General mechanism for modulating immunoglobulin effector function

Abstract

IgG and IgE mediate their proinflammatory properties through the crosslinking of the 1:1 complex of the Fc receptor (FcR) monomer in the Fc dimer cleft (1, 2). By contrast, both IgG and IgE can engage a second class of receptors, the evolutionarily related, C-type lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) (3) and CD23 (4), respectively, resulting in anti-inflammatory and immunosuppressive responses (5, 6). The structural basis for the ability of IgE to interact either with one or the other of these two disparate classes of receptors has recently been defined (7). The intrinsic flexibility of the IgE Cε3 domain results in both open and closed conformations of the IgE Fc, resulting in the binding of either FcεRI or CD23, respectively. Binding of either receptor induces an allosteric change in the IgE Fc to the alternative conformation, thus precluding the interaction with the other receptor (7). Binding of IgE to the type II, C-type lectin CD23 is neither carbohydrate- nor calcium-dependent, mediated exclusively through protein–protein interactions, generating a 2:1 complex of CD23 with the Cε3–Cε4 interface (7). DC-SIGN is a structurally homologous, calcium-dependent, carbohydrate-binding, type II lectin, tightly linked to CD23 on chromosome 19 (8), displaying ligand specificity for mannose-containing glycoconjugates and fucose-containing Lewis antigens. Binding of DC-SIGN to IgG requires that the complex, biantennary glycan, attached to the evolutionarily conserved glycosylation site Asn-297 and enclosed within the cavity formed by the Cγ2 domains of the A and B chains of the Fc dimer, be processed to the α2,6 sialylated form (9, 10). Importantly, no evidence has been found for DC-SIGN binding to sialylated glycans or glycoconjugates (11), suggesting that the binding interaction between sialylated Fc and DC-SIGN may not involve the canonical glycan interactions previously defined for this lectin and bind to sialylated Fc in a manner analogous to CD23 binding to IgE.

Results and Discussion

Because the Cγ2 domain of IgG lacks the intrinsic flexibility of Cε3 (12, 13), we hypothesized that sialylation may induce this flexibility, allowing it to engage DC-SIGN and limiting its binding to FcγRs. We therefore investigated the effect of sialylation on the Fc structure by circular dichroism, thermal and chemical denaturation, and anilinonaphthalene sulfonates (ANS) binding (Fig. 1). The CD spectrum of neuraminidase-treated and therefore asialylated human IgG1 Fc (NAse Fc, G2F glycoform) yields the classical spectral pattern associated with β-sheet structure with a minimum at 216 nm and a peak near 203 nm. Deglycosylation of the Fc induces dramatic shifts in the CD spectrum consistent with the structural changes observed in crystal structures of aglycosyl Fc (14, 15). Upon sialylation (A2F form), however, we observe a small shift in the spectra for sFc (Fig. 1A). Sialylation may alter the β-sheet content in the Fc because this spectral shift includes a ∼14% decrease in the ellipticity (Δε) value at 216 nm compared with NAse Fc. Deglycosylation of the Fc abrogated the CD differences between these two glycoforms (Fig. S1), indicating that the observed spectral differences were the result of a change in the Fc structure induced by sialylation.

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Fig. 1.

Sialylation destabilizes the Cγ2 domain of IgG1 Fc. (A) Circular dichroism spectra of IgG1 Fc glycoforms recorded in the far UV range (190–260 nm). Spectra represent an average of four scans with buffer spectra subtracted from sample spectra. (B) Thermal denaturation spectra of Fc glycoforms measured at 206.5 nm. (C) Change in free energy of unfolding, ΔG_U, with increasing concentration of chemical denaturant (GnHCl) calculated by the linear extrapolation method. Y-intercept estimates ΔG_H2O in aqueous solvent; m value (slope) correlates to solvent-accessible surface area exposed upon unfolding. (D) Fluorescence spectra of sFc and NAse Fc bound to hydrophobic probe ANS.

We next measured the thermal stability of these Fc glycoforms to evaluate their thermodynamic properties. Ellipticity values (Δε) for Fcs were recorded at 206.5 nm as a function of temperature (16). As shown in Fig. 1B, we observe a transition phase as the Cγ2 domain denatures above 65 °C, as described previously (17). Sialylated Fc has a lower melting temperature (T_M) than asialylated Fc, differing by 0.9 °C. Fully deglycosylated Fc is further reduced in its T_M by 3.1 °C, as previously reported (17). A second transition phase occurs above 80 °C, which corresponds to the denaturation of the Cγ3 domain (17, 18). The decrease in T_M^Cγ2 correlates with the CD spectral shifts illustrated in Fig. 1A. This suggests that structural changes induced by sialylation occur in the Cγ2 domain. As with T_M^Cγ2, the change in van’t Hoff enthalpy, ΔH_V°, and the free energy of folding, ΔG°, also decreased with sialylation and deglycosylation relative to NAse Fc (Fig. S2), which is indicative of decreased protein stability (Fig. S2).

GnHCl-induced denaturation (Fig. 1C and Fig. S3) also revealed a decrease in ΔG_H2O° values for sFc relative to NAse Fc (−6.4 kcal/mol and −7.125 kcal/mol, respectively). However, in contrast to thermal denaturation, GnHCl-induced denaturation revealed similar ΔG_H2O° values between deglycosylated and NAse Fc −7.27 and −7.125 kcal/mol, respectively. M values (Fig. 1C; SI Materials and Methods) for sFc, NAse Fc, or deglycosylated Fc were 1.2 kcal⋅mol⁻¹⋅M⁻¹, 0.575 kcal⋅mol⁻¹⋅M⁻¹, and 0.319 kcal⋅mol⁻¹⋅M⁻¹, respectively. The greater m value for sFc relative to NAse Fc or deglycosylated Fc suggests that sFc contain more solvent accessible surface area, which may result in more hydrophobic residues exposed to the aqueous solvent (19). To more directly measure hydrophobic surface area, sFc and NAse Fc were incubated with ANS, a chemical probe that fluoresces upon binding to hydrophobic surfaces on proteins (20). Fig. 1D shows that fluorescence intensity of ANS increases substantially with sFc and that the peak wavelength is blue-shifted by 30 nm compared with ANS alone. In contrast, NAse Fc only slightly increase fluorescence intensity of ANS and shifts the peak wavelength by 10 nm. Thus, the greater solvent-accessible surface area associated with sFc appears to increase hydrophobic surface area as well. Although it is known that the glycan at Asn-297 is required to maintain the quaternary structure of the Cγ2 dimer (14, 21), the results shown here indicate that effect of sialylation differs from deglycosylation on Fc structure and stability. Because GnHCl denaturation and ANS binding give comparable results for NAse or deglycosylated Fc (Fig. 1), although strikingly different for sialylated Fc, it suggests that sialylation of the glycan induces structural perturbations in the Fc that differ from deglycosylation that are required for DC-SIGN binding. Consistent with this interpretation, deglycosylated Fc does not bind DC-SIGN (3, 9).

The similarities in the structures of the FcεRI-IgE Fc complex (2) to the FcγRIII-IgG Fc complex (1) and the structures of the carbohydrate recognition domains (CRDs) of CD23 and DC-SIGN (Fig. S4) suggested that these Ig isotypes may have evolved similar structural modes to modulate their effector functions. Additional support for this hypothesis comes from the observations that, similar to the allosteric change in Fc conformation observed when IgE engages FcεRI or CD23, the effect of sialylation on the IgG Cγ2 conformation results in reduction in FcR binding (3, 9, 22) and the acquisition of DC-SIGN binding (3). As shown in Fig. 2 and Fig. S4, structural alignment of CD23 with DC-SIGN and IgE with that of IgG was performed. Overlaying of the predicted complexes with the IgE-CD23 complex (Fig. 2 A and B) demonstrates significant concordance of the molecules with no sterical clashes of the protein backbone. Overall, the interaction surface for the IgG/DC-SIGN complex (Fig. 2C, Left) yields productive but no prohibitive contacts between both molecules, and the IgG/CD23 complex model (Fig. 2C, Right) reveals a significant hydrophobic cluster within the interface. In addition to the protein–protein interaction, the α2,6-linked sialic acid on the α1,3-arm can contact DC-SIGN and may form an important hydrogen bond in the IgG/DC-SIGN complex, specifically to Asn370. The sialic acid is wedged between the residues Lys340 and Glu318 to the Cγ2 domain surface and stabilized by three hydrogen bonds. Besides these carbohydrate–amino acid interactions, the salt bridge triad between Gln342 in the Fc-Cγ3 domain and Asn350 and Glu353 of DC-SIGN as well as several hydrogen bonds mainly located in the Fc Cγ2–DC-SIGN interface (Lys338/Gly341→Glu353; Asn315→His278) and a hydrophobic contact (Leu314 in Fc-Cγ2; Leu321 in DC-SIGN) support the IgG/DC-SIGN interaction.

