GENETIC BACKGROUND OF THE VARIABILITY OF ANTIGEN

RECOGNIZING RECEPTORS

The total number of antibody specificities available to an individual is known as the antibody repertoire, and in humans is at least 1011, perhaps many more.

BUTThere are an estimated only 20,000-25,000 human protein-coding genes.

Genetic background of antibody diversity

Germline theory (separate genes for each different Ab)

V2 C2 Vn CnV1 C1

1 GENE

Somatic diversification theory

(high rate of somatic mutations in the V-region)

V C

Gen

Protein

1 GENE = 1 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

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

Tools:

• cDNA probes to distinguish V from C regions

C

VV

VV

V

V

VV

V

Germline DNA

• Germline (e.g. placenta) and rearranged B cell DNA (e.g. from a myeloma B cell)

• DNA restriction enzymes to fragment DNA

CV

V

VV

V

Rearranged DNA

Approach

CV

V

VV

V

CV

V

V

VV

Size fractionate by gel

electrophoresis

VV

V V

CV

Blot with a V region probe

Blot with a C region probe

Cut myeloma B cell DNA with restriction enzymes

V and C probes detect the same fragmentSome V regions missing

C fragment is larger cf germline DNA

VV

V V

CV

Evidence for gene recombination

V V V

Blot with a V region probe

Blot with a C region probe

C

V V

VV

V V

Size fractionate by gel

electrophoresis

Germline DNA

There are many variable genes but only one constant gene

V CV V V

GERM LINE

V and C genes get close to each other in B-cells only

CV V V

B-CELL

Conclusion

PROTEIN

GENE Rearrangement of gene segments into a single functional unit (gene)

The gene rearrangement concept

• Germline configuration

• Gene segments need to be reassembled for expression

• Sequentially arrayed

• Occurs in the B-cells precursors in the bone marrow (soma)

• A source of diversity BEFORE exposure to antigen

Ig gene sequencing complicated the model

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?

CLVL

~ 95 aa ~ 100 aa

L CLVL

~ 95 aa ~ 100 aa

JL

Extra amino acids provided by one of a

small set of J or JOINING regions

L

CLVL

~ 208 aa

L

The germline organization of the human immunoglobulin light-chain loci

Figure 2-15 part 1 of 2J-joining

CDR1 and CDR2 CDR3

During B-lymphocyte development

Jk Jκ Jκ JκVκ Vκ VκB-cell 1

JκVκB-cell 2

35 Vκ 5 Jκ

Vκ Vκ Vκ Vκ Jκ Jκ Jκ JκGermline

Somatic rearrangement of kappa (κ) chain gene segments

DNA

pACκEJJ

Vκ-Jκ

VκVκ P

CκJVκ Protein

mRNACκJVκ AAAA

Translation

Expression of the kappa chain

Primary RNA transcript

CκEJJVκLeader

• In developing B cells, the immunoglobulin genes undergo

structural rearrangements that permit their expression.

• The V domains of immunoglobulin light chains are encoded

in two (V and J) different kinds of gene segments, that are

brought into juxtaposition by recombination.

Further diversity in the Ig heavy chain

VL JL CLL

CHVH JHDHL

Heavy chain: between 0 and 8 additional amino acids between JH and CH

The D or DIVERSITY region

Each light chain requires one recombination event:VL to JL

Each heavy chain requires two recombination events:DH to JH and VH to DHJH

Heavy-chain V regions are constructed from three gene segments

The germline organization of the human immunoglobulin heavy-chain loci

*

During B-cell development

VH2 JH JH

40 VH 6 JH

VH1 VH3 D JH JH

23 D

D DD

JH JH

JH JHD D

SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS

D DVH1 VH2 VH3

VH1 VH2

• The V domains of immunoglobulin heavy chains are encoded

in three (V, D and J) different kinds of gene segments, that are

brought into juxtaposition by recombination.

The numbers of functional gene segments available to construct the variable and constant regions of human

immunoglobulin heavy chains and light chains

VH D JH

VL JL

V-Domains

C-Domains

VH-D-JH VL-JL

Variability of B-cell antigen receptors and antibodies

B cells of one individual 1 2 3 4

Estimates of combinatorial diversityTaking account of functional V D and J genes:

46 VH x 23 D x 6JH = 6,348 combinations

38 V k x 5 Jk = 190 combinations33 V l x 5 Jl = 165 combinations

= 355 different light chains

If H and L chains pair randomly as H2L2 i.e. 6,348 x 355 = 2,253,540 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.

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?

-Special - Recombitation Signal Sequences (RSS)

- Recognized by Recombination-Activating Genes coded proteins (RAGs)

12-23 rule

V, D, J flanking sequences

Vl 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

Vk 7 12 9 Jk7239

Jl7129

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 9712

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

V6 V5

V9 D J

V1 D J

V2

V3V4

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

Gene segments encoding the variable region are joined by recombination at recombination signal sequences

23-mer

12-mer

V1 D J

V2

V3V4

V8

V7

V6

V5

V9

7

9

97

Consequences of recombinationGeneration of P-nucleotides

23-mer

12-mer

Loop of interveningDNA is excised

V1 D J

V2

V3V4

V8

V7

V6

V5

V9

7

9

97

Terminal deoxynucleotidyl Transferase (TdT)

Generation of N-nucleotides

Junctional diversity increases diversity by 6 orders of magnitude

Hipervariable and framework regions exist within the variable domains of Igs

HV3 in the light-chain is at the junction between rearranged V and J segments

In the heavy chain HV3 is formed by the D segment and the residues between the rearranged V and D segments and the D andJ segments .

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)