The generation of lymphocyte antigen receptors (Chapter 5): 1. Ig Gene Rearrangement (somatic recombination). 2. Ig Somatic Hypermutation. 3. Ig Class Switching 4. T cell Receptor Gene Rearragement
1. Immunoglobulin gene rearrangement (in the Bone Marrow): - Abs have different specificity and affinity for an epitope. - The antibody repertoire: The total number of antibody specificities available to an individual (10 11 in humans). - Theories for the immunoglobulin diversity: 1. The germline theory: There is a separate gene for each different immunoglobulin chain, and the antibody repertoire is largely inherited. 2. Somatic diversification theory: The antibody repertoire is generated from a limited number of inherited V-region sequences that undergo alteration within B cells during the life time.
In mature B lymphocytes, the V region sequences lie close to the C region by somatic recombination
Generation of immunoglobulins by the somatic recombination of gene segments:
Excluding Pseudogenes (Non-functionalgene segments)
Gene segments for V, J, D and C: μ: IgM γ: IgG δ: IgD α: IgA ε: IgE
Flanking DNA sequences guide V-region rearrangement (V(D)J recombination): 7 hepta 9 nona 12/23 rule: A gene segment flanked by an RSS with a 12-bp spacer typically joins to one flanked by an RSS with a 23-bp spacer
- CDR1 & 2: encoded in the V gene - CDR3: sequences created by the joining of the V and J gene segments (light chain) or the V, D and J gene segments (heavy chain). CDR (=HV): complementarity-determining regions
Same Transcriptional Orientation (Deletional Joining) Opposite Transcriptional Orientation (Inversional Joining)
The molecular mechanism of V(D)J recombination: V(D)J Recombinase 1. RAG-1 and RAG-2 (Recombination Activating Gene): Recognize RSS, RAG-1 has endonuclease activity 2. Ku (Ku70:Ku80) + DNA-Pkc: DNA-PK (Phosphorylates Artemis) 3. Artemis: has nuclease activity to generate ss nicks randomly within the hairpin 4. DNA ligase IV and XRCC4 (X-ray repair comp lementing defective repair in Chinese hamster cells 4: DNA repair enzyme) 5. TdT (Terminal deoxynucleotide transferase): add nucleotide to the ss ends randomly Janeway s Immunobiology, 8 th Ed. Severe Combined Immune Deficiency (SCID): lack of V(D)J recombinase-no RAG, DNA-PK, Ku or Artemis; No or deficient lymphocyte development
Addition or deletion of nucleotides at the junctions between gene segments: Contributes to the diversity of the CDR Addition? 1. P-nucleotides: palindromic sequences added to the ends of the gene segments 2. N-nucleotides: non-template-encoded nucleotides added by TdT Janeway s Immunobiology, 8 th Ed. Deletion? By Unknown exonuclease Addition or deletion of nucleotides may disrupt the reading frame and produce a non-functional protein Nonproductive rearrangement Animation
Ways to generate the diversity of the immunoglobulin repertoire 1. Combinatorial diversity: rearrangement of gene segments for V-region and combination of V H and V L 2. Junctional diversity: addition or deletion of nucleotides at the joints 3. Somatic hypermutation: Generation of point mutations into the rearranged V-region genes
2. Immunoglobulin Somatic Hypermutation (in peripheral lymphoid organs): - Secondary diversification of the antibody repertoire in response to Ag and in aid of T cells to obtain the Affinity Maturation : by Somatic Hypermutation: point mutation into the V region by Gene converstion (some animals): The V region is replace by the V pseudo-gene segment
Enzymes for Somatic Hypermutation: 1. Activation-induced cytidine deaminase (AID): active only in activated B cells, bind to ssdna and deaminate Cystidine to Uridine. 2. Uracil-DNA glycosylase (UNG): the base-exision repair enzyme, removes pyrimidine base to form an abasic site in the DNA. 3. Apurinic/apyrimidinic endonuclease 1 (APE1): excise the abasic site generated by UNG.
1) AID:
2) UNG:
3) APE1:
Purine to Purine or Pyrimidine to Pyrimidine, C to T (non-templated replication, transition is also possible) Purine to Pyrimidine or Purine to Pyrimidine (Using homologous pseudogenes) (V region) (C region)
Gene Conversion:
3. Immunoglobulin Class Switching (or Isotype Switching): -Naïve B cells: Cµ and Cδ (IgM and IgD) -Activated B cells can switch to the expression of C H other than Cμ and Cδ by a type of somatic recombination known as class switching
Human Immunoglobulin Isotypes:
IgM and IgD on B cells IgG, IgA, IgE
A long primary mrna is cleaved at the poly(a) site and spliced to produce IgM and IGD.
Mechanism of Class Switching: - Utilize same machinery for somatic hypermutation - Uses Switch (S) region - S region consists of 150 repeats of (GAGCT)n(GGGGGT) - No AID, No Class Switching R-loops (extended regions of single-stranded DNA) Janeway s Immunobiology, 8 th Ed. dsdna break is introduced by an unknown mechanism
Immunoglobulin: Transmembrane bound or secreted (antibody) MC: Membrane-Coding
SC: Secretion-Coding
IgM and IgA can form polymers: -Disulfide bond between the cysteins of a tailpiece (18aa) in the secreted forms of the μ and α. -Multiple binding sites give the antibody a better total binding strength, or avidity
4. T cell receptor gene rearrangement: - Just like Immunoglobulin (same mechanism) - In the thymus - Simple C region (No secreted form of TCR): one Cα and two β
TCR diversity is generated by 1. combinatorial and 2. junctional diversity 3. No Somatic Hypermutation