IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 8. Sources of Diversity in Antigen-Recognizing Structures

Ig GENE RECOMBINATION

The Gene encoding the light chain V domain is formed by the recombination of V and J segments

The Study of antibody genes was made possible by The Development of Recombinant DNA technology. This method revealed that the constant and variable regions of light chains are encoded by two separate DNA segments. In Cells that do not produce Antibodies at all, these gene segments are located far apart on the chromosome, but during antibody synthesis, they are brought into close proximity. However, even in a fully differentiated B Cell, these two segments do not join directly; they are separated by a region approximately 1500 bp long. Between the V and C segments in the unrearranged chromosome, as well as immediately adjacent to the V segment following intrachromosomal recombination, lies a short stretch of DNA known as the joining (J) segment. (The J segment should not be confused with the J chain found in IgM and dimeric IgA molecules.)

The gene system encoding light chains. The VK gene segment encodes the V region of light chains from position 1 to 95, while the JK segment encodes the remaining portion of the V region (Fig. 8.8). The Human Genome contains only a single constant region gene for the k-type light chain and 76 VK segments, of which only 35 are potentially functional, 16 carry minor defects, and 25 are pseudogenes. As a result of DNA recombination during lymphoid Cell Differentiation, one of the V segments joins with one of five J segments. This can subsequently yield at least 175 (i.e., 35 x 5) different variable regions of k chains. Each V segment is preceded by a DNA stretch encoding a short signal (leader) peptide composed of hydrophobic amino acid residues, which serves as an "address tag" directing the synthesized chain to The Endoplasmic reticulum, where the leader peptide is cleaved off. Intracellular Processing of the immunoglobulin molecule then begins, leading to The formation of its secreted form and its transport along the cellular export pathway.

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Fig. 8.8. During pre-B cell maturation, one segment from the VK group (V1-Vn) of germline DNA is brought into proximity with one of five JK segments (JK1-JK5) through recombination. In a mature B cell, the rearranged DNA segment is transcribed into a primary RNA transcript containing a long intermediate sequence of additional J segments and introns. Through exon splicing, the primary RNA transcript is converted into mature mRNA, which is translated on Ribosomes into kappa (k) chains. Gene rearrangement is illustrated here as just one of many possible recombination pathways.

The human lambda ($\lambda$) light chain gene locus contains a set of V genes and seven C genes, with each C gene accompanied by an adjacent J segment. Despite this difference from the k chain locus, the V-J recombination process in the $\lambda$ chain locus is generally very similar to that in the k chain locus (Fig. 8.9).

Fig. 8.9. During B cell differentiation, one of the germline V$\lambda$ segments joins with one of the J segments to form a V-J gene. The resulting rearranged gene is transcribed into a primary RNA transcript containing introns (non-coding segments), exons (protein-coding segments), and a polyadenylate tail (poly-A). Splicing removes the introns from the primary RNA transcript, while the exons assemble into an mRNA molecule that is subsequently translated into protein.

Following the recombination of V and J segments, an intron (a non-coding intervening sequence) still remains between the rearranged VJ genes and the C gene. This entire stretch of DNA (from the leader sequence to the 3' end of the C gene, including the introns) is fully transcribed into heterogeneous nuclear RNA (hnRNA), an mRNA precursor. After intron removal and exon joining, this precursor yields an mRNA molecule capable of being translated into protein.

The gene encoding the heavy chain V domain is formed by the recombination of V, D, and J segments

Like those of light chains, heavy chain variable regions are encoded by V and J gene segments. An additional contribution to antibody diversity is made by a third gene segment, D (diversity) (Fig. 8.10). It is highly variable in both nucleotide sequence and codon number. In antibodies against dextran, it specifies two amino acid residues; in antibodies against phosphorylcholine, it accounts for an insertion of up to eight additional Amino Acids; while in antibodies against levan, this segment of the Amino Acid Sequence is completely absent. Several D segments joined in series can form an expanded D region with three potential reading frames lacking stop codons, further increasing heavy chain diversity. To date, only 30 D segments have been identified in germline genes. Chromosome 14 has been shown to contain 87 VH segments, of which at least 32 are pseudogenes. Notably, additional "orphan" VH and D segments have been found on Chromosomes 15 and 16; some of these may be functional, though there is as yet no evidence of their contribution to heavy chain diversity. Recombinational joining of V, D, and J segments is the primary source of hypervariability in the third complementarity-determining region, CDR-3, which constitutes an essential part of the antigen-binding site. In the Immune Response to certain Antigens, such as dextrans, almost all differences among antibody molecules of varying Specificity reside precisely in CDR-3.

