Where is the antigenic determinant located

T cells do not recognize polysaccharide or nucleic acid antigens. This is why polysaccharides are generally T-independent antigens and proteins are generally T-dependent antigens. The determinants need not be located on the exposed surface of the antigen in its original form, since recognition of the determinant by T cells requires that the antigen be first processed by antigen presenting cells.

Free peptides flowing through the body are not recognized by T cells, but the peptides associate with molecules coded for by the major histocompatibility complex MHC. This combination of MHC molecules and peptide is recognized by T cells. Antigen-Binding Site of an Antibody : Antigen-binding sites can recognize different epitopes on an antigen. In order for an antigen-presenting cell APC to present an antigen to a naive T cell, it must first be processed so itacan be recognized by the T cell receptor.

This occurs within an APC that phagocytizes an antigen and then digests it through fragmentation proteolysis of the antigen protein, association of the fragments with MHC molecules, and expression of the peptide-MHC molecules at the cell surface. There, they are recognized by the T cell receptor on a T cell during antigen presentation.

MHC molecules must move between the cell membrane and cytoplasm in order for antigen processing to occur properly. However, the pathway leading to the association of protein fragments with MHC molecules differs between class I and class II MHC, which are presented to cytotoxic or helper T cells respectively. There are two different pathways for antigen processing:. Some viral pathogens have developed ways to evade antigen processing.

For example, cytomegalovirus and HIV-infected cells sometimes disrupt MHC movement through the cytoplasm, which may prevent them from binding to antigens or from moving back to the cell membrane after binding with an antigen. The immune system will try to destroy or neutralize any antigen that is recognized as a foreign and potentially harmful invader. Antibodies tend to discriminate between the specific molecular structures presented on the surface of the antigen.

Antigens are usually either proteins, peptides, or polysaccharides. This includes parts coats, capsules, cell walls, flagella, fimbrae, and toxins of bacteria, viruses, and other microorganisms. Lipids and nucleic acids are antigenic only when combined with proteins and polysaccharides.

For example, the combination of lipids and polysaccharides are lipopolysaccharides LPS , which are the primary component of gram negative bacterial endotoxin. LPS forms the cell wall of gram negative bacteria and causes a powerful immune response when bound. Cells present their immunogenic-antigens to the immune system via a major histocompatibility MHC molecule. Depending on the antigen presented and the type of the histocompatibility molecule, several types of immune cells can become activated due to an antigen.

Antigens have several structural components of interaction that may be bound by different classes of antibodies. Each of these distinct structural components is considered to be an epitope, also called an antigenic determinent. Therefore, most antigens have the potential to be bound by several distinct antibodies, each of which is specific to a particular epitope. The antigen binding receptor on an antibody is called a paratope, and is specific to the epitope of the antigen.

Antigens are categorized into broad classes of antigens based on their origin. So many different molecules can function as an antigen in the body, and there is considerable diversity even within these categories. These are the main classes of antigens that are involved in immune system activation. Their diversity is analogous to the immense diversity of the diseases that the immune system works to overcome.

Exogenous antigens are antigens that have entered the body from the outside, for example by inhalation, ingestion, or injection. Exogenous antigens are the most common kinds of antigens, and includes pollen or foods that may cause allergies, as well as the molecular components of bacteria and other pathogens that could cause an infection. Endogenous antigens are that have been generated within previously-normal cells as a result of normal cell metabolism or because of viral or intracellular bacterial infection which both change cells from the inside in order to reproduce.

The fragments are then presented on the surface of the infected cells in the complex with MHC class I molecules. These antigens should, under normal conditions, not be the target of the immune system, but due to mainly genetic and environmental factors, the normal immunological tolerance for such an antigen has been lost.

These antigens result from a tumor-specific mutation during malignant transformation of normal cells into cancer cells. Despite expressing this antigen, many tumors have developed ways to evade antigen recognition and immune system killing. A native antigen is an antigen that is not yet processed by an APC to smaller parts.

T cells cannot bind native antigens, but require that they be digested and processed by APCs, whereas B cells can be activated by native ones without prior processing. Antigens are basic molecules that induce an immune response when detected by immune system cells. Antigens may be either complete or incomplete based on the nuances of their molecule structure.

