Predicting antigenic sites on proteins.

The ability to predict antigenic sites on proteins is of major importance for the production of synthetic peptide vaccines and synthetic peptide probes of antibody structure. Many predictive methods, based on various assumptions about the nature of the antigenic response have been proposed and tested. This review will discuss the principles underlying the various approaches to predicting antigenic sites and will attempt to answer the question of how well they work.     

Two antigenic sites of tissue transglutaminase.

The immunization of rabbits with purified guinea pig liver transglutaminase resulted in the appearance of two antibody populations against the enzyme: one which reacted only with the Ca2+-enzyme complex and another which reacted with the intact as well as the Ca2+-enzyme. The Ca2+-induced confomrational change of the enzyme molecule exposes a new antigenic determinant which initiates the production of a specific antibody population. When the glutamine substrate of the enzyme was a dipeptide, the result of the interaction of the Ca2+-enzyme and its isolated specific antibody was an apparent activation of the catalytic activity. However, when protein substrates were used, an inhibition was observed. The characterization of the mechanism of the activation and the inhibition has led to the conclusion that the consequence of the interaction of Ca 2+-enzyme and its specific antibody is not only a limited steric hindrance of the active center but, besides that, a stabilization of the otherwise labile Ca2+-enzyme. The other antibody population reacts with both forms of the enzyme and its inhibitory effect, which has been observed in each assay, could be due to a prevention of the Ca2+-induced formation of the active enzyme.     

Lysine conjugate of acrylonitrile as antigenic sites in hemoglobin adducts.

For biomonitoring environmental exposure to acrylonitrile (AN), a monoclonal antibody (mAb) A2D1, was developed to recognize specifically the hemoglobin (Hb) adduct, Hb-AN, but not Hb itself. This appears to be the first example that a small molecule-like AN may introduce new antigenicity into hemoglobin, which already exhibits multiple antigenic determinants. This report addresses the localization of the newly formed antigenic sites in human Hb-AN. As antigenic probes, the AN conjugates of 10 amino acids, six dipeptides, and four tripeptides were prepared as monitored by 1H NMR, and their antigenicity was evaluated by competitive inhibition immunoassay. A Lys-epsilonNH-AN was found essential to inhibiting activity. The potent peptide-AN inhibitors, containing a sequence of His and Lys, showed IC50 at the micromolar concentration, thus implicating human Hbalpha-89,90 and Hbbeta-143,144 in the distal heme pocket region as the new antigenic sites.     

Structurally inherent antigenic sites. Localization of the antigenic sites of the alpha-chain of human haemoglobin in three host species by a comprehensive synthetic approach.

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.     

The conformational stabilities of tropomyosins.

The stability to denaturation by heat and guanidine hydrochloride of seven vertebrate (including skeletal, cardiac and smooth muscle) tropomyosins and three invertebrate tropomyosins was examined. The transition profiles were discontinuous and in many cases distinct plateaux were observed which indicated the presence of unique partially unfolded states at intermediate temperatures and guanidine hydrochloride concentrations. The denaturation by guanidine hydrochloride could be described in the majority of cases by a model in which the native state unfolds to a partially unfolded stable intermediate which then unfolds to the completely denatured state. On this basis it was possible to estimate the free energies of unfolding in water. It was shown that part of the alpha-helical structure of tropomyosin is only marginally stable and the free energy of unfolding in water of this segment is less than values found for globular proteins, whereas another segment (or segments) has a stability comparable to that found for globular proteins. The stepwise unfolding may be explained in terms of the coiled-coil interactions in tropomyosin. Differences in stability were found between tropomyosins from different muscles of the same species as well as between species, no two tropomyosins giving the same denaturation profiles. The invertebrate tropomyosins showed a wider range of stabilities, that from scallop striated muscle being far more easily denatured than all the others. No correlation was found between the stability of tropomyosin and the type of regulatory system of the muscle. A comparison of the results from vertebrate and invertebrate species suggests that there has been no selection for proteins of higher or lower stability during the evolutionary time scale.     

