The Genetic Basis of Antigen Receptor Diversity

Antibodies are proteins, and proteins are synthesized following the instructions encoded in genes. Thus the antibody repertoire of an animal must represent the expression of genetic information encoded in its DNA. The vertebrate immune system draws on a collection of gene segments that are assembled into complete genes. These are then transcribed and translated into antibody molecules. However, the ability of B cells to assemble a limited number of gene segments in a virtually unlimited number of different ways makes possible the seemingly limitless number of antigen-binding specificities of antibodies. An IgG molecule consists of four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains linked by disulfide bonds (Figure 1). The H and L chains are each divisible into two regions: the variable (V) region, which is located at the N terminal end of the oligopeptide chain, and the constant (C) region, located at the C-terminal end. The V regions are so named because the amino acid sequence in this portion of the molecule varies considerably between different antibodies. In contrast, the C region shows much less sequence variation. In the fully folded IgG molecule the H and L V regions associate to form the V domain, which comprises the antigen-combining site. An IgG molecule has two identical antigen-combining sites. The effector capabilities, such as complement binding and attachment to phagocytic cells, are located in the C-terminal portions of the molecule and are mediated by the C domain of the H chains. Two Genes-One polypeptide Chain Hypothesis Hints as to the nature of the genes encoding immunoglobulin molecules came from a variety of experimental observations. Genetic and serological investigations had shown that antibodies were divisible into classes, e.g. IgM and IgG, and that class determined effector capability. Antibodies of the same specificity could occur in different classes. Furthermore, particular allotypes and idiotypes, serological markers associated with the N-terminal end of the immunoglobulin molecule, were shown to associate with different classes. Amino acid composition studies of myeloma proteins (homogeneous immunoglobulin molecules derived from plasma cell tumors) revealed a striking disparity in the distribution of amino acid residues in the

Ig molecule. The N-terminal half of the H and L polypeptide chains had a much more diverse amino acid composition than did the C-terminal region of the molecule. Together, these findings contributed to the idea that an immunoglobulin polypeptide chain may be encoded by more than one gene (Koshland et al., 1969). Direct support for this idea was obtained in 1965 with the publication of the entire amino acid sequence of two human myeloma protein L chains (Hilschmann and Craig, 1965). A clear pattern of amino acid sequence variation was revealed. The primary sequence of the Nterminal portion of the L chain was highly variable between the two chains, whereas the sequence of the C-terminal half of the molecule was nearly invariant. Variability in primary structure could account for antigen-binding specificity differences among antibodies, and the shared constant regions were responsible for the class of the molecule. These observations prompted the hypothesis that two genes encoded a single immunoglobulin polypeptide chain; one gene encoded the V region and one gene encoded the C region (Dreyer and Bennett, 1965). Many immunoglobulin sequencing studies followed this initial report, and they confirmed that both H and L polypeptide chains contained V and C regions. Furthermore, three regions of hypervariability were found to occur within V regions. These areas, called hypervariable or complementarity-determining regions, were later shown to be areas that contact antigen and play a pivotal role in determining the chemical character of the combining site.

Fig 1. An immunoglobulin G (IgG) molecule.

Structure of immunoglobulinrelated genes The H chain V region is encoded by an exon that is assembled upstream of C region coding sequences by the joining of three component segments: the variable (VH), diversity (D) and joining (JH) segments (Fig 2). A VH segment encodes a 5 hydrophobic leader sequence and approximately 98 amino acids of the V region that extend from the

amino-encoded by the CDR3 and include CDR1 and CDR2. CDR3 is encoded by the D segment (3-7 amino acids) and by sequences at the V H D and D JH junctions. The remainder of a JH segment encodes the fourth framework region, which consists of 12-17 amino acids. The V region of each of L chain isotype is encoded by an upstream exon that is assembled from analogous variable (VL) and joining (JL) segments, which are joined directly to each other; there are no L chain D segments. The basic V-(D)-J structure of these segments has been conserved among species, but their number and organization varies widely. In mice, there are present as clusters with multiple members (Fig 2). During B lymphocyte differentiation, H chain genes are assembled in two steps: D and J H segments are joined first and on both allelic chromosomes, followed by V H to DJH rearrangement (Fig 3). Assembly of L chain genes follows H chain gene assembly, usually after production of an H chain polypeptide. Joining between Ig V region gene segments has been observed only in B lymphocytes, with the exception that D and J H segments have been joined in some cells of the T and myeloid lineages. Each C region domain is encoded by a separate exon. H chain C regions are encoded by exon clusters that also include hinge region and alternate carboxy-terminal sequences. In mice and humans, the C region (C ) exon cluster is located closest to the JH segments, and is followed downstream first by C sequences and then by exon clusters encoding the other H chain isotypes (Fig 2). Processing of RNA transcripts joins V and C region exons and permits simultaneous expression of and H chains that contain the same V region (Fig 4). B lymphocytes express H chain first during differentiation. Most virgin B lymphocyte express on their surface both IgM and IgD, which have identical antigen-binding specificities because their respective and H chains are encoded by the same allele. The function of IgD is unknown, but it has been proposed that it might be a particularly effective antigen receptor because it contains an unusually large hinge region. During an immune response, a B lymphocyte can express a different H chain isotype (i.e. C or C 2), by replacing the C coding sequences with those of a different C region by a DNA recombination mechanism (class switching) that is distinct from the mechanism which assembles the V region exon. Class-switching allows the progeny of a given B lymphocyte to produce antibodies that retain their original specificity but can perform different effector functions.
Ig Gene Organization:

