The Molecular Genetics of Immunoglobulins

Recall Structure • Numerous V region genes are preceded by Leader or signal

sequences (60-90 bp) exons interspersed with introns.• Heavy chain contains V (Variable), D (Diversity), J (Joining) and C

(Constant) region gene segments. V - D - J – C• Light chain contains V, J, and C region gene segments. V - J - C• Constant region genes are sub-divided into exons encoding

domains (CH1,CH2, CH3, CH4)

CHARACTERISTICS OF IMMUNOGLOBULIN GENERE-ARRANGEMENT

1. Involves Allelic Exclusion.– Only one of two parental alleles of Ig is expressed in a B cell.– Either kappa or lambda light chain is expressed by a B cell (light chain

isotype exclusion).2. Ig rearrangement occurs prior to antigen exposure.A. Heavy chain re-arrangement– Re-arrangement occurs in a precise order:– Heavy chain re-arranges before Light chain.– D-J joining occurs first to form DJ and is followed by V-DJ joining to form VDJ.– Just as in light chain the production of μ heavy chain by re-arrangement of

one allele inhibits re-arrangement on other allele. If re-arrangement on first allele is non-productive (due to mutations, deletions or frame shifts that generate stop codons), then re-arrangement on the second allele is stimulated.

Allelic exclusion: only one chromosome is active in any one lymphocyte

Light chain re-arrangement

• Kappa chain (κ) rearranges before lambda (λ) chain V-joining occurs.• Productive arrangement on one allele blocks re-arrangement on other

allele.• If kappa protein is produced, re-arrangement of lambda chain is

blocked. • Otherwise lambda chain undergoes re-arrangement.

Questions?

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

2. How can the same specificity of antibody be on the cell surface and secreted?

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

4. How does the DNA break and rejoin?

Proof of the Dreyer - Bennett hypothesis

VV

VV

V

V

VV

V

V

VV

V

Rearranging V and C genesCV

C

Single germline C gene separate from multiple V genes

Aim: to show multiple V genes and rearrangement to the C gene

Proof of the Dreyer - Bennett hypothesis

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

V V V

C

V V

VV

V V

Size fractionate by gel

electrophoresis

C

V

V

V

V

V

V

VV

V CV

V

V

V

V

V

V

V V

Cut germline DNA with restriction enzymes

V V

V V V

VV

V V

C

A range of fragment sizes is generated

Blot with a V region probe

Blot with a C region probe

N.B. This example describes events on only ONE of the chromosomes

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 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

- compare the pattern of bandswith germline DNA

V and C probes detect the same fragmentSome V regions missing

C fragment is larger cf germline

VV

V V

CV

Evidence for gene recombination

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

~ 95aa ~ 100aa

L CLVL

~ 95aa ~ 100aa

JL

Extra amino acids provided by one of a small set of J

or JOINING regions

L

CLVL

~ 208aa

L

Further diversity in the Ig heavy chain

VL JL CLL

CHVH JH DHL

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

The D or DIVERSITY region

Each light chain requires two recombination events:VL to JL and VLJL to CL

Each heavy chain requires three recombination events:VH to JH, VHJH to DH and VHJHDH to CH

Diversity: Multiple Germline Genes

• 123 VH genes on chromosome 14• 40 functional VH genes with products identified• 79 pseudo VH genes• 4 functional VH genes - with no products identified• 24 non-functional, orphan VH sequences on chromosomes

15 & 16

VH Locus:

JH Locus: • 9 JH genes• 6 functional JH genes with products identified• 3 pseudo JH genes

DH Locus: • 27 DH genes• 23 functional DH genes with products identified• 4 pseudo DH genes• Additional non-functional DH sequences on the chromosome 15

orphan locus• reading DH regions in 3 frames functionally increases number

of DH regions

Diversity: Multiple germline genes

• 132 Vk genes on the short arm of chromosome 2• 29 functional Vk genes with products identified• 87 pseudo Vk genes• 15 functional Vk genes - with no products identified• 25 orphans Vk genes on the long arm of chromosome 2• 5 Jk regions

Vk & Jk Loci:

• 105 Vl genes on the short arm of chromosome 2• 30 functional genes with products identified• 56 pseudogenes• 6 functional genes - with no products identified• 13 relics (<200bp Vl of sequence)• 25 orphans on the long arm of chromosome 2• 4 Jl regions

Vl & Jl Loci:

Genomic organisation of Ig genes(No.s include pseudogenes etc.)

