Antibodies to steroid receptor deoxyribonucleic acid binding domains and their reactivity with the human glucocorticoid receptor

Functional properties of the DNA-binding domain of the human glucocorticoid receptor were investigated using high titer polyclonal antibodies produced against single synthetic peptides or a mixture of peptides whose sequences were derived from the DNA-binding domain of steroid receptor proteins. Three of seven antisera recognized both native and denatured forms of the glucocorticoid receptor, although considerably lower antisera dilutions were required for antibody binding to native receptor. Activation of the glucocorticoid receptor to its DNA-binding form was required for antibody recognition of the native receptor. Antisera to the second finger region of the DNA-binding domