Download Antibody structure : the early studies

Antibody structure : the early studies
Albumin
Globulins
Untreated antiserum
Absobanc
A
Ab
rbance
bsor
c
Antiserum upon
p
incubation with the Ag
Electrophoretic migration
Antibody structure : the early studies (cont.)
Fab
Fab
papain
Fc
F(ab')2
pepsin
pFc
pFc'
Light chain
reduction Heavy
chain
Antibody structure by electron microscopy
Angle between arms is 0
degrees
Angle between arms is 90
degrees
Angle between arms is 60
degrees
Antibody arms are joined by a flexible hinge
How Antibodyy Binds to Antigen
g
The top part of this figure shows how different shaped antigens can
fit into the binding site of antibodies: left, pocket; center, groove;
right, extended surface. The panels below show space-filling or
computer-generated
t
t d models
d l iindicating
di ti where
h
contact
t tb
between
t
th
the
peptide antigen and antibody occurs.
How an Antibody Works
When an Ab finds its Ag on an invader, it will bind there and
act as a “trash tag”, marking it for destruction by “killer”
cells,
ll macrophages
h
or complement
l
Antibody binds to
target antigen
Receptor for
constant region of
antibody on NK
cell recognizes a
bound antibody
After binding, the NK
cell is signaled to kill
the target cell
The target cell dies
by apoptosis and/or
membrane damage
Antibody structure by modern techniques
Antigenbinding sites
Antigenbindingg sites
VH
VH
CH1
CH1
VL
h
hinge
disulfide
bonds
carbohydrate
hinge
g
CL
CH2
CH3
VL
CL
carbohydrate
CH2
CH3
disulfide
bonds
Diagrammatic IgG structure
Light
Light
chains
chains
Antigen
binding site
hinge
Papain
Pepsin
carbohydrate
carbohydrate
Heavy
chains
Immunoglobulin G (IgG)
• L chains have 2 domains each (VL and CL).
• H chains of IgG (IgA and IgD also) have 4 domains
(VH, CH1, CH2, CH3).
• The Hinge region are cys- and pro-rich; it is flexible,
open accessible and located between CH1 and CH2
open,
domain.
• There are carbohydrate moieties attached to the CH2
domain in the two H chains.
• Pairing: VL/VH, CL/CH1, CH2/CH2 and CH3/CH3.
• H chains of IgM and IgE have 5 domains (VH, CH1, CH2,
CH3 and CH4).
4) There is no hinge region in these two
isotypes.
Antibody architecture
IgD
IgM
IgG
IgE
IgA
κ
or
λ
μ
γ
δ
α
ε
Heavy chain constant domains determine isotype
J chain
J chain
IgM and IgA can form polymers
Light chain (approx. 25 kDa)
Two types of L chains: Kappa (κ) and Lamda (λ) encoded by
genes located in different chromosomes.
Ratio of κ to λ chains can vary (mouse: 20/1 κ/λ;
human: 2/1 κ/λ, cattle: 1:20 κ/λ).
The two L chains of any one Ig molecule contain either κ
or λ,
λ i.e.
i e ne
never
er one κ with
ith the other λ.
λ
Heavy chain (approx.
(approx 50 kDa)
Five classes or isotypes of antibodies defined by the H chains:
Ig class (isotype)
H chain
IgM
μ
IgG
γ
IgA
α
IgD
δ
IgE
ε
The five H chains differ in their primary structures with approx.
60% homology.
Four subclasses (subtypes) of IgG:
Human: IgG1 (γ1), IgG2 (γ2),
IgG3
g
(γ
(γ3)) and IgG4
g
(γ
(γ4).
)
Mouse: IgG1 (γ1), IgG2a (γ2a),
IgG2b (γ2b) and IgG3 (γ3).
Two IgA subclasses (IgA1 (α1)
and IgA2 (α2))
(α2)).
• Extensive
E t
i h
homology
l
((approx. 90%) among th
the subclasses
b l
off
H chains that belong to the same isotype.
• Both H chains are identical in any one Ig molecule.
Reading: Chapters 3 and 4.
Antibody fine structure
Light chain C domain
Light chain V domain
beta strands
beta strands
disulfide bond
A
Arrangement
t off bbeta
t strands
t d
Antibody fine structure (cont.)
(cont )
CDR
Heavy-chain V region
Light-chain V region
HV3
HV2
HV1
Variability
HV3
HV1
HV2
Variabilityy
FR1
FR1
FR2
FR2
FR3
FR3
Residue #
HV : hypervariable region (= CDR : complementarity determining
g )
region)
FR : framework region
Residue #
• Three Hypervariable regions, HV1, HV2 and HV3,
present in
each of the H and L chains.
• They are franked by less variable regions called
Framework
regions, FR1, FR2, FR3 and FR4.
• The combination/folding in HV regions bet
between
een H
and L chains
constitutes the antigen binding sites that are
complementary in
structure to the Ag epitope, termed
complementarity-determining regions (CDRs),
CDR1, CDR2 and CDR3.