DH1-27 JH 1-9 CmLH1-123VH 1-123

Lk1-132Vk1-132 Jk 1-5 Ck

Ll1-105Vl1-105 Cl1 Jl1 Cl2 Jl2 Cl3 Jl3 Cl4 Jl4

Ig light chain gene rearrangement by somatic recombination

Germline

Vk Jk Ck

SplicedmRNA

Rearranged1° transcript

Questions?

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

2. How can the same specificity of antibody be on the cell surface and secreted?

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

4. How does the DNA break and rejoin?

•Cell surface antigen receptor on B cellsAllows B cells to sense their antigenic environmentConnects extracellular space with intracellular signalling machinery

•Secreted antibodyNeutralisationArming/recruiting effector cellsComplement fixation

Remember

How does the model of recombination allow fortwo different forms of the protein?

Primary transcript RNA AAAAA

Cm

Polyadenylation site (secreted)

pAs

Polyadenylation site (membrane)

pAm

The constant region has additional, optional exons

Cm1 Cm2 Cm3 Cm4

Each H chain domain (& the hinge) encoded by separate

exons

h

Secretioncoding

sequence

Membranecoding

sequence

mRNACm1 Cm2 Cm3 Cm4 AAAAAh

Transcription

Membrane IgM constant region

Cm1 Cm2 Cm3 Cm41° transcriptpAm

AAAAAh

Cm1 Cm2 Cm3 Cm4DNA h

Membrane coding sequence encodes

transmembrane regionthat retains IgM in the cell

membrane

Fc

Protein

Cleavage & polyadenylation at pAm and RNA splicing

mRNA

Secreted IgM constant region

Cm1 Cm2 Cm3 Cm4 AAAAAh

Cm1 Cm2 Cm3 Cm4DNA h

Cleavage polyadenylation at pAs and RNA splicing

1° transcriptpAs

Cm1 Cm2 Cm3 Cm4

Transcription

AAAAAh

Secretion coding sequence encodes the C

terminus of soluble, secreted IgM

Fc

Protein

Why do V regions not join to J or C regions?

IF the elements of Ig did not assemble in the correct order, diversity of specificity would be severely compromised

Full potential of the H chain for diversity needs V-D-J-C joining - in the correct order

Were V-J joins allowed in the heavy chain, diversity would be reduced due to loss of the imprecise join between the V and D regions

DIVERSITY

2x

DIVERSITY

1x

VH DH JH C

Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences

V, D, J flanking sequences

Vl 7 23 9

Sequencing up and down stream of V, D and J elementsConserved 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

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

V 7 23 9

D7 12 9JV 7 23 9

7 23 9

7 12 9D7129 J

7 23 9

7 12 9

VDJRecombination activating gene products, (RAG1 & RAG 2) and ‘high mobility group proteins’ bind to the RSS

The two RAG1/RAG 2 complexes bind to each other and bring the V region adjacent to the DJ region

• The recombinase complex makes single stranded nicks in the DNA. The free OH on the 3’ end hydrolyses the phosphodiester bond on the other strand.

• This seals the nicks to form a hairpin structure at the end of the V and D regions and a flush double strand break at the ends of the heptamers.

• The recombinase complex remains associated with the break

Steps of Ig gene recombination

V

DJ

7 23 9

7 12 9

A number of other proteins, (Ku70:Ku80, XRCC4 and DNA dependent protein kinases) bind to the hairpins and the heptamer ends.

V D J

The hairpins at the end of the V and D regions are opened, and exonucleases and transferases remove or add random nucleotides to the gap between the V and D region

V D J 723

97

129

DNA ligase IV joins the ends of the V and D region to form the coding joint and the two heptamers to form the signal joint.

