Fundamentals of Biochemistry - A. A. Anisimov 1986

Protein and Amino Acid Metabolism
Structure of antibodies and the mechanism of their formation

The appearance of antibody Proteins in human and vertebrate serum and Tissues in response to the Introduction of genetically foreign compounds (Antigens) forms The basis of the HUMORAL Immune Response. Antigens are defined as substances that carry markers of genetic foreignness and, upon entering the body, trigger specific immunological reactions. Typical antigens include foreign proteins and microbial Polysaccharides.

Antigenic properties can also be exhibited by Nucleic Acids, Complex Lipids, certain low-molecular-weight compounds (such as various Hormones and drugs), and chemically synthesized compounds, provided they are bound to a carrier such as a protein, polysaccharide, etc. Such substances are termed haptens. The minimum Molecular Weight of substances against which Antibodies have been successfully raised without coupling them to larger molecules is approximately 1,000 (for instance, oligopeptides containing more than 8 amino acid residues).

Antibodies are glycoprotein substances produced in response to the introduction of an antigen and capable of specific reaction with it. In addition to Blood serum, antibodies are found in excretory fluids (milk, tear secretions, saliva, etc.) as well as On the surface of certain types of Cells in The Lymphatic system. The encounter between an antigen and its corresponding antibody leads to The formation of a complex. When this complex precipitates, a precipitation reaction occurs. If antibodies interact with antigens to cause Cell clumping, they are called agglutinins, whereas if they cause lysis, they are referred to lysins.

Antibodies exhibit a high degree of Specificity toward the foreign proteins that induced their formation. Different proteins from the same Organism elicit The production of distinct antibodies. For example, if a rabbit is immunized with horse Hemoglobin, the resulting antibodies are incapable of reacting with other horse proteins. Furthermore, Homologous proteins from different organisms are immunologically distinct, with Hemoglobins serving as another prime example. Antibodies produced in a rabbit in response to horse hemoglobin react most strongly with horse hemoglobin and significantly weaker with the hemoglobins of other mammals.

Antibody specificity reflects the Phylogenetic relationships among species. Homologous proteins of closely related species show greater immunological similarity than do proteins from distantly related species. The narrow selectivity of the antigen–antibody interaction is utilized in laboratory biochemical research to identify individual proteins. Due to its exceptionally high specificity, the immunochemical method (in combination with Electrophoresis, i.e., Immunoelectrophoresis) is highly accurate and in many cases indispensable.

Antibodies are soluble Plasma Proteins belonging to IMMUNOGLOBULINS (predominantly y-globulins). Based on sedimentation and electrophoretic characteristics, as well as their marked elevation in certain physiological states, human immunoglobulins are divided into five classes: IgG, IgM, IgA, IgD, and IgE. The first three are present in significantly greater quantities, while the latter two are referred to as minor classes. The relative concentrations of IgG, IgM, and IgA in the blood of a healthy individual account for 70–80%, 5–10%, and 10–20% of the total immunoglobulin content, respectively.

As Glycoproteins, immunoglobulins invariably contain CARBOHYDRATES, The amount of which relative to the total mass ranges from 2–3% in IgG to 10–12% in IgA and IgM. These covalently linked carbohydrates are primarily hexose- and hexosamine-containing Oligosaccharides with minor amounts of sialic acid and fucose. The carbohydrate prosthetic groups determine The rate of antibody degradation by hepatocytes. The molecular weight of immunoglobulins ranges from 150,000 to 900,000; for IgM macroglobulins it is ≈ 900,000, for IgA ≈ 170,000–500,000, for IgG ≈ 150,000, and for IgD and IgE ≈ 180,000.

Most immunoglobulin molecules are dimers consisting of two light (L) chains and two heavy (H) chains. IgA and IgM contain more than four chains in their Structure. H- and L-chains differ substantially in their Primary Structure. Within an individual molecule, both L-chains are structurally identical to each other, and both H-chains share the same Amino Acid Sequence. Their length varies across different immunoglobulin classes. The molecular weight of light chains is 20,000–25,000, while that of heavy chains is 50,000–55,000. Interchain Disulfide Bonds link the four chains into a single covalently bound structure. Following the reduction of interchain disulfide bonds, additional and rather stringent conditions—such as The addition of urea or a decrease in pH—are required to disrupt the strong non-covalent contacts between the L- and H-chains and separate them.

