Antibodies (Immunoglobulins)

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1 Antibodies (Immunoglobulins) The immune system plays a major role in the body s defense mechanisms against pathogens and other foreign bodies. It protects organisms from infection with a layered defense wall of increasing specificity as it is divided into 3 lines of defense which are classified into two types according to the Specificity of the mechanism through which they fight the pathogen : 1- Non-specific immunity: Also called INNATE immunity, which means that it is always present in our bodies, and it is found by nature (NOT as a response to exposure to any antigen!). As mentioned, the immune system is built up of 3 lines of defense, and this type of immunity represents the front lines (The first and second lines). Being non-specific, the 1 st and 2 nd lines of defense hinder the penetration by pathogens WITHOUT requiring specialized cells to fight them. Thus these cells exert their immunologic tasks in the same mechanisms in all organisms. These cellular biochemical defense mechanisms are considered Non-adaptive which means that these mechanisms are undergone in the same way every time the body gets exposed to the same pathogen. Being adaptive also means that this immunity type responses only for microbial pathogens (It only recognizes microbes), so a tissue damage cannot be detected by the innate immunity! If a pathogen breaches this outer layer of defense, the cellular warriors (fighters) come into play... In reality, you discover that you are sick only after a pathogen has managed to beat your front-lines defense..! BUT what are these lines composed of?? The 1 st line of defense: includes PHYSICA BARRIERS, such as: hair, skin and mucous layers which line body cavities. It also includes CHEMICAL BARRIERS, such as: tears, sweat and all other secretions of the body. The 2 nd line of defense: includes phagocytes and antimicrobial proteins. 2- Specific immunity: Also called ACQUIRED immunity, since our bodies develop it during life in response to exposures to a variety of antigens. It is formed of the 3 rd line of defense which is dependent on both types of lymphocytes (T cells & B cells). It is Adaptive which means that it adapts to certain pathogens, this adaptation includes the increase in its magnitude (By magnitude, I guess the doc meant the number of cells required for the immunologic response!), and increasing in its specificity in responding This increase is detectable every time the body exposes to the antigen, (the second exposure is higher in magnitude and specificity than the first exposure, and so on ). Unlike the innate immunity, the acquired immunity recognizes both microbial and antimicrobial antigens. The general scheme of the immune system :

2 Innate Humoral antibodies Cellular Acquired Humoral antibodies Cellular The innate immunity stimulates the acquired, since the 1 st & 2 nd lines are reached before the 3 rd. Humoral: related to body fluids, it can excrete substances through pores or react through cells (literally as the doctor said!). From Wikipedia: Humoral immune response (HIR) is the aspect of immunity that is mediated by secreted antibodies. Immune system cells : 1- Myeloid cells. 2- Lymphoid cells : B-lymphocytes : These cells are secreted & maturate in the Bone marrow. Once these cells maturate, they are called plasma cells which secret the Antibodies, (So B-lymphocytes are the stem cells of the plasma cells). Thus the way of action of these cells is the secreted antibodiesmediated action. B-lymphocytes can also maturate into memory cells. T-lymphocytes: These cells are synthesized in the bone marrow, and then translocated to the Thymus gland where maturation occurs. The way of action of T-lymphocytes is Cell-mediated immunologic process, so its mechanism of defense is through the action of the cell itself NOT its secreted products as in the action of B-lymphocytes. T-lymphocytes are responsible for every immunologic process that requires a cellular action, such as: Graft Rejection (Transplant Rejection) زراعة األنسجة المناعي,رفض Hypersensitivity reactions and defense against Malignant Cells. There are 4 different types of T-lymphocytes, one of these types is Memory cells type. Genetic Deficiencies in the genome of T-or B-lymphocytes: These genetic deficiencies are reported in both T & B cells, but mainly in T cells, cells of the cellular action of the specific immunity. In the specific (acquired) immunity, although the system is slow to respond the first time it encounters an antigen, it s much quicker in the next time..! This is because it is an adaptive type of immunity, as we have already mentioned. And since it is adaptive, the magnitude of immune cells should be increased every time the body exposes to the antigen, one of the main types of these immune cells is T memory cells type.