A hapten is essentially an incomplete antigen. These small molecules can elicit an immune response only when attached to a large carrier such as a protein; the carrier typically does not illicit an immune response by itself. Many hapten carriers are normal molecules that circulate through the body.

When haptens and carriers combine, the resulting molecule is called an adduct, the combination of two or more molecules. Haptens cannot independently bind to MHC complexes, so they cannot be presented to T cells.

The first haptens used were aniline and its carboxyl derivatives o-, m-, and p-aminobenzoic acid. One well-known hapten is urushiol, the toxin found in poison ivy and a common cause of cell-mediated contact dermatitis. When absorbed through the skin from a poison ivy plant, urushiol undergoes oxidation in the skin cells to generate the actual hapten, a reactive molecule called a quinone, which then reacts with skin proteins to form hapten adducts.

The size of the combining sites has also been estimated by using simple synthetic oligopeptides of increasing length, such as oligolysine. In this case, a series of elegant studies suggested that the maximum length of chain that a combining site could accommodate was six to eight residues, corresponding closely to that found earlier for oligosaccharides 13 , 14 , discussed previously.

Several types of interactions contribute to the binding energy. Many of the amino acid residues exposed to solvent on the surface of a protein antigen are hydrophilic. These are likely to interact with antibody contact residues through polar interactions. For instance, an anionic glutamic acid carboxyl group may bind to a complementary cationic lysine amino group on the antibody, or vice versa, or a glutamine amide side chain may form a hydrogen bond with the antibody.

However, hydrophobic interactions can also play a major role. Proteins cannot exist in aqueous solution as stable monomers with too many hydrophobic residues on their surface. The hydrophobic residues that are on the surface can contribute to binding to antibody for exactly the same reason.

When a hydrophobic residue in a protein antigenic determinant or, similarly, in a carbohydrate determinant 8 interacts with a corresponding hydrophobic residue in the antibody-combining site, the water molecules previously in contact with each of them are excluded.

The result is a significant stabilization of the interaction. These aspects of the chemistry of antigen-antibody binding were thoroughly reviewed by Getzoff et al. Mapping Epitopes: Conformation Versus Sequence. The other component that defines a protein antigenic determinant, besides the amino acid residues involved, is the way these residues are arrayed in three dimensions.

Because the residues are on the surface of a protein, this component can also be thought of as the topography of the antigenic determinant. Sela 37 divided protein antigenic determinants into two categories, sequential and conformational, depending on whether the primary sequence or the three-dimensional conformation appeared to contribute the most to binding. On the other hand, because the antibody-combining site has a preferred topography in the native antibody, it seems a priori that some conformations of a particular polypeptide sequence would produce a better fit than others and therefore would be energetically favored in binding.

Thus, conformation or topography must always play some role in the structure of an antigenic determinant. Moreover, by looking at the surface of a protein in a space-filling model, it is not possible to ascertain the direction of the backbone or the positions of the helices contrast Figs.

It is hard to recognize whether two residues that are side by side on the surface are adjacent on the polypeptide backbone or whether they come from different parts of the sequence and are brought together by the folding of the molecule. If a protein maintains its native conformation when an antibody binds, then it must similarly be hard for the antibody to discriminate between residues that are covalently connected directly and those connected only through a great deal of intervening polypeptide.

Thus, the probability that an antigenic determinant on a native globular protein consists of only a consecutive sequence of amino acids in the primary structure is likely to be rather small. Even if most of the determinant were a continuous sequence, other nearby residues would probably play a role as well.

Only if the protein were cleaved into fragments before the antibodies were made would there be any reason to favor connected sequences. The a helices are labeled A through H from the amino terminal to the carboxy terminal. Side chains are omitted, except for the two histidine rings F8 and E7 involved with the heme iron. Methionines at positions 55 and are the sites of cleavage by cyanogen bromide CNBr , allowing myoglobin to be cleaved into three fragments.

