genetics of diversity

1. GENETICSOFANTIBODYDIVERSITY
Submitted By: Surekha Hirdesh
2204641
GD (ZOM-805)

2. Antibody structure and function
◦ Antibodies recognize foreign material and trigger its elimination.
◦ They are Y‐ or T‐shaped molecules
◦ The molecule (Fab) recognize foreign material and the stem (Fc)
interacts with immune molecules that lead to the elimination of the
antibody‐decorated foreign material.
◦ Antibodies are based on a four‐chain structure consisting of two
identical heavy chains and two identical light chains.
◦ Differences in the Fc regions lead to different classes and subclasses
of antibodies or immunoglobulins (Igs).

3. Immunoglobulin classes
1. IgG - It is monomeric and the major antibody in serum and non-mucosal tissues, where it inactivates
pathogens directly and through interaction with triggering molecules such as complement and Fc receptors.
2. IgA - It exists mainly as a monomer in plasma, but in the seromucous secretions, where it is the major Ig
concerned in the defense of the external body surfaces, it is present as a dimer linked to a secretory
component.
3. IgM - It is most commonly a pentameric molecule. It is essentially intravascular and is produced early in the
immune response. Because of its high valency it is a very effective bacterial agglutinator and mediator of
complement‐dependent cytolysis and is therefore a powerful first‐line defense against pathogen.
4. IgD - It is largely present on the lymphocyte and functions together with IgM as the antigen receptor on naive
B‐cells.
5. IgE - It binds very tightly to mast cells and contact with antigen leads to local recruitment of antimicrobial
agents through degranulation of the mast cells and release of inflammatory mediators. IgE is of importance in
certain parasitic infections and is responsible for the symptoms of atopic allergy.

4. Genetics of Antibody Diversity
Antibody diversity is the ability of the immune system to produce a wide range of different antibodies,
each with a unique binding specificity for a specific antigen.
This diversity is essential for the immune system to be able to recognize and respond to a vast array of
pathogens and foreign substances
1. SOMATIC RECOMBINATION - Immunoglobulin genes are composed of variable (V), diversity (D),
and joining (J) gene segments.
These gene segments can be randomly combined during the development of B cells to produce a
unique antibody molecule. The process of combining these segments is known as V(D)J recombination,
and it is the primary mechanism for generating antibody diversity.
2. SOMATIC HYPERMUTATION - During somatic hypermutation, B cells undergo random mutations
in the variable region of their antibody genes, resulting in changes to the amino acid sequence of the
antibody.
These mutations can increase the affinity of the antibody for its target, allowing for more efficient
recognition and elimination of the foreign substance.

5. 3. CLASS SWITCHING - It is a process that occurs in activated B cells, where the constant region of
the antibody gene is altered to switch the type of antibody produced from one isotype to another.
This process allows the immune system to produce antibodies with different effector functions that are
better suited for different stages of an immune response.
Class switching occurs through a process called recombination, which is mediated by the enzyme
activation-induced cytidine deaminase (AID).
The mechanism of class switching is controlled by signals from the surrounding environment, such as
cytokines and other immune system proteins, which direct the B cells to switch to the appropriate
isotype for the type of pathogen encountered.
◦ The combination of V(D)J recombination, somatic hypermutation and Class switching allows the
immune system to generate a vast repertoire of different antibodies, each with a unique binding
specificity for a particular foreign substance.
◦ This diversity is essential for the immune system to recognize and respond to a wide range of
pathogens and foreign substances such as viruses, bacteria, and toxins, and marking them for
destruction by other immune cells.

6. The immunoglobulin variable gene segments and loci
3 sets of Immunoglobulins chain
◦ Kappa (κ) light chain
◦ lambda (λ) light chain
◦ heavy (H) light chain
3 Immunoglobulin Loci
◦ Kappa (κ) light chain locus – Chromosome no. 2
◦ lambda (λ) light chain locus – Chromosome no. 22
◦ heavy (H) light chain locus – Chromosome no. 14

7. Human immunoglobulin loci

8. 1. V – Variable gene segment
◦ At 5’ end of each locus a cluster of Variable gene segment is present
◦ It code for the portion of V region of Ig chain
◦ The no. of Variable gene segment in each locus is different
◦ Kappa (κ) light chain – 40 Variable gene segment in light chain
◦ lambda (λ) light chain – 30 Variable gene segment in light chain
◦ heavy (H) light chain – 40 Variable gene segment in light chain
 L - Leader sequence
◦ Each variable segment is associated with leader sequence at it’s 5’ side
◦ leader sequence encodes a signal or leader peptide that facilitates the transport of Ig protein through
endoplasmic reticulum
◦ leader sequence are later cleaved and therefore they are not present in mature Ig protein.

