Adaptive immunity - B cells & antibodies

Question 1

Name 5 key features of adaptive immunity

Answer 1

• Adaptive immune responses are initially slower to develop than innate responses, but show SPECIFICITY and MEMORY.
• B and T lymphocytes recognise ANTIGENS through specific ANTIGEN RECEPTORS
• B lymphocytes acquire their antigen receptors in the bone marrow, but T lymphocytes acquire theirs in the THYMUS
• Single B or T cells express single antigen receptors. Cells (B or T) with appropriate receptors undergo CLONAL SELECTION in response to antigen.
• B and T cell receptors are extremely diverse (>10^9)
• What is the structural and genetic basis of this diversity?

Question 2

What are immunoglobulins

Answer 2

antibodies

Question 3

In which 2 ways may antibodies exist as?

Answer 3

• Integral (intrinsic) membrane proteins on B lymphocytes
  
  • antibodies act as antigen receptors
• soluble proteins secreted by plasma cells
  
  • antibodies act as antigen eliminators

Question 4

Describe the structure of antibodies
Draw this structure with appropriate naming

Answer 4

• often made up of 4 polypeptide chains (4 chain structure)
  
  • often 2 light chains, 2 heavy chains
• Light (L) chain = 25kD : (outer - only on fab arms)
• Heavy (H) chain - 50kD : (innter with hinge)
• Immunoglobulin G (IgG) = L2H2 - 150kD
  
  • linked by disulfide bonds at the hinge region
  • flexible region, allows FAB arm flexibility
• Fab arms, Fc tail. C terminus at Fc tail

Question 5

Describe how they discovered the FAB and FC regions

Answer 5

• Cleavage of antibodies by papain (enzyme) yields two types of fragments
  
  • Hinge region is succesptible to proteolysis, when treated with pepsin enzyme → 2 types of fragments. one was able to bind to antigen monovalently (binds 1 antigen) (FAB)
    
    • other part didn’t bind antigen (Fc)
  • Fragment antigen binding (FAB)
    
    • binds antigen
  • Fragment crystallisable (FC)
    
    • interacts with elements of the innate immune system (antigen elimination)
    • e.g. bind to phagocytes
    • e.g. complement
  • Treating with papain enzyme, cleaves different site → larger bind antigen divalently (bind 2 antigens at once) → called the Fab 2 fragment)
  • The F(ab’)2 fragment can bind antigen divalently, as in the intact molecule.
  • Rodney Porter and colleagues studied immunoglobulin G (IgG), the most abundant class of antibody in serum.

Question 6

Describe the immunoglobulin classes

Answer 6

• There are five classes of immunoglobulin that differ in the amino acid sequence of their heavy chains.
• IgG
  
  • main class in serum and tissues important in secondary responses
  • i.e. when we encounter pathogen for second time
• IgM
  
  • important in primary responses
    
    • the one we make first
• IgA
  
  • alfa
  • in serum & secretions protects mucosal surfaces
    
    • found in mucus, milk etc.
    • most infections occur through mucosal surfaces
• IgD
  
  • not much known
• IgE
  
  • Present at very low levels
  • involved in protection against parasites and allergy
• There are also two light chain types: kappa and lambda
• These are not class restricted i.e. can have IgG-kappa or IgG-lambda light chains but all Fab arms will have the same light chain

Question 7

Using what methods did they discover info about antibodies

Answer 7

• Further information on the structure of antibodies came from protein sequencing.
• used a type of leukemia cells
  
  • Myeloma proteins (myeloma = cancer of plasma cells)

Question 8

What are 3 important features of antibodies discovered from protein sequencing

Answer 8

1. Antibodies contain constant and variable regions
2. Antibodies are comprised of homologous domains
3. The variable region domains contain 3 HYPERVARIABLE regions

Question 9

Describe what V and C regions of antibodies are

Answer 9

• VARIABLE (V) regions - bind antigen. Differ between antibodies with different specificities.
• CONSTANT (C) regions - same for antibodies of a given Hchain class or L chain type.
  
