Properties of BCell Epitopes Are Determined by the Nature of the Antigen Binding Site

Several generalizations have emerged from studies in which the molecular features of the epitope recognized by B cells have been established.

The ability to function as a B-cell epitope is determined by the nature of the antigen-binding site on the antibody molecules displayed by B cells. Antibody binds to an epitope by weak noncovalent interactions, which operate only over short distances. For a strong bond, the antibody's binding site and the epitope must have complementary shapes that place the interacting groups near each other. This requirement poses some restriction on the properties of the epitope. The size of the epitope recognized by a B cell can be no larger than the size of the antibody's binding site. For any given antigen-antibody reaction, the shape of the epitope that can be recognized by the antibody is determined by the shape assumed by

Comparison of antigen recognition by T cells and B cells

Characteristic

B cells

T cells

Interaction with antigen

Binding of soluble antigen Involvement of MHC molecules Chemical nature of antigens

Epitope properties

Involves binary complex of membrane Ig and Ag

None required

Protein, polysaccharide, lipid

Accessible, hydrophilic, mobile peptides containing sequential or nonsequential amino acids

Involves ternary complex of T-cell receptor, Ag, and MHC molecule

Required to display processed antigen

Mostly proteins, but some lipids and glycolipids presented on MHC-like molecules

Internal linear peptides produced by processing of antigen and bound to MHC molecules the sequences of amino acids in the binding site and the chemical environment that they produce.

Smaller ligands such as carbohydrates, small oligonucleotides, peptides, and haptens often bind within a deep pocket of an antibody. For example, angiotensin II, a small octapeptide hormone, binds within a deep and narrow groove (725 A2) of a monoclonal antibody specific for the hormone (Figure 3-2). Within this groove, the bound pep-tide hormone folds into a compact structure with two turns, which brings its amino (N-terminal) and carboxyl (C-termi-nal) termini close together. All eight amino acid residues of the octapeptide are in van der Waals contact with 14 residues of the antibody's groove.

A quite different picture of epitope structure emerges from x-ray crystallographic analyses of monoclonal antibodies bound to globular protein antigens such as hen egg-white lysozyme (HEL) and neuraminidase (an envelope glycoprotein of influenza virus). These antibodies make contact with the antigen across a large flat face (Figure 3-3). The interacting face between antibody and epitope is a flat or undulating surface in which protrusions on the epitope or antibody are matched by corresponding depressions on the antibody or epitope. These studies have revealed that 15-22 amino acids on the surface of the antigen make contact with a similar number of residues in the antibody's binding site; the surface area of this large complementary interface is between 650 A2 and 900 A2. For these globular protein antigens, then, the shape of the epitope is entirely determined by the tertiary conformation of the native protein.

Thus, globular protein antigens and small peptide antigens interact with antibody in different ways (Figure 3-4). Typically, larger areas of protein antigens are engaged by the antibody binding site. In contrast, a small peptide such as an-giotensin II can fold into a compact structure that occupies less space and fits into a pocket or cleft of the binding site. This pattern is not unique to small peptides; it extends to the binding of low-molecular-weight antigens of various chemical types. However, these differences between the binding of small and large antigenic determinants do not reflect fundamental differences in the regions of the antibody molecule that make up the binding site. Despite differences in the binding patterns of small haptens and large antigens, Chapter 4 will show that all antibody binding sites are assembled from the same regions of the antibody molecule—namely, parts of the variable regions of its polypeptide chains.

Octapeptide Angiotensin Fab

FIGURE 3-2

Three-dimensional structure of an octapeptide hormone (angiotensin II) complexed with a monoclonal antibody Fab fragment, the antigen-binding unit of the antibody molecule. The angiotensin II peptide is shown in red, the heavy chain in blue, and the light chain in purple. [From K. C. Garcia et al, 1992, Science 257:502.]

FIGURE 3-2

Three-dimensional structure of an octapeptide hormone (angiotensin II) complexed with a monoclonal antibody Fab fragment, the antigen-binding unit of the antibody molecule. The angiotensin II peptide is shown in red, the heavy chain in blue, and the light chain in purple. [From K. C. Garcia et al, 1992, Science 257:502.]

