V(D)J Recombination (Part 8- Antibody Basics)
Welcome to the 8th part of the 13-part series on Antibody basics.
Previous parts: Part 1, Part 2, Part 3, Part 4, Part 5, Part 6, and Part 7
VJ recombination in κ light chain DNA
As discussed in Part 7, the variable region of each of the light chains of an immunoglobulin molecule is encoded by multiple V and J gene segments. These gene segments are first rearranged to form an exon VJ segment that encodes the variable region of the light chain. Though there are multiple copies of each type of gene segment in the germline DNA, only one gene segment is expressed. In the case of the κ light chain, during the process of VJ recombination, any of the Vκ gene segments out of 40 segments can join with any of the functional Jκ gene segments.
In the below Fig, assume the 2nd Vκ gene segment has to join with the 4th Jκ gene segment. This VκJκ joining creates an exon that encodes the whole variable region of the κ light chain. In the rearranged DNA, a leader sequence is present at the 5' end of the joined VκJκ gene segment. Upstream from each of the leader sequences, a promoter is present.
The RNA polymerase binds on the promoter present upstream of the leader sequence at the 5’ end of the joined VκJκ gene segment. Once bound, RNA polymerase initiates the transcription from the L sequence through the C segment to the stop signal, generating a κ light chain primary RNA transcript. Next, the non-coding sequences referred to as introns present in the light chain primary RNA transcript are removed by RNA processing enzymes. This process is called RNA splicing. It creates the joining of the variable region VκJκ exon to the constant region exon to form VκJκCκ exon.
The polyA tail is then added at 3’, resulting in the formation of messenger RNA, also written as mRNA. This resulting light chain messenger RNA exits from the nucleus, binds to the ribosome, and is translated into the light chain polypeptide. The leader sequence present at the amino terminus of the polypeptide pulls the growing polypeptide chain into the endoplasmic reticulum and is then cleaved. Therefore, the leader sequence is not present in the finished light chain protein product. This is how the gene arrangements occur to generate a functional κ light chain. To summarize, the variable region VL of the κ light chain is formed by Vκ and Jκ gene segments, and Cκ gene segments form the C region.
VJ recombination in ƛ light chain DNA
The VJ rearrangement of gene segments in the ƛ light chain is slightly different from that of the κ light chain. In the case of the κ light chain, during VJ recombination, any of the Vκ gene segments out of 40 segments can join with any of the functional Jκ gene segments. But in the case of ƛ light chain, any of the Vƛ gene segment out of 31 segments can join with any of the Jƛ-Cƛ gene segment combination. Since the ƛ light chain locus contains multiple Cƛ gene segments and each Jƛ gene segment is associated with 1 Cƛ gene segment. Therefore, to form a functional VƛJƛ segment that encodes the variable region of ƛ light chain, functional Vƛ gene segment combines with any of the Jƛ-Cƛ gene segment combinations.
Then the RNA polymerase binds on the promoter present upstream of the leader sequence at the 5’ end of the joined VƛJƛCƛ gene segment. Once bound, RNA polymerase initiates the transcription of the gene segment from the L sequence through the C segment to the stop signal, generating a ƛ light chain primary RNA transcript.
In the next step, the non-coding sequences, also known as introns present in the light chain primary RNA transcript, are removed by RNA processing enzymes. The polyA tail is then added at 3’, resulting in the formation of messenger RNA. This resulting ƛ light chain messenger RNA exits from the nucleus binds to the ribosome and is translated into the light chain polypeptide.
The leader sequence present at the amino terminus of the polypeptide pulls the growing ƛ polypeptide chain into the endoplasmic reticulum and is then cleaved. This is how the gene arrangements occur to generate a functional ƛ light chain. To summarize, the variable region VL of the ƛ light chain is formed by Vƛ and Jƛ gene segments, and Cƛ gene segments form the C region.
Another essential point to know is that the presence of multiple Cƛ gene segments in the germline DNA gives rise to the four subtypes of lambda light chain ƛ1, ƛ2, ƛ3, and ƛ4.
VDJ recombination in heavy chain DNA
Similar to light chains, the rearrangements occur in gene segments of heavy chain DNA to generate a functional heavy chain. The rearrangements occur sequentially. First, any of the DH segments can join any of the JH gene segments to form the DHJH segment. Assume in the below Fig, DH2 segment has to join the JH3 gene segment to form the DHJH segment. In the next rearrangement step, the resulting DHJH segment can join with any of the randomly selected VH gene segments, say VH1 gene segment, to generate a VHDHJH unit. This VHDHJH segment encodes the entire variable region of the heavy chain of an antibody molecule.