Human immunoglobulins may contain L-chains of one of two types: κ (kappa) or λ (lambda).

There are five types of antibody heavy chains: α, γ, δ, ε, and μ. They determine the antibody classes: IgM contains the μ-chain, IgG contains the γ-chain, IgD contains the δ-chain, IgE contains the ε-chain, and IgA contains the α-chain. Each heavy chain can associate with any of the light chains. The blood serum of a healthy individual contains a mixture of numerous diverse immunoglobulins. Their exceptionally high heterogeneity makes it impossible to isolate and study The structure of any single individual immunoglobulin from the mixture. However, a known disease—multiple myeloma (a Bone Marrow tumor)—involves the malignant proliferation (rapid division, expansion, and transformation) of a single specific type of antibody-producing cell that synthesizes one type of immunoglobulin in quite high concentrations. Myeloma proteins have been shown to closely resemble normal immunoglobulins in A number of Structural and functional features. Myeloma Igs can be isolated in relatively large quantities and are easily purified to homogeneity. Bence-Jones proteins, which represent Ig L-chains, have been isolated from the urine of myeloma patients.

In the 1970s, research breakthroughs made it possible to solve The problem of obtaining highly specific antibodies in large quantities. By fusing a B lymphocyte (a cell with a short antibody production period) and a tumor cell, researchers successfully generated a hybrid that manifested the most crucial properties of both parental cells: the tumor cell's capacity for unlimited proliferation and the lymphocyte's ability to synthesize antibodies. Because researchers used a myeloma cell as the tumor partner, the hybrid was named a hybridoma. Cloned (propagated) hybridoma cells synthesize Monoclonal Antibodies in large quantities that are homogeneous in structure and specificity.

Determining The amino acid sequence of immunoglobulins revealed that the antibody molecule can be divided into segments, or domains. Some of these are characterized by a constant amino acid sequence and are termed constant regions (C), whereas others feature highly variable sequences known as variable regions (V).

Both C- and V-regions are present in all light and heavy chains. The variable regions are located at the N-terminal PARTS OF THE L- and H-chains. In L-chains, the V-region comprises about 100 amino acid residues, while in heavy chains it comprises about 120. Within the variable regions of immunoglobulin chains lie hypervariable domains. These exact regions form the antigen-binding sites and are referred to as the antigen-binding centers (active sites) of antibody molecules. Their amino acid sequence varies in accordance with differences in antibody specificity. These structural features enable interaction with various antigens.

The constant regions of antibodies function in Complement binding and mediate the transplacental transfer of antibodies (in humans and certain mammals, maternal-fetal Immunity can be conferred during embryonic development via The transport of antibodies from maternal blood across the Placenta).

Studies on immunoglobulin structure have established that Cleavage into fragments upon partial proteolysis occurs within the hinge region of the H-chains. This region is located on the molecular surface and possesses a degree of flexibility. Hydrolysis of IgG by Papain yields three fragments: two Fab (antigen-binding) fragments and one Fc (crystallizable) fragment with a constant Amino Acid Composition. The molecular weight of the Fab fragment is 50,000–52,000. It retains the immunological activity of the parent IgG but behaves as a monovalent antibody, meaning it possesses a single antigen-binding site. The Fab fragment comprises an entire L-chain (either κ or λ) and the N-terminal half of an H-chain, connected by a disulfide bond. The Fc fragment has an M of 48,000 and consists of the remaining halves of the H-chains (Fig. 5.8). The primary Secondary structure motif of immunoglobulins is the antiparallel β-sheet. It alternates with α-helices and forms "loops" generated by the cross-linking of amino acid residues via disulfide bridges within each chain. These loops are designated as domains.

Class="center">

Fig. 5.8. Diagram of a human Ig molecule

An IgG molecule contains a total of 12 domains: 4 on each heavy chain and 2 on each light chain. The molecular weight of each domain is approximately equal, around 12,500. The first domains (V_L and V_H) are formed by the variable Regions of the light and heavy chains, whereas the remaining domains are formed by the constant regions of the light or heavy chains (C_L and C_H). The active sites of Antibodies Are Formed by the variable region domains. This involves 4 to 8 amino acid residues that exhibit wide Variability in amino acid types and combinations. These regions are called the hypervariable domains of the light and heavy chains.

