L10 – Antibody Structure & Function

Question 1

How is Ig transported in body?

Answer 1

group of glycoproteins in serum and tissue fluids in body:

 Carried on surface of B cells as receptors for specific antigens; or

 Free in blood / lymph antibodies

Question 2

Gross composition/ structure of Ig?

Answer 2

Typical Y-shaped IgG antibodies consist of 4 polypeptide chains:

 2 heavy (H) chains (50 KD)
 2 light (L) chains (25 KD)

 Held by disulphide bonds between hinge regions of 2 H chains + between L and H chains

Question 3

Define the 2 structural regions of Ig?

Answer 3

Both heavy and light chains are composed of constant and variable regions

Question 4

Compare the background variation and function between the 2 structural regions on both H and L chians of Ig?

Answer 4

Constant region:

• Carboxy-terminal sequences are relatively constant between Ig chains (0% background variation)
• Engage in effector functions

Variable region:

• Amino-terminal acid sequences vary greatly (5-15% background variation
• Involved in antigen recognition

Question 5

Describe the variable region on both H and L chains of Ig. (is the genetic variation localized or widespread throughout the variable regions)

Answer 5

In the same position on both VL and VH domains = 3 areas of maximum variation:
»> Hypervariable region (HV)/ Complementarity Determining region (CDR)

The rest of the VL and VH domains with less variation = Framework Regions (FR)

Question 6

Function of the framework regions in the VH and VL chains in Ig?

Answer 6

form the b-sheets for structural framework of the variable domain

Question 7

Function of the hypervariable regions in the VH and VL chains in Ig?

Answer 7

Hypervariable regions from both VH and VL come together to form a single hypervariable surface

> > > Antigen-binding site **

Question 8

2 factors that determine the antigen specificity of an Ig?

Answer 8

1. the amino acid sequences of the V regions (largely determined by the Hypervariable regions)
2. the 3-dimensional shape of the antigen-binding site

Question 9

One Ig has 2 antigen binding sites with different specificity. T or F?

Answer 9

False

One Ab has two Ag-binding sites, with the SAME specificity

Question 10

Compare the gene segments that code for VH and VL chains?

Answer 10

VH:
- encoded by 3 gene segments: V (variable), D (diversity) and J (joining)

VL:
- Encoded by 2 gene segments: V ( variable) and J (joining)

Question 11

One gene with the same exons codes for both the constant and variable regions in an H or L chain in Ig. True or False?

Answer 11

False

Each constant region = encoded by a separate exon

Each variable region = encoded by a cluster of exons*** that form a functional gene

> > > TWO GENES, ONE POLYPEPTIDE CHAIN

Question 12

List the 4 major mechanisms that produce antibody diversity?

Answer 12

Make numerous peptides with diff. V regions:
1. Numerous Ig region genes (inherited)

2. Rearrangement of V-J (light chain) and V-D-J (heavy chain) segments&raquo_space; make primary RNA transcript

Further enhance diversity:
3. Junctional inaccuracies during recombination

4. Reassortment of H and L chains

Question 13

List all the Ig isotypes?

Answer 13

IgG
IgA
IgM
IgE
IgD

Question 14

Describe the organization/ sequence of the light chain locus.

Answer 14

2 types of Light chain locus:

KAPPA = clearly defined V, J and C segments

Lambda = VJCJC repeated segments

Question 15

Describe the organization/ sequence of the heavy chain locus.

Answer 15

Clearly defined V, D, J and C segments

C segments contain different genes (μ, , γ1, γ2, γ3, γ4, ε, α) that determine the antibody type (i.e. μ for IgM)

Question 16

Describe the mechanism of heavy and light chain rearrangement?

Answer 16

V-J and V-D-J segments rearrange:

Gene segments recombine randomly = functional genes encoding many different V regions

> > > primary RNA transcript is produced*****

(i.e. each V segment on kappa chain locus has 40 genes to choose from to make combo)

Question 17

Enzymes involved in the VJ and VDJ rearrangement process?

