Molecular Biology of the Cell - Volume 3 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

From Cells to Multicellular Organisms
The Immune System
Functional Properties of Antibodies

Vertebrates quickly succumb to infections if they are unable to produce Antibodies. Antibodies protect us against infections by inactivating Viruses or Bacterial toxins and by mobilizing The Complement System and various types of leukocytes, which destroy invading microorganisms and larger parasites. Synthesized exclusively by B lymphocytes, antibodies are produced in millions of varieties, each with a unique Amino Acid Sequence and a distinct antigen-binding site. Collectively known as IMMUNOGLOBULINS (abbreviated as Ig), they constitute one of the major protein Components of the Blood, accounting for roughly 20% of the total plasma protein by weight. In this section, we will examine the five classes of antibodies found in higher vertebrates, each of which triggers a characteristic biological response following antigen binding.

18.2.1. Antigen-Specific Receptors on B Cells Are Antibody Molecules [12]

As predicted by clonal Selection theory, all antibody molecules produced by a given B Cell possess the identical antigen-binding site. The initial antibodies synthesized by a newly formed cell are not secreted; instead, they are inserted into The Plasma Membrane, where they function as receptors for antigen. Each B cell carries approximately 10⁵ such molecules on its plasma membrane.

When an antigen binds to antibody molecules On the surface of a naive or memory B cell, this typically triggers a complex cascade of events leading to cell proliferation and the maturation of memory cells or active (antibody-secreting) cells. Active cells produce large quantities of soluble (non-membrane-bound) antibodies with the exact same antigen-binding site as The Cell-surface antibodies, releasing these into the bloodstream. Active B cells may begin secreting antibodies while still small lymphocytes, but The final stage of this differentiation pathway is the large plasma cell (see Fig. 18-4, C), which secretes antibodies at a rate of about 2,000 molecules per second. Apparently, plasma cells devote such a massive fraction of their protein-synthesizing machinery to antibody production that they lose the capacity for further growth and division, dying within a few days.

18.2.2. Antibody Production by B Cells Can Be Stimulated in Culture [13]

The 1960s witnessed breakthroughs that opened up new avenues for studying B cells. The first of these was The Development of the plaque assay,

which made it possible to identify and enumerate individual active B cells producing antibodies against a specific antigen. In the simplest version of this assay, lymphocytes (usually from the Spleen) are harvested from an animal immunized with sheep red Blood Cells (SRBCs). They are then suspended in Agar together with an excess of SRBCs. As a result, a lawn of immobilized SRBCs interspersed with lymphocytes forms in the culture dish. Under these conditions, the cells cannot migrate, but any antibodies secreted by a B cell will diffuse outward and coat The surface of nearby SRBCs. Such antibody-coated red blood cells can be lysed by adding complement (Section 18.5). Consequently, the presence of each antibody-secreting cell is revealed by a clear zone (a plaque) in the opaque layer of SRBCs. A similar approach can be used to count cells producing antibodies against other Antigens, such as Proteins or Polysaccharides, by coupling these antigens to the surface of sheep red blood cells.

The second major advance was the realization that B cells can be induced to produce antibodies by exposing them to antigen in culture, where cell interactions and environmental conditions can be precisely controlled. This research revealed that The stimulation of antibody secretion by B lymphocytes in response to most antigens requires T Lymphocytes and specialized antigen-presenting cells; the underlying intercellular interactions will be described later (Section 18.6.12).

18.2.3. Antibodies Have Two Identical Antigen-Binding Sites [11]

The simplest antibody molecules have a Y-shaped Structure with two identical antigen-binding sites—one at the tip of each of the two "arms" (Fig. 18-12). Because they possess two such sites, these antibodies are termed bivalent. Such antibodies can cross-link antigen molecules into an extensive network, provided each antigen molecule bears three or more antigenic determinants (Fig. 18-13). Once it reaches a critical size, this network precipitates out of solution. The tendency of large immune complexes to precipitate is a useful property for detecting antibodies and antigens. The efficiency of antigen binding and cross-linking is greatly enhanced by the flexible hinge region of the antibody, which allows the distance between the two antigen-binding sites to vary (Fig. 18-14).

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Fig. 18-13. Because antibodies have two identical antigen-binding sites, they are able to cross-link antigens. The types of antigen-antibody complexes formed depend on the number of antigenic determinants on the antigen. This diagram illustrates the binding of a single species of antibody (a monoclonal antibody) to antigens bearing one, two, or three identical antigenic determinants. Antigens with two determinants can form small cyclic complexes or linear chains with antibodies, whereas antigens with three or more determinants can form extensive three-dimensional networks that readily precipitate.

Fig. 18-14. The hinge region of an antibody molecule enhances the efficiency of antigen binding and cross-linking.

The protective action of antibodies extends far beyond their mere ability to bind antigen. They perform a variety of other Functions mediated by the "tail" of the Y-shaped molecule. This region determines The Fate of the antigen once it is bound. Due to the peculiarities of immunoglobulin Biosynthesis, antibodies with identical antigen-binding sites can feature markedly different tail regions (Section 18.4.7), each conferring distinct functional properties, such as The ability to activate complement (Section 18.5.1) or bind to phagocytic cells (Section 18.2.5).

18.2.4. An Antibody Molecule Consists of Two Identical Light Chains and Two Identical Heavy Chains [14]

The fundamental structural unit of an antibody molecule is formed by four polypeptide chains—two identical light chains (L chains, each containing approximately 220 Amino Acids) and two identical heavy chains (H chains, each containing approximately 440 amino acids). All four chains are held together by noncovalent and covalent (disulfide) bonds. The molecule is composed of two symmetrical halves, each with an identical antigen-binding site; the antigen-binding surface is typically formed by the collaboration of one L and one H chain (Fig. 18-15).

The Proteolytic Enzymes Papain and Pepsin cleave antibody molecules into distinct characteristic fragments. Papain yields two separate, identical Fab fragments (Fab stands for fragment antigen binding), each possessing a single antigen-binding site, and one Fc fragment (Fc stands for crystallizable fragment). Pepsin yields one large F(ab')₂ fragment, composed of two covalently linked F(ab') fragments (each slightly larger than a Fab fragment), along with many small Peptides (Fig. 18-16). Because F(ab') fragments are bivalent, unlike monovalent Fab fragments, they retain the ability to cross-link antigens and form precipitates. None of these fragments exhibit the other biological properties of native antibody molecules because they lack the tail (Fc) region responsible for those functions.

Fig. 18-15. A typical antibody molecule is composed of two identical heavy (H) chains and two identical light (L) chains. Note that the antigen-binding sites are formed by the N-terminal domains of both L and H chains, whereas the tail and hinge regions are formed exclusively by H chains. Each H chain carries one or more oligosaccharide (carbohydrate) side chains of unknown function.

Fig. 18-16. Fragments generated by the proteolytic Cleavage of antibody molecules with papain and pepsin.

18.2.5. There are five Classes of H Chains, Each with Distinct Biological Properties [11, 15]

Higher vertebrates possess five classes of antibodies—IgA, IgD, IgE, IgG, and IgM—each characterized by a distinct class of H chains: α, δ, ε, γ, and μ, respectively. IgA molecules contain α chains, IgG molecules contain γ chains, and so forth (Table 18-1). In addition, there are several subclasses of IgG and IgA immunoglobulins; for instance, humans have four IgG subclasses (IgG1, IgG2, IgG3, and IgG4), which contain the heavy chains γ₁, γ₂, γ₃, and γ₄, respectively. Different H chains impart distinct Conformations to the hinge and tail regions of antibodies, thereby dictating the specific functional properties of each class and subclass (see Table 18-1).

IgM is invariably the first antibody produced by developing B cells, although many B cells eventually switch to synthesizing antibodies of other classes (Section 18.4.7). The immediate precursor of a B cell, known as a pre-B cell, initially synthesizes and accumulates only μ chains. Later on, light chains begin to be produced within the cell; they assemble with μ chains to form four-chain IgM molecules (each comprising two μ chains and two light chains), which are then incorporated into the plasma membrane. In this way, the cell acquires surface receptors enabling it to bind antigen, at which point it is designated a naive B lymphocyte. Many of these naive B cells soon begin expressing surface IgD molecules as well, which share the same antigen-binding site as the IgM molecules.