Fig. 8.10. In the heavy chain gene locus, joining three DNA segments forms the V-D-J exon encoding the VH domain. Of the 80 VH genes, about 50 are functional, while the rest are pseudogenes. Recombination of a V gene with one of 30 D segments and one of six J segments in a B cell produces a functional V-D-J gene.

Rearrangement and expression of V region genes follow a specific developmental program early in Embryogenesis

When animals are immunized prenatally or immediately after birth, the capacity to respond to each antigen develops in a well-defined, presumably programmed manner. According to this program, humans first utilize V segments closest to the J segments; intriguingly, among such segments, V61 represents the sole conserved sequence stretch. In all primates whose VH genes have been studied, this gene segment is represented by a single copy without intraspecific variants, and differences between humans and other primates involve only 2% of the NUCLEOTIDES.

The fetal specificity repertoire is prominently represented in autoantibodies, suggesting that autoimmunity may partly result from delayed rearrangement of germline V segments. Similarly, unrearranged V segments are abundantly represented in immature B-cell lymphomas, with 20 such V segments identified in 85% of chronic Lymphocytic Leukemia cases.

Nucleotide sequences flanking unrearranged V, D, and J genes direct their recombination

Gene segment recombination is the pivotal event in generating functional variable-region genes for light and heavy chains. The intricate mechanism of these gene rearrangements is now understood, and specific nucleotide sequences acting as recombination signal sequences (RSS) in this process have been identified (Fig. 8.11).

Fig. 8.11. Recombination involves splicing (joining) V and J segments (e.g., in the case of k light chains) (1) or splicing V, D, and J segments (for heavy chains) (2). This process is facilitated by RAG-1 and RAG-2 Proteins recognizing nucleotide sequences in the introns at the 3' ends of V and D segments and the 5' ends of J and D segments. The 12-bp and 23-bp spacers have non-conserved sequences but a fixed length corresponding to approximately one and two turns of the DNA double helix, respectively. This allows the heptamers and nonamers of both strands to align adjacently, enabling them to interact simultaneously with RAG proteins.

The signal sequence located downstream (at the 3' side) of V and D segments consists of a CACAGTG heptamer (or its analogue), followed by a non-conserved 12-bp spacer, which in turn is adjacent to an ACAAAAACC nonamer or its analogue. The signal sequence directly flanking (at the 5' side) all germline D and JH segments consists of a nonamer, an upstream non-conserved 23-bp spacer, and a further upstream heptamer. The heptamers and nonamers following Vl, VH, and D segments are complementary to the corresponding sequences preceding Jl, D, and JH segments and undergo recombination with them. The 12-bp spacer corresponds to approximately one turn, and the 23-bp spacer to approximately two turns of the DNA helix.

The recombination process is regulated (at least in part) by two recombination-activating genes, RAG-1 and RAG-2. Mice lacking these genes fail to produce T-cell receptors and IMMUNOGLOBULINS and therefore lack mature T AND B cells. The expression levels of RAG-1 and RAG-2 are influenced by interleukin-7 (IL-7), a cytokine secreted by Bone Marrow stromal cells. Purified RAG-1 and RAG-2 proteins, when acting on synthetic DNA substrates containing heptamers and nonamers together with 12-bp and 23-bp spacers, are capable of promoting the formation of a synaptic complex of two RSSs. First, RAG-1 binds to the nonamer; then RAG-2, which cannot bind DNA directly, joins the RAG-1/DNA complex immediately adjacent to the 12-bp spacer. Subsequently, a synapse forms between the 12-bp and 23-bp spacers, apparently resulting from interactions between the RAG-1-nonamer-spacer (23 bp) and RAG-2-RAG-1-nonamer-spacer (12 bp) complexes. Following binding, heptamer-directed DNA Cleavage occurs at the boundaries between the heptamers and the joining V-(D)-J gene segments.

