Organization of genetic material
Genes encoding immunoglobulin (Ig) molecules have a mosaic structure. Coding regions are separated by long, non-informative nucleotide sequences.
There are four main types of gene segments:
- V segments (Variable) — encode most of the variable domain.
- D segments (Diversity) — diversity segments, present only in heavy chain genes.
- J segments (Joining) — joining regions.
- C segments (Constant) — contain information on the structure of constant regions and determine the antibody class (e.g., $C_\mu$ for IgM or $C_\gamma$ for IgG).
Through the random combination of these fragments, the body can synthesize millions of different antibody variants, generating a colossal diversity of protective molecules.
Light and heavy chain synthesis
The formation of a complete gene occurs within the B lymphocyte DNA via somatic recombination.
Light chain assembly (using the $\varkappa$-chain as an example):
- At the DNA level, one variable (V) and one joining (J) segment unite. The intervening DNA between them is deleted.
- The formed VJ complex approaches the constant C segment.
- Transcription occurs, producing a pre-mRNA that contains the VJ region, extra J segments, and the C region.
- During splicing (processing), introns and unused J fragments are excised to form mature mRNA.
Heavy (H) chain assembly: The process is more complex due to the presence of D segments and requires two stages of recombination. First, a mixed exon is assembled from random D and J segments. Then, one of the $V_H$ segments joins the resulting DJ complex. The final $V_H D_H J_H$ exon is positioned next to the constant region, after which transcription begins.
Class switch recombination
During a primary immune response, B lymphocytes always begin by synthesizing IgM. This is because the $C_\mu$ segment gene is physically located at the 5' end of the DNA coding region, meaning it comes first before all other constant genes.
As cells differentiate, switching to other classes (IgG, IgA, etc.) occurs. This process is carried out via additional specific recombination. Enzyme systems remove the C segments located between the assembled variable gene and the C region of the immunoglobulin class to be newly synthesized.
Alternative splicing: from membrane to secretion
Immunoglobulins can exist both as cell membrane receptors and as free molecules in the blood. Regulation of this process depends on alternative splicing and polyadenylation of the primary transcript.
- In pre-B lymphocytes (membrane form): An mRNA including a specific exon is synthesized. It encodes a hydrophobic region in the C region that acts as an "anchor," securely fixing the IgM molecule in the cell membrane.
- In plasma cells (secretory form): During terminal differentiation, transcription stops earlier—at a stop codon within the intron. Due to alternative polyadenylation, the exon with the hydrophobic region is excluded from the mature mRNA. As a result, shortened antibodies without the membrane "anchor" are produced and secreted into the bloodstream.
Briefly: answers to topical questions
Which enzymes (recombinases) carry out V(D)J recombination?
V(D)J recombination is triggered by the expression of genes belonging to the V(D)J recombination complex. By source, the complex includes 6 components:
- RAG-1/RAG-2 recombinase (exonuclease) dimer;
- DNA-dependent protein kinase;
- DNA ligase IV;
- terminal deoxynucleotidyl transferase (TdT), associated with non-templated synthesis of oligodeoxynucleotides / non-templated nucleotide addition;
- HMG 1/2 heterodimer;
- Ku70/Ku80 heterodimer.
At the synapse stage, RAG-1 and RAG-2 bind to the ends of the 12 and 23 spacer sequences of the joined gene regions, dimerize, and bring the regions close together. Double-stranded DNA breakage is catalyzed by the RAG recombinase. HMG1/2 facilitates the approximation of the segments.
What additional mechanisms (besides V(D)J recombination) ensure antibody diversity?
In addition to recombination / DNA segment combinatorial diversity itself, antibody diversity is provided by the following mechanisms:
- junctional inaccuracy: imprecise localization of DNA strand breaks during V, D, and J segment rearrangement;
- nucleotide insertion at joints: P- and N-insertions, addition of extra nucleotides at junctions;
- use of 3 reading frames in the D segment, providing a threefold increase in variability;
- somatic hypermutation of V genes.
What antibody classes can be formed during isotype switching following IgM?
During isotype switching following primary IgM synthesis, other antibody classes can be formed via specific DNA recombination under the influence of cytokines. These include:
- Immunoglobulin G (IgG) — subclasses IgG1, IgG2, IgG3, and IgG4 are formed.
- Immunoglobulin A (IgA) — subclasses IgA1 and IgA2 are formed.
- Immunoglobulin E (IgE) — synthesized, for example, under the influence of interleukin-4.
Switching occurs by removing the constant C segments located between the formed variable gene and the C region of the new isotype.
It is easy to remember the chromosomes for antibody chains: kappa — 2, heavy — 14, lambda — 22. They form a sequence with a step of about a dozen: 2, 14, 22.
Class and exam questions
Why is IgM specifically synthesized during the primary immune response?
The gene for the $C_\mu$ segment, which encodes the constant region of immunoglobulin M, is located at the 5' end of the DNA. It is physically positioned first before all other C segments.
How does heavy chain assembly differ from light chain assembly?
The heavy chain contains additional D (diversity) segments. Therefore, recombination occurs in two stages: first, D and J fuse, and only then is the V segment added to them.
How does a B lymphocyte start secreting antibodies into the blood instead of anchoring them to the membrane?
During differentiation into a plasma cell, mRNA processing changes. Due to termination at an early stop codon, the exon encoding the hydrophobic "anchor" is spliced out, and the molecule becomes soluble.
What else to study on this topic
- The mechanism of alternative splicing using the calcitonin gene as an example
- The role of stop codons in pre-mRNA processing
- Post-translational modification and oligosaccharide attachment
- Statistics and the number of V, D, and J segments in the human genome
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