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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
How can antibody spcificity and diversity be explained?
Paul Ehrlich: side chain theory (1897)
There are receptors for nutrients. There may be receptors (side chains) for
foreign entities.
BUT: the diversity is huge, hard to explain at that time
What about pre-existing antibodies?
Antibody is not produced by the host
Information of the large antibody repertoire comes from the antigen itself.
Direct: antibody comes from the antigen… BUT antibody level does not decline
together with that of the antigen!!
Instruction Theories:
Antigen is produced by the host but the antigen teaches antibody to fold properly
or it is synthesized on the antigen
Problem: antibody is also produced by the descendents of the activated B cells
1950: Protein is created on nucleic acid template, but the paradox of coding for
such diversity remained a mystery
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
Variábilis
Constans
MOLECULAR GENETICS OF IMMUNOGLOUBLINS
GERMLINE VARIATION OR SOMATIC MUTATION?
How can the diversity of antibodies be explained genetically?
In 1965, William Dreyer & Claude Bennett
proposed that :
• 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
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
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)
Experiment of Susumu Tonegawa 1976 Basel
DNA-extraction
Digestion by restriction enzyme
Gel electrophoresis
Southern blot
Kb
C probe V
C probe V
Liver cell 6,0
4,0
1,5
V
C
V-probe
6,0
4,0
1,5
C-probe
VC
6.0 Kb
4.0 Kb
V
C
1.5. Kb
V
C
B-cell
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
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