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