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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 33.4Diversity Is Generated by Gene Rearrangements

A mammal such as a mouse or a human being can synthesize large amounts of specific antibody against virtually any foreign determinant within a matter of days of being exposed to it. We have seen that antibody specificity is determined by the amino acid sequences of the variable regions of both light and heavy chains, which brings us to the key question: How are different variable-region sequences generated?

The discovery of distinct variable and constant regions in the L and H chains raised the possibility that the genes that encode immunoglobulins have an unusual architecture that facilitates the generation of a diverse set of polypeptide products. In 1965, William Dreyer and Claude Bennett proposed that multiple V (variable) genes are separate from a single C (constant) gene in embryonic (germ-line) DNA. According to their model, one of these V genes becomes joined to the C gene in the course of differentiation of the antibody-producing cell. A critical test of this novel hypothesis had to await the isolation of pure immunoglobulin mRNA and the development of techniques for analyzing mammalian genomes. Twenty years later, Susumu Tonegawa found that V and C genes are indeed far apart in embryonic DNA but are closely associated in the DNA of antibody-producing cells. Thus, immunoglobulin genes are rearranged in the differentiation of lymphocytes.

33.4.1. J (Joining) Genes and D (Diversity) Genes Increase Antibody Diversity

Sequencing studies carried out by Susumu Tonegawa, Philip Leder, and Leroy Hood revealed that V genes in embryonic cells do not encode the entire variable region of L and H chains. Consider, for example, the region that encodes the κ light-chain family. A tandem array of 40 segments, each of which encodes approximately the first 97 residues of the variable domain of the L chain, is present on human chromosome 2 (Figure 33.14).

Figure 33.14. The κ Light-Chain Locus.

Figure 33.14

The κ Light-Chain Locus. This part of human chromosome 2 includes an array of 40 segments that encode the variable (V) region (approximately residues 1 – 97) of the light chain, an array of 5 segments that encode the joining (J) region (more...)

However, the variable region of the L chain extends to residue 110. Where is the DNA that encodes the last 13 residues of the V region? For L chains in undifferentiated cells, this stretch of DNA is located in an unexpected place: near the C gene. It is called the J gene because it joins the V and C genes in a differentiated cell. In fact, a tandem array of five J genes is located near the C gene in embryonic cells. In the differentiation of an antibody-producing cell, a V gene becomes spliced to a J gene to form a complete gene for the variable region (Figure 33.15). RNA splicing generates an mRNA molecule for the complete L chain by linking the coding regions for the rearranged VJ unit with that for the C unit (Figure 33.16).

Figure 33.15. VJ Recombination.

Figure 33.15

VJ Recombination. A single V gene (in this case, V2) is linked to a J gene (here, J4) to form an intact VJ region. The intervening DNA is released in a circular form. Because the V and J regions are selected at random and the joint between them is not (more...)

Figure 33.16. Light-Chain Expression.

Figure 33.16

Light-Chain Expression. The light-chain protein is expressed by transcription of the rearranged gene to produce a pre-RNA molecule with the VJ and C regions separated. RNA splicing removes the intervening sequences to produce an mRNA molecule with the (more...)

J genes are important contributors to antibody diversity because they encode part of the last hypervariable segment (CDR3). In forming a continuous variable-region gene, any of the 40 V genes can become linked to any of five J genes. Thus, somatic recombination of these gene segments amplifies the diversity already present in the germ line. The linkage between V and J is not precisely controlled. Recombination between these genes can take place at one of several bases near the codon for residue 95, generating additional diversity. A similar array of V and J genes encoding the λ light chain is present on human chromosome 22. This region includes 30 Vλ gene segments and four Jλ segments. In addition, this region includes four distinct C genes, in contrast with the single C gene in the κ locus.

In human beings, the genes encoding the heavy chain are present on chromosome 14. Remarkably, the variable domain of heavy chains is assembled from three rather than two segments. In addition to VH genes that encode residues 1 to 94 and JH segments that encode residues 98 to 113, this chromosomal region includes a distinct set of segments that encode residues 95 to 97 (Figure 33.17). These gene segments are called D for diversity. Some 27 D segments lie between 51 VH and 6 JH segments. The recombination process first joins a D segment to a JH segment; a VH segment is then joined to DJH. A greater variety of antigen-binding patches and clefts can be formed by the H chain than by the L chain because the H chain is encoded by three rather than two gene segments. Moreover, CDR3 of the H chain is diversified by the action of terminal deoxyribonucleotidyl transferase, a special DNA polymerase that requires no template. This enzyme inserts extra nucleotides between VH and D. The V(D)J recombination of both the L and the H chains is executed by specific enzymes present in immune cells. These proteins, called RAG-1 and RAG-2, recognize specific DNA sequences called recombination signal sequences (RSSs) adjacent to the V, D, and J segments and facilitate the cleavage and religation of the DNA segments.