The a.a.
a a in the CDRs provide multi-point contacts
and different types of interaction with those in the
Ag epitope: H-bonds,
H-bonds electrostatic forces,
forces
hydophobic forces and van der Waals forces.
Together they provide stable noncovalent Ab/Ag
association.
Antibody fine structure (cont.)
Variability
N
C
residue #
N
C
Ag-binding
site
N
C
The nature of antigen-antibody interactions
Antigen
Antibody
Gln
Ser
Lys
Hydrogen
y g bond
Ionic bond
F~1/d 7
Glu
Val
Val
Leu
Asp
Hydrophobic bond &
Leu Van der Waals
interactions
Lys Ionic bond
F~1/d
These are allll REVERSIBLE forces.
Th
f
They only act on SHORT DISTANCE.
Th can yield
They
i ld either
ith attracion
tt i or repulsion.
li
2
Like a key in a lock
poor fit
good fit
Ab
Ab
Ag
Ag
high attraction - low repulsion high repulsion - low attraction
Ab
Ag
Ab
Ag
affinity =Σ attractive and repulsive forces
Affinity
Affi
it M
Maturation:
t
ti
The Ag-specific abs
produced in secondary
response exhibit greater
affinity than those seen
in the primary response.
Antibody-Antigen interaction : a dynamic equilibrium
association (k1)
Ab + Ag
AgAb
dissociation (k-1)
K = k1/ k-1
K = [AbAg] /[Ab] [Ag] (association constant)
If [[AbAg]
g] = [Ab],
[ ], that is,, if half of all the Ab is
occupied, K = 1/[Ag] ( dimension of K: liter per mole)
If K iis hi
high,
h lilittle
l A
Ag iis needed
d d to occupy hhalf
lf off the
h Ab
Ab.
p
on the reaction conditions!
K is dependent
Affinity determined by equilibrium dialysis
%
labeled
antigen
distribution
semipermeable
membrane
A
B
A
without antibody
B
%
with antibody
antigen
g
bound by
antibody
Initial state
antibody
labeled
antigen
Equilibrium
Affinity determined by equilibrium dialysis (cont.)
Equilibrium dialysis of diffusible antigen
Add Ab to low
Ag concentration
No antibody
concIN = concOUT
Add Ab to high
Ag concentration
concIN = concOUT + concBOUND
Scatchard analysis
Single antibody
Mixture
of antibodies
Mixture
of Abs
Mixture of Abs
r
conc of
free Ag
slope = - KA
Moles Ag bound per mole Ab (= r)
Affinity and Avidity
Fab
IgG
IgG
IgM
Eff ti Ab valence
Effective
l
1
1
2
Antigen valence
1
1
n
n
Association const.
104
104
107
1011
Cooperative
advantage
-
-
103
107
up to 10
Definition of binding
intrinsic affinity
avidity or
functional affinity
Applications
liquid-phase
techniques
solid-phase
techniques
Antibody Variability
There are several reasons why there are an enormous number of
different antibodies:
• different combinations of heavy and light chains which are
encoded by different genes
• recombination
• others
The Number Dilemma
• You have about a trillion different antibodies able to react with
millions of different types of Ag
• b
butt you only
l have
h
about
b t 30,000-40,000
30 000 40 000 genes which
hi h code
d for
f all
ll
the proteins you need in your entire body, most of which are not
Ab
• so there cannot be one gene for one antibody to code for these –
we wouldn’t have enough antibodies!
S hhow can your bbody
So
d produce
d
Ab to so many antigens,
i
even those
h
it’s never seen?
Antibody Genes
Genes for antibodies aren’t like most other genes - they come
in pieces ((“gene
gene-lets
lets”):
):
•
•
•
•
variable
i bl segments (V)
( ) – many different
diff
versions
i
diversity segments (D) – several different versions
joining segments (J) – a few different versions
constant segments (C) – a few different versions that
are nearly
l identical
id ti l
Other sources of variability
• when V, D, and J pieces are joined, they may not always
be joined perfectly – if some base-pairs
base pairs are lost or added,
added
the Ab will end up with a different amino acid sequence
• variable region genes mutate at a higher rate than other
genes in your body
Lecture 7 to 9 (cont.II)
Generation of Antibody Diversity
Overview:
Gene rearrangement in the generation of antibody diversity.
Junctional and insertional diversity.
Somatic hypermutation.
Allelic exclusion
Genes encoding
g human κ L chain ((chromosome 2))
λ L chain (chromosome 22)
H chain (chromosome 14)
The genes encoding the H and L chains are arranged in exons
separated by the intervening introns.
L chains have gene segments of V (variable),
(variable) J (joining)
and C (constant) gene segments.
y), J and C g
gene segments.
g
H chains have V,, DH ((diversity),
Antigenic determinants on immunoglobulins
mouse IgG1
mouse IgM
"What makes human IgG1 different
f
from
human
h
IgM."
