Humoural Immunity - Antibody structure and Function

Question 1

Briefly describe what antibodies are

Answer 1

• Y shaped gamma immunoglobulins made up of 2 x heavy chains and 2 x light chains
• Can be membrane bound or secreted
  
  • Membrane bound is also known as BCR
  • Secreted form is the final fully secreted functional form secreted by mature plasma cells

Question 2

How many domains does the heavy chain have and what are the different classes they can have?

Answer 2

• Heavy chain has four domains (V_H1 + C_H1-3)
  
  • Variable heavy chain domain + constant heavy chain domain
• There are 5 classes
  
  • μ, δ, γ, α, ε
    
    • These can be further subdivided into gamma 1-4 and alpha 1-2 subclasses
    • So in total there are 9 different heavy chains altogether

Question 3

How many domains does the light chain have and what are the different classes?

Answer 3

• The light chain has two domains (V_L and C_L)
• The classes are divided into (κ) kappa and lambda (λ) chains

Question 4

What is the importance of the constant region of the antibody?

Answer 4

• It is important for biological activity
  
  • The constant region is the same for all antibodies of the same class
    
    • E.g All IgM will have the same μ heavy chain and all IgG will have the γ chain

Question 5

How are the heavy and light chains held together?

Answer 5

They are held together through disulphide bonds held together through cysteine residues and intramolecular disulphide bonds to stabilise each of the domains

Question 6

What is the purpose of the hinge region between C_H1 and C_H2?

Answer 6

Provides flexibillity

Question 7

What is the importance of the carbohydrate glycosylations on the antibody?

Answer 7

They will promote the interaction between antibodies and other immune cells

Question 8

What are the two regions of the antibody which were discovered through proteolysis?

Answer 8

• Fab region (fragment antigen binding region) = Made up of variable fragment and first constant region
• Fc region (fragment crystalline) = Constant fragments made up of C_H2-C_H3 domains
  
  • Because this fragment is identical to all antibodies of a particular type it will crystallise in solution hence its name
• Fv region = Made up of variable fragment only

Question 9

What are the regions of the antibody called which directly interact with your antigen?

Answer 9

• Complementarity determining regions
  
  • The antibody is like the hand and the CDRs are like your three fingers
    
    • CDRs is where your antibody will interact with your antigens

Question 10

Summarise the functions of antibodies

Answer 10

1. Variable fragment can bind to the part where the pathogen docks the host cell and prevents the pathogen from entering the cell
2. Antibodies can bind to and active sites of toxins produced by pathogens and neutralise them
3. Opsonisation = tagging of pathogens so that it will become visible to other immune cells (macrophages and NK cells)
   
   • Antibody dependant Cellular Phagocytosis (ADCP) performed by macrophages (engulfs smaller pathogens
   • Antibody dependant Cellular Toxicity (ADCC) performed by NK cells (for infected/ cancerous cells)
4. Antibodies can also forms immune complexes
   
   • Can involve complement proteins
     
     • This will lead to a series of events promoting inflammation, phagocytosis and formation of MAC (membrane attack complex) causing cell lysis

Question 11

What are the different classes of antibodies and summarise the main differences between them.

Answer 11

• 5 Classes
  
  • IgM, IgD, IgG, IgE, IgA
    
    • Each antibody class will express a different heavy chain constant but light chain and heavy chain variable region is the same for the antibodies produced in the same B-cell
• IgM (µ) = Fixes complement, First Ab of primary response, forms immune complexes, monomer serves as BCR
• IgD = BCR indicates mature B-cell, only non-secreting Ab!!!
• IgG = Fixes complement, main Ab of Secondary response, neutralises toxins + opsonisation
• IgA = Secreted into mucous, tears + saliva
• IgE = Allergies and parasites (Fc will bind to basophils and mast cells)

Question 12

What indicates that the B-cell is now a mature B-cell?

Answer 12

When they can express both IgM and IgD which is obtained through alternative splicing of their mRNA (also known as minor class switching)

Question 13

Describe class switching. What is its purpose?

Answer 13

• ONLY AFFECTS HEAVY CHAIN CONSTANT REGION
  
  • Heavy chain variable region and light chain remain the same!
• This is where the Ab chain will change its class
  
  • e.g from IgM to IgD or IgM→ IgG, IgA →IgE, IgG → IgA + IgE
• Purpose = Different effector functions, allows the body to be more versatile in dealing with different pathogens

Question 14

What are the two types of class switching?

Answer 14

• Minor class switching: Differential splicing (mRNA level)
  
  • This occurs between IgM and IgD
    
    • Occurs at the mRNA level (differential splicing)
      
      • Called minor because it does not affect the DNA of the B cell itself
• Major class switching: DNA recombination
  
  • IgM→ IgG, IgA →IgE, IgG → IgA + IgE

Question 15

How is class switching stimulated?

Answer 15

It is carried out by cytokines produced by T-helper cells and indicates the type of pathogen we are dealing with

• An increase of IL-4 will favour a class switch to IgG1, IgG4 and IgE
• An increase of IL-5 and TGF-beta will favour IgA
• We also need a CD40L on T cells which interacts with CD40 on B cells + cytokine signalling = this confirms it is interacting with T-cells
  
  • This occurs in the T-cell dependant B-cell activation stage (of the antigen dependant stage of the B-cell life cycle)

Question 16

What mechanism occurs in major class switching and what does it require?

Answer 16

• Major class switch uses a mechanism called class switch recombination (CSR)
  
  • This requires
    
    • Cytokine signals
    • Switch regions (black diamonds between constant gene regions)
    • AID and DSB repair proteins

Question 17

Describe the mechanism of class switching

Answer 17

1. The B-cell with this genome will be expressing the gene IgM and IgD in two forms through differential splicing of the mRNA
   
   1. It will recieve cytokine signals to class switch to e.g IgA1
2. AID and DSB repair proteins will pull the two class switch regions betwen the Cμ segment and the Cα1 segment together
3. This part will be cleaved and joined into a switch DNA circle while the heavy chain loci will be also be re-joined
4. The V(D)J region plus the constant gene segment (in this case the α1 segment) will be transcribed and translated
   
   • The B-cell will now express IgA1

Question 18

Describe the difference between membrane and secreted Ig

Answer 18

• There are two types of antibodies

1. Secreted form (generated by plasma cells)
2. Membrane bound B cell receptor

• Both the secreted and membrane bound antibody have the same heavy and light chain variable regions and constant region
  
  • The secreted version – has a tail piece
  • Membrane bound – transmembrane region + cytoplasmic tail as an anchor

Question 19

How are membrane bound vs cytoplasmic Abs synthesised?

Answer 19

• The Cμ region which was previously presented as one rectangle is actually made up of μ1-4 with a tail piece located at μ4.
  
  • There is a stop codon and a poly-A tail (poly-A site 1) after the genes coding for the tail piece.
    
    • This is followed by M1 and M2 coding for the transmembrane region and the cytoplasmic tail and another stop codon + poly-A tail
• To form:
• Secreted antibodies
  
  • The whole region will be transcribed into mRNA which is spliced out forming the RNA to code for secreted antibodies
• Membrane bound antibodies
  
  • The whole region up to the second poly-A tail will be transcribed and 8 regions (including genes coding for tail piece and stop signal) will be spliced out