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GENETIC BACKGROUND OF ANTIBODY DIVERSITY

GENETIC BACKGROUND OF ANTIBODY DIVERSITY. STRUCTURE OF IMMUNOGLOBULINS/ANTIBODIES. Heavy chain (H). VH. VL. CH. Light chain (L). CL. Antigen. Antigen binding. Variable domains. COMPLEMENT ACTIVATION BINDING TO CELLS DEGRADATION TRANSPORT. Constans domains. Effector functions.

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GENETIC BACKGROUND OF ANTIBODY DIVERSITY

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  1. GENETIC BACKGROUND OF ANTIBODY DIVERSITY

  2. STRUCTURE OF IMMUNOGLOBULINS/ANTIBODIES Heavy chain (H) VH VL CH Light chain (L) CL Antigen Antigen binding Variable domains COMPLEMENT ACTIVATION BINDING TO CELLS DEGRADATION TRANSPORT Constans domains Effector functions

  3. AMINO ACID SEQUENCE OF IMMUNOGLOBULINS Multiple myeloma (MM) Plasma cell tumors – tumor cells reside in the bone marrow Produce immunoglobulins of monoclonal origin,serum concentration 50-100mg/ml Rodney Porter & Gerald Edelman 1959 – 1960 myeloma protein purification L H Reduction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Variable Constant Gel electrophoresis 50 kDa Heavy chain 25 kDa Light chain

  4. GENETIC BACKGROUND OF ANTIBODY DIVERSITY VH VH VL VL S – S S – S Mechanism of the generation of variability? Different rules for encoding the variable and constant regions? Symmetric molecule  two identical VH and VL  both chromosomes encode for the same sequence?

  5. DOGMA OF MOLECULAR BIOLOGY CHARACTERISTICS OF IMMUNOGLOBULIN SEQUENCE 1 GEN = 1 PROTEIN THEORIES 1 GEN High rate of somatic mutations in the V-region Gen V C Many GENES (10000 – 100000) Protein V1 C V2 C Vn C

  6. MOLECULAR GENETICS OF IMMUNOGLOUBLINS How can the bifunctional nature of antibodies be explained genetically? In 1965, Dreyer & Bennett proposed that for a single isotype of antibody there may be: • A single C region gene encoded in the GERMLINE and separate from the V region genes • Multiple choices of V region genes available • A mechanism to rearrange V and C genes in the genome so that they can fuse to form a complete Immunoglobulin gene. This was genetic heresy as it violated the then accepted notion that DNA was identical in every cell of an individual

  7. A single C region gene is encoded in the germline and separated from the multiple V region genes V V V V V V V V V C V V V C A mechanism to rearrange V and C genes in the genome exists so that they can fuse to form a complete Immunoglobulin gene V V Proof of the Dreyer - Bennett hypothesis Find a way to show the existence of multiple V genes and rearrangement to the C gene

  8. V V Germline DNA V V V V V V V C V V V C Rearranged DNA V V Approach • Tools: • A set of cDNA probes to specifically distinguish V regions from C regions • DNA restriction enzymes to fragment DNA • Examples of germline (e.g. placenta) and mature B cell DNA (e.g. a plasmacytoma/myeloma)

  9. The experiment of Susumi Tonegawa 1976 V-CmRNS probe CmRNS probe * * * * V C * * * Embryonal cell V B-cell C

  10. Gén Gén V V V V V V V C C GÉN SZEGMENSEK SZOMATIKUS ÁTRENDEZŐDÉSE EGY GÉNNÉ GÉN SZEGMENSEK SZOMATIKUS ÁTRENDEZŐDÉSE EGY GÉNNÉ Fehérje Fehérje CONCLUSION V and C genes get close to each other in B-cells only B-CELL There are many variable genesbut only one constant gene GERM LINE

  11. L VL JL CL L VL CL ~ 95aa ~ 100aa ~ 95aa ~ 100aa VL CL L Some of the extra amino acids are provided by one of a small set of J or JOINING regions ~ 208aa Ig gene sequencing complicated the model The structures of germline VL genes were similar for Vk, and Vl, However there was an anomaly between germline and rearranged DNA: Where do the extra 13 amino acids come from?

  12. SOMATIC REARRANGEMENT OF KAPPA (κ) CHAIN GENE SEGMENTS B-cell 2 5 Jκ 40 Vκ Germ line Vκ Jκ Jκ Jκ Jκ Vκ Vκ Vκ Vκ Vκ Vκ Vκ During B-lymphocyte development Jk Jκ Jκ Jκ B-cell 1 Jκ DNA

  13. Leader pA Vκ P Vκ Vκ Vκ Vκ J J J J J J E E Vκ-Jκ Primary RNAtranscript Cκ Cκ Cκ Cκ mRNA AAAA Translation Protein EXPRESSION OF THE KAPPA CHAIN Efficiency of somatic gene rearrangement?

  14. Ig light chain rearrangement: Rescue pathway Vk Jk Ck Non-productive rearrangement Light chain has a second chance to make a productive join using new V and J elements Spliced mRNA transcript There is only a 1:3 chance of the join between the V and J region being in frame

  15. L VL JL CL Further diversity in the Ig heavy chain DH JH L VH CH The heavy chain was found to have further amino acids (0 – 8) between the JH és CH genes D (DIVERSITY) region Each heavy chain requires 3 recombination events JH to DH, VH to JHDH,and VHJHDH to CH Each light chain requires 2 recombination events VL to JL and VLJL to CL

  16. SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS During B-cell development VH1 VH1 VH1 D D D D D D D D JH JH VH2 JH JH VH2 VH3 120 VH 12 D 4 JH VH2 VH3 JH JH JH JH

  17. IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE GENE SEGMENTS Gene segments Light chain Heavy chain kappa lambda Variable (V) 132/40 105/30 123/65 Diversity (D) 0 0 27 Joining (J) 5 4 9 ORGANIZATION OF IMMUNOGLOBULIN GENE SEGMENTS Chromosome 2 kappa light chain gene segments Chromosome 22 lambda light chain gene segments Chromosome 14 heavy chain gene segments HOW MANY IMMUNOGLOBULIN GENE SEGMENTS

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