The functional characteristics conserved in tropomyosins.

1. Tropomyosin, one of the regulatory proteins in muscle contraction, was prepared from chickens, rabbits, frogs, shrimps, and shellfish, and conserved characteristics were studied using an enzymological technique. 2. All tropomyosins tested, irrespective of their sources, were found to have the ability to mediate the inhibitory activity of rabbit troponin toward rabbit Mg2+-activated actomyosin ATPase (Mg2+-ATPase) activity in the absence of Ca2+ ions. 3. The effect of tropomyosin on the Mg2+-ATPase activity in the presence of Ca2+ ions varied, depending on the source, and this variation appeared to reflect the evolutionary course of this protein. 4. Tropomyosin from shellfish adductor muscle had the ability to bind to rabbit skeletal muscle troponin and actin. This ability is also considered to be a basic characteristic of tropomyosin which has been conserved during evolution.     

Purification and characterization of cardiac tropomyosins.

A new procedure was developed to purify tropomyosin. The procedure was an adaptation of that described for purification of myosin. By eliminating troponin before precipitating with (NH4)2 SO4, it was possible to obtain pure tropomyosin from the same preparation from which myosin was purified. When tropomyosin was subjected to isoelectrofocusing two tropomyosins were present, having similar isoelectric points of pH 5.4 and 5.6; two tropomyosin subunits were resolved in the presence of 6 M urea. The two subunits had very similar isoelectric points, pH 4.7 and 5.0. According to Ouchterlony analyses the tropomyosins from canine skeletal and cardiac tissue were immunologically identical when incubated with goat gammaG antitropomyosin (cardiac).     

A common antigenic epitope expressed on human Pneumocystis carinii.

Recently antigenic heterogeneity in human Pneumocystis carinii (Pc) isolates was observed in several laboratories. Monoclonal antibodies (MAb) were produced to human Pc (PcH) from a lung autopsy sample from a non-AIDS patient (MAb Group I, n = 10), or from bronchoalveolar lavage (BAL) fluid from AIDS patients (MAb Group II, n = 8). To detect Pc antigen from specimens, indirect immunofluorescence and immunoblotting techniques were used. The reactivity was evaluated by using one autopsy sample from the non-AIDS patient and 14 BAL samples from AIDS patients. The MAb in group I (C5-9, E9) stained a part of PcH from all isolates. On the other hand, several MAb in group II (L20-5, M34-2, M78-3, M79-5, N23-4) stained all PcH from all isolates. Some MAb (C5-9, E9, M34-2, M78-3) stained cysts as well as trophozoites. Immunoblot studies detected a 92 kDa molecule as a common antigen by all of these MAb. Therefore, we have found a common antigenic epitope on PcH and MAb that recognize this epitope may become useful for diagnosis of infection and for biological characterization studies on the organism.     

The existence of two completely distinct antigenic sites within a decapeptide.

A decapeptide (1182-1191) derived from the bovine interphotoreceptor retinoid-binding protein (IRBP) was found to contain two completely distinct antigenic sites when tested in Lewis rats. One site, localized in sequence 1182-1191, is the core of the immunodominant and highly uveitogenic determinant of IRBP. The second site localizes within sequence 1183-1191 and becomes detectable only when tryptophan at position 1182 is deleted. Lymphocytes sensitized against the first, larger site recognized all longer peptides within sequence 1169-1191, as well as whole IRBP. In contrast, lymphocytes sensitized against the second, short epitope recognized only two peptides, 1184-1191 and (to a lesser degree) 1183-1191. The responses to both sites were restricted by the same major histocompatibility complex (MHC) product (I-A), as shown by monoclonal antibody blocking and by the finding that the lymphocyte response to 1184-1191 was competitively inhibited by peptide 1181-1191. The unique finding of two completely distinct antigenic sites within a decapeptide could be explained by the hypothesis that peptides of the two sites combine with the MHC molecule on antigen-presenting cells by different configurations, thus forming two distinct antigenicities.     

Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites

Three monoclonal antibodies that react with previously undefined antigenic determinants on the clathrin molecule have been produced and characterized. They were isolated from a fusion between myeloma cells and popliteal lymphocytes from SJL mice that had received footpad injections of human brain clathrin. This protocol was chosen to favor the production of antibodies to poorly immunogenic proteins and thereby increase the repertoire of anti-clathrin monoclonal antibodies. One antibody (X16) reacts preferentially with the heavier of the two clathrin light chains (LCa) when it is not associated with heavy chain. This specificity is different from that of the anti-LCa antibody, CVC.6, which has preferential reactivity with heavy chain-associated LCa. In addition, X16 and CVC.6 bound simultaneously to LCa, confirming that they react with different sites. The other two antibodies produced, X19 and X22, react with two different determinants on the clathrin heavy chain, based on immunoprecipitation, Western blot, and binding studies. Competitive binding studies with anti-clathrin monoclonal antibodies showed that they define a total of five distinct antigenic determinants on bovine clathrin.     

DAPI-inducible common fragile sites

DAPI, a compound specific for the AT bases of DNA, causes gaps and breaks in three human chromosome sites, at the 1q41-1q42 interface, 2q31, and 7p22. It also induces undercondensation of a chromosome site at the 13q21-13q22 interface. The first three sites have the characteristics of "common fragile sites" and are present as gaps and breaks on the chromosomes of seven individuals.     

Identification of three continuous antigenic sites in horse muscle acylphosphatase

The main antibody-combining sites of horse skeletal muscle acylphosphatase were mapped by preparing and purifying CNBr, tryptic and peptic peptides from the pure enzyme, and looking for the immunoreactivity of each peptide by the dot-immunobinding assay using specific polyclonal antienzyme antibodies previously purified by immunoaffinity chromatography. The immunoreactive peptides were identified on the basis of either their elution times in the fingerprint analysis or amino acid composition, or both, by comparison with the known enzyme amino acid sequence. All the CNBr as well as two tryptic and two peptic peptides were immunopositive, leading to identification of three main continuous antigenic sites on the enzyme molecule. The strong inhibition (92%) of the antigen-antibody reaction carried out in the presence of antibodies previously incubated with the immunoreactive peptide mixture supports the possibility that, at our experimental condition, the three identified antigenic domains contain the main antigenic determinants of the enzyme. The relationship between structure and antigenicity of the immunoreactive peptides is discussed in detail.     

Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus.

Human convalescent serum and bacterial fusion proteins constructed from overlapping open reading frames of the nucleotide sequence encoding the human cytomegalovirus gp55 component of the major envelope glycoprotein complex, gp55-116 (gB), were used to localize antigenic regions recognized by human antibodies. All donor serum analyzed contained antibody reactivity for an antigenic site(s) located between amino acids (AA) 589 and 645, a region containing a previously defined linear site recognized by neutralizing monoclonal antibodies (U. Utz, B. Britt, L. Vugler, and M. Mach, J. Virol. 63:1995-2001, 1989). Furthermore, in-frame insertion of two different synthetic oligonucleotides encoding four amino acids into the sequence at nucleotide 1847 (AA 616) eliminated antibody recognition of the fusion protein. A second antibody binding site was located within the carboxyl terminus of the protein (AA 703 through 906). A competitive binding inhibition assay in which monoclonal antibodies were used to inhibit human antibody reactivity with recombinant gp55-116 (gB) suggested that the majority of human anti-gp55-116 (gB) antibodies were directed against a single antigenic region located between AA 589 and 645. Furthermore, inoculation of mice with fusion proteins containing this antigenic site led to a boostable antibody response. These results indicated that the antigenic site(s) located between AA 589 and 645 was an immunodominant antibody recognition site on gp55 and likely the whole gp55-116 (gB) molecule. The enhanced immunogenicity of this region in vivo may account for its immunodominance.