How Antigen-Binding Receptors Are Made

Key Points

• Antigen molecules bind to a T cell receptor (TCR) or B cell receptor (BCR) when their shape matches the conformation of the groove in the antigen-binding receptor.

• The shape of the antigen-binding groove depends on the sequence of the amino acids that line the groove. The sequence of these amino acids depends on the genes encoding the variable domains of the receptor.

• Because of the ways by which nucleotide sequences can be rearranged in these genes, an enormous number of different BCRs and TCRs can be generated.

• In some mammals, variable regions may be constructed by means of gene recombination. Different genes selected at random from a large library are joined to generate great sequence diversity.

• In other mammals, receptor diversity is generated by gene conversion. Small blocks of donor nucleotides are inserted into V-region genes to generate sequence changes.

• The genes coding for antigen-binding sites in BCRs, but not TCRs, also undergo random somatic mutation, resulting in even more sequence changes.

• These mechanisms collectively enable an animal to make the millions of different receptors that can bind to almost all foreign antigens.

One of the central problems encountered in understanding adaptive immunity is to explain how lymphocytes recognize the enormous diversity of microbes that may invade the body. Given that microorganisms change rapidly, the immune system must be able to respond not only to existing organisms but also, within reason, to newly evolved organisms. The ability of the adaptive immune responses to respond specifically to an enormous number of foreign antigens implies the existence of an enormous number of different lymphocytes, each with its own specific antigen receptors. This then raises the question, How do lymphocytes generate such an enormous diversity of antigen-specific receptors?

The ability of a receptor to bind an antigen is determined by the shape of its binding site. This shape depends on the folding of its peptide chains, which is governed, in turn, by their amino acid sequences. Each amino acid in a peptide chain exerts an influence on its neighboring amino acids, which determines their relative configuration. The shape of a peptide chain therefore represents the contributions of all amino acids in the chain as the peptide assumes its most energetically favorable conformation. The folding of a protein is determined by its amino acid sequence, and that sequence is determined by the sequence of bases in the DNA coding for that protein. The diversity of antigen receptors implies either a corresponding diversity in the genes coding for these receptors or a mechanism that generates diversity from a limited pool of receptor genes. This second mechanism is now known to be the method employed by the adaptive immune system.

Receptor-Antigen Binding

When an antigen and its receptor bind, they interact through the chemical groups on the surface of the antigen and on the complementarity determining regions (CDRs) of the receptor. In classic chemical reactions, molecules are assembled through the establishment of firm, covalent bonds. These bonds can be broken only by the input of a large amount of energy, energy that is not readily available in the body. In contrast, the formation of noncovalent bonds provides a rapid and reversible way of forming complexes and permits reuse of molecules in a way that covalent bonding would not allow. However, noncovalent bonds act over short intermolecular distances and, as a result, form only when two molecules approach each other very closely. The binding of an antigen to a BCR or TCR is exclusively noncovalent, so the strongest binding occurs when the shape of the antigen and the shape of the receptor conform to each other. This requirement for a close conformational fit has been likened to the specificity of a key for its lock.

The major bonds formed between an antigen and its receptor are hydrophobic (Figure 17-1). When antigen and antibody molecules come together, they exclude water molecules from the area of contact. This frees some water molecules from constraints imposed by the proteins and is therefore energetically stable. (The bond can be likened to two wet glass microscope slides stuck together. Anyone who has tried to separate two wet glass slides can confirm the effectiveness of this type of bonding.)

FIGURE 17-1 Noncovalent bonds that link an antigen with its receptor arranged in order of relative importance. All these bonds are effective only over a very short distance. It is therefore essential that the shape of the antigen and its receptor site match very well if strong binding is to be achieved.

A second type of binding between an antigen and its receptor is through hydrogen bonds. When a hydrogen atom covalently bound to one electronegative atom (e.g., an–OH group) approaches another electronegative atom (e.g., an O=C—group), the hydrogen is shared between the two electronegative atoms. This situation is energetically favorable and is called a hydrogen bond. The major hydrogen bonds formed in antigen-receptor interaction are O–H–O, N–H–N, and O–H–N. Hydrogen bonds are already present between proteins and water molecules in aqueous solution, so the binding of an antigen to its receptor by hydrogen bonds requires relatively little net energy change.

Electrostatic bonds formed between oppositely charged amino acids may contribute to antigen-receptor binding, but the charge on many protein groups is commonly neutralized by electrolytes in solution. As a result, the importance of electrostatic bonding is unclear.

When two atoms approach very closely, a nonspecific attractive force, called a van der Waals force, becomes operative. It occurs as a result of a minor asymmetry in the charge of an atom because of the position of its electrons. This force, although very weak, may become collectively important when two large molecules come into contact. It can therefore contribute to antigen-receptor binding.

The binding of a receptor to its antigen is therefore mediated by multiple noncovalent bonds. Each bond is relatively weak in itself, but collectively the bonds may have a significant binding strength. All these bonds act only across short distances and weaken rapidly as that distance increases. Electrostatic bond and hydrogen bond strengths are inversely proportional to the square of the distance between the interacting molecules; the van der Waals forces and hydrophobic bonds are inversely proportional to the seventh power of that distance. Thus the strongest binding between an antigen and its receptors occurs when their shapes match perfectly and multiple noncovalent bonds form. Antigens can bind to receptors when they fit less than perfectly, although the strength of this binding will be much reduced.

