V(D)J recombination is a process of constructing various unique receptors by different gene segments such as variable (V), diversity (D) and joining (j) through recombinase enzyme.
Understanding the process of antibody diversity is quite difficult as it is the most complicated subject in genetics yet it is interesting. Varied receptors or antigen-binding sites are produced by different antibody coding gene segments.
An interdisciplinary subject of biology studies immunity and related things is known as immunology. The immune system is our defense mechanism, protecting our body through varied pathways and the synthesis of molecules.
Those molecules are immunoglobulins- a kind of proteins encoded by specific gene segments. Immunogenetics comprises the study of how different antigens and immunoglobulins are formed from genes or gene segments.
Any of the foreign particles which possibly harm our cells are characterized as antigen and the protein which helps to destroy antigens is termed as an antibody. The antibody is a specialized antigen-specific protein. Each antibody is specific for a single type of antigen which means that for different antigens, antigen-specific different antibodies are required.
Varied DNA sequences (not entire genes but gene segments) located on different chromosomes govern the whole process through recombination.
In this article, we are majorly focusing on an overview of how antibody diversity is generated, what are the antibody coding gene segments, their structure and their importance.
Vdj recombination and antibody diversity
Susumu Tonegawa won the Nobel Prize for finding the principle of generation of antibody diversity in 1987. First, let’s understand the structure of antibodies.
Structurally, an antibody is a Y-shaped protein molecule that contains two light chains and two heavy chains (identical). Each light chain is made up of 220 amino acids, and each heavy chain is made up of about 440-450 amino acids and these chains are allied by disulfide bonds.
A variable region present on each heavy and light chain at the N terminus end makes it antigen-specific. At the C terminus end, a constant region is present on both chains which are non-variable and it provides structural support to the antibody.
A constant region constructs an effector functional domain, which is responsible for the interaction of that antibody with other immune components. Depending upon the functionality of the antibody, it is characterized into 5 major classes: IgM, IgD, IgG, IgE and IgA.
IgM and IgD are referred to as surface antibodies and are embedded into the cell membrane. Every antigen initially comes in contact with this surface antibody.
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The light chain of the antibody is of two types: kappa light chain and lambda light chain and is determined by the constant region of a light chain, now we can understand that though the constant region remains unchanged it plays an important role in the formation of each type of light chain.
The arrangement is hard to determine but it is easy to understand. Different DNA sequences for antibody (protein) light chains are located on different chromosomes.
Instead of saying a gene it is known as gene sequences or gene segments because different sequences within the gene are allotted for the formation of different polypeptide chains of the variable region.
Different heavy chains, light chains, constant regions and lambda and kappa chain polypeptides are synthesized by different gene segments. Now firstly we will discuss the arrangements of different gene segments for each chain.
Depending upon the function and location, two types of antibodies are present: The membrane-bound antibodies, present on the membrane of the cell and the second is secreted antibodies, secreted from the B lymphocyte cell, induced by the antigen attack.
Arrangements of gene segments for Generation of antibody diversity:
The light chain is of two types: kappa light chain or lambda light chain.
Let’s denote variable region as V,
The variable regions are actually coding exons that make different polypeptides.
A constant region as C and
Joining segments as J (joining segments are just non-coding intervening sequences).
Keep in mind one point that each chain (whether it is a light or a heavy chain) is made up of a long chain of polypeptides with N terminus and C terminus, it starts with N terminus at the variable region and ends at C- terminus towards the constant region. See the figure to understand it properly.
A number of gene segments for the different type of polypeptide chains:
|Kappa gene segments||Lambda gene segments||Heavy chain|
|Variable region||Vk=38||Vλ 33||VH=38-46|
|Joining region||Jk=5||Jλ =5||JH=6|
|Constant region||CK=1||Cλ =4||CH=9|
Kappa light chain:
At the N terminus end the Vk gene segment is code for 95 amino acids, Thee Jk gene segments code for 13 amino acids, and at C terminus end, the Ck gene segments code for the remaining amino acid of a light chain.
One chain of hydrophobic leader sequences is also present at the N terminus end of the kappa light chain which helps in the transportation of antibodies at the site of action. It is made up of 17-20 amino acids.
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During the development of B lymphocyte cells recombination of the different Lk-Vk segment and Vk-Jk segments construct the different types of antigen-specific antibody. The entire segment of Lk-(Vk)Jk-non coding sequence-Ck is transcribed and the noncoding sequences and other non-functional sequences are removed from the mature kappa chain. Only Vk-Jk-Ck sequences remain intact for the development of a particular antibody.
Lambda light chain
These gene segments are assembled during B lymphocyte maturation as well and create different antibodies through recombination of different gene segments. The maturation process is the same as kappa chain gene segments. Here the segments are denoted as Jλ, Vλ, Cλ.
The genetic information for coding heavy chain gene segments is arranged on LH-VH, JH and CH gene segments. Here the kappa or lambda variable region remains the same as a light chain, nevertheless, one additional gene segment is present in the heavy chain segment, a “diversity segment” or “D segment”.
D segment awards additional diversity to the antibody which is present in the variable region of a heavy chain.
The D segment is made up of the 2-13 amino acid long polypeptide chain. Here 4 separate CH gene segments are present for each Ig class heavy chain.