9. 2. J – Joining gene segment
◦ At 3’ end of cluster of V gene segment is present the cluster of Joining gene segment.
◦ Joining gene segment also encodes the portion of variable region of the Ig chain
◦ Distance between cluster of V & J segment varies at each locus.
◦ Kappa (κ) light chain – 5 Joining gene segment
◦ lambda (λ) light chain – 4 Joining gene segment
◦ heavy (H) light chain – 6 Joining gene segment
3. D – Diversity gene segment
◦ Additional gene segment are present between V & J gene segment called Diversity gene segment
present in case of heavy chain locus
◦ Diversity gene segment also encodes some portion of variable region of heavy chain along with V & J
gene segment.
◦ 23 Diversity gene segment are present on heavy chain locus.

10. Constant region genes organization
◦ Constant region genes located at 3’ end of the J gene segment in each Ig loci
◦ The number and rearrangement of Constant region genes vary in each case
1. Kappa (κ) light chain locus – single Cκ gene which encodes the entire constant region
◦ No subtype of κ light chain are found
2. Lambda (λ) light chain locus – 4-5 Cλ are present
◦ Each J gene segment is associated with 1Cλ gene
◦ Presence of multiple C gene in case of λ give rise to subclasses of λ chain
3. Heavy (H) light chain locus – 9 CH are found
◦ Arranged in following order Cμ Cδ Cγ3 Cγ1 Cα1 Cγ2 Cγ4 Cε Cα2
◦ C genes are arranged in order they appear in an immune response

11. V(D)J recombination
A process by which B- cells randomly assemble Ig gene segments (i.e V, D & J gene segment) to from
functional variable region exon
This process occurs during early development of lymphocytes as we know each Ig loci has multiple
gene segment and these can be rearranged in many possible combinations, this contributes to Ab
diversity.
The process of V(D)J recombination involves the recognition and cleavage of the recombination signal
sequences (RSSs) located at the borders of the V, D, and J gene segments by the recombination-
activating genes (RAGs) enzymes. This cleavage creates a double-strand break at the border between
the gene segments.
The broken ends of the DNA are then brought together and joined by non-homologous end-joining
(NHEJ) enzymes, resulting in the formation of a functional V(D)J exon.
The V(D)J exon encodes the variable region of the Ig, which determines the antigen-binding specificity
of the receptor.

12. V(D)J recombination of light chain
◦ The process of V(D)J recombination for the light chain of immunoglobulins involves the rearrangement of variable (V)
and joining (J) gene segments.
◦ Unlike the heavy chain, there are no diversity (D) gene segments involved in the light chain rearrangement.

13. STEPS
1) The RAG complex recognizes the recombination signal sequences (RSSs) located at the border of the V and J
gene segments in the light chain locus.
2) The RAG complex introduces a double-strand break at the border between the V and J gene segments by
cleaving the DNA at the RSSs.
3) The ends of the broken DNA strands are then processed and modified by a series of enzymes, such as DNA
polymerases and nucleases, to form blunt or staggered ends suitable for rejoining.
4) The recombination process is completed by non-homologous end joining (NHEJ), a repair pathway that joins
the ends of the broken DNA strands together to form a functional VJ exon. This process can involve the
trimming or addition of nucleotides at the junctions to generate diversity in the light chain sequence.
5) The newly formed VJ exon is then transcribed and spliced to produce a functional light chain mRNA, which is
translated into a mature light chain protein.
6) The mature light chain protein is assembled with the heavy chain protein to form a complete immunoglobulin
molecule, which is secreted by the B-cell and can recognize and bind to specific antigens.

14. V(D)J recombination of heavy chain
 V, D & J gene segment encodes the variable domain of Ig heavy chain
 Two gene rearrangement events ---- 1. D-J joining 2. V- DJ joining

15. STEPS
1. Recognition of the RSS sequences:
◦ The RAG complex recognizes the recombination signal sequences (RSSs) located at the border of the D and J gene
segments in the heavy chain locus. The RSSs consist of a conserved heptamer and nonamer separated by a spacer of
12 or 23 base pairs.
2. Cleavage of DNA by RAG complex:
◦ The RAG complex introduces a double-strand break at the border between the gene segments by cleaving the DNA
at the RSSs. This cleavage creates a hairpin loop on the coding end of the broken DNA strand.
3. Recruitment of DNA repair enzymes:
◦ The ends of the broken DNA strands are then processed and modified by a series of enzymes, such as DNA
polymerases and nucleases, to form blunt or staggered ends suitable for rejoining. Additionally, the Ku70/80
heterodimer is recruited to protect the DNA ends from further damage.
4. Formation of the signal joint:
◦ If the RSSs are different from each other in terms of their spacer, then the RAG complex recruits the Artemis nuclease
to open the hairpin loop and generate a clean break at the coding end. This results in the formation of a "signal
joint" between the coding end and the RSS of the gene segment that was not cleaved.