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Fig. 2.

Structural homology of the DC-SIGN/IgG1 Fc and the CD23/IgE Fc complex. (A) Structure-based sequence alignment of human DC-SIGN and IgG1-Fc to the CD23/IgE-Fc complex. The amino acid residues depicted in the one-letter code are colored according to their property (i.e., acidic residues in red, basic in blue, hydrophobic in green). Cysteine residues, all involved in disulfide bridges, are depicted in gray. The interactions between the complex partners are indicated by black lines connecting the involved residues with salt bridges relevant for the CD23/IgE-Fc complex shown in red (7). Hydrogen bonds between IgG and DC-SIGN or CD23 are shown in blue and green lines, respectively. (B) Model of the DC-SIGN/sFc complex. The protein main chains are represented as tubes. The CD23/IgE-Fc complex [thin gray tube, Protein Data Bank (PDB) code 4EZM] is overlaid with the model of the IgG Fc fragment (red and blue) in complex with DC-SIGN (magenta and green). Disulfide bridges and the carbohydrate moiety associated to the Fc fragment are shown as sticks. The square depicts the region shown in detail in C. The generation of the model is described in the supplementary materials. (C) Detailed interaction of DC-SIGN and CD23 with hIgG1-Fc. A close-up of some of the residues expected to be involved in the DC-SIGN/IgG1-Fc (Left) and CD23/IgG1-Fc complex (Right), respectively. The depicted clipping of the complex model is indicated in B.

The contribution of sialylation to Cγ2 structure and predicted binding interactions is proposed in Fig. 3. In the absence of sialylation, the α1,3 arm of the glycan protrudes into the internal space of the A-B cleft where it contacts the α1,3 arm of the second Cγ2 domain, thereby maintaining the “open” conformation of the Fc, whereas the α1,6 arm makes extensive noncovalent interactions with the amino acid backbone (23). Upon sialylation, the Fc fragment transitions into the “closed” conformation that—apart from the changes in the secondary structure of the Cγ2 domain—is essential for DC-SIGN or CD23 binding (Fig. 3). The structural changes observed within the sialylated Fc fragment are most likely induced by the interaction of the carbohydrate tree with the Cγ2 domain. The terminal sialic acid on the α1,3 arm is predicted to position in a pocket of the Fc formed by amino acids Lys340 and Glu318 (Fig. 2C), thereby approximating this sugar to the DC-SIGN interface. It is notable that only the α2,6 configuration of the sialic acid–galactose linkage can reasonably accommodate this interaction and bring the carbohydrate potentially in contact with the receptor, consistent with the absolute requirement for α2,6 sialylation of the Fc to facilitate DC-SIGN binding (9, 10). The extension of the carbohydrate on the α1,6 arm with a sialic acid would position it in a similar way in a pocket formed by Glu258 and Lys290. These data are consistent with the association of both carbohydrate chains with the protein core at physiological temperatures although only minor changes were observed upon sialylation (24).

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Fig. 3.