In humans, a total of 9 to 10 functional CH gene segments are present. The list of different segments is given in the table. In CH gene segments of the heavy chain, the intervening sequences are typically pseudogenes ( sequences which are similar to the functional gene but are non-function), during maturation of B lymphocyte pseudogenes are removed
Depending upon the function and location, two types of antibodies are present: The membrane-bound antibodies, present on the membrane of the cell and secreted antibodies, secreted from the B lymphocyte cell, induced by the antigen attack.
Now the membrane-bound antibodies are fixed on the cell membrane and embedded into the lipid layer of the membrane. The C terminus end of a heavy chain antibody is hydrophobic in nature which helps them to fix in the lipid layer, whereas the C terminus end of circulating antibodies have the hydrophilic end which makes them circulate freely.
IgM and IgD are fixed antibodies present on the surface of the cell, others are freely circulating in the circulatory system and depending upon the type of antigen, the specific antibody is secreted.
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D(V)J recombination process-
In the process of recombination, first, the recombinase enzyme came in the picture that recognizes the RSS sequences- recombination signal sequences. Interestingly RSS are located adjacent to each gene segment, viz near to Variable, joining and diversity segments.
The RSS is very important for the process in vivo as well as in vitro. Recombination between varied gene segments only occurs when the RSS has a varied length of a spacer sequence.
The spacer sequences are present between the heptane and nonamer elements of RSS. The heptamer and nonamer sequences are nearly conserved.
Different types of recombinase are RAG1- recombination activating gene 1, RAG2- recombination activating gene 2, Artemis nuclease, TdT- terminal deoxynucleotidyl transferase is involved in the NHEJ DNA repair pathway.
Other enzymes besides these are DNA protein kinase, DNA ligase IV, non-homologous end-joining factor 1 and X-ray repair cross-complementing protein 4. All perform different functions in the whole process.
For example, ligase IV joins or ligates the final recombined fragment to construct a receptor.
The RAG1 protein initiates the process by recruiting the recombinase to the site of recombination at where it creates a single-strand nick between the first base of RSS and coding segment DNA sequence.
At this recombination center, a hairpin loop at the coding segment occurs due to free 3’ and 5’ ends on the same strand. The hairpin formation is crucial to manufacturing different combinations.
The rest are the signal sequences- non-coding ones, ligated and form circular DNA which later incorporated into the genomic DNA.
Other proteins mentioned above also function in a sequential and complicated process to ligate a varied gene segment of V(D)J.
In the last step, the exonuclease removes some non-useful nucleotides and DNA polymerase inserts nucleotides to make segments compatible for rejoining.
At the end of the process, a highly variable antigen-binding region is formed to invading pathogens.
In this manner, millions of different antigen-binding receptors are formed every day, novel and known too. However, in order to produce a specific receptor or antigen-binding segment, DNA sequences explained above and are involved in this process must be in a sequential manner to form the correct chain of amino acids. If not, the cell will die soon, because an abrupt combination is no longer needed.
Note that there are many different enzymes, DNA sequences and elements are involved in the whole pathway, some are known, some aren’t but to make you understand, we have explained it in simple language and elaboratively.
Generation of antibody diversity by class switching:
IgM and IgD are called primary antibodies because both are present on the surface of the cell membrane and primarily interact with antigens. The secondary antibodies IgE, IgA and IgG are secreted by B cells depending upon the antigen present on the cell membrane hence this class of antibodies is termed secondary antibodies.
Depending upon the type of antigen, instead of making one class of antibody, the mature B cell starts making another class of antibody, this phenomenon is called antibody class switching.
Once the primary class of antibody recognizes the antigen, the B cell differentiation is initiated depending upon the type of antigen present on the cell membrane. Even though it synthesizes some specific antibodies, here it synthesizes antigen-specific antibodies.
The Constant segment of antibody decides which class of antibody will produce the immune response, here as the primary antibody is IgM, the CH gene segment is preliminarily the same for all antibodies.
In class switching instead of producing IgM antibodies, some mature B lymphocytes produce another class of antibody depending upon the gene segment arrangement of CH.
This antigen-induced secondary antibody production is called class switching. The mechanism is governed by the rearrangement of different DNA segments of V, D, J and C regions followed by class switching recombination.
Importance of V(D)J recombination:
Our body faces many brutal attacks by virus, phase, bacteria, fungus or other chemical particles every day. A body needs to invade through different mechanisms.
Foreign particles broadly categorized into “antigens” initially come in contact with Antigen Presenting Cells. Antigens are modified and synthesized as per the requirements.
To invade many attacks, different gene segments of the heavy chain, light chain, constant region, joining region combine by the process of recombination and create different combinations.
Nonetheless, some combinations can’t work, whilst memories for some common combination saved for future use. This means, when the same pathogen or foreign antigen attacks, cells immediately form immunoglobulins by using the memory.
But On the downsides, chromosomal DNA portions, especially those involved in antibody diversity, break and rejoin many times, in fact, thousands or millions of times during the lifetime to manufacture various antibody receptors. Some fragments are repaired and some remain unrepaired which causes serious genetic problems.
The mechanism of class switching is as important as gene segment re-arrangements in the generation of antibody diversity. In the next article, we will discuss the VDJ joining, junctional flexibility, nucleotide addition and somatic hypermutation.
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