L10 – Antibody Structure & Function Flashcards
How is Ig transported in body?
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
Gross composition/ structure of Ig?
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
Define the 2 structural regions of Ig?
Both heavy and light chains are composed of constant and variable regions
Compare the background variation and function between the 2 structural regions on both H and L chians of Ig?
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
Describe the variable region on both H and L chains of Ig. (is the genetic variation localized or widespread throughout the variable regions)
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)
Function of the framework regions in the VH and VL chains in Ig?
form the b-sheets for structural framework of the variable domain
Function of the hypervariable regions in the VH and VL chains in Ig?
Hypervariable regions from both VH and VL come together to form a single hypervariable surface
> > > Antigen-binding site **
2 factors that determine the antigen specificity of an Ig?
- the amino acid sequences of the V regions (largely determined by the Hypervariable regions)
- the 3-dimensional shape of the antigen-binding site
One Ig has 2 antigen binding sites with different specificity. T or F?
False
One Ab has two Ag-binding sites, with the SAME specificity
Compare the gene segments that code for VH and VL chains?
VH:
- encoded by 3 gene segments: V (variable), D (diversity) and J (joining)
VL:
- Encoded by 2 gene segments: V ( variable) and J (joining)
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?
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
List the 4 major mechanisms that produce antibody diversity?
Make numerous peptides with diff. V regions:
1. Numerous Ig region genes (inherited)
- Rearrangement of V-J (light chain) and V-D-J (heavy chain) segments»_space; make primary RNA transcript
Further enhance diversity:
3. Junctional inaccuracies during recombination
- Reassortment of H and L chains
List all the Ig isotypes?
IgG IgA IgM IgE IgD
Describe the organization/ sequence of the light chain locus.
2 types of Light chain locus:
KAPPA = clearly defined V, J and C segments
Lambda = VJCJC repeated segments
Describe the organization/ sequence of the heavy chain locus.
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)
Describe the mechanism of heavy and light chain rearrangement?
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)
Enzymes involved in the VJ and VDJ rearrangement process?
Initiated by recombination-activating genes (RAG-1, RAG-2)»_space; deletional joining (looping)
Mediated by DNA-dependent protein kinase, DNA ligase»_space; DNA cutting ,rejoining
What is the sequence of gene arrangement between heavy and light chains in Ig?
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
Compare the B cell maturation stage in heavy chain vs light chain rearrangement?
H chain = Pro-B cell
L chain = Pre B cell
How is Primary RNA transcript of H or L chain of Ig turned into mRNA?
Splicing recombines C region genes with VJ/VDJ complex
> > produce mRNA»_space; polypeptide chain
Which isotype of Ig is produced first? How are other isotypes made?
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
Disease caused by Impaired VDJ rearrangement in B, T cells?
severe combined immunodeficiency disease (SCID)
Disease caused by defective CD40/CD40L signaling ?
hyper IgM syndrome: frequent infection
Describe the process of junctional inaccuracies during recombination.
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
Describe the process of resortment of H and L chains.
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
List the Ig rearrangement events in Pro-B cells?
Intranuclear
μH chain gene rearrangement
List the Ig rearrangement events in Pre-B cells?
Intracellular
μH chain expression + L chain gene rearrangement
List the Ig rearrangement events in B cells?
Combine H + L chains to make Ig
μH chain always expressed first»_space;> IgM
Prevalence, distribution of IgG?
80% of total Ig pool
Distributed evenly between the intravascular and extravascular pool
Structure and function of IgG?
- 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
Prevalence, distribution of IgM?
10% of pool
Confined to intravascular pool
Structure and function of IgM?
- 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
Prevalence, distribution of IgA?
15-20% in serum pool
Structure and function of IgA?
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…)
Prevalence, distribution of IgD?
<1%
Clustered on B cells
Function of IgD?
- Ag-triggered lymphocyte differentiation
- Enhance basophil function
Prevalence and function of IgE?
Rare in serum, clustered on basophils and mast cells
Type IV sensitivity reaction
Immune response against helminthic parasites