I M"
a. Isotypic determinants
mouse IgG1
strain A
mouse IgG1
strain B
"What makes my IgG1 different
from yours."
b. Allotypic determinants
"What
What makes my anti-tetanus
different from my anti-pertussis"
mouse IgG1
against Ag a
mouse IgG1
against Ag b
c. Idiotypic determinants
During
g B cell development
p
in the bone marrow,, the gene
g
segments undergo gene rearrangement by bringing together
one V, (one D), and one J gene for joining to a C gene
segment, a process known as somatic recombination.
This process involves excision and splicing of gene segments.
The DNA-splicing
DNA splicing enzymes are encoded by recombination
activator genes, RAG-1 and RAG-2. RAG knock-out mice
has no functional T and B cells.
H chain gene rearrangement occurs early in B cell development
at the Pro-B cell stage (before κ chain gene rearrangement).
κ chain
h i gene rearrangementt occurs before
b f
λ chain
h i
rearrangement at the immature B cell stage in human and mice.
A unique recombination
occurs in each B cell
•
each B cell combines these gene
segments to make an Ab chain like
shuffling a deck of cards
- V, D, and J for the heavy chain, V
and
d J ffor th
the li
light
ht chain
h i
•
since
i
there
th are multiple
lti l types
t
off eachh
gene segment, there are many
thousands of possible V-D-J
combinations so that each B cell gets a
unique combination of segments!
Unique combination of segments becomes joined by
somatic gene rearrangement
Combinatorial Diversity
Human κ chain gene:
(40 Vκ) (5 Jκ) = 200 possible distinct κ V regions.
Human λ chain gene:
(30 Vλ) (4 Jλ) = 120 possible distinct λ V regions.
Total possible L chain V regions:
200 + 120 = 320
Human H chain gene:
(40 VH) (25 DH) (6 JH) = 6,000 possible distinct H V regions.
(Each of the VH region can join to any one of the C gene
segments)
Each of the 320 different L chains could combine with each
of the
,
possible
p
heavyy chains.
6,000
(6,000 H) (320 L) = 1.92 x 106 different ab specificities.
Junctional and Insertional Diversity
• When the 3' end of V and 5' end of J join together
together, the point
of joining does not always need to be the same.
In this way, the nucleotide triplet which encodes one a.a. may
be different each time the same V and J segments are joined.
• Similarly in H chains, additional variability occurs at the
D-J and V-D-J joining points.
• In addition to imprecise joining, additional nucleotides may
also be inserted at the junction during rearrangement
rearrangement.
The inserted nucleotides are found in the CDR3 of both
H and L chains, and are called P
P- or N
N-nucleotides.
nucleotides.
P-nucleotides are template-based.
N-nucleotides are nontemplate-based, i.e. not encoded in the
gene segment.
The endpoint is additional nucleotides and therefore
new amino acid sequences are generated in CDR3
CDR3.
Somatic hypermutation
yp
After boosting immunization, point mutations may occur in
the CDRs.
Thi diversity
This
di
it is
i created
t d as a result
lt off point
i t mutation
t ti iin
certain "hot spots" and less-defined secondary structural
features in the V region.
region
Junctional Diversity and Somatic Hypermutation together
increases the B cell repertoire to 10 11 different specificities.
Allelic Exclusion
Chromosomes are diploidy.
One H chain will undergo rearrangement.
If completed, rearrangement of the other (allele) will be
prohibited.
prohibited
If it fails, the other (allele) will continue.
The same for L chains.
The end result is that each B cell will use only one parental
chromosome. This phenomenon is known as allelic exclusion.
This is why no B cell will express both κ and λ L chain.
Transcription
p
and Class Switching
g
Transcription can stop at 3' end of μ gene segment.
This yields VDJCμ chain (μ chain).
Alt
Alternatively,
ti l ttranscription
i ti can go th
through
h th
the Cμ and
d stops
t
after the δ gene segment. The intervening Cμ will then be
spliced out and yield a VDJCδ chain (δ chain).
chain) This occurs at
mature B cell stage of development. Therefore, mature B cells
can express both IgM and IgD sharing the same VHDHJH and
the same VJ of the L (κ or λ) chain. This means that the same
Ag specificity of different isotypes can be found on a given
mature
t
B cell.
ll
A single B cell may produce Ig of different isotypes but it
always produces an Ig of only ONE antigenic specificity
specificity.
Class (Isotype) Switching
B cells
ll mature
t
iin b
bone marrow express b
both
th IIgM
M and
d IIgD.
D
They then join the blood and recirculate in the periphery.
In the periphery,
periphery B cells can potentially switch isotypes
by producing sequentially IgM→IgG →IgA →IgE.
The switching of isotype is mediated by the Switch (S)
region in the introns 5' to each of the C gene segment
(
(except
t for
f δ).
δ)
Therefore, the same specificity can be expressed as distinct
Therefore
isotypes starting always with IgM.
Class switching is largely regulated by cytokines (IFN-γ for
IgG2 class, and IL-4 for IgG1 and IgE) produced upon Ag
stimulation.
ti l ti