Basics of Immunology - Lecture Course by M. V. Skok - Kyiv 2002

Chapter I. Immunochemistry

Lecture 3. Antibodies: Structure and Properties

In the late 19th century, E. von Behring discovered antibodies (antitoxins) against diphtheria and tetanus toxins, demonstrating that Immunity can be transferred humorally. P. Ehrlich was the first to prove that antibodies can be raised not only against pathogens, but also against purely chemical substances, as well as plant poisons such as ricin and abrin. M. Pfeiffer observed that antibodies cause antigen-bearing substances to precipitate or agglutinate, converting them from a soluble to an insoluble state.

The true nature of antibodies was only elucidated in the mid-20th century. In 1937, Arne Tiselius performed serum Protein Electrophoresis, identifying (from positive to negative) albumin, alpha-1, alpha-2, beta, and gamma globulins. Twenty-two years later, Kabat discovered that following animal immunization, the peak of gamma globulins in the serum increases, and that this peak decreases again after absorbing out the antibodies with the specific antigen. This conclusively proved that antibodies belong to the gamma globulin fraction. To emphasize The Role of antibodies in immune responses, they came to be called IMMUNOGLOBULINS.

The Study of antibody Structure was greatly facilitated by the availability of myeloma immunoglobulins. Myelomas are malignancies characterized by the clonal expansion of B lymphocytes of a single Specificity, leading to the accumulation of massive amounts of completely identical monoclonal antibodies (up to 70 mg/mL of serum).

The Structure of antibodies was elucidated by Edelman and Porter in the 1960s, for which they were awarded the Nobel Prize in 1972.

An antibody molecule (Fig. 1) consists of Two Types of polypeptide chains: heavy (H) and light (L). Heavy chains are divided into classes: α, μ, γ, δ, ε, corresponding to five immunoglobulin classes: IgA, IgM, IgG, IgD, and IgE. Light chains exist in two types: k and λ, with k being more prevalent than λ. An immunoglobulin molecule contains two heavy and two light chains. The molecule is composed of identical halves containing matched pairs of chains: γ₂k₂, γ₂λ₂, α₂k₂, α₂λ₂, and so on. Heavy chain subclasses (in humans) include 4 for γ (γ1, γ2, γ3, and γ4), 2 for μ (μ1 and μ2), 2 for α (α1 and α2), 1 for ε, and 1 for δ. Light chain subtypes include 1 for k, and 4 for λ (along with λ₅, which is expressed during embryonic development).

A heavy chain consists of 440–450 amino acid residues (the μ-chain has about 100 residues more) and has a Molecular Weight of approximately 50 kDa, whereas a light chain consists of 220–230 amino acid residues with a molecular weight of about 25 kDa. The chains are linked together by interchain Disulfide Bonds, which are concentrated primarily in the so-called hinge region, or the "waist" of the molecule.

Intrachain disulfide bonds form loops that define domains (comprising approximately 80 amino acid residues each); There are two domains in a light chain and four in a heavy chain (five in the μ-chain). Domains with this structural motif are found in many receptor molecules and are appropriately termed immunoglobulin-like domains.

When comparing the Amino acid sequences of dozens of myeloma Proteins, researchers noticed that differences between them (Amino Acid Substitutions) are not distributed stochastically along the chain, but are instead localized in the first (N-terminal) domains of both heavy and light chains. Consequently, scientists distinguished between constant (C) and variable (V) domains. Thus, a light chain is composed of one V and one C domain, while a heavy chain consists of one V and three C domains (four in the μ-chain), designated as V_L, V_H, C_L, and C_H, respectively. A single set of C-domains can be paired with many different V-domains. The immunoglobulin Class is determined precisely by the C-domain; in other words, the same V-domain can be part of either an IgM or an IgA molecule, and immunoglobulins of the same class can feature a broad spectrum of V-domains.

