generation of diversity · 2013. 6. 25. · different protein sequences lead to antibody diversity...

Immunoglobulins

Generation of Diversity

Introduction• Immunologist estimate that each person has

the ability to produce a range of individual

antibodies capable of binding to a total of well

over 1010 epitopes

• According to the germline theory, a unique

gene encodes each antibody

• Unfortunately, for this theory to be true the

number of antibody genes would need to be

100-1000-fold greater than the entire human

genome

Theories

• An alternative theory, the somatic mutation theory, holds that a single germline immunoglobulin gene undergoes multiple mutations that generate immunoglobulin diversity. This scheme, however, requires an unimaginable mutation rate

• The immune system has developed a much more elegant solution- the chromosomal rearrangement of separate gene segments, which employs some elements of the germlineand somatic mutation theories

Gene Rearrangement

• Each light and heavy chain is encoded by a series of

genes occurring in clusters along the chromosome

• In humans, the series of genes encoding the k light

chain, λ light chain, and the heavy chain are located

on chromosomes 2, 22, and 14 respectively

• When a cell becomes committed to the B lymphocyte

lineage, it rearranges the DNA, encoding its light and

heavy chains by cutting and splicing together some of

the DNA sequences, thus modifying the sequence of

the variable region gene

Tonegawa’s demonstration• 1976—used restriction enzymes and DNA probes to show that

germ cell DNA contained several smaller DNA segments compared to DNA taken from developed lymphocytes (myeloma cells)

Hk l

1 gene 1 transcript 1 protein

Antibody specificities more than 1,000,000,000,000

Human genome about 30,000 genes

Human Antibody genesH: chromosome 14 k: chromosome 2 l: chromosome 22

VH1 VH65VH2 DH1-------27 JH1-----6 Cg

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 up to 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:

JH to DH, JHDH to VH and JHDH VHto 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

• 132 Vk genes on the short arm of chromosome 2

• 29 functional Vk genes with products identified

• 87 pseudo Vk genes

• 16 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

22

• 30 functional genes with products identified

• 56 pseudogenes

• 6 functional genes - with no products identified

• 13 relics (<200bp of Vl sequence)

• 25 orphans on the long arm of chromosome 22

• 4 Jl regions

Vl & Jl Loci:

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

GGGACAGGGGGCGlyGlnGly

GGGACAGGGGGCAspArgGly

Analysis of D regions from different antibodies

One D region can be used in any of three frames

Different protein sequences lead to antibody diversity

Frame 1

Frame 2

Frame 3

Estimates of combinatorial diversity

Using functional V, D and J genes:

40 VH x 27 DH x 6JH = 5,520 combinations

D can be read in 3 frames: 5,520 x 3 = 16,560 combinations

29 Vk x 5 Jk = 145 combinations

30 Vl x 4 Jl = 120 combinations

= 265 different light chains

If H and L chains pair randomly as H2L2 i.e.

16,560 x 265 = 4,388,400 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 genes

JUNCTIONAL 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?




Mathematically, Combinatorial Diversity can

account for some diversity – how do the elements

rearrange?

Genomic organisation of Ig genes(Numbers include pseudogenes etc.)

DH1-27 JH 1-9 Cm

LH1-123

VH 1-123

Lk1-132

Vk1-132 Jk 1-5 Ck

Ll1-105

Vl1-105 Cl1 Jl1 Cl2 Jl2 Cl3 Jl3 Cl4 Jl4

Ig light chain gene rearrangement by somatic

recombination

Germline

Vk Jk Ck

Spliced mRNA

Rearranged

1° 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

Vk Jk Ck

Non-productive

rearrangement

Spliced mRNA transcript

Light chain has a second chance

to make a productive join using

new V and J elements

Ig heavy chain gene rearrangement

DH1-27 JH 1-9 CmVH 1-123

Somatic recombination occurs at the level of DNA

which can now be transcribed

Cm1 Cm2 Cm3 Cm4

pAs

AAAAAhJ8 J9DV

Primary transcript RNA

Cm1 Cm2 Cm3 Cm4 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?








Combinatorial Diversity and genomic organisation

can account for some diversity

• Cell surface antigen receptor on B cells

Allows B cells to sense their antigenic environment

Connects extracellular space with intracellular signalling

machinery

• Secreted antibody functions

Neutralisation

Arming/recruiting effector cells

Complement fixation

Remember These Facts?

How does the model of recombination allow for

two different forms of the same 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

Secretion

coding

sequence

Membrane

coding

sequence

mRNACm1 Cm2 Cm3 Cm4 AAAAAh

Transcription

Membrane IgM constant region

Cm1 Cm2 Cm3 Cm41° transcript

pAm

AAAAAh

Cm1 Cm2 Cm3 Cm4DNA h

Membrane coding

sequence encodes

transmembrane region

that 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° transcript

pAs

Cm1 Cm2 Cm3 Cm4

Transcription

AAAAAh

Secretion coding

sequence encodes the

C terminus of soluble,

secreted IgM

Fc

Protein

Alternative RNA processing generates transmembrane or secreted Ig

Secreted & membrane forms of the heavy chain by alternative ( differential ) RNA processing of primary transcript.

(a)

Synthesis, assembly, and secretion of the immunoglobulin molecule.