Steps of Ig gene recombination

7D 12 9J

Junctional diversity: P nucleotide additions

7V 23 9

D7 12 9J

V 7 23 9TC CACAGTGAG GTGTCAC

AT GTGACACTA CACTGTG

The recombinase complex makes single stranded nicks at random sites close to the

ends of the V and D region DNA.

7D 12 9J

7V 23 9CACAGTGGTGTCAC

GTGACACCACTGTG

TCAG

ATTADJ

V TCAG

ATTA

UU

The 2nd strand is cleaved and hairpins form between the complimentary bases at ends of the V and D

region.

V2V3

V4

V8

V7V6

V5

V9

7 23 9CACAGTGGTGTCAC

7 12 9GTGACACCACTGTG

V TCAG U

DJ ATTA U

Heptamers are ligated by DNA ligase IV

V and D regions juxtaposed

V TCAG U D JAT

TA

U

V TCAG U D JAT

TA

U Endonuclease cleaves single strand at random sites in V and D segment

V TC~GAAG D JAT

TA~TAThe nucleotides that flip out, become part of the complementary DNA strand

Generation of the palindromic sequence

In terms of G to C and T to A pairing, the ‘new’ nucleotides are palindromic.The nucleotides GA and TA were not in the genomic sequence and introduce

diversity of sequence at the V to D join.

V TCAG U D JAT

TA

U Regions to be joined are juxtaposed

The nicked strand ‘flips’ out

Junctional Diversity – N nucleotide additions

V TC~GAAG D JAT

TA~TA

Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNA

CACTCCTTATTCTTGCAA

V TC~GAAG D JAT

TA~TACACACCTTA

TTCTTGCAA Complementary bases anneal

V D JDNA polymerases fill in the gaps with complementary nucleotides and DNA ligase IV joins the strands

TC~GAAG

ATTA~TA

CACACCTTATTCTTGCAA

D JTA~TA Exonucleases nibble back free endsV TC~GACACACCTTATTCTTGCAA

V TCDTA

GTT AT ATAG C

V D JTCGACGTTATATAGCTGCAATATA

Junctional Diversity

Germline-encoded nucleotides

Palindromic (P) nucleotides - 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.

Problems?

3. How do V region find J regions and why don’t they join to C regions?The 12-23 rule

1. How is an infinite diversity of specificity generated from finite amounts of DNA?Combinatorial Diversity, genomic organisation and Junctional Diversity

2. How can the same specificity of antibody be on the cell surface and secreted?Use of alternative polyadenylation sites

4. How does the DNA break and rejoin?Imprecisely to allow Junctional Diversity

Variable addition and subtraction of nucleotides at the junctions between gene segments contributes to

diversity in the third hypervariable region

• Of the three hypervariable loops in the protein chains of immunoglobulins, two are encoded within the V gene segment DNA. The third (HV3 or CDR3) falls at the joint between the V gene segment and the J gene segment, and in the heavy chain is partially encoded by the D gene segment.

• In both heavy and light chains, the diversity of CDR3 is significantly increased by the addition and deletion of nucleotides at two steps in the formation of the junctions between gene segments. The added nucleotides are known as P-nucleotidesand N-nucleotides 

• As the total number of nucleotides added by these processes is random, the added nucleotides often disrupt the reading frame of the coding sequence beyond the joint.

• Such frameshifts will lead to a nonfunctional protein, and DNA rearrangements leading to such disruptions are known as nonproductive rearrangements.

• As roughly two in every three rearrangements will be nonproductive, many B cells never succeed in producing functional immunoglobulin molecules, and junctional diversity is therefore achieved only at the expense of considerable wastage.

Imprecise joining generates diversity

Some rearrangements are productive, others are non-productive: frame shift

alterations are non-productive

V D J712

9

723

9

7 12 97239

V D J

Imprecise and random events that occur when the DNA breaks and rejoins allows new nucleotides to be inserted or lost from the sequence at and around the coding

joint.

Junctional diversity

Mini-circle of DNA is permanently lost from the

genome

Signal jointCoding joint

V1 V2 V3 V4 V9 D J

Looping out works if all V genes are in the same transcriptional orientation

V1 V2 V3 V9 D J

Non-deletional recombination

D J7129V47239

V1 7 23 9 D7129 J

How does recombination occur when a V gene is in opposite orientation to the DJ region?

