Transcript Document

GENETIC BACKGROUND OF
ANTIBODY DIVERSITY
STRUCTURE OF
IMMUNOGLOBULINS/ANTIBODIES
Heavy chain (H)
VH
VL
CH
Light chain (L)
CL
Antigen
Antigen
binding
antigénkötés
s
VL
s
s
s
s
s
s
ss
ss
Constans
konstadomains
ns dom ének
CH2 ss
s
s
CH3 ss
s
s
BINDING TO CELLS
DEGRADATION
TRANSPORT
effektor funkc iók
Effector functions
s
s
s
CL
s
COMPLEMENT ACTIVATION
s
CH1
s
va riábilis
d om ének
Variable
domains
s
s
S
s
S
s
s
s
s
VH
AMINO ACID SEQUENCE OF IMMUNOGLOBULINS
Multiple myeloma (MM)
Plasma cell tumors – tumor cells reside in the bone marrow
Produce immunoglobulins of monoclonal origin, serum concentration 50-100mg/ml
Rodney Porter & Gerald Edelman 1959 – 1960 myeloma protein purification
Gel electrophoresis
L
H
Reduction
50 kDa
Heavy chain
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
25 kDa
Light chain
Variable
Constant
GENETIC BACKGROUND OF ANTIBODY DIVERSITY
VH
VH
VL
VL
S–S
S–S
Mechanism of the generation of variability?
Different rules for encoding the variable and constant regions?
Symmetric molecule  two identical VH and VL  both chromosomes encode
for the same sequence?
DOGMA OF MOLECULAR BIOLOGY
CHARACTERISTICS OF IMMUNOGLOBULIN SEQUENCE
1 GEN = 1 PROTEIN
THEORIES
1 GEN
Gen
High rate of somatic mutations in the V-region
V
C
Many GENES (10 000 – 100 000)
V1 C
Protein
V2 C
Vn C
MOLECULAR GENETICS OF IMMUNOGLOUBLINS
How can the bifunctional nature of antibodies be explained genetically?
In 1965, Dreyer & Bennett proposed that for
a single isotype of antibody there may be:
• 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.
This was genetic heresy as it violated the then accepted
notion that DNA was identical in every cell of an
individual
Proof of the Dreyer - Bennett hypothesis
V
V
V
V
V
V
V
V
V
V
V
V C
V
V
A single C region gene is
encoded in the germline
and separated from the
multiple V region genes
C
A mechanism to rearrange V and C
genes in the genome exists so that
they can fuse to form a complete
Immunoglobulin gene
Find a way to show the existence of multiple V genes and
rearrangement to the C gene
Approach
V
V
V
V
V
V
V
V
V
V
V
V C
V
Germline DNA
C
Rearranged DNA
V
Tools:
• A set of cDNA probes to specifically distinguish V regions from C regions
• DNA restriction enzymes to fragment DNA
• Examples of germline (e.g. placenta) and mature B cell DNA (e.g. a
plasmacytoma/myeloma)
The experiment of Susumi Tonegawa 1976
*
*
V-CmRNS probe
CmRNS probe
*
*
V
C
Embryonal cell
*
*
V
C
B-cell
CONCLUSION
V and C genes get close to each other in B-cells only
V
V
V
C
B-CELL
There are many variable genes but only one constant gene
V
V
V
V
C
GERM LINE
Gén
Fehérje
GÉN SZEGMENSEK SZOMATIKUS
ÁTRENDEZŐDÉSE EGY GÉNNÉ
Ig gene sequencing complicated the model
The 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
LV
L
CL
~ 208aa
Where do the extra 13
amino acids come from?
LV
L
~ 95aa
JL
CL
~ 100aa
Some of the extra amino
acids are provided by
one of a small set of J or
JOINING regions
SOMATIC REARRANGEMENT OF KAPPA (κ) CHAIN GENE
SEGMENTS
Vκ Jκ
B-cell 2
Vκ
Vκ
40 Vκ
Vκ
Vκ
5 Jκ
Germ line
Jκ Jκ Jκ Jκ
During B-lymphocyte
development
Vκ
B-cell 1
DNA
Vκ
Vκ
Jk Jκ Jκ Jκ
EXPRESSION OF THE KAPPA CHAIN
Vκ
P
Vκ J
pA
J
E
Cκ
J
E
Cκ
Vκ-Jκ
Leader
Vκ J
Primary RNA transcript
Vκ J
Cκ
AAAA
mRNA
Translation
Vκ J
Cκ
Protein
Efficiency of somatic gene rearrangement?
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
Non-productive rearrangement
Light chain has a second chance to make
a productive join using new V and J elements
Spliced mRNA transcript
Ck
Further diversity in the Ig heavy chain
L VH DH JH
CH
The heavy chain was found to have further amino acids (0 – 8)
between the JH és CH genes
D (DIVERSITY) region
Each heavy chain requires 3 recombination events
JH to DH , VH to JHDH, and VHJHDH to CH
L VL
JL
CL
Each light chain requires 2 recombination events
VL to JL and VLJL to CL
SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE
SEGMENTS
120 VH
VH1
VH2
12 D
VH3
D
D
D
4 JH
D
JH JH JH JH
During B-cell development
VH1
VH2
VH3
VH1
D
D JH JH
VH2
D
D JH JH
IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE
GENE SEGMENTS
ORGANIZATION OF IMMUNOGLOBULIN GENE SEGMENTS
Chromosome 2
kappa light chain gene segments
Chromosome 22
lambda light chain gene segments
Chromosome 14
heavy chain gene segments
HOW MANY IMMUNOGLOBULIN GENE SEGMENTS
Gene segments
Light chain
Heavy chain
kappa
lambda
Variable (V)
132/40
105/30
123/65
Diversity (D)
0
0
27
Joining (J)
5
4
9
The key experiment of Nobumichi Hozumi and Susumu Tonegawa