Table 18-1. Properties of the Major Classes of human antibodies

IgM is not only the first class of antibodies to appear on the surface of developing B cells; it is also the primary class of antibodies secreted into the blood during the Cytology/cytology/16.html">Early stages of a primary Immune Response. In its secreted form, IgM consists of five four-chain units, giving the molecule a total of 10 antigen-binding sites. Each pentamer contains an additional polypeptide chain called the J-chain (joining chain). The J-chain is synthesized by IgM-secreting cells and is covalently inserted between two adjacent Fc regions, where it closes the cyclic STRUCTURE OF THE oligomer and prevents further polymerization (Fig. 18-17).

The binding of an antigen to the Fab regions of a secreted pentameric IgM molecule induces the binding of its Fc regions to the first component of the complement system, thereby activating it. If the antigen is located on the surface of an invading microorganism, complement activation triggers a biochemical attack that leads to the destruction of the pathogen (Section 18.5). Unlike IgM, IgD molecules are rarely secreted by active B cells, and no functions are known for them other than serving as cell-surface receptors for antigens.

Fig. 18-17. Pentameric IgM molecule. Five subunits are linked by Disulfide Bonds. A single J-chain (molecular mass ~15,000), connected by disulfide bridges to two heavy μ-chains, closes the ring structure and prevents further polymerization. The J-chain is homologous to a single Ig domain (Section 18.3.3).

Fig. 18-18. A bacterium coated with IgG antibodies is efficiently phagocytosed by a macrophage or neutrophil bearing surface receptors capable of binding the Fc region of the IgG molecule. The binding of the antibody-coated bacterium to these Fc receptors triggers the phagocytic process (Section 6.5.14).

IgG is the principal class of immunoglobulins found in the blood, produced in large quantities during a secondary immune response. In addition to activating the complement system, the Fc region of IgG molecules binds to specific receptors on macrophages and neutrophils. To a large extent, these Fc receptors enable phagocytic cells to bind, engulf, and destroy invading microorganisms coated with IgG antibodies generated in response to an infection (Fig. 18-18). Various types of leukocytes bearing Fc receptors can also destroy foreign Eukaryotic cells coated with IgG without engulfing them. This process, known as antibody-dependent cellular cytotoxicity, can be carried out by macrophages, neutrophils, and eosinophils (see below), as well as by killer cells (K cells). Killer cells are lymphocyte-like cells specialized, apparently, for destroying abnormal Cells of the Organism's own body (Section 18.6.4).

IgG molecules are the only antibodies that can cross the Placenta from mother to fetus. Placental cells in contact with maternal blood possess Fc receptors that bind IgG molecules, thereby facilitating their transfer into the fetal Circulatory system. The antibodies are first internalized by receptor-mediated endocytosis, then transported across the cell in vesicles, and finally released into the fetal blood by exocytosis (a process known as transcytosis; see Section 6.5.11). Antibodies of other classes do not bind to these receptors and therefore cannot cross the placenta.

IgA is the predominant class of antibodies in secretions (milk, saliva, tears, and the secretions of the respiratory and intestinal tracts). It occurs mainly as four-chain monomers (similar to IgG) or as dimers containing one J-chain and an additional polypeptide chain called the secretory component (Fig. 18-19). In secretions, IgA exists as a dimer. It is transported from the extracellular fluid into the secreted fluid in the same manner as IgG molecules pass from maternal blood to fetal blood—that is, via transcytosis. In this case, transport involves specialized Fc receptors located on the basal surface of epithelial cells lining the intestine, Bronchi, or the ducts of mammary, salivary, or lacrimal glands. Here, the Fc receptors bind IgA dimers from the extracellular fluid (Fig. 18-20).

