Immunoglobulin structure, function and types

1. STRUCTURE OF
ImmUnOglObUlInS

2. Immunoglobulin Super Family

3. The Antibody

4. •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

5. 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.

6. Binds Ab still
Binds
complement,
ppt’s in the cold,
only constant
region

12. Unfolded VL region showing 8 antiparallel β-pleated
sheets connected by loops.
NH2
COOH
S S
The Immunoglobulin Fold

13. Hypervariable=
CDR= Ag binding
regions- towards the
outside of V domain.
Complementarity
determining region

14. CDRs

15. View structures

17. CL VL
S
S
S
S
S
S
S
S
C
H3
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 Fc
Papain cleavage sites - 2 x Fab 1 x Fc
Light chain C
domains
κ or λ
Heavy chain C
domains
α, δ, ε, γ, or µ

18. CH3

19. CH3
CH2

20. CH3
CH2
CH1

21. CH3
CH2
CH1
VH1

22. CH3
CH2
CH1
VH1
VL

23. CH3
CH2
CH1
VH1
CLVL

24. CH3
CH2
CH1
VH1
CLVL

25. Hinge
CH3
CH2
CH1
VH1
VLCL
Elbow

26. C
H3
CH2Fb
Fv
Fv
FvFb
Fv
Hinge
Elbow
C
H3
CH2Fb
Fv
Flexibility and
motion of
immunoglobulins

27. Hinge
Fv
Fb
Fab
CH3
CH2
CH1
VH1
VLCL
Fc
Elbow
Carbohydrate

28. View structures

29. Unfolded VL region showing 8 antiparallel β-pleated
sheets connected by loops.
NH2
COOH
S S
The Immunoglobulin Fold

30. View structures

31. Hypervariable regions
Hypervariable CDRs are located
on loops at the end of the Fv regions

32. 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

33. •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

34. Antigens vary in size and complexity
Protein:
Influenza haemagglutinin
Hapten:
5-(para-nitrophenyl
phosphonate)-pentanoic acid.

35. 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

36. View structures

38. C
H3
CH2Fb
Fv
Fv
FvFb
Fv
Hinge
Elbow
C
H3
CH2Fb
Fv
Flexibility and
motion of
immunoglobulins

39. 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

40. 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

41. Antibody + complement- mediated
damage to E. coli
Healthy E. coli
Electron micrographs of the effect of antibodies and
complement upon bacteria

42. 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)

43. 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

45. Structure and function of the Fc region
C
H3
CH
2
IgA IgD IgG
C
H4
CH
3
CH2
IgE IgM
The 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

46. Monomeric IgM
IgM only exists as a monomer on the surface of B cells
Cµ4 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
C
µ
4
Cµ3
Cµ2 Cµ1
N.B. Only constant
heavy chain
domains are shown

47. Cµ3 binds C1q to initiate activation of the classical
complement pathway
Cµ1 binds C3b to facilitate uptake of opsonised antigens by
macrophages
Cµ4 mediates multimerisation (Cµ3 may also be involved)
C
µ
4
Cµ3
Cµ2 Cµ1
N.B. Only constant
heavy chain
domains are shown
Polymeric IgM
IgM forms pentamers and hexamers

48. C
C
C
C
C C
Multimerisation of IgM
Cµ4
Cµ3
Cµ2
C
C
C
µ
4
Cµ3
Cµ2
C
C
Cµ 4
Cµ3
C
µ2
C
C
Cµ4
Cµ3
Cµ2
C
C
Cµ4
Cµ3
C
µ2
C
C
s s
ss
s
s
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.

49. 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

50. IgM facts and figures
Heavy chain: µ - 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

51. 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: δ - 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

52. 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
S
S
S
S
C
C
S
S
S
S
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

53. Epithelial
cell
JC C
SS
S
S
C
C
SS
S
S
C
C
ss
Secretory IgA and transcytosis
B
JC C
SS
SS
C
C
SS
SS
C
C
ss
JC C
SS
S
S
C
C
SS
S
S
C
C
ss
JC C
SS
S
S
C
C
SS
S
S
C
C
ss
pIgR & IgA are
internalised
‘Stalk’ of the pIgR is degraded to release IgA
containing part of the pIgR - the secretory
component
JC C
SS
S
S
C
C
SS
S
S
C
C
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

54. IgA facts and figures
Heavy chains: α1 or α2 - 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

55. 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: ε - 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

56. IgG facts and figures
Heavy chains: γ 1 γ 2 γ3 γ4 - 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 ++

57. Fcγ receptors
Receptor Cell type Effect of ligation
FcγRI Macrophages Neutrophils,
Eosinophils, Dendritic cells Uptake, Respiratory burst
FcγRIIA Macrophages Neutrophils,
Eosinophils, Platelets
Langerhans cells Uptake, Granule release
FcγRIIB1 B cells, Mast Cells No Uptake, Inhibition of stimulation
FcγRIIB2 Macrophages Neutrophils,
Eosinophils Uptake, Inhibition of stimulation
FcγRIII NK cells, Eosinophils,
Macrophages, Neutrophils
Mast cells Induction of killing (NK cells)
High affinity Fcγ receptors from the Ig superfamily: