Antibody Diversity Flashcards
chains of antibodies
- 2 heavy chains
- 2 light chains
- identical
domains of antibodies held together by
- disulfide linkage
secreted IgG antibody:
- L chains
- H chains
How many Ig Domains?
- 2
- 4
variable region
- sequence varies among different antibodies
- antigen binding pocket
constant region
- sequence is the same among different antibodies
light chains (VL and CL)
1 VL + 1 CL for each chain
heavy chain (VH and CH)
- 1 VH + 3 or more CH
CL domain functions
- no effector functions
hypervariable regions
- CDR1
- CDR2
- CDR3 (most variability)
- complementarity-determining regions
how do hyper variable regions interact with antigen
- stick away from the protein so they can contact antigen
each antigen binding pocket consists of
- VH and VL chain
each antibody has how many antigen binding pockets?
- 2
IgA in what form?
- dimer
- connect by J chain
H chain isotypes
- IgG
- IgA
- IgM
- IgE
- IgD
L chain isotypes
- kappa
- lambda
secreted and membrane associated antibodies differ how
- at the C-terminal tail
chain structure of T cell receptor
- has two chains
- alpha-beta
- gamma-delta
- each chain has a V and C region
CDRs in variable region of T cell receptor
- bind antigen
T cell receptor recognizes antigen in what context
- context of MHC
mechanisms of antibody diversity
- Multiple VH and VL genes
- combinatorial association of different V,D, and J gene segments
- junctional diversity by nucleotide addition
- combinatorial association of VH and VL regions
- somatic hypermutation
heavy chain gene
- V
- D
- J
- C
light chain gene
- V
- J
- C
VH CDR1,2 derived from
- VH segment
VH CDR3 derived from
- DH and JH segments
VL CDR1,2 derived from
- VL segment
VL CDR3 derived from
- JL segment
leader sequence on each V gene segment
- codes for N-terminal signal peptide
- targets the proteins to the ER
VDJ recombination
- two gene segments brought together and double strand break made
- somatic recombination of V-D-J joining
- N and P nucleotides added or removed and ends ligate together
- C regions separated from variable regions by introns
- DNA splicing of introns bring exons back together
what mediates the recombination
- recombination signal sequences
- heptameter separated by 12 or 23 nt spacer
- followed by AT-rich nonamer
prime ends of gene segement
- 3’ end of V segment
- 5’ end of J segment
- both sides of D segment
- think 3’VDJ5’
which segments can pair up
- only 12 bp segment with a 23 bp segment
V(D)J recombination by deletion
- deleting D in this case
- 12 bp segment synapses with 23 bp segment
- ensure D segment not skipped or that two of the same segment don’t join together
- cut between gene segment and heptamer
- ligate gene segments together
V(D)J recombination by inversion
- intervening DNA not removed by a circle but remains present
- RNA splicing remove intervening sequences
rearrangement and transcription
- rearrangement brings enhancers close to promoter allowing transcription to occur
- enhancer can be at 3’ end or in intervening sequence
non-homologous end joining
- uses 1-3 nt single stranded regions of homology to enhance repair
non-homologous end joining synapsis
- chromatin opens in developing lymphocyte to allow recombination enzymes access to DNA
- chromosome looping brings gene segments together
RAG1 and RAG2 in synapsis
- form a complex and recognize RSS sequences
- RAG2 binds to hypermethylated H3K4 sites in chromatin and recruits/activates RAG1
non-homologous end joining; cleavage
- RAG1 nicks one strand
- single stranded DNA forms hairpin with other strand
non-homologous end joining; hair pin opening end processing an joining
- Ku70/80 binds to break
- recruits DNA-PK (protein kinase)
- DNA-PK phosphorylates and activates Artemis endo-exonuclease
- Artemis endonuclease opens the hairpins
- DNA polymerase fills in nucleotides (P nucleotides)
- TdT (terminal transferase) adds nucleotides onto broken ends (N nucleotides)
- DNA ligase joins ends together
P nucleotide addition
