slot-blotting and surface plasmon resonance technology. Furthermore, EndoS hydrolysis of the IgG glycan influences the binding of IgG to immobilized soluble FcγR and to an erythroleukemic cell line, K562, expressing FcγRIIa. Incubation of whole blood with EndoS results in a dramatic decrease of IgG binding to activated monocytes as analyzed by flow cytometry. Moreover, the IgG bound to K562 cells dissociates when cells are treated with EndoS. Likewise, IgG bound to immobilized FcγRIIa and subsequently treated with EndoS, dissociates from the receptor as analyzed by surface plasmon resonance and Western blot. Conclusions/Significance We provide novel information about bacterial enzymatic modulation of the IgG/FcγR interaction that emphasizes the importance of glycosylation for antibody effector functions. Moreover, EndoS could be used as a biochemical tool for specific IgG N-glycan hydrolysis and IgG purification/detection, or as a potential immunosuppressing agent for treatment of antibody-mediated pathological processes.


INTRODUCTION
The immunoglobulin G (IgG) class of antibodies plays an important role in the adaptive immune defense of the human host against pathogens. IgG consists of two identical heavy chains and two identical light chains, which in turn are composed of variable and constant domains. Papain treatment of the IgG molecule generates two separate monovalent Fab fragments recognizing antigens and an intact Fc fragment, a recognition site for host receptors and a site of interaction with a number of effector molecules, including the classical complement pathway starting with factor C1q [1,2]. IgG is a glycoprotein containing a conserved complex carbohydrate structure attached to the asparagine 297 residue in the CH2 domain of each IgG Fc part. It consists of a biantennary core of Nacetylglucosamine and mannose with added terminal and branching carbohydrate residues such as N-acetylglucosamine, fucose, sialic acid, and galactose ( Fig. 1A) [3]. The presence of this carbohydrate is crucial for proper antibody structure and for interactions with cellular immunoglobulin G Fcc receptors (FccRs) and the complement system [1,4]. Altered glycosylation of IgG have been associated with autoimmune disorders like rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Crohns disease [5][6][7]. Several forms of autoimmune vasculitis show a differentiating glycosylation with decreased Fc galactosylation and sialylation [8][9][10][11][12]. Additionally, it has been suggested that sialylated IgG in vivo switch from its anti-inflammatory activity with subsequent reduced antibody effector activity, to a pro-inflammatory/toxic activity upon decreased Fc sialylation [13]. IgGs, classified into four subclasses, IgG1, IgG2, IgG3 and IgG4, are described to interact with different types of FccRs giving different activation profiles [14,15].
FccRs provide a linkage between the humoral and cellular immune responses. Phagocytic cells express members of three classes of IgG-Fc receptors, FccRI, FccRII and FccRIII, charac-terized by structural and functional homology and by the specific recognition site on the CH2 region of IgG [1, 16,17]. Binding of pathogen-IgG complexes to FccRs mediates an essential response from the host against pathogens by initiating a cascade of signals causing antibody-dependent-cellular-cytotoxicity (ADCC), complement-dependent-cellular-cytotoxicity (CDCC), endocytosis, phagocytosis, oxidative burst, the release of inflammatory mediators, etc. [2,18]. Complexed IgG-FccR can besides activation of the C1q component of complement also activate other ligands e.g. mannan binding lectin (MBL), the neonatal receptor FcRn, the mannose receptor (MR), etc. [2,4]. FccRs may be expressed constitutively on haematopoietic cells and may also be induced or up-regulated by cytokines and other agents. FccRs are responsible for balancing activation (FccRI, FccRIIa and FccRIIIa) and inhibitory signals (FccRIIb) of the immune system with the ability of both activating and inhibiting the IgG mediated effector stimulation [1,19].
Streptococcus pyogenes is one of the most common human pathogens causing pharyngitis, scarlatina and more severe infections like necrotizing fasciitis and sepsis [20,21]. Like other bacteria it expresses several virulence factors and uses several immune evasion strategies to successfully invade its host [22][23][24][25]. The recently discovered enzyme Endoglycosidase S (EndoS) is secreted by Streptococcus pyogenes and has a specific endoglycosidase activity on native IgG by hydrolyzing the conserved asparaginelinked glycans on the heavy chains of IgG (Fig. 1A) [24,26]. This 108 kDa-enzyme is encoded by the gene ndoS that is highly conserved and is present in virtually all examinated isolates. EndoS is the first known bacterial enzyme with a unique specificity for native IgG [24]. This is in contrast to other related endoglycosidases as EndoF1-3 from Elisabethkingia meningoseptica (previously Flavobacterium meningosepticum), which show enhanced hydrolytic activities on the denaturated forms of basically any glycoproteins with the appropriate N-linked glycan, or EndoE from Enterococcus faecalis that in addition to activity on the glycan of native IgG also hydrolyzes high-mannose glycans on other denatureted glycoproteins [27,28]. EndoS is N-terminally processed by the cysteine proteinase SpeB that could be of importance in regulating EndoS activity [29]. Furthermore, the molecular requirements for EndoS glycosidase activity have recently been elucidated revealing the importance of glutamic acid 235 (Glu-235) and tryptophans [29].
EndoS activity affects the functionality of opsonizing IgG by decreased binding to Fc-receptors on a monocyte-like cell line and impaired classical complement activation in vitro [26].
In the present study we elucidated the effect(s) of EndoS on IgG subclasses and IgG-FccR interactions. The results revealed that EndoS hydrolyses the heavy chain of all four human IgG subclasses (IgG1-4), both soluble and in a plasma environment. Additionally, we found that EndoS hydrolysis of the IgG glycan dramatically influences the binding of IgG to soluble, immobilized FccRIIa and FccRIIb as well as to FccR-expressing cells. Moreover, IgG pre-bound to these cells dissociates due to treatment of cells with EndoS. Furthermore, an inactive form of EndoS generated by site-directed mutagenesis binds with high affinity to IgG1-4, while the active form only transiently interacts with its substrates. These results provide novel information about the mechanisms behind enzymatic modulation of the host immune defense by bacteria, provide novel information about the molecular interactions between an IgG glycan-hydrolyzing enzyme and IgG, and emphasize the importance of IgG glycosylation for correct antibody effector functions.

