Structural Biology and Functionsof Immunoglobulins
•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
Topic 1
Immunoglobulin Structure-Function Relationship
Outline of Lectures
•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
Immunoglobulin Structure-Function Relationship
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.
•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.
Immunoglobulin domains
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
Ig gene superfamily - IgSF
CL VL
S
S
S
S
SS
SS
CH3
CH2 CH1
VH
Fc Fab
F(ab)2
Domains are folded, compact, protease resistant structures
Domain Structure of Immunoglobulins
Pepsin cleavage sites - 1 x (Fab)2 & 1 x FcPapain cleavage sites - 2 x Fab 1 x Fc
Light chain Cdomains or
Heavy chain Cdomains
or
CH3
CH3
CH2
CH3
CH2
CH1
CH3
CH2
CH1VH1
CH3
CH2
CH1VH1
VL
CH3
CH2
CH1VH1
CLVL
CH3
CH2
CH1VH1
CLVL
Hinge
CH3
CH2
CH1VH1
VLCL
Elbow
CH3
CH2
Fb
Fv
Fv
FvFbFv
Hinge
Elbow
CH3
CH2
Fb
Fv
Flexibility andmotion of immunoglobulins
Hinge
Fv
Fb
Fab
CH3
CH2
CH1VH1
VLCL
Fc
Elbow
Carbohydrate
View structures
The Immunoglobulin Fold
The characteristic structural motif of all Ig domains
Barrel under construction
A barrel made of a sheet of staves arranged in a folded
over sheet
A barrel of 7 (CL) or 8 (VL) polypeptide strands connected
by loops and arranged to enclose a hydrophobic interior
Single VL domain
Unfolded VL region showing 8 antiparallel-pleated sheets connected by loops.
NH2
COOH
S S
The Immunoglobulin Fold
View structures
•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?
Immunoglobulins are Bifunctional Proteins
Amino acid No.
Variability80
100
60
40
20
20 40 60 80 100 120
Cytochromes C
Variability of amino acids in related proteinsWu & Kabat 1970
Amino acid No.
Variability80
100
60
40
20
20 40 60 80 100 120
HumanIg heavy
chains
FR1 FR2 FR3 FR4CDR2 CDR3CDR1
•Distinct regions of high variability and conservation led to the
concept of a FRAMEWORK (FR), on which hypervariable regions were
suspended.
Framework and Hypervariable regions
Amino acid No.
Variability80
100
60
40
20
20 40 60 80 100 120
•Most hypervariable regions coincided with antigen contact points -
the COMPLEMENTARITY DETERMINING REGIONS (CDRs)
Hypervariable regions
Hypervariable CDRs are locatedon loops at the end of the Fv regions
Space-filling model of (Fab)2, viewed from above,illustrating the surface location of CDR loops
Light chains Green and brownHeavy chains Cyan and blueCDRs Yellow
•The framework supports the hypervariable loops
•The framework forms a compact barrel/sandwich with a
hydrophobic core
•The hypervariable loops join, and are more flexible than, the
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
Hypervariable loops and framework: Summary
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
CH3
CH2
Fb
Fv
Fv
FvFbFv
Hinge
Elbow
CH3
CH2
Fb
Fv
Flexibility andmotion of immunoglobulins
30 strongly neutralising McAb
60 strongly neutralising McAb Fab regions 60 weakly neutralising McAb Fab regions
Human Rhinovirus 14- a common cold virus
30nm
Models of Human Rhinovirus 14 neutralised by monoclonal antibodies
Electron micrographs of Antibodies and complement opsonising Epstein Barr Virus (EBV)
Negatively stained EBV
EBV coated with a corona ofanti-EBV antibodies
EBV coated with antibodies and activated complement components
Antibody + complement- mediated damage to E. coli
Healthy E. coli
Electron micrographs of the effect of antibodies and complement upon bacteria
Non-covalent forces inantibody - 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?