DNA cleavage begins with a single-strand nick at the boundary between the 5' end of the signal heptamer and the functional gene. RAG proteins then extend this initial nick into a complete double-strand break, creating a hairpin Structure at the end of the functional gene, which is readily observable in vitro. Tracking this process in vivo is more challenging because the hairpins often undergo further modifications even before the gene segments are joined (Fig. 8.12).

Fig. 8.12. a. Following the Formation of the synaptic complex, the recombination signal sequences (RSS) of the joining gene segments are excised along with the intervening DNA segment, and then (b), with their opposite ends joined, form a circle. c. The ends of the two strands of The Double Helix of each functional gene are covalently joined, adopting a hairpin-like conformation. To remove the hairpins, an endonuclease nicks one of the DNA strands at some point (d). The gaps in The nucleotide sequence are filled in via DNA Synthesis, and the two ends are thereby "stitched" together (e). Single-strand Cleavage of the DNA leads to the formation of palindromic sequences (P) that extend the length of the junctional region (blue square). Additional diversity is generated by the template-independent addition of nucleotides (N-region diversity) mediated by terminal deoxynucleotidyl transferase; notably, this has been observed during heavy-chain gene rearrangement, but may also occur during the synthesis of one of the light-chain subgroups (f).

The joining site of the V and J gene segments can vary slightly

Minor variations in the joining positions of gene segments during recombination serve as an additional source of antibody diversity. For example, the last codon of the Vκ segment typically encodes the 95th amino acid residue, and the first Jκ triplet encodes the 96th amino acid residue of the light κ chain. However, the 96th residue is sometimes encoded by a composite triplet in which the second and third nucleotides (or only the third) belong to the first Jκ triplet, while the first (or the First and Second) originates from the intron at the 3' end of the Vκ segment (Fig. 8.13), leading to variations in The amino acid sequence. Naturally, synthesis of a functionally competent light chain requires the correct reading frame; if a reading frame shift occurs during gene segment recombination, the resulting antibodies may be inactive.

Fig. 8.13. As a result of junctional Variability, the identical germline Vk21 and J1 segments encode three distinct proteins: PC2880, PC6684, and PC7940. Due to recombination involving the end of the CCC codon, positions 95 and 96 in protein PC2880 are occupied by Proline and Tryptophan residues, respectively. Recombination of a single nucleotide located downstream of the CCC codon results in Proline and Arginine residues at these positions in protein PC6684. Following recombination of two nucleotides located downstream of the end of the Vk21 segment, protein PC7940 contains two proline residues at these positions.

Similar joining inaccuracies are observed during the recombination of heavy-chain D and J gene segments, where deviations can reach up to 10 nucleotides (Fig. 8.14). Furthermore, several additional nucleotides may be inserted between the D and JH segments, as well as between the VH and D segments, through the template-independent action of terminal deoxynucleotidyl transferase (Fig. 8.12). The addition of such "N-nucleotides" generates further amino-terminal antibody diversity. In mice, terminal deoxynucleotidyl transferase activity increases with age, leading to the accumulation of long N-segments in adult animals. As a consequence, the recombination Variability of the D region can be so extensive that every single germline D gene segment is altered.

In severe combined immune deficiency (SCID), functional T or B cells fail to develop due to a defect in V-(D)-J recombination.

Fig. 8.14. The nucleotide sequence of the coding genes (1) is aligned with the amino acid sequence (2) of the heavy chains from three anti-phosphorylcholine antibody samples. Variable recombinations among the germline VH, D, and JH segments give rise to amino acid sequence variations (highlighted in brown). In some cases (such as protein M167), additional codons appear to be inserted. However, the overall reading frame is not shifted because the number of inserted nucleotides is a multiple of three.