EndoS has glycosidase activity on all four IgG subclasses
It has previously been shown that EndoS hydrolyzes the chitobiose core of the conserved N-linked glycan on the c-chain of human polyclonal IgG [24] (Fig. 1A). It was therefore of interest to elucidate whether EndoS has activity on all four subclasses of human IgG (IgG1-4). Purified recombinant EndoS was incubated with purified human IgG1-4. SDS-PAGE analysis revealed that EndoS-treated IgG of all subclasses migrated at an apparent molecular weight of approximately 3 kDa lower than untreated IgG (Fig. 1B), which is consistent with hydrolysis of the chitobiose core of the IgG glycan. To confirm glycan hydrolysis, samples were also analyzed by lectin blot using a Lens culinaris agglutinin (LCA) lectin recognizing alinked mannose residues (Fig. 1A). Lectin blot analysis of the samples revealed that all IgG subclasses lose the reactivity with LCA after incubation with EndoS consistent with complete or nearly complete hydrolysis of the glycan (Fig. 1C). Additionally, the glycosidase activity of EndoS on IgG1-4 in a plasma environment was investigated. In this experiment human plasma was incubated with purified EndoS or buffer, followed by affinity purification of the IgG fraction. These fractions were subsequently subjected to a LCA ELISA using immobilized monoclonal antibodies against IgG1-4 to capture IgG. This revealed that all four IgG subclasses reacted with lectin when plasma was incubated with buffer, indicating presence of the glycan (data not shown). In contrast, when plasma was treated with EndoS, a dramatically reduced IgG1-4 reactivity with LCA lectin was observed. IgG1 was hydrolyzed to 87611%, IgG2 was hydrolyzed to 81613%, IgG3 was hydrolyzed to 74623%, and IgG4 was hydrolyzed to 7263% (Fig. 2). Taken together, these results clearly show that EndoS has the ability to hydrolyze human IgG of all subclasses, in purified form as well as in whole plasma.