Antigen Receptor Genes

The information needed to make all proteins, including antigen receptors, is stored in an animal’s genome. All that is required for the production of these molecules is that the necessary genes be turned on. Once the appropriate genes are activated, they can be transcribed into RNA and translated into the appropriate receptor protein on B or T cells. It has been estimated that mammals can produce up to 10¹⁵ different antigen receptors to be expressed on B and T cells. In order to produce this enormous diversity, they use fewer than 500 genes!

Multiple genes code for each receptor peptide chain. Several genes code for each variable region, whereas only one codes for a constant region. As a result, the single constant-region gene can be combined with any one of several different variable-region genes to make a complete receptor chain (Figure 17-2). Instead of having genes for all possible receptor chains, it is only necessary to have genes for all the variable regions and to join these to an appropriate constant-region gene as required. In addition, antigen receptor chains may be paired in different combinations to yield even greater diversity, a process called combinatorial association.

FIGURE 17-2 Antigen receptor chains are coded for by three genes originating in three widely separated groups. The genes for a complete receptor chain are assembled by joining one gene selected from each group.

Immunoglobulin/B Cell Receptor Diversity

To make as many different antibodies as possible, it is necessary to diversify the amino acid sequences of the variable domains in both light and heavy chains. Since these amino acid sequences are determined by the nucleotide sequences in the genes coding for these variable regions, mechanisms must exist for generating this nucleotide sequence diversity. In practice, gene diversity is generated through three distinct mechanisms: gene recombination, somatic mutation, and gene conversion. All three mechanisms alter and diversify antibody gene sequences in such a way that an incredibly diverse array of antigen receptors is generated. The relative importance of each of these mechanisms differs among species, and the diversity-generating mechanisms that operate in humans and mice are not the same as those that operate in domestic mammals. The assembly of these diverse antigen receptors is carefully controlled during lymphocyte development by such factors as DNA methylation, chromatin structure, and location within the nucleus.

Gene Recombination

Gene recombination results from the random selection of one gene from each of several groups of genes followed by recombining these selected genes to generate sequence diversity. It is well seen in the genes that code for immunoglobulins.

Three gene loci code for immunoglobulin peptide chains, and each is found on a different chromosome (Figure 17-3). One locus, called IGK, codes for κ light chains; one, called IGL, codes for λ light chains; and one, called IGH, codes for heavy chains.

FIGURE 17-3 Genes coding for immunoglobulin light chains and heavy chains. Note that there are two distinct light chain loci, one coding for kappa chains and one coding for lambda chains. These are located on different chromosomes. The precise number of V, D, and J genes varies among species.

Generation of Junctional Diversity

Gene Rearrangement

The most obvious way to generate V-region diversity is to randomly select one V gene from the available pool and join it to one randomly selected J gene—a process called recombination. Since many different V and J genes are available, the number of possible combinations can be very large. For example, if there are 100 V genes and 10 J genes, then 100 × 10 = 1000 different V regions can be constructed.

Light chain assembly requires the combination of one V, one J, and one C gene. During B cell development, the intervening genes are looped out, excised, and discarded. The V and J genes have sites at each end that guide the cutting enzymes (Figure 17-4). The looped-out genes are chopped off, and the free ends of the DNA are rejoined so that the V and J genes form a continuous sequence. Two sets of enzymes are used in this process. Recombinases cut the DNA at two points, thus excising unwanted genes. Following this, DNA repair enzymes join the two free ends to reform a continuous sequence. If these enzymes are defective, antibodies (and TCRs) cannot be made. In foals with severe combined immunodeficiency, for example, there is a defect in the DNA repair enzyme that joins the cut ends. As a result, these foals cannot make either TCRs or BCRs and thus have no functional B or T cells (Chapter 37).

FIGURE 17-4 One of the most important mechanisms of deleting unwanted genes is by looping out. In this case, unwanted V genes form a loop that is then cut off by a recombinase enzyme and the cut ends joined together. As a result, the desired V gene is linked directly to a J gene. The excised loop is destroyed.

Light chain gene recombination occurs in two stages. Randomly selected V and J genes are first joined to form a complete V-region gene. The joined V-J genes remain separated from the C gene until messenger RNA (mRNA) is generated. At that time the unwanted J genes are excised, and the complete V-J-C mRNA is then translated to form a light chain (Figure 17-5).

FIGURE 17-5 Construction of an immunoglobulin light chain. Selected V and J genes are first joined as the intervening genes are deleted. The VJ and C genes remain separated until RNA splicing occurs. At that time the intervening RNA segments are deleted leaving the V, J, and C genes together in the mRNA. DNA rearrangement occurs during early B cell development so that each individual B cell is committed to making a single form of light chain for its antigen receptor.

When a heavy chain V region is assembled, its construction requires the splicing together of IGHV, IGHD, and IGHJ genes (Figure 17-6). This use of three randomly selected genes enormously increases the amount of variability. For example, if a pool of 100 V, 10 J, and 10 D genes are recombined, then 100 × 10 × 10 = 10,000 different V regions can be constructed. The recombination of these genes also occurs in a specific order. Thus IGHD is first joined to IGHJ, then V genes are attached to make a complete V-region gene. After transcription, any unwanted J genes are deleted, the C gene mRNA is attached, and the completely assembled V-D-J-C mRNA is translated to form a heavy chain.