Introduction to Antibodies and their basic structure (Part 1- Antibody Basics)
Welcome to the 1st part of the 13-part series on Antibody basics.
Antibodies are one of the crucial warriors produced by our immune system in response to the presence of foreign agents that enter our bodies. These foreign agents are called antigens. A wide range of substances are recognized as antigens by our bodies, such as viruses, bacteria, fungi, and some non-living substances such as toxins, chemicals, allergens, etc. As a result, our immune system generates billions of different antibodies, each capable of recognizing a specific antigen. When an antibody binds to a particular antigen, it sends a signal to the immune system indicating that this antigen is marked for destruction.
Antibodies are also called Immunoglobulins
In 1890 two immunologists: Emil von Behring and Shibasabura Kitasato, showed that the serum of the animals immunized against diphtheria contains a component that can neutralize diphtheria toxin. Thus, the transfer of serum from immunized animals to the animals infected with diphtheria could cure the infected animals. The serum is the liquid part of the blood that remains after blood cells and clotting factors have been removed. For this discovery, Behring was later awarded the Nobel Prize in 1901. This serum component was later named “antibody” by Paul Ehrlich in 1901.
The first evidence that antibodies are present in the gamma (𝛾) globulin fraction of serum proteins came from the experiment performed by two scientists: Tiselius and Kabat, in 1939. The scientists immunized rabbits with the antigen protein ovalbumin, i.e., the albumin of egg whites. After immunization, the serum of rabbits was harvested and was divided into two aliquots. Out of two aliquots, when electrophoresis of one aliquot of serum proteins was done, it showed four peaks (represented as solid peaks) corresponding to albumin and alpha, beta, and gamma globulins.
On the other hand, the second aliquot of serum was reacted with ovalbumin before electrophoresis. The precipitate formed was discarded, and the remaining serum proteins were electrophoresed. When the electrophoresis profiles of the two serum aliquots were compared, the scientists observed that there was a significant drop in the 𝛾 globulin peak (represented as dotted peaks) in the aliquot that had reacted with ovalbumin. It suggested that the antibodies are contained in the 𝛾 globulin fraction of the serum. Proteins unrelated to antibodies may also migrate with the electrophoretic mobility of 𝛾-globulins. Therefore, antibodies were named “immunoglobulins” (symbol Ig) to distinguish them from any other proteins that might be contained in the 𝛾 globulin fraction.
How are antibodies produced?
Antibodies are produced by specialized white blood cells called B cells, specifically plasma cells. When a B cell encounters an antigen, it gets activated and proliferates into a group of identical cells called a clone. Some of the cells differentiate into antibody-producing plasma cells, and others become long-lived memory B cells. Both the plasma cells and memory B cells are specific to the antigen, which the mature B cell initially encountered. Plasma cells secrete millions of antibodies into the bloodstream and lymphatic system, where they attack and neutralize antigens that are identical to the ones that triggered the immune response.
B cells distinguish antigens through the B-cell receptors found on their surfaces. A B-cell receptor is basically an antibody protein that is not secreted but is anchored to the B-cell membrane. All B-cell receptors located on a particular B cell are identical, but receptors present on other B cells differ.
Basic structure of immunoglobulins or antibodies
All antibodies have the same core structure, consisting of 4 polypeptide chains: two identical light (L) chain polypeptides and two identical heavy (H) chain polypeptides, which assemble to form a Y-shaped molecule. Heavy chains are longer ones, and light chains are shorter ones. The term heavy and light chains refer to their molecular weight. Heavy chains have a molecular weight of 50,000 Da or more, and on the other hand, the light chains have a low molecular weight of about 25000 Da. Since these are polypeptide chains, the N terminal of the polypeptide chain is present at the tip end, and the C terminal at the base of each polypeptide chain.
Each light chain is bound to a heavy chain by a disulfide bond and several non-covalent interactions such as salt bridge, hydrogen bonds, and hydrophobic bonds. Similar non-covalent interactions and disulfide bonds also connect the heavy chains in the mid-region to form the basic four polypeptide chain antibody structure.
Each light chain and heavy chain contain two distinct regions:
· Variable regions (V) and
constant regions (C).
