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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
DEGRADATION
TRANSPORT
s
ss
ss
Constans
domains
konsta
ns dom ének
effektor funkc iók
Effector functions
CH2 ss
s
s
CH3 ss
s
s
s
BINDING TO CELLS
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
VL
S–S
VH
VL
S–S
Mechanism of the generation of variability?
Different rules for encoding the variable and constant regions?
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 – 30 000)
V1 C1
Protein
V2 C2
Vn Cn
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
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-CmRNA probe
CmRNA probe
*
*
V
C
Embryonal cell
*
*
V
C
B-cell
The key experiment of Nobumichi Hozumi and Susumu Tonegawa
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
GENE
PROTEIN
REARRANGEMENT OF GENE
SEGMENTS INTO A SINGLE
FUNCTIONAL UNIT (GENE)
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
5 Jκ
40 Vκ
Vκ
Vκ
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?
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 2 recombination events
JH to DH , VH to JHDH,
L VL
JL
CL
Each light chain requires 1 recombination events
VL to JL
SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS
65 VH
VH1
VH2
27 D
VH3
D
D
D
6 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
kappa
Heavy chain
lambda
Variable (V)
40
30
65
Diversity (D)
0
0
27
Joining (J)
5
4
6
VARIABILITY OF B-CELL ANTIGEN RECEPTORS AND ANTIBODIES
B cells of one individual
2
3
1
4
V-Domains
C-Domains
VH
D
JH
VL
VH-D-JH
JL
VL-JL
Estimates of combinatorial diversity
Taking account of functional V D and J genes:
65 VH x 27 DH x 6JH = 10,530 combinations
40 Vk x 5 Jk = 200combinations
30 Vl x 4 Jl = 120 combinations
= 320 different light chains
If H and L chains pair randomly as H2L2 i.e.
10,530x 320 = 3,369,600 possibilities
Due only to COMBINATORIAL diversity
In practice, some H + L combinations do not occur as they are unstable
Certain V and J genes are also used more frequently than others.
There are other mechanisms that add diversity at the junctions between genes
- JUNCTIONAL diversity
GENERATES A POTENTIAL B-CELL REPERTOIRE
Somatic recombination to generate
antibody diversity
Severe combined immunodeficiency (SCID)
Omenn syndrome - RAG deficiency
Lack of T-cells and B cells
Early manifestation
red rash on the face and shoulders,
infections with opportunistic pathogens. (Candida albicans, Pneumocystis carnii
pneumonia)
Lack of palpable lymph nodes
How does somatic gene rearrangement
(recombination) work?
1.
How is an infinite diversity of specificity generated from finite amounts of
DNA?
Combinatorial diversity
2.
How do V region find J regions and why don’t they join to C regions?
12-23 rule
-Special - Recobnitation Signal Sequences (RSS)
- Recognized by Recombination Activation Gene
coded proteins (RAGs)
PALINDROMIC SEQUENCES
HEPTAMER
NONAMER
CACAGTG
GTGACAC
ACAAAAACC
TGTTTTTGG
CACAGTG
GTGACAC
GGTTTTTGT
CCAAAAACA
V, D, J flanking sequences
Sequencing upstream and downstream of V, D and J elements revealed
conserved sequences of 7, 23, 9 and 12 nucleotides in an arrangement
that depended upon the locus
Vl
7
Vk
7
23
12
7
23
9
12
9
7
12
9
9
9
VH
9
D
23
7
12
9
7
Jl
7
Jk
9
23
7
JH
Recombination signal sequences (RSS)
HEPTAMER - Always contiguous
with coding sequence
9
VH 7
23
9
VH
7
12
23
7
D 7
9
9
12
9
9
12
7
D
NONAMER - Separated from
the heptamer by a 12 or 23
nucleotide spacer
7 JH
23
7
12
9
9
23
7
JH
12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked
to a segment flanked by a 12mer RSS
Molecular explanation of the 12-23 rule
12-mer = one turn
23-mer = two turns
23
V7
Intervening DNA
of any length
9
12
9
7D J
Molecular explanation of the 12-23 rule
V4
V1
V8
V9
V3
V2
V7
V6
V3
V4
V2
V5
9
9
23-mer
• Heptamers and nonamers
align back-to-back
V6
Loop of
intervening
DNA is
excised
DJ
V7
V8
V9
V1
7
12-mer
7
• The shape generated by the
RSS’s acts as a target for
recombinases
V5
DJ
• An appropriate shape can not be formed if two 23-mer flanked elements
attempted to join (i.e. the 12-23 rule)
CONSEQUENCES OF RECOMBINATION
Generation of P-nucleotides
V4
V5
V3
V6
V2
9
9
23-mer
7
V1
7
12-mer
DJ
V7
V8
V9
Generation of N-nucleotides
V4
Terminal
deoxynucleotidyl
Transferase (TdT)
V3
V2
9
9
23-mer
7
7
12-mer
V1
V5
DJ
V6
Loop of
intervening
DNA is
excised
V7
V8
V9
Junctional Diversity
V
TCGACGTTATAT
AGCTGCAATATA D
J
TTTTT Germline-encoded nucleotides
TTTTT Palindromic (P) nucleotides - not in the germline
(N) encoded nucleotides - not
TTTTT Non-template
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
How does somatic gene rearrangement
(recombination) work?
1.
How is an infinite diversity of specificity generated from finite amounts of
DNA?
Combinatorial diversity
2.
How do V region find J regions and why don’t they join to C regions?
12-23 rule
3.
How does the DNA break and rejoin?
Imprecisely, with the random removal and addition of nucleotides to
generate sequence diversity
Junctional diversity (P- and N- nucleotides, see above)