The inactive form of EndoS binds IgG
We have previously partly elucidated the molecular requirements for EndoS hydrolysis of IgG. Site directed mutagenesis of glutamic acid 235 to glutamine (EndoS(E235Q)) at the proposed orifice of the catalytic tunnel abolishes enzymatic activity. In addition, chemical blocking of tryptophanes revealed that these amino acid residues are important for activity [29]. To further investigate the physical interaction between enzyme and substrate, the binding of EndoS and EndoS(E235Q) to immobilized polyclonal IgG and IgG1-4 subclasses was studied using slot-binding experiments with immobilized IgG probed with EndoS and EndoS(E235Q). Purified, soluble IgG subclasses 1-4, each immobilized onto a nitrocellulose membrane, were probed with EndoS and Endo-S(E235Q) followed by incubation with antibodies against EndoS. This experiment revealed a strong binding of EndoS(E235Q) to polyclonal IgG, IgG1 and IgG2, and a weaker association to IgG3 and IgG4, while only very weak interactions between active EndoS and all subclasses could be seen (Fig. 3A). To calculate the affinity constants between EndoS and immobilized IgG1-4, surface plasmon resonance technology was used. Similarly to slot-binding results, this showed that EndoS(E235Q) binds all IgG subclasses with high affinity, while there is no detectable binding of EndoS to IgG. (Fig. 3B, Table 1). The kinetic parameters of EndoS (E235Q) binding to immobilized IgG subclasses were of similar character and the strongest interaction was demonstrated between IgG1 and EndoS(E235Q) with a binding affinity constant (K D ) of 0.42 mM. No binding of either EndoS or EndoS(E235Q) to IgG1-4 subclasses, which were hydrolysed by EndoS before immobilization, was detected. These findings indicate that the intact IgG glycan is necessary for the interaction between EndoS and IgG. Furthermore, the experiments comparing the interactions between EndoS, EndoS(E235Q) and IgG indicates that EndoS binds IgG with a high affinity, but the active enzyme is instantly released after glycan hydrolysis in a ''touch and go'' manner.

EndoS influences the binding of IgG1-4 to FccRs
Since the nature of the interactions between FccRs and the Fc domain of IgG is highly dependent on the IgG glycosylation state [1, 3,4] we explored the effects of EndoS activity on IgG interactions with FccRs. Thus, in an ELISA experiment the soluble FccRIIa, FccRIIb and FccRIIIa were immobilized and probed with purified IgG1-4 subclasses. In line with other observations it was here seen that FccRIIa and FccRIIb binds IgG1 [14]. This binding was nearly abolished after treatment of IgG1 with EndoS. The binding of the other IgG subclasses to these receptors was weak and was even more reduced after treatment with EndoS. In general, ELISA studies revealed the IgG subclass binding affinity pattern IgG1.IgG3.IgG4.IgG2 for FccRIIa and IgG1.IgG4.IgG3.IgG2 for FccRIIb (Fig. 4A). Furthermore, we observed that the EndoS hydrolysed IgG2 had a different outcome regarding the binding to FccRIIa/FccRIIb with more extensive binding ability, compared to the untreated IgG2. FccRIIIa was negative in binding of all IgG subclasses (data not shown). The interaction between IgG1-4, with or without EndoS treatment, with FccRs was further analyzed by surface plasmon resonance. Each IgG subclass was tested for binding to a surface with an immobilized FccRIIa, FccRIIb or FccRIIIa. Consistent with the ELISA data, the results showed that IgG1 had the strongest affinity for both FccRIIa and FccRIIb with similar binding affinity constants, 97 nM and 170 nM respectively (Fig. 4B, Table 2). In agreement with our previous findings, no binding of IgG1 to these receptors was detectable when EndoS treated IgG1 was used. There was no detectable interaction between FccRIIa/ FccRIIb and IgG2 or IgG3, or between IgG4 and FccRIIa ( Table 2). No binding of soluble IgG1-4 subclasses to immobilized FcRIIIa could be detected. These results indicate that EndoS hydrolysis dramatically decreases IgG's affinity for FccRs.