Variable regions refer to the first 110 amino acids of the amino-terminal region in each heavy and light chain. The variable regions are named because the amino acid sequences in these regions vary significantly among antibodies of different specificities. V regions are called VL in light chains and VH in heavy chains. It is the variable region in the light and heavy chain which together forms the antigen-binding site, also called a paratope.
In an antibody molecule, there are two antigen-binding sites. The variable region is responsible for giving the antibody its specificity to bind to a particular antigen. Or in other words, all the differences in specificity displayed by different antibodies to bind to different antigens can be traced to differences in the V regions’ amino acid sequences.
Constant regions: By contrast, within the same antibody molecule, the regions beyond the variable regions of both heavy and light chains are called constant regions. The amino acid sequence in these regions shows less variation among the different antibodies. There is a single constant region present in each light chain, which is designated as CL. On the other hand, multiple constant regions are present in each heavy chain and are represented as CH1, CH2, CH3, etc.
The constant region of the heavy chain of the antibodies forms the basis of the classification of antibodies. In a particular class of antibodies, all antibody molecules have almost the same constant region. But the constant region of the antibodies of one class is different from the constant region of the antibodies of another class. The classification of antibodies will be discussed in Part 3.
Another essential thing to know about antibodies is that they are glycoproteins. Glycoproteins are proteins that have carbohydrates attached to the polypeptide chain. To each heavy chain, short carbohydrate molecules are attached, which serve various functions such as increasing antibody molecules’ solubility and facilitating removal of the antigen and death of the pathogen.
Functional components of Antibodies
The intact Y-shaped antibody molecule has three components: two fragment antigen-binding domains abbreviated as Fab and the fragment crystallizable written as Fc. The knowledge of functional components of an antibody molecule was derived from various enzymatic and chemical methods conducted by Gerald M. Edelman and Rodney R. Porter, for which they were awarded the Nobel Prize in 1972. In the enzymatic method, the antibody molecule is digested with enzymes papain and pepsin. And in the chemical method, the antibody molecule is treated with mercaptoethanol.
- When the antibody molecule is digested with the enzyme papain, it cleaves the antibody molecule just above the interchain disulfide bonds linking heavy chains. As a result, three fragments are produced, two of which have identical structures consisting of variable and constant regions of light chains (VL and CL) and heavy chains (VH and CH1) linked by disulfide bonds.
Since these fragments contain V regions and have antigen-binding sites, they are called fragments of antigen binding, abbreviated as Fab. There are 2 Fab fragments in an antibody molecule, and each of the Fab fragments can bind only one antigen. The third fragment has no antigen-binding activity. It consists of constant regions of heavy chains held together by disulfide bonds.
It was found that this fragment crystallized during cold storage, so it was called fragment crystallizable abbreviated as Fc fragment. The Fc fragment plays a role in opsonization and complement activation. The functions of antibodies are discussed in detail in parts 4 and 5.
2. When the enzyme pepsin digests the antibody molecule, it cleaves the antibody just below the disulfide bonds linking the heavy chains. The resulting fragment consists of two antigen-binding arms (Fabs) of the antibody linked together by disulfide bonds. This fragment is represented as F(ab’)2.
Here F stands for the fragment, and ab stands for antigen binding. The prime symbol represents variation in this fragment’s structure as it contains few more amino acids than the Fab fragment. The number 2 represents the two Fabs that are linked together. The Fc fragment was not recovered because it gets digested into multiple peptide fragments by the pepsin enzyme.
3. When the antibody molecule is subjected to mercaptoethanol reduction and alkylation, it cleaves disulfide bonds. The cleavage of all disulfide bonds leads to splitting the antibody molecule into two identical heavy chains with a molecular weight of 50,000 Daltons each and two other identical light chains with 25,000 Daltons MW each.
Thus the enzymatic and chemical methods can determine that each antibody molecule comprises two identical heavy chains of 50,000 MW and two identical light chains of 25,000 MW linked by disulfide bonds. Also, there are two antigen-binding fragments called Fabs in an antibody molecule consisting of variable and constant regions of light (VL and CL) and heavy chains (VH and CH1) linked by disulfide bonds.
Antibody sequencing
It was essential for researchers to know the amino acid sequence of heavy and light chain polypeptides to understand their different types. Unfortunately, the unavailability of sufficient numbers of homogeneous antibodies hindered the researchers’ initial attempts to determine their amino acid sequences. Although the basic structure and chemical properties of different antibodies are similar, the amino acid sequences of their heavy and light chain polypeptides are quite different.
