Organization of immunoglobulin genes (Part 7- Antibody Basics)
Welcome to the 7th part of the 13-part series on Antibody basics.
Previous parts: Part 1, Part 2, Part 3, Part 4, Part 5 and Part 6
As discussed in Part 1, each antibody molecule comprises four polypeptide chains: two light chains and two heavy chains. Each light and heavy chain is made up of 2 regions: variable region and constant region. The variable region of a light chain and a heavy chain together form the antigen-binding site. We have a countless number of antibodies that can recognize a countless number of antigens. It is estimated that we can recognize more than 100 million different epitope sequences by our B cell receptors or immunoglobulins.
Each B cell can recognize and bind specific antigen, which triggers its activation, resulting in the generation of plasma cells that secrete antibodies.
For several decades researchers sought to investigate a genetic mechanism that could explain the tremendous diversity of antibodies.
Different theories were proposed more than 50 years ago.
Germline & somatic hypermutation Theory
1. First is the germline theory, which suggests that the genome contributed by the germ cells: egg and sperm contains an extensive repertoire of immunoglobulin genes that correspond to 100 million different specificities. This could not hold true because the body cannot invest such a large number of genes being dedicated only to the immune system and that also to B cells in specific.
2. So, the second theory proposed was somatic hypermutation, according to which genome contains a relatively smaller no of Ig genes, from which a large no of antibody specificities can be generated by mutation or recombination in the somatic cells. As more and more immunoglobulins were sequenced, it became evident to immunologists that there must be some mechanisms that not only account for generating antibody diversity but also for maintaining consistency.
Neither the germline nor somatic-mutation theory gives a reasonable explanation for the mechanism that could generate diversity in the variable region of heavy and light chains while preserving the amino acid sequences of constant regions of both the chains.
Dreyer and Bennett’s theory
In 1963, Dreyer and Bennett proposed that two separate genes encode for a single heavy or light chain; one gene encodes for the variable region, and the second gene encodes for the constant region. Thus, the two genes must somehow come together at the DNA level to form a continuous message and get translated to form a single immunoglobulin heavy or light chain.
Additionally, they proposed that hundreds or thousands of Variable region genes and single gene copies of class and subclass of Constant region of antibodies were carried in the germline. This could account for the conserved constant region of immunoglobulins while allowing evolutionary diversification of variable region genes. But the suggestion that two genes encoded a single polypeptide contradicted the existing one gene-one polypeptide principle.
Tonegawa’s Theory- Immunoglobulin genes rearrange
Thirteen years later, in 1976, two scientists named Tonegawa and Hozumi found the first direct evidence that separate genes encode V and C regions of immunoglobulins. They suggested that during the differentiation of lymphocytes from the embryonic state to the fully differentiated plasma stage, V and C genes undergo rearrangement. In the embryo, V and C genes are separated by a large DNA segment containing restriction endonuclease sites. But during the B cell differentiation, the V and C genes are brought closer together and are rearranged.
Let us look at the experiment they carried out. They took DNA from two types of cells. First was the embryonic cell that contributes germline DNA. The other source of DNA was the myeloma cell, which is a cancerous plasma cell. The DNA isolated from two cells was then digested with a restriction endonuclease enzyme. The DNA fragments obtained by digestion were electrophoresed separately.
Electrophoresis is the technique to separate the DNA fragments by size. For this, the digested DNA is loaded into wells at one end of the gel, and then the electric current is applied. Since DNA fragments are negatively charged, they start migrating towards the positive electrode. Smaller DNA fragments migrate through the gel more quickly and, therefore, travel further than larger fragments that migrate more slowly and travel a shorter distance.
As a result, the molecules are separated based on size by electrophoresis. Then the germline and myeloma DNA fragments separated by electrophoresis are analyzed by Southern Blotting. In the Southern Blotting technique, a radiolabelled probe (containing one of the Ig gene segments J) is allowed to hybridize to the electrophoresed fragments of DNA isolated from both cells.
The pattern obtained from restriction digestion followed by hybridization was different in the DNA of 2 cells. The hybridized restricted fragment of myeloma DNA is of larger size than that of germline DNA. This experiment suggested that there has been a change in the DNA of the myeloma cell.
It is because several coding sequences separated by non-coding sequences in the germline DNA are brought together at the DNA level during rearrangement in the myeloma cell. Thus, the size of the hybridized restricted fragment of myeloma DNA is larger than that of germline DNA. This experiment concluded that rearrangement of immunoglobulin genes occurs during B cell differentiation.
Organization of Ig genes
The genes encoding the immunoglobulin chains are present as multiple gene segments and are present on different chromosomes. There are three immunoglobulin loci on human DNA. These are κ chain locus, ƛ chain locus, and heavy chain locus. Locus is defined as the specific and fixed position on a chromosome where a particular gene is found.
The κ chain locus is present on chromosome 2 and contains the gene segments encoding the κ chain. The ƛ chain locus is present on chromosome 22 and contains the gene segments encoding the ƛ chain, and the heavy chain locus is present on chromosome 14 and has the gene segments encoding the heavy chain.
Assume the bold black line represents germline DNA. At the 5' end of any immunoglobulin locus on germline DNA, there is a cluster of variable gene segments. Variable gene segments are designated as V and are numbered from 1 to n. Each V gene segment is preceded at its 5’ end by a leader sequence designated as L. The leader sequence encodes a leader peptide that facilitates the transport of growing immunoglobulin polypeptide chains into the endoplasmic reticulum.
At the 3’ of the cluster of V gene segments, another cluster of J gene segments are present. J stands for joining. Like V gene segments, J gene segments are also numbered from 1 to n.
