IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 6. Antibodies and Cellular Receptors Therefor

ANTIBODY STRUCTURE

The basic four-chain structural unit (monomer) of immunoglobulin molecules (Fig. 6.5) is formed by polypeptide chains of two different types. The smaller light (L) chains have a molecular mass of 25 kDa and are identical across all classes, whereas the larger heavy (H) chains, with a molecular mass of 50–77 kDa, vary structurally among different classes and subclasses of IMMUNOGLOBULINS. The polypeptide chains are held together by covalent and non-covalent bonds.

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Fig. 6.5. The N-terminal sequences of both light (L) and heavy (H) chains of IgG1 exhibit Variability (V), which is why these regions are designated Vl and Vh, respectively. The remaining PARTS OF THE molecule have a relatively invariant (constant - C) Structure. The constant region of the light chain is designated Cl. The constant region of the heavy chain is further subdivided into three structurally distinct regions: Ch1, Ch2, and Ch3. Both the Variable and constant Regions of the light and heavy chains form globular structures called domains, stabilized by intrachain Disulfide Bonds (shown in red). The antigen-binding sites of the immunoglobulin molecule are formed by the variable domains Vl and Vh. The segment of the heavy chain between the Ch1 and Ch2 domains is called the "hinge" region; it provides flexibility, allowing the two antigen-binding sites to function independently of each other. With the exception of the Ch2 domain, the domains of one heavy chain lie in close proximity to the homologous (V and C) domains of the light chain and to the Ch3 region of the other heavy chain (see Fig. 6.7). Carbohydrate moieties are attached to the Ch2 domains.

Each chain contains a variable and a constant region. In most vertebrates, light chains exist in two different isotopic forms, designated kappa (k) and lambda (λ). Pairs of light and heavy chains of any type can combine in an immunoglobulin molecule, but both chains in a given pair belong to the same type.

As established by Hilschmann, Craig, et al. (1965), light chains consist of two distinct regions. The C-terminal half (approximately 107 amino acid residues) of the chain is identical (constant) in light chains of all types (excluding certain allotypic and isotypic variants, see below); it is designated the constant, or Cl region (constant light chain). Meanwhile, the N-terminal half of this chain exhibits numerous variations in Amino Acid Sequence, which is why it is termed the variable, or Vl region (variable light chain).

IgG molecules possess a "typical" antibody structure

An IgG molecule can be considered a "typical" antibody (Fig. 6.5). It contains two intrachain disulfide bonds in each light chain—one in each of the variable and constant regions (Fig. 6.6)—and four such bonds in each heavy (γ) chain, which is twice as long as the light chain. Each disulfide bond encloses a peptide loop of 60–70 amino acid residues; comparing the Amino acid sequences of these loops reveals a strikingly high degree of Homology. Largely for this reason, each immunoglobulin polypeptide chain forms several globular domains with very similar secondary and tertiary structures, as illustrated for the light chain in Fig. 6.6.

The peptide loop enclosed by a disulfide bond forms the central part of a "domain," which contains a total of approximately 110 amino acid residues. In both light and heavy chains, the domains closest to the N-terminus are formed by the variable regions Vl and Vh, respectively (Fig. 6.7). The heavy chains of IgG, IgA, and IgD have three additional domains—Ch1, Ch2, and Ch8 [Note: Ch3], which constitute the constant region. In the μ and ε chains, a single additional domain immediately follows Ch1 (see Fig. 6.10); thus, the C-terminal domains of IgM and IgE [Note: IgC] heavy chains (designated Cμ4 and Cε4) are homologous to the Ch3 domain of IgG (Cγ3).

Fig. 6.6. The immunoglobulin light chain folds stereotypically such that its linear polypeptide segments form two domains—variable and constant—running parallel to the longitudinal axis of the domain and forming two layers with oppositely directed amino acid sequences. Numerous hydrophobic amino acid side chains face the space between the layers. One of the layers in each domain is formed by four polypeptide segments (indicated by white arrows), and the other by three (indicated by black arrows); these two layers are connected by a single disulfide bond (red bar). The segments of the Vl domain are arranged such that the hypervariable regions project onto the surface as three separate (but spatially close) loops. The position number of one of The amino acid residues is indicated for each hypervariable region.

Fig. 6.7. Model of an IgG1 molecule showing the globular domains of the heavy (H) and light (L) chains. Note the close apposition of the Ch3 domains and the Separation of the Ch2 domains, between which carbohydrate moieties are located (shown in blue). In this drawing (as well as in Fig. 6.10), disulfide bonds between the H and L chains are not shown.

X-ray crystallographic data have made it possible to reconstruct the α-carbon backbone and build computer models of entire IgG molecules (Fig. 6.8). Model IgGs appear as Y- and T-shaped structures, and similar shapes of IgG have been observed using Electron Cell/15.html">Microscopy.

Fig. 6.8. Computer model of a human IgG1 molecule, constructed from X-Ray Diffraction data of the Dob protein, which lacks the hinge region. Protein molecules of this type do not exhibit the flexibility characteristic of normal human IgG molecules. It is precisely their structural rigidity that allows for higher resolution analysis. One heavy chain is colored blue, the other red, and both light chains are green. Carbohydrate chains attached to Polypeptides in the Fc region of the molecule are shown in turquoise. According to this model, the Cγ2 domains interact very weakly with each other, whereas the Cγ3 domains interact quite strongly. (After David R. Davies et al. 1977. Proc. Natl. Acad. Sci. USA 74. Computer graphics generated using a system developed by Richard J. Feldmann, National Institutes of Health, USA).

In the Fab region of the immunoglobulin molecule, homologous domains of the light and heavy chains are paired (as shown in Fig. 6.7); the Cγ3 domains of the two heavy chains also form a pair, but the Cγ2 domains are separated by carbohydrate moieties.

Despite the structural similarity of homologous domains, interdomain interactions differ significantly between various pairs. For example, variable domains contact each other via layers consisting of three chain segments, whereas constant domains contact via layers of four segments (for a schematic of the two-layer segment arrangement in the two domains of a light chain, see Fig. 6.6). The IgG1 molecular model presented in Fig. 6.7 generally provides an adequate representation of the elementary units within all immunoglobulin isotypes, although each class and subclass has its own characteristic structural nuances.

IgG. The four human IgG subclasses differ only slightly in the amino acid sequence of their heavy chains. These differences, which mainly pertain to the hinge region, account for the isotypic variations in the position and number of interchain disulfide bonds. Among the four subclasses, IgG3 possesses the most pronounced structural feature—an elongated hinge region—which explains its higher molecular mass and, in part, its enhanced biological activity (Fig. 6.9).

Fig. 6.9. Schematic representation of a human IgG3 molecule. Note the elongated hinge region of the heavy chains.

IgM. In humans, IgM is typically found as a pentamer of the basic four-chain structural unit (Fig. 6.10). Its μ chain differs from IgG γ chains by having a different amino acid sequence and an additional constant domain with a C-terminal peptide of 18 amino acid residues. The pentamer subunits are linked by disulfide bonds between the Cμ3 domains and, likely, between the C-terminal Peptides. Electron microscopy reveals that the IgM molecule has a tightly packed central core from which five branches radiate.

Fig. 6.10. The heavy chains of IgM fold spatially to form five domains, with the Cμ3 and Cμ4 domains of each chain linked by a disulfide bond to the corresponding domain of the adjacent monomer. The carbohydrate side-chain attachment sites (in blue) and the putative position of the J chain are also indicated. While IgM molecules lack extended hinge regions, they retain The ability to flex in the region of the Cμ2 domains.

Micrographs show that IgM Antibodies bound to a bacterial flagellum (or to a poliovirus virion, as seen in Fig. 6.11) adopt a crab-like conformation. This shape indicates that the heavy chains in the region between Cμ2 and Cμ3 can readily flex, although this region is structurally non-homologous to the hinge region of IgG. The relative spatial arrangement of the different parts of the IgM molecule in this crab-like conformation is presumably related to Complement activation.

