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Antibody Structure & Functions
Antibody structure – key features?
Antibody structure
H
LL
H
IgA a chains IgE e IgM m IgG g IgD d
k, l
2 Heavy chains- different for each class
2 Light chains- k or l for all classes
COOH COOH
NH3 NH3 NH3
NH3
Polypeptide chains or glycopeptide (decorated with CHO)Held together with disulphide bonds
Antibody fragments
H
LL
H
HLL
HPepsin
F(ab)2
LL
Fab
HH
Papain
Fc
•Detect antigen•Precipitate antigen•Block the active sites of toxins or pathogen-associated
molecules•Block interactions between host and pathogen-associated
molecules
(Fab)2 fragment
•Inflammatory and effector functions associated with cells•Inflammatory and effector functions of complement•The trafficking of antigens into the antigen processing
pathways
Fc fragment
Antibody is organised into domains
antigen antigen
Variable region (V)
Constant region (C)
Light chain (L)21-22KDa
Heavy chain (H) 52-64KDa
V regionControls antigen specificity
Fc regionControls effector function
m and e have 4 CH domains and no hinge regionAb usually have 3CH domains
A domain is the characteristic structural motif of all Ig domains• is a b barrel of 7 (CL) or 8 (VL) polypeptide strands • connected by loops • arranged to enclose a hydrophobic interior
eg. Single VL domain
Hinge
CH3
CH2
CH1VH1
VLCL
Elbow
Crystal structure showing antibody domains
•Structurally antibodies are arranged into domains.
•Domains are derived from a single ancestral gene that has duplicated, diversified & modified.
•Ig-like Domains are not restricted to immunoglobulins.
•The characteristic of the immunoglobulin domain is a disulphide bond (110aa)
Immunoglobulin domains
• The genes encoding Ig domains are not restricted to Ig genes.
• They were subsequently found in a superfamily of related genes
• They encode proteins for cell-cell interactions and molecular recognition.
• Found in most cell types and different animal species
Ig gene superfamily
Immunoglobulins as Bifunctional Proteins
•Immunoglobulins must interact with a small number of specialised molecules (common Fc to each class)
Fc receptors on cells
Complement proteins
Intracellular cell signalling molecules
•whilst simultaneously recognising an infinite array of antigenic determinants (? Mechanism).
FR1 FR2 FR3 FR4CDR2 CDR3CDR1
•Distinct regions of high variability and conservation led to the concept of a framework 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 coincide with antigen contact points - the COMPLEMENTARITY DETERMINING REGIONS
(CDRs)
The sequences of the hyper-variable loops :-
•are highly variable amongst antibodies of different specificities
•influence the shape, hydrophobicity and charge at the tip of the antibody
•accounts for the diversity of antigens that can be recognised
Hypervariable loops
L & H chain folds to yield 3 CDR in each chain to form walls of Ag binding groove
Hypervariable regions
Hypervariable CDRs are located on loops at the end of the Fv regions
Activates C1q complement (when bound to Ag)
Opsonises (by binding to phagocyte and Ag)
Helps kill infected cells (eg.via ADCC, eg. phagocytosed organisms)
Activates immune cells(by binding to cell and Ag)
Summary of antibody effector functions?
IgM IgG
IgD IgM - B cell receptorIgE – binds to mast cellsIgG- macrophages
IgG - bloodIgA - mucosa
Mainly IgG
EFFECTOR FUNCTIONS of antigen-bound antibody
Effector function is determined by Antibody class or subclass How? This is largely determined by the distribution of specific Fc receptors for each class of Ab
Fcg – HIGH AFFINITY Fc receptors
Receptor Cell type Effect of ligationFcgRI Macrophages Neutrophils,
Eosinophils, Dendritic cells Uptake, Respiratory burstFcgRIIA Macrophages, Neutrophils,
Eosinophils, PlateletsLangerhans cells Uptake, Granule release
FcgRIIB1 B cells, Mast Cells No Uptake, Inhibition of stimulationFcgRIIB2 Macrophages ,Neutrophils,
Eosinophils Uptake, Inhibition of stimulationFcgRIII NK cells, Eosinophils,
Macrophages, Neutrophils,Mast cells Induction of killing (NK cells)
Monomeric IgM
IgM only exists as a monomer on the surface of B cells
Cm4 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
Cm4
Cm3Cm2 Cm1
N.B. Only constant heavy chain
domains are shown
Cm1 binds C3b to facilitate uptake of opsonised antigens by macrophages
Cm4 mediates multimerisation (Cm3 may also be involved)
Cm3 binds C1q to initiate activation of the classical complement pathway
Polymeric IgMIgM forms pentamers and hexamers
Non-antigen binding sites
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 receptorsEpithelial 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 splicingIgM > IgD on naïve B cells
function of IgD in host defence is unknown - knockout mice inconclusiveLigation of IgD with antigen can activate, delete or anergise B cells
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
IgA dimerisation and secretion
IgA is the major isotype of antibody secreted at mucosal surfaces
Exists in serum as a monomer, but also as a J chain-linked dimer
IgA1 is mostly found in serum (monomer), made by bone marrow B cells
IgA2 is mostly found in mucosal secretions (dimer), colostrum and milk; made by mucosal B cells
Body eg gut or nasal mucosa
Mucosalepithelium
1. IgA2 binds to pIgR
2. Bound IgAis transportedacross epithelium
3. sIgAis released via cleavage of pIgR
IgA facts and figuresHeavy chains: a1 or a2 - 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 - 14IgA2 1 - 4
Complement activation: IgA1 - by alternative and lectin pathwayIgA2 - No
Interactions with cells: Epithelial cells by pIgRPhagocytes by IgA receptor
Transplacental transfer: No
IgA is inefficient at causing inflammation and elicits protection by excluding, binding, cross-linking microorganisms and facilitating phagocytosis
IgE facts and figures
IgE has a role in protecting against parasite infectionsMost IgE is absorbed onto the high affinity IgE receptors of effector cellsIgE is also closely linked with allergic diseases
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
The high affinity IgE receptor (FceRI)
a chain
b chaing2
S S S S
S S
Ce1Ce1Ce2 Ce2Ce3Ce3
Ce4 Ce4
Ce1Ce1Ce2 Ce2Ce3 Ce3Ce4 Ce4
The IgE - FceRI interaction is the highest affinity of any Fc receptor with an extremely low dissociation rate.
CH3 of IgE interacts with the FceRI causing a conformational change.
Binding of IgE to FceRI thus increases the half life of IgE
IgG facts and figures
Heavy chains: g 1 g 2 g3 g4 - 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 - 6
IgG3 0.5 - 1IgG4 0.2 - 1
% of Ig in serum: IgG1 45 - 53IgG2 11 - 15
IgG3 3 - 6IgG4 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 also essential for complement activation
C1q binding motif on the Cg2 domain
Subtle differences in hinge region between subclasses accounts for differing abilities to activate complement
a, m, d, e, gk, l chains
Species specific C (Hyper)-variable region (antigen binding site)
ANTIBODY variability isotypic allotypic idiotypic