Generation of antibody diversity (Part 9- Antibody Basics)
Welcome to the 9th part of the 13-part series on Antibody basics.
Previous parts: Part 1, Part 2, Part 3, Part 4, Part 5, Part 6, Part 7, and Part 8
The antibody repertoire has the specificity to recognize more than 100 million different antigen molecules. There are various sources that generate diversity in antibody molecules because of which they can do so.
Combinatorial VJ and VDJ joining
The first source is the presence of multiple V, D, and J segments in germline DNA. In humans, there are 51 VH, 27 DH and 6 JH gene segments for immunoglobulin heavy chain variable region and 31 Vƛ, 4 Jƛ, 40 Vκ and 5 Jκ gene segments for immunoglobulin light chain variable regions.
The presence of multiple germline V, D and J gene segments and their random rearrangement contribute to the diversity of antigen-binding sites in an antibody molecule. For instance, in the heavy chain locus, 51 VH gene segments can combine with any of the 27 DH gene segments and any of the 6 JH gene segments. It allows 51*27*6= 8262 possible combinations of the heavy chain variable region that significantly contributes to the generation of the heavy chain diversity. Similarly, in κ light chain, 40 Vκ gene segments can randomly combine with any of the 5 Jκ gene segments and therefore has the potential to generate 40*5=200 possible combinations of κ light chain variable region. On the other hand, the 31 Vƛ gene segments can combine with any of the 4 Jƛ gene segments to generate 31*4=124 possible combinations of the ƛ light chain variable region.
Since it is the variable region in the light and heavy chain that together form the antigen-binding site, combining a heavy chain with any of the two light chains further leads to diversity in an antibody. As a result of this combination, 8262*200*124, which approximately equates to 10⁸ different antigen-binding sites, can be generated.
Junctional Flexibility
The second source that generates antibody diversity is junctional flexibility. The enormous diversity generated through V, D, and J combinations is further augmented by junctional flexibility. As already discussed in Part 8, the joining of V and J segments involves joining recombination signal sequences and coding sequences. However, the joining of these coding sequences is often imprecise. For example, let’s analyze the joining of Vκ21 and Jκ1 coding sequences in 4 B cell lines.
After joining V and J gene segments by recombination process, different B cell lines exhibit sequence variability in their coding sequences.
This is called junctional flexibility & because of junctional flexibility, VJ segments encode alternate amino acids sequences in different cell lines, thus increasing antibody diversity.
The amino acid sequence variation generated by junctional flexibility in the coding joints is primarily in the third hypervariable region, or CDR3 in the immunoglobulin heavy and light chain. Since CDR3 makes a major contribution to the diversity in the antigen-binding site of the antibody molecule, amino acid changes in CDR3 generated by junctional flexibility lead to the generation of further antibody diversity. Sometimes, the junctional flexibility can also cause non-productive rearrangements in some of the B cell lines. If V, D, and J gene segments are joined out of phase, it leads to the occurrence of stop codons in the resulting VJ or VDJ unit.
These stop codons are TAG, TGA, and TAA (in RNA, stop codons are designated as UAG, UGA, and UAA). The stop codons signal the end of the light or heavy chain polypeptide synthesis, thus leading to the death of B cells by apoptosis.
P-addition
The third source that generates antibody diversity is P-addition. As already discussed in Part 8, during recombination of VJ gene segments, RAG-1 and RAG-2 recognize the recombination signal sequences or RSS of the gene segments V and J, followed by bringing the two signal sequences and the adjacent coding sequences in proximity. Then RAG-1 and RAG-2 cause single-stranded DNA cleavage at the junctures of signal sequences and coding sequences.
After that, the free 3’OH group of the cut DNA attacks the phosphodiester bond linking the opposite strand to the signal sequence, thus forming a hairpin-like structure at the cut end of the coding sequence of the V and J segment. The hairpin structure is then cleaved by the endonuclease. Remember that the nicking of DNA occurs randomly on both hairpin structures. This cleavage of the hairpin structure leaves a short strand at one end of both V and J gene segments. To this short strand, the repair enzymes add the nucleotides complementary to the nucleotides of the opposite strand.
This addition of complementary nucleotides generates a palindrome sequence in the coding joint, because of which these added nucleotides are called P nucleotides.
The term palindrome refers to a string of letters with the same meaning whether you read it from left or from the right. For example, some palindrome words are “CIVIC,” “LEVEL,” “NOON,” “ROTOR,” etc. Palindrome examples also exist in phrases or sentences where punctuation, capitals, and spacing are ignored. For instance, in the case of “NEVER ODD OR EVEN.” The sequence of letters remains the same whether read from right to left or from left to right.
Similar is the case with palindromic repeat sequences in double-stranded DNA. A sequence of nucleotides is said to be palindromic if the sequence on one DNA strand is identical to the opposite strand’s sequence when both are read in their respective 5’ to 3’ directions. An example of a palindromic sequence is 5’-GGATCC-3’, which has a complementary strand, 3’-CCTAGG-5’. When either strand is read from the 5’ to 3’ direction, the nucleotide sequence remains the same, i.e., GGATCC.
Similarly, the sequence of P nucleotides added to the coding joint after the endonuclease activity on the hairpin structure remains the same when either strand is read from the 5’ to 3’ direction. For instance, the sequence of nucleotides added to the V segment remains TCGA when either strand is read from 5’-3’ direction. On the other hand, the sequence of nucleotides added to the J segment remains TATA when either strand is read from 5’-3’ direction. Thus, these added nucleotides are called P or palindromic nucleotides.
Further, the endonuclease enzyme that cleaves the hairpin structure does not recognize any specific sequence; instead, it recognizes the hairpin structure. Because of this, nicking of DNA occurs randomly on both hairpin structures.