Figure 33.17. V( D ) J Recombination.

Figure 33.17

V( D ) J Recombination. The heavy-chain locus includes an array of 51 V segments, 27 D segments, and 6 J segments. Gene rearrangement begins with D-J joining, followed by further rearrangement to link the V segment to the DJ segment.

33.4.2. More Than 108 Antibodies Can Be Formed by Combinatorial Association and Somatic Mutation

Let us recapitulate the sources of antibody diversity. The germ line contains a rather large repertoire of variable-region genes. For κ light chains, there are about 40 V-segment genes and five J-segment genes. Hence, a total of 40 × 5 = 200 kinds of complete Vκ genes can be formed by the combinations of V and J. A similar analysis suggests that at least 120 different λ light chains can be generated. A larger number of heavy-chain genes can be formed because of the role of the D segments. For 51 V, 27 D, and 6 J gene segments, the number of complete VH genes that can be formed is 8262. The association of 320 kinds of L chains with 8262 kinds of H chains would yield 2.6 × 106 different antibodies. Variability in the exact points of segment joining and other mechanisms increases this value by at least two orders of magnitude.

Even more diversity is introduced into antibody chains by somatic mutation — that is, the introduction of mutations into the recombined genes. In fact, a 1000-fold increase in binding affinity is seen in the course of a typical humoral immune response, arising from somatic mutation, a process called affinity maturation. The generation of an expanded repertoire leads to the selection of antibodies that more precisely fit the antigen. Thus, nature draws on each of three sources of diversity — a germ-line repertoire, somatic recombination, and somatic mutation — to form the rich variety of antibodies that protect an organism from foreign incursions.

33.4.3. The Oligomerization of Antibodies Expressed on the Surface of Immature B Cells Triggers Antibody Secretion

The processes heretofore described generate a highly diverse set of antibody molecules — a key first step in the generation of an immune response. The next stage is the selection of a particular set of antibodies directed against a specific invader. How does this selection occur? Each immature B cell, produced in the bone marrow, expresses a monomeric form of IgM attached to its surface (Figure 33.18). Each cell expresses approximately 105 IgM molecules, but all of these molecules are identical in amino acid sequence and, hence, in antigen-binding specificity. Thus, the selection of a particular immature B cell for growth will lead to the amplification of an antibody with a unique specificity. The selection process begins with the binding of an antigen to the membrane-bound antibody.

Figure 33.18. B-Cell Receptor.

Figure 33.18

B-Cell Receptor. This complex consists of a membrane-bound IgM molecule noncovalently bound to two Ig-α-Ig-β heterodimers. The intracellular domains of each of the Ig-α and Ig-β chains include an immunoreceptor tyrosine-based (more...)

Associated with each membrane-linked IgM molecule are two molecules of a heterodimeric membrane protein called Ig-α-Ig-β (see Figure 33.18). Examination of the amino acid sequences of Ig-α and Ig-β is highly instructive. The amino terminus of each protein lies outside the cell and corresponds to a single immunoglobulin, and the carboxyl terminus, which lies inside the cell, includes a sequence of 18 amino acids called an immunoreceptor tyrosine-based activation motif (ITAM) (see Figure 33.18). As its name suggests, each ITAM includes key tyrosine residues, which are subject to phosphorylation by particular protein kinases present in immune-system cells.

