Structural characterization of the Man5 glycoform of human IgG3 Fc

Abstract

Immunoglobulin G (IgG) consists of four subclasses in humans: IgG1, IgG2, IgG3 and IgG4, which are highly conserved but have unique differences that result in subclass-specific effector functions. Though IgG1 is the most extensively studied IgG subclass, study of other subclasses is important to understand overall immune function and for development of new therapeutics. When compared to IgG1, IgG3 exhibits a similar binding profile to Fcγ receptors and stronger activation of complement. All IgG subclasses are glycosylated at N297, which is required for Fcγ receptor and C1q complement binding as well as maintaining optimal Fc conformation. We have determined the crystal structure of homogenously glycosylated human IgG3 Fc with a GlcNAc2Man5 (Man5) high mannose glycoform at 1.8 Å resolution and compared its structural features with published structures from the other IgG subclasses. Although the overall structure of IgG3 Fc is similar to that of other subclasses, some structural perturbations based on sequence differences were revealed. For instance, the presence of R435 in IgG3 (and H435 in the other IgG subclasses) has been implicated to result in IgG3-specific properties related to binding to protein A, protein G and the neonatal Fc receptor (FcRn). The IgG3 Fc structure helps to explain some of these differences. Additionally, protein-glycan contacts observed in the crystal structure appear to correlate with IgG3 affinity for Fcγ receptors as shown by binding studies with IgG3 Fc glycoforms. Finally, this IgG3 Fc structure provides a template for further studies aimed at engineering the Fc for specific gain of function.

A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress

“…A previous study indicated that the CH3 and CH2 domains of the IgG2 antibody interacted via L251 in the CH2 region and residues M428, H429, E430, and H435 in the CH3 domain . This is supported by another study that suggested that the R435 residue in the IgG3 CH3 domain may influence the CH2 domain pivot . Based on these data and our own results, we hypothesized that the substitutions made in the IgG3 CH3 domain affected the CH2 domain structure allosterically, impacting FcγRIIIa binding.…”

“…We further modified IgG3KV by introducing an R435H substitution that was previously shown to be important for protein A binding . R435H substitution led to the creation of IgG3KVH, with three amino acid mutations introduced into the IgG3 antibody (N392K, M397V, and R435H).…”

“…In contrast, the H435 residue is located some distance away from the I253 in the IgG1 CH2 domain. Therefore, the R435 and I253 residues may actually bring the CH2 domain closer . We hypothesized that the R435H substitution affects the CH2 pivot, increasing the thermal stability of the CH2 domain.…”

Immunoglobulin G Subclass-Specific Glycosylation Changes in Primary Epithelial Ovarian Cancer

“…Despite being relatively underrepresented (approximately 5-8% of total IgG), IgG 3 deserves special attention, as in many ways, it is a unique IgG subclass. Its separation can be accomplished due to a particular feature of its amino acid sequence, that is, the presence of arginine instead of histidine at position 435 that prevents its binding to Protein A (42). Moreover, due to its superior affinity toward activating FcγRs and C1q, IgG 3 was shown to be the strongest inducer of Fc-mediated effector functions, including ADCC and CDC (23).…”

Effects of Glycan Structure on the Stability and Receptor Binding of an IgG4-Fc

“…IgG3-based mAbs are usually not developed because of their susceptibility to proteolysis, the existence of many allotypes, and their shorter half-life in vivo. 4 The 4 IgG isotypes are highly conserved with respect to their primary sequences, presenting more than 90% similarity. 5 However, each isotype reveals a distinctive profile with regard to biological effector functions and immune complexes.…”