Answer 17

Initiated by recombination-activating genes (RAG-1, RAG-2)&raquo_space; deletional joining (looping)

Mediated by DNA-dependent protein kinase, DNA ligase&raquo_space; DNA cutting ,rejoining

Question 18

What is the sequence of gene arrangement between heavy and light chains in Ig?

Answer 18

Heavy chain genes rearranged first:

• 1 V + 1 D + 1 J segment recombine
• Pro-B cells: D-J > V > DJ > VDJ

Light chain genes rearranged later:

• 1 V + 1 J segment recombine
• Pre-B cells: V-J > VJ-C

Question 19

Compare the B cell maturation stage in heavy chain vs light chain rearrangement?

Answer 19

H chain = Pro-B cell

L chain = Pre B cell

Question 20

How is Primary RNA transcript of H or L chain of Ig turned into mRNA?

Answer 20

Splicing recombines C region genes with VJ/VDJ complex

> > produce mRNA&raquo_space; polypeptide chain

Question 21

Which isotype of Ig is produced first? How are other isotypes made?

Answer 21

1st transcript is always μ (i.e. IgM)

Later in the immune response, the H chain transcript will change: plasma cell will “switch” to produce a different isotype of Ig

Question 22

Disease caused by Impaired VDJ rearrangement in B, T cells?

Answer 22

severe combined immunodeficiency disease (SCID)

Question 23

Disease caused by defective CD40/CD40L signaling ?

Answer 23

hyper IgM syndrome: frequent infection

Question 24

Describe the process of junctional inaccuracies during recombination.

Answer 24

terminal deoxynucleotidyl transferase (TdT):

cause base substitution / add N-nucleotides at ssDNA recombination junctions

> > cause imprecise joining of V gene segments
100-fold of diversity of variable regions in both H and L chains
10^4- fold increase when paired together

Question 25

Describe the process of resortment of H and L chains.

Answer 25

VH and VL form antigen-binding site

> > combination of different VH and VL regions from different H and L chains
Large number of possible antigen-binding sites
Diversity

Question 26

List the Ig rearrangement events in Pro-B cells?

Answer 26

Intranuclear

μH chain gene rearrangement

Question 27

List the Ig rearrangement events in Pre-B cells?

Answer 27

Intracellular

μH chain expression + L chain gene rearrangement

Question 28

List the Ig rearrangement events in B cells?

Answer 28

Combine H + L chains to make Ig

μH chain always expressed first&raquo_space;> IgM

Question 29

Prevalence, distribution of IgG?

Answer 29

80% of total Ig pool

Distributed evenly between the intravascular and extravascular pool

Question 30

Structure and function of IgG?

Answer 30

• simple 4 chain molecule: 2H + 2L: monomer
• 4 IgG subclasses with different γ chain: IgG1, IgG2, IgG3, IgG4
• Major Ab of secondary immune response
• Maternal IgG can cross placenta for Neonatal immunity

Question 31

Prevalence, distribution of IgM?

Answer 31

10% of pool

Confined to intravascular pool

Question 32

Structure and function of IgM?

Answer 32

• Monomeric form on B cell surface
• Pentameric IgM secreted by Plasma cells (J chain)
• Primary response to Ag
• J chain allow transport across epithelial cells to external secretions

Question 33

Prevalence, distribution of IgA?

Answer 33

15-20% in serum pool

Question 34

Structure and function of IgA?

Answer 34

In serum:
- Monomer (major)
- Dimer or trimer (minor) with J chain 
Secretory IgA:
- Dimer or tetramer + J chain and secretory component 

• External seromucous secretions (i.e. tears, saliva, breast milk…)

Question 35

Prevalence, distribution of IgD?

Answer 35

<1%

Clustered on B cells

Question 36

Function of IgD?

Answer 36

• Ag-triggered lymphocyte differentiation

- Enhance basophil function

Question 37

Prevalence and function of IgE?

Answer 37

Rare in serum, clustered on basophils and mast cells

Type IV sensitivity reaction
Immune response against helminthic parasites