3 These cells represent the Archive of our immune system, so a T-Cell Memory is the cell responsible for saving the genetic material of an antigen it encounters in the first exposure in order to stop it directly in the next time, so the body won t be infected by that antigen again. Recall that in the 2 nd exposure, number of immune cells is increased, so number of T-cells (of the memory type) is much larger than was present in the initial encounter with the antigen. By the action of these cells, the infection is considered as an infrequent/ not recurrent infection, because the body is infected only after the first exposure. A Genetic Deficiency during the differentiation of these memory cells leads to a Recurrent infection, since the body will be infected by that antigen every time it encounters, because it lacks full differentiated T memory cells required for avoiding the frequency of that infection. And this is what happens with Cancer Patients! Cancer is a Recurrent infection because the weak immune system of the body lacks T memory cells. Related Definitions : Epitope: Is a binding site for the antibody on the antigen, so there is a different antibody for every epitope. These epitopes are Antigenic determinants, since they help the antibody to recognize and determine whether this foreign body is an antigen or not. Hapten: A small molecule that cannot elicit an immune reaction by itself, but only if it was attached to a large carrier, such as proteins. E.g. Dinitrophinol (C6H4N2O5) is a hapten since it is a small molecule that cannot elicit an immune response unless it is attached to The Bovine Serum Albumin (large protein). Binding with a large molecule makes haptens larger in size as an antigen, and thus, easier to be detected by the immune system. What is an antibody? An antibody, also known as an Immunoglobulin, is a glycoprotein secreted by Plasma cells (Mature B- Lymphocytes), has the ability to bind a foreign substance, even the ones it hasn t encountered before. Such foreign body is called an Antigen if it was an Immunogen!!!, which is any substance that can elicit an immune reaction. The Antigen-Antibody affinity is high, so this binding process is highly specific. This binding enhances the Effector Functions of antibodies. These functions include: the inactivation or degradation of the foreign material. They could also include binding of the antibody to the foreign cell and decomposing it. The antibody is Y-shaped, with binding of antigen occurring on both tips (prongs) of the Y. Each single antibody contains a minimum of 2 identical light (L) chains (each of about 25 kda), and 2 identical heavy (H) chains (each of about 50 kda), so we can conclude that the average weight of any antibody is about 150 kda. H & L chains are synthesized separately inside Plasma cells, and then, they are held together, forming a tetramer, by both: Inter- and Intra-chain disulfide bridges before being excreted as a single unit-immunoglobulin.

4 Each chain (whether H or L) is a polypeptide (with a C-terminal and an N-terminal), and is divided into specific multiple domains. Recall that a Domain is a supra secondary structure that has structural and functional significance. The Light chain is divided into two domains (two halves). The half toward the C-terminal is referred to as the Constant region of the L chain (C L), while the other half (toward the N-terminal) is the Variable region of the L chain (V L). Constant means that it is relatively variable with the antigen it encounters, whereas Variable means the place where variability occurs according to the antigen it encounters (As the doc said!!). It was noticed that in each Variable region, there are 3 stretches (3 lobes) of amino acids called The Hypervariable region. Every lobe is composed of 7-12 amino acids which may differ from one immunoglobulin to another. This difference contributes to the hyper variability of that region. On the other hand, the Heavy chain is divided into 4 domains (4 quarters), where the 3 quarters toward the C-terminal are referred to as the Constant regions of the heavy chain (CH1, CH2, CH3), and the 4 th quarter toward the N-terminal is referred to as the Variable region of the heavy chain (V H). Notice that the portion of the antibody molecule that binds the specific antigen is formed by the N-terminals (Variable regions) of both H & L chains. The immunoglobulin could be digested into multiple fragments by the effect of two enzymes (Proteases): 1- Papain, which cleaves the immunoglobulin molecule into 3 fragments as it cleaves it in the site between the CH1 domain and the inter-chain disulfide bonds that link the 2 heavy chains