Antibody specificity during antigen binding is governed by both primary and tertiary structure. The Tertiary Structure of immunoglobulins is complementary to the antigen conformation, ensuring the close approach and mutual orientation of specific polypeptide chain segments to form the binding site. Nevertheless, the narrow selectivity of the antibody–antigen interaction is determined primarily by differences in the Primary structure of the variable regions.

The antigen–antibody reaction proceeds analogously to the interaction between a substrate and an enzyme's Active Site. This mechanism was investigated using the binding of the Fab fragment to haptens. The interaction between the antigen and the Fab fragment is mediated by Hydrogen Bonds, hydrophobic interactions, and electrostatic forces. THE CONTRIBUTION OF individual amino acid residues or monosaccharide units of the antigen to the antibody interaction can vary widely. For instance, the substitution of even a single amino acid in bradykinin (a nonapeptide) leads to an almost complete cessation of its antibody binding. A different picture is observed during the interaction of blood group substance A with IgM antibodies. The latter complementarily bind only N-acetylglucosamine; other monosaccharide residues of blood group substance A do not affect the affinity of the antibody for the antigen in this case.

The synthesis of immunoglobulins is carried out via standard Protein Synthesis mechanisms, but the L- and H-chains are produced by two distinct types of polyribosomes. The synthesis of antibodies involves the cooperative interaction of three cell types originating in the bone marrow: B lymphocytes, T lymphocytes, and macrophages.

B lymphocytes are potentially capable of producing antibodies. They migrate from the bone marrow to peripheral Lymph Nodes AND the Spleen. Another group of bone marrow cells migrates either to the Thymus, lymph nodes, or spleen, where they transform into T lymphocytes under METABOLISM/18.html">The Influence of thymic hormones. Macrophages play a role in immunoglobulin synthesis through their ability to phagocytose and partially degrade particulate foreign objects (Bacteria, protein structures). The products of macrophage activity serve as additional antigenic stimuli for B AND T lymphocytes.

Nature contains a vast array of diverse compounds that, upon entering the body, act as antigens. This raises questions that are both fascinating and complex, remaining poorly explored within immunochemistry: does DNA in its fully formed state contain an equally immense number of genes for different antibody species as there are diverse antigens? Since simple calculations and logic suggest a negative answer, by what other means is the colossal variability of antibodies achieved?

According to current concepts, The Immune System is capable of distinguishing approximately 10⁵–10⁷ different antigens. Immunoglobulins are encoded by A large number of mini-genes (subgenes) separated by non-coding sequences. Originally unlinked mini-genes encoding various parts of immunoglobulin chains join together at the DNA and mRNA levels during The Development of B lymphocytes, which produce specific antibodies. Embryonic lymphocytes contain many hundreds of subgenes for the variable parts of light (V_L) and heavy (V_H) chains. Furthermore, thousands of nucleotide pairs upstream of the constant region genes lie several short sequences known as J (joining) segments. The chains also contain very short D (diversity) segments, which likewise participate in generating diversity (Fig. 5.9).

Fig. 5.9. Correspondence between immunoglobulin polypeptide regions and their encoding subgenes (after R. B. Khesin, 1984):

regions encoding variable regions; С_1-n — regions encoding constant regions of Polypeptides; J_1-4 — short regions connecting V and C regions; D_1-n — "very short regions"

During the formation of complete genes, one of the V_L subgenes joins with one of the J_L regions to form a single Gene. Following its Transcription and subsequent splicing, a single mRNA is produced, encoding a unified VJC polypeptide of the immunoglobulin light chain. Rearrangement during the formation of H-chain genes is even more complex: V_H regions fuse with J_H, D_H, and potentially C_H. The immense variability of lymphocyte clones is thus explained by the colossal number of possible combinations of V, V, D, J, and C subgenes.

The Selection of specific genetic elements that "construct" functioning immunoglobulin genes in a given lymphoid cell appears to be stochastic. Since both H and L chains participate in forming the active site, assuming an equal probability of any H chain joining with any L chain, the number of potential active-site variants increases dramatically.

Overall, the DIVERSITY OF ANTIBODY specificity is currently attributed to the fact that, throughout evolution, organisms encountered a vast array of foreign agents, which drove the accumulation of a large pool of antibody genes. Somatic Mutations within genes and the assembly of the genes themselves from segments (subgenes or minigenes) exponentially increase antibody diversity. Antigens act as selective agents: they recognize immunoglobulin receptors on The Cell surface, bind to them, and stimulate the proliferation of that specific cell, facilitating the rapid expansion of its numerous clone.