EndoS decreases IgG binding to blood cells
Based on results from ELISA and surface plasmon resonance, we continued to analyze the effect(s) of EndoS glycosidase activity on the interaction between FccRs and IgG. For this purpose we used an erythroleukemic cell line (K562) exclusively expressing FccRIIa [14,30]. Since soluble FccRI was not available to us, we also investigated human monocytes that predominantly bind IgG through this receptor [14]. Thus, IgG was purified from plasma treated with EndoS or PBS, labeled with 125 I and incubated with the K562 cells. The radioactivity of the cell pellets was measured. This revealed significantly decreased binding of radioactive IgG, originally purified from plasma treated with EndoS, to K562 cells (Fig. 5A). In a control experiment, the specific IgG binding to these cells was calculated by addition of cold human IgG, which inhibited the binding of radioactive IgG to 93% (data not shown). A strong binding of IgG to K562 cells after incubation of cells with human plasma was confirmed by Western blot and the reactivity of cell lysates with antibodies against human IgG. In contrast, there was a clear decrease in binding of IgG to K562 cells  incubated with plasma pre-treated with EndoS (Fig. 5B). Likewise, the binding of 125 I-IgG to monocytes as analyzed by SDS-PAGE was totally inhibited when IgG was treated with EndoS ( Fig. 6A).
To further analyze EndoS' influence on the interaction between FccRs on monocytes and IgG, flow cytometry analysis of whole blood was performed. Human blood was pre-incubated with EndoS before addition of the leukocyte activator fMLP.
Monocytes were gated based on forward and side scatter and the reactivity of monocytes with monoclonal anti-human IgG was evaluated. The result revealed that 87% of monocytes were positive for IgG binding, while only 43% of monocytes in blood incubated with EndoS were positive (Fig. 6B). These results indicate that EndoS-hydrolyzed IgG is significantly reduced in its binding capacity to human cells expressing different sets of FccRs.

IgG dissociates from FccRIIa upon treatment with EndoS
Our experiments this far have revealed that EndoS hydrolysis of IgG inhibits binding to FccRs on cells and surfaces, but it remained unclear if EndoS has activity on IgG already bound to FccRs and if such activity could release the IgG bound to FccRs. Therefore, we investigated the effects of EndoS on IgG bound to K562 cells that had been exposed to human plasma and subsequently treated with EndoS. The cell lysates were analyzed by SDS-PAGE and Western blot using an antibody against human IgG. There was a significant binding of IgG to K562 cells as judged by the results presented in figure 7A. Interestingly, no IgGband was visible on a blot when cells were treated with EndoS, suggesting a total IgG dissociation from the cells (Fig. 7A). A control experiment, using EndoS(E235Q), revealed an IgG signal on the surface of K562 cells comparable to the untreated cells.
These results strongly suggest that IgG dissociates from the cell surface due to N-glycan hydrolysis of IgG by EndoS. Similarly, the effect of EndoS on IgG bound to monocytes was analyzed. This showed that most of the monocyte-bound IgG dissociated from cells due to the treatment with EndoS as compared to untreated cells (Fig. 7B). As expected, monocytes treated with EndoS, in contrast to control cells, showed no reaction with the LCA lectin, indicating that the minute amounts of IgG remaining on the cells as detected in the IgG blot had most likely been hydrolyzed by EndoS (Fig. 7B). The results demonstrated above were further confirmed by surface plasmon resonance experiments. Soluble IgG1 and FccRII receptor were chosen because of our earlier observation that IgG1 is the strongest binder of FccRII. After binding of IgG1 to pre-immobilized FccRIIa and reaching a steady-state dissociation phase, the IgG1 injection was aborted and replaced by EndoS injection or running buffer. This revealed that EndoS injection causes the dissociation of IgG1 from immobilized FccRIIa receptor while the IgG1 dissociation from FccRIIa was unaffected when adding running buffer (Fig. 7C). Taken together, these results clearly demonstrate that EndoS by IgG glycan hydrolysis can release IgG bound to FccRs on cells and surfaces.