V4

D J7129V47239 V4 and DJ in opposite transcriptional orientations

DJ

712

9V47239

1.

DJ

712

9

V47239

3.

DJ7

129

V47239

2.

D J7129

V472394.

Non-deletional recombination

D J7129

V47239

1.

D J

V4

71297239

3.

V to DJ ligation - coding joint formation

D J7129

V47239

2.

Heptamer ligation - signal joint formation

D JV47 12 97239

Fully recombined VDJ regions in same transcriptional orientationNo DNA is deleted

4.

Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences

• A system is required to ensure that DNA rearrangements take place at the correct locations relative to the V, D, or J gene segment coding regions.

• V gene segment joins to a D or J and not to another V.• DNA rearrangements are in fact guided by conserved noncoding DNA sequences that

are found adjacent to the points at which recombination takes place. • These sequences consist of a conserved block of seven nucleotides—

the heptamer 5′CACAGTG3′—which is always contiguous with the coding sequence, followed by a nonconserved region known as the spacer, which is either 12 or 23 nucleotides long.

• This is followed by a second conserved block of nine nucleotides—the nonamer 5′ACAAAAACC3′ .

• The spacer varies in sequence but its conserved length corresponds to one or two turns of the DNA double helix.

• This brings the heptamer and nonamer sequences to the same side of the DNA helix, where they can be bound by the complex of proteins that catalyzes recombination. The heptamer-spacer-nonamer is called a recombination signal sequence (RSS).

12/23 Rule• Recombination only occurs between gene segments located

on the same chromosome.• It generally follows the rule that only a gene segment flanked

by a RSS with a 12-base pair (bp) spacer can be joined to one flanked by a 23 bp spacer RSS. This is known as the 12/23 rule.

• For the heavy chain, a DH gene segment can be joined to a JH gene segment and a VH gene segment to a DH gene segment, but VH gene segments cannot be joined to JH gene segments directly, as both VH and JH gene segments are flanked by 23 bp spacers and the DH gene segments have 12 bp spacers on both sides

The diversity of the immunoglobulin repertoire is generated by four main processes

• Antibody diversity is generated in four main ways. • The gene rearrangement that combines two or three gene segments

 to form a complete V-region exon generates diversity in two ways. – First, there are multiple different copies of each type of gene

segment, and different combinations of gene segments can be used in different rearrangement events. This combinatorial diversity is responsible for a substantial part of the diversity of the heavy- and light-chain V regions.

– Second, junctional diversity is introduced at the joints between the different gene segments as a result of addition and subtraction of nucleotides by the recombination process.

• A third source of diversity is also combinatorial, arising from the many possible different combinations of heavy- and light-chain V regions that pair to form the antigen-binding site in the immunoglobulin molecule.

• Somatic mutation

Rearranged V genes are further diversified by somatic hypermutation

• The mechanisms for generating diversity described so far all take place during the rearrangement of gene segments in the initial development of B cells in the central lymphoid organs.

• There is an additional mechanism that generates diversity throughout the V region and that operates on B cells in peripheral lymphoid organs after functional immunoglobulin genes have been assembled.

• This process, known as somatic hypermutation.• Introduces point mutations into the V regions of the rearranged

heavy- and light-chain genes at a very high rate, giving rise to mutant B-cell receptors on the surface of the B cells.

• Some of the mutant immunoglobulin molecules bind antigen better than the original B-cell receptors, and B cells expressing them are preferentially selected to mature into antibody-secreting cells. This gives rise to a phenomenon called affinity maturation of the antibody population,

Somatic hypermutation • Occurs when B cells respond to antigen along with signals from

activated T cells. • The immunoglobulin C-region gene, and other genes expressed in

the B cell, are not affected, whereas the rearranged VH and VL genes are mutated even if they are nonproductive rearrangements and are not expressed.

• The pattern of nucleotide base changes in nonproductive V-region genes illustrates the result of somatic hypermutation without selection for enhanced binding to antigen.

Somatic hypermutation

FR1 FR2 FR3 FR4CDR2 CDR3CDR1

Amino acid No.