Upon the interaction of antigens with antibodies, The Complement System is triggered. It consists of several (approximately 10) Serum proteins. Their cascade activation mechanism is initiated by the Formation of the antigen-antibody complex. Ultimately, the action of complement leads to the lysis of foreign substances and the activation of leukocytes. Thus, complement, together with antibodies and specialized cells, participates in protecting the host organism against infections.

The phenomenon of transplanted tissue rejection observed in mammals and birds is caused by the presence of Histocompatibility Antigens on The surface of virtually all cells (with the exception of embryonic cells and erythrocytes). These have been studied in greatest detail in humans and mice. They are highly hydrophobic proteins encoded by a series of distinct gene loci (similar to isozymes). Often, these genes are located close to one another on the chromosome, forming gene regions. Numerous alleles (alternative forms of a gene) exist for each gene locus, determining a high degree of polymorphism.

The most active histocompatibility antigens are encoded by genes clustered within a specific region of the chromosome known as the MHC (Major Histocompatibility Complex). In humans, this region is designated as HLA, and in mice, as H-2. The Functions of histocompatibility antigens are not yet fully understood, though there is good reason to believe they play a role in regulating the immune and complement systems, as well as in mediating cell-to-cell interactions.

Throughout its lifetime, the organism encounters a vast array of different bacteria and antigens; consequently, normal plasma contains a huge variety of different antibodies. Specifically, immunity to certain bacteria or Viruses is conferred by the presence of corresponding 'self' specific antibodies. Artificial active immunity is achieved by introducing killed bacteria into the body, as well as toxoids—diphtheria or tetanus toxins that have been treated with formaldehyde (and thereby rendered harmless). As a result of vaccination with Vaccines obtained in this manner, the body produces specific antibodies capable of reacting with a specific type of live bacteria or natural toxin, thereby establishing immunity.

The most effective vaccines for establishing robust artificial immunity are those derived from live attenuated microorganisms. These are produced by culturing pathogens under adverse conditions (such as sub-optimal temperatures or the addition of specific substances to the nutrient medium). For a number of infections, live vaccines are virtually the only means of specific prophylaxis (e.g., anthrax, tularemia, plague). Live vaccines are also widely used against smallpox, rabies, poliomyelitis, and many other diseases. Recently, chemical vaccines have come into use, prepared by extracting antigenic fractions from cultures of the respective microorganisms. Research into the Chemical synthesis of antigens—which have significantly simpler structures than natural ones—is progressing successfully, bringing the production of synthetic vaccines ever closer to reality.

Temporary passive immunity is established by administering antibodies produced by another, immune organism, typically an animal. For the Treatment of diphtheria and tetanus, for example, plasma from immunized horses is used, which contains antibodies against the respective toxins.

Antibodies are produced exclusively in vertebrates, but this does not mean that invertebrates lack immunity. The immune system evolved from mutual Cell Recognition mediated by complementary surface receptors, allowing the organism to distinguish 'self' from 'non-self'. Because the immune system functions in close coordination with the histocompatibility complex, their evolution must be considered in tandem.

In starfish and Annelids, transplanted tissue undergoes rejection and destruction. Arthropods possess non-inducible Mechanisms for the agglutination of foreign substances. The thymus and spleen—and with them, antibodies—appear only in vertebrates. The structure of antibody chains has evolved over the course of vertebrate history. Agnathans (or cyclostomes, which include lampreys and hagfishes) possess immunoglobulins with μ-like heavy chains.

Even the most primitive immunoglobulins already consist of multiple, albeit uniform, polypeptide chains. However, their subunits are not yet linked by disulfide bridges. IgM is one of the most ancient classes of immunoglobulins, present in all cartilaginous and bony Fishes. Class M antibodies have also been found in amphibians, alongside heavy chains resembling the α or δ types in certain anuran amphibians. Only in reptiles do ϰ- and λ-chains first appear. α-Chains are found in birds and mammals, while ε-chains are exclusive to mammals. Mammals also possess diverse forms of α-, γ-, δ-, and ε-chains, and may exhibit the expression of more than one class within a single cell.