The generation of lymphocyte antigen receptors Flashcards
The mechanism of generating antigen receptors
- Individual lymphocytes bear numerous copies of a single antigen receptor with a unique-binding site.
These cells collectively enable a response to a great variety of antigens. - Initial evidence of the immense size of the antibody repertoire:
Use of synthetic molecules to stimulate antibody production;
Antibodies can discriminate between small synthetic molecules differing in as little in the position of an amino or hydroxyl group on a phenyl ring.
Paradigm-shifting experiment
Hozumi and Tonegawa
The human antibody repertoire is at least 10^11.
There are ~ 20 000 - 25 000 protein-coding genes.
During the experiment they tried to answer the following questions:
- Does the DNA encoding Ig light chain C and V regions exist in separate segments in non-antibody producing cells?
- Can a piece of DNA change its place on a chromosome in a somatic cell?
The experiment that answered these questions is described below:
They harvested cells from non-producing and producing antibodies tissues in the the same animal. Then they extracted the DNA of those cells, cut them with a restriction enzyme and blotted using radioactive sequences complementary to the antibody’s constant and variable regions. They saw differences in the band patterns of the two types of cells, meaning that the DNA reconfigured in the cells that produce antibodies.
- Explanation:
Segments of the genomic DNA within the immunoglobulin genes are rearranged in cells of the B-lymphocyte lineage, but not in other cells.
Primary immunoglobulin gene rearrangement
V regions of the receptors are encoded in gene segments.
Gene segments are assembled in the developing lymphocyte by somatic DNA recombination to form a complete V-region sequence, a mechanism known as gene rearrangement.
A fully assembled V-region sequence is made up of two or three types of gene segment, each of which is present in multiples copies in the germline genome.
The selection of a gene segment of each type during gene rearrangement occurs at random.
The gene rearrangement segments
There are the variable regions that are encoded in segments. One type of segment is called the V segment, the other one is called a J segment. These two segments are only for the light chain. The heavy chain will have an extra segment - the D segment.
- The V segment is the variable segment - the bulk of the domain (from 97 AA);
- The J-segment is the joining segment (about 12 AA)
- The D-segment is the diversity segment (about 4 AA)
The variable region is made of these segments, but they need to be assembled together.
The assembly can happens in the following way:
- The segments need to stick together into a functional exon. By the somatic recombination, the V and J segments will go together in the light chain;
- In the heavy chain we will need an extra step of somatic recombination, because we have three segments there. First DJ go together, and then the V segment is also attached.
- The functional exon is then used for transcription of the proteins.
There are multiple copies of all gene segments in germline DNA.
Multiple contiguous V gene segments are present at each immunoglobulin locus.
It is the random selection of just one gene segment of each type that makes possible the great diversity of V regions among Igs.
- There are multiple variable segments for the light chain, either kappa or lambda, and for the heavy chain - there is only one type.
- The diversity segment is only in the heavy chain.
- The joining segments are found in all of them;
- For the constant region for kappa only one, for lambda 4-5, and for H we have 9.
This diversity is only for the functional part. - The immunoglobulin gene segments that encode these chains
are organised into three clusters or genetic loci. Each locus is on a different chromosome.
The human V gene segments can be grouped into families in which each member shares at least 80% DNA sequence identity with all others in the family.
Combinatorial diversity
All the segments can combine for the different parts of the light and heavy chains. According to their abundance, the combinations can be calculated:
- Kappa:
38 (V)5 (J)=190
- Lambda:
33(V)5 (J)=165
- Heavy:
46(V)23(D)6(J)=6348
They come from a different part of the chromosome, they rearrange separately, so when the functional part comes together, there can be as many combinations as above.
Now the Heavy chain can be multiplied by the Light chain, and that would give 2 million combinations.
So the combinatorial diversity would give us a lot of space to live up to the antibody repertoire of 10^11.
Diversity considerations
Pseudogenes.
Not all the gene segments discovered are functional.
A proportion have accumulated mutations that prevented from encoding a functional protein.
Because there are many V, D, and J gene segments in germline DNA, no single one is essential. This reduces the evolutionary pressure on each gene segment to remain intact, resulting in pseudogenes.
Pseudogenes undergo rearrangement just like normal gene segments, a significant proportion of rearrangements incorporate a pseudogene rendering a nonfunctional product.
