Evolution Of Antibody Recombination

As this is my first molecular evolution post, I would appreciate any feedback on clarity. No question is too trival either.

Vertebrates have an amazing capacity to produce proteins called antibodies. These form an important part of our immune system. It has been estimated that humans can produce up to 10 billion different antibodies. In our life time, we will produce antibodies that neutralise toxins (eg tetanus toxin) and recognise components of bacteria, viruses and parasites. If we are unlucky, we can produce antibodies to our own molecules (eg systemic lupus erythyamatosis and some forms of arthritis). We may also produce antibodies to food substances or pollen and develop allergies. We can even produce antibodies to chemicals that do not occur in nature. This is even more amazing when we consider that antibodies are proteins, and proteins are encoded by genes. Why is this amazing? Well, estimates suggest that humans may contain as few as 25 000 genes. How this large antibody repertoire is possible will be explained shortly. But I better introduce these molecules first.

Antibody structure

Antibodies are generally made up of 4 protein chains that are stuck together to form a Y shaped molecule. Each antibody consists of two identical heavy and two identical light chains. This molecule shown in Figure 1 is called an immunoglobulin and has two binding sites for recognising other molecules (called antigens). It is differences these sites (also called the variable regions) that determine what molecules an antibody can bind to. Some types of immunoglobulin (called Ig for short) can also consist of 2 (Ig A) or 5 (IgM) copies of these Y shaped immunoglobulins stuck together. This can increase their ability to keep antigens bound.


Figure 1

Generation of Antibody Diversity
As mentioned above, it is the chemistry of the variable regions that determines which antigens a particular antibody can bind. Both the heavy and light chains contribute to antigen binding, so, juggling different heavy and light chain combinations is one way of increasing the number of possible different antibodies an organism can produce. This however is not the main mechanism employed.

Antibodies are the products of different gene segments that are stuck together in different combinations at random. These segments fall into three different classes called Variable (V) Diversity (D) and Junctional/Joining (J) genes. The human heavy chain variable region has 50 functional V segments, 30 D segments, and six J segments to choose from. These segments all occur on the same chromosome. Also on the same chromosome, are the constant region genes of the heavy chain (basically the non variant part of the heavy chain). The light chains also have a constant region. They however do not use D segments. Their V and J segments also occur on the same chromosomes as their constant regions. The light gene situation however is slightly more complicated. There are two types of constant chain, termed the kappa and lambda chains - each antibody contains only one or the other. The Kappa chain can choose from 40 V and 5 J genes and the lambda chain can choose from 30 V and 4 J genes.
So, to summarise, and antibody is made up from a light chain that is stuck together from either a kappa or a lambda gene stuck to a randomly chosen J gene, which is then stuck to a randomly chosen V gene. The protein chain that this codes for is then stuck to a heavy chain that is coded for by a constant region gene (one of either a,b,g,d or e) fused to a randomly chosen V,D and J gene segment. These joining events can also be imprecise, further increasing the antibody repertoire. This can be increased yet further by an enzyme called Terminal Deoxynucleotidyl Transferase, which can incorporate up to 15 new nucleotides in to the heavy chain V and J gene segments. The cell then turns these chains into a Y shaped immunoglobulin molecule. Once the genes have been rearranged, further mutations can occur. This can help select for B cells producing higher affinity antibodies by a natural selection like process.
The intervening DNA that is not used is cut out and discarded (figure 2)


Figure 2


Michael Behe uses the above process as an example as an irreducibly complex system that he claims could not arise by natural means and must require a “designer” (euphemism for the Christian god). At the Dover county intelligent design trial, he boasted in his ignorance that “the scientific literature has no detailed testable answers on how the immune system could have arisen by natural selection and random mutation”. A problem with claims made from the argument from incredulity (I prefer the term personal ignorance) is that it is easily dismissed in an instant – not to mention that it shows a distinct lack of imagination and intellectual laziness. Behe was presented with a tiny fraction of the scientific literature that he claimed did not exist. This included a pile of peer reviewed research papers and text books on the subject.

Behe claims that to generate antibodies, you need the gene segments, recombination signals and the Recombination Activating Genes (called RAG1 and RAG2) all present at once, or the system cannot work. This also displays a lack of understanding about how evolution works – it tinkers and refines what is already there. Behe’s claim displays as big a misunderstanding of evolution as the creationists who claim the eye evolved in one step.
After the long introduction, it is the RAG1 protein that I wish to concentrate on.


Mechanism of RAG action

As mentioned previously, when antibody gene segments are chosen at random, the intervening DNA is cut out. This is achieved by the RAG proteins, and animals with function destroying mutations in these genes are severely immuno-deficient. Figure 3 shows the mechanism of action. The enzyme recognizes the Recombination Signal Sequences (yellow or orange triangles) that are located before or after the gene segments to be spliced together. Any combination of segments can occur and the process is random. The RAG proteins then cut out the intervening stretch of DNA and other DNA repair enzymes stick the two ends of the gene segments together (in this case V49 to D28).


Figure 3


Evolution of recombination

So, what would we need to evolve such a system? Firstly, an antibody like molecule. Interestingly, antibodies belong to an ancient family of proteins called the immunoglobulin superfamily. The family is defined on the basis of of a common structural feature called the immunoglobulin domain. Furthermore, this class of protein is found throughout the animal kingdom from sponges up, where their function is molecular recognition. So, we have a starting material to evolve an antibody gene. Michael Behe has claimed that since the chance that an antibody of a single specificity would be unlikely to recognise anything of importance, it would be of little advantage and could not evolve (Darwin’s black box pp. 130-131) . This is where the ignorance and intellectual laziness of the creationist is exposed. The immune system is packed with single specificity proteins, such as the Toll like receptors that recognise repeating molecular patterns. Secondly, If the ancestral antibodies recognised a more restricted set of molecules like a particular carbohydrate structure that is only found in pathogens (and there are plenty), it would be of significant advantage. Many invertebrates use a group of carbohydrate recognising proteins called lectins to recognise infectious agents.

The next thing a primitive recombination system would need would be the RAG genes. It has been hypothesised for about 25 years that the RAG genes may have evolved from pieces of selfish (or if you wish, parasitic) DNA called transposons. There are two interesting features of transposons; they insert into genomes and often encode genes that allow them to cut themselves out and paste themselves elsewhere in the genome. They often contain structures at either end called Terminal Inverted Repeats, which are essential for function. These are structurally very similar to Recombination Signal Sequences. Many transposons also make copies of themselves and these become incorporated throughout the genome – this is also a possible source of V,D and J gene segments.
To test this hypothesis, we would predict that RAG like sequences exist in transposons elsewhere and that they pre-date the evolution of jawed vertebrates. Both these predictions have recently been realised. A family of transposons (called Transib) that contain structural similarities to RAG1 and Recombination Signal like Sequences has been described from the sea slug Aplysia californica. These Transib family members have now been shown to be present in insects, nematodes, fungus and hydra, so they clearly are of ancient origin.

One further line of evidence confirms the link between RAG and transposons: RAGs have recently been shown to be able to act as transposons.

Interestingly, if RAG genes and antibody gene fragments are put into an organism that does not rearrange antibody genes – like yeast, they are perfectly able to. This points out that the rest of the machinery needed to stick the broken ends of gene segments together was already present – that’s what evolution does, it uses material already there and builds upon it.

Finally, it would appear that the ancestral RAG gene was originally of viral origin.

This illustrates the weakness of the argument of personal ignorance. Behe could not conceive how antibody rearrangement could evolve – shame he did not look at the available evidence.