Genetics of antibody diversity and function

Antibody genes are produced by somatic recombination

● Developing B-cells
⇒ Gene segments that undergo somatic diversification
⇒ Immunoglobulin repertoire

● Basic components of antibody are inherited

● Individual’s mature antibody repertoire is essentially formed by alteration of the
inherited germline genes

● Rearrangement of DNA in somatic rather than gamete cells

● Further modified during an individual’s lifetime by their exposure to different
antigens
Immunoglobulin variable gene segments
and loci
● Variable light and heavy chain loci
● Multiple gene segments: joined by somatic
recombination producing the final V region exon

● Heavy chain variable region

● Locus on chromosome 14
● Joining of three gene segments: V (variable), D (diversity), and J (joining)
● 40 VH genes, 23 DH genes, 6 JH genes
L: leader sequence.
Immunoglobulin variable gene segments and loci

● Light chain Lambda

● Locus on chromosome 22
● 30 Vλ genes, 5 Jλ gene, each J segment followed by a constant segment

● Light chain Kappa

● Locus on chromosome 2
● 40 Vκ genes, 5 Jκ genes
● J segments clustered upstream of the constant region
 Immunoglobulin variable gene segments and loci

● VH locus
○ Highly polymorphic
○ Evolved through the repeated duplication, deletion, and recombination of DNA
○ High frequency of insertion or deletion of gene segments
○ Occurrence of different alleles of the same segment

● Immunoglobulin loci also contain regulatory elements

○ (Next slide)
 Regulatory elements of immunoglobulin loci.

● Leader sequence:
○ Each VDJ segment encoding the variable region
○ Transporting the newly synthesized H chains into the endoplasmic reticulum
● TATA box of the promoter
○ RNA polymerase II
● Octamer motif
○ Transacting regulatory transcription factors
● Enhancers
○ Binding nuclear proteins
○ Increasing the transcription rate to levels typical of actively secreting B-cells
Overview of V(D)J recombination.

1. Diversity (D) and joining (J) gene

segments in the germline DNA are
joined together (somatic
recombination)
2. Variable (V) gene segment is then
joined to the recombined D–J gene
(somatic recombination)
⇒ Fully recombined heavy chain exon
⇒ At the light chain loci, somatic
recombination occurs with V and J
segments only.
1. Recombined DNA is transcribed
2. Primary RNA transcript
3. Spliced (bringing together the V and
constant (C) regions)
4. Spliced mRNA molecule is translated to
produce the immunoglobulin protein
 V(D)J recombination and combinatorial diversity

● Heavy chain repertoire

○ Approximately 40 VH × 23 DH × 6 JH = 5500

● Light chain repertoire

○ Approximately 30 Vλ × 5 Jλ = 150
○ Approximately 40 Vk × 5 Jk = 200
⇒ Total of 350 light chain combinations

● Each heavy chain could potentially pair with each light chain

● **Total diversity of the immunoglobulin repertoire

○ Total of 2 million possible combinations.
 Recombination signal sequence (RSS)

• Noncoding sequence
• Conserved heptamer and nonamer sequences
• Separated by an unconserved 12- or 23-nucleotide spacer
• Efficient recombination occurs between segments with a 12-nucleotide spacer and
a 23-nucleotide spacer
 The V(D)J recombinase.

1. RAG-1 and RAG-2 proteins bind to the

recombination signal sequences(RSS).
2. Single-stranded nick is introduced
between the 5ʹ-heptameric end of the
recombination signal sequence and the
coding segment
⇒ Free 3ʹ-OH group
⇒ Transesterification reaction
1. Formation of DNA hairpins at the coding
ends
2. Hairpin cleavage and resolution of the
post-cleavage complex
⇒ by NonHomologous End
Joining (NHEJ) proteins
⇒ Formation of separate coding
and signal joints

● RAG-1 and RAG-2 activity is specific to lymphoid cells

Example
Somatic hypermutation ● Variable regions of Ig heavy and light chains
are diversified by somatic hypermutation
● Nontemplated point mutations into V
regions
● Occurs at a high rate
○ ⇒ Approximately 106 times higher than
mutation rate of cellular housekeeping
genes
● Affinity maturation
○ Increasing average affinity of the
antibodies
○ Increasing binding affinity of the B-cell
receptor for its cognate ligand
● Activation-induced cytidine deaminase (AID)
are essential for both somatic
hypermutation
○ Cytidine deaminase capable of carrying
out targeted deamination of C to U
Sepulveda-Yanez et.al DOI:10.5772/intechopen.72122
Junctional diversity further diversifies the immune repertoire.
 Junctional diversity further
diversifies the immune repertoire.

● Immunoglobulin repertoire is further diversified

during cleavage and resolution of the coding-end
hairpins
(1) By deletion of a variable number of
coding-end nucleotides
(2) By the addition of N-nucleotides by
terminal deoxynucleotidyl transferase (TdT)
(Previous slide) By palindromic (P) nucleotides
that arise owing to template-mediated fill-in of
the asymmetrically cleaved coding hairpins

(1) Exonuclease trimming, to remove unpaired nucleotides, and the DNA repair machinery act to
repair the DNA joint.
(2) TdT randomly adds nucleotides to the DNA ends (N-nucleotides), and the single-stranded
ends pair, possibly but not necessarily, through complementary nucleotides (TG on top strand
and AC on bottom strand).
Class switch recombination allows expression of different
antibody isotypes.
 Class switch recombination allows expression of different
antibody isotypes.

● B-cells in germinal centers of secondary lymphoid organs (spleen and lymph nodes)
○ Antigen-stimulated IgM expressing B cells
● Class switch recombination (CSR) allows the IgH constant region exon of a given
antibody to be exchanged for an alternative exon, giving rise to the expression of
antibodies with the same antigen specificity but of differing isotypes
○ CSR occurs through a deletional DNA recombination event at the IgH locus
● Constant region exons for IgD, IgG, IgE, and IgA isotypes are located downstream of
the IgM (Cμ) exon, and CSR occurs between switch or S regions.
● DNA recombination at repetitive sequences termed switch or S regions
 Class switch recombination allows expression of different
antibody isotypes.

• DNA recombination at repetitive sequences termed switch or S regions

• (Ex in the figure) IgM to IgG2a switch at the mouse heavy chain locus
○ Begins with germline transcription from the promoter upstream of the constant region
exon and recombination between the Sμ and Sγ2a regions
○ DNA recombination reaction brings the IgG2a constant region exon downstream of the
variable region exon.
○ Remaining switch regions and constant region exons are deleted and form an episomal
circle.
○ Transcription of the rearranged DNA yields IgG2a mRNA, which can be translated to give
rise to the IgG2a immunoglobulin protein.