DISCUSSION
In the present study we attempted to elucidate the physical interaction between EndoS and IgG and the physiological relevance of EndoS IgG N-glycan hydrolyzing activity for IgG-FccR interactions. We present for the first time that EndoS specifically acts as an endoglycosidase on all human IgG subclasses, both in purified form and in a plasma environment. As expected, there is a physical interaction between the enzyme and all IgG subclasses, that we successfully demonstrated using an enzymatically inactive, mutated form of EndoS. In this study we could not separately investigate the EndoS effects on the isolated binding of IgG to FccRI. However, we observed that EndoShydrolyzed IgG did not bind to monocytes, and that there was a nearly complete dissociation of IgG from monocytes upon hydrolysis by EndoS. Since monocytes express FccRI and this receptor has the highest affinity for IgG, we conclude that EndoS influences IgG binding even to FccRI because the effects of EndoS observed must be predominantly due to involvement of FccRI.
EndoS seems to have an effect on both isotypes of FccRII receptors, thus influencing both activating and inhibiting IgG mediated effector stimulation. Interestingly, IgG2 treated with EndoS, in opposite to what was observed for the other subclasses of IgG, showed increased binding to FccRIIb, and slightly also to FccRIIa, immobilized to microtiter plate. One possible explanation for this could be aggregation of IgG2 upon hydrolysis by EndoS leading to increased binding to FccRs. However, no binding of IgG2 to FccRs was detected using surface plasmon resonance which is in agreement with earlier publications [14]. This could be explained by the constant flow of IgG2 over the immobilized receptors in the case of surface plasmon resonance, in contrast to ELISA where IgG2 is allowed to aggregate and interact with the receptor. The activity of EndoS on IgG has obvious benefits for S. pyogenes expressing this enzyme with potential modulation and/or evasion of an IgG-mediated response against the bacteria. We have previously demonstrated that EndoS treatment of human After incubation for 30 minutes at room temperature, the proteins from cell lysates, 10 mg total protein, were separated by 10% SDS-PAGE. The gel was dried and analyzed by phosphorimaging. Panel B. Flow cytometry analysis showing the decreased binding of IgG to activated monocytes in blood treated with EndoS. Human blood was treated with EndoS before addition of leukocyte activator fMLP. The IgG binding to monocytes was detected using mouse anti-human IgG and FITC-labelled goat anti-mouse IgG as a secondary antibody. doi:10.1371/journal.pone.0001413.g006 opsonizing IgG antibodies directed towards the cell-wall anchored M protein significantly enhances the bacterial survival in blood [26]. Therefore, in the context of an intact infecting S. pyogenes, EndoS is a potentially harmful molecule to the human host that contributes to the bacterial virulence. In contrast to this, the purified form of EndoS has substantial potential as a biotechnological tool and/or a therapeutical agent that could be beneficial for future experimental science and possibly also health care.
Our results reveal that EndoS possesses a capacity to inhibit the IgG binding to FccRs and detach IgG bound to FccRs on cell surfaces. We have recently been able to show that pre-treatment of arthritogenic antibodies abrogates development of arthritis in a mouse model of collagen-induced arthritis [31]. This suggests that EndoS may have potential for being further developed as a therapeutical agent in other antibody-mediated autoimmune disorders.
We suggest two principally different biotechnological uses of EndoS, one based on the IgG-glycan hydrolyzing activity of the wild-type enzyme, and the other based on the high affinity IgGbinding of EndoS(E235Q). The active enzyme could be used for in vitro treatment of whole blood or purified blood cells in order to remove IgG already bound to various FccRs on these cells. This could facilitate the analysis of effects of specific IgG preparations added to the cells, regarding receptor binding and cellular activation, without the interference of pre-bound IgG. The inactive form of EndoS (EndoS(E235Q)) has a great potential as a specific IgG purification and detection tool. In this study we have demonstrated that EndoS(E235Q) interacts equally well with all subclasses of IgG. This is comparable to what can be seen for protein G, one of the major molecules currently used for IgG preparation and detection [32], but advantageous compared to protein A that does not bind IgG3 [33]. Protein A also binds IgM and IgA to a certain extent. We have previously shown that there is no interaction between EndoS and IgM or IgA [24]. Furthermore, we could show here that EndoS(E235Q) does not interact with IgG lacking its heavy chain glycans. This is in contrast to both protein G and protein A that bind IgG irrespective of its glycosylation state [24]. This could be especially important when only intact IgG with a certain functional effector region is required. When using currently available reagents like protein G, a second purification step using for instance a lectin column is required to obtain only the glycosylated fraction of IgG. This property of EndoS(E235Q) could be used in combination with for instance protein G to assess the glycosylation state and the functional quality of an IgG preparation.
In conclusion, EndoS is a bacterial immunomodulatory protein with a great potential. Our results provide novel information about bacterial pathogenesis, i.e. how the pathogens evade the immune system of the host organism by affecting the functions of IgG/ FccRs. Moreover, EndoS could be used as an important biochemical tool for specific IgG N-glycan hydrolysis and IgG purification/detection, or perhaps as a potential immunosuppressing agent that could be used to interfere with antibody-mediated pathological processes.