Within V-domains, there are specific regions known as hypervariable regions, where substitutions occur much more frequently than elsewhere. These are also referred to as CDRs ( complementarity-determining regions ), meaning the areas that determine complementarity in antigen binding. These very regions form the Active Site of the antibody—three in the heavy chain and three in the light chain. The intervening segments are framework regions, which are evolutionarily more conserved and maintain the spatial orientation of the CDRs. Due to the symmetrical STRUCTURE OF THE immunoglobulin molecule, each antibody molecule has two antigen-binding sites formed by the V-domains of each heavy-light chain pair.

Immunoglobulin fragments.

Proteolytic Cleavage can be used to generate immunoglobulin fragments, some of which retain The ability to bind antigen (Fig. 2). The antibody structure was largely deciphered through the study of such fragments. The hinge region is particularly sensitive to proteolysis due to a high concentration of Cysteine and Proline residues, which provide flexibility to The polypeptide chains and render them accessible to Proteolytic Enzymes. Because of this flexibility in the hinge region, an immunoglobulin molecule can alter the angle between its two antigen-binding arms.

Papain cleaves the immunoglobulin molecule just above the hinge region into two Fab (Fragment antigen-binding) fragments and one Fc (Fragment crystallizable, constant) fragment. The Fab fragments (approximately 50 kDa each) retain antigen-binding activity because they contain the variable domains of both heavy and light chains, and they are monovalent. The Fc fragment (50 kDa) does not bind antigen but mediates effector Functions characteristic of the given immunoglobulin class. Treatment with plasmin can further cleave a Fab fragment into Fv and Fd fragments, each of about 25 kDa.

Pepsin cleaves the immunoglobulin molecule below the hinge region, yielding a divalent F(ab')₂ fragment and pFc' fragments, with molecular weights of 100 kDa and 25 kDa, respectively.

By reducing disulfide bonds (for example, using 2-mercaptoethanol), an immunoglobulin molecule can be dissociated first into two halves of 75 kDa each, and subsequently into individual heavy and light chains. The halves retain full antigen-binding capacity, whereas the heavy chains retain it only partially.

Immunoglobulins are Glycoproteins containing up to 12% carbohydrate (IgG contains 2–3%), predominantly mannose and galactose, which are attached to the C_H2 domain at Asp residues via N-acetylglucosamine.

The Secondary structure of immunoglobulins is predominantly composed of antiparallel β-pleated sheets, with virtually no helical regions. Regarding the tertiary structure, It is important to highlight their distinct domain architecture, The flexibility of the Fab fragments relative to the molecular axis, and the specific architecture of the antigen-binding site, which will be discussed separately.

Certain classes of immunoglobulins form oligomeric structures. For instance, IgA forms dimers, and IgM forms pentamers. The pentamer is stabilized by disulfide bonds between individual IgM molecules, along with an additional J-chain (12 kDa) that cyclizes the entire oligomer into a star-like shape. IgG, IgD, and IgE occur exclusively as monomers. Their molecular weight is approximately 150 kDa, whereas that of the IgM pentamer is 890–900 kDa.

Immunoglobulins can themselves act as Antigens. Three types of antigenic determinants can be distinguished on them: isotypic, allotypic, and idiotypic. Isotypic antigenic determinants are localized on the Fc regions of heavy chains and essentially reflect the classes and subclasses of heavy chains. Allotypic determinants arise from allelic variations (allelic genes at a single locus) of immunoglobulins and differ among genetically distinct individuals of the same species. At the protein level, these are determined by substitutions of 1–2 amino acid residues. For example, 25 allotypic markers are known on human γ-chains, and three on the k-chain. Unlike iso- and allo-determinants, idiotypic determinants are associated with the Fab fragments of antibodies, specifically with their active site. In effect, the idiotype defines the Specificity of the antibody; it is unique to each individual molecule and comprises amino acid residues from the hypervariable regions.