•Detect antigen
•Precipitate antigen
•Block the active sites of toxins or pathogen-associated
molecules
•Block interactions between host and pathogen-associated
molecules
The (Fab)2 fragment can -
•Inflammatory and effector functions associated with cells
•Inflammatory and effector functions of complement
•The trafficking of antigens into the antigen processing
pathways
but can not activate
Structure and function of the Fc region
CH3
CH2
IgA IgD IgG
CH4
CH3
CH2
IgE IgMThe 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
C4 contains the transmembrane and cytoplasmic regions. These are
removed by RNA splicing to produce secreted IgM
Monomeric IgM has a very low affinity for antigen
C4
C3C2 C1
N.B. Only constant heavy chain
domains are shown
C3 binds C1q to initiate activation of the classical
complement pathway
C1 binds C3b to facilitate uptake of opsonised antigens by
macrophages
C4 mediates multimerisation (C3 may also be involved)
C4
C3C2 C1
N.B. Only constant heavy chain
domains are shown
Polymeric IgM
IgM forms pentamers and hexamers
CC
C
C
C C
Multimerisation of IgM
C4 C
3
C2
C
C
C4
C3
C2
C C
C4
C3
C2
C
C
C4
C3C2
C
C
C4
C3
C2
C
C
s s
ss
ss
C
C
ss
1. Two IgM monomers in the ER(Fc regions only shown)
2. Cysteines in the J chain form disulphide bonds with cysteines from each monomer to form a dimer
3. A J chain detaches leaving the dimer disulphide bonded.
4. A J chain captures another IgM monomer and joins it to the dimer.
5. The cycle is repeated twice more
6. The J chain remains attached to the IgM pentamer.
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: - 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
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
Heavy chain: - 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
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 chain-
linked dimer, that is formed in a similar manner to IgM pentamers.
JC C
SS
SS
C
C
SS
SS
C
C
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
Epithelialcell
JC C
SS
SS
C
C
SS
SS
CC
ss
Secretory IgA and transcytosis
B
JC C
SS
SS
CC
SS
SS
CCss
JC C
SS
SS
C
C
SS
SS
CC
ss
JC C
SS
SS
C
C
SS
SS
CC
ss
pIgR & IgA areinternalised
‘Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR - the secretory component
JC C
SS
SS
C
C
SS
SS
CC
ss
IgA and pIgR are transported to the apical surface in vesicles
B cells located in the submucosaproduce 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: 1or2 - Alpha 1 or 2
Half-life: IgA1 5 - 7 daysIgA2 4 - 6 days
Serum levels (mgml-1): IgA1 1.4 - 4.2IgA2 0.2 - 0.5
% of Ig in serum: IgA1 11 - 14
IgA2 1 - 4
Complement activation: IgA1 - by alternative and lectin pathwayIgA2 - No
Interactions with cells: Epithelial cells by pIgRPhagocytes 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
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 cellsIgE is also closely linked with allergic diseases
Heavy chain: - 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 cellsVia low affinity IgE receptor on B cells and monocytes
Transplacental transfer: No
The high affinity IgE receptor (FcRI)
chain
chain2
S SS S
S S
C1C1
C2C2C3C3
C4C4
C1
C1C2C2
C3C3
C4C4
The IgE - FcRI interaction
is the highest affinity of any
Fc receptor with an
extremely low dissociation
rate.
Binding of IgE to FcRI
increases the half life of IgE
C3 of IgE interacts with the
chain of FcRI causing a
conformational change.
IgG facts and figures
Heavy chains: 123 4 - Gamma 1 - 4
Half-life: IgG1 21 - 24 days IgG2 21 - 24 days
IgG3 7 - 8 days IgG4 21 - 24 days
Serum level (mgml-1): IgG1 5 - 12 IgG2 2 - 6IgG3 0.5 - 1 IgG4 0.2 - 1
% of Ig in serum: IgG1 45 - 53 IgG2 11 - 15IgG3 3 - 6 IgG4 1 - 4
Complement activation: IgG1 +++ IgG2 + IgG3 ++++ IgG4 No
Interactions with cells: All subclasses via IgG receptors on macrophages and phagocytes
Transplacental transfer: IgG1 ++ IgG2 +IgG3 ++ IgG4 ++
Carbohydrate is essential for complement activation
Subtly different hinge regions between subclasses accounts for differing abilities to activate complement
C1q binding motif is located on the C2 domain
Fc receptors
Receptor Cell type Effect of ligation
FcRI Macrophages Neutrophils,
Eosinophils, Dendritic cells Uptake, Respiratory burst
FcRIIA Macrophages Neutrophils,
Eosinophils, Platelets
Langerhans cells Uptake, Granule release
FcRIIB1 B cells, Mast Cells No Uptake, Inhibition of stimulation
FcRIIB2 Macrophages Neutrophils,
Eosinophils Uptake, Inhibition of stimulation
FcRIII NK cells, Eosinophils,
Macrophages, Neutrophils
Mast cells Induction of killing (NK cells)
High affinity Fc receptors from the Ig superfamily:
The neonatal Fc receptor
The FcRn is structurally related to MHC class I
In cows FcRn 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.
Human FcRn Human MHC
Class I
Molecular Genetics of Immunoglobulins
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