Variability80

100

60

40

20

20 40 60 80 100 120

Wu - Kabat analysis compares point mutations in Ig of different specificity.

What about mutation throughout an immune response to a single epitope?How does this affect the specificity and affinity of the antibody?

Clone 1Clone 2Clone 3Clone 4Clone 5Clone 6Clone 7Clone 8Clone 9Clone 10

CD

R1

CD

R2

CD

R3

Day 6

CD

R1

CD

R2

CD

R3

CD

R1

CD

R2

CD

R3

CD

R1

CD

R2

CD

R3

Day 8 Day 12 Day 18

Deleterious mutationBeneficial mutationNeutral mutation

Lower affinity - Not clonally selectedHigher affinity - Clonally selectedIdentical affinity - No influence on clonal selection

Somatic hypermutation leads to affinity maturation

Hypermutation is T cell dependentMutations focussed on ‘hot spots’ (i.e. the CDRs) due to double stranded breaks repaired

by an error prone DNA repair enzyme.

Cells with accumulated mutations in the CDR are selected for high antigen binding capacity – thus the affinity matures throughout the course of the response

Antibody isotype switching

Throughout an immune response the specificity of an antibody will remain the same (notwithstanding affinity maturation)

The effector function of antibodies throughout a response needs to change drastically as the response progresses.

Antibodies are able to retain variable regions whilst exchanging constant regions that contain the structures that interact with cells.

J regions Ca2CeCg4Cg2Ca1Cg1Cg3CdCm

Organisation of the functional human heavy chain C region genes

Ca2CeCg4Cg2Ca1Cg1Cg3CdCm

Switch regions

• The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7.

• Switching is mechanistically similar in may ways to V(D)J recombination.• Isotype switching does not take place in the bone marrow, however, and it

will only occur after B cell activation by antigen and interactions with T cells.

Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2Sm

• Upstream of C regions are repetitive regions of DNA called switch regions. (The exception is the Cd region that has no switch region).

Ca2CeCg4Cg2Ca1Cg1Cg3CdCm

Cm

Cd

Cg3VDJ

Sg3

Cm

Cd

Cg3

VDJ

Cg1

Sg1

Ca1

Cg3

VDJ Ca1

Cg3VDJ

IgG3 produced.Switch from IgM

VDJ Ca1

IgA1 produced.Switch from IgG3

VDJ Ca1

IgA1 produced.Switch from IgM

Switch recombination

At each recombination constant regions are deleted from the genomeAn IgE - secreting B cell will never be able to switch to IgM, IgD, IgG1-4 or IgA1

Summary• Diversity within the immunoglobulin repertoire is achieved by

several means. • Perhaps the most important factor that enables this extraordinary

diversity is that V regions are encoded by separate gene segments, which are brought together by somatic recombination to make a complete V-region gene.

• Many different V-region gene segments are present in the genome of an individual, and thus provide a heritable source of diversity. Additional diversity, termed combinatorial diversity, results from the random recombination of separate V, D, and J gene segments to form a complete V-region exon. 

Summary• Variability at the joints between segments is increased by the

insertion of random numbers of P- and N-nucleotides and by variable deletion of nucleotides at the ends of some coding sequences.

• The association of different light- and heavy-chain V regions to form the antigen-binding site of an immunoglobulin molecule contributes further diversity.

• Finally, after an immunoglobulin has been expressed, the coding sequences for its V regions are modified by somatic hypermutation upon stimulation of the B cell by antigen.

• The combination of all these sources of diversity generates a vast repertoire of antibody specificities from a relatively limited number of genes.

MECHANISMS FOR GENERATING ANTIBODY DIVERSITY

• Presence of multiple V genes in the germ line.• Combinatorial Diversity - due to potentially different

associations of different V, D and J gene segments.• Junctional Diversity • Somatic Hypermutation• Random Assortment of H and L chains.

Understanding of immunoglobulin structure and formation has opened up a new world of possibilities

• Monoclonal antibodies• Engineering mice with human immune

systems• Generating chimeric and hybrid antibodies

for clinical use• Abzymes: antibodies with enzyme

capability