Transcript Immunoglobulin Structure-Function Relationship Immunoglobulins

Structural Biology and Functions
of Immunoglobulins
©Dr. Colin R.A. Hewitt
2005-2006
Topic 1
Immunoglobulin Structure-Function Relationship
Outline of Lectures
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Signalling antigen receptors on B cells - bifunctional antigen-binding
secreted molecules
Structural conservation and infinite variability - domain structure.
The Immunoglobulin Gene Superfamily
The immunoglobulin fold
Framework and complementarity determining regions - hypervariable
loops
Modes of interactions with antigens
Effector mechanisms and isotype – role of the Fc.
Multimeric antibodies and multimerisation
Characteristics and properties of each Ig isotype
Ig receptors and their functions
Immunoglobulin Structure-Function Relationship
• Cell surface antigen receptor on B cells
Allows B cells to sense their antigenic environment
Connects extracellular space with intracellular signalling
machinery
• Secreted antibody
Neutralisation
Arming/recruiting effector cells
Complement fixation
Immunoglobulins are Bifunctional Proteins
• Immunoglobulins must interact with a small number of
specialised molecules Fc receptors on cells
Complement proteins
Intracellular cell signalling molecules
• - whilst simultaneously recognising an infinite array of
antigenic determinants.
Immunoglobulin domains
•
Structural conservation and a capacity for infinite variability in a
single molecule is provided by a DOMAIN structure.
•
Ig domains are derived from a single ancestral gene that has
duplicated, diversified and been modified to endow an
assortment of functional qualities on a common basic structure.
•
Ig domains are not restricted to immunoglobulins.
•
The most striking characteristic of the Ig domain is a disulphide
bond - linked structure of 110 amino acids long.
Ig gene superfamily - IgSF
The genes encoding Ig domains are
not restricted to Ig genes.
Although first discovered in
immunoglobulins, they are found in a
superfamily of related genes,
particularly those encoding proteins
crucial to cell-cell interactions and
molecular recognition systems.
IgSF molecules are found in most
cell types and are present across
taxonomic boundaries
Domain Structure of Immunoglobulins
Domains are folded, compact, protease resistant structures
Fab
Fc
Light chain C
domains
k or l
S
S
S S
S S
S
Heavy chain C
domains
a, d, e, g, or m
Pepsin cleavage sites
Papain cleavage sites
S
F(ab)2
- 1 x (Fab)2 & 1 x Fc
- 2 x Fab 1 x Fc
CH3
CH2
CH3
CH1
CH2
CH3
VH1
CH1
CH2
CH3
VH1
CH1
VL
CH2
CH3
VH1
CH1
VL
CH2
CH3
CL
VH1
CH1
VL
CH2
CH3
CL
VH1
CH1
CL
VL
CH2
Elbow
Hinge
CH3
Flexibility and
motion of
immunoglobulins
Elbow
Hinge
Fv
VH1
CH1
Fb
VL
CL
Fab
CH2
Elbow
Hinge
Fc
Carbohydrate
CH3
View structures
The Immunoglobulin Fold
The characteristic structural motif of all Ig domains
A b barrel of 7 (CL) or 8 (VL)
polypeptide strands connected
by loops and arranged to
enclose a hydrophobic interior
Single VL domain
A barrel made of a sheet of
staves arranged in a folded
over sheet
Barrel under construction
The Immunoglobulin Fold
COOH
S S
NH2
Unfolded VL region showing 8 antiparallel b-pleated
sheets connected by loops.
View structures
Immunoglobulins are Bifunctional Proteins
• Immunoglobulins must interact with a finite number of
specialised molecules Easily explained by a common Fc region irrespective of specificity
• - whilst simultaneously recognising an infinite array of
antigenic determinants.
In immunoglobulins, what is the structural basis for the
infinite diversity needed to match the antigenic universe?
Variability of amino acids in related proteins
Wu & Kabat 1970
100
Variability
80
Cytochromes C
60
40
20
20
40
60
80
100
120
Amino acid No.
100
Variability
Human
Ig heavy
chains
80
60
40
20
20
40
60
80
100
120
Amino acid No.
Framework and Hypervariable regions
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Distinct regions of high variability and conservation led to the concept
of a FRAMEWORK (FR), on which hypervariable regions were
suspended.
•
Most hypervariable regions coincided with antigen contact points the COMPLEMENTARITY DETERMINING REGIONS (CDRs)
FR1
CDR1 FR2 CDR2
FR3
CDR3
FR4
100
Variability
80
60
40
20
20
40
60
80
100
120
Amino acid No.
