Lecture 2 - B lymphocyte Biology and Disease (2) Flashcards
What is RSS?
Recombination signal sequence: a unique nucleotide sequence that is the RAG recognition sequence, is adjacent to each minigene segment
What is RAG1/RAG 2?
Recombination Activating Genes, essential enzymes for Ig gene recombination, they provide the recognition and DNA cleave activity – to START the process – binds to RSS
What is HMG1?
High mobility group 1, chromatin-binding structural protein important to but not unique to Ig gene rearrangement
What is Artemis/DNA-PK/Ku79/Ku80?
Essential proteins for recognition and synapsis of the DNA ends
What is DNA ligase/XRCC4?
X-ray repair for complementing Chinese hamter cells 4, enzymes responsible for joining DNA ends
What is TdT?
Terminal deoxynucleotidyl transferase, enzyme unique to B cells that adds extra (random) nucleotides to the broken ends at V-D-J junctions (mostly at heavy chain)
What is a basic overview of VDJ?
Immunoglobin genes are composed of separated segments of DNA that become joined together by a process known as somatic recombination/VDJ recombination to make a functional gene.
What do the V,D and J stand for?
V = variable minigene segment
D = diversity minigene segment
J = joining minigene segment
D is exclusive to heavy chains and NOT present on light chains
Describe the process of VDJ recombination?
STEP 1: Selection of V and J regions to recombine
* Each V,D,J gene segment is flanked by RSS specific DNA motifs
* RAG1, RAG2 proteins together form a nuclease that initiates V(D)J recombination by cutting DNA at randomly selected RSS, making double-stranded DNA breaks
* HGM1 enhances binding of RAG1/RAG2/RSS
Cut ends are ligated to form:
1. Coding joint (hairpins)
a. D-J, then V-DJ for Heavy chains
b. V-J for light chains
2. Signal end joints:
a. Loop of DNA deleting all intervening DNA
STEP 2: Coding ends are processed (repaired) before ligation
* DNA protein kinases (DNA-PKs) bind to each broken DNA end and recruits Artemis, Ku70 and Ku80 to form a complex which closes the DNA ends
o Autophosphorylation of DNA-PKs activates Artemis
o Artemis = nuclease that opens coding hairpins (like a scissor) is essential for non-homologous end joining (NHEJ) DNA repair
o Ku70/Ku80 is a dimer that binds double-stranded DNA ends, its role is to form a bridge between broken DNA ends, as structural support to allow access by polymerases, nucleases and ligases to promote end joining
STEP 3: DNA ends are ligated together
* DNA-Protein Kinase catalytic subunits (DNA-PKcs) and XRCC4 (DNA ligase) align DNA ends and recruits TdT enzyme
o TdT = DNA polymerase that adds random (N) nucleotides to coding end in a 5’ to 3’ direction. Continues until complementary sequence is achieved – creates more diversity on top of random VDJ/somatic recombination.
* Exonucleases remove bases from coding ends and DNA polymerases fill in nucleotides compatible for joining
* Processed coding ends ligated together by DNA ligase IV
* Signal joints = well preserved pieces of DNA used to detect the development of B & T cells during screening for PIDs
Why are N-nucleotides most prevalent in V-D and D-J junctions of the heavy chains?
There is no N-region addition at the light chain junctions because TdT is no longer expressed in the Pre-B cells undergoing light chain rearrangement. Yes, there is a reduction in diversity as a consequence.
At what points in Ig Variable gene rearrangement does signalling success occur?
- Ig heavy chain pairs with 2 surrogate light chains (SLC) to form Pre-BCR
a. Pre-BCR sends success signal into the cell which requires signalling proteins downstream of BCR (BtK, BLNK) - Real Ig light chain pairs with the Ig heavy chain to form a complete Ig on surface
a. Ig heavy chain + light chain complex pairs with Iga/IgB to form BCR
b. BCR sends success signal into cell using signalling proteins
What are the final checkpoints before immature B cell is released out of the bone marrow?
- Light chain gene rearrangement has occurred
- Allows coupling of u heavy chain with kappa or lambda light chains on cell surface, BCR
- Btk (Bruton’s TYR kinase) required – signalling protein
What are the population of naïve B-cells in the spleen?
o LN B cells (lymph node B cells): LN B cells are typically involved in the production of antibodies against systemic infections, and they play a critical role in the adaptive immune response.
o MALT (Mucosa-associated lymphoid tissue): MALT B cells produce antibodies that provide protection against infections that enter through mucosal surfaces.
o GALT (gut-associated lymphoid tissue): GALT B cells are a type of MALT B cell that specifically reside in the gut-associated lymphoid tissue. They produce antibodies that provide protection against pathogens that enter through the gastrointestinal tract.
* Here, naïve B cells await antigen (and T cell help) before differentiating further
What is tolerance?
- Since BCR is randomly generated there is a possibility for self-specifcity
- Prior to exiting BM, immature B cells are screened for auto-reactivity