Peptide Modeling Angiotensin Binding

FIGURE 3-3

(a) Model of interaction between hen egg-white lysozyme (HEL) and Fab fragment of anti-HEL antibody based on x-ray diffraction analysis. HEL is shown in green, the Fab heavy chain in blue, and the Fab light chain in yellow. A glutamine residue of lysozyme (red) fits into a pocket in the Fab fragment. (b) Representation of HEL and the Fab fragment when pulled apart showing complementary surface features. (c) View of the interacting surfaces of the Fab fragment and HEL obtained by rotating each of the molecules. The contacting residues are numbered and shown in red with the protruding glutamine (#14) in HEL now shown in white. [From A. G. Amit et al, 1986, Science 233:747.]

FIGURE 3-3

(a) Model of interaction between hen egg-white lysozyme (HEL) and Fab fragment of anti-HEL antibody based on x-ray diffraction analysis. HEL is shown in green, the Fab heavy chain in blue, and the Fab light chain in yellow. A glutamine residue of lysozyme (red) fits into a pocket in the Fab fragment. (b) Representation of HEL and the Fab fragment when pulled apart showing complementary surface features. (c) View of the interacting surfaces of the Fab fragment and HEL obtained by rotating each of the molecules. The contacting residues are numbered and shown in red with the protruding glutamine (#14) in HEL now shown in white. [From A. G. Amit et al, 1986, Science 233:747.]

The B-cell epitopes on native proteins generally are composed of hydrophilic amino acids on the protein surface that are topographically accessible to membrane-bound or free antibody. A B-cell epitope must be accessible in order to be able to bind to an antibody; in general, protruding regions on the surface of the protein are the most likely to be recognized as epitopes, and these regions are usually composed of predominantly hydrophilic amino acids. Amino acid sequences that are hidden within the interior of a protein often consist of predominantly hydrophobic amino acids, and cannot function as B-cell epitopes unless the protein is first denatured. In the crystallized antigen-antibody complexes analyzed to date, the interface between antibody and antigen shows numerous complementary protrusions and depressions (Figure 3-5). Between 15 and 22 amino acids on the antigen contact the antibody by 75-120 hydrogen bonds as well as by ionic and hydrophobic interactions.

B-cell epitopes can contain sequential or nonsequential amino acids. Epitopes may be composed of sequential contiguous residues along the polypeptide chain or nonsequential residues from segments of the chain brought together by the folded conformation of an antigen. Most antibodies elicited by globular proteins bind to the protein only when it is in its native conformation. Because denaturation of such antigens usually changes the structure of their epitopes, antibodies to the native protein do not bind to the denatured protein.

Five distinct sequential epitopes, each containing six to eight contiguous amino acids, have been found in sperm whale myoglobin. Each of these epitopes is on the surface of the molecule at bends between the a-helical regions (Figure 3-6a). Sperm whale myoglobin also contains several nonsequential epitopes, or conformational determinants. The residues that constitute these epitopes are far apart in the primary amino acid sequence but close together in the tertiary structure of the molecule. Such epitopes depend on the

FIGURE 3-4

Models of the variable domains of six Fab fragments with their antigen-binding regions shown in purple. The top three antibodies are specific for lysozyme, a large globular protein. The lower three antibodies are specific for smaller molecules or very small segments of macromolecules: McPC603 for phosphocholine; BV04 for a small segment of a single-stranded DNA molecule; and 17/9 for a peptide from hemagglutinin, an envelope protein of influenza virus. In general, the binding sites for small molecules are deep pockets, whereas binding sites for large proteins are flatter, more undulating surfaces. [From I. A. Wilson and R. L. Stanfield, 1993, Curr. Opin. Struc. Biol. 3:113.]

FIGURE 3-4

Models of the variable domains of six Fab fragments with their antigen-binding regions shown in purple. The top three antibodies are specific for lysozyme, a large globular protein. The lower three antibodies are specific for smaller molecules or very small segments of macromolecules: McPC603 for phosphocholine; BV04 for a small segment of a single-stranded DNA molecule; and 17/9 for a peptide from hemagglutinin, an envelope protein of influenza virus. In general, the binding sites for small molecules are deep pockets, whereas binding sites for large proteins are flatter, more undulating surfaces. [From I. A. Wilson and R. L. Stanfield, 1993, Curr. Opin. Struc. Biol. 3:113.]

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