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?

S – S S – S

MMany GENEany GENESS (10 (10 000 – 000 – 3300 000)000)

VV22 CC22 VVnn CCnnVV11 CC11

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

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-CmRNA probeCmRNA probe

CCmRNA probemRNA probe

**

The experiment of Susumi Tonegawa 1976

The key experiment of Nobumichi Hozumi and Susumu Tonegawa

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

PROTEINPROTEIN

GGENEENE

REARRANGEMENT OF GENE SEGMENTS INTO A SINGLE FUNCTIONAL UNIT (GENE)

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?

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 1 recombination eventsVL to JL

Each heavy chain requires 2 recombination events JH to DH , VH to JHDH,

During B-cell developmentDuring B-cell development

VH2 JH JH

6565 VH VH 66 JH JH

VH1 VH3 D JH JH

2727 D 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) 40 30 65

Diversity (D) 0 0 27

Joining (J) 5 4 6

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

VHVH DD JHJH

VLVL JLJL

V-DV-Domainsomains

C-DC-Domainsomains

VHVH--DD--JHJH VLVL--JLJL

VARIABILITY OF B-CELL ANTIGEN RECEPTORS AND ANTIBODIES

B cells of one individual 1 2 3 4

Estimates of combinatorial diversity

Taking account of functional V D and J genes:

65 VH x 27 DH x 6JH = 10,530 combinations

40 Vx 5 J = 200combinations30 Vx 4 J = 120 combinations

= 320 different light chains

If H and L chains pair randomly as H2L2 i.e. 10,530x 320 = 3,369,600 possibilities Due only to COMBINATORIAL diversity

In practice, some H + L combinations do not occur as they are unstableCertain V and J genes are also used more frequently than others.

There are other mechanisms that add diversity at the junctions between genes - JUNCTIONAL diversity

GENERATES A POTENTIAL B-CELL REPERTOIRE

Somatic recombination to generate antibody diversity

Severe combined immunodeficiency (SCID)

Early manifestationred rash on the face and shoulders, infections with opportunistic pathogens. (Candida albicans, Pneumocystis carnii pneumonia)Lack of palpable lymph nodes

Omenn syndrome - RAG deficiencyLack of T-cells and B cells

How does somatic gene rearrangement(recombination) work?

1. How is an infinite diversity of specificity generated from finite amounts of

DNA?

Combinatorial diversity

2. How do V region find J regions and why don’t they join to C regions?

12-23 rule-Special - Recobnitation Signal Sequences (RSS)

- Recognized by Recombination Activation Gene coded proteins (RAGs)

PALINDROMIC SEQUENCES

HEPTAMER CACAGTG CACAGTGGTGACAC GTGACAC

NONAMER ACAAAAACC GGTTTTTGTTGTTTTTGG CCAAAAACA

V, D, J flanking sequences

V 7 23 9

Sequencing upstream and downstream of V, D and J elements revealed conserved sequences of 7, 23, 9 and 12 nucleotides in an

arrangement that depended upon the locus

V 7 12 9 J7239

J7129

D7129 7 12 9

VH 7 23 9 JH7239

Recombination signal sequences (RSS)

12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS

VH 7 23 9

D7129 7 12 9

JH7239

HEPTAMER - Always contiguous with coding sequence

NONAMER - Separated fromthe heptamer by a 12 or 23

nucleotide spacer

VH 7 23 9

D7129 7 12 9

JH7239

23-mer = two turns 12-mer = one turn

Molecular explanation of the 12-23 rule

Intervening DNAof any length23

V 97

12

D J79

23-mer

12-mer

Loop of intervening

DNA is excised

• Heptamers and nonamers

align back-to-back

• The shape generated by the

RSS’s acts as a target for

recombinases

7

9

97

V1 V2 V3 V4

V8V7

V6V5

V9 D J

V1 D J

V2

V3

V4

V8

V7

V6

V5

V9

• An appropriate shape can not be formed if two 23-mer flanked elements

attempted to join (i.e. the 12-23 rule)

Molecular explanation of the 12-23 rule

23-mer

12-mer

V1 D J

V2

V3

V4

V8

V7

V6

V5

V9

7

9

97

CONSEQUENCES OF RECOMBINATIONCONSEQUENCES OF RECOMBINATION

Generation of P-nucleotidesGeneration of P-nucleotides

23-mer

12-mer

Loop of interveningDNA is excised

V1 D J

V2

V3

V4

V8

V7

V6

V5

V9

7

9

97

Terminal deoxynucleotidyl Transferase (TdT)

Generation of Generation of NN-nucleotides-nucleotides

V D JTCGACGTTATATAGCTGCAATATA

Junctional Diversity

TTTTTTTTTTTTTTT

Germline-encoded nucleotides

Palindromic (P) nucleotides - not in the germline

Non-template (N) encoded nucleotides - not in the germline

Creates an essentially random sequence between the V region, D region and J region in heavy chains and the V region and J region in light chains

How does somatic gene rearrangement(recombination) work?

1. How is an infinite diversity of specificity generated from finite amounts of

DNA?

Combinatorial diversity

2. How do V region find J regions and why don’t they join to C regions?

12-23 rule

3. How does the DNA break and rejoin?

Imprecisely, with the random removal and addition of nucleotides to

generate sequence diversity

Junctional diversity (P- and N- nucleotides, see above)