Post on 24-Feb-2016
description
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
1 in 3 in phase VL to VJ
1 in 3 in phase VH to DHJH
Only 11% of cells mature and leave
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.
Promoter, Enhancer and Silencer
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