Iso-, allo-, and idiotype-specific antibodies can be produced artificially and used to identify corresponding determinants on an immunoglobulin molecule. It is also possible that such antibodies are generated naturally. This is particularly true for anti-idiotypic antibodies, which may be induced following the appearance of large amounts of antibodies of a specific titer due to immunization. The logical chain of events involving The formation of anti-idiotypes and anti-anti-idiotypes formed The basis of the idiotype network theory (N. Jerne). According to this theory, anti-idiotypic antibodies and corresponding Cells of matching specificity act as a powerful regulatory mechanism controlling the Immune Response. This theory was extremely popular in the 1970s and 1980s, but it lacked robust experimental confirmation and is virtually unmentioned in modern literature.

The immunoglobulin molecule is structured in such a way that its different regions mediate distinct functions. Thus, the function of the Fab fragment is to form the antigen-binding site. The biological outcome of antigen binding—that is, the downstream consequence of immune recognition—depends on The Nature of the Fc fragment. These include binding to Fc receptors present On the surface of numerous Cell types, Complement fixation, and various other so-called effector functions. Different classes of immunoglobulins possess distinct Fc structures and consequently mediate different biological functions.

Functions of different immunoglobulin classes.

IgM

This is The structure of antigen-specific receptors on B cells. On the B cell membrane, IgM exists as a monomer with an additional hydrophobic domain. Upon activation, B lymphocytes initially secrete pentameric IgM and subsequently switch to IgG or other immunoglobulin classes. Thus, IgM serves as the first line of defense against infection. It exhibits relatively low specificity (affinity) for the antigen, but due to its pentameric structure, it can simultaneously bind five antigen molecules, resulting in high overall binding avidity. Furthermore, owing to its oligomeric nature, it easily induces agglutination (clumping) of microbial cells, which facilitates their destruction by macrophages. The so-called "normal" antibodies present in the Blood of healthy individuals are also predominantly IgM. Evolutionarily, this class appeared earlier than other immunoglobulin classes.

IgG

This is the principal class of serum antibodies during a secondary immune response. It crosses the Placenta, making maternal IgG the primary means of protecting the newborn from infections During the first weeks of life. It is also found in colostrum. It plays a crucial role in the opsonization of Bacterial toxins and microorganisms. Opsonization is the formation of immune complexes that facilitate the destruction of an antigen by non-immune cells. This occurs through the binding of the complex: 1) to Fc receptors on macrophages and natural killer cells, or 2) to complement components. Antigen binding induces a conformational change in the Fc fragment, significantly enhancing its capacity for 1) and 2) compared to free antibodies.

IgG subclasses differ in their properties, such as the propensity for spontaneous aggregation and complement binding. Their Biosynthesis is regulated by various mechanisms involving different T cell types. Sometimes the presence of a specific IgG subclass is critically important. For example, in autoimmune Pemphigus, autoantibodies are produced against desmoglein 3, one of the Skin proteins. In healthy individuals and patients in remission, antibodies of the IgG1 subclass are found. When the disease flares up and begins to affect the skin and mucous membranes, both IgG1 and IgG4 antibodies are detected in patients. Thus, IgG4 antibodies are pathogenic, whereas IgG1 are not.

Among all immunoglobulins present in blood serum, the concentration of IgG is the highest, reaching approximately 10 mg/mL (5x10¹⁶ molecules) in an unimmunized animal. For comparison, the serum concentration of IgM is 0.1 mg/mL.

IgA

It is found primarily in mucosal secretions: saliva, tears, nasal mucus, sweat, colostrum, as well as pulmonary, gastrointestinal, and genitourinary secretions. It exists as a dimer joined by a J chain (15 kDa). It is protected from proteolysis through complexation with the so-called secretory component (60 kDa). The secretory component is a part of the IgA receptor on epithelial cells, through which IgA is transported from blood or Lymphatic vessels to mucosal surfaces. It is internalized along with IgA and subsequently exocytosed into the secretion.

