STRUCTURE OF Immunoglobulins

Immunoglobulin Super Family

The Antibody

• 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

FUNCTIONS

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.

Binds Ab still

Binds

complement,

ppt’s in the cold,

only constant

region

Unfolded VL region showing 8 antiparallel b-pleated

sheets connected by loops.

NH2

COOH

S S

The Immunoglobulin Fold

Hypervariable=

CDR= Ag binding

regions- towards the

outside of V domain.

Complementarity

determining region

CDRs

View structures

S

S

S

S

S

S

S

S

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 Fc

Papain cleavage sites - 2 x Fab 1 x Fc

Light chain C

domains

k or l

Heavy chain C

domains

a, d, e, g, or m

CH3

CH3

CH2

CH3

CH2

CH1

CH3

CH2

CH1

VH1

CH3

CH2

CH1

VH1

VL

CH3

CH2

CH1

VH1

CLVL

CH3

CH2

CH1

VH1

CLVL

Hinge

CH3

CH2

CH1

VH1

VLCL

Elbow

Hinge

Elbow

Flexibility and

motion of

immunoglobulins

Hinge

Fv

Fb

Fab

CH3

CH2

CH1

VH1

VLCL

Fc

Elbow

Carbohydrate

View structures

Unfolded VL region showing 8 antiparallel b-pleated

sheets connected by loops.

NH2

COOH

S S

The Immunoglobulin Fold

View structures

Hypervariable regions

Hypervariable CDRs are located

on 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 brown

Heavy chains Cyan and blue

CDRs Yellow

• The framework supports the hypervariable loops

• The hypervariable loops join, and are more flexible

• 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 haemagglutininHapten:

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

Hinge

Elbow

Flexibility and

motion 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 of

anti-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 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?

• 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

IgA IgD IgG

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

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

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)

Cm2

N.B. Only constant

heavy chain

domains are shown

Polymeric IgM

IgM forms pentamers and hexamers

Multimerisation of IgM

Cm

2

C C

Cm4

Cm

4 s s

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: 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

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: 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 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.

J

SS

S

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

Epithelial

cell

J

SS

SS

SS

SS

ss

Secretory IgA and transcytosis

B

JSS

SS

SS

SS

ss

J

SS

SS

SS

SS

ss

J

SS

SS

SS

SS

ss

pIgR & IgA are

internalised

‘Stalk’ of the pIgR is degraded to release IgA

containing part of the pIgR - the secretory

component

J

SS

SS

SS

SS

ss

IgA and pIgR

are transported

to the apical

surface in

vesicles

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

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

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

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 - 1 IgG4 0.2 - 1

% of Ig in serum: IgG1 45 - 53 IgG2 11 - 15

IgG3 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 ++

Fcg receptors

Receptor Cell type Effect of ligation

FcgRI Macrophages Neutrophils,

Eosinophils, Dendritic cells Uptake, Respiratory burst

FcgRIIA Macrophages Neutrophils,

Eosinophils, Platelets

Langerhans cells Uptake, Granule release

FcgRIIB1 B cells, Mast Cells No Uptake, Inhibition of stimulation

FcgRIIB2 Macrophages Neutrophils,

Eosinophils Uptake, Inhibition of stimulation

FcgRIII NK cells, Eosinophils,

Macrophages, Neutrophils

Mast cells Induction of killing (NK cells)

High affinity Fcg receptors from the Ig superfamily: