Generating Antibody Diversity

Antibodies are proteins manufactured by vertebrate immune systems that aid in defense against infectious agents and other substances foreign to the animal. It is estimated that a human is capable of synthesizing more than 10 million distinct antibodies. Most of this great diversity is generated through the action of precisely controlled gene rearrangements which occur during differentiation of many individual clones of cells, each clone specialized for the synthesis of one and only one antibody. The immune response involves proliferation of clones of cells that produce antibodies reacting with a specific antigen, or immunogen. This clonal expansion allows large-scale production of the specific antibodies needed to combat infection or other challenges to the immune system.

The immunoglobulin G, or IgG, class of proteins consists of two heavy chains and two light chains (Figure 25.32). Each chain comprises two distinct segments--a domain of variable polypeptide sequences and a constant domain, which is virtually invariant among different IgG light or heavy chains.

Figure 25.32 shows the organization of the precursor genes to chains in germ-line cells, undifferentiated for antibody formation, and the rearrangements leading to one such gene in a differentiated antibody-producing cell.

Each light chain is encoded by DNA sequences that are noncontiguous in the genome of undifferentiated cells but are all in the same chromosome. These sequences are called V (variable), C (constant), and J (joining). The human genome contains about 300 different V sequences, each of which encodes the first 95 amino acids of the variable region; 4 different J sequences, each of which encodes the last 12 residues of the variable region and joins it to the constant region; and one C sequence, which encodes the constant region. In an embryonic cell the V sequences, each preceded by a leader sequence containing a transcriptional activator that is not expressed, form a tight cluster; the J sequences form another cluster some distance away; and the C sequence follows shortly after the J cluster. Each J sequence is flanked by nonexpressed spacer sequences.

In differentiation of one antibody-forming clone of cells, a gene rearrangement links one of the approximately 300 V sequences with one of the 4 J sequences. All of the DNA that lies between these two spliced sequences is deleted in this rearrangement and disappears from all progeny of this cell line. Any upstream V sequences (on the 5' side, to the left in Figure 25.32) and downstream J sequences (on the 3' side, to the right) remain in these cells but are not used in antibody synthesis.

Additional diversity is provided by the way in which the V and J sequences recombine. The cutting and splicing can occur within the terminal trinucleotide sequences of V and J in any way that yields one trinucleotide sequence in the spliced product (Figure 25.33). This increases the total number of different light chain sequences by about 2.5 (the average number of different amino acids encoded by four random triplets). Thus, the total number of possible light chain sequences that can be formed from 300 V sequences and 4 J sequences is about 3000 (300 X 4 X 2.5).

Related DNA sequences are found to the 3' side of each V sequence and to the 5' side of each J sequence, and they represent recognition sites for the enzymes involved in the joining reaction. Those sequences, which are called recognition signal sequences, are as follows:

5'...V...CACAGTG...12 bases...ACAAAAAC...3'

3'...J...GTGTCAC...23 bases...TGTTTTTG...5'

Note the presence of nearly identical seven-base palindromic sequences and nearly complementary eight-base AT-rich regions in these segments. These features allow the alignment of distant regions of the chromosome, with a process akin to that in phage integration, recombining the sequences and excising the intervening DNA. In addition, the DNA joining reactions are imprecise, with nucleotides removed from one or both ends, creating additional diversity.

The final step in producing a light chain polypeptide involves the joining of the C and J segments (see Figure 25.32). This occurs not at the DNA level but at the level of splicing of messenger RNA (see here). In this case, transcription yields an RNA molecule extending from the 5' side of the V gene that is spliced to J to the 3' side of C. Depending on which J region has been spliced to V in this cell, the RNA excised during splicing may contain sequences corresponding to other J regions.

Heavy chains are formed similarly-from V sequences, J sequences, and a class of sequences called D. In addition, there are eight different C sequences, which are also involved in the synthesis of other antibody classes. The total number of possible IgG heavy chains is about 5000. Because any light chain can combine with any heavy chain to form a complete IgG, the total number of IgG molecules possible is 3000 X 5000, or 1.5 X 107. In this way, enormous diversity can be generated from a very small fraction of the total DNA in germ-line cells. Even further diversity arises from the high rate of V sequence mutation during development of the antibody-producing cell. By this somatic mutation process (mutation not involving germ-line cells), cells that undergo the same V-J joining event may still differentiate to produce different IgGs.

It is not clear whether both of the homologous chromosomes in a diploid antibody-forming cell undergo identical rearrangements. However, given that each cell produces only one type of antibody, that must occur or one chromosome must be silenced after the other has completed its rearrangement.

See also: Gene Rearrangements, Site-Specific Recombination, Homologous Recombination, Recombination, Antibody Structure (from Chapter 7), The Immune Response (from Chapter 7), Generation of Antibody Diversity (from Chapter 7)

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