In blood serum, IgA is present in monomeric form. Two IgA subclasses are known: IgA1 (80-90%) and IgA2, which lacks disulfide bonds between heavy and light chains.

IgE

It is present in blood serum in low concentrations. Its main function is to induce an acute inflammatory response by releasing inflammatory mediators— vasoactive amines such as histamine and serotonin—from mast cells. This process is triggered following the interaction of IgE with an antigen. Normally, inflammatory mediators induce an influx of IgG antibodies, complement, neutrophils, and eosinophils to the site of infection, aiding in infection clearance. However, this entirely normal mechanism frequently malfunctions, causing the Organism to suddenly mount an excessive, inappropriate response to a minor and harmless antigen such as plant pollen or bee venom. This results in an allergic reaction: anaphylactic Shock, hay fever, or Bronchial Asthma. IgE molecules are the primary mediators of allergies.

IgD

They exist exclusively in a membrane-bound form as receptors on B lymphocytes. During B cell development, they appear after IgM. Their specific functions remain largely unknown.

Structure of the antibody active site.

The structure of the antibody active site has been studied using various Methods.

1. Photoaffinity and spin labeling. Principle: a chemical group was introduced into the antigen, which upon irradiation formed covalent bonds with surrounding amino acid residues. Subsequently, the antigen-antibody complex was hydrolyzed to determine precisely which amino acid the label was bound to. This approach revealed that hypervariable amino acid residues comprise the active site of the antibody.

2. It was observed that the antigen-antibody bond is disrupted by changes in pH, Ionic strength, and upon The addition of organic Solvents or chaotropic ions. This led to the Conclusion that the bond between antigen and antibody is non-covalent, specifically ionic, hydrogen-bonded, or hydrophobic.

3. The dimensions of the active site were investigated using Amino Acid and carbohydrate oligomers of various sizes. It was found that 5 to 6 amino acid or glucose residues can fit into the antibody active site, with dimensions corresponding approximately to 15x6x8 Å (12x20 Å), which may vary among different antibodies.

4. The fact that the active site is a cavity recessed within the Fv fragment was discovered using a bivalent hapten (two dinitrophenol molecules separated by varying numbers of methylene radicals). Upon adding antibodies to such an antigen, characteristic ring structures formed by the convergence of multiple antibody molecules bound to the antigen were observed under an Electron microscope. Such structures were not formed if the spacer length was less than 5(CH₂), meaning that the active site was recessed into the Fv fragment by approximately 15 Å.

5. Finally, in the 1970s, X-Ray Diffraction Analysis of myeloma immunoglobulins confirmed earlier data and demonstrated that the antibody active site is a cavity formed by hypervariable Regions of the Fab fragment brought into close proximity in the tertiary structure.

Why does an antigen bind specifically within the active site rather than on the exterior, even though identical combinations of amino acid residues are entirely possible on The surface of a protein molecule? This question also applies to the active sites of enzymes. The work of Richards (1974) showed that atomic density at the enzyme-substrate binding center is significantly lower than in other PARTS OF THE molecule: chemical groups can move with a greater number of degrees of freedom. The same pattern was found for antibody active sites. Considering that increased atomic mobility is also a characteristic of B epitopes, one can conclude that the most flexible, mobile regions exhibit mutual affinity. Binding leads to mutual stabilization, making it energetically favorable (occurring with a decrease in Free energy). The resulting complex is more stable than its components. This has been confirmed experimentally: antibodies in a complex with an antigen require twice as much guanidine hydrochloride for Denaturation as the same antibodies in their free form.

In the 1970s, modeling of the antibody active site was performed through surface-mimicking synthesis. Thus, according to X-ray crystallography data, the following amino acid residues were involved in forming the active site of the phosphocholine-specific antibody (myeloma IgM-603):

A peptide containing these residues linked by Glycine spacers was synthesized: Ser-Tyr-(Gly)₂-Arg-Tyr-Gly-Glu-Trp-Val. The synthetic peptide bound to phosphocholine and competed with natural antibodies.

