Recap

1
Recap Antibody structure Ig
domains Antibody Antigen interactions Affin
ity versus avidity Antibody isotype (class and
subclass)
Ig gene rearrangement (Tonegawa)
2
Ig light chain gene structure (germ line)
L leader sequence required for protein
secretion V gene encodes most of the VL protein
domain
J joining segment encodes VL framework region 4
C constant region encodes CL protein domain
3
Ig gene rearrangement (Ig light chain)
VJ rearrangement
4
Production of primary RNA transcript
transcription
(primary RNA transcript)
5
Production of mature mRNA
primary RNA transcript
RNA splicing requirement for splicing signals
mature mRNA
6
Production of primary protein product
translation
7
Production of mature Ig light chain
Mature Ig light chain
8
Ig heavy chain gene structure (germ line)
D segment encodes a few acids in CDR3
9
2 rearrangement events occur at the heavy chain
locus
10
The heavy chain protein is generated in the same
way as the light chain
11
So how does this generate diversity?
40 x 5 200 different combinations of V?J?
12
The human ? light chain locus
Light chains using a particular J? use the paired
C? 30 x 4 120 different combinations of
V?J?
13
The human heavy chain locus
About 65 VH genes about 27 DH 6 JH
segments segments
Each heavy chain VH protein domain includes one
VH gene, one DH and one JH
65 x 27 x 6 combinations of VDJ 10,530
possibilities
14
Antibody diversity Combinatorial diversity
use of different V, D and J elements 200
V?J? 120 V?J? 320 different light chain
combinations 10,530 different VDJ combinations
Assortment of heavy and light chains 320 x
10, 530 3.3 x 106 different
possibilities from less than 200 gene
segments
15
Recombination signal sequences (RSS) heptamer-nona
mer sequences
Sequences that undergo V(D)J recombination are
flanked by recombination signal sequences
conserved palindromic heptamer (CAGAGTG) conserved
AT rich nonamer (ACAAAAACC)
spacer sequence is not conserved but length
is conserved (12 or 23 base pairs)
16
The 12/23 base pair rule
Rearrangement occurs between a sequence
flanked by an RSS(12) and a sequence flanked by
an RSS(23)
17
Looping out of intervening DNA
18
Inversion and integration (less common)
19
V(D)J recombination
RAG-1/RAG-2 and HMG proteins bind RSS sequences
20
V(D)J recombination
21
V(D)J recombination
V?-A-T-C-A-G-T-C-A-C-A-G-T-G-
T-A-G-T-C-A-G-T-G-T-C-A-C-
22
V(D)J recombination
Protection of ends by Ku complex,
Ku70/Ku80/DNA-PKcs Lack of any of these
components leads to failure to complete
rearrangement
23
V(D)J recombination
A-T-C-A-G-T-A-C-T-G-C T-A-G-T nicked hairpin
24
V(D)J recombination
A-T-C-A-G-T-A-C-T-G-C T-A-G-T nicked hairpin
(V?)
A-T-C-A-G-T-A-C-T-G-C-C-G-A T-A-G-T
G-A-C-G-G-C-T nicked hairpin (V?) nicked
hairpin (J?)
25
P and N nucleotides in the VJ junction
P nucleotides are due to the asymmetric nicking
of a hairpin end
A-T-C-A-G-T-A-C-T-G-C T-A-G-T
N nucleotides are added by the enzyme
terminal deoxyribonucleotidyl transferase (Tdt)
26
Recombinational inaccuracy
V? - ATC AGT GCT CGA - J?
ATC AGT GCT CGA V?J? junction
ATC AGT CT CGA nucleotide deletion
ATC AGTAGCT CGA nucleotide insertion
Nucleotide insertions defined as N and P
nucleotides
27
Recombinational inaccuracy
V? - ATC AGT GCT CGA - J?
ATC AGT CGA GCT CGA N nucleotide additions
ATC A CT CGA nucleotide deletion
ATC AGT ACT CGA P nucleotide additions
ATC AGT CGA AGC GCT CGA N and P additions
Different length of junctions --- all in frame
and productive
28
Consequences of recombinational inaccuracy
Increased variability of CDR3
2/3 of rearrangements are non-productive (out of
frame) this leads to in frame stop
codons.... TAA, TAG or TGA in J or C region
A trade off between greater diversity and loss
of efficiency
29
Somatic hypermutation as an added source of
diversity
30
Somatic hypermutation
Antigen driven, induced by the expression of
Activation Induced Cytidine Deaminase (AID)
Focused to V(D)J region but not restricted to
coding sequence
Random but with hotpots often in CDRs
Can be silent or replacement
Can change CDR sequence and therefore change
affinity of antibody
Part of the process of affinity maturation
31
Sources of antibody diversity
Multiple V, D and J elements Combinatorial
diversity Assortment of heavy and light
chains Recombinational inaccuracy Somatic
hypermutation
32
Organization of constant region genes
C? always next to JH locus C? next to C? (if it
is present) Evolution of locus by gene duplication
33
Expression of IgM
Each domain encoded by a separate exon
RNA splicing removes introns
34
Expression of IgD
35
Co-expression of IgM and IgD
Many cells express IgM and IgD at the same time.
Some transcripts are spliced to produce
IgM mRNA, other transcripts are spliced to
produce IgD mRNA.
The same VDJ is used for both so IgM and IgD
from the same cell has the same specificity.
36
Organization of a C? gene
Each domain encoded by a separate exon
The hinge is encoded by an exon
RNA splicing removes introns
37
Isotype switching
Switch sequences upstream of all C regions genes
(except ?)
Alignment of switch sequences
38
Isotype switching
Deletion of sequences between S? and the switch
sequence for the downstream isotype induced by AID
39
Ig can be expressed on the cell surface or
secreted
40
Ig can be expressed on the cell surface or
secreted
The same VDJ is used for both membrane and
secreted forms of IgM.
This follows a prediction of the clonal
selection hypothesis.