As with the light chain, the heavy chain gene also contains a leader sequence that is present at the 5' end of the joined VHDHJH segment, followed by the J segments that were not joined to V and D, and then the series of C gene segments at the 3’ end. In addition, a promoter sequence is located upstream of the leader sequence. Thus, once the heavy chain gene rearrangements are accomplished, RNA polymerase can bind to the promoter region and transcribe the entire heavy chain gene, including introns. Initially, both Cµ and Cδ gene segments are transcribed. Then the introns are removed, and the V region exon is joined to the Cµ and Cδ exons.
After that, polyadenylation of the RNA transcript occurs to generate two mRNAs; one mRNA contains Cµ, while the other mRNA contains Cδ transcribed gene segment. These two mRNAs are then translated into µ and δ heavy chains. The leader peptide present in the nascent polypeptides is cleaved, generating finished µ and δ heavy chains.
Thus, the production of two different heavy chains allows a mature immunocompetent B cell to express both IgM and IgD immunoglobulins with identical antigen specificity on its surface.
Mechanism of variable region DNA rearrangement
Each germline segment V, D, and J is flanked by recombination signal sequences, abbreviated as RSS. Recombination signal sequences are present adjacent to the points at which recombination takes place. Therefore, one RSS is located 3’ to each V gene segment, one RSS is located 5’ to each J segment, and two RSS are present on both 5’ and 3’ of D segment.
The RSS sequences function as signals for the recombination process that rearranges the gene segments. Each RSS contains a conserved palindromic heptamer and a conserved AT-rich nonamer separated by an intervening sequence of 12 or 23 base pairs.
If the intervening sequence is 12 base pairs, RSS is called a one turn recombination signal sequence. If the intervening sequence is of 23 base pairs, then RSS is called a two turn recombination signal sequence.
In the case of the κ light chain, the Vκ gene segment RSS is one turn spacer, and the Jκ signal sequence has a two turn spacer.
Whereas in the case of ƛ light chain, the order is reversed Vƛ gene segment gene signal has a two turn spacer, and the Jƛ signal sequence has one turn spacer.
In heavy chain DNA, VH and JH gene segments have two turn spacer signal sequences, and signals on either side of the DH gene segment have one turn spacers.
The most important rule in gene rearrangements is that a gene segment containing a signal sequence with one turn spacer can join only the gene segments with a two-turn spacer. Thus, it is also called the one turn/two turn joining rule. For example, the joining rule ensures Vκ segment joins only to a Jκ segment but not to another Vκ segment.
The rule likewise ensures that VH, DH, and JH segments of heavy chain join in the proper order.
VJ or VDJ recombination is catalyzed by the recombinase enzyme and terminal deoxynucleotidyl transferase enzyme. There are two types of recombinase enzymes: RAG-1 and RAG-2. The recombination of the variable region gene segments involves the following steps:
- Firstly, the recombinase enzymes recognize the recombination signal sequences or RSS of the gene segments V and J, then bring the two signal sequences and the adjacent coding sequences in proximity.
2. RAG-1 and RAG-2 then cleave one strand of DNA. This cleavage occurs at the juncture between RSS and the coding sequence of V and J segments. Mutations or defects in RAG-1 and RAG-2 enzymes do not allow rearrangement of immunoglobulin genes, leading to a lack of generation of B cells.
3. After single-strand cleavage by recombinase enzyme, the free 3’OH group on the cut DNA strand attacks the phosphodiester bond linking the opposite strand to the signal sequence. Thus forming a hairpin-like structure at the V and J segment.
4. Further, a specific endonuclease enzyme known as Artemis cleaves the hairpin loops. This endonuclease does not recognize any specific sequence. Instead, it recognizes the hairpin structure. As a result, the nicking of DNA occurs randomly on both hairpin structures, after which a series of palindromic or ‘P’ nucleotides are added.
5. Then, the enzyme terminal deoxynucleotidyl transferase or TdT adds up to 15 nucleotides at the cut ends of V, D, and J coding sequences of the heavy chain, but not on the light chain. Tdt is one of the marker enzymes present in B cells.
6. After adding P and N nucleotides, ligation occurs, which joins the coding sequences and the signal sequences. It is performed by the double-strand break repair enzymes abbreviated as DSBR enzymes. Thus, the recombination event results in a coding joint formed by the combination of V and J coding gene segments and a signal joint formed by joining 2 RSS.
It is important to note that if the two gene segments are in the same transcriptional orientation, the joining of the gene segments results in the deletion of the signal joint, and the intervening DNA is obtained as a circular excision product.
But if the two gene segments are in reverse orientation, their joining results in retention of both the coding joint and the signal joint on the chromosome. For instance, this occurs in the human κ locus, where about half of the Vκ gene segments are in reverse orientation with respect to the Jκ gene segments.
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