Immunoglobulin structures and antibodies functions from University of Portsmouth

Learning Objectives

On completion of this session you should be able to

1. Describe the basic structure of immunoglobulins.
2. Explain the diverse functions of antibodies in immune defence.
3. Define antibody response upon encountering antigens.

Ig Structure

1. Structure and function are interlinked.
2. Immunoglobulins or antibodies have 2 functions:
   a) To recognise antigen
   b) To engage effector mechanisms to dispose of antigen
3. Recognition sites vary between antibodies and constitute the variable region or V region
4. Antibodies of the same class elicit the same effector mechanisms through the constant region or C region

Ig Structure - Polypeptide Chains

1. Antibody molecules are Y-shaped and consist of 2 heavy (H) and 2 light (L) chain polypeptides held together by disulphide bonds
2. M.W. is approximately 150 kDa
3. The 2 heavy chains (M.W. 50 kDa) and 2 light chains (M.W. 25 kDa) are identical
4. Light chains can be either kappa (k) or lambda (2)
5. No functional difference between k and 2 chains and ratio in humans is 2: 1 and 20:1 in mice

Ig Structure - Antigen-Binding Sites

antigen-binding sites
VL
CL
VH
CH1
hinge
disulfide
bonds
CH2
a
carbohydrate .
CH3
b
N terminus
Variable region
VH
CH1
VL
CL
disulfide
bonds
CH2
Constant
region
CH3
C
C terminus
Figure 4.1 Janeway's Immunobiology, 8ed. (@ Garland Science 2012)

Ig Structure - Constant Region

1. Constant region, heavy chains (u, 8, y, a, &) determine the class and function of antibody either IgM, IgD, IgG, IgA or IgE
2. H and L chains comprise both variable and constant domains
3. Each chains consists of 110 amino acid sequence repeats
4. L chains have 2 sequences and H chains 4 sequences and these correspond to structural domains
5. N-terminal domains vary considerable in sequence and are designated the VH and V domains

Ig Structure - Domains

1. Vi and V_ domains form the V region of the antibody
2. C-terminal domains (CL, C 1, C 2, C_3) are constant for each isotype and form the C region of the molecule
3. H and L chains associate such that V_ and V, domains are paired, as are the C and C_1 domains and C_3 domains
4. C_2 domains do not interact as carbohydrate is present between them
5. Antibodies are glycosylated (glycoproteins)

Ig Structure - Proteolytic Cleavage

1. Antibody molecules can be cleaved into three portions with proteases
2. Papain cleaves the H chains on the N-terminal side of the S=S bonds in the hinge region
3. This releases 2 x Fab fragments (fragment antigen binding) and 1 x Fc fragment (fragment crystallisable)
4. Pepsin cleaves and antibody on the C-terminal side of the S=S bond releasing 1 x F(ab')2 fragment
5. Pepsin cleaves the Fc fragment into several peptides

Proteolytic cleavage
by papain
Proteolytic cleavage
by pepsin
amino terminus
carboxy terminus
F(ab') 2
Fab
Fab
Fc
pFc'
Figure 4.3 Janeway's Immunobiology, 8ed. (@ Garland Science 2012)

lg Structure - Hinge Region

The polypeptide chain linking C 1 and C_2 domains forms a flexible hinge

1. This allows the angle between the two Fab regions to vary
2. Some flexibility is also found at the junctions between the V and C domains
3. This enables both Fab arms to bind sites different distances apart and for Fc portions to engage effector mechanisms

(Micrograph X300,000)
Angle between arms is 60°
Angle between arms is 90°
Figure 4.4 Janeway's Immunobiology, 8ed. (@ Garland Science 2009)

Ig Structure - Domain Structure

1. V and C domains form similar structures consisting of ß-pleated sheets arranged as a ß-sandwich
2. A ß-sandwich is 2 ß-sheets lying face-to-face
3. ß-sandwiches are stabilised by S=S bonds
4. The V domain has a 4-strand + 5-strand structure and the C-domain a 4 strand + 3-strand structure
5. These structures constitute the immunoglobulin fold which is found in many proteins including the T cell receptor & MHC proteins (immunoglobulin superfamily)

Antigen Binding Sites - V-domains

1. Made up from the VH and V_ domains
2. V region has ß-pleated sheet structure
3. How is variability produced?
4. Amino acids comprising V-domains show 3 areas of variability designated the hypervariable regions, HV1, HV2 and HV3.
5. For both chains these regions approximately comprise residues:
   HV1 28 - 35
   HV2
   49 - 59
   HV3 92 - 103

Heavy-chain V region
Light-chain V region
50
Variability
00
Variability
40
80
30
60
20
40 -
20
10
0
0
0
20
40
60
80
100
120
0
20
40
60
80
100 120
Residue
Residue
FR1
FR2
FR3
FRA
FR1
FB2
FR3
FR4
HV1
HV2
HV3
HV1
HV2
HV3

Antigen Binding Sites - Framework Regions

1. Remaining residues in V region are less variable and termed the framework regions (FR1, FR2, FR3, FR4)
2. FR regions form ß-strands and the characteristic structure
3. Residues in HV regions form loops BC, C'C" and FG that are adjacent in folded protein
4. When V_ and V, domains associate, HV loops create the antigen binding site

Antigen Binding Sites - Complementarity

1. As they form a surface complementary to the antigen, they are also referred to as the complementarity-determining regions or CDRs (CDR1, CDR2, CDR3)
2. The combination of different H and L chains increases the diversity of antigen binding sites, a concept called combinatorial diversity

Light-chain V region
Variability
50
40
30-
20-
10-
0
. O
0
20
40
60
80
100
Residue
FR1 HV1 FR2 HV2
FR3
HV3 FR4
N
C
N
HV3
(CDR3)
HV1
(CDR1)
C
HV2
(CDR2)
antigen-
binding
site

Antigen:Antibody Interactions

1. The part of an antigen to which an antibody binds is called the epitope
2. Epitopes can be continuous or discontinuous
3. Antigen:antibody interactions are mediated by topological and chemical factors
4. Interactions are stabilised by non-covalent bonds (electrostatic, hydrogen bonds, Van der Waals and hydrophobic interactions)
5. B cells can recognise lipid, polysaccharide, proteins, small molecules

epitope of an
antigen
antigen
Fab
variable
region
Fab
light
chain
variable
region
(V)
constant
region
(C)
(C) constant
region
Fc
heavy
chain
S-S bond

Antibody Function - Classes

1. Five classes (isotypes) of antibody IgM, IgD, IgG, IgA, IgE
2. Differ in:
   no & location of S=S bonds
   no of glycans
   no of C domains
   length of hinge region
3. Two subclasses (allotypes) of IgA - IgA1 and IgA2
4. Four subclasses (allotypes) of IgG - IgG1, IgG2, IgG3, IgG4

IgG

1. Most abundant isotype in serum and extracellular fluid (80% immunoglobulin)
2. Monomer of MW 150 kDa
3. Four subclasses numbered according to serum concentration:
   IgG1
   9 mg/mL
   IgG2
   3 mg/mL
   IgG3
   1 mg/mL
   IgG4
   0.5 mg/mL
4. Subclasses vary in hinge region and number and position of disulphide bonds between heavy chains

IgG - Hinge Structure

The four subclasses of IgG differ in the structure of the hinge
antigen-binding sites
variable
region
hinge
region
constant
region
lgG1
lgG2
lgG3
lgG4
Figure 9.37 The Immune System, 3ed. (@ Garland Science 2009)

IgG - Biological Activities

1. IgG neutralises or opsonises pathogens and activates complement
2. Subclasses have differing biological activities
3. IgG2 and IgG4 are poor activators of complement and poor opsonins - function as neutralising antibodies
4. lgG1 and lgG3 activate complement and phagocytosis - promote inflammatory responses
5. All IgG subclasses cross the placenta via binding to FcRn

IgM

1. Accounts for 5 - 10% serum immunoglobulin
2. Monomeric form on B cell surface
3. Pentamer in serum (MW 900 kDa) linked by S=S bonds between CH4 and C_3 domains
4. Each pentamer has one J chain required for polymerisation
5. IgM is first antibody class to be released during an immune response

IgM
Pentamer
VL
VH
CH
CL
ICH
disulfide
bond
CH3
CHA
J chain

IgM - Binding and Activation

1. Antibodies are low affinity but pentameric structure allows strong binding
2. Bind to viruses and cellular targets and cause neutralisation by agglutination
3. Very efficient activators of complement
4. Low concentrations in extracellular fluid but IgM is found in external secretions

IgA

1. 10 - 15% total serum immunoglobulin
2. Predominant immunoglobulin in external secretions (breast milk, saliva, tears, mucous of respiratory, GU and GI tracts)
3. In external secretions IgA is a dimer or tetramer comprising a J-chain and secretory component (MW 150 - 600 kDa)
4. 5 - 15 g IgA is secreted daily
5. IgA functions primarily as a neutralising antibody
6. Weak opsonin and activator of complement
7. Two subclasses have similar functions but IgA1 is most predominant

Light chain
Heavy chain
J chain
Secretory
component

IgE

1. Low serum concentration (0.3 ug/mL)
2. Monomeric immunoglobulin (MW 190 kDa)
3. Mediate hypersensitivity reactions via binding to Fce receptors on mast cells and basophils
4. IgE triggers release of chemicals that induce reflex reactions (coughing, sneezing, vomiting)

Heavy
chain
Fab
Light
chain
FcERI
receptor
binding site
Fc

IgD

1. B cell surface associated IgD mediates B cell activation
2. Only functions as BCR

88%
Tail Piece

Antibody Responses

Three kinds of antibody response occur

Initial IgM Production

1. Initial IgM production from B1 cells and marginal zone B cells

• T cell- independent (TI) response
• Occurs within 48 hours
• Shared with innate response
• Predominantly low affinity IgM antibodies
• Antigens stimulating TI responses comprise repetitive protein/carbohydrate sequences

Antibody Responses - Early T Cell Dependent

1. Early T cell dependent (TD) response

• From marginal zone and follicular B cells
• Clonal expansion of B cells
• High levels of antibody produced
• Mainly IgM, some lgG
• Produced 3 - 4 days after infection

Antibody Response - Germinal Centre Reaction

1. Germinal Centre Reaction

• TD response
• Forms germinal centre in B cells follicles
• 1-3 weeks to develop fully
• IgG, IgA and/or IgE produced
• Secreted by long-lived plasma cells in bone marrow or other site
• Viable for many years