Immunoglobulins

Immunoglobulin
Je_at_uwo 2006, WikiMedia Commons

Introduction

Also called antibodies

Immunoglobulins (Ig) are the soluble form of B cell receptors (BCR) released by plasma cells after they have been activated. Immunoglobulins have to bind to a number of different antigens in a variety of environments and as such there are several different immunoglobulin classes. Each class has an optimum environment of action.

Structure

Immunoglobulin
Asher Allison 2008

Antibody consists of two identical heavy (H) chains and two identical light (L) chains
- Each H chain is linked to an L chain
The heavy chain consists of 4 or 5 protein domains
- 1 amino terminal variable domain and 3 or 4 constant domains
Each light chain has one variable and one constant domain
Antibody is "Y-shaped"
- Each arm of the molecule consists of one L chain and part of an H chain
- There are two identical antigen-binding sites on each antibody molecule
  - One at the tip of each arm
  - Made up of a combination of the variable domains of 1H and 1L
Single cells only have receptors of one specificity
The antigen binding site of antibody is determined by a combination of the amino acid sequence of both the heavy and light chain variable domains (VH and VL)
- Antibodies of different specificities have different amino acids within one of three regions of the variable domains
  - These are called the hypervariable regions
- The variable domains are folded in such a way as to form a pocket into which antigens fit
The constant domains do not bind antigen
- They determine the biological functions of the antibody molecules
At the carboxyl terminus of B-cell expressed Ig is a region that holds the antibody molecule in the cell membrane
- Secreted antibodies do not possess this region
The Fab fragments of the immunoglobulin is the antigen binding fragment generated from the light chains when digested by papain (proteolytic enzyme)
The Fc fragment is the fragment produced by papain from the heavy chains
- The Fc fragment mediates phagocytosis, triggers inflammation and targets Ig to certain tissues.

Function

Antibody binds 2 whole antigen molecules - B. Catchpole, RVC 2008

For an overview of the role of immunoglobulins in the adaptive immune response, see here
All antibody classes bind antigen in a similar manner:
- The initial trigger for all antibody function is the association of antigen with the antigen-binding sites of the immunoglobulin molecules
However, different classes serve different physiological functions
Functional differences between antibody classes are reflected in the structural differences in their heavy chain constant regions
- These regions comprise the effector domains of antibody molecules
Antibodies have four major effector functions:

Blockade and agglutination

The action of antibody binding to antigen can be protective
Antibody may bind to biologically active sites on toxins, inhibiting their binding sites
In a similar manner, antibody can bind the surface of viruses and prevent them from infecting cells
Antibody molecules are at least divalent, and many are greater than divalent
- This means that antibody can cross-link antibody: agglutination
  - The ability to cross-link can be the primary function of the antibody
In order to be pathogenic, many micro-organisms must be able to contact viable host cells
- If antibody molecules agglutinate these micro-organisms, cell contact is prevented
  - The pathogenic effect is removed or reduced
The main biological function of IgA is to cross-link potentially pathogenic molecules

Promotion of phagocytosis

Phagocytes hace receptors for the Fc portion of antibody
- Fc receptors - FcR
FcR bind the Fc portion of antibody on antigen/antibody complexes
- Antibody alone does not interact with FcR
Interaction of FcR with Ab/Ag complexes stimulates:
- Cellular activation
  - Causes greatly increased efficiency of phagocytosis - opsonisation
- Production of intracellular enzymes
  - Promotes the killing and digestion of internalised micro organisms
  - Systems important in intracellular killing include:
    - Free radicals
      - Oxygen and chlorine free radicals
    - Hydrogen peroxidase
    - Nitric oxide
      - Produced by nitric oxide synthase
- Secretion of certain enzymes and cytotoxic molecules
- The degranulation of phagocytes
These systems are also activated by the interferons, especially the immune interferon – IFNγ
Antibody-mediated opsonisation of micro organisms is much more efficient that innate receptors
Phagocytes also have receptors for iC3b, a complement component
- A major initiator of opsonisation

Degranulation of mast cells/ eosinophils

Complement fixation

Classes

There are five classes of Immunoglobulins, which vary due to the composition of their heavy chains. These are common across mammals but subclasses of them do vary between species. IgY is present in yolk.

	IgA	IgD	IgE	IgG	IgM
Weight (kDa)	360	180	200	180	900
No. subunits	2	1	1	1	5
Heavy chain	α	δ	ε	γ	µ
Mainsite of production	Alimentary/Respiratory tracts	Spleen/Lymph nodes	Alimentary/Respiratory tracts	Spleen/Lymph nodes	Spleen/Lymph nodes

Variation

Subclasses

Subclasses of the five major classes also exist. These occur in the heavy chains and are coded for by the IGH gene. For examples horses have six subclasses of the IgG immunoglobulin coded for by the genes IGHG1→ IGHG6 while cattle and sheep only have three subclasses (IGHG1→ IGHG3).

Within the IgG class there are several subclasses depending on the species
- Humans and rodents have four IgG subclasses
- Dogs have three subclasses
- Ruminants have two or three subclasses
In some species there are also two IgA subclasses
Specificity is unrelated to class
- Different classes of antibody can be associated with the same V domains and have the same specificity
- It is possible for a single B-cell to produce antibody of one specificity but two or more classes
  - A mature B-cell has the genetic capacity to produce antibody of all classes and subclasses but of one specificity only

Allo & Idiotypes

Allotypes are differences in immunoglobulins between individuals of a particular species. They are inheritable. Idiotypes are variations in the sequences of amino acids in the variable regions of the light and heavy chains.

Immunoglobulin Class Switching

Different classes of antibody differ from each other in:
- Size
- Charge
- Amino acid composition
- Associated carbohydrate
- Function
Immunoglobulin heavy and light chains are encoded by separate genes
- The first 300 bases of each gene encodes the variable part of each protein chain
  - The combination of the VH and VL contribute to the antigenic specificity
    - Any individual has the capability of producing over 100 million different antibody specificities
- The rest of the antibody gene encodes all the constant domains.
  - There are four different types of heavy chain constant domains - μ, γ, ε, α
    - Different heavy chains are associated with different classes of antibody
      - μ chain - IgM
      - γ chain -[ [IgG]]
      - ε chain - IgE
      - α chain - IgA
  - In humans and mice there exists a fifth Ig class, IgD (δ chain)
    - IgD is always expressed on B-cell membranes with IgM but is never secreted
The sequence of constant region genes on the chromosome is M, G, E and A
- In humans/ rodents the Cδ (IgD) is immediately downstream of the M gene
Prior to antigenic stimulation, B-cells express cell membrane-associated IgM
- The first immunoglobulin produced during an immune response is always IgM because the Cμ gene is the first constant H chain gene downstream of the the variable domain
After antigenic exposure the IgM+ B-cells differentiate
- Begin to synthesise other classes of immunoglobulin
  - Only under the direct influence of a T-cell
  - Known as immunoglobulin heavy chain switch
- Retain the same variable domains expressed by the parental B-cell
There are also two possible light chains
- There are no functional consequences for either type
The IgG antibodies produced in both primary and secondary responses originate from the same clones of B-cells as the IgM antibodies
The different antibody classes have the same variable domain combinations, and therefore the same antigenic specificity
- Under the influence of T-cells, B-cells undergo immunoglobulin heavy chain switch
  - The B-cells mature from IgM to IgG producers
IgE and IgA producers can be generated
- These immunoglobulins tend to be produced within lymphoid tissue associated with mucosal surfaces

Clinical Uses

Vaccination

Immunological Testing