X-ray crystallography has provided structural data on complete IgG molecules (Fig. 3.5). Mobility around the hinge region of IgG allows for the generation of the Y- and T-shaped structures visualized by electron microscopy.

Fig. 3.5 Model of an IgG molecule

A model of an IgG molecule showing the polypeptide backbones of the four chains as a ribbon. Heavy chains are shown in dark blue and dark green. The antigen-binding sites are at the tips of the arms of the Y-shaped molecule and are formed by domains from both the heavy and light chains. The extended, unfolded hinge region lies at the center of the molecule. Carbohydrate units are shown as ball and stick structures, covalently linked to the Fc region.

For all antibody isotypes there is pairing between VH/VL and CH1/CL domains through extensive non-covalent interactions to form the antigen binding (Fab) region.

Antigen binding is a common feature for IgG-Fab regions of each of the four human IgG subclasses; however, although there is >95% sequence homology between the IgG-Fc regions each IgG subclass exhibits a unique profile of effector activities.

The hinge regions are structurally distinct and determine the relative mobilities of the IgG-Fab and IgG-Fc moieties within the intact molecule. The equivalent of the IgG hinge region is present in all isotypes, except IgM.

In addition to the pairing of the VL/VH and CL/CH1 domains the CH3 domains of the IgG-Fc are also paired through non-covalent interactions.

The CH2 domains are not paired and, potentially, present a hydrophobic surface to solvent. This unfavorable property is avoided by interactions with a hydrophilic N-linked oligosaccharide moiety.

The N-linked oligosaccharide of the CH2 domain, although accounting for only 2–3% of the mass of the IgG molecule, is crucial to the expression of effector functions. The conformation of the CH2 domain protein moiety, and ultimately the IgG-Fc, results from reciprocal interactions between the CH2 protein and the oligosaccharide. The oligosaccharide exhibits structural heterogeneity and effector functions may be modulated depending on the particular oligosaccharide structure (glycoform) attached.

Assembled IgM molecules have a ‘star’ conformation

IgM is present in human serum as a pentamer of the basic four-chain structure (see Fig. 3.w3). Each heavy chain is comprised of a VH and four CH domains. One advantage of this pentameric structure is that it provides 10 identical binding sites, which can dramatically increase the avidity with which IgM binds its cognate antigen. Given that serum IgM commonly functions to eliminate bacteria containing low affinity, polysaccharide antigens, the increased avidity provided by the pentameric structure provides an important functional advantage.

Fig. 3.w3 Structural characteristics of various human immunoglobulins

Carbohydrate side chains are shown in blue. Inter heavy (H) chain disulfide bonds are shown in red, but interchain bonds between H and L chains are omitted. (1) A model of IgG1 indicating the globular domains of H and L chains. Note the apposition of the CH3 domains and the separation of the CH2 domains. The carbohydrate units lie between the CH2 domains. (2) Polypeptide chain structure of human IgG3. Note the elongated hinge region. (3) IgM H chains have five domains with disulfide bonds cross-linking adjacent CH3 and CH4 domains. The possible location of the J chain is shown. IgM does not have extended hinge regions, but flexion can occur about the CH2 domains. (4) The secretory component of sIgA is probably wound around the dimer and attached by two disulfide bonds to the CH2 domain of one IgA monomer. The J chain is required to join the two subunits. (5) This diagram of IgD shows the domain structure and a characteristically large number of oligosaccharide units. Note also the presence of a hinge region and short octapeptide tailpieces. (6) IgE can be cleaved by enzymes to give the fragments F(ab′)₂, Fc, and Fc′. Note the absence of a hinge region.

Covalent disulfide bonds between adjacent CH2 and CH3 domains, the C terminal 18-residue peptide sequence, referred to as the ‘tailpiece’, and J chain link the subunits of the pentamer.

J chain is synthesized within plasma cells, has a mass of ~15 kDa and folds to form an immunoglobulin domain. Each heavy chain bears four N-linked oligosaccharide moieties, however, the oligosaccharides are not integral to the protein structure in the same way as in IgG-Fc. Oligosaccharides present on IgM activate the complement cascade via binding to the mannose binding lectin (see Chapter 4).

In electron micrographs the assembled IgM molecule is seen to have a ‘star’ conformation with a densely packed central region and radiating arms (Fig. 3.6); however, electron micrographs of IgM antibodies binding to poliovirus show molecules adopting a ‘staple’ or ‘crab-like’ configuration (see Fig. 3.6), which suggests that flexion readily occurs between the CH2 and CH3 domains, though this region is not structurally homologous to the IgG hinge. Distortion of this region, referred to as dislocation, results in the ‘staple’ configuration of IgM required to activate complement.

Fig. 3.6 Electron micrographs of IgM molecules

(1) In free solution, deer IgM adopts the characteristic star-shaped configuration. ×195 000. (2) Rabbit IgM antibody (arrow) in ‘crab-like’ configuration with partly visible central ring structure bound to a poliovirus virion. × 190 000.

((1) Courtesy of Drs E Holm Nielson, P Storgaard, and Professor S-E Svehag. (2) Courtesy of Dr B Chesebro and Professor S-E Svehag.)

Secretory IgA is a complex of IgA, J chain and secretory component

IgA present in serum is produced by bone marrow plasma cells and secreted as a monomer with the basic four-chain structure. Each heavy chain is comprised of a VH and three CH domains.

The IgA1 and IgA2 subclasses differ substantially in the structure of their hinge regions:

• the hinge of IgA1 is extended and bears O-linked oligosaccharides;

• the hinge of IgA2 is truncated, relative to IgA1.

A deficit in the addition of O-linked sugars within the hinge region of IgA1 protein has been linked with the disease IgA nephropathy.

IgA is the predominant antibody isotype in external secretions but is present as a complex secretory form. IgA is secreted by gut localized plasma cells as a dimer in which the heavy chain ‘tailpiece’ is covalently bound to a J chain, through a disulphide bond (see Fig. 3.w3).

Electron micrographs of IgA dimers show double Y-shaped structures, suggesting that the monomeric subunits are linked end-to-end through the C terminal Cα3 regions (Fig. 3.7).

Fig. 3.7 Electron micrographs of human dimeric IgA molecules

The double Y-shaped appearance suggests that the monomeric subunits are linked end to end through the C terminal Cα3 domain. × 250 000.

(Courtesy of Professor S-E Svehag.)

The dimeric form of IgA binds a poly-Ig receptor (Fig. 3.8) expressed on the basolateral surface of epithelial cells. The complex is internalized, transported to the apical surface where the poly-Ig receptor is cleaved to yield the secretory component (SC) that is released still bound to the IgA dimer. The released secretory form of IgA is relatively resistant to cleavage by enzymes in the gut and is comprised of:

• two units of IgA;

• J chain; and

• a secretory component (mass 70 kDa) (see Figs. 3.w3 and 3.8).