Download 14 Diversity of BCR BA

GENETIC BACKGROUND
OF THE VARIABILITY OF ANTIGEN
RECOGNIZING RECEPTORS
Genetic background of antibody diversity
The total number of antibody specificities available to an individual is known as the
antibody repertoire, and in humans is at least 1011, perhaps many more.
BUT
There are an estimated only 20,000-25,000 human protein-coding genes.
Dogma of molecular biology
Characteristics of immunoglobulin sequence
THEORIES
1 GENE = 1 PROTEIN
Gen
1 GENE
Somatic diversification theory
(high rate of somatic mutations in the V-region)
V
C
Germline theory (separate genes for each different Ab)
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
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
V
Aim: Find a way to show the existence of multiple V genes and
rearrangement to the C gene
Approach
V
V
V
V
V
Germline DNA
V
V
V
V
V
V
V
V C
C
Rearranged DNA
V
Tools:
• cDNA probes to distinguish V from C regions
• DNA restriction enzymes to fragment DNA
• Germline (e.g. placenta) and rearranged B cell DNA (e.g. from a myeloma B cell)
Evidence for gene recombination
V
Cut myeloma B cell V
DNA with restriction
enzymes
V
V
V C
V
V
V
V
V C
Germline DNA
Size fractionate
by gel
electrophoresis
Blot with a V
region probe
Blot with a C
region probe
V C
Size fractionate
by gel
electrophoresis
V
V
V
C
V
V
V
V
V
V
V
V and C probes detect the same fragment
Some V regions missing
C fragment is larger cf germline DNA
V
V
V
Blot with a V
region probe
Blot with a C
region probe
Conclusion
There are many variable genes but only one constant gene
V
V
V
C
V
GERM LINE
V and C genes get close to each other in B-cells only
V
V
V
C
B-CELL
GENE
PROTEIN
Rearrangement of gene segments into a
single functional unit (gene)
The gene rearrangement
concept
•
Germline configuration
•
Gene segments need to be reassembled
for expression
•
Sequentially arrayed
•
Occurs in the B-cells precursors in the bone
marrow (soma)
•
A source of diversity BEFORE exposure to
antigen
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:
VL
CL
~ 95 aa
~ 100 aa
L
LV
L
CL
~ 208 aa
Where do the extra
13 amino acids
come from?
LV
L
~ 95 aa
JL
CL
~ 100 aa
Extra amino acids
provided by one of a
small set of J or
JOINING regions
The germline organization of the human immunoglobulin
light-chain loci
J-joining
Figure 2-15 part 1 of 2
CDR1 and CDR2
CDR3
Somatic rearrangement of kappa (κ) chain gene segments
Vκ Jκ
B-cell 2
Vκ
Vκ
5 Jκ
35 Vκ
Vκ
Vκ
Germline
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
• In developing B cells, the immunoglobulin genes undergo
structural rearrangements that permit their expression.
• The V domains of immunoglobulin light chains are encoded
in two (V and J) different kinds of gene segments, that are
brought into juxtaposition by recombination.
Further diversity in the Ig heavy chain
L VH
DH JH
CH
Heavy chain: between 0 and 8 additional amino acids between JH and CH
The D or DIVERSITY region
Each heavy chain requires two recombination events:
DH to JH and VH to DHJH
L VL
JL
CL
Each light chain requires one recombination event:
VL to JL
Heavy-chain V regions are constructed from
three gene segments
The germline organization of the human immunoglobulin
heavy-chain loci
*
SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS
40 VH
VH1
VH2
23 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
• The V domains of immunoglobulin heavy chains are encoded
in three (V, D and J) different kinds of gene segments, that are
brought into juxtaposition by recombination.
The numbers of functional gene segments available to
construct the variable and constant regions of human
immunoglobulin heavy chains and light chains
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:
46 VH x 23 D x 6JH = 6,348 combinations
38 Vk x 5 Jk = 190 combinations
33 Vl x 5 Jl = 165 combinations
= 355 different light chains
If H and L chains pair randomly as H2L2 i.e.
6,348 x 355 = 2,253,540 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.
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 - Recombitation Signal Sequences (RSS)
- Recognized by Recombination-Activating Genes
coded proteins (RAGs)
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
9
9
12
9
9
12
7
7
D
NONAMER - Separated from
the heptamer by a 12 or 23
nucleotide spacer
23
7
12
9
7
JH
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
Intervening DNA
of any length
23
V 7
9
12
9
7D J
Molecular explanation of the 12-23 rule
V4
V1
V8
V9
V2
V7
V3
V6
V3
V4
V2
V5
9
23-mer
• Heptamers and nonamers
align back-to-back
V7
V8
V9
V1
7
12-mer
7
• The shape generated by the
RSS’s acts as a target for
recombinases
V6
Loop of
intervening
DNA is
excised
D J
9
V5
DJ
• An appropriate shape can not be formed if two 23-mer flanked elements attempted
to join (i.e. the 12-23 rule)
Gene segments encoding the variable region are joined
by recombination at recombination signal sequences
Consequences of recombination
Generation of P-nucleotides
V4
V3
V5
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
V6
Loop of
intervening
DNA is
excised
V7
V8
V9
12-mer
V1
V5
DJ
Junctional diversity increases diversity by
6 orders of magnitude
Hipervariable and framework regions exist within
the variable domains of Igs
HV3 in the light-chain is at the junction
between rearranged V and J segments
In the heavy chain HV3 is formed
by the D segment and the residues between the
rearranged V and D segments and the D and
J segments .
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)