The Rag-1: The Human Immune System

Introduction
The human immune system consists of an immense amount of proteins, cells, and systems that all simultaneously work together to defend the body against any pathogens that come its way. One of these important proteins is the recombinase activating gene, Rag-1. Rag-1 plays a major part in lymphocyte production, and when this gene is knocked out, it can cause a major negative effect in the immune system. When Rag-1 is knocked out, the process in making T and B cell receptors is completely disabled. Because of this, all of the lymphocytes in a Rag-1 deficient organism are immature, and unable to participate in normal immune system functioning. Fortunately, there are no physical or neurological deformities in the organism because of

this deficiency. In humans, if even the smallest mutation occurs in the Rag-1 gene, as in the Omenn syndrome, the consequence is fatal if not treated properly. Symptoms of this deficiency are often severe because of the immune system’s inability to fight back. At the molecular level, some mature lymphocytes are able to be made, but they are few in quantity and have almost no variability in receptor type. The Rag-1 protein is critical for a normal functioning immune system and for survival.

Background
The protein Rag-1 is structurally sound for its function consisting of around 1000 peptides (Godderz and Rodgers).

The Rag-1 comes in different forms such as a dimer, tetramer, or octomer. The octomer is the most stable form in a not as strong ionic solution, but the most common form found in a biological environment is the dimer form. Each subunit of the Rag-1 protein contains many alpha helices also. The active site in Rag-1 is soluble, and it houses the most important and largest domains of the protein: the N-terminis domain, the C-terminis domain, and the core domain. A combination of these domains could perform all of the functions of the protein without the other residues. The N-terminis region and the core domain bind to RSS site at the nonomer region and the 12 or 23 spacer portion respectively. In binding to the RSS site, the core domain instigates a conformational change in the DNA which allows the complex to interact and cut the DNA appropriately. It is theorized that the Rag-1 gene along with its partner, Rag-2, were once a transposable element (Agrawal et al.). Scientists have found that the transposon containing these two genes was solidified into the genome once jaw containing vertebrates split from jawless vertebrates. From then on, the Rag genes have been in mammalian DNA, and have played a major part in their

immunity.

V(D)J Recombination is an immunological process in which Rag-1 participates in a recombinase complex that begins maturation of immature T and B cells into full-functioning lymphocytes (Godderz and Rodgers). V(D)J Recombination, or variable, diversity, and joining, occurs in the bone marrow in B cells and in the thymus in T cells (Hewitt et al.). For B cells, V(D)J recombination creates the heavy and light chain immunoglobulin receptors on the surface of the B cells, and for T cells, it creates the CD4 and CD8 receptors on immature T cells. To start this process illustrated in Figure 1, Rag-1 and Rag-2 bind at the same time to the recombination signal sequence, or RSS site, of one of the coding regions V, D, or J (Godderz and Rodgers). The RSS site consists of a heptamer region, a spacer region of either 12 or 23 base pairs, and a nonomer region (Akamatsu and Oettinger). For the best results in V(D)J recombination, different size spacers must be joined together because this gives the most diversity. This constituent is called the 12/23 rule. Once both Rag proteins bind, they jointly cleave a single strand of the DNA at the junction of the RSS site and the coding region. The free single stranded 3’OH now attacks the opposite strand to form a DNA hairpin which will be important in the rejoining to another V, D, or J region. Since the 3’OH catalyzes the nick on the other strand, no ATP is necessary in this reaction (Melek et al.). Rag-2 continues to bind the complex to the DNA at this point even though the strands have been cleaved (Godderz and Rodgers). Once the DNA hairpin has been formed, rejoining occurs with the help of accessory proteins such as the Ku protein and DNA dependent protein kinase (Melek et al.).

There are three types of rejoining: signal joining, coding joining, and hybrid joining. In the targeted region of the DNA strand that Rag-1 and Rag-2 cleave, each coding region is flanked by two RSS sites (Godderz and Rodgers). In signal rejoining, the blunt cut ends of the RSS sites made from the Rag complex, are rejoined via non-homologous end joining. In coding rejoining, after the DNA hairpin forms, a DNA transferase can add extra nucleotides to the ends of the coding regions for added diversity, and the two separate coding joints are joined together (Melek et al.). Lastly, in hybrid joining, the Rag complex generates a coding end rejoined to either its own RSS site or another RSS site. Through each of these rejoining mechanisms, V(D)J recombination is able to bring forth many diverse types of receptors.

Figure 1.

The Process of V(D)J Recombination
a) The V, D, or J double-stranded DNA sequence is flanked by an RSS site. b) Rag-1 and Rag-2 bind simultaneously on the RSS joints. c) The Rag complex initiates a single stranded nick between the RSS and the coding region. d) Through an attack by the 3’ OH end, the DNA forms a hairpin leaving the RSS site with a double stranded break. e) With the help of accessory proteins, the newly cleaved coding region is joined to a separate coding region with added nucleotides in between, that were added by a DNA transferase. Note: Rag-1 and Rag-2 interact with both strands of DNA.
A scientist would want to knockout the Rag-1 gene for viewing the effects at the molecular level as well as using these type of organisms for research on human diseases. A main reason to knockout the Rag-1 gene is to see how it will affect the entire V(D)J recombination process. By knocking the Rag-1 gene out, one will be able to see where the process is interrupted, if it is interrupted. Researchers will also be able to see if other receptors on lymphocytes, such as IL2, are affected. In V(D)J recombination, Rag-1 interacts closely with Rag-2 to instigate recombination (Godderz and Rodgers). If one were to knock out this gene, the results would show if the expression of the Rag-2 gene would be affected. Researchers could also see if both Rag proteins are crucial for recombination, or if just one is needed. Rag-1 transcripts are also found in the brain in mice (Mombaerts

et al.). By knocking out Rag-1, scientists can evaluate if the Rag-1 transcripts affect the brain’s activity by observing any abnormalities from the wild type.

Also, by knocking out the Rag-1 gene, researchers will be able to use these organisms for future medical research. A few diseases such as Omenn syndrome and types of Severe Combined Immunodeficiency (SCID) involve mutations with Rag-1, but it is unethical and unreasonable to do research on patients affected by these diseases. If scientists were able to generate a Rag-1 knockout organism, they would be able to test the effects of specific diseases, and how an organism would react to those changes (Ménoret et al.). In terms of patients with SCID or Omenn syndrome, medical researchers could test the effects of implanting proper Rag-1 genes into knockout organisms and viewing the effects of doing so (Lagresle-Peyrou et al.). Lastly, knocking out Rag-1 can allow for scientists to attempt to make an organ similar to the organs in humans (Tsuchida et al.). Research has already been done in attempting to make human-like organs in other organisms, and immune-deficient organisms have been used in trying to do so. Generating human-like organs allows scientists to break through with new research and findings that directly relate to the human body.