In this essay we will discuss about the molecular structure of Immunoglobulin in human body.
The human body is capable of synthesising more than a million different kinds of immunoglobulin molecules, each capable of reacting with a different antigen, but all of them appear to share the same fundamental quaternary (globular) structure.
Typically, an immunoglobulin molecule is a Y-shaped heteromer and composed of two identical heavy (H) polypeptide chains and two smaller identical light (L) chains. Each arm of the Y contains a complete L chain and a part of an H chain and the leg of the Y contains the remaining parts of the H chains.
Near its C-terminus, each L chain is linked to an H chain by Disulphide Bridge, and two additional disulphide bridges link the H-chains together. The H chains possess antigenic determinants in the “tail” segments by which they can be classified as Ig G, Ig M, Ig A, Ig D or Ig E, each with its own class of H chain, such as, γ (gamma), µ (mu), α (alpha), δ (delta) and ɛ (epsilon) respectively.
Light chains can likewise be typed as kappa (k) or lambda (λ). Within a H chain or L chain, C- termini segments exhibit very little variation in primary structure from one individual to another and are called constant regions (C).
The amino ends or N-termini of both heavy and light chains, however, are extremely diverse in primary structure, even within a class and are called variable (V) regions. The VH and VL regions together form antibody-combining site for specific interaction with a homologous antigen molecule. Thus, each Y- shaped antibody has two identical antigen-binding sites, one at the tip of each arm of the Y.
Because of their two antigen-binding sites, antibodies are said to be bivalent. The efficiency of antigen binding and cross-linking of antibodies is greatly increased by the flexible hinge regions in antibody molecules, which allow the distance between the two antigen- binding sites to vary.
Further, the proteolytic enzyme papain splits antibody molecule into different characteristic fragments: two separate and identical Fab (= fragment antigen-binding) fragments, each with antigen-binding site and one Fc fragment (so called because it is readily crystallised).
Each of the four polypeptide chains of an immunoglobulin is also divided into repeating segments, called domains, each of which folds independently to form a compact functional unit. Thus, there are two domains in the L chains (i.e., VL and CL) and four in the H chains (i.e. VH, CH, CH1, CH2 and CH3).
Recently, it has been found that the antigen-binding site of the antibodies is formed by only about 20 to 30 of the amino acid residues in the variable regions of both L and H chains. In fact, the variability in the variable regions of both L and H chains is for the most part restricted to three small hyper-variable regions in each chain.
The remaining parts of the variable regions, know as, framework regions, are relatively constant. Those parts of an antigen that combine with the antigen-binding site on an antibody molecule or on a lymphocyte receptor, are called antigenic determinants or epitopes. Molecules that bind specifically to such an antigen-binding site but cannot induce immune responses, are called haptens.
Haptens are usually small organic molecules, they become antigenic if they are coupled to a suitable macromolecule, called carrier. Haptens such as dinitrophenyl (DNP) group have been important tools in experimental immunology.
During the early stages of an infection, the response to the antigen involves the production of a specific class of immunoglobulins, called IgM. IgM is the first class of antibody which appears on the surface of a developing B lymphocyte and is, secreted into the blood during the primary antibody response.
In its secreted form, IgM is composed of five four-chain units and has a total of 10 antigen binding sites. It also contains one copy of another polypeptide chain, called J chain (J=joining) which is produced by secretory B lymphocyte.
Later on, the amount of IgM gradually declines as another class or isotype of immunoglobulin, called IgG, appears. IgG represents the most abundant form of body’s antibodies (75 per cent) and is one of the most extensively studied immunoglobulins.
IgG has a molecular weight of 1,50,000 and is composed of four polypeptide chains in its Y- shaped quaternary molecule-two identical H chains and two identical L chains. The L chains have the molecular weight of 20,000 to 25,000 and consist of about 450 amino acids.
Each L chain is covalently linked to an H chain by a disulphide bridge, and two light chain-heavy chain pairs are covalently linked by two disulphide bridges (near the hinge). There are also 12 intra-chain disulphide bridges, four in each H chain and two in each L chain.
Lastly, an asparagine residue in each H chain is bonded to carbohydrate, since an immunoglobulin is also a glycoprotein.
IgG is produced in large quantities during secondary responses and is involved in antibody dependent cell-mediated killing. IgG molecules are the only antibodies that can pass from mother to foetus via the placenta.
The cells of the placenta that are in contact with FC receptors beating IgG molecules mediate their passage to the foetus. The antibodies are first ingested by receptor- mediated endocytosis and then transported across the cell in vesicles and released by exocytosis into the foetal blood. This process is called transcytosis.
