GENETIC BACKGROUND OF ANTIBODY DIVERSITY

s

s

s

s

s

s

s

s

s ss s

CH2

CH3

s

s

s

s

s

s

s

s

ss

VL

VH

CL

CH1 ss

ss

s

s

s

s

ss

effektor funkciók

konstans domének

antigénkötés

variábilis domének

STRUCTURE OF IMMUNOGLOBULINS/ANTIBODIES

SS

COMPLEMENT ACTIVATION

BINDING TO CELLS

DEGRADATION

TRANSPORT

Light chain (L)

Heavy chain (H)

VL

CL

VH

CH

Antigen binding

Variable domains

Antigen

Constans domains

Effector functions

MMultiple myeloma (MM)ultiple myeloma (MM)PPlasmlasmaa cell tumors – tumor cells reside in cell tumors – tumor cells reside in tthe bone marrowhe bone marrowProduce immunoglobulins of monoclonal origin,Produce immunoglobulins of monoclonal origin, serum concentration 50-100mg/mlserum concentration 50-100mg/mlRodney Porter & Gerald Edelman 1959 – 1960Rodney Porter & Gerald Edelman 1959 – 1960 myeloma protein purification myeloma protein purification

AMINO ACID SEQUENCE OF IMMUNOGLOBULINS

50 kDa50 kDaHeavy chainHeavy chain

25 kDa25 kDaLight chain Light chain

Gel electrophoresisGel electrophoresis

Variable Constant

123456789101112131415161718

Reduction

L H

GENETIC BACKGROUND OF ANTIBODY DIVERSITY

VLVLVHVH

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?

S – S S – S

MMany GENEany GENESS (10 (10 000 – 100000 – 100 000)000)

VV22 CC VVnn CCVV11 CC

1 GEN1 GEN

HHigh rate of somatic mutations in igh rate of somatic mutations in tthe he VV-region-region

VV CC

GGeenn

ProteinProtein

1 G1 GENEN = 1 = 1 PROTEIN PROTEIN

DOGMA OF MOLECULAR BIOLOGY

CHARACTERISTICS OF IMMUNOGLOBULIN SEQUENCE

THEORIES

MOLECULAR GENETICS OF IMMUNOGLOUBLINS

• 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.

In 1965, Dreyer & Bennett proposed that for a single isotype of antibody there may be:

How can the bifunctional nature of antibodies be explained genetically?

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

VV

VV

V

V

VV

V

V

VV

V

A mechanism to rearrange V and C genes in the genome exists so that they can fuse to form a complete

Immunoglobulin gene

CV

C

A single C region gene is encoded in the germline and separated from the multiple V region genes

Find a way to show the existence of multiple V genes and rearrangement to the C gene

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)

C

VV

VV

V

V

VV

V

Germline DNA

CV

V

VV

V

Rearranged DNA

* *

*

*

*BB-cell-cellV C

V C

Embryonal cellEmbryonal cell

V-V-CmRNS probeCmRNS probe

CCmRNS probemRNS probe

**

The experiment of Susumi Tonegawa 1976

There are many vThere are many vaariable genesriable genes but only one constant genebut only one constant gene

V CV V V

GERM LINEGERM LINE

V aV and nd CC g geenes nes gget close to each other in B-ceet close to each other in B-cellls onlyls only

CV V V

BB-CELL-CELL

CONCLUSION

FehérjeFehérje

GGéénn

GÉN SZEGMENSEK SZOMATIKUS ÁTRENDEZŐDÉSE EGY GÉNNÉ

Ig gene sequencing complicated the model

The structures of germline VL genes were similar for V, and V,However there was an anomaly between germline and rearranged DNA:

Where do the extra 13 amino acids come from?

CLVL

~ 95 ~ 100

L CLVL

~ 95 ~ 100

JL

Some of the extra amino acids are provided by

one of a small set of J or JOINING regions

L

CLVL

~ 208

L

?

DDuuring ring BB-lymphocyte -lymphocyte developmentdevelopment

Jk Jκ Jκ JκVκ Vκ VκB-cell 1

JκVκB-cell 2

440 V0 Vκκ 55 J Jκκ

Vκ Vκ Vκ Vκ Jκ Jκ Jκ JκGerm lineGerm line

SOMATIC REARRANGEMENT OF KAPPA (κ) CHAIN GENE SEGMENTS

DNA

pACCκEJJ

Vκ-Jκ

VκVκ P

CCκJVκ ProteinProtein

mRNAmRNACCκJVκ AAAA

TransTranslationlation

EXPRESSION OF THE KAPPA CHAIN

PrimaPrimary ry RNRNAA transcripttranscript

CCκEJJVκLeader

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

V J C

Non-productive rearrangement

Spliced mRNA transcript

Light chain has a second chance to makea productive join using new V and J elements

Further diversity in the Ig heavy chain

VL JL CLL

CHVH JHDHL

The heavy chain was found to have further amino acids (0 – 8) between the JH és CH genes

D (DIVERSITY) region

Each light chain requires 2 recombination events

VL to JL and VLJL to CL

Each heavy chain requires 3 recombination events JH to DH , VH to JHDH, and VHJHDH to CH

During B-cell developmentDuring B-cell development

VH2 JH JH

120 VH120 VH 4 JH4 JH

VH1 VH3 D JH JH

12 D12 D

D DD

JH JH

JH JHD D

SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS

D DVH1 VH2 VH3

VH1 VH2

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

Chromosome 2 kappa light chain gene segments

Chromosome 22 lambda light chain gene segments

Chromosome 14 heavy chain gene segments

IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE GENE SEGMENTS

ORGANIZATION OF IMMUNOGLOBULIN GENE SEGMENTS

The key experiment of Nobumichi Hozumi and Susumu Tonegawa