Problems?







accounts for some diversity



Use of alternate polyadenylation sites

V, D, J flanking sequences

Vl 7 23 9

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

Vk 7 12 9 Jk7239

Jl7129

D7129 7 12 9

VH7 23 9 JH

7239

Gene rearrangements are made at recombination signal

sequences (RSS). RSSs are heptamer-nonamer sequences

Each RSS contains a conserved heptamer, a conserved nonamer and a

spacer of either 12 or 23 base pairs.

Generic light chain locus

Generic heavy chain locus

There is a RSS downstream of every V gene segment,

upstream of every J gene segment and flanking every D gene

segment

V JD

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 sequenceNONAMER - Separated from

the heptamer by a 12 or 23

nucleotide spacer

VH7 23 9

D7129 7 12 9

JH7239

√ √

1. Rearrangements only occur between segments on the same

chromosome.

2. A heptamer must pair with a complementary heptamer; a nonamer

must pair with a complementary nonamer.

3. One of the RSSs must have a spacer with 12 base pairs and the

other must be 23 base pairs (the 12/23 rule).

- RSS having a one-turn spacer can join only with RSS having a two-turn spacer

: one-turn / two-turn joining rule

- This ensures that V,D,J segments join in proper order & that segments of the

same type do not join each other.

- The enzymes recognizing RSS : recombination-activating genes.

( RAG-1, -2), lymphoid-specific gene products

23-mer = two turns 12-mer = one turn

Molecular explanation of the 12-23 rule

Intervening DNA

of 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

V47239

1.

V47239

3.

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 J

7129

V47239

2.

Heptamer ligation - signal

joint formation

D JV47 12 97239

Fully recombined VDJ regions in same transcriptional orientation

No DNA is deleted

4.

Problems?



The 12-23 rule




accounts for some diversity


the cell surface and secreted?Use of alternative polyadenylation sites


V 7 23 9

D7 12 9J

V 7 23 9

7 23 9

7 12 9

D7129 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 9

TC CACAGTG

AG GTGTCAC

AT GTGACAC

TA 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 9CACAGTG

GTGTCAC

GTGACAC

CACTGTG

TC

AG

AT

TADJ

VTC

AG

AT

TA

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 9CACAGTG

GTGTCAC

7 12 9GTGACAC

CACTGTG

VTC

AG

DJAT

TA

Heptamers are ligated by

DNA ligase IV

V and D regions juxtaposed

VTC

AGD J

AT

TA

VTC

AG D JAT

TA

Endonuclease cleaves single

strand at random sites in V and D

segment

VTC~GA

AG 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.

VTC

AGD J

AT

TA Regions to be joined are juxtaposed

The nicked strand ‘flips’ out

(Palindrome - A Santa at NASA)

Junctional Diversity – N nucleotide additions

VTC~GA

AG 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

VTC~GA

AG D JAT

TA~TA

CACACCTTA

TTCTTGCAAComplementary bases anneal

V D JDNA polymerases fill in the

gaps with complementary

nucleotides and DNA ligase IV

joins the strands

TC~GA

AGAT

TA~TA

CACACCTTA

TTCTTGCAA

D JTA~TAExonucleases nibble back free endsV

TC~GACACACCTTA

TTCTTGCAA

VTC

DTA

GTT AT AT

AG C

P-nucleotide and N-nucleotide addition during joining.

Generation of Antibody Diversity

P and N region nucleotide alteration

adds to diversity of V region

• During recombination some nucleotide bases are cut

from or add to the coding regions (p nucleotides)

• Up to 15 or so randomly inserted nucleotide bases are

added at the cut sites of the V, D and J regions (n

nucleotides_

• TdT (terminal deoxynucleotidyl transferase) a unique

enzyme found only in lymphocytes

• Since these bases are random, the amino acid

sequence generated by these bases will also be

random

V D JTCGACGTTATATAGCTGCAATATA

Junctional Diversity

TTTTT

TTTTT

TTTTT

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?



The 12-23 rule



Combinatorial Diversity, genomic organisation

and Junctional Diversity



Use of alternative polyadenylation sites


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

Additional Degrees of Variation

• Somatic hypermutation: Stimulated memory B

cells accumulate small mutations on the VL or

VH leading to affinity maturation to antigens that

are frequently or chronically present

• Isotype switching

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 1

Clone 2

Clone 3

Clone 4

Clone 5

Clone 6

Clone 7

Clone 8

Clone 9

Clone 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 selected

Higher affinity - Clonally selectedIdentical affinity - No influence on clonal selection

Somatic hypermutation leads to affinity maturation

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.

Cells with

accumulated

mutations in

the CDR are

selected for

high antigen

binding

capacity –

thus the

affinity

matures

throughout

the course of

the response

Allelic Exclusion• A single B cell can express only one VL and one VH

allele to the exclusion of all others

• Both must be on the same member of the chromosome pair-either maternal or paternal

• The restriction of VL and VH expression to a single member of the chromosome pair is termed allelic exclusion

• The presence of both maternal and paternal allotypes in the serum reflects the expression of different alleles by different population of B cells

Allelic exclusion: only one chromosome is

active in any one lymphocyte

Model to account for allelic exclusion:If one allele arranges nonproductively, a B cell still can rearrange the other allele productively; once a productive rearrangement( 33%) have occurred, the recombination machinery is turned off. ( the protein product acts as a signal to prevent further gene rearrangement)

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).

7 means of generating antibody

diversity

Generation of Antibody Diversity

• Germ line diversity.

• Combinatorial diversity.

• Junctional diversity.

• Somatic hypermutation ( affinity maturation)

generation of diversity · 2013. 6. 25. · different protein sequences lead to antibody diversity...

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