For amino acid sequencing of heavy and light chain polypeptides, researchers wanted enough homogeneous antibodies with similar antigen specificity because of the similar amino acid sequences of light and heavy chains, making it easier to determine their amino acid sequence. The population of antibodies in serum gamma globulin fraction does not serve the purpose of amino acid sequencing because of the heterogeneous spectrum of antibodies with different antigen-binding specificities. Even if immunization is done with one antigen, the antibodies formed just to one antigen are heterogeneous because they recognize different epitopes of the antigen. An epitope is the specific part of an antigen to which the antibody binds. This heterogneity of antibodies made them unsuitable for amino acid sequencing studies.
Antibody sequencing eventually became feasible with the discovery of multiple myeloma, a cancer of antibody-producing plasma cells. In a normal individual, plasma cells, in response to an antigen, secrete antibodies for a limited period and then die. But in multiple myeloma, plasma cells escape normal controls of cell proliferation and even do not require any activation by antigen to induce proliferation. Thus, the cancerous plasma cell continues to secrete a large number of homogeneous antibodies.
Most patients afflicted with multiple myeloma excrete excessive amounts of light chains in their urine, named Bence-Jones proteins on their discoverer’s name.
Types of light chains
To know the different types of light chains, Bence-Jones proteins, or light chains, were isolated from the patients’ urine, and then their amino acid sequences were determined. When the amino acid sequences of Bence-Jones proteins from different individuals were compared, it was observed that the amino-terminal half of the chain consisting of 110 amino acids was found to vary among different Bence-Jones proteins. This region was called the variable region designated as V.
On the other hand, the other 110 amino acids in the carboxy-terminal half of the molecule comprise the light chain’s constant region.
The constant region’s amino acid sequencing revealed two sequence patterns based on the subtle differences in the amino acid sequences. And based on these differences in amino acid sequences of constant regions, the light chain can be divided into two types: kappa (κ) chain and lambda (ƛ) chain. These are similar in structure and function but are coded by different genes. The genes coding for Kappa chains are present on chromosome 2, and the genes coding for lambda chains are present on chromosome 22.
It is important to note that each antibody molecule produced by a plasma cell will either have a kappa or lambda light chain but can never have both. 60% of the light chains are kappa in humans, and 40% are lambda. Further, the amino acid sequences of the constant region of lambda light chains also show minor differences. Thus, lambda light chain can be classified into subtypes; in humans, there are four subtypes of lambda light chain ƛ1, ƛ2, ƛ3, and ƛ4.
Types of heavy chains
To know the different types of heavy chains of antibody molecules, antibodies produced by cancerous plasma cells were reduced with mercaptoethanol. The resulting heavy chains were separated by gel filtration. When the amino acid sequences of several heavy chains were compared, a pattern similar to that of the light chains emerged. The amino-terminal part, or variable region consisting of 110 amino acids, showed significant sequence variation among the heavy chains.
By contrast, the amino acid sequencing of the regions beyond the variable region called the constant region of heavy chains revealed five basic sequence patterns based on the differences in the amino acid sequences. Based on the amino acid differences in the constant regions, the heavy chains can be divided into five types, named as δ, 𝛾, α, µ, and ε heavy chains. Each of the five different heavy chains is called an isotype. The length of the constant regions in δ, 𝛾, and α heavy chains is approximately 330 amino acids, and the length of the constant regions is 440 amino acids for µ and ε heavy chains.
The presence of the specific type of heavy chain determines the class of that antibody: IgD (δ), IgG (𝛾), IgA (α), IgM (µ), or IgE (ε). Further, each class of antibody can have either kappa or lambda light chains.
Minor differences in the amino acid sequences of the α and 𝛾 heavy chains lead to the further classification of heavy chains into subisotypes that determine the subclass of antibody molecules they constitute. In humans, there are two sub isotypes of α heavy chain α1 and α2 and thus two subclasses of IgA- IgA1 and IgA2 whereas there are four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4 based on four subisotypes of heavy chain: 𝛾1, 𝛾2, 𝛾3, and 𝛾4 .
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