Antibody Diversity (Genetic Basis of Antibody Diversity):
Past attempts to explain the genetic basis of antibody diversity can be classified into the following three hypotheses:
1. The “germ line” hypothesis states that there is a separate germ line gene for each antibody.
2. The “somatic mutation” hypothesis states that there is only one or a few germ line genes specifying each major class of antibodies and that the diversity is generated by a high frequency of somatic mutation (i.e., mutations occurring in the antibody-producing somatic cells or in cell lineages leading to antibody-producing cells).
3. The “minigene” hypothesis states that the diversity is generated by the shuffling of many small segments of a few genes into a multitude of possible combinations. This shuffling would occur by recombination processes in somatic cells.
All three hypotheses have been found connect in certain respects. Thus, it is now known that the minigene hypothesis explains a great deal of the observed diversity. It is also true that somatic mutation contributes additional diversity. Finally, it is also well known that one segment (the “constant” region) of immunoglobulin or antibody chain is specified by a “gene” or “gene segment” that is present in the genome in only a few copies.
During differentiation of B lymphocytes, antibody diversity can be originated by the following three ways:
1. by genome rearrangement (or DNA splicing);
2. By alternate pathways of transcript or RNA splicing; and
3. By variable joining sites and somatic mutation.
The following brief discussion regarding mode of origin of antibody diversity would illuminate our readers:
Here we will consider how the enormous number of amino acid sequences of the V regions (variable chains) are formed from a comparatively small numbers of genes. To be clear, we will only describe how the kappa type light (L) chain is produced: Many genes can be used to form a kappa-type L chain; they are of three types-V, J and C.
There are roughly 300 different V genes (which are responsible for the synthesis of the first 95 amino acids of the variable region), 4 different J genes (which encode the final 12 amino acids of the variable region and join the V and C regions; J = junction of joining sequences) and 1 copy of the C gene (which encodes the constant region).
In an embryonic cell the V genes form a tight cluster, the J genes form a second tight cluster quite far from the V gene cluster, and the C gene follows not far after the J gene cluster. Note that each V gene is preceded by leader regions where transcription can be initiated; the 7 and C genes are not preceded by leaders.
Regions encoding particular IgG molecules have been cloned (by recombinant DNA techniques) from various mouse cell lines, each producing a particular IgG molecule. The V-J-C region has also been cloned from mouse embryo cells, which have not yet been committed to antibody synthesis and thus, presumably contain an unaltered master set of genes for antibody synthesis.
For each clone obtained from an antibody-producing cell line it has been found that a large segment of the embryonic DNA sequence is not present and that the missing segment is always a sequence between the particular V gene that encodes the first 95 ammo acids of the V region and the J gene that encodes the last 12 amino acids of the V region.
This is explained by a gene rearrangement in which DNA between the particular V and J genes is deleted (i.e., looping out of the intervening segment as in interstitial deletion). Many different gene sequences encoding particular IgG proteins have been cloned and it has been found that in each clone a different segment of DNA is not present.
For example, in the figure the DNA between V16 and J3 is absent; in another clone there might be a V210 – J1 junction instead. In both cases, a V gene and a J gene have become spliced together to form a complete gene for the variable region.
Studies of the base sequence of cloned IgG DNA sequences show that the junction between a particular V gene and a particular J gene is not always the same. That is, the two terminal triplets of juxtaposing V and J genes can exchange at any one of four sites that yields a triplet. In the example shown, there are three possible amino acids at the joint, which add diversity to the number of possible regions.
Since there are 300 different V genes, 4 different J genes, and (on the average) 2.5 different amino acids at the junction, there are, then, 300 x 4 x 2.5 = 3000 different variable regions. The H chain genes are organised in a similar but not identical way (therefore four different types of genes) and there are 5000 variable regions in the H chains.
Thus, since each IgG molecule contains two identical H chains and two identical L chains, there are about 3000 x 5000 = 1.5 x 107 different IgG molecules can be formed from the VL JL VH and JH genes. This is sufficient reason to explain the diversity of antibody molecules.
DNA splicing does not fully generate a L chain sequence, since;
(1) The spacer between the J and C genes remains and
(2) The actual L chain has amino acids derived from only one V gene and one J gene and the spliced DNA usually contains many V and J genes. The correct amino acid sequence is obtained by a final RNA-splicing event.
Note how the particular V-J joint determines the RNA splicing pattern. For example, the RNA removed is always a segment between the leader and the V gene and between C gene and the right end of the J gene in the V-J joint.