Topic (7): Antibodies and Antigens

Size: px
Start display at page:

Download "Topic (7): Antibodies and Antigens"

Transcription

1 Topic (7): Antibodies and Antigens INTRODUCTION Antibodies (Abs) are one of the three classes of molecules able to differentiate between antigens [Ags] (the other two are T-cell receptor [TCR] and major histocompatibility complex [MHC]) {Figure 1}. Antibodies have a very special reputation among these antigenrecognition molecules because they have the broadest range of antigen specificities and they possess a bigger affinity (strength of binding) to antigens compared to the two others. An antigen (Ag) is any substance that is specifically recognized and bound by the antigen receptors of T or B lymphocytes. The antigen that is used to immunize the host and induce antibody production is called the cognate (i.e. related) antigen. An epitope (antigenic determinant) is a small region of an immunogenic molecule that binds to an antigen receptor. While B and T cells carry receptors that are similar from the functional point of view, in that they both bind antigen, these receptors recognize epitopes in fundamentally different ways. The TCR recognizes an epitope composed of an antigenic peptide presented in association with an MHC molecule, while B cell receptors recognize epitopes on macromolecules in solution, or on macromolecules bound to cell surfaces. Almost every kind of organic molecule (including proteins, carbohydrates, lipids, and nucleic acids) can be an antigen. Proteins and polysaccharides have the size and properties necessary to be the most prevalent antigens to induce an immune response. 1

2 IMMUNOGLOBULIN (ANTIBODY STRUCTURE) Electrophoresis is a technique used for characterizing proteins. Antibodies were identified in the third peak (the γ-peak) of electrophoretically fractionated serum globulin proteins, thus they are also termed gamma globulins. Basic structure of an Ab responsible for certain functional features was inferred using digestion by two different enzymes. If papain was used as the proteolytic enzyme, the antibody molecules were reproducibly cleaved into three parts. Of these, two were identical to each other and retained the ability to bind antigen. The third fragment did not bind antigen and tended to crystallize. The first two fragments Fab (fragment, antigen-binding), and the third fragment Fc (fragment, crystalline). If another enzyme, pepsin, was used in for proteolytic cleavage, the results were different: the Fc fragment was unstable and degraded quickly, while the two Fab fragments were bonded to each other. These doublets of Fab fragments F(ab )2 (Figure 2). Each Ab molecule consists of four polypeptide chains: Two heavy chains that are identical to each other and have a molecular weight of approximately 55 to 70 kda. The other two chains are also identical to each other and have a molecular weight of approximately 24 kda and they are called antibody light chains. Each light chain is attached to a heavy chain, and the two heavy chains are attached to each other through covalent disulfide-bonds. Each heavy and each light chain has its own variable (V) and its own constant region (C). This macromolecule consisting of four polypeptide chains is called a monomeric antibody molecule because, antibodies of some isotypes form multimeric aggregates, in which several monomeric molecules are joined together. 2

3 The entity that can physically bind an epitope (the antibody variable region), is the heavy chain variable region and the light chain variable region that are juxtaposed in space (Figure 3). Now, we will discuss the concept of antibody domain (Figure 3). A domain is a portion of an Ab chain that has the length of about 110 amino acids and a sphere-like shape of the fold. The Ab domains follow each other along the length of individual heavy and light chains. The light chains always have two Ig domains, of which the N-terminal domain is the variable region of the light chain (or the VL region), and the C-terminal domain is its C-region (or the CL region). The heavy chains consist of four or five domains, of which the N-terminal domain is the variable region of the heavy chain (or the VH region), and the remaining three or four domains, comprise the heavy chain constant region (or the CH region) and are called the CH1, CH2, CH3 and CH4 (in IgM and IgE) domains. The portion of Ab between the CH1 and CH2 domains is very flexible and forms the so-called hinge, the region where the VH and the CH1 domains together with the VL and CL domains that are attached to them form an angle, allowing the variable regions to deviate in space from the longitudinal axis. The Ab interaction with epitopes is always mediated through portions of the VH and VL domains. The interaction with complement and Fc receptors (to confer the Ab effector functions), is mediated through the CH2 domains. At this point we need to understand What gives each Ab molecule a particular specificity to interact with a sole specific epitope? 3