V gene segments usage
They are used at different frequencies. Some are common in antibodies, while others are found only rarely.
Heavy chain – light chain pairs
Not every heavy chain can pair with every light chain. Certain combinations of VH and VL regions are not stable.
CDR1 and CDR2 are encoded in the V gene segment itself.
CDR3 is encoded by the additional DNA sequence that is created by the joining of the V and J segments for the light chain, and the V, D, and J gene segments for the heavy chain.
Additional diversity can result from the generation of CDR3 that can be the result of joining one D segment to another D segment.
D-D joining is found is ~ 5% of antibodies and is the major mechanism accounting for unusually long CDR3 loops.
12/23 rule
Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences called recombination signal sequences.
There is a heptamer close to the segment, a spacer(12 or 23bp) in the middle and nonamer at the end on the segment. The spacer will dictate which segments recombine together.
12/23 rule:
Recombination occurs between gene segments located on the same chromosome.
A gene segment flanked by an RSS with a 12-bp spacer typically can be joined only to one flanked by a 23-bp spacer RSS.
12 bp correspond to one DNA double helix turn.
23 bp correspond to two turns.
Bringing the heptamer and nonamer sequences to the same side of the DNA helix to allow interactions with proteins catalyzing recombination.
- Heptamers and nonamers align back-to-back;
- The shape generated by the RSS’sacts as a target for recombinases;
- An appropriate shape can not be formed if two 23-mer flanker elements attempted to join.
Combinational diversity
The rearrangements happen like this:
- lambda: V (23bp) joins J(12bp);
- kappa: V(12bp) joins J(23bp);
- H chain: V(23bp) joins D(12bp) joins J(23bp).
The extra DNA can be removed from the sequence by looping out and deletion. Rearrangement by inversion
Intervening DNA. This mode of recombination accounts for half of all Vκ to Jκ joints.
For both mechanisms we start with a linear configuration. The way the DNA is configured n a loop, while for other it is in a coil.
Enzymatic recombination. Combinational diversity.
- V(D)J recombination is a multistep enzymatic process
- Involves V(D)J recombinase
- RAG-1 and RAG-2 are the lymphoid-specific components of the recombinase along with the Terminal deoxynucleotidyl transferase (TdT).
- RAG-1 and RAG-2 are expressed in developing lymphocytes only when they are engaged in assembling their antigen receptors.
The way it practically works in steps is:
1)RAG1/2 binds RSS - synapsis of RAG complexes.
(RAG1/2 and HMG proteins bind to the RSS and catalyze synapse formation between a V and J gene segment);
2) Cleavage of RSS to coding joints and signal joints.
(RAG1/2 performs a single straded nick at the exact 5’ border of the heptameric RSSs bordering both the V and the J segments);
3) The coding Joints are kept together by the Ku70:Ku80 by joining the DNA ends.
(The hydroxyl group attacks the phosphate group on the non-coding strand of the V segment to yield a covalently-sealed hairpin coding end and a blunt signal end);
4) DNA-PK:Artemis opens a hairpin
*Artemis makes a single-strand nick, this nicking can happen at various points along the hairpin, which leads to sequence variability in the final joint.
(Opening of the hairpin can result in a 5’ overhang, a 3’ overhang, or a blunt end)
5) TdT processes the DNA ends
*DNA repair enzymes modify the opened hairpins by removing nucleotides, at the same time TdT adds nucleotides randomly to the single-strand ends.
Addition and deletion of nucleotides can occur in any order.
(Cleavage of the hairpin generates sites for P nucleotide addition)
6) DNA ligase ligates the DNA ends
( Ligation of light chain V and J regions)
*They make up palindromic sequences added to the ends of the gene segments
7) In heavy chain VD and DJ joints only: Exonuclease cleavage results in loss of coding nucleotides at joint - can occur on either or both sides of joint
8) Non-templated nucleotides are added to the coding joint by TDT. Up to 20nu.
9) Ligation of the heavy chain by DNA ligase IV and NHEJ proteins
10) Imprecise coding joints.
Side) The signal Joints are also held together by the Ku70:Ku80, which ends up in the circular DNA.
Other proteins of the recombinase complex are mainly ubiquitous DNA-modifying proteins involved in the repair of DNA double-strand breaks and the modification of the ends of broken DNA strands.