5 together. Two fragments are identical and called Antigen binding fragments (Fab), each one of them is composed of (CH1,VH,CL and VL), and the 3 rd fragment is called the crystallizable fragment (Fc) which is composed of 2 (CH2) and 2 (CH3) domains. 2- Pepsin, which cleaves the immunoglobulin molecule into only 2 fragments as it cleaves it in the site between the CH2 domain and the inter-chain disulfide bonds that link the 2 heavy chains together. The two resulted fragments are not identical. One fragment is called the Fab which consists of the 2 antigen binding sites held together as a single subunit, and the other fragment is called the Fc, which is composed of 2 (CH2) and (CH3) domains. BUT,, what are the functions of these fragments? Each Fab is responsible for detecting and binding the epitope of the antigen, and for blocking the antigen binding site against toxic foreign bodies. The Fc, on the other hand, is responsible for functions of the Immunoglobulins other than direct binding of antigens, such as guiding the immunoglobulin toward other certain cells, proteins or any other molecules involved in the immunogenic response mechanisms, other than the antigen. It is also responsible for delivering signaling messages to the immune system once the Fab recognizes and binds an antigen. In other words, the Fc mediates the response reactions by the previously mentioned functions as the Fab cannot mediate it by itself. Here you have to remember that every Fc is composed of CH2 & CH3 domains (CH4 domain is also found in the Fc of certain classes of Immunoglobulins), so it is acceptable to say that CH2 & CH3 (And CH4) mediate the immunogenic response. The area in which Papain and Pepsin cleave the immunoglobulin (The whole area between CH1 & CH2 domains) is referred to as The Hinge Region. The hinge region confers flexibility and allows both Fab arms (Y prongs) to move freely, thus helping them to bind to antigenic sites (epitopes) that may be variable distances apart. The Fab is the area where the Antigen-antibody reactions occur in order to elicit the immune response, since it is the fragment that contains the antigen binding site, which contains both: an H chain and an L one as well. In a 3D graph of an immunoglobulin, you can find some linked carbohydrates, which were added to the structure of the immunoglobulin during posttranslational processes, and that s why an immunoglobulin is considered as a Glycoprotein. The domains are composed of number of anti-parallel β pleated sheets that wrap around each other forming a barrel-like structure. A domain in the constant region is composed of 7 β- sheets, whereas a domain in the variable region is composed of 8 β-sheets. What contributes to the high diversity of Immunoglobulins? Two scientist (Sry I couldn t get their names! :P) have discovered that there are 10^8 Immunoglobulins in humans with only types of genes! And according to the one gene- one

6 protein principle, each protein is a product of one gene!, so how did we get that large number of immunoglobulins from this limited number of genes?! Later, they found that: There are two general types of light chains, kappa (κ) & lambda (λ), which can be distinguished on the basis of structural differences in their Constant Light regions. And a given immunoglobulin molecule always contains 2 kappa or 2 lambda light chains, NEVER a mixture of both. There are 5 types of heavy chains have been found in humans, distinguished by differences in their Constant Heavy regions. They are designated μ,α,ɤ,ɛ,ϭ. Each immunoglobulin light chain is the product of at least 3 different structural genes: a Variable region (V L) gene, a Joining region (J L) gene and a constant region (C L) gene. On the other hand, each heavy chain is the product of at least 4 different genes: a Variable region (V H), a Joining region (J H) gene, a Diversity region (D H) and a Constant region (C H) gene. So, a single polypeptide chain of an immunoglobulin is a product of a mixture of genes, not a single gene, and thus the one-gene one-protein principle is NOT valid with immunoglobulins. Antibody Diversity depends on genes rearrangement. These genes were rearranged during the maturation of B-lymphocytes into Plasma cells. They also hypothesized that each genetic material has variable & constant segments. The constant segment gets separated by itself, then the variable segments start rearranging themselves so as to produce a diverse mixture of genes. One more way to produce different polypeptide chains is to splice different genes. Getting a large number of immunoglobulins depends upon a number of factors including the existence of multiple gene segments (V,C,D &J segments). Thus, a large number of immunoglobulins can be synthesized from several hundred gene segments,, Where: - In the L chain, there are 40 V genes, 5 J genes & C genes are constant with both types kappa & lambda! So we can get 200 (40*5) combination of the kappa type. And 120 (40*3) combinations of the lambda type. As a final result we have 320 ( ) combinations from the L chain. - In the H chain, there are 51 V genes, 27 D genes, 6 J genes and C genes are also constant with kappa & lambda types. So we have 8262 (27*6*51) combinations, resulted from the combination of D genes with J genes first, then they combine with V genes and finally with the C genes to get that huge number of H chain combinations. - The total number of combinations of immunoglobulins is the final result we can get from both chains which equals 2.6*10^6 (8262*320). By Lna Malkawi