Lec 5- antibody structure and B cell diversity Flashcards
Abs in summary
- Clear extracellular pathogens and neutralise toxins
- Are produced by activated, effector B lymphocytes (plasma cells)
- Circulate in plasma and lymph and are found at mucosal surfaces
- Are specific- bind 1 antigen
- The antibodies repertoire is high- 1x109 - 1x10(16)
B cells have surface antibodies (known as immunoglobulins)
- Resting B cells with there immunoglobulins
- Encounter with antigen (bacteria binds with immunoglobulins)
- Stimulated B cell give rise to anti-body secreting plasma cells
- There is also helper T cells with MHC
Antibody structure
- Important features
- 2 identical heavy chains (Green)
- 2 identical light chains (there are delta or kappa)
- Variable regions are the tips of the light and heavy chains which form the antigen binding sites
- Constant chain
- Hinge region, this allows flexibility
- Having 2 arms gives our Ab bivalence (this can bind to a bacteria in 2 places or 2 different bacteria)
Ab dissection
-Papain digestion release 3 fragments
-2 FAB (Fragment Antigen Binding)- if w
-1 Fc (Fragment Crystallisable)
Fc region
-Allows Ab to interact with other parts of the immune system
-Needs to be constant
The hinge region gives flexibility
- Withought the ability of the hinge, we would have to find them at a set distance
- However with a hinge we can find the Ag more easily over a larger distance
Structure of human Ig classes
- Heavy chain change (Constant regions) leads to different sub-classes with different functions
- Light chain can either be Kappa or Lambda: no difference if functionality between them
Ig domain in 3d- light chain
- Beta pleated sheets give the Ab structural properties
- Loops give the variability
Antigen binding- hypervariable regions
- HV regions are flanked by framework regions
- There are 3 HV regions in each Variable domain
- Come together at the tip
- Since antibodies are bivalent and the antigen binding region is made of variable regions of heavy and light chains, there are 12 CDR per antibody
Epitopes are exposed on pathogen surfaces
- Ab bind to epitopes (A small part of the whole Ag)
- Most often carbohydrate or protein
- These are the sections of the Ag that the Ab’s variable region will recognise
Ag binding shapes
1 of 4 shapes
- Groove
- Extended surface
- Projection
Epitopes can be linear or conformational
Linear
-Ammino acids are lined up one after another, folding is irrelevant
Conformational
-Folding of the protein Ag has to be folded in a particular way so certain amino acids are next to each other
The challenge for the immune system
- The immune system has to be able to respond to any foreign Ag
- We need to produce a different specific Ab for each of these Ag
- We need one gene for each protein
- But we only have a about <30,000 genes
- And only 1% of these genes are available for Ab production
- Thats only 300 Ab, but the antibody repertoire in humans is 100 billion
Generating Ab diversity
- Immunoglobulin genes are made up of gene segments- pick and mix
- Each set of segments contains alternative versions
- Gene segments must be rearranged to form functional genes
- This occurs during B cell development in the bone marrow
Recombination is random
-Segments are cut and spliced by somatic recombination
-Heavy chains: 3 types of segment; V,D,J
-Light chain: 2 types of segment: V,J
-One segment of each type is brought together to form an intact sequence
+Light chains need one recombinant (VL and JL)
=Heavy chains need 2 recombinant (D joins JH the DJ joins VH0
-All segments are selected at random
Functional sequence are constructed by rearranging gene segments
1)Germline DNA (V, J, C) Somatic recombination 2) Rearranged DNA (V,J, C) transcription 3)Primary RNA transcript (V,J, C) splicing 4) mRNA (V,J,C) translation 5) Polypeptide chain (VL and CL)
Number of gene segments in human immunoglobulin gene: different
Light chains -Varible: K=31-36 and Lambda = 39-33 -Diversity= 0 -Joining: K=5; L=4-5 -Constant: K=1; L= 4-5 Heavy chain -Varible= 38-46 -Divercity= 23 Joining= 6 -Constant= 9
How we create such a large amount of variation
- We have the variable and joining region we want with a large sections of variable DNA that we don’t want to use
- The unwanted DNA forms a loop
- V(D)J recombinase then splices the unwanted DNA
- And the coding region joins
- The cutting of the Unwanted DNA is not accurate to the amino acid this further adds to the variability of the Ab
Immunoglobulins alter after antigenic encounter
-Ag binding to the membrane-bound immunoglobulin triggers
+Proliferation, activation and differentiation
+Secretion of Ab
-Ultimately, activated B cells become plasma cells
-As immune response progress, both Ag binding and effector functions of the Ab will change
Secreted Ab are the same specificity as membrane bound
-After antigenic encounter, soluble IgM is the first antibody secreted (primary immune response)
-Plasma cells have no surface immunoglobulins; they only make soluble Ab
-Differential RNA splicing adds hydrophilic sequence to the tail of soluble Ab’s
+Permits Ab secretion
Somatic hypermutation refines Ag binding
- Somatic hypermutation introduces point mutations to the variable regions of the Heavy and light chain
- NO alterations to the constant region
- Improve or die
- i.e. if the hypermutation has improved the Ab then they will be kept, if the mutations have decreased the effectiveness of the Ab then they will be destroyed
Isotype switching- change of heavy chain
- More DNA recombination, keep the same V region but change the C region
- Removal of DNA between switch sequence
- Same Ag binding BUT different effector function
- 2ndary immune response