Structural changes upon glycosylation of IgG Fc fragments. (A) Schematic view of the principal glycosylation structures attached to Asn297 in the Fc fragment including the respective linkage. Gray shaded parts of the carbohydrate moiety are not displayed in the cartoon. (B) Cartoon of the proposed structural changes within the Fc fragment upon sialylation. The nongalactosylated (G0F) Fc-fragment (top) maintains an open conformation that allows the binding of FcγRs and precludes binding of DC-SIGN or CD23. In the fully α2,6-sialylated Fc (G2FS2), the α1,3-arm (orange/blue) associates with the protein core of the Cγ2 domain, inducing a closed conformation. The resulting closed conformation of the Fc fragment with a changed tertiary structure reveals the binding site for DC-SIGN, whereas that for FcγRs is blocked. The coloring of the carbohydrate moiety is according to the schematic view in A. (C, Upper) Front view of the Fc fragment with the individual domains colored separately with CHOs of the G0F form (PDB code 3AVE). (C, Lower) Front view of the model of fully sialylated Fc (G2FS2) with DC-SIGN bound to it. The coloring of the domains is according to the Fc fragment at top. The right side shows the top view of the respective structures.

The structural predictions of these models were tested by determining the ability of CD23 to bind to sialylated IgG, an interaction that had not been anticipated or predicted. CD23 expressing CHO-K1 cells bound sialylated but not asialylated IgG (Fig. 4A) in a dose-dependent manner and could be competed by soluble DC-SIGN (Fig. 4B). Conversely, sialylated IgG binding to DC-SIGN expressing cells could be competed by soluble CD23 (Fig. 4C). These low-affinity binding complexes are stabilized by the high avidity of the interactions formed by the 2:1 DC-SIGN-sFc or CD23-sFc complexes with the cell surface–expressed tetrameric DC-SIGN or trimeric CD23 molecules. This requirement may explain the lack of binding observed when soluble DC-SIGN is used in ELISA binding studies (25) and is thus not reflective of the physiological state of these complexes. In support of this notion, stabilizing the trimeric form of soluble CD23 through a leucine zipper motif linked to the N-terminus of the receptor (lz-CD23) allows for enhanced detection of sialylated IgG binding in a solid-phase ELISA format at physiological temperature relative to the extracellular region of CD23 alone (Fig. S5), as has been reported for IgE (26). However, in contrast to IgE, binding of sialylated IgG to trimeric CD23 is not enhanced at 4 °C.

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Fig. 4.

CD23 expressing cells preferentially bind α2,6-sialylated IgG. (A) In cell-based ELISA binding assays, DC-SIGN, CD23, and/or mock transfected CHO-K1 cells were pulsed with an increasing amount of sialylated (sIgG) or asialylated IgG. (A and B) For competition binding assays, DC-SIGN–expressing cells (B) or CD23-expressing cells (C) were pulsed with a constant amount of sIgG and an increasing amount of soluble CD23 or DC-SIGN. In all panels, bound IgG was detected using HRP-labeled anti-human antibody. ECD, extracellular domain.

The structural model and data reported here support the conclusion that a common mechanism for regulating the effector activity of immunoglobulins is accomplished through the alternation of Fc conformations between open and closed states, thereby regulating Fc binding to FcRs or SIGN/CD23, respectively. Regulation of these conformations may be intrinsic, as observed for IgE, resulting from the disordered Cε3 “molten globular” domain, or extrinsic, the result of modification of the IgG Asn-297 N-linked glycan. Pathogens have exploited this common mechanism to avoid host defense by shifting the equilibrium of the Fc conformation to the closed state, either by modulating the glycan composition (27) or stabilizing the closed conformation (28). Sialylation of the N-linked glycan of IgG induces a conformation that will also bind CD23 in addition to DC-SIGN, thereby providing a mechanism for suppressing follicular B-cell activation by regulating IgG sialylation to maintain homeostasis through feedback regulation by IgG of its synthesis.

Materials and Methods

Monoclonal human IgG1 preparations were used for biophysical characterization of Fc glycoforms. Sialylated Fc produced in two-step in vitro reaction with β1,4-GalT and ST6Gal. Binding assays performed in cell-based ELISA format with CHO-K1 cells transiently expressing full-length DC-SIGN or CD23. Detailed experimental and analytical procedures are presented in SI Materials and Methods.

Supplementary Material

Acknowledgments

Footnotes

References