Proteins and reagents
Blood was drawn from healthy individuals and collected in heparin-containing tubes. Full-length EndoS with glutathione-Stransferase (GST) as a fusion was recombinantly expressed and purified from Escherichia coli harboring the plasmid pGEXndoS. When appropriate, the GST-tag was removed using Factor Xa as previously described [23]. The mutation of glutamic acid 235 of EndoS into glutamine was performed using QuickChange II Site-Directed Mutagenesis Kit according to manufacturer's instructions (Stratagene, La Jolla, CA) with following verification of the mutation by sequencing [29]. Soluble purified Fc-receptors were generated by co-transfection of CHO-K1(CHO) cells with pNTneo-FccRII or pNT-neo-FccRIII plasmids with subsequent selection in 1 mg/ml genetecin [34]. IgG-subclasses were produced by transient transfection in 293T cells [34]. RPMI 1640 medium and Hanks balanced salt solution (HBSS) were from GIBCO, Paisley, U.K. All other reagents were purchased from Sigma-Aldrich unless indicated otherwise.

EndoS-treatment of antibodies
Purified human IgG1-4 was hydrolyzed with GST-EndoS purified as previously described [23]. Enzyme/substrate molar ratio was 1:20 in PBS and samples were incubated for 2 h at 37uC. GST-EndoS was removed from the samples by passing three times over a glutathione-Sepharose column (Amersham Biosciences, Uppsala, Sweden). 1 mg of treated and untreated IgG1-4 was separated on 10% SDS-PAGE followed by staining with Coomassie Blue or Lens culinaris agglutinin (LCA)-lectin (Vector Laboratories, Burlingame, CA, USA) blot analysis (se below).

Treatment of human plasma with EndoS and purification of IgG
A volume of 2 ml human plasma was incubated with 20 mg EndoS or a PBS equivalent for 1.5 hours at 37uC. The IgG fraction was purified using Protein G Sepharose (GE Healthcare Bio-sciences AB, Uppsala, Sweden). Briefly, 200 ml Protein G Sepharose suspended 1:1 in PBS (phosphate-buffered saline; 10 mM phosphate buffer, pH 7.4, 120 mM NaCl, 3 mM KCl) was added to plasma samples and incubated at 4uC for 2 hours or over night. After centrifugation for five minutes at 80006g, the supernatant was discarded and the pellet washed three times with PBS. IgG was eluated with 0.1 M glycine pH 2.0 and neutralized with 1 M Tris-HCl pH 8.0. The IgG concentration was determined to 8 mg/ml using the Advanced Protein Assay (Cytoskeleton, Denver, CO, USA).

Cell preparations
The K562 cell line was cultured in RPMI 1640 medium supplemented with Glutamax-I, 100 mg/ml antibiotics (penicillin and streptomycin) and 10% fetal calf serum at 37uC in an atmosphere containing 5% CO 2 and 95% humidity. Nunclon flasks for cell culture were used (Nunc A/S, Roskilde, Denmark). Cells were cultured in a serum free medium for 20 hours before being used in experiments. Monocytes were isolated from human whole blood using the Polymorphprep kit (AXIS-SHIELD, Oslo, Norway) or Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) according to instructions provided by the manufacturers. After isolation, the cells were counted and resuspended in PBS or RPMI-medium.

