Lecture 13 - Aby / B Cell

Question 1

Describe the structure of and Aby.

Answer 1

Y-shaped glycoprotein
4 polypeptide chains
2 heavy chains (~50kDa)
2 light chains (~25kDa)
Chains linked by disulphide bonds
Variable region at N terminus (antigen binding) 
2 Fab and 1 Fc region
Hinge region allowing different spatial orientation for Fab portions

Question 2

What are the different antibody isotypes?

Answer 2

GAMED for heavy chain

Kappa / lambda for light chain.

Question 3

What are the main structural difference between the isotypes?

Answer 3

GAD have 3 heavy chain C domains per polypeptide and 1 variable chain. GAD gave a linker / hinge region.
ME have 4 heavy chain C domains and 1 variable chain domain per polypeptide. ME do not have a hinge region and are thus less flexible.
All light chains for GAMED have a single variable and constant domain.

Question 4

Describe an Ig domain.

Answer 4

100-110 aa in length
Vh and Vl domains form the antigen binding site.
Constant domains show little sequence diversity.
Domains contain beta sheets held together by strong hydrophobic interactions between the side chains and disulphide bonds.
The HV loops of the Vh and Vc domains confer specificity.

Question 5

Describe the antigen binding site of an Ig.

Answer 5

The HV loops of the Vh and Vc form this.
AKA paratrope.
Less variable framework regions flank the HV or CDR which provide a brining surface complementary to a specific antigen or antigenic determinant.
3 HV regions per V domain.
Localized variability (HV) within framework (FR)

Question 6

What is an antigenic determinant?

Answer 6

The part of the Ag that binds the Ig.
Epitope
usu carb, protein, both
clusters of aa or a small part of a polysac chain
Antigens usually have many potential antigenic determinants or epitopes.

Question 7

What are the common ways that epitopes bind to paratopes?

Answer 7

The epitope can bind to a pocket/hole, groove, surface, or nob/projection

Question 8

What is the difference between mono and polyclonal antiserum?

Answer 8

Antiserum - serum containing Aby - can be monoclonal (have only one Aby against Ag) or polyclonal (have many Aby against different epitopes of the same antigen)

Question 9

What is the difference between plasma and serum?

Answer 9

Plasma: fluid from blood with cells removed containing Aby.
Serum: clotted blood where cells and fibrin have been removed.

Question 10

What is the difference between a linear and discontinuous epitopes?

Answer 10

A linear epitope binds a linear stretch within an Ag.

A discontinuous epitope binds to aa within an antigen that are not in linear sequence.

Question 11

Describe Germline Ig Genes

Answer 11

The different Ig heavy and light chains are present on different loci on different chromosomes. The light chain kappa(2) and lambda (22) loci contain variable, joining, and constant segments.
The heavy chain locus (14) contains variable, join gin, diversity and constant segments
All loci contain leader peptides.

Question 12

Describe gene segment recombination.

Answer 12

The leader and constant regions do not require recombination thus are ready to transcribe.
V regions coded for by 2 or 3 gene loci require rearrangement.
Junctions between V and J or VJD create diversity and the V region of an Ig.
In the light chain loci, there is a single somatic recombination event to join a V and J chain.
In the heavy chain loci, there are two. A random D then J are joined. A random V in then joined with the DJ segment to create VDJ.
RAG enzymes are involved.

Question 13

Describe the recombination process that occurs within a Ig Gene.

Answer 13

Process involved recombination signal sequences (7 and 9mer) separated by 12 and 23 bp
(7-12-9 and 7-23-9)
A 7-12-9 will only recombine with a 7-23-9.
A RAG unit associates with each region, then comes together to form a RAG complex. The RAG complex then cuts at the 7mer sequence joining the D and J or V and DJ together. Ligase repairs the DNA cut forming the coding joint and excised signal joint.

Question 14

Which form of Ig do naive B cells express and why?

Answer 14

M,D
Full mRNA is transcribed then translated into either M or D via differential mRNA splicing. Expression of one isotype excludes the expression of the other isotype. Differential splicing either cuts out the Cm or Cd exons. Transcription of the G,A,E C regions is not done as TXN stops before these can be transcribed.

Question 15

Describe Ig Assembly.

Answer 15

Ig polypeptides enter ER.
Self-Assemble into Ig mlcs.
Presence of a hydrophobic MC region means Ig will be membrane bound.
Ig associates with transmembrane Iga and IgB which act as signal transducers. IgB and Iga have long cytoplasmic tails that can interact with intracellular signalling proteins.
This association is necessary for membrane translocation of Ig.
Differential splicing created Ig without a MC region and containing a soluble hydrophilic sequence. These will be secreted as Aby.

Question 16

Describe Aby affinity maturation.

Answer 16

This occurs once a B cell has been activated by an antigen.
Somatic hypermutation occurs by introduction of single nt mutations randomly through the V regions by AID (activation-induced cytidine deaminase).
Mutations that confer a better Ag binding will preferentially be activated and undergo subsequent somatic hypermutation.
Progressive selection for Ig that bind an Ag the best is affinity maturation; over time and Aby becomes more specific for a certain ag.

Question 17

Describe isotype switching.

Answer 17

IgM secreted first as a pentamer: the first baby in primary adaptive response.
IgA is secreted as a dimer.
IgG,E,D secreted as monomers.
Isotype -switched Ig will have the same antigen specificity but different effector functions.
Switch regions act similar to RSS and act to excise unwanted DNA and C sections.

Question 18

Describe IgM

Answer 18

First class made in primary adaptive response.
Surface IgM monomeric.
Secreted IgM is pentameric.
No hinge region.
Pentamer requires J chain for assembly.
Has 10 paratopes brining preferentially to pathogens with multiple repetitive epitopes.
Low affinity binding by themselves. Disulphide bonds involved in pentameric form.
Activated Complement.

Question 19

Describe IgA

Answer 19

Dimer of IgA joined by J chain.

Especially present in the GALT

Question 20

Describe IgE

Answer 20

Mast cells, eosinophils, basophils bind IgE in absence of Ag

Type 1 hypersensitivity, defence against parasites.

Question 21

Describe IgD

Answer 21

Common in upper airways.

Binds basophils to orchestrate a local immune response to eliminate pathogens.

Question 22

Describe IgG

Answer 22

Most abundant Ag in blood and lymph.
Binds in a more flexible way than IgM.
Facilitated phagocytosis by acting as an opsonin.
Activated Complement

Question 23

What changes occur to an Ig Gene during the life of a B cell?

Answer 23

V region assembly from a selection of fragments. brings promoter and enhancer closer together so that txn can start.
Junctional diversity by VDJ recombination results from mutations at the site of joining.
Initial alternative splicing of RNA create IgM or IgD,and membrane bound vs secreted Ig.
Activated B cells undergo somatic hypermutation and isotype switching.