L09: BCR and TCR structure and generation of diversity Flashcards
BCR and TCR - antigen receptors of B and T cells
- Structurally and evolutionarily related
- Both have variable region which binds antigen and constant region
- BCR has secreted form of Ab called immunoglobulin.
- BCR contains hydrophobic C terminal which anchors it to membrane
- Each B/T cell has BCR/TCR single binding specificity
Antibody structure
Basic structure: 2 identical heavy chains (50-77kD) and light chains (25kD) linked by disulphide bonds. 2 important domains separated by hinge region - Fab (fraction antigen binding) and Fc (fraction crystallisable) generated with protease digestion.
Antibody classes
Defined by constant region. Ig exists in 5 different Ig classes/isotypes which differ in use of heavy chain constant region and have different functions - IgM, IgD, IgG, IgA, IgE.
Ig variable region
Hypervariable (HV1, HV2, HV3)
Complementarity determining regions (CDR1, CDR2, CDR3) - regions comprise loops which dominate the atnigen recognition site, in both heavy and light chain. Loop cluster on one end of site make up antigen binding sites.
B/T cell diversity (4)
- Random recombination of V, D and J gene segments (cutting and joining of DNA in developing B and T cells to generate diversity
- Insertion of extra nucleotides at recombination sites
- Combinations of two different chains to make Ag-binding site
- Somatic hypermutation - in B cells only, extra random mutations occur after B cell activation which are selected for if they increase the affinity for antigens
Immunoglobulin Gene Loci
Rearranged in B cells through somatic recombination. Two loci encoding light chains (kappa/lambda) and one for heavy chain. In light chains, one recombination to give V-J joining. In heavy chain, there is D-J joining, then V-D joining to give recombined V-D-J
Functional gene segment calculation
Number of H chain combinations Number of kappa chain combinations Number of lambda chain combinations H and kappa combinations H and lambda combinations
12/23 rule in heavy chain
DH can be joined to VH and JH
VH cannot join to JH
VH cannot join to another VH
Recombination with Rag1/Rag2
RAG = recombination activating genes, only expressed in lymphocytes. Helps fibroblasts rearrange DNA. Deficiency of RAG genes leads to lack of T and B cells (SCID). Coding joint leads to productive CDJ recombination. Signal joint contains RSS (recombination signal sequences) forms a circular molecule which is lost from the chromosome. Artemis cleaves the hairpin and TdT randomly adds nucleotides to the junction.
Junctional diversity
- Formation of hairpins mediated by Rag complex
- Artermis nuclease cleaves DNA hairpin at random site near the hairpin to yield a ssDNA end - the P nucleotides - more diversity
- TdT randomly adds nucleotides to ends - the N nucleotides - more diversity
- Nucleotide removal can also occur
- Many rearranged receptors are non-functional. Insertion of nucleotides puts the J region out of frame 66% of time. The D region can usually be read in 3 reading frames.
- B cells with both BCR heavy chain loci or all 4 light chain loci rearranged to yield non-functional proteins will die.
P & N nucleotides
P nucleotides - generated by cleavage of artermis
N nucleotides - random nucleotides added by TdT
Somatic hypermutation
- Introduces point mutations into the V regions of rearranged heavy and light chain genes
- Occurs in germinal centre of secondary lymphoid tissues where B cells are responding to antigen (with aid of Thelper cells)
- Activation-induced cytidine deaminase (AID) converts cytosine to uracil in DNA. Mutations are introduced during DNA repair
- Base changes are distributed along V region
- Resulting new BCR sequences are tested for binding to Ag
- Outcomes: mutations may make BCR non functional, increase affinity for Ag and amino acid changes clusters in the CDRs