Enzyme linked immunosorbent assay (ELISA)
For glycan detection, microtiter plates (NUNC, Roskilde, Denmark) were coated with 100 mL monoclonal mouse anti-human IgG1, IgG2, IgG3 or IgG4 (SIGMAH, Saint Louis, MO, USA) diluted to final concentrations of 1.5-0.5 mg/ml in a coating buffer containing 16 mM Na 2 CO 3 and 35 mM NaHCO 3, pH 9.6 and kept at 4uC overnight. The plates were washed three times with lectin buffer containing 10 mM HEPES, pH 7.5, 0.15 M NaCl, 0.01 mM MnCl 2 , 0.1 mM CaCl 2 and 0.1% v/v Tween 20 and blocked in the same buffer for one hour at room temperature. In the next step purified IgG fraction (dilution 1:100) was added and the incubation proceeded for another 2 hours at 37uC. After three washes with lectin buffer, 1 mg/ml biotinylated LCA-lectin was added and incubation continued for 1 hour at 37uC. Following three more washes, 0.1 mg/ml peroxidase-labeled streptavidin (Vector Laboratories) was added and the plate was incubated for 1 hour at 37uC. The color reaction was developed with 0.1 M citric acid monohydrate, 0.1 M Na 2 HPO 4 62H 2 O buffer pH 4.5 containing 0.012% v/v H 2 O 2 and 1.7 mM 2,29-azino-bis(3ethylbenzthiazoline-6-sulphonic acid) (ABTS). The absorbance was read on a model 550 micro plate reader (BIO-RAD, Hercules, CA, USA) at 415 nm. For detection of binding of human IgG subclasses to FccRs, the plate was coated with soluble FccRIIa or FccRIIb or FccRIIIa at a concentration 5 mg/ml for 20 hours at 4uC. Next day, the plate was blocked with PBS supplemented with 0.05% v/v Tween 20 (PBST) and 2% w/v bovine serum albumin for 2 hours at room temperature. After this step, the purified IgG subclasses, 0.1 mg of each, were added. The plate was washed three times with PBST after the coating step and between each of the following incubation steps. A peroxidase-conjugated protein G (dilution 1:5000) (BIO-RAD, Hercules, CA, USA) was used for detection. The color reaction was performed as above. All experiments were made in triplicates.

Radioactive labeling
Proteins were labelled with 0.2 mCi Na 125 I (PerkinElmer, Upplands-Vä sby, Sweden) using the IODE-BEADS Iodination reagent kit (PIERCE, Rockford, IL, USA) according to the manufacturer's instructions. The unbound radioactivity was removed by desalting the proteins on PD-10 Sepharose (Pharmacia, Sweden). The activity of the labeled proteins was estimated to 4 mCi/mg protein.

Detection of IgG binding to cells
IgG was purified from human plasma treated with EndoS or PBS as described above and thereafter labeled with 125 iodine ( 125 I). For detection of 125 I-IgG binding to K562 cells, 2610 6 cells were incubated with 0.5610 6 cpm of 125 I-IgG or 125 I-deglycosylated IgG for 30 minutes at room temperature. After five washes with PBS and centrifugations at 10006g for three minutes, the radioactivity of the cell pellets was detected using Wallac Wizard TM 1470 Automatic Gamma Counter (PerkinElmer, Waltham, MA, USA). To evaluate the specificity of the binding of radioactive IgG to K562 cells a control experiment was performed. The cells were incubated with 20 mg human IgG in addition to radioactive IgG during the similar incubation conditions as mentioned above. In another experiment, 1610 6 monocytes were incubated with 0.5610 6 cpm of 125 I-IgG or 125 I-EndoS treated IgG for 30 minutes at room temperature. After five repeated washes of cells with PBS and final pelleting of cells by centrifugation at 10006g for five minutes, the cells were resuspended in lysis buffer containing 20 mM Tris-HCl pH 7.4, 0.150 M NaCl, 1% v/v Triton-100 and 0.25% v/v NP40 for ten minutes at 4uC. Next, the samples were centrifuged for ten minutes at 140006g and supernatants applied on a polyacrylamide gel. After separation, the gel was dried and samples analyzed by phosphoimaging in a Fujix BAS 2000 Bioimaging analyzer (Fujifilm Sverige AB, Stockholm, Sweden). In an experiment where the binding of IgG to cells was analyzed by Western blot, 0.5-1610 6 cells were incubated with plasma treated with either EndoS or buffer (as described above), at 37uC for 1 hour. Afterwards, the cells were washed three times with PBS or RPMI medium, resuspended in 100 mL lysis buffer and the bound IgG in cell lysates analyzed by Western blot.

