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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 GENE
RE-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
V
V
V
V
V
V
V
V
V
V
Single germline C gene
separate from multiple V genes
V
V C
V
C
Rearranging V and C genes
V
Aim: to show multiple V genes and rearrangement to the C gene
Proof of the Dreyer - Bennett hypothesis
V
V
V
V
V
Germline DNA
V
V
V
V
V
V
V
V C
C
Rearranged DNA
V
Tools:
• cDNA probes to distinguish V from C regions
• DNA restriction enzymes to fragment DNA
• Germline (e.g. placenta) and rearranged B cell DNA (e.g. from a myeloma B cell)
N.B. This example
describes events on
only ONE of the
chromosomes
V
Cut germline DNA with
restriction enzymes
V
V
V
V
V
C
V
V
V
V
C
V
V
V
V
V
Size fractionate
by gel
electrophoresis
V
V
V
V
V
C
A range of fragment
sizes is generated
V
C
V
V
V
V
V
V
Blot with a V
region probe
Blot with a C
region probe
Evidence for gene recombination
V
Cut myeloma B cell V
DNA with restriction
enzymes
V
Size fractionate
by gel
electrophoresis
Blot with a V
region probe
V
V C
V
V
Blot with a C
region probe
V C
V
V
V C
Size fractionate
by gel
electrophoresis
V
V
Blot with a V
region probe
Blot with a C
region probe
V
C
V
V
V
V
V
V
V
V and C probes detect the same fragment
Some V regions missing
C fragment is larger cf germline
V
V
V
- compare the pattern of bands
with germline DNA
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:
VL
CL
~ 95aa
~ 100aa
L
L
VL CL
~ 208aa
Where do the extra
13 amino acids
come from?
L
VL JL
~ 95aa
CL
~ 100aa
Extra amino acids provided
by one of a small set of J
or JOINING regions
Further diversity in the Ig heavy chain
L
VH JH DH
CH
Heavy chain: between 0 and 8 additional amino acids between JH and CH
The D or DIVERSITY region
Each heavy chain requires three recombination events:
VH to JH, VHJH to DH and VHJHDH to CH
L
VL JL
CL
Each light chain requires two recombination events:
VL to JL and VLJL to CL
Diversity: Multiple Germline Genes
VH Locus:
•
•
•
•
•
JH Locus:
• 9 JH genes
• 6 functional JH genes with products identified
• 3 pseudo JH genes
DH Locus:
•
•
•
•
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
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
Vk & Jk Loci: •
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
Vl & Jl 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
•
•
•
•
•
•
•
•
•
•
•
Genomic organisation of Ig genes
(No.s include pseudogenes etc.)
LH1-123
VH 1-123
DH1-27
Lk1-132
Vk1-132
Ll1-105
Vl1-105
JH 1-9
Jk 1-5
Cl1 Jl1
Cl2 Jl2
Cm
Ck
Cl3 Jl3
Cl4 Jl4
Ig light chain gene rearrangement by somatic
recombination
Vk
Germline
Rearranged
1° transcript
Spliced mRNA
Jk
Ck
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?
Remember
• Cell surface antigen receptor on B cells
Allows B cells to sense their antigenic environment
Connects extracellular space with intracellular signalling machinery
• Secreted antibody
Neutralisation
Arming/recruiting effector cells
Complement fixation
How does the model of recombination allow for
two different forms of the protein?
The constant region has additional, optional exons
Cm
Primary transcript RNA
AAAAA
Each H chain domain (& the
hinge) encoded by separate
exons
Cm1
h
Cm2
Secretion
coding
sequence
Cm3
Polyadenylation
site (secreted)
pAs
Polyadenylation
site (membrane)
pAm
Cm4
Membrane
coding
sequence
Membrane IgM constant region
DNA
Cm1
h
Cm2
Cm3
Cm4
Transcription
1° transcript
pAm
Cm1
Cleavage &
polyadenylation at pAm
and RNA splicing
mRNA
h
Cm2
Cm3
Cm1 h Cm2 Cm3 Cm4
Cm4
AAAAA
AAAAA
Membrane coding
sequence encodes
transmembrane region
that retains IgM in the cell
membrane
Protein
Fc
Secreted IgM constant region
DNA
Cm1
h
Cm2
Cm3
Cm4
Transcription
1° transcript
pAs
Cm1
h
Cm2
Cm3
Cm4
AAAAA
Cleavage polyadenylation
at pAs and RNA splicing
mRNA
Cm1 h Cm2 Cm3 Cm4
Protein
AAAAA
Secretion coding
sequence encodes the C
terminus of soluble,
secreted IgM
Fc
Why do V regions not join to J or C regions?