Therefore, the variation in the position at which endonuclease cuts the hairpin structure and further addition of different P nucleotides by the repair enzymes in the coding joint leads to variations in the sequence of the coding joint, thus leading to the diversity in the variable region of the antibody molecule.
N-addition
The fourth source that generates antibody diversity is N-addition. N- addition generates additional diversity in the variable region coding joint VHDHJH in the rearranged heavy chains. The mechanism of VDJ recombination is similar to that of VJ recombination.
First, any of the DH segments join any of the JH gene segments to form the DHJH segment. Then the resulting DHJH segment joins with any of the randomly selected VH gene segments to form the VHDHJH segment. Let’s take the scenario of recombination of the DHJH segment. RAG-1 and RAG-2 recognize the recombination signal sequences or RSS of the gene segments D and J, followed by bringing the two signal sequences and the adjacent coding sequences in proximity. Then RAG-1 and RAG-2 cause single-stranded DNA cleavage at the junctures of signal sequences and coding sequence of D and J segments.
Then the free 3’OH group of the cut DNA attacks the phosphodiester bond linking the opposite strand to the signal sequence, thus forming a hairpin-like structure at the cut end of the coding sequence of the D and J segment. The hairpin structure is then cleaved by the endonuclease. This cleavage of the hairpin structure leaves a short strand at one end of both D and J gene segments. To this short strand, the repair enzymes add the nucleotides complementary to nucleotides of the opposite strand. This addition of complementary nucleotides generate a palindrome sequence in the coding joint, because of which these added nucleotides are called P nucleotides.
After adding P nucleotides by repair enzymes, the enzyme terminal deoxynucleotidyl transferase, abbreviated as TdT, adds up to 15 nucleotides at the DHJH coding joints. Then the resulting DHJH segment joins with any of the randomly selected VH gene segments through a similar mechanism. Also, the enzyme terminal deoxynucleotidyl transferase adds up to 15 nucleotides at the VHDHJH coding joints. Therefore, the diversity generated by adding N nucleotides is quite large because of the addition of random nucleotide sequences at the coding joints.
The diversity generated by N-addition at the VDJ joints is localized in the CDR3 region of the heavy chain.
The addition of N-nucleotides is not found in the VL-JL coding joints.
Somatic Hypermutation
The fifth source that generates antibody diversity is somatic hypermutation. Somatic hypermutation is a process in which mutations accumulate in the rearranged VDJ or VJ gene segments that encode the variable regions of heavy and light chains of an antibody molecule.
The mutations may lead to amino acid changes in the variable region of the antibody molecules that increase the antibody’s affinity for antigen. Most of these mutations are nucleotide substitutions, with deletions or insertions being less common.
Nucleotide substitution is a type of mutation where one base pair is replaced by a different base pair. For instance, A is replaced with T. This may lead to replacing one amino acid in a protein with another amino acid.
These mutations mainly occur within the CDRs of the VH and VL domains, where they most likely influence the affinity of antibodies for antigens.
When a B cell contacts antigen through the B cell receptor (BCR), the B cell proliferates. During the proliferation of B cells, the somatic hypermutation process generates mutations in the genes encoding the variable regions of both heavy and light chains of an antibody molecule. The introduction of mutations in the proliferating B cells leads to the production of thousands of B cells that possess slightly different B cell receptors and varying affinity for the antigen.
Then the B cells with the highest affinities for the antigen are selected. After this, the selected B cells with the highest affinity differentiate into plasma cells producing antibody and long-lived memory B cells with high-affinity B cell receptors. The memory B cells then provide enhanced immune responses upon reinfection.
Let’s understand it through an experiment that was conducted on a rabbit. On day 1, the rabbit was injected with an antigen. After a lag phase of 7 days, antibodies were generated against the antigen. Then, when the antibody titers came down, the rabbit was again immunized, and then the secondary response to Ag was developed in the rabbit. Similarly, when the antibody titers came down during secondary immune response, the rabbit was again immunized with the same antigen a third time, thus generating the tertiary immune response against the Ag.
It was observed that the amplitude of antibody titer during a primary immune response was small. In contrast, the secondary immune response showed a larger amplitude of antibody titer than that of the primary immune response. The antibody titer amplitude was even larger than that of the secondary immune response during the third immune response. It is because, during the primary immune response to antigen, the memory cells were generated, thus on second exposure to antigen, memory cells formed during the primary response begin to proliferate without any lag phase. Therefore antibody titer was higher than that of the primary immune response. The same was the case during the tertiary immune response. The memory cells formed during secondary immune response proliferated without any lag phase, thus resulting in a high antibody titer during the tertiary immune response.
When the scientists compared the B cell receptors on memory cells versus those on the naive B cells, they observed that the B cell receptors present on the naive B cells have a predefined sequence of CDRs determined by the rearrangement of VDJ segments. On the other hand, it was observed that mutations occurred in the B cell receptors of the memory cells after the secondary and tertiary immune response. Most of the mutations were clustered within the CDR region or antigen-binding site region of the antibodies. The number of somatic hypermutations progressively increases following the primary, secondary and tertiary immunizations, thus contributing to the overall increase in the affinity of antibodies for that antigen. During the tertiary immune response, the affinity of antibodies increases even 100–1000 fold.
Thus, it can be concluded from the experiment that during subsequent exposure to antigens, the affinity of the antibodies increases.
To summarize, the presence of numerous germline VDJ gene segments, combinatorial joining of VJ and VDJ gene segments, junctional flexibility, P and N region nucleotide addition, and somatic hypermutation contribute to overall antibody diversity.
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