- hairpin cleavage and repair
- allows nucleotides to be added to junction by DNA polymerase
N nucleotide addition
- terminal transferase adds additional nucleotides at junction
why is there greater diversity at CDR3
- junctional diversity
- multiple gene segments
importance of CDR3
- most important region for antibody binding specificity
allelic exclusion
- if one chromosome undergoes rearrangement successfully, it inhibits rearrangement on the other chromosome
- each B cell makes only one antibody
allelic exclusion: chromatin on the successful chromosome
- open
- acetylated
allelic exclusion: chromatin on excluded locus
- heterochromatized
- methylated
light chain isotype exclusion
- if the kappa locus undergoes a productive rearrangement, it inhibits lambda rearrangement
how does higher affinity arise
- due to somatic hypermutation in V region by CD40:CD40L signaling
- at DNA level
- during an immune response, B cells produce antibodies that progressively have a higher affinity for antigen
mechanism of class switching
- cytokine stimulation triggers transcription through I exon, switch region, and CH exons
- different cytokines dictate which CH gene is switched to
- double stranded breaks occur at mu switch region and downstream switch region
- intervening DNA lost
- RNA splicing yields transcript with new CH
activation-induced cytidine deaminase activated by
- activated by CD40
activation-induced cytidine deaminase process
- R loop on nontemplate strand
- AID converts C’s to U’s by deamination
- Uracil-N-glycosylase removes U residues
- Ape1 endonuclease nicks regions without bases
- some nicks generated on the template strand
- double strand breaks into two switch regions and repair by non-homologous end joining
in proliferating B cells, mutation rate for which genes is 1000x higher than for other genes
- V genes
- due to somatic hypermutation
As somatic mutation of antibody increases
- Kd decreases
- which means affinity for antigen increases
mechanism of somatic mutation
very similar process to isotype switching
- AID converts C-> in U in V region
MECHANISM - U’s changed to T’s (C->T mutation)
OR - Uracil-N-glycosylase exists bases and repairs with an error prone repair system
OR - mismatch repair enzymes remove the U nucleotides and replaces surrounding DNA using an error prone DNA polymerase which generates mutations
why do membrane versus secreted forms of antibodies differ in C terminal tails?
- due to RNA splicing
antibody membrane form structure
- Cu4 followed by hydrophobic tail and cytoplasmic tail
antibody secreted form structure
- Cu4 followed by hydrophilic tail piece
- utilizes a poly A cleavage site before the exons encoded the hydrophobic and cytoplasmic tail so those exons aren’t included in primary transcript
difference of T cell receptor diversity and antibody diversity
- T cell receptors DO NOT UNDERGO SOMATIC MUTATION
severe combined immunodeficiency (SCID) caused by
- mutations in recombination enzymes
- RAG
- Artemis
- DNA-PK
- DNA ligase 4
SCID result
- complete loss of B and T cells
Omenn syndrome
- less severe than SCID
- due to mutations with reduced function of RAG and Artemis genes
Hyper IgM syndrome caused by
- genetic defects in AID or uracil-N-glycoslyase
- defective in class switching and somatic mutation
- ALSO CAUSED BY DEFECTS IN CD40
Hyper IgM syndrome result
- all antibody is IgM
- susceptibility to infection
AID deficiency in somatic mutation
- completely defective
AID deficiency in UNG deficiencies
- preserved but reduced
lymphoid tumors and translocations
- B and T cell tumors commonly have translocations of oncogenes into antibody loci
OR
- RAG-RAG and RAG-AID translocations
lymphoid tumors and translocations process
- oncogenes suffer double stranded break by AID at sequences related to switch site
- join to AID induced class switch recombination break in antibody gene locus
lymphoid tumors and translocations result
- over expression of oncogene in B or T cells due to active antibody or TCR promoters
