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