The Molecular Genetics Of Immunoglobulins
Transcript of The Molecular Genetics Of Immunoglobulins
The Molecular Genetics of Immunoglobulins
©Dr. Colin R.A. Hewitt
The molecular genetics of immunoglobulins
• 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.
Dreyer & Bennett (1965)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
Genetic models of the 1960’s were also unable to explain:
• How B cells shut down the Ig genes on just one of their chromosomes.
All other genes known at the time were expressed co-dominantly. B cells expressed a light chain from one parent only and a heavy chain from one parent only (evidence from allotypes).
• A genetic mechanism to account for increased antibody affinity in an immune response
• How a single specificity of antibody sequentially switched isotype.
• How the same specificity of antibody was secreted and simultaneously expressed on the cell surface of a B cell.
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 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
Extra amino acids provided by one of a
small set of J or JOINING regions
L
CLVL
~ 208
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
Problems?
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?
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
Reading D segment in 3 frames
GGGACAGGGGGCGlyThrGlyGly
GGGACAGGGGGC GlyGlnGly
GGGACAGGGGGC AspArgGly
Analysis of D regions from different antibodiesOne D region can be used in any of three frames
Different protein sequences lead to antibody diversity
Frame 1
Frame 2
Frame 3
Diversity: Multiple germline genes
• 132 V genes on the short arm of chromosome 2• 29 functional V genes with products identified• 87 pseudo V genes• 15 functional V genes - with no products identified• 25 orphans V genes on the long arm of chromosome 2• 5 J regions
V & J Loci:
• 105 V 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 V of sequence)• 25 orphans on the long arm of chromosome 2• 4 J regions
V & J Loci:
Estimates of combinatorial diversity
Using functional V D and J genes:
40 VH x 27 DH x 6JH = 6,480 combinationsD can be read in 3 frames: 6,480 x 3 = 19,440 combinations
29 Vx 5 J = 145 combinations30 Vx 4 J = 120 combinations
= 265 different light chains
If H and L chains pair randomly as H2L2 i.e. 19,440 x 265 = 5,151,600 possibilities Due only to COMBINATORIAL diversity
In practice, some H + L combinations are unstable.Certain V and J genes are also used more frequently than others.
Other mechanisms add diversity at the junctions between genesJUNCTIONAL diversity
Problems?
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?
1. How is an infinite diversity of specificity generated from finite amounts of DNA?Mathematically, Combinatorial Diversity can account for some diversity – how do the elements rearrange?
Genomic organisation of Ig genes(No.s include pseudogenes etc.)
DH1-27 JH 1-9 CLH1-123VH 1-123
L1-132V1-132 J 1-5 C
L1-105V1-105 C1 J1 C2 J2 C3 J3 C4 J4
Ig light chain gene rearrangement by somatic recombination
Germline
V J C
SplicedmRNA
Rearranged1° transcript
Ig light chain rearrangement: Rescue pathway
There is only a 1:3 chance of the join between the V and J region being in frame
V J C
Non-productive rearrangement
Spliced mRNA transcript
Light chain has a second chance to makea productive join using new V and J elements
Ig heavy chain gene rearrangement
DH1-27 JH 1-9 CVH 1-123
Somatic recombination occurs at the level of DNA which can now be transcribed
BUT:
Problems?
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?
1. How is an infinite diversity of specificity generated from finite amounts of DNA?Combinatorial Diversity and genomic organisation can account for some diversity
•Cell surface antigen receptor on B cellsAllows B cells to sense their antigenic environment
Connects extracellular space with intracellular signalling
machinery
•Secreted antibodyNeutralisation
Arming/recruiting effector cells
Complement fixation
Remember Lecture 1, Slide 1?
How does the model of recombination allow fortwo different forms of the protein?
Primary transcript RNA AAAAA
C
Polyadenylation site (secreted)
pAs
Polyadenylation site (membrane)
pAm
The constant region has additional, optional exons
C1 C2 C3 C4
Each H chain domain (& the hinge) encoded by
separate exons
h
Secretioncoding
sequence
Membranecoding
sequence
mRNAC1 C2 C3 C4 AAAAAh
Transcription
Membrane IgM constant region
C1 C2 C3 C41° transcriptpAm
AAAAAh
C1 C2 C3 C4DNA 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
C1 C2 C3 C4 AAAAAh
C1 C2 C3 C4DNA h
Cleavage polyadenylation at pAs and RNA splicing
1° transcriptpAs
C1 C2 C3 C4
Transcription
AAAAAh
Secretion coding sequence encodes the C terminus of soluble,
secreted IgM
Fc
Protein
Primary transcript RNA AAAAA
C
Polyadenylation site (secreted)
pAs
Polyadenylation site (membrane)
pAm
The constant region has additional, optional exons
C1 C2 C3 C4
Each H chain domain (& the hinge) encoded by
separate exons
h
Secretioncoding
sequence
Membranecoding
sequence
C1 C2 C3 C4pAs
AAAAAhJ8 J9DV
Primary transcript RNA
C1 C2 C3 C4 AAAAAhJ8DVmRNA
The Heavy chain mRNA is completed by splicing the VDJ region to the C region
RNA processing
VL JL CL AAAAA
CH AAAAAhJHDHVH
The H and L chain mRNA are now ready for translation
Problems?
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?
1. How is an infinite diversity of specificity generated from finite amounts of DNA?Combinatorial Diversity and genomic organisation accounts for some diversity
2. How can the same specificity of antibody be on the cell surface and secreted?Use of alternate polyadenylation sites
V, D, J flanking sequences
V 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
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
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 J7129V47239V4 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
7129
7239
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.
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 and genomic organisation accounts for some 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?
V 7 23 9
D7 12 9J
V 7 23 9
7 23 9
7 12 9D7129 J
7 23 9
7 12 9
V
DJRecombination 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 72
39
71
29
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
(Palindrome - A Santa at NASA)
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
CACTCCTTA
TTCTTGCAA
V TC~GAAG D JAT
TA~TA
CACACCTTA
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
CACACCTTA
TTCTTGCAA
D JTA~TAExonucleases nibble back free endsV TC~GACACACCTTA
TTCTTGCAA
V TCDTA
GTT AT AT
AG C
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.
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
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
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 C2CC4C2C1C1C3CC
Organisation of the functional human heavy chain C region genes
C2CC4C2C1C1C3CC
Switch regions
• The S 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.
S3 S1 S1 S2 S4 S S2S
• Upstream of C regions are repetitive regions of DNA called switch regions. (The exception is the C region that has no switch region).
C2CC4C2C1C1C3CC
C
C
C3VDJ
S3
C
C
C3
VDJ
C1
S1
C1
C3
VDJ C1
C3VDJ
IgG3 produced.Switch from IgM
VDJ C1
IgA1 produced.Switch from IgG3
VDJ C1
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