  • important for Fc region, as it can interact with elements of the innate immune system easily as its the same in all antibodies
• V regions of H and L chain make up the antigen binding site

Question 10

What is meant by: Antibodies are comprised of homologous domains

Answer 10

• Regions of sequence homology identified (~110 amino acids each, always including 2 cysteines (thought to be involved for intermolecular disulfide bonds).
• Hypothesized that sequence homology form a series of globular domains, each stabilised by an intra-chain disulphide bond
• Image:
  
  • Domain structure confirmed by X-ray crystallography of Fab and Fc fragments
  • The folding pattern of the domains is known as the Immunoglobulin Fold
  • VL + VH → rest of domains constant
  • hinge region not compactly folded, more extended, lots of proline residues → more susceptible to proteolysis

Question 11

Describe the immunoglobulin fold

Answer 11

• based on exo structure of light chain (simpler)
• each domain is bade up of beta-sheets/strands stabilised by intra-chain disulfide bonds
• C domain has 7 beta strands (2 layers), V domain has 9 beta strands
  
  • slight differences in the structure of constant and variable domains
• Found in all members of Immunoglobulin Gene Superfamily.
  
  • i.e. not only found in immunoglobulins we talk about
• Beta strands for all domains are joined by loop regions (less structured)

Question 12

Describe the Immunoglobulin Gene Superfamily

Answer 12

• lots of the members are involved in immunity e.g. CD4 and CD8, T cell receptor, MHC 1 and 2 proteins too. Found on nervous system too
• Generally involved in recognition, binding, adhesion
• 700 members in the human genome (biggest gene family in humans)
• Domains can be V-like (Ig-V) or C-like (Ig-C)

Question 13

Describe hypervariable regions, where they are found and their importance

Answer 13

• The variable region domains contain 3 HYPERVARIABLE regions
  
  • hypervariable regions mainly involved in binding to antigen (the tip of the variable regions of FAB arm H and L chains are hypervariable)
  THE STRUCTURE OF THE ANTIGEN COMBINING SITE.
  • Image:
    
    • most variable regions in red, correspond to the loops found at the end of the variable region. The hypervariable loops make up the antigen binding site.
  • In red: Hypervariable regions (~ 7-12 amino acids) (3 regions in red)
    
    • separated by the framework regions (less variable)
  • in yellow: Framework regions
  • Hypervariable regions = Complementarity Determining Regions (CDRs)
    
    • reflects the fact that they have to be complimentary to antigen in order to bind

Question 14

Describe the interaction between antibody and antigen

Answer 14

• The interactions between the antibody and antigen are non-covalent (weak)
  
  • (Electrostatic interactions, Hydrogen bonds, Van der Waals forces, Hydrophobic interactions)
  • individually, these interactions are weak, but a lot occurring at the same time results in strong binding
  • (i.e. if the antigen combining site and antigen contain many COMPLEMENTARY residues)
  • Individually weak, but if many form simultaneously (i.e. if the antigen combining site and antigen contain many COMPLEMENTARY residues), the antibody:antigen interaction is specific and of high affinity.

Question 15

What is epitope

Answer 15

The specific part of an antigen that an antibody binds to is known as the epitope.

Question 16

Describe the B cell Receptor

Answer 16

• • NB! different to soluble antibodies as it is inserted into the lipid bilayer
  • Membrane immunoglobulins (antibodies) contain and extra ~ 26 hydrophobic amino acids at the C-terminus.
  • Most B-cells express IgM and/or IgD as B-cell receptors.
    
    • when developing
  • Recognise and bind to antigen, but cannot generate a signal itsself!
  • Membrane immunoglobulins are associated with two other proteins, IgALPHA and IgBETA.
  • Image:
    
    • Iga and Igb contain a single ITAM (Immunoreceptor Tyrosine Activation Motif) in their long cytoplasmic domains.
    • monomer of IgM
    • region that passes lipid bilayer, only a few amino acids ~3 pertrude past the lipid bilayer which is not enough to transduce a signal
    • through the Ig-alpha and Ig-beta which protrude further into the cell allows signalling