Humoral Immunity and the Generation of Antibody Diversity Flashcards
Describe the tetrameric protein structure of antibody
- 2 identical heavy chains
- 2 identical light chains (either kappa or lambda)
Each chain of the antibody has a variable region. What is it for?
- amino acid sequence varies from one Ig molecule to another
- binds antigen (fab - fraction antigen binding)
Each chain also has a constant region (Fc) - what is this responsible for?
- for effector functions eg. activating complement, binding to phagocytes
What are the complementarity determining regions?
- CDRs also known as the hypervariable regions
- Parts of the variable region that bind the antigen
- CDR 3 is the most variable region (you can get CDR1,2,3)
In the primary structure of the protein the CDRs are separated. What about in the tertiary structure?
- tertiary structure = 3D structure
- here, the CDRs lie adjacent to each other
- the rest of the V-region forms a framework, allowing the 3 CDRs to face the antigen
Apart from binding a complementary antigen to it’s antibody, how else is a B cell activated?
- direct involvement of CD4+ T helper cells (Th1)
- cytokines released by Th1 cells
How many Ig genes are inherited?
- no complete Ig gene is inherited
- only bits of genes - “gene segments”
- but by arranging these segments in diff combos, many diff Ig sequences can be generated
Describe the segments of the germline kappa light chain gene
- 1 constant region (C) segment
- 35 variable region (V) segments
- 5 short joining (J) segments
The J segments are quite close to the C segment, but the V segments are a long way away on the DNA
Where will an endonuclease enzyme cut on the germline DNA of the kappa light chain gene?
- binding site for the endonuclease enzyme after each V segment
- and in front of each J segment
- so enzyme will cut randomly @ 1 V and 1 J
For example, if the endonuclease enzyme cuts after V24 and before J2 what will happen next?
- free ends are ligated together joining V24 + J2
- this is NOT RNA splicing, but double stranded DNA joining
- V24, J2 and CK now form a functional gene
Describe/summarise what happens in the kappa light chain from germline DNA to forming the actual protein
- start with germline DNA
- somatic rearrangement -> Kappa producer DNA - active gene
- transcription -> precursor RNA (nucleus)
- splicing -> mRNA (cytoplasm)
- translation -> K chain (VxJYCK) - protein (ER + secreted)
(occurs in B cell)
How many number of different variable region structures can be generated from the kappa light chain?
- 1C, 35V, 5J
- 1 x 35 x 5
- = 175
BUT modifications to the junction add at least 10x more variation
-> 175 x 10 = >1750
What are the events during V(D)J recombination?
- 1V segment + 1J segment are brought together via their recognition sequences
- RAG recombinases cut + remove intervening DNA
- ends are processed before rejoining
An exonuclease will mess around with the free ends, before they are ligated together. What will the enzyme deoxynucleotidyl transferase (TdT) do here?
randomly adds a few nucleotides (these are called N nucleotides) to the free ends before they are ligated together
How is the most variable region of the antibody created? What is it?
- the V-J joins
- this further variation at the joins create the most variable region of the antibody
- CDR 3
What is the importance of TdT?
- for generating Ig + TCR gene diversity
- as a leukaemia marker
- useful enzyme in genetic engineering/recombinant DNA work
Which bits of DNA (gene segments) encode which bits of the kappa light chain polypeptide?
- polypeptide means the variable/constant regions
- gene segments = V, J, C
- C codes for constant region
- V codes for most of variable region
- VJ region join to make CDR 3 (most variable region)
How is lambda light chain gene diversity generated, in comparison to the kappa light chain?
Slightly more complex, but generates a similar number of possibilities
For the heavy chain, what gene segments are inherited?
- 1C, 45V, 6J
- also 20 diversity (D) segments
- J&C part are called “H chain”
- number of diff variable region structures that can be generated = 45 x 6 x 20 = 5,400, x10 due to modification so = 540,000 structures
What is the process of gene segment rearrangement for generating diversity in the heavy chain?
- same process as light chain
- but 1 V can join with 1 D and 1 J so more combos
- exonuclease + TdT can add further variation both to V-D and D-J junctions
Which bits of DNA (gene segments) encode which bits of the heavy chain polypeptide?
- V = variable
- C = constant
- J + D = CDR 3
What is allelic exclusion, in terms of heavy chains?
- any B cell is diploid
- it has 2 alleles of the Ig heavy chain gene
- in theory, could make 2 diff heavy chain proteins
- this never happens - “allelic exclusion”
What is the mechanism for allelic exclusion?
As soon as 1 allele successfully rearranged + starts making heavy chain protein, the gene rearrangement process for heavy chains is switched off.
Similarly for light chains as well, B cell only makes either lambda or kappa. Has 2 alleles for each so could make 4 diff light chain proteins but only makes one.
What is the mechanism for the T Cell Receptor rearrangement?
Absolutely the same as antibody genes
