GENETIC BACKGROUND OF ANTIBODY DIVERSITY

STRUCTURE OF IMMUNOGLOBULINS/ANTIBODIES COMPLEMENT ACTIVATION 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

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 AMINO ACID SEQUENCE OF IMMUNOGLOBULINS Gel electrophoresis Reduction L H 50 kDa Heavy chain 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 25 kDa Light chain Variable Constant

GENETIC BACKGROUND OF ANTIBODY DIVERSITY VL VH 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?

V C V2 C Vn V1 DOGMA OF MOLECULAR BIOLOGY Gen Protein 1 GEN = 1 PROTEIN DOGMA OF MOLECULAR BIOLOGY CHARACTERISTICS OF IMMUNOGLOBULIN SEQUENCE THEORIES 1 GEN High rate of somatic mutations in the V-region V C Many GENES (10 000 – 100 000) V2 C Vn V1

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

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

Approach C V Germline DNA C V Rearranged DNA 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)

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

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

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: ? CL VL ~ 95aa ~ 100aa L CL VL ~ 95aa ~ 100aa JL Some of the extra amino acids are provided by one of a small set of J or JOINING regions L CL VL ~ 208aa L Where do the extra 13 amino acids come from?

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

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

Ig light chain rearrangement: Rescue pathway There is only a 1:3 chance of the join between the V and J region being in frame 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

Further diversity in the Ig heavy chain L VH DH JH 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 VL JL CL L Each light chain requires 2 recombination events VL to JL and VLJL to CL

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

IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE GENE SEGMENTS 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 Variable (V) 132/40 105/30 123/65 Diversity (D) 0 0 27 Joining (J) 5 4 9 Gene segments Light chain Heavy chain kappa lambda

The key experiment of Nobumichi Hozumi and Susumu Tonegawa