Most of the helicity and other features of the native conformation are lost when the molecule is cleaved. A less drastic change in conformation is produced by removal of the heme to form apomyoglobin, because the heme interacts with several helices and stabilizes their positions in relation to one another. The other labeled residues 4 Glu, 79 Lys, 83 Glu, Lys, Ala, and Lys are residues that have been found to be involved in antigenic determinants recognized by monoclonal antibodies The "sequential" determinant of Koketsu and Atassi 39 residues 15 to 22 is located at the elbow, lower right, from the end of the A helix to the beginning of the B helix.

Adapted from Dickerson 40 , with permission. This orientation, which corresponds to that in Fig. The heme and aromatic carbons are shaded darkest, followed by carboxyl oxygens, then other oxygen molecules, then primary amino groups, then other nitrogens, and finally side chains of aliphatic residues.

The backbone and the side chains of nonaliphatic residues, except for the functional groups, are shown in white. Note that the direction of the helices is not apparent on the surface, in contrast to the backbone drawing in Fig. The residues Glu 4, Lys 79, and His 12 are believed to be part of a topographic antigenic determinant recognized by a monoclonal antibody to myoglobin This stereo pair can be viewed in three dimensions with an inexpensive stereoviewer such as the "stereoscopes" sold by Abrams Instrument Corp.

Adapted from Berzofsky et al. This concept was analyzed and confirmed quantitatively by Barlow et al. As the radius increases, the probability that all the atoms within the sphere are from the same continuous segment of protein sequence decreases rapidly.

Correspondingly, the fraction of surface atoms that would be located at the center of a sphere containing only residues from the same continuous segment falls dramatically as the radius of the sphere increases. These residues are primarily in regions that protrude from the surface.

Assembled topographic sites of lysozyme illustrated by the footprints of three nonoverlapping monoclonal antibodies. Shown are the a carbon backbones of lysozyme in the center and the Fv portions of three antilysozyme monoclonal antibodies D1. The footprints of the antibodies on lysozyme and lysozyme on the antibodies d that is, their interacting surfaces d are shown by a dotted representation. Note that the three antibodies each contact more than one continuous loop of lysozyme and thus define assembled topographic sites.

From Davies and Padlan 32 , with permission. Antigenic sites consisting of amino acid residues that are widely separated in the primary protein sequence but brought together on the surface of the protein by the way it folds in its native conformation have been called assembled topographic sites 44 , 45 because they are assembled from different parts of the sequence and exist only in the surface topography of the native molecule.

In contrast, the sites that consist of only a single continuous segment of protein sequence have been called segmental antigenic sites 44 , In contrast to T-cell recognition of "processed" fragments retaining only primary and secondary structures, there is overwhelming evidence that most antibodies are made against the native conformation when the native protein is used as immunogen.

For instance, antibodies to native staphylococcal nuclease were found to have about a 5,fold higher affinity for the native protein than for the corresponding polypeptide on which they were isolated by binding to the peptide attached to Sepharose An even more dramatic example is that demonstrated by Crumpton 47 for antibodies to native myoglobin or to apomyoglobin. Antibodies to native ferric myoglobin produced a brown precipitate with myoglobin but did not bind well to apomyoglobin, which, without the heme, has a slightly altered conformation.

On the other hand, antibodies to the apomyoglobin, when mixed with native brown myoglobin, produced a white precipitate. These antibodies so strongly favored the conformation of apomyoglobin, from which the heme was excluded, that they trapped the molecules that vibrated toward that conformation and pulled the equilibrium state over to the apo form.

It could almost be said, figuratively, that the antibodies squeezed the heme out of the myoglobin. Thermodynamically, it is clear that the conformational preference of the antibody for the apo versus native forms, in terms of free energy, had to be greater than the free energy of binding of the heme to myoglobin.

Thus, in general, antibodies that are very specific for the conformation of the protein used as immunogen are made. Synthetic peptides corresponding to segments of the protein antigen sequence can be used to identify the structures bound by antibodies specific for segmental antigenic sites.

To identify assembled topographic sites, more complex approaches have been necessary. The earliest was the use of natural variants of the protein antigen with known amino acid substitutions, in which such evolutionary variants exist Thus, substitution of different amino acids in proteins in the native conformation can be examined.