A fundamental observation with regard to the mechanism by which the binding of antigen to membrane-bound antibody triggers the subsequent steps of the immune response is that oligomerization or clustering of the antibody molecules is required (Figure 33.19). The requirement for oligomerization is reminiscent of the dimerization of receptors triggered by growth hormone and epidermal growth factor encountered in Sections 15.4 and 15.4.1; indeed, the associated signaling mechanisms appear to be quite similar. The oligomerization of the membrane-bound antibodies results in the phosphorylation of the tyrosine residues within the ITAMs by protein tyrosine kinases including Lyn, a homolog of Src (Section 15.5). The phosphorylated ITAMs serve as docking sites for a protein kinase termed spleen tyrosine kinase (Syk), which has two SH2 domains that interact with the pair of phosphorylated tyrosines in each ITAM. Syk, when activated by phosphorylation, proceeds to phosphorylate other signal-transduction proteins including an inhibitory subunit of a transcription factor called NF-κB and an isoform of phospholipase C. The signaling processes continue downstream to activate gene expression, leading to the stimulation of cell growth and initiating further B-cell differentiation.

Figure 33.19. B-Cell Activation.

Figure 33.19

B-Cell Activation. The binding of multivalent antigen such as bacterial or viral surfaces links membrane-bound IgM molecules. This oligomerization triggers the phosphorylation of tyrosine residues in the ITAM sequences by protein tyrosine kinases such (more...)

Image caduceus.jpg Drugs that modulate the immune system have served as sources, of insight into immune-system signaling pathways. For example, cyclosporin, a powerful suppressor of the immune system, acts by blocking a phosphatase called calcineurin, which normally activates a transcription factor called NF-AT by dephosphorylating it.

Image ch33fu2.jpg

The potent immune supression that results reveals how crucial the activity of this transcription factor is to the development of an immune response. Without drugs such as cyclosporin, organ transplantation would be extremely difficult because transplanted tissue expresses a wide range of foreign antigens, which causes the immune system to reject the new tissue.

The role of oligomerization in the B-cell signaling pathway is illuminated when we consider the nature of many antigens presented by pathogens. The surfaces of many viruses, bacteria, and parasites are characterized by arrays of identical membrane proteins or membrane-linked carbohydrates. Thus, most pathogens present multiple binding surfaces that will naturally cause membrane-associated antibodies to oligomerize as they bind adjacent epitopes. In addition, the mechanism accounts for the observation that most small molecules do not induce an immune response; however, coupling multiple copies of the small molecule to a large oligomeric protein such as keyhole limpet hemocyanin (KLH), which has a molecular mass of close to 1 million daltons or more, promotes antibody oligomerization and, hence, the production of antibodies against the small-molecule epitope. The large protein is called the carrier of the attached chemical group, which is called a haptenic determinant. The small foreign molecule by itself is called a hapten. Antibodies elicited by attached haptens will bind unattached haptens as well.

33.4.4. Different Classes of Antibodies Are Formed by the Hopping of VH Genes

The development of an effective antibody-based immune response depends on the secretion into the blood of antibodies that have appropriate effector functions. At the beginning of this response, an alternative mRNA splicing pathway is activated so that the production of membrane-linked IgM is supplanted by the synthesis of secreted IgM. As noted in Section 33.1, secreted IgM is pentameric and has a relatively high avidity for multivalent antigens. Later, the antibody-producing cell makes either IgG, IgA, IgD, or IgE of the same specificity as the intially secreted IgM. In this switch, the light chain is unchanged, as is the variable region of the heavy chain. Only the constant region of the heavy chain changes. This step in the differentiation of an antibody-producing cell is called class switching (Figure 33.20). In undifferentiated cells, the genes for the constant region of each class of heavy chain, called Cμ, Cδ, Cγ, Cϵ, and Cα, are next to each other. There are eight in all, including four genes for the constant regions of γ chains. A complete gene for the heavy chains of IgM antibody is formed by the translocation of a VH gene segment to a DJH gene segment.

Figure 33.20. Class Switching.

Figure 33.20

Class Switching. Further rearrangement of the heavy-chain locus results in the generation of genes for anti-body classes other than IgM. In the case shown, rearrangement places the VDJ region next to the Cγ1 region, resulting in the production (more...)

How are other heavy chains formed? Class switching is mediated by a gene-rearrangement process that moves a VDJ gene from a site near one C gene to a site near another C gene. Importantly, the antigen-binding specificity is conserved in class switching because the entire VHDJH gene is translocated in an intact form. For example, the antigen-combining specificity of IgA produced by a particular cell is the same as that of IgM synthesized at an earlier stage of its development. The biological significance of CH switching is that a whole recognition domain (the variable domain) is shifted from the early constant region (Cμ) to one of several other constant regions that mediate different effector functions.

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Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22477