4 Within the V domains of both the L and H chains are three short hypervariable regions that exhibit extreme amino acid variability. These regions of five to seven amino acids are responsible for the diversity that allows the total repertoire of Abs to recognize almost any molecule in the universe of antigens. The antigen-binding sites are formed by 3D juxtaposition of the three hypervariable regions in the VL domain with those in the VH domain. Because these sequences result in a structure complementary to an antigenic epitope, the hypervariable regions are also called complementarity-determining regions (CDRs). [Figure 4]. The CDR loops contribute to unique regions within the antigen-binding site that form the paratope of the Ab which binds specifically to the corresponding epitope of the antigen. In contrast to the variability and hypervariability of the V domains, the C domains exhibit very little variation among antibodies, which is expected for a structure that carries out the same effector action in response to a wide variety of antigens. So, what are these effector functions of Abs? 1. Neutralization of pathogens and interference with pathogen attachment. 2. Activation of the complement system with subsequent lysis of the microbe. 3. Opsonization and aiding in phagocytosis. 4. Aiding NK cell killing through ADCC (do you still remember that?). 4

5 So, these functions are carried out by the CH domains and the structure of these CH domains will determine the particular function of each Ab group that can be classified as separate classes or isotypes. There are five classes of human Ab heavy chains, called: α, γ, δ, ε, and µ corresponding to the five isotypes of Abs, which are called IgA, IgG, IgD, IgE, and IgM,. The amino acid differences in these different chains can affect the size, charge, solubility, and structural features of a particular Ab, which in turn influence where an Ig goes in the body and how it interacts with surface receptors and other molecules. A mechanism called isotype switching occurs later in the life span of a B cell clone that allows its individual members to produce Ab of different constant region sequences. All of these differences can influence how a given antibody will clear its Ag. For the light chain constant region, two classes are present that are referred to as κ (kappa) or λ (lambda) chains. In an individual human s repertoire of Ab-producing B cells, 60% produce Abs with κ light chains, and 40% produce Abs with λ chains. IMMUNOGLOBULIN ISOTYPES: STRUCTURE AND FUNCTION I. IgM: Monomeric IgM is always the first form and isotype of Ig generated by naïve B cells. Following its initial activation by Ag, the naïve B cell proliferates and differentiates, and its progeny produce the pentameric secreted form of IgM. All other Ab isotypes (except IgD) are generated by isotype switching, a process that occurs only late in a primary response. Thus, it is IgM that is expressed first in any primary immune response, and those that are synthesized first in the newborn (which has just begun to encounter antigens on its own). The detection of increased IgM levels in an adult indicates a recent exposure to a novel antigen. It is also the Ab produced in response to Ti Ags that can activate a B cell in the absence of T cell help. IgM comprise only about 5 10% of normal serum Ig. However, the low absolute numbers of IgM are balanced by the number their binding sites. Because of its pentameric nature, the IgM displays 10 Fab sites that can theoretically bind to a pathogen. IgM are large pentamers of total molecular mass 970 kda with J-chain (joining chain, a small polypeptide that regulates the multimerization of IgM and IgA) and they are generally concentrated in the blood. IgM is the most efficient isotype in activating complement. IgM is not an isotype prominent in either opsonization or ADCC. II. IgD (the enigma): IgD is the second Ig isotype to be synthesized by a B cell, and first appears on its surface early in B cell development. Recent studies have shown that IgD plays an important role in the regulation of tolerogenic and protective B cell responses at mucosal sites of antigen entry, including the respiratory route. This mucosal homeostasis may augment immune adaptations following post natal exposure of the mucosa to airborne and food antigens, 5