Subsequently, this approach was applied to model the active site of protein-specific antibodies, specifically those directed against the Lysozyme epitope:

116 113 114 34 33

Lys Asn Arg Phe Lys

Lys-Asn-Arg-Gly-Phe-Lys - synthetic peptide

Asp-Gln-Asp-Gly-Leu-Asp - complementary peptide

The complementary peptide bound to lysozyme and competed with specific antibodies. This did not imply that the peptide completely mirrored the structure of the active site, but rather that it was built upon the same design principle.

Thus, it became clear that the basis for the high specificity of antibodies lies in the proper arrangement of amino acid residues within the active site; any alteration in charge or dimensions of the antigenic determinant diminishes binding efficiency.

Further studies of myeloma proteins revealed that the active site is not monospecific: structurally diverse antigens can bind within a single active site. This was first demonstrated using the MOPC 450 protein, which, alongside dinitrophenol, bound the structurally unrelated menadione. Investigation into a broad spectrum of unrelated haptens showed that the probability of their binding within the same active site reaches 1 in 140. They bind at various regions of the active site cavity, which is sufficiently large to accommodate several modes of hapten binding.

It was thus proven that a limited antibody repertoire can bind a vast, virtually limitless array of antigens. Consequently, the universal Nature of the immune response requires a finite (10⁴-10⁶) repertoire of antibody variants.

The high probability of structurally distinct antigens binding to a single antibody molecule raised questions about the foundation of immune response specificity as a whole: why do specific responses avoid attacking self-antigens or other unrelated antigens? The answer emerged through the identification of extensive antibody heterogeneity. Immunization with a specific antigen triggers The production of a wide array of antibody variants. Each of these antibodies, while recognizing the target antigen, may also cross-react with a few others; however, its fraction within the total mixture is so small that the likelihood of effectively recognizing unrelated antigens remains low. Conversely, a common feature among all produced antibody variants is their shared recognition of the target antigen, making the probability of its efficient binding very high. Thus, the specificity of the immune response is an aggregate value composed of numerous contributions from various antibodies, each of which is multifunctional. The greater the number of stimulated B cell clones, the higher the proportion of antibodies directed against a given antigen and the lower the proportion targeting unrelated ones. This indicates that immune response specificity is fundamentally a population-level phenomenon rather than a property of any single member of that population. Integral specificity is achieved precisely through antibody heterogeneity.

Summary.

Antibodies are immunoglobulins by nature. They possess a symmetrical molecular architecture consisting of two types of chains: heavy and light. The chain regions are folded into domains, which serve as the fundamental structural units of these proteins. A distinction is made between Variable and constant domains. The variable domains form the antibody's active site responsible for antigen binding. The constant domains mediate the EFFECTOR FUNCTIONS OF antibodies: toxin precipitation, bacterial agglutination, opsonization, complement activation, and mast cell degranulation. The active site of an antibody is a cavity recessed within the Fv fragment, capable of binding structurally diverse antigens.

There are four striking Features of the HUMORAL IMMUNE RESPONSE that, at first glance, appear mutually exclusive:

1) universality: antibodies can be generated against virtually any chemical group. It is hard to fathom how many antibody variants would be required if each antigen demanded a completely dedicated antibody;

2) specificity: antibodies distinguish between ortho- or para-positions of functional groups, stereoisomers, and proteins differing by just a single amino acid residue;

3) heterogeneity: multiple types (populations) of antibodies are produced in response to a single antigen, occasionally exceeding 100 per single antigenic determinant;

4) multifunctionality: a single antibody is capable of binding more than one antigen.

It turns out that it is precisely the multifunctionality of antibodies that underlies the universality of the immune response, whereas their heterogeneity accounts for its high specificity.