Structural Analysis of Human IgG-Fc Glycoforms Reveals a Correlation Between Glycosylation and Structural Integrity

Abstract

Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defence mechanisms. Thus, when antigen-specific IgG antibodies form antigen/antibody immune complexes the effectively aggregated IgG can activate a wide range of effector systems. Multiple effector mechanisms result from cellular activation mediated through a family of IgG-Fc receptors differentially expressed on leucocytes. It is established that glycosylation of IgG-Fc is essential for recognition and activation of these ligands. IgG antibodies predominate in human serum and most therapeutic antibodies are of the IgG class.

The IgG-Fc is a homodimer of N-linked glycopeptide chains comprised of two immunoglobulin domains (Cγ2, Cγ3) that dimerise via inter-heavy chain disulphide bridges at the N-terminal region and non-covalent interactions between the C-terminal Cγ3 domains. The overall shape of the IgG-Fc is similar to that of a “horseshoe” with a majority of the internal space filled by the oligosaccharide chains, only attached through asparagine residues 297.

To investigate the influence of individual sugar (monosaccharide) residues of the oligosaccharide on the structure and function of IgG-Fc we have compared the structure of “wild-type” glycosylated IgG1-Fc with that of four glycoforms bearing consecutively truncated oligosaccharides. Removal of terminal N-acetylglucosamine as well as mannose sugar residues resulted in the largest conformational changes in both the oligosaccharide and in the polypeptide loop containing the N-glycosylation site. The observed conformational changes in the Cγ2 domain affect the interface between IgG-Fc fragments and FcγRs. Furthermore, we observed that the removal of sugar residues permits the mutual approach of Cγ2 domains resulting in the generation of a “closed” conformation; in contrast to the “open” conformation which was observed for the fully galactosylated IgG-Fc, which may be optimal for FcγR binding. These data provide a structural rationale for the previously observed modulation of effector activities reported for this series of proteins.

Introduction

Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defence mechanisms. Antigen-specific recognition by antibody results in the formation of immune complexes that may activate multiple effector mechanisms, resulting in the removal and destruction of the complex. The immunoglobulin G (IgG) molecule is comprised of three independent protein moieties connected through a flexible linker or hinge region. Two of these moieties are of identical structure and each expresses an antigen-specific binding site, the Fab regions; the third, or IgG-Fc region, expresses interaction sites for ligands that activate clearance mechanisms. The hinge region allows for segmental flexibility such that each Fab may access and bind its target antigen whilst the Fc remains accessible to effector ligands. The principal ligands through which effector pathways are initiated are cellular Fc receptors and the C1 component of complement. The aggregated forms of IgG-Fc present in immune complexes engage and cross-link the membrane bound Fcγ-Receptors (FcγRs), to initiate an activating or inhibitory signal. Activating signals are mediated through receptors expressing an immunoreceptor tyrosine-based activation motif (ITAM) or inhibitory signals through receptors expressing an immunoreceptor tyrosine-based inhibitory motif (ITIM), within or associated with the cytoplasmic part of the FcγR.¹ FcγRs are constitutively expressed on leucocytes, e.g. B-cells, T-cells, NK-cells, macrophages, eosinophils, neutrophils and mast cells, etc. The consequences of cellular activation by immune complexes depend on the nature of the immune complex formed, the isotype distribution of the specific antibody and the cell type; effector responses include antibody-dependent cellular cytotoxicity (ADCC),² secretion of inflammatory mediators,³ enhanced antigen presentation, regulation of antibody production,4., 5. the oxidative burst⁶ and phagocytosis.⁷ FcγRs are type I transmembrane proteins and belong to the immunoglobulin superfamily. In humans there are three classes of FcγRs: the high affinity IgG receptor FcγRI (K_D≈10⁻⁸ M) and two low affinity receptors FcγRII and FcγRIII (K_D≈10⁻⁶–10⁻⁷ M), which are responsible for the clearance of immune complexes.⁸ They occur in various isoforms (FcγRIa, -b1, -b2, -c; FcγRIIa1, -2, -b1-3, -c) and there exists a significant polymorphism within the human population (FcγRIIa1-HR, -LR; FcγRIIIb-NA1, -NA2).⁹

Comparison of intact murine and human IgG structures reveals extreme asymmetry and strong interdomain flexibility within the antibody.¹⁰ The homodimeric Fc fragment of IgG has a horseshoe-like structure and is glycosylated at the Asn297 residues, located in the Cγ2 domains. The oligosaccharide is of the complex type and is comprised of a “core” heptasaccharide (Figure 1) that may be extended by the addition of fucose to the primary N-acetylglucosamine (GlcNAc) residue and galactose to the arm GlcNAc residues. Galactosylated oligosaccharides may be further extended by the addition of sialic acid. The predominant core structure observed for human IgG is the octasaccharide FucGlcNAc₂Man₃GlcNAc₂ which may be extended by the variable addition of galactose and sialic acid residues¹¹ (Figure 1). The structure of the oligosaccharide moiety reveals branching oligosaccharide arms at the Man4 residue (Figure 1). The α(1–6) arm (Figure 1), branching from Man4, is directed towards the Cγ2–Cγ3 interface region and makes multiple interactions with the surface of the Cγ2 domain, whereas the α(1–3) arm extends into the space between the Cγ2 domains and contacts the α(1–3) arm of the opposing Cγ2 domain (Figure 2(a) and (b)). The sugars of the oligosaccharide trunk and the α(1–6) arm make multiple non-covalent interactions with the protein surface of the Cγ2 domain, predominantly through hydrophobic amino acid residues.12., 13., 14.

Complete N-deglycosylation of IgG1 results in a loss of its capacity to bind FcγRs15., 16., 17., 18. and consequently to a failure in the initiation of the corresponding effector functions.¹⁹ The analysis of the binding affinity of various truncated glycoforms of IgG-Fc demonstrated a progressive decrease in the affinity of binding to soluble recombinant FcγRIIb.20., 21. Although these biochemical data characterise the effects of particular sugar residues, the precise manner in which the oligosaccharide influences the Fc-fragment structure and function remains unclear.

Given a virtually infinite repertoire of antibody molecules we can anticipate the development of a huge catalogue of antibody therapeutics. Optimal therapeutic efficacy will depend on the specificity and affinity for antigen recognition and the effector activities triggered in vivo. Some treatment protocols may attempt to maximise effector function activation whilst for others down regulation may be considered to be of benefit.

Detailed information about the interaction of the IgG-Fc with recombinant soluble FcγRIII was obtained from the crystal structure of the IgG1-Fc-fragment in complex with the FcγRIII.²² Interestingly only one minor contact between a sugar residue and the FcγRIII was observed. Since the activation of FcγRIII is dependent on IgG-Fc glycosylation it is apparent that the oligosaccharide moiety exerts its influence, indirectly, through modulation of IgG-Fc conformation. We have, therefore, investigated the crystal structure of a series of homogeneous, truncated glycoforms of IgG-Fc and interpreted our findings in relation to our previous studies of their influence on Fc receptor recognition and activation.