IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 9. Antigen Recognition

■ Antibodies are highly specific for the three-dimensional conformation of epitopes on the antigen that induced their production.

■ Antibody affinity is a measure of the binding strength between an antibody combining site and a single antigenic epitope. Functional affinity, or avidity, of antibody-antigen interactions is also determined by the number of antigen-binding sites on the antibody molecule and their ability to bind multiple epitopes of a given antigen.

■ T Cells recognize Antigens that are bound (presented) by other cells in association with Class I or class II MHC molecules. Peptide fragments of processed antigens bind within a specialized cleft of MHC molecules.

■ Class I and class II MHC molecules present Peptides derived from endogenous and exogenous antigens, respectively. Depending on their origin, processed antigens encounter and bind to MHC molecules in different intracellular Organelles.

■ Antigenic peptides bound by class I MHC molecules are generated in the Cytoplasm through the Cleavage of antigens by organelles called proteasomes. Transport Proteins belonging to the ABC superfamily mediate the translocation of these peptides across The Endoplasmic reticulum membrane. A tripartite complex consisting of a class I heavy chain, β₂-microglobulin, and a peptide is then transported to The Cell surface.

■ Antigenic peptides bound by class II MHC molecules originate from exogenous antigens taken up by endocytosis and subsequently processed in endosomes or Lysosomes. Class II MHC molecules complexed with the invariant chain (Ii polypeptide) are transported through the Golgi apparatus to endosomes, where the Ii chain is dissociated and replaced by antigenic peptides.

■ Complexes of antigenic peptides with MHC molecules displayed on the cell surface can be recognized by specific T-cell receptors. However, subsequent T-cell activation requires a series of additional interactions involving accessory molecules.

Antibodies and antigen-recognizing receptors of T cells share several common features. Both possess constant (C) and variable (V) domains, and the V-domain-encoding genes undergo similar V-, D-, and J-segment recombination (see Ch. 8). Nevertheless, the mechanisms of antigen recognition by B AND T cells are fundamentally different. Antibodies can recognize antigens both in solution and on the cell surface, but always in their native conformation. In contrast, T-cell receptors require the antigen to be associated with cell-surface MHC molecules. T-cell antigens are often subjected to prior cleavage, or Processing, such that the determinant recognized by the TCR is merely a small fragment of the original antigen.

Another distinction between antibodies and TCRs is that antibodies exist in two forms—as B-cell antigen-binding receptors and as secreted molecules—whereas TCRs are invariably complex membrane-bound proteins. Secreted antibodies are typically bifunctional molecules: their V domains are designed for antigen binding, while their C domains interact with host cell receptors or Complement components.

This chapter examines The Structure of antigen-binding sites in antibodies and TCRs, as well as their interactions with specific antigens or antigen-MHC complexes. The selectivity of these interactions forms The basis of acquired Immunity Specificity.

ANTIBODY-ANTIGEN BINDING

Numerous non-covalent bonds are formed between an antibody and an antigen

X-ray crystallographic analysis of V domains has revealed that hypervariable Regions of the polypeptide chain are clustered at the tips of the Fab arms of antibody molecules (see Ch. 6 and 8). Specific amino acid residues within these regions interact directly with antigenic epitopes (Fig. 9.1). Framework residues within the same regions, which generally do not participate in direct antigen binding, play a crucial role in maintaining the V-domain folding required for an appropriate conformation of the antigen-binding site.

Fig. 9.1. An antigen molecule fits into the cleft formed between the light and heavy chains of an antibody—the antigen-binding site. An example of such an interaction is the binding of the γ-hydroxylated form of vitamin K to immunoglobulin G (NEW myeloma protein). According to X-ray crystallography data, 10 to 12 amino acid residues located in the hypervariable regions of the heavy and light chains contact the antigen; these residues are marked with numbers indicating their positions in the figure.

When specific antibodies contact an antigen, numerous non-covalent bonds are formed between The amino acid residues of the antigen-binding site and the antigenic epitope. Compared to covalent bonds, individual non-covalent Intermolecular Forces (Hydrogen Bonds, electrostatic, Van der Waals, and hydrophobic interactions) are relatively weak; however, A large number of these weak interactions result in a substantial net binding energy.

The Conformations of the antibody antigen-binding site and the target antigen are complementary

The strength of a non-covalent bond depends primarily on the distance (d) between the interacting chemical groups. For Electrostatic Interactions, it is proportional to 1/d², and for van der Waals forces, to 1/d⁷, meaning they become significant only when the molecules are brought into close proximity (Fig. 9.2). The binding of an antigenic determinant (epitope) to an antibody combining site (paratope) (Fig. 9.3) requires mutual attraction between atomic groups on the contacting surfaces, facilitated by the conformational complementarity of the epitope and paratope, and the concurrent formation of multiple non-covalent bonds. At a certain level of complementarity, the attractive energy becomes sufficient to prevent thermal dissociation. Conversely, when the electron clouds of the antigen and antibody molecules overlap, repulsive forces arise, the magnitude of which is inversely proportional to the 12th power of the intermolecular distance: F ~ 1/d¹². It is precisely these forces that determine antibody specificity (i.e., The ability to distinguish between antigens), since any deviation from ideal conformational complementarity leads to a decrease in overall binding energy due to sharply increasing repulsive forces and decreasing attractive forces (Fig. 9.3).