16. 5. Formation of the coding joint:
◦ The RAG complex then moves upstream and recognizes the RSS located at the border of the V and DJ gene segments and introduces
another double-strand break at the border between the gene segments by cleaving the DNA at the RSS. This cleavage creates a
hairpin loop on the coding end of the broken DNA strand.
6. Opening of the hairpin loop:
◦ The hairpin loop on the coding end is then opened by the Artemis nuclease, generating a clean break at the coding end.
7. Addition of nucleotides:
◦ The recombination process is completed by non-homologous end joining (NHEJ), a repair pathway that joins the ends of the broken
DNA strands together to form a functional VDJ exon. This process can involve the trimming or addition of nucleotides at the
junctions to generate diversity in the heavy chain sequence.
8. Transcription and translation:
◦ The newly formed VDJ exon is then transcribed and spliced to produce a functional heavy chain mRNA, which is translated into a
mature heavy chain protein.
9. Assembly of heavy and light chains:
◦ The mature heavy chain protein is assembled with the light chain protein to form a complete immunoglobulin molecule, which is
secreted by the B-cell and can recognize and bind to specific antigens

17. Recombinationsignalsequence (RSS)
 Rearrangement of V, D & J gene segment is guided by DNA segment called RSS
 Found at the border of immunoglobulin gene segments that undergo V(D)J recombination.
 They are non-coding DNA sequence
 Located at –
◦ 3’ end of each V gene segment
◦ 5’ end of each J gene segment
◦ Both side of each D gene segment

18. RSS is composed of three components
1. HEPTAMER - It is a 7-nucleotide sequence
◦ It is highly conserved among different RSS sequences.
◦ The heptamer sequence is essential for the recognition of the RSS by the RAG1/RAG2 complex and for
the initiation of the V(D)J recombination process.
2. NONAMER - It is a 9-nucleotide sequence
◦ It is also highly conserved among different RSS sequences.
◦ The nonamer sequence is located at the other end of the spacer region and is also important for the
recognition of the RSS by the RAG complex.
3. SPACER - It region is the region between the heptamer and nonamer sequences
◦ It is either 12 nucleotides or 23 nucleotides long.
◦ The 12/23 rule ensures that only gene segments with different spacer lengths can be joined together,
thus promoting diversity in the rearranged genes.

20. RECOMBINASE MACHINERY
◦ The recombinase machinery involved in V(D)J recombination is a complex system that includes
several key components, such as the recombination activating genes (RAG) 1 and 2, the high-
mobility group (HMG) proteins, and the non-homologous end joining (NHEJ) proteins.
1) RAG1 and RAG2 – These proteins are responsible for recognizing and binding to the
Recombination Signal Sequence (RSS) located at the border of the gene segments that are
being rearranged.
Once bound, the RAG complex initiates double-strand breaks at the border of the gene segment
and the RSS, leading to the activation of the cellular DNA repair machinery
2) HMG proteins – It helps to stabilize the RAG complex on the DNA and facilitate its
interaction with the RSS.
These proteins also help to create a distorted DNA structure that is required for efficient V(D)J
recombination.

21. 3) NHEJ proteins - These proteins recognize and bind to
the broken ends of the gene segments and help to repair
the DNA by joining the broken ends together.
NHEJ is the main pathway for repairing the broken ends of
DNA during V(D)J recombination.
The recombinase machinery is tightly regulated to ensure
that the rearrangement process occurs efficiently and
accurately.

22. reference
◦ Roitts Essential Immunology 13th Ed 2017 by Peter J. Delves, Seamus J.
Martin, Dennis R. Burton, Ivan M. Roitt (z-lib.org)
◦ Kuby Immunology [7 Ed, 2013]
◦ https://www.britannica.com/science/antibody
◦ https://www.ncbi.nlm.nih.gov/books/NBK546670/

23. THANKYOU