Enzyme functions:
- RAG-1/2 and HMG:
Recognize and align two RSS
- Ku70:Ku80:
Ring around DNA
- DNA-PKcs:
Phosphorylates Artemis
-Artemis:
Nuclease activity
-Terminal deoxynucleotidyl transferase (TdT):
Adds nucleotides randomly to the single-strand ends
-DNA ligase IV:
Joins DNA
-XRCC4 (turquoise):
DNA repair protein
-DNA pol μ and λ:
DNA-end fill-in synthesis
-DNA pol μ:
Independent addition of nucleotides
Junctional Diversity
It is estimated that at least 10^11 different receptors could make up the repertoire of receptors expressed by naive cells, and diversity could be several orders of magnitude greater, depending on how one calculates junctional diversity.
The total number of antibody specificities available to an individual is known as the antibody repertoire or immunoglobulin repertoire.
Nonproductive rearrangements
As the total number of nucleotides added by TdT or deleted by exonuclease activity is random, the added nucleotides often disrupt the reading frame of the coding sequence beyond the joint. Therefore nonfunctional proteins are produced.
2/3 rearrangements are nonproductive, many B-cell progenitors never succeed in producing functional immunoglobulin and never become mature B cells.
Junctional diversity is achieved only at the expense of considerable cell wastage.
T-cell receptor gene rearrangement
The T-cell receptor gene segments are arranged in a similar pattern to immunoglobulin gene segments and are rearranged by the same enzymes
- For alpha, there will be V and J segments;
- For beta, there will be V, D, and J segments.
Here all the segments are clustered together, and there will be a region for the constant part (in alpha);
In beta there will be two constant portions.
There are more alpha than beta segments.
They have ore J segments and therefore there will be more diversity.
T-cell receptor rearrangement takes place in thymus, while in the B-cell it happens in the bone marrow.
Gene recombination deficiencies
All known defects in genes that control V(D)J recombination affect T cells and B cells equally, affected individuals with these genetic defects lack functional lymphocytes.
- Severe combined immune deficiency (SCID):
Lymphocyte specific defects
KOs(knock-outs): RAGs DNA-PKcs, Ku, Artemis
No lymphocyte development at the gene rearrangement stage or produce trivial numbers of B and T cells.
Tdt-/- do not add extra nucleotides to the joints between segments. - SCID:
Bubble boy syndrome: nonfunctional RAG1 or RAG2. Basically, people with this deficiency don’t have an adaptive immunity.
Omenn syndrome: RAG-1 and RAG2 mutations that result in partial V(D)J recombinase activity. No circulating B cells and infiltration of skin by activated oligoclonal T lympocytes. - Irradiation-Sensitive SCID (IR-SCID)
Mutations in the DNA repair pathways. Defects in Artemis produce a combined immunodeficiency of B and T cells that is associated with increased radio sensitivity. - Ataxia Telangiectasia
Radiosensitivity with some degree of immunodeficiency
Due to mutations in ATM which encodes a protein kinase of the DNA-PKcs family.
The number of segments for the T-cell receptors
Alpha and beta:
- V segments: 70 and 52
- D segments: 0 and 2
- J segments: 61 and 13
Calculating the combinatorial diversity, we have more diversity compared to the B cell receptors. This is because the T-cell receptors have more alpha V and J segments.
All together the total diversity gives a number of 10^13 (B cell) and 10^18 (T cells) possible combinations.
T-cell receptors concentrate diversity in the third hyper-variable region.
The structurally equivalent CDRs of the T-cell receptor α and β chains, to which the D and J segments contribute, also form the center of the antigen-binding site of T-cell receptor.
γ:δ T-cell receptors
γ:δ T-cell receptors are also generated by gene rearrangement.
There are substantially fewer V gene segments at the TCRγ and TCRδ than other variable loci.
Gamma receptors also have V and J segments between the constant and variable regions.
Delta receptors are located between the segments of the alpha locus. It is just after the variable alpha and before the J alpha. And this is probably why there are less gamma-beta subunits. These subunits will recognize molecules from nonclassical MHC, which is even more specificity.
Conclusion:
- T-cell receptors are structurally similar to immunoglobulins and are encoded by homologous genes.
- T-cell receptors genes are assembled by somatic recombination from sets of gene segments in the same way that the immunoglobulins genes are.