Fig. 9.2. Close apposition of interacting atomic groups is required for binding forces to develop between an antibody and an antigen. Hydrogen bonds are formed via hydrogen bridges between such groups. Electrostatic interactions result from the attraction between oppositely charged atomic groups located on the side chains of the binding proteins. Van der Waals bonds arise from interactions between the electron clouds of molecules (in this case, between induced oscillating dipoles). Hydrophobic interactions, which can account for up to half of the total binding energy between an antigen and an antibody, represent a strong attraction in Water between nonpolar (hydrophobic) groups that virtually eliminates their contact with water. The optimal interaction distance between groups varies depending on the type of bond.

Fig. 9.3. Conformational match between the antibody binding site and the antigenic determinant favors intermolecular attractive forces while strongly discouraging repulsive forces. In contrast, incomplete conformational matching leads to the dominance of repulsive forces. When the electron clouds of epitopes and paratopes overlap, significant repulsive forces are generated that outweigh any weak attractive forces.

Studies of the interaction between Lysozyme and Fab fragments of antilysozyme antibodies have shown that the surfaces of the epitope and paratope are complementary even beyond the hypervariable regions. A total of 17 amino acid residues of the antibody molecule contact 16 residues of the lysozyme molecule (Fig. 9.4). All hypervariable regions of both the light and heavy immunoglobulin chains contribute to The formation of the antigen-binding site, though the CDR-3 region, encoded by the heavy-chain V-D-J segment, appears to play a particularly prominent role. This is likely due to its greater Variability generated by V, D, and J segment recombination.

Fig. 9.4. Immune complex formed by the Fab fragment of an anti-lysozyme antibody and lysozyme (space-filling model). Top: lysozyme (shown in green) bound to the hypervariable regions of the heavy (blue) and light (yellow) chains of the D1.3 antibody Fab fragment. Center: dissociated complex; the glutamine residue at position 121 of the lysozyme, highlighted in red, is deeply embedded in the cavity between the antibody's heavy and light chains. Bottom: the same molecules rotated by 90° to reveal the interacting amino acid residues of both antibody and antigen. (Reprinted with permission from R. J. Poljak. Science 1986; 233: 747-753.)

Antibody affinity is the binding strength between a single antigen-binding site and an individual epitope of an antigen

Antibody affinity, or the strength of the antigen-antibody interaction, refers to the binding force resulting from the aforementioned attractive and repulsive forces (Fig. 9.5). The interaction between an antigen-binding site and an antigen can be investigated thermodynamically. To measure the affinity of an individual antigen-binding site, a monovalent antigen—or more precisely, an isolated antigenic determinant (hapten)—is used. Because the non-covalent bonds between paratopes and epitopes can dissociate, the formation of immune complexes is a reversible process; applying the law of mass action to this equilibrium yields the Equilibrium Constant $K$, which essentially represents the affinity constant (Fig. 9.6).

Fig. 9.5. Antibody affinity is the net result of attractive and repulsive forces operating between the antibody and antigen. High-affinity antibodies exhibit a precise conformational complementarity to the antigen, whereas low-affinity antibodies do not.

Fig. 9.6. All antigen-antibody reactions are reversible and therefore governed by the law of mass action, allowing antibody affinity to be calculated as the equilibrium constant, $K$. (Square brackets denote reactant concentrations.)

Antibody avidity is the overall binding strength of an antibody-antigen interaction

The basic immunoglobulin molecule unit, consisting of four polypeptide chains, contains two antigen-binding sites, making antibodies potentially multivalent with respect to the antigen. Furthermore, antigens themselves can be monovalent (such as haptens) or multivalent (such as microbial cells). Unlike affinity, which measures the binding strength between a single antigenic determinant and an antigen-binding site, avidity describes the interaction force between multivalent antibodies and a multivalent antigen. Avidity depends on the affinities of the individual antigen-binding sites for the corresponding epitopes, but it always exceeds their arithmetic sum if both sites bind to the antigen. Antigen and antibody multivalency substantially enhances binding stability because dissociation of such immune complexes requires the simultaneous rupture of all bonds (Fig. 9.7). Under physiological conditions, it is more appropriate to consider avidity rather than affinity, as natural antigens are typically multivalent. However, studying the immunochemical aspects of antibody-antigen interactions requires precise measurement of antibody affinity for haptens.

Fig. 9.7. As reflected by the equilibrium constant, multivalent antibody-antigen binding (functional affinity, or avidity) is significantly stronger than simple monovalent binding (true affinity, arbitrarily set here at 10⁴ L · mol^-1). Formerly termed the multivalency enhancement effect, this phenomenon increases binding energy—for instance, 10³-fold for IgG when both binding sites of the molecule are engaged, and up to 10⁷-fold for IgM.

Kinetics of antigen-antibody reactions

Antibody affinity values pertain to equilibrium conditions and reflect the ability of antibodies to form stable immune complexes. However, for many biological activities of antibodies, the reaction kinetics with the antigen are likely just as crucial as affinity.

Kinetically, antibody-antigen binding is characterized by the association and dissociation rate constants, denoted respectively as K_1.2 (mol^-1 · s^-1) and K_2.1 (s^-1). At equilibrium, The ratio of these two rate constants yields the equilibrium constant, which defines the affinity of a given antibody preparation. It was previously thought that variations in antibody affinity were primarily driven by differences in the dissociation rate of immune complexes, but more recent findings show that the association rate is also affinity-dependent.

Recent studies have revealed that during the maturation of the HUMORAL Immune Response, B cells are selected based on their ability to bind antigen rapidly (kinetic Selection) as well as the strength of antigen binding (thermodynamic selection).