GENERATION OF DIVERSITY
An individual can make a multitude of different antibodies, even though only a small portion of the genome is devoted to encoding antibody molecules. Diversity stems from the fact that several genes are necessary to encode a single antibody molecule, and these genes can shuffle their order within the germline DNA.
B. l light chain
The synthesis of chains is similar to that of k chains, involving rearrangements of V, J, and C exons. There is a smaller set of V genes,
and each of seven C genes is accompanied by just one J gene.
C. Heavy (H) chain
The genetic rearrangement mechanisms for H chains are similar to those
just discussed. However, H chains are more complex than light chains
because there are many more exons involved in producing the final
polypeptide.
Each of the antibody chains is encoded by a cluster of genes on a separate chromosome: k on chromosome 2, l on chromosome 22, and the heavy chain on chromosome 14.
I. Review of nonimmunoglobulin gene structure Regions of DNA that code for amino acids are called exons. Exons are interrupted by non-coding regions of DNA called introns. The DNA is transcribed into a primary RNA transcript within the nucleus of the cell.
Enzymes splice out the introns and ligate the exons together to yield a mature messenger RNA (mRNA) that is transported to the cytoplasm of the cell.
The mRNA is translated to protein on ribosomes of the rough endoplasmic reticulum.
Attached to each membrane protein is a signal peptide of about 20 amino acids. This signal peptide is encoded by a leader sequence preceding the membrane protein's gene. The signal peptide is used to transport the nascent polypeptide chain from the ribosome through the membrane of the
endoplasmic reticulum and into the Golgi apparatus. The signal peptide is
then cleaved off and the protein is inserted into the cell membrane.
II. Immunoglobulin gene structure
All of the events that occur during expression of non-immunoglobulin genes also occur when immunoglobulin genes are expressed. However, a unique
additional event occurs in the pre-B cell: immunoglobulin DNA rearranges
itself before it is transcribed into a primary RNA transcript. Large portions of germline DNA loop out and are excised by recombinase enzymes as the
lymphoid cell matures. As a consequence of this transpositional event, different exons are ligated together in a DNA molecule that is much shorter than that with which the cell started.
A. k light chain
The constant (C) and variable (V) regions of immunoglobulin chains are
encoded by different exons. There are 100-200 separate exons for the V region of the k light chain and a single C exon. The V exon encodes the first 95 amino acids of the V region polypeptide, while the J (joining)
region exon codes for the remaining 13 amino acids of the V region.
During transposition of k light chain DNA, one of the V exons moves to a position adjacent to one of the J region exons. Any V exon can combine with any J exon (except J3, a pseudogene). After transposition, the entire sequence is transcribed into the primary RNA transcript. All of the introns and the unused J chains are spliced out of the transcript, and the remaining coding sequences are ligated into a continuous strand of mRNA. In the cytoplasm the mRNA is translated to polypeptide which associates with heavy chain polypeptide. Carbohydrate residues are added in the rough endoplasmic reticulum (RER), then modified in the Golgi body, to complete the antibody molecule.
B. light chain
The synthesis of chains is similar to that of k chains, involving rearrangements of V, J, and C exons. There is a smaller set of V genes, and each of seven C genes is accompanied by just one J gene.
C. Heavy (H) chain
The genetic rearrangement mechanisms for H chains are similar to those
just discussed. However, H chains are more complex than light chains because there are many more exons involved in producing the final polypeptide.
Heavy chain DNA contains about 50 functional V region genes, 30 diversity (D) genes, 6 J genes, and multiple constant region genes. The small D genes provide an additional degree of variability in the possible permutations of genetic sequences. Although the figure above shows only a single C region gene for each isotype of antibody, in reality there is a separate C exon for each constant domain found in the heavy chain. The g genes also have a separate exon coding for the hinge, and all the genes have one or more exons coding for membrane-bound immunoglobulin. The precise positions at which the genes for the V, J and D segments are spliced together are not constant, and imprecise
recombination can lead to changes in the amino acids at these junction
sites, contributing another source of antibody diversity.
Recombination of immunoglobulin genes is controlled by two recombination-activating genes called RAG-1 and RAG-2. The recombinase enzymes encoded by these genes catalyze the formation of
a stem-and-loop structure in the immunoglobulin DNA that brings together distant exons. The enzymes cleave off the loop, and the gap in the DNA is filled in by nucleotides under the direction of terminal deoxyribonucleotidyl transferase. The nucleotides that are added are not encoded by the germline DNA, hence they contribute what is known as N-region diversity to the antibody molecule.
III. Class switching
While a single B cell makes antibody of a single specificity, the particular
class of antibody that the cell makes can change from IgM to IgG, IgA, or IgE. This class switch can occur at either the DNA or RNA level. Class-switching at the DNA level employs switch sequences that precede the
exons for all constant region genes except the d gene. Initially, B cells
transcribe a VDJ gene and a m heavy chain that is spliced to produce mRNA
for IgM. Under the influence of T cells and cytokines, class-switching to
other classes may occur, illustrated below as a switch from IgM to IgG1. All intervening constant region genes—in this case m, d, and g 3—are lost. The cytokines that affect class switching are believed to induce the loosening of the structure of the DNA double helix at only certain points along the immunoglobulin genes, allowing the recombinase access only to specific C regions.
Class switching may also occur at the RNA level through differential splicing
of the primary transcript at different polyadenylation sites. When this happens, it is possible for a single cell to produce two (or more) different
isotypes simultaneously, but both isotypes will share the same variable region exon and will therefore have the same antigen specificity.
The first time an antigen is encountered, there is a primary immune response that consists mostly of IgM. Subsequent contact with the same antigen usually esults in a secondary, or anamnestic, immune response that consists mostly of IgG, especially when the antigen contacts B cells in the peripheral lymph or blood. Antigens contacting B cells in the MALT usually induce a class switch to IgA, while allergens most often induce IgE synthesis.
IV. Somatic mutation
Often a class switch is accompanied by somatic mutations in the immunoglobulin hypervariable region. Most somatic mutations are caused by
point mutations in the DNA that lead to a single amino acid change. The
somatic mutations usually result in greater affinity of the antibody for its antigen (affinity maturation).
V. The TCR
The chains of the TCR are encoded on chromosomes 7 (b, g ) and 14 (a, d).The general arrangement of T cell receptor genes is very similar to that of
immunoglobulin heavy chains in that multiple genes encode V, D, J and C
polypeptides for each chain. T cell receptor and antibody genes recombine
by similar mechanisms; however, T cell receptor genes do not undergo productive somatic mutations. |
