TEMA 7. LA GENERACIÓN DE LA DIVERSIDAD

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1 TEMA 7. LA GENERACIÓN DE LA DIVERSIDAD Ehrlich's side-chain theory. Ehrlich proposed that the combination of antigen with a preformed B-cell receptor (now known to be antibody) triggered the cell to produce and secrete more of those receptors. Although the diagram indicates that he thought a single cell could produce antibodies to bind more than one type of antigen, it is evident that he anticipated both the clonal selection theory and the idea that the immune system could generate receptors before contact with antigen. Cómo se genera la diversidad estructural de las inmunoglobulinas? 1. Múltiples genes (V, J, D) en la línea germinal 2. Recombinación génica (VJ en las ligeras y V, J, D en las pesadas 3. Imprecisiones en las recombinaciones 4. Mutación somática puntual 5. Existencia de dos tipos de cadenas (pesada y ligera) Since each mechanism can occur with any of the others, the potential for increased diversity multiplies at each step of immunoglobulin production. 1

2 Variability is calculated by comparing the sequences of many individual chains and, for any position, is equal to the ratio of the number of different amino acids found at that position, to the frequency of the most common amino acid. The areas of greatest variability, of which there are three in the Vl domain, are termed the hypervariable regions. In some sequences studied, extra amino acids have been found, but these are excluded here to enhance comparison; their positions are indicated by arrows. The areas shaded pink denote regions of hypervariability (CDR), and the most hypervariable positions are shaded red. The four framework regions (FR) are shown in yellow. (Courtesy of Professor E. A. Kabat.) 2

3 76 genes 35 funcionales 16 con defectos leves 25 pseudogenes During differentiation of the pre-b cell one of several Vκ genes on the germ line DNA (V1-Vn) is recombined and apposed to a Jκ segment (Jκ1-Jκ5). The B cell transcribes a segment of DNA into a primary RNA transcript that contains a long intervening sequence of additional J segments and introns. This transcript is processed into mrna by splicing the exons together, and is translated by ribosomes into kappa (κ) chains; B-cell DNA is coloured light brown; RNA is coloured green; and immunoglobulin peptides are coloured yellow. The rearrangement illustrated is only one of the many possible recombinations. During B-cell differentiation, one of the germ line Vλ genes recombines with a J- segment to form a V-J combination. The rearranged gene is transcribed into a primary RNA transcript complete with introns (non-coding segments occurring between the genes), exons (which code for protein) and a poly-a tail. This is spliced to form messenger RNA (mrna) with loss of the introns, and then translated into protein. 3

4 The heavy chain gene loci combine three segments to produce the exon (V-D-J gene) which codes for the Vh domain. Of some 80 V genes, about 50 are functional, and the others are pseudogenes. The V gene recombines with one of 30 D segments and one of six J segments, to produce a functional V-D-J gene in the B cell. 4

5 The same Vκ21 and J1 sequences of the germ line create three different amino acid sequences in the proteins PC2880, PC6684 and PC7940 by variable recombination. PC2880 has proline and tryptophan at positions 95 and 96, caused by recombination at the end of the CCC codon. Recombination one base down produces proline and arginine in PC6684. Recombination two bases down from the end of Vκ21 produces proline and proline in PC

6 The DNA sequence (1) and amino acid sequence (2) of three heavy chains of anti-phosphorylcholine are shown. Variable recombination between the germ line, V, D and J regions and N region insertion causes variation (orange) in amino acid sequences. In some cases (e.g. M167) there appear to be additional inserted codons. However, these are in multiples of three, and do not alter the overall reading frame. DNA of two IgG antibodies to phosphorylcholine are illustrated. Both antibodies share the T15 idiotype. Black lines indicate positions where the sequence has mutated from the germ line sequence. There are large numbers of mutations in both the introns and exons of both genes, but particularly in the second hypervariable region (HV2). By comparison, there are no mutations in the regions encoding the constant genes, which implies that the mutational mechanism is highly localized. 6

7 The constant-region genes of the mouse are arranged 8.5 kb downstream from the recombined VDJ segment. Each C gene (except Cδ) has one or more switching sequences at its start (red circles), which correspond to a sequence at the 5' end of theµgene. This allows any of the C genes to be expressed together with the VDJ segment. δ genes appear to use the same switching sequences as µ, but theµgene transcript is lost in RNA processing to produce IgD. The C genes (expanded below forµandγ2a) contain introns separating the exons for each domain (C1, C2, etc.). Theγgenes also have a separate exon coding for the hinge (H), and all the genes have one or more exons coding for membrane-bound immunoglobulin (M). The human immunoglobulin heavy chain gene locus (IGH) is shown. Initially, B cells transcribe a VDJ gene and a µ heavy chain that is spliced to produce mrna for IgM. Under the influence of T cells and cytokines, class switching may occur, illustrated here as a switch from IgM to IgG2. Each heavy chain gene except CD (which encodes IgD) is preceded by a switch region. When class switching occurs, recombination between these regions takes place, with the loss of the intervening C genes - in this case CM, CD, CG3, CG1 and CA1. (Note that pseudogenes have been omitted from this diagram.) 7

8 Initially the VDJ region is transcribed together with the M gene for the IgM heavy chain (left). After removal of introns during processing, mrna for secreted IgM is produced. During B-cell maturation, class-switch recombination occurs between the Sµ recombination region and a downstream switch region (G1 in this example). The intervening region (containing genes for IgM, IgD and IgG3 in this instance) is looped out and then cut, with deletion of the intervening regions and joining of the two switch regions. Single B cells produce more than one antibody isotype from a single long primary RNA transcript. A transcript containingµandδis shown here. Polyadenylation can occur at different sites, leading to different forms of splicing, producing mrna for IgD (top) or IgM (bottom). Even within this region, there are additional polyadenylation sites that determine whether the translated immunoglobulin is the secreted or membrane-bound form. 8

9 C-terminal amino acid sequences for both secreted and membrane-bound IgM are identical up to residue 556. Secreted IgM has 20 further residues. Residue 563 (asparagine) has a carbohydrate unit attached to it, while residue 575 is a cysteine involved in the formation of interchain disulphide bonds. Membrane IgM has 41 residues beyond 556. A stretch of 26 residues between 568 and 595 contains hydrophobic amino acids sandwiched between sequences containing charged residues. This hydrophobic portion may traverse the cell membrane as two turns ofα helix. A short, positively charged section lies inside the cytoplasm. Mouse IgM is shown in this example. Part of the DNA coding for IgM is shown diagrammatically. The exons for theµ3 andµ4 domains (H3 and H4) and the transmembrane and cytoplasmic segment of membrane IgM (M) are indicated. The 3' untranslated sequence is present at the end of the H4 and second membrane segments (S = stop codons). The DNA can be transcribed in two ways. If transcription stops after S, the transcript with a poly-a tail is processed to produce mrna for secreted IgM. If transcription runs through to include the membrane segments, processing removes the codons for the terminal amino acids and the stop signal of H4, so that translation yields a protein with a different C terminus. 9

10 The genes of the murine TCR chains are shown. Theδchain loci are embedded within theαloci and tandem duplication has occurred in theβ chain loci. The last of each set of Jβ genes and the Vγ3 gene are pseudogenes. 10