Fig 2: Organization of mouse Ig genes. Shaded boxes indicate the chromosomal locations of six of the nine known VH segment families. D segment families and JH segments are differently and are not drawn to scale. Separate H chain C region exons are shown for C sequences only. The mouse light chain V segments are shaded differently to indicate that there are separate V families, the chromosomal organization of which has not yet been described. The H chain and L chain enhancer elements are each indicated by E.

Heavy chain Gene Assembly:

Fig 3: Assembly of the V region of a H chain gene. Open triangles denote recognition elements separated by a 23 bp spacer and filled triangles denote recognition elements separated by a 12 bp spacer. The shaded triangle shown within V1 in the diagram of VH replacement represents the heptamer that is conserved near the 3 end of VH segments. Heavy Chain Gene Assembly:

Fig 4: H chain gene expression. Coding sequences are represented, except that 5 leader (L) and C sequences are shown. B lymphocytes which express both and H chains produce transcripts which contain both C and C sequences, but in plasma

cells transcription is apparently terminated upstream of the C exons. These indicated primary transcripts are processed to generate the mRNA species.

Genomic Structure Of Ig Genes Human immunoglobulin genes comprise three linkage groups. The genes encoding the kappa (k) and lambda (l) L chain genes are located on chromosome 2 and 22, respectively, and the H chain genes are found on chromosome 14. Although these gene families share general features, their respective organization is unique, and therefore they will be discussed separately. L chain genes The genomic organization of the human k locus is depicted in Figure . A single C gene is located towards the centromere. A cluster of five J joining) regions occurs immediately 5 to the C gene. A large cluster of 76 V gene segments is located further upstream of the J regions. They are organized into two groups: one group which is the most proximal to the J cluster contains 40 V gene segments, and another group which is distal to the J cluster contains 36 V gene segments. The proximal and distal groups appear to be derived from one another, as the sequences of many of the genes in one cluster are closely related to members in the other cluster. This organization indicates that the clusters are likely to have arisen from a common ancestral group of genes that underwent a duplication event. The majority of the V genes in the proximal group are in the same transcriptional polarity as the J regions and the C region, whereas the V gene segments in the distal segment are in the opposite transcriptional polarity. Thus, the primordial duplication event probably occurred by inversion. Approximately one-half of the V segments in each group are nonfunctional genes, as they contain errors in their sequence that prevents transcription or translation. However, some of these nonfunctional V genes have minor defects, such as single base changes resulting in a stop codon, and, therefore, reversion to functionality could occur relatively frequently. Indeed, the precise number of functional V gene segments is likely to vary between different individuals. The V gene segments can be grouped into seven families or subgroups based upon sequence similarities.

Fig 5: Genomic organization of human immunoglobulin genes. (a) k Light chain locus (chromosome 2); (b) light chain locus (chromosome 22); (c) heavy chain locus (chromosome 14). Coding sequences are indicated by open boxes. Nonfunctional or pseudogenes are indicated by shading. Arrows indicate transcriptional polarity. Not drawn to scale.

L chain genes The locus resembles the k locus in that the V gene segments, the J regions and the C regions exist as separate entities. However, in contrast to the k locus, the locus contains seven C genes and each has its own J region gene (Figure ). Three of these seven C-J clusters are considered pseudogenes because they contain either in-frame stop codons or frame-shifting deletions that prevent their expression. The V gene segment cluster is located upstream of the J-C cluster and consists of approximately 70 members, of which 3035 are classified as pseudogenes. V genes are classified into 10 subgroups of related genes. Unlike V genes, the V genes are not duplicated into proximal and distal clusters. A single V gene segment may recombine with either of the four functional J-C genes to form a complete L chain gene. H chain genes The H chain locus is more complexthan either L chain locus (Figure ). There are nine functional CH region genes: , , 1, 2, 3, 3, 1, 2 and . These encode the different classes or isotypes of immunoglobulin polypeptide H chains: IgM, IgD, the four subclasses of IgG, the two subclasses of IgA and IgE. A nonfunctional pseudogene, je, lies between the 1 and 1 C genes. The duplicated pattern, ---, suggests that the C H locus evolved by

duplication of this group of genes. The VH gene segments are located upstream from the CH genes. There are approximately 130 VH gene segments but only about 45 are functional. The V H gene segments have been assigned to seven subgroups based upon their sequence relatedness. Individual members of a family are not grouped together but are interspersed throughout the V gene cluster. The H locus contains six J regions. In contrast to either L chain locus, there is an additional cluster of elements called D regions that are located between the V and J clusters. Approximately 25 functional D region genes have been so far described.