Incubation of cells with EndoS
K562 cells or monocytes, 2610 6 and 8610 6 respectively, were incubated with two ml of human plasma for 30 minutes at 37uC. The cells were washed five times with PBS and centrifuged at 10006g for ten minutes after every wash. EndoS, 40 mg in PBS or PBS alone was added to cells and incubation followed for one hour at 37uC. Cells were washed three times with PBS and resuspended in 100 mL lysis buffer. Samples were centrifuged for five minutes at 140006g, pellets discarded and supernatants analyzed for IgG and glycan contents using SDS-PAGE and Western blot.

Slot-blotting analysis
IgG1-4, 0.3, 015 and 0.075 mg of each in PBS were applied to PVDF membranes using a slot-blot apparatus from Schleicher and Schuell, Inc., Kene, NH 03431, USA. The membranes were incubated with PBST and 5% skim milk for 1 hour, washed with PBST and incubated with EndoS or EndoS (E235Q), 0.05 mg/ml in PBST and 5% skim milk for 1 hour. After washing, the membranes were incubated with rabbit EndoS-antiserum and subsequently with peroxidase conjugated goat anti-rabbit antibodies. The color development was made using ABTS as peroxidase substrate. All incubation steps were performed at room temperature.

Surface plasmon resonance interaction analysis
Receptors, IgGs and deglycosylated IgGs were diluted with 10 mM sodium acetate pH 4 and immobilized via amine coupling to different flow cells of CM5 sensorchips (BIAcore, Uppsala, Sweden). Immobilization levels were optimized to around 8000-10000 response units. After determining EndoS(E235Q) as a nonbinder to all deglycosylated IgG variants, these flow cells were considered as controls for bulk refraction index changes for EndoS(E235Q) binding to IgG1 throughout IgG4, respectively. In experiments determining IgG1-IgG4 affinity for the receptors FccRIIa, FccRIIb and FccRIIIa, a flow cell subjected to the immobilization protocol but without addition of protein was used as control. For affinity measurements, the binding and dissociation phases were monitored in a BIAcore 2000 instrument. In control experiments for possible mass transfer limitations, the IgGs were injected over the receptors and the EndoS variants over the IgG sub-classes at different flow rates. No differences in initial binding were observed at 5 ml/min or above indicating no limitations to any combinations. Interactants were injected in different concentrations (typically 10-1.25 mg/ml) at 35 ml/min and 25uC over the different coated surfaces (flow cells) (in running buffer: 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.005% surfactant P20, and 3.4 mM EDTA). Between experiments, the surfaces were strictly regenerated with pulses of running buffer containing 2 M NaCl followed by an extensive wash procedure after reaching baseline. For EndoS digestion of IgG bound to pre-immobilized FccRIIa, an IgG1 concentration (10 mg/ml) was chosen to give a suitable steady-state dissociation phase at a time point were the IgG1 injection was aborted and replaced by running buffer. This experiment was considered as a control and as such compared to an EndoS injection at the same time point after IgG1 binding to FccRIIa. After X and Y normalization of data, the blank curves from control flow cells of each injected concentration were subtracted. Where applicable, the association (k a ) and dissociation (k d ) rate constants were determined simultaneously using the equation for 1:1 Langmuir binding in the BIA Evaluation 4.1 software (BIAcore). The binding curves were fitted locally and the equilibrium dissociation constants (K D ) were calculated from mean values of the obtained rate constants.