Fig. 6.11. Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF IgM molecules. 1. A deer IgM molecule in solution assuming its characteristic star-like shape. x 195,000. (Photographs kindly provided by Dr. E. Holm, Dr. Nielson P. Storgaard, and Prof. S.-E. Svehag.) 2. A rabbit IgM molecule bound to a poliovirus virion (indicated by the arrow) in a crab-like conformation; the central rounded region of the molecule is partially visible, x 190,000. (Photographs kindly provided by Dr. V. Chesebro and Prof. S.-E. Svehag.)

The IgM molecule is characterized by two key features: numerous Oligosaccharides attached to the μ chain and an additional peptide J chain (from 'joining'), which is thought to participate in the polymerization of monomeric units prior to secretion from the producing cell. The J chain is a 137-amino-acid polypeptide that forms an immunoglobulin-type domain. Each IgM molecule contains only a single J chain. It is linked via disulfide bonds to the C-terminal 18-amino-acid Peptides of the heavy chains of individual monomers (the disulfide bond is formed by the Cysteine residue at the penultimate position). Notably, it has been observed that Cells secreting IgM predominantly as hexamers lack free J chains.

IgA. Comprising 472 amino acid residues, the α chain folds into four domains: Vh, Cα1, Cα2, and Cα3 (Fig. 6.12). Similarly to IgM, the IgA heavy chain contains an additional C-terminal 18-amino-acid peptide with a cysteine residue at the penultimate position. This residue can covalently interact with a J chain to join two molecules into a dimer. In electron micrographs, IgA dimers appear as double Y-shapes, indicating an end-to-end joining of two monomeric subunits mediated by their C-terminal Cα3 regions (Fig. 6.13).

Fig. 6.12. Domain structure of IgA1 and the putative arrangement of carbohydrate chains (shown in blue). The 18-amino-acid 'tail' peptide at the C-terminus (a feature shared with IgM) and the hinge region are indicated.

Fig. 6.13. Electron micrographs of human dimeric IgA molecules. The double Y-shapes demonstrate the end-to-end joining of two monomeric subunits within the Cα3 domain region. x 250,000. (Photograph kindly provided by Prof. S.-E. Svehag.)

Secretory IgA (sIgA) occurs predominantly as an 11S dimers (molecular mass ~380 kDa). A fully assembled molecule consists of two IgA monomers, one secretory component (molecular mass ~70 kDa), and one J chain (molecular mass ~15 kDa) (Fig. 6.14). The exact Nature of the linkages among all these peptide chains remains incompletely understood. Unlike the J chain, the secretory component is synthesized not by plasma cells, but by epithelial cells. IgA molecules held in a dimeric configuration by the J chain and secreted by subepithelial plasma Cells of the mucosal membranes actively bind the secretory component as they traverse the epithelial layer. This component facilitates the delivery of sIgA antibodies into bodily secretions and protects them from proteolysis.

Fig. 6.14. The secretory component presumably wraps around the sIgA dimer, attaching via two disulfide bonds to the Cα2 domain of one of the monomers; the J chain is required to hold the two monomers together.

The predominant IgA subclass in both serum and bodily secretions (nasal mucus, saliva, tears, milk) is IgA1 (~90% and 70–95%, respectively). However, in the lumen of the Large Intestine, approximately 60% of IgA belongs to the IgA2 subclass. Many commensal Bacteria of the Upper Respiratory Tract microflora secrete proteases capable of cleaving IgA1.

IgD. IgD accounts for less than 1% of serum immunoglobulins. This protein is significantly more sensitive to proteolysis than IgG1, IgG2, IgA, or IgM, and also exhibits a strong tendency toward spontaneous proteolysis. Apparently, its δ chains are held together by a single disulfide bond and are associated with A large number of carbohydrate (oligosaccharide) chains (Fig. 6.15).

Fig. 6.15. Domain structure of IgD, featuring the high number of oligosaccharide chains typical of this isotype. The hinge region and short C-terminal octapeptides are indicated.

IgE. The IgE molecule (Fig. 6.16) is composed of larger ε chains (72.5 kDa compared to other isotypes), which contain a greater number of amino acid residues (approximately 550) and form five domains (Vh, Cε1, Cε2, Cε3, and Cε4).

Fig. 6.16. The IgE molecule can be cleaved by Enzymes into F(ab')₂, Fc, and Fc' fragments. A hinge region is absent.