Computer simulation of an interaction between antibody and influenza virus antigen, a globular protein. (a) The antigen (yellow) is shown interacting with the antibody molecule; the variable region of the heavy chain is red, and the variable region of the light chain is blue. (b) The complementarity of the two molecules is revealed by separating the antigen from the antibody by 8 A. [Based on x-ray crystallography data collected by P. M. Colman and W. R. Tulip. From G.J. V. H. Nossal, 1993, Sci. Am. 269(3):22.]
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Protein antigens usually contain both sequential and nonsequential B-cell epitopes. (a) Diagram of sperm whale myoglobin showing locations of five sequential B-cell epitopes (blue). (b) Ribbon diagram of hen egg-white lysozyme showing residues that compose one nonsequential (conformational) epi-tope. Residues that contact antibody light chains, heavy chains, or both are shown in red, blue, and white, respectively. These residues are widely spaced in the amino acid sequence but are brought into proximity by folding of the protein. [Part (a) adapted from M. Z. Atassi and A. L. Kazim. 1978, Adv. Exp. Med. Biol. 98:9; part (b) from W. G. Laver et al., 1990, Cell 61:554.]
native protein conformation for their topographical structure. One well-characterized nonsequential epitope in hen egg-white lysozyme (HEL) is shown in Figure 3-6b. Although the amino acid residues that compose this epitope of HEL are far apart in the primary amino acid sequence, they are brought together by the tertiary folding of the protein.
Sequential and nonsequential epitopes generally behave differently when a protein is denatured, fragmented, or reduced. For example, appropriate fragmentation of sperm whale myoglobin can yield five fragments, each retaining one sequential epitope, as demonstrated by the observation that antibody can bind to each fragment. On the other hand, fragmentation of a protein or reduction of its disulfide bonds often destroys nonsequential epitopes. For example, HEL has four intrachain disulfide bonds, which determine the final protein conformation (Figure 3-7a). Many antibodies to HEL recognize several epitopes, and each of eight different epitopes have been recognized by a distinct antibody. Most of these epitopes are conformational determinants dependent on the overall structure of the protein. If the intrachain disul-fide bonds of HEL are reduced with mercaptoethanol, the nonsequential epitopes are lost; for this reason, antibody to native HEL does not bind to reduced HEL.
The inhibition experiment shown in Figure 3-7 nicely demonstrates this point. An antibody to a conformational determinant, in this example a peptide loop present in native HEL, was able to bind the epitope only if the disulfide bond that maintains the structure of the loop was intact. Information about the structural requirements of the antibody combining site was obtained by examining the ability of structural relatives of the natural antigen to bind to that antibody. If a structural relative has the critical epitopes present in the natural antigen, it will bind to the antibody combining site, thereby blocking its occupation by the natural antigen. In this inhibition assay, the ability of the closed loop to inhibit binding showed that the closed loop was sufficiently
(a) Hen egg-white lysosome
(b) Synthetic loop peptides 80
(b) Synthetic loop peptides 80
Experimental demonstration that binding of antibody to conformational determinants in hen egg-white lysozyme (HEL) depends on maintenance of the tertiary structure of the epitopes by intrachain disulfide bonds. (a) Diagram of HEL primary structure, in which circles represent amino acid residues. The loop (blue circles) formed by the disulfide bond between the cysteine residues at positions 64 and 80 constitutes one of the conformational determinants in HEL. (b) Synthetic open-loop and closed-loop peptides corresponding to the HEL loop epitope. (c) Inhibition of binding between HEL loop epitope and anti-loop antiserum. Anti-loop antiserum was first incubated with the natural loop sequence, the synthetic closed-loop peptide, or the synthetic open-loop peptide; the ability of the antiserum to bind the natural loop sequence then was measured. The absence of any inhibition by the open-loop peptide indicates that it does not bind to the anti-loop antiserum. [Adapted from D. Benjamin et al., 1984, Annu. Rev. Immunol. 2:67.]
(c) Inhibition of reaction between HEL loop and anti-loop antiserum
l Natural loop Closed synthetic loop Open synthetic loop
8 16 Ratio of loop inhibitor to anti-loop antiserum similar to HEL to be recognized by antibody to native HEL. Even though the open loop had the same sequence of amino acids as the closed loop, it lacked the epitopes recognized by the antibody and therefore was unable to block binding of HEL.
B-cell epitopes tend to be located in flexible regions of an immunogen and display site mobility. John A. Tainer and his colleagues analyzed the epitopes on a number of protein antigens (myohemerytherin, insulin, cytochrome c, myoglo-bin, and hemoglobin) by comparing the positions of the known B-cell epitopes with the mobility of the same residues. Their analysis revealed that the major antigenic determinants in these proteins generally were located in the most mobile regions. These investigators proposed that site mobility of epitopes maximizes complementarity with the antibody's binding site, permitting an antibody to bind with an epitope that it might bind ineffectively if it were rigid. However, because of the loss of entropy due to binding to a flexible site, the binding of antibody to a flexible epitope is generally of lower affinity than the binding of antibody to a rigid epitope.
Complex proteins contain multiple overlapping B-cell epitopes, some of which are immunodominant. For many years, it was dogma in immunology that each globular protein had a small number of epitopes, each confined to a highly accessible region and determined by the overall conformation of the protein. However, it has been shown more recently that most of the surface of a globular protein is potentially antigenic. This has been demonstrated by comparing the antigen-binding profiles of different monoclonal antibodies to various globular proteins. For example, when 64 different monoclonal antibodies to BSA were compared for their ability to bind to a panel of 10 different mammalian albumins, 25 different overlapping antigen-binding profiles emerged, suggesting that these 64 different antibodies recognized a minimum of 25 different epitopes on BSA. Similar findings have emerged for other globular proteins, such as myoglobin and HEL.
The surface of a protein, then, presents a large number of potential antigenic sites. The subset of antigenic sites on a given protein that is recognized by the immune system of an animal is much smaller than the potential antigenic repertoire, and it varies from species to species and even among in dividual members of a given species. Within an animal, certain epitopes of an antigen are recognized as immunogenic, but others are not. Furthermore, some epitopes, called immunodominant, induce a more pronounced immune response than other epitopes in a particular animal. It is highly likely that the intrinsic topographical properties of the epi-tope as well as the animal's regulatory mechanisms influence the immunodominance of epitopes.
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