The use of this method, which is illustrated later, is limited to the study of the function of amino acids that vary among homologous proteins: that is, those that are polymorphic.

Its use may now be extended to other residues by use of site-directed mutagenesis. A second method is to use the antibody that binds to the native protein to protect the antigenic site from modification 48 or proteolytic degradation A related but less sensitive approach makes use of competition with other antibodies A third approach, taking advantage of the capability of producing thousands of peptides on a solid-phase surface for direct binding assays 53 , is to study binding of a monoclonal antibody to every possible combination of six amino acids If the assembled topographic site can be mimicked by a combination of six amino acids not corresponding to any continuous segment of the protein sequence but structurally resembling a part of the surface, then a "mimotope" defining the specificity of that antibody can be produced Myoglobin also serves as a good model protein antigen for studying the range of variation of antigenic determinants from those that are more sequential in nature to those that do not even exist without the native conformation of the protein Fig.

A good example of the first, more segmental type of determinant is that consisting of residues 15 to 22 in the amino-terminal portion of the molecule. Crumpton and Wilkinson 54 first discovered that the chymotryptic cleavage fragment consisting of residues 15 to 29 had antigenic activity for antibodies raised to either native or apomyoglobin.

Two other groups 39 , 55 then found that synthetic peptides corresponding to the shorter sequence 15 to 22 bind antibodies made to native sperm whale myoglobin, even though the synthetic peptides were only seven to eight residues long. Peptides of this length do not spend much time in solution in a conformation corresponding to that of the native protein. On the other hand, these synthetic peptides had a several hundred-fold lower affinity for the antibodies than did the native protein.

Thus, even if most of the determinant was included in the consecutive sequence 15 to 22, the antibodies were still much more specific for the native conformation of this sequence than for the random conformation peptide.

Moreover, there was no evidence to exclude the participation of other residues, nearby on the surface of myoglobin but not in this sequence, in the antigenic determinant 1. A good example of the importance of secondary structure is the case of the loop peptide residues 64 to 80 of hen egg-white lysozyme This loop in the protein sequence is created by the disulfide linkage between cysteine residues 64 and 80 and has been shown to be a major antigenic determinant for antibodies to lysozyme The isolated peptide 60 to 83, containing the loop, binds antibodies with high affinity, but opening of the loop by cleavage of the disulfide bond destroys most of the antigenic activity for antilysozyme antibodies At the other end of the range of conformational requirements are the determinants involving residues far apart in the primary sequences that are brought close together on the surface of the native molecule by its folding in three dimensions.

Myoglobin also provides a good example of these determinants, called assembled topographic determinants 44 , Of six monoclonal antibodies to sperm whale myoglobin studied by Berzofsky et al. These were studied by comparing the relative affinities for a series of native myoglobins from different species with the known amino acid sequences of these myoglobins.

With the myoglobins available, this approach allowed the definition of some of the residues involved in binding to three of these antibodies. The striking result was that two of these three monoclonal antibodies were found to recognize topographic determinants, as defined previously.

The other antibody recognized a determinant involving Glu 83 in the E-F corner and Ala and Lys on the H helix of the myoglobin molecule Fig. Similar examples have been reported for monoclonal antibodies to human myoglobin 62 and to lysozyme 32 , Other examples of such conformation-dependent antigenic determinants have been suggested with the use of conventional antisera to such proteins as insulin 63 , hemoglobin 64 , tobacco mosaic virus protein 65 , and cytochrome c Moreover, the crystallographic structures of lysozyme-antibody 29 , 31 , 32 and neuraminidase-antibody 30 complexes show clearly that, in both cases, the epitope bound is an assembled topographic site.

In the case of the three monoclonal antibodies binding to nonoverlapping sites of lysozyme Fig. This result beautifully illustrates the concept that the majority of antibody combining sites must interact with more than a continuous loop of polypeptide chain and thus must define assembled topographic sites Another important example is represented by neutralizing antibodies to the human immunodeficiency virus HIV envelope protein that similarly bind assembled topographic sites 67 , 68 see also the end of this section.

How frequent are antibodies specific for topographic determinants in comparison with those that bind consecutive sequences when conventional antisera are examined?