Lecture 2 - Antibodies and Diversity

Question 1

What is an agonist?

Answer 1

a chemical that can combine with a receptor on a cell to produce a physiological reaction

Question 2

What is the general process of the adaptive immune response?

Answer 2

1) Activation of tissue dendritic cells
2) migration of tissue dendritic cells to the lymph node
3) Activation of antigen specific T helper cells
4) Assist B cells in the production of antibodies

Question 3

What are the advantages of an adaptive immune response?

Answer 3

1) Immunological diversity - immune system has large repetoire of lymphocytes with unique receptors
2) Immunological Diversity - can respond quicker and more effectively to second infection, e.g. vaccines

Question 4

What are the four principles of B and T cell clonal selection?

Answer 4

1) Each lymphocyte has a single type of receptor with a unique specificity
2) Immature cells with potential to be self reactive removed
3) Interaction between antigen and lymphocyte capable of binding activates that lymphocyte
4) daughter cells derived from activated cells have identical receptors to parent cells

Question 5

What is the function of a B lymphocyte?

Answer 5

• neutralises toxins and viruses
• helps in phagocytosis of pathogens
• destroys bacteria and viruses

Question 6

What is the structure of an antibody on a B lymphocyte?

Answer 6

2 heavy chains joined by disulphide bond
Light chains that pair with heavy chains
Hinge region

Question 7

What is the hinge region and what is it’s function?

Answer 7

A flexible domain on an antibody

-makes antibody able to grab onto antigens and form a variety of shapes when binding to other antibodies

Question 8

What are the four forces by which an antibody can bind with high affinity?

Answer 8

1) Electrostatic forces (NH3+ -OOC) -attraction between opposite forces
2) Hydrogen bonds (N-H&+ -&O=C) hydrogen shared between electronegative atoms
3) Van der Waals (electron &-/+ clouds) fluctuations in electron clouds around molecules oppositely polarise neighbouring molecules
4) Hydrophobic forces - hydrophobic groups interact unfavourably with water and pack together to exclude water

Question 9

How did PORTER and EDDMAN discover the chemical structure of antibodies?

Answer 9

Used papin enzyme to cleave antibodies into either Fab2 fragments (2 arms) or into Fab fragments (1 arm)
Separated fragments and showed that Fab2 and Fab fragments were capable of binding to an antigen and that there must be two arms to make Fab 2 fragments

Question 10

What did PORER and EDDMAN identify about antibodies?

Answer 10

The FC domain determines antibody function and identified two chains come together on and antibody and where binding capacity lay

Question 11

How is diversity shown in antibodies at the protein level?

Answer 11

through protein sequencing of antibodies show high level of amino acid variability in hyper variable regions (contain hypervariable loops which stick out and take up different structure) -> different amino acid sequence, different shape IN BOTH LIGHT AND HEAVY CHAINS

Question 12

What is the immunoglobulin repetoire of an adult human?

Answer 12

10^11 different antibodies

Question 13

Why do sharks have an inefficient antibody repertoire?

Answer 13

inefficient process of creating diversity = duplicate the antibody gene leading to only slight differences in structure through gene conversion, requires genome to get hugely bigger

Question 14

What are the regions of the antibody light chain?

Answer 14

VJC Variable Joining Constant region

Lots of V regions, Lots of J regions and a constant region

Question 15

In the human immune system which are the only cells that do not contain lots of V regions + J regions and a constant region?

Answer 15

B cells

Question 16

What are the regions of the antibody heavy chain?

Answer 16

V J D C regions

Question 17

What is the measure of diversity after recombination between V J C regions in light chain and V J D C regions in the heavy chain are combined?

Answer 17

1.8 X 10^6

Question 18

Who discovered the genetic principle for antibody diversity and how?

Answer 18

TONEGWA 1987

B cell DNA different sized bands on southern blot than for germline DNA -> showed that DNA changed size

Question 19

What is the 12, 23 rule that RSS elements function by?

Answer 19

every V, J , D C region has an RSS.
a 23 base pair spaced must bind to a 12 base pair spacer e.g. in heavy chain a V region cannot come together a J region as both have 23 RSS bp

Question 20

Why is there a 12, 23 rule?

Answer 20

Need to get rid of intervening DNA e.g. at a random frequency one, in light chain, of the V regions and one of the J regions come together to form loop type structure, through alignment of the 23 bp RSS and 12 bp RSS which leads to intervening piece of DNA being excised leaving with piece of recombined DNA

Question 21

Why does recombination only occur in B lymphocytes?

Answer 21

requires the RAG1 and RAG2 recombinases only active inB cells

Question 22

How does recombination of light and heavy chains occur enzymatically?

Answer 22

1) RAG binds to DNA and forms a complex with 12 and 23 base pair RSS
2) cleavage of RSS elements and circular interfering DNA
3) leads to closed hairpin ends

Question 23

How is junctional diversity created?

Answer 23

1) nuclease digests hairpin ends to allow them to come together
2) during the process of the pairing of the strands, the TdT enzyme (expressed by B cell) adds random base pairs leading to a different protein sequence
3) exonuclease and ligase repair DNA

Question 24

What is junction diversity?

Answer 24

When diversity is increased through TdT adding base pairs when the hairpin ends created in combinational diversity are processed

Question 25

What two mechanisms create 10^11 levels of diversity found in human adults?

Answer 25

1) Combinational diversity and heavy and hight chain pairing

2) Junctional diversity

Question 26

How does immunological memory confer the ability to respond more effectively to a second encounter with a pathogen?

Answer 26

Affinity maturation through somatic hyper mutation

Question 27

What is affinity maturation (somatic hyper mutation)?

Answer 27

The formation of higher affinity antibodies through somatic hyper mutation

Question 28

What is the process of somatic hyper mutation?

Answer 28

1) during an immune response lots of antibodies are manufactured
2) Transcription induces ssDNA
3) AID enzyme converts cytosine to uridine (transition mutation)
4) UNG enzyme replaces uracil with abasic residue (transversion mutation)
5) EPE1 enzyme induces breaks (Gene conversion)

Question 29

What does RSS stand for?

Answer 29

Recombination signal sequences

Question 30

What does CDR stand for and what is it?

Answer 30

Complimentarity defining region

the hypervariable region of an antibody at the genome level

Question 31

When does somatic hypermutation occur and how is this shown?

Answer 31

Occurs only in secondary infection

• mutations build as secondary infection occurs and cluster around CDR which code for the hyper variable regions
• muations are specifically selected for in CDR to lead to higher affinity

Question 32

Why does somatic hypermutation only occur in secondary immune response?

Answer 32

AID is only expresssed in B cells during an immune reponse