VH
DH
JH
C
IF the elements of Ig did not assemble in the correct order, diversity of specificity
would be severely compromised
2x
DIVERSITY
Full potential of the H
chain for diversity needs
V-D-J-C joining - in the
correct order
1x
DIVERSITY
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
Rearrangement of V, D, and
J gene segments is guided
by flanking DNA sequences
V, D, J flanking sequences
Sequencing up and down stream of V, D and J elements
Conserved sequences of 7, 23, 9 and 12 nucleotides in an arrangement that
depended upon the locus
Vl
7
Vk
7
23
12
7
23
9
12
9
7
12
9
9
9
VH
9
D
23
7
12
9
7
Jl
7
Jk
9
23
7
JH
Recombination signal sequences (RSS)
HEPTAMER - Always contiguous with
coding sequence
9
VH 7
23
√
VH
9
7
12
23
7
D
9
9
12
9
9
12
7
7
D
NONAMER - Separated from
the heptamer by a 12 or 23
nucleotide spacer
23
7
12
9
7 JH
√
9
23
7
JH
12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment
flanked by a 12mer RSS
Molecular explanation of the 12-23 rule
12-mer = one turn
23-mer = two turns
23
V7
Intervening DNA
of any length
9
12
9
7D J
Molecular explanation of the 12-23 rule
V4
V1
V8
V9
V2
V7
V3
V6
V3
V4
V2
V5
9
9
23-mer
• Heptamers and nonamers
align back-to-back
7
7
V7
V8
V9
12-mer
V1
V6
Loop of
intervening
DNA is
excised
D J
• The shape generated by the
RSS’s acts as a target for
recombinases
V5
DJ
• An appropriate shape can not be formed if two 23-mer flanked elements
attempted to join (i.e. the 12-23 rule)
Steps of Ig gene recombination
V
7 23
V
7 23
D J
9 12 7
7 23
9 12 7
9
7 23
D J
The two RAG1/RAG 2 complexes
bind to each other and bring the V
region adjacent to the DJ region
9
9 12 7 9 12 7
V
9
Recombination activating
gene products, (RAG1 & RAG
2) and ‘high mobility group
proteins’ bind to the RSS
9
• 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
D J
Steps of Ig gene recombination
V
7
23
9
D J
9 12 7
V D J
D
J
9 12 7 7 23 9
V
A number of other proteins, (Ku70:Ku80,
XRCC4 and DNA dependent protein
kinases) bind to the hairpins and the
heptamer ends.
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
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.
Junctional diversity: P nucleotide additions
7 23
V
AT GTGACAC
J D TA CACTGTG
9
9 12 7
V
TC CACAGTG
AG GTGTCAC
7
7
9
23
12
9
The recombinase complex makes single
stranded nicks at random sites close to the
ends of the V and D region DNA.
TC
AG
TC CACAGTG
AG GTGTCAC
7
GTGACAC
CACTGTG
7
V V
AT
AT
J JDTA DTA
9
23
12
9
The 2nd strand is cleaved and hairpins form between
the complimentary bases at ends of the V and D
region.
D J
V3
V2
V4
CACAGTG
GTGTCAC
7
GTGACAC
CACTGTG
7
9
23
12
V5
9
V9
Heptamers are ligated by
DNA ligase IV
TC
AG
V
AT
J DTA
V
V8
V6
V7
TC
AG
V and D regions juxtaposed
AT
TA
D J
Generation of the palindromic sequence
V
V
V
TC
AG
TC
AG
TC~GA
AG
AT
TA
D J
AT
TA
D J
Regions to be joined are juxtaposed
Endonuclease cleaves single strand at
random sites in V and D segment
The nicked strand ‘flips’ out
AT
TA~TA
D J
The nucleotides that flip out, become
part of the complementary DNA strand
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.