6 including allergens. An early dysregulation of these adaptation processes could contribute to the pathogenesis of common disorders such as allergies by disrupting mucosal homeostasis. III. IgG: This Monomeric isotype is the most common in the circulation and tissues. In the blood of normal adult, 70 75% of serum Ig is IgG. It can be classified into four subclasses with the following proportions of total IgG in serum: IgG1 (67%), IgG2 (22%), IgG3 (7%), IgG4 (4%). These subclasses have minor amino acid differences in the CH domains, however, these minor differences confer different half-lives and effector functions for the different subclasses. The diffusibility and high serum concentration make IgG the most prevalent Ab in the extracellular fluid. IgG is a key opsonin and is important in ADCC. The IgG1 and IgG3 subclasses are particularly good opsonins and mediators of ADCC because FcγRs bind IgG1 and IgG3 antibodies with high affinity. This is related to the length of the hinge region in the CH2 domain of the Ig structure, since IgG3 has an extended hinge, and IgG4 has a very short hinge. IgG is an important activator of complement (though not as efficient as IgM). IgG3 is the most efficient complement-activating IgG followed by IgG1 and IgG2. IgG4 cannot fix the complement. In addition, the only Abs to cross the placenta is of IgG class. Maternal IgG1, IgG3, and IgG4 molecules have been found to readily cross the placenta via binding to neonatal Fc receptor (FcRn), whereas IgG2 crosses the placenta with much lower efficiency. IV. IgA: 85 90% of IgA antibodies are found in the secretory form in the external secretions of the body, which include tears, saliva, mucous secretions of the gastrointestinal, urogenital, and respiratory tracts and breast milk. Secretory IgA antibodies are of enormous importance because they facilitate antigen removal right at the mucosal surface, the most common site of initial pathogen attack. The remaining 10 15% of IgA antibodies occurs in blood. There are two subclasses, designated IgA1 and IgA2 that differ in content by 22 amino acids, 13 of which are located in the hinge region and are deleted in IgA2. The lack of this region appears to make IgA2 more resistant to some bacterial proteases that are able to cleave IgA1. Hence IgA2 is the predominant form in secretions at mucosal surfaces, while IgA1 is mainly found in serum. IgA2 is present as a dimer with the two monomers held together by a J chain. A secretory component (SC), is later attached to the FC region around the hinge portion of α chains of the dimeric IgA. This protein, is derived from epithelial cells found in close proximity to the plasma cells. Secretory IgA has antiviral activity through neutralization. IgA also plays a role in the elimination of helminth worms, in that IgA-coated parasites can be dispatched by ADCC carried out by eosinophils bearing FcαR. IgA does not have the ability to fix the complement. V. IgE: IgE is present in the serum at the lowest concentration of all Ab isotypes. IgE antibodies do not cross the placenta, cannot fix complement, and do not function as opsonins. The serum concentration of IgE rises dramatically in response to worm infections. IgE 6

7 antibodies are also responsible for the symptoms experienced in allergic reactions such as hay fever, and more severe conditions such as asthma and anaphylactic shock. Shortly after synthesis, IgE attaches to basophils and tissue mast cells by means of specific surface proteins, termed high-affinity FCε receptors. When two adjacent IgE molecules on a mast cell bind specific antigen, a cascade of cellular events is initiated that results in degranulation of the mast cells with release of vasoactive amines such as histamine and heparin. ANTIGENS AND ITS INTERACTION WITH ABS AND B CELLS B cells provide Ag receptors that can recognize soluble or cell-bound Ags without prior modification (compared to T cells that fail to do so, and need MHC presentation of a modified Ag). Thus, the B lymphocyte population is able to recognize a broad range of molecules, including proteins in their native or denatured conformations, lipids, carbohydrates and nucleic 7

8 acids. However, most B cells cannot proceed beyond recognition of the antigen without T cell help. The term T cell help is used to describe the cooperation between helper T cells and B cells that is necessary for most B cell responses. T cell help takes the form of direct intercellular T-B cell contacts, and the binding to the B cell of specific cytokines produced by a T cell responding to the same antigen. Without T cell help, the majority of B cells are unable to achieve complete activation. Antigens that bind to B cell receptors but cannot activate B cells without T cell help are called T-dependent (Td) antigens. For some non-protein Ags, interaction between the B cell receptors and the Ag alone is sometimes sufficient to activate the B cell. These Ags, which are often polymers, are called T-independent (Ti) antigens, because no T cell help is required for lymphocyte activation. Ti antigens generally are large polymeric proteins or polysaccharides (and sometimes lipids or nucleic acids) whose structure is composed of repetitive elements, as occur in many bacterial and viral products and structural elements. Ti antigens are only a small fraction of the immunogens that attacks the host. A high proportion of the molecules making up a pathogen are proteins of unique amino acid sequences that lack the large repetitive structures needed to cross-link BCRs and trigger B cell activation. Obviously a Td antigen must contain protein, since it must supply at least one peptide that can act as a T cell epitope. The immunogenicity of a Td antigen also depends on other physical properties such as its foreignness, conformation, and molecular complexity. The nature of the antigenic challenge must also be taken into account (i.e. the immunogenicity of a particular molecule may be affected by its dosage and its route of entry or administration). Finally, host factors may influence the immunogenicity of a molecule. Each step in the immune response is controlled by the expression of the host s genes, and subtle allelic differences in the genetic constitution of an individual can alter the type, as well as the intensity, of the immune response to a given immunogen. END of TEXT 8

9 References 9