Humoral Immunity and the Generation of Antibody Diversity

Question 1

Describe the tetrameric protein structure of antibody

Answer 1

• 2 identical heavy chains
• 2 identical light chains (either kappa or lambda)

Question 2

Each chain of the antibody has a variable region. What is it for?

Answer 2

• amino acid sequence varies from one Ig molecule to another
• binds antigen (fab - fraction antigen binding)

Question 3

Each chain also has a constant region (Fc) - what is this responsible for?

Answer 3

• for effector functions eg. activating complement, binding to phagocytes

Question 4

What are the complementarity determining regions?

Answer 4

• 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)

Question 5

In the primary structure of the protein the CDRs are separated. What about in the tertiary structure?

Answer 5

• 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

Question 6

Apart from binding a complementary antigen to it’s antibody, how else is a B cell activated?

Answer 6

• direct involvement of CD4⁺ T helper cells (Th1)
• cytokines released by Th1 cells

Question 7

How many Ig genes are inherited?

Answer 7

• 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

Question 8

Describe the segments of the germline kappa light chain gene

Answer 8

• 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

Question 9

Where will an endonuclease enzyme cut on the germline DNA of the kappa light chain gene?

Answer 9

• 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

Question 10

For example, if the endonuclease enzyme cuts after V24 and before J2 what will happen next?

Answer 10

• 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

Question 11

Describe/summarise what happens in the kappa light chain from germline DNA to forming the actual protein

Answer 11

• start with germline DNA
• somatic rearrangement -> Kappa producer DNA - active gene
• transcription -> precursor RNA (nucleus)
• splicing -> mRNA (cytoplasm)
• translation -> K chain (V_xJ_YC_K) - protein (ER + secreted)

(occurs in B cell)

Question 12

How many number of different variable region structures can be generated from the kappa light chain?

Answer 12

• 1C, 35V, 5J
• 1 x 35 x 5
• = 175

BUT modifications to the junction add at least 10x more variation

-> 175 x 10 = >1750

Question 13

What are the events during V(D)J recombination?

Answer 13

1. 1V segment + 1J segment are brought together via their recognition sequences
2. RAG recombinases cut + remove intervening DNA
3. ends are processed before rejoining

Question 14

An exonuclease will mess around with the free ends, before they are ligated together. What will the enzyme deoxynucleotidyl transferase (TdT) do here?

Answer 14

randomly adds a few nucleotides (these are called N nucleotides) to the free ends before they are ligated together

Question 15

How is the most variable region of the antibody created? What is it?

Answer 15

• the V-J joins
• this further variation at the joins create the most variable region of the antibody
• CDR 3

Question 16

What is the importance of TdT?

Answer 16

1. for generating Ig + TCR gene diversity
2. as a leukaemia marker
3. useful enzyme in genetic engineering/recombinant DNA work

Question 17

Which bits of DNA (gene segments) encode which bits of the kappa light chain polypeptide?

Answer 17

• 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)

Question 18

How is lambda light chain gene diversity generated, in comparison to the kappa light chain?

Answer 18

Slightly more complex, but generates a similar number of possibilities

Question 19

For the heavy chain, what gene segments are inherited?

Answer 19

• 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

Question 20

What is the process of gene segment rearrangement for generating diversity in the heavy chain?

Answer 20

• 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

Question 21

Which bits of DNA (gene segments) encode which bits of the heavy chain polypeptide?

Answer 21

• V = variable
• C = constant
• J + D = CDR 3

Question 22

What is allelic exclusion, in terms of heavy chains?

Answer 22

• 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”

Question 23

What is the mechanism for allelic exclusion?

Answer 23

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.

Question 24

What is the mechanism for the T Cell Receptor rearrangement?

Answer 24

Absolutely the same as antibody genes