Junctional Diversity – N nucleotide additions
V
TC~GA CACTCCTTA
AT
AG
TTCTTGCAA
TA~TA
D J
Terminal deoxynucleotidyl transferase
(TdT) adds nucleotides randomly to
the P nucleotide ends of the singlestranded V and D segment DNA
V
TC~GA CACACCTTA
AT
AG
TTCTTGCAA TA~TA
D J
Complementary bases anneal
V
TC~GACACACCTTA
D J
Exonucleases nibble back free ends
V
TC
CACACCTTA
TC~GA
GTT ATAT
AT
AGC
TTCTTGCAA
TA
TA~TA
AG
D J
DNA polymerases fill in the gaps
with complementary nucleotides
and DNA ligase IV joins the strands
TTCTTGCAA TA~TA
Junctional Diversity
V
TCGACGTTATAT
AGCTGCAATATA D
J
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?
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
3.
How do V region find J regions and why don’t they join to C
regions?
The 12-23 rule
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
Junctional diversity
Mini-circle of DNA is
permanently lost from the
genome
9
7
V
7
12
23
9
9
23
Coding joint
7
7
12
9
Signal joint
DJ
VDJ
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.
Non-deletional recombination
V1
V1
V1
V3
V2
7
V2
V3
9
23
V4
23
V9
9
V4
7
D J
V9
V4
Looping out works if all V
genes are in the same
transcriptional orientation
9
D J
12
7
D J
How does recombination occur
when a V gene is in opposite
orientation to the DJ region?
9
12
7
D J
Non-deletional recombination
9
23
7 V4
9 12 7 D J
1.
2.
9
7 V4
23
9
3.
V4 and DJ in opposite
transcriptional orientations
23
9
23
7 V4
7 V4
4.
9
23
7 V4
9 12 7 D J
2.
1.
9
9
23
7
12
9
23
9
V4
12
7
7
V4
D J
Heptamer ligation - signal
joint formation
7 D J
3.
V4
9
23
9
4.
9
12
23
7
7
D J
7
7
12
9
V to DJ ligation coding joint
formation
V4 D J
Fully recombined VDJ regions in same transcriptional orientation
No DNA is deleted
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
CDR1 FR2 CDR2
FR3
CDR3
FR4
100
Variability
80
60
40
20
20
40
60
80
100
120
Amino acid No.
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?
Somatic hypermutation leads to affinity maturation
Day 6
Day 8
Day 12
Day 18
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
Clone 1
Clone 2
Clone 3
Clone 4
Clone 5
Clone 6
Clone 7
Clone 8
Clone 9
Clone 10
Cells with
accumulated
mutations in
the CDR are
selected for
high antigen
binding
capacity –
thus the
affinity
matures
throughout
the course of
the response
Deleterious mutation Lower affinity - Not clonally selected
Beneficial mutation Higher affinity - Clonally selected
Neutral mutation
Identical affinity - No influence on clonal selection
Hypermutation is T cell dependent
Mutations focussed on ‘hot spots’ (i.e. the CDRs) due to double stranded breaks
repaired by an error prone DNA repair enzyme.
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.
Organisation of the functional human heavy chain C region genes
J regions
Cm
Cd
Cg3
Cg1
Ca1
Cg2
Cg4
Ce
Ca2
Switch regions
Cm
Sm
Cd
Cg3
Sg3
Cg1
Sg1
Ca1
Sa1
Cg2
Sg2
Cg4
Sg4
Ce
Se
Ca2
Sa2
• Upstream of C regions are repetitive regions of DNA called switch
regions. (The exception is the Cd region that has no switch region).
• 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.
Switch recombination
Cm
Cd
Cg3
Cg1
Ca1
Cg2
Cg4
Cd
Ce
Cd
Ca2
Sg3
Cg3
Cg3
Cm
Sg1
Cm
Cg1
VDJ
Cg3
VDJ
Ca1
VDJ
Ca1
VDJ
Cg3
VDJ
Ca1
VDJ
Ca1
IgG3 produced.
Switch from IgM
IgA1 produced.
Switch from IgG3
IgA1 produced.
Switch from IgM
At each recombination constant regions are deleted from the genome
An 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