Hypervariable CDRs are located
on loops at the end of the Fv regions
Hypervariable regions
Space-filling model of (Fab)2, viewed from above,
illustrating the surface location of CDR loops
Light chains
Heavy chains
CDRs
Green and brown
Cyan and blue
Yellow
Hypervariable loops and framework: Summary
• The framework supports the hypervariable loops
• The framework forms a compact b barrel/sandwich with a
hydrophobic core
• The hypervariable loops join, and are more flexible than, the b
strands
• The sequences of the hypervariable loops are highly variable
amongst antibodies of different specificities
• The variable sequences of the hypervariable loops influences
the shape, hydrophobicity and charge at the tip of the antibody
• Variable amino acid sequence in the hypervariable loops
accounts for the diversity of antigens that can be recognised by
a repertoire of antibodies
Antigens vary in size and complexity
Protein:
Influenza haemagglutinin
Hapten:
5-(para-nitrophenyl
phosphonate)-pentanoic acid.
Antibodies interact with
antigens in a variety of ways
Antigen inserts into a
pocket in the antibody
Antigen interacts
with an extended
antibody surface
or a groove in
the antibody
surface
View structures
Flexibility and
motion of
immunoglobulins
Elbow
Hinge
Models of
Human
Rhinovirus 14
neutralised by
monoclonal
antibodies
30nm
Human Rhinovirus 14
- a common cold virus
60 strongly neutralising McAb Fab regions
30 strongly neutralising McAb
60 weakly neutralising McAb Fab regions
Electron micrographs of Antibodies
and complement opsonising
Epstein Barr Virus (EBV)
Negatively stained EBV
EBV coated with a corona of
anti-EBV antibodies
EBV coated with antibodies and
activated complement components
Electron micrographs of the effect of antibodies and
complement upon bacteria
Healthy E. coli
Antibody + complement- mediated
damage to E. coli
Non-covalent forces in
antibody - antigen interactions
Electrostatic forces
Attraction between opposite charges
Hydrogen bonds
Hydrogens shared between electronegative atoms
Van der Waal’s forces Fluctuations in electron clouds around molecules
oppositely polarise neighbouring atoms
Hydrophobic forces
Hydrophobic groups pack together to exclude
water (involves Van der Waal’s forces)
Why do antibodies need an Fc region?
The (Fab)2 fragment can •
Detect antigen
•
Precipitate antigen
•
Block the active sites of toxins or pathogen-associated
molecules
•
Block interactions between host and pathogen-associated
molecules
but can not activate
•
Inflammatory and effector functions associated with cells
•
Inflammatory and effector functions of complement
•
The trafficking of antigens into the antigen processing
pathways
Structure and function of the Fc region
IgA IgD IgG
IgE IgM
CH2
The hinge region is replaced
by an additional Ig domain
Fc structure is common to all specificities of antibody within an ISOTYPE
(although there are allotypes)
The structure acts as a receptor for complement proteins and a ligand
for cellular binding sites
Monomeric IgM
IgM only exists as a monomer on the surface of B cells
Monomeric IgM has a very low affinity for antigen
Cm2
N.B. Only constant
heavy chain
domains are shown
Cm4 contains the transmembrane and cytoplasmic regions. These are
removed by RNA splicing to produce secreted IgM
Polymeric IgM
IgM forms pentamers and hexamers
Cm2
N.B. Only constant
heavy chain
domains are shown
Cm3 binds C1q to initiate activation of the classical
complement pathway
Cm1 binds C3b to facilitate uptake of opsonised antigens by
macrophages
Cm4 mediates multimerisation (Cm3 may also be involved)
Multimerisation of IgM
Cm2
1. Two IgM monomers in the ER
(Fc regions only shown)
C
3. A J chain detaches
leaving the dimer
disulphide bonded.
C
2. Cysteines in the J chain
form disulphide bonds
with cysteines from each
monomer to form a dimer
4. A J chain captures another
IgM monomer and joins it
to the dimer.
6. The J chain remains
attached to the IgM
pentamer.
Cm4
5. The cycle is repeated
twice more
ss
Cm4
Antigen-induced conformational changes in IgM
Planar or ‘Starfish’ conformation found in
solution.
Does not fix complement
Staple or ‘crab’ conformation of IgM
Conformation change induced by
binding to antigen.
Efficient at fixing complement
IgM facts and figures
Heavy chain:
m - Mu
Half-life:
5 to 10 days
% of Ig in serum:
10
Serum level (mgml-1):
0.25 - 3.1
Complement activation:
++++ by classical pathway
Interactions with cells:
Phagocytes via C3b receptors
Epithelial cells via polymeric Ig receptor
Transplacental transfer:
No
Affinity for antigen:
Monomeric IgM - low affinity - valency of 2
Pentameric IgM - high avidity - valency of 10
IgD facts and figures
Heavy chain:
d - Delta
Half-life:
2 to 8 days
% of Ig in serum:
0.2
Serum level (mgml-1):
0.03 - 0.4
Complement activation: No
Interactions with cells:
T cells via lectin like IgD receptor
Transplacental transfer: No
IgD is co-expressed with IgM on B cells due to differential RNA splicing
Level of expression exceeds IgM on naïve B cells
IgD plasma cells are found in the nasal mucosa - however the function of IgD in
host defence is unknown - knockout mice inconclusive
Ligation of IgD with antigen can activate, delete or anergise B cells
Extended hinge region confers susceptibility to proteolytic degradation
IgA dimerisation and secretion
IgA is the major isotype of antibody secreted at mucosal sufaces
Exists in serum as a monomer, but more usually as a J chainlinked dimer, that is formed in a similar manner to IgM pentamers.
S
S
S
J
S
ss
S
S
S
S
IgA exists in two subclasses
IgA1 is mostly found in serum and made by bone marrow B cells
IgA2 is mostly found in mucosal secretions, colostrum and milk and is made
by B cells located in the mucosae
Secretory IgA and transcytosis
S
S
SS
SS
SS
SS
ss
ss
S
S
J
S
S
S
S
S
S
J
ss
S
S
S
S
SS
S
S
B
J
J
Epithelial
cell
pIgR & IgA are
internalised
ss
SS
S
S
SS
J
SS
S
S
ss
IgA and pIgR
are transported
to the apical
surface in
vesicles
SS
‘Stalk’ of the pIgR is degraded to release IgA
containing part of the pIgR - the secretory
component
SS
B cells located in the submucosa
produce dimeric IgA
Polymeric Ig receptors
are expressed on the
basolateral surface of
epithelial cells to
capture IgA produced
in the mucosa
IgA facts and figures
Heavy chains:
a1 or a2 - Alpha 1 or 2
Half-life:
IgA1 5 - 7 days
IgA2 4 - 6 days
Serum levels (mgml-1):
IgA1 1.4 - 4.2
IgA2 0.2 - 0.5
% of Ig in serum:
IgA1 11 - 14
IgA2 1 - 4
Complement activation: IgA1 - by alternative and lectin pathway
IgA2 - No
Interactions with cells:
Epithelial cells by pIgR
Phagocytes by IgA receptor
Transplacental transfer: No
To reduce vulnerability to microbial proteases the hinge region of IgA2 is truncated,
and in IgA1 the hinge is heavily glycosylated.
IgA is inefficient at causing inflammation and elicits protection by excluding, binding,
cross-linking microorganisms and facilitating phagocytosis
IgE facts and figures
Heavy chain:
e - Epsilon
Half-life:
1 - 5 days
Serum level (mgml-1):
0.0001 - 0.0002
% of Ig in serum:
0.004
Complement activation: No
Interactions with cells:
Via high affinity IgE receptors expressed
by mast cells, eosinophils, basophils
and Langerhans cells
Via low affinity IgE receptor on B cells
and monocytes
Transplacental transfer: No
IgE appears late in evolution in accordance with its role in protecting against
parasite infections
Most IgE is absorbed onto the high affinity IgE receptors of effector cells
IgE is also closely linked with allergic diseases
The high affinity IgE receptor (FceRI)
The IgE - FceRI interaction
is the highest affinity of any
Fc receptor with an
extremely low dissociation
rate.
Binding of IgE to FceRI
increases the half life of IgE
a chain
S
g2
S
S
b chain
S
S
S
Ce3 of IgE interacts with the
a chain of FceRI causing a
conformational change.
IgG facts and figures
Heavy chains:
g 1 g 2 g3 g4 - Gamma 1 - 4
Half-life:
IgG1
IgG3
21 - 24 days
7 - 8 days
IgG2
IgG4
21 - 24 days
21 - 24 days
Serum level (mgml-1):
IgG1
IgG3
5 - 12
0.5 - 1
IgG2
IgG4
2-6
0.2 - 1
% of Ig in serum:
IgG1
IgG3
45 - 53
3-6
IgG2
IgG4
11 - 15
1-4
+++
++++
IgG2
IgG4
+
No
Complement activation: IgG1
IgG3
Interactions with cells:
All subclasses via IgG receptors on macrophages
and phagocytes
Transplacental transfer: IgG1
IgG3
++
++
IgG2
IgG4
+
++
C1q binding motif is
located on the Cg2
domain
Carbohydrate is essential for
complement activation
Subtly different hinge regions
between subclasses accounts
for differing abilities to activate
complement
Fcg receptors
High affinity Fcg receptors from the Ig superfamily:
Receptor
FcgRI
FcgRIIA
FcgRIIB1
FcgRIIB2
FcgRIII
Cell type
Effect of ligation
Macrophages Neutrophils,
Eosinophils, Dendritic cells
Uptake, Respiratory burst
Macrophages Neutrophils,
Eosinophils, Platelets
Langerhans cells
Uptake, Granule release
B cells, Mast Cells
No Uptake, Inhibition of stimulation
Macrophages Neutrophils,
Eosinophils
Uptake, Inhibition of stimulation
NK cells, Eosinophils,
Macrophages, Neutrophils
Mast cells
Induction of killing (NK cells)
The neonatal Fcg receptor
Human FcgRn
Human MHC
Class I
The FcgRn is structurally related to MHC class I
In cows FcgRn binds maternal IgG in the colostrum at pH 6.5 in the gut.
The IgG receptor complex is trancytosed across the gut epithelium and
the IgG is released into the foetal blood by the sharp change in pH to 7.4
Some evidence that this may also happen in the human placenta,
however the mechanism is not straightforward.
Molecular Genetics of Immunoglobulins