Organisation and expression of immune genes (antibody diversity) Flashcards
(25 cards)
Non-immunoglobulin gene structure and expression
Splicesomal proteins come in and loop the RNA and join the needed sections together
Post-translational modifications can occur after the protein is produced – see slide 4
Epigenetics – every diploid cell contains the same DNA but express different gene patterns
Heterochromatin – closed DNA structure not actively transcribed
Euchromatin – open and allows for the expression of this gene by transcription machinery
Cell specific methylation patterns are responsible for these different gene patterns
Methylation and acetylation account for the genome post-transcriptional events
(RNA) editing also accounts for differential gene expression
Immunoglobulin gene structure and expression
IgD is expressed on the B cell surface and engages with the Ag during specific immune response activation
Ig have enormous diversity and there a many B cells expressing different Ag specificities
The number of genes is only in the 100s not the billions
Genetic recombination
Genetic recombination during meiosis 1 creates genetically diverse (2n) daughter cells
Meiosis 2 creates 4 genetically distinct (1n) cells
Immunoglobulin genomic arrangement
Kappa chains have different gene segments
Gamma chains are different
Heavy gain also have a different arrangement
The Greek letters represent the different isotypes on the heavy chain C segments
Organisation and rearrangement of light-chains
2 light chains (kappa and lambda)
The Ig has 2 major light chain domains
Variable region (Vl)
Constant region (Cl)
Vl = 108 amino acid residues
One V gene segment and one J segment are brought together in the genome which combine with a C (constant) gene segment
This is known as V(D)J recombination
VJ recombination of kappa light-chains
First step of recombination is the joining of the V segment to the J segment (VJ complex) - any DNA in between this is deleted
VJ recombination of lambda light-chains
Lambda gene recombination is similar to kappa – it is only expressed when kappa are not recombined correctly
Principle step involves rearranging the genomic DNA to join the V and the J segment together
Organisation of these differ to kappa
There are 4 different four different C(lambda) gene segments than the J segment can associate with
Therefore 4 different lambda chain isotypes produce
Organisation of heavy chains
There are 3 gene segments involved in the heavy-chain variable region – VH (variable), DH (diversity) and JH (joining)
The D and J segments encode for the residues that constitute the complementary determining region 3 (CDR3) of the heavy chainThe mechanism for heavy-chain synthesis using the same gene rearrangements as light-chains
Early stages of B cell differentiation 2 gene arrangement processes occur
One D segment associate with one J segment
One V segment associate with the DJ complex
The new V(D)J complex is closest to C(mew) and C(delta) segments which for the primary transcripts
Alternative splicing of the primary transcript yields 2 different mature mRNA – V(D)J-mew and V(D)J-delta
When resting a B cell may express both IgM and IgD with identical Ag specificity
V(D)J recombination of heavy-chains
1st step of recombination is the joining of a D segment to a J segment (DJ complex)
this joins upstream with a V segment
primary RNA contains mew and delta constant segments
produced through alternative splicing making 2 Ab classes
V(D)J recombination mechanism
Recombination signal sequence (RSS) +/- the V and J gene segments co-ordinate recombination activating gene (RAG) binding
RSS has 3 elements
A conserved heptamer
A less conserved spacer (either 12 or 23 bp)
A 2nd conserved nonamer (5’-ACAAAAACC-3’)
RAG1/2 proteins will bind to DNA at the RSS and mediate VDJ recombination by bringing together (see slide 18)
Step 1 RAG1/2 complexes with the heptamer and RAG1 with nonamer
Step 2 RAG1/2 create a single strand nicks
Step 3 V and J hairpin structures form due to RAG1/2 mutual affinity and a blunt end cut formed
Step 4 ligation of 12 and 23 bp signal end
Step 5 hairpin cleavage
Step 6 overhang extension (palindromic hairpins)
Step 7 ligation of V and J segments
What is the only antibody B cells can produce prior to class switching
Only IgD and IgM
FAB regions do not change upon class switching
Regulation of immunoglobulin gene expression
B cells may create one single Ag specific Ig – VJ (light) and VDJ (heavy) are fixed
This is Ag independent
However B cells can switch class to make different Ig’s –> IgA, IgG or IgE while retaining the same Ag specificity (isotype switching)
This involves rearranging the VDJ gene segment complex
Juxtaposes the rearranged VDJ complex with different C chain gene segment
Isotype class switching
Double stranded DNA breaks occur at consensus regions known as switch-regions – this is up stream of C gene segments
Switch-regions occur adjacent all C genes except delta
Activation induced deaminase mediates at 2 selected switch-regions, eliminating unwanted C genes
Non-homologous end joining links VDJ with the new C region
This only occurs in mature B cells and is Ag stimulation dependent
Factors (like cytokines) that regulate this process are secreted by T-cells
Cytokines are peptides that bind cognate cell surface receptors and alter cell activity and function
Other mechanisms to create antibody diversity
Prescence of multiple V genes in the germ line
This represents the minimum number of different Ig that can be produced and forms the baseline from which Igs are derived
Random assortment of H and L chains
Any distinct H chain may associate with any distinct L chain
Junctional and insertional diversity
The precise position at which the V and J or the V,D and J segments are fused together are not constant –> imprecise DNA recombinations can cause frame shift mutations –> alter amino-acid sequence at the regions –> diversifying further
Somatic hypermutation
After secondary Ag exposure , Ag:Ab affinity increases –> analysis shows SNPs in VDJ region –> fine tuning of the immune system
Bonds involved in antibody-antigen interactions
This depends on 4 types of non-covalent forces
Ionic bonding
Hydrogen bonding – electromagnetic interactions
London dispersion forces
Hydrophobic interactions
Dissociation of the Ab:Ag complex
Low pH (<3.5) alters the protonation and charge depending on residue pKa
High pH (>10.5) - alters protonation and charge depending on residue pKa
High salt concentration (NaCl > 0.3M) causes ionic displacement
Chaotrophic agents (cyanates) interfere with H-bonding in water molecules
Affinity
Strength of total non-covalent interactions between a single Ag binding site on an Ab and a single epitope – affinity of the Ab for that epitope
Low affinity binds weakly and tends to dissociate
High affinity binds tightly and remain associated
Avidity
(co-operation)
The interaction of the Ab and Ag at one site will increase the probability of a reaction between Ab and Ag at a different site
The strength of multiple of such interactions between a multivalent Ab (like IgM) and multiple epitopes of an Ag is called avidity
Valence
The number of epitopes the antibody interacts with – IgG has a valence of 2 and IgM has a valence of 5 (rigid structure does not allow more than this to bind) - a single fab fragment has a valence of 1
Cross reactivity
Interactions between Ag-Ab are usually specific
Cross reactivity can occur with Ag’s that share an identical epitope or if the Ab specific for one epitope also binds to an unrelated epitope possessing similar chemical properties
Precipitation reactions
IgG, IgA and IgM can precipitate antigen at equivalence zone –> solid particle –> phagocytosis
IgG, IgM and IgA can agglutinate whole cells at “equivalence zones”
Haemagglutination –> erythrocytes (ABO blood group)
Polyclonal vs monoclonal antibodies
Antibodies that interact with the same Ag may do so through distinct epitopes (improves avidity)
In polyclonal antiserum, epitope specific Ab’s recognise the same Ag (same Ag, different epitopes recognised)
Monoclonal antibodies interact with the same epitope on the same Ag
Crude generation of polyclonal antisera
Inject rabbit with human Ag of choice (50-1000 µg of AgX – triggers primary response
Repeat after 30 days to induce the secondary response
Adjuvant boosts response
Harvest serum and purify AgX specific antibodies
Affinity chromatography can be used to purify – AgX is immobilised to sephadex resin to form the stationary phase – until antibodies are pulled out
Monoclonal antibody generation
Mouse immunised with AgX
Harvest spleen cells and fused with immortalised myeloma cells (hybridoma)
Expand in cell culture
Screen each cell clone antibody for AgX reactivity