The C gene segments are located at the 3’ of the J gene segments in each immunoglobulin loci. The number of gene segments varies in each immunoglobulin locus.
In the case of heavy chain locus, additional D gene segments are present between V and J segments. Here D stands for diversity. Therefore, light chain polypeptide is encoded by V, J, and C gene segments. On the other hand, heavy chains are encoded by V, D, J, and C gene segments.
During B cell maturation, these gene segments undergo rearrangement and are brought together to form a functional and continuous V region exon and C region exon. Exons are the coding sequences of the gene, which upon transcription and translation, produce Variable and Constant regions of the heavy and light chains of antibodies. It is essential to understand that though there are multiple copies of each type of gene segment in the germline DNA; still, only one gene segment is expressed at a time to synthesize one immunoglobulin molecule.
For instance, 1 V segment rearranges with 1 J segment to produce a VJ exon. This VJ exon encodes the variable region of the light chain.
And on the other hand, 1 V segment rearranges with 1 D and 1 J segment to produce a VDJ exon. This VDJ exon encodes the variable region of the heavy chain of the antibody. The C gene segments encode the constant regions of the antibody.
The gene segments V, J, D, and C, along with the L sequence, are the coding sequences; therefore, they are also called exons. These exons are separated by some non-coding sequences, which are referred to as introns. This entire process of rearrangement and organization of the gene segments gives our immune system its capabilities to generate a repertoire of antibodies that are able to recognize and respond to a variety of antigens.
Organization of gene segments encoding light chains
Organization of gene segments encoding the ƛ chain: In humans, the ƛ light chain locus, present on chromosome 22, contains 31 Vƛ gene segments. Each Vƛ gene segment is preceded at the 5’ end by a leader sequence that encodes a leader peptide designated as L. The leader peptide transports the growing ƛ polypeptide chain into the endoplasmic reticulum. Leader peptides are later cleaved; therefore, they are not present in the mature ƛ chain.
The cluster of 4 Jƛ segments is present at the 3’ of the Vƛ gene segments. The rearranged VƛJƛ segment encodes the variable region of ƛ light chain.
Since the variable region comprises 110 amino acids, the Vƛ gene segment encodes the first 97 amino acids, and Jƛ encodes the remaining 13 amino acid residues of the variable region.
Additionally, 4 Cƛ gene segments are located at the 3’ of the Jƛ gene segments. Each Jƛ gene segment is associated with 1 Cƛ gene segment. The presence of multiple Cƛ gene segments gives rise to the four subtypes of ƛ light chain ƛ1, ƛ2, ƛ3, and ƛ4.
Organization of gene segments encoding the κ chain: The κ light chain locus present on chromosome 2 contains 40 Vκ gene segments. Similar to Vƛ gene segments, each Vκ gene segment is preceded by a leader sequence at the 5’ end. The 5 Jκ segments are present at 3’ of Vκ gene segments. Out of 5 Jκ gene segments, 4 Jκ gene segments are functional. The non-functional Jκ segment is represented by Ѱ. The rearranged VκJκ segment encodes the variable region of the κ light chain. The Vκ gene segment encodes the first 97 amino acids, and Jκ encodes the remaining 13 amino acid residues of the variable region.
The κ light chain locus has a single Cκ gene segment present at the 3’ of the Jκ gene segments. The single Cκ gene segment encodes for the entire constant region of the κ light chain. Since there is only one C gene segment, therefore, no subtypes of κ light chains are present.
Heavy chain multigene family
The organization of immunoglobulin heavy chain genes is similar to κ and ƛ light chain genes but is more complex. Here in the case of heavy chains, D gene segments are also present in addition to V, J, and C gene segments. In other words, the heavy chain family contains VH, DH, JH, and CH gene segments. In humans, there are 51 VH, 27 DH, 6 JH, and 8 CH gene segments. The rearranged VHDHJH gene segments encode the variable region of the heavy chain of an antibody.
The VH gene segment encodes 1–94 amino acids, the DH gene segment encodes 95–97 amino acids, and the JH segment encodes 98–113 amino acids of the variable region of the heavy chain.
As discussed in Part 2, within the variable domain of each heavy and light chain, there are 3 CDRs. The CDRs from both VH and VL domains contribute to the antigen-binding site. In this case, the DH segment encodes amino acids within the third complementarity determining region CDR3 of the antigen-binding site of the antibody.
Also, the CDRs within the variable region account for the diversity of antigens that a repertoire of antibodies can recognize. Since the DH segment encodes amino acids within the third complementarity determining region CDR3, thus the segment DH is designated D because of its contribution to the generation of antibody diversity. The CH gene segments are arranged in the order Cµ, Cδ, C𝛾3, C𝛾2, C𝛾1, C𝛾4, Cε, Cα1, and Cα2. The constant gene segments encode the constant domain of the classes and subclasses of antibodies — for instance, Cµ gene segment codes for the constant domain of IgM antibody.
During the B cell maturation, the heavy chain variable region gene segments rearrange first, then the light chain variable region rearranges. After the rearrangement, each B cell contains a single functional variable region DNA sequence for its heavy chain and another for its light chain. Then the B cell becomes committed to produce a membrane-bound antibody with a unique antigen-binding site encoded by a particular sequence of its rearranged variable region genes. Later, the rearrangements of heavy chain constant region genes occur. The rearrangements in the gene segments of the heavy chain constant region generate changes in the immunoglobulin class or isotype expressed by a cell. These changes, however, do not affect the cell’s antigenic specificity.
If you liked this article and want to know more about Antibodies and their role in Therapeutics and Diagnosis, click the below links.
For book lovers:
For video lovers:
Happy learning!