The Fc region of IgE molecules binds to yet another type of Fc receptor, which exhibits an unusually high affinity for it ($K_a \sim 10^{10}\text{ L/mol}$). These receptors are found on the surface of tissue mast cells and blood basophils (Section 17.5.1), and the IgE molecules bound to them serve, in turn, as receptors for antigens. Antigen binding triggers the release of biologically active amines by these cells (particularly histamine, and serotonin in some species) (Fig. 18-21). These amines cause vasodilation and increase the permeability of blood vessel walls, being largely responsible for the clinical manifestations of allergic reactions such as hay fever, asthma, and urticaria. Presumably, under normal circumstances, these vascular changes make the site of inflammation more accessible to leukocytes, antibodies, and complement components. Mast cells also secrete factors that attract and activate a specialized class of leukocytes—eosinophils (Section 17.5.1)—which can destroy various parasites, especially when the latter are coated with IgG antibodies.

Fig. 18-19. Structure of a dimeric secretory IgA antibody molecule (highly simplified diagram). In addition to two IgA monomers linked by a single disulfide bridge between their heavy $\alpha$-chains, the complex also contains a J-chain and an additional polypeptide chain with a molecular mass of 70,000 daltons, termed the secretory component. This chain is believed to protect IgA molecules from Digestion by proteolytic enzymes present in secretions.

Fig. 18-20. Mechanism of Transepithelial Transport of a dimeric IgA molecule. IgA binds at the basal surface of an epithelial cell to a specialized transmembrane protein, the Fc receptor. The receptor-IgA complexes are internalized via receptor-mediated endocytosis, transported in vesicles across the epithelial Cell Cytoplasm, and secreted into the glandular lumen on the opposite side of the cell via exocytosis. At this point, the portion of the Fc receptor bound to the IgA dimer (the secretory component) is cleaved from the transmembrane "tail," thereby releasing the antibody complex into the lumen. Dimeric IgA enters the extracellular fluid of secretory Organs from two sources: some is produced locally within these organs by IgA-secreting plasma cells, while the rest is formed in the spleen and Lymph Nodes, enters the bloodstream, and subsequently leaks out from capillaries in various Tissues.

18.2.6. Antibodies contain either $\kappa$ or $\lambda$ chains, but not both

In addition to the five classes of heavy chains, antibodies in higher vertebrates possess Two Types of light chains, $\kappa$ and $\lambda$, either of which can associate with any heavy chain. An individual antibody molecule always consists of two identical light chains and two identical heavy chains; consequently, its two antigen-binding sites are always completely identical. Such Symmetry is crucial for the cross-linking function of secreted antibodies. Therefore, an Ig molecule may contain either $\kappa$ or $\lambda$ light chains, but never both simultaneously. No significant Functional differences between these two types of light chains have yet been established.

Fig. 18-21. Mast cells (and basophils) passively acquire antigen-binding surface receptors. IgE antibodies produced by active B lymphocytes enter the tissues and bind to Fc receptors on the surface of mast cells, which specifically recognize the Fc region of these antibodies. Consequently, unlike B cells, individual mast cells (and basophils) bear surface antibodies with A wide variety of different antigen-binding sites. When an antigen molecule attaches to these membrane-bound IgE antibodies and thereby cross-links adjacent IgE molecules, it activates the mast cell, which then releases histamine via exocytosis.

18.2.7. The strength of antigen-antibody binding depends on both affinity and the number of binding sites [16]

The binding of an antigen to an antibody, much like that of a substrate to an enzyme, is reversible. It is determined by the sum of many relatively weak noncovalent interactions, including hydrophobic and Hydrogen Bonds, Van der Waals forces, and ionic interactions. These weak interactions are effective only when the antigen and antibody molecules are sufficiently complementary in shape that certain atoms of the antigen fit into corresponding depressions on the antibody surface. The antigen-complementary regions of a four-chain antibody molecule are its two identical antigen-binding sites, whereas the corresponding region on the antigen is its antigenic determinant (epitope) (Fig. 18-22). Most antigenic macromolecules possess many different determinants; if two or more of these determinants are identical (as in certain polymers), the antigen is said to be multivalent (Fig. 18-23).

The reversible reaction between an antigen with a single antigenic determinant (Ag) and a single antigen-binding site (Ab) can be represented as follows:

The equilibrium point depends on both the concentrations of Ab and Ag and the strength of their interaction. Obviously, as the concentration of Ag increases, a greater fraction of Ab will become bound to Ag. The strength of the interaction is usually expressed by the affinity constant ($K_a$) (see Fig. 3-7):

K_a = [AgAb]/[Ag][Ab]

(the expressions in square brackets indicate the concentration of each component at equilibrium).

The affinity constant, sometimes called the association constant, can be determined by measuring the concentration of free Ag required to occupy half of the antibody's antigen-binding sites. When half of the sites are occupied, [AgAb] = [Ab] and K = 1/[Ag]. Thus, the antibody-antigen affinity constant is equal to the reciprocal of the antigen concentration that yields half-maximal binding. Typical values vary widely, ranging from 5·10⁴ to 10¹¹ L/mol. The affinity constant below which an immunoglobulin molecule is no longer considered an antibody against a given antigen is somewhat arbitrary, but an antibody with a K_a below 10⁴ is unlikely to be biologically effective; furthermore, it is improbable that B cells with receptors having such a weak affinity for the antigen would be activated by it.

Fig. 18-22. A highly simplified diagram of the binding of a macromolecule's antigenic determinant to the antigen-binding sites of two different antibodies with high and low affinity for the given antigen. The antigenic determinant is held within the binding site by various weak noncovalent interactions. Note that both the light and heavy chains of the antibody molecule typically contribute to The formation of this site.

Fig. 18-23. Molecules with multiple antigenic determinants. A. A globular protein featuring a series of distinct antigenic determinants. Note that different Regions of the polypeptide chain can approach each other in the folded structure to form a single determinant on the protein surface. B. A polymeric structure with repeating identical antigenic determinants; such a molecule is referred to as a multivalent antigen.

Antibody affinity reflects the strength of interaction between an antigenic determinant and an individual antigen-binding site, regardless of the total number of such sites. In contrast, the overall avidity of an antibody for a multivalent antigen (such as a polymer with repeating subunits) characterizes the cumulative strength of interaction of all binding sites combined. When a multivalent antigen interacts with more than one antigen-binding site of an antibody, the binding strength increases dramatically: for the antigen and antibody to dissociate, all of their mutual bonds must be broken simultaneously. Therefore, a typical IgG molecule, when both antigen-binding sites are engaged, will bind to a multivalent antigen at least 1,000 times more strongly than when only a single site is involved.

For the same reason, if the affinities of individual antigen-binding sites of IgG and IgM are equal, an IgM molecule (possessing 10 such sites) will exhibit an incomparably greater avidity for a multivalent antigen than an IgG molecule (possessing 2 sites). This difference in avidity, often 10,000-fold or greater, is highly significant because antibodies produced in the early stages of an immune response typically have a significantly lower affinity for the antigen than those produced later. (The increase in the average affinity of produced antibodies over time following immunization is termed affinity maturation — see Section 18.4.4.) Owing to their high overall avidity, IgM antibodies (the main Ig class produced early in the immune response) can function effectively even with a low affinity of individual binding sites.

Summary

An antibody molecule is a Y-shaped protein with two identical antigen-binding sites at the tips of its arms (Fab regions) and sites for binding complement components and/or various cell-surface receptors on its "stem" (Fc region). Antibodies protect vertebrates against infections by neutralizing viruses or bacterial toxins and by mobilizing complement and various cells that destroy and engulf invading microorganisms.

Each B-cell clone produces antibody molecules with a unique antigen-binding site. Initially, these molecules are incorporated into the cell's plasma membrane, where they serve as receptors for the antigen. When an antigen binds to these receptors, the B cells are activated (typically with the help of T cells), begin to proliferate, and mature into either memory cells or effector cells that secrete antibodies with the same antigen-binding site as the membrane-bound antibodies.

Each antibody molecule is composed of two identical heavy (H) chains and two identical light (L) chains. Typically, portions of both H and L chains form the antigen-binding sites. There are five classes of antibodies (IgA, IgD, IgE, IgG, and IgM), which possess distinct H chains (α, δ, ε, γ, and μ, respectively). The H chains also form the Fc region of the antibody, which dictates which other proteins will bind to the antibody, thereby determining the biological properties of that antibody class. L chains of either type (ϰ or λ) can associate with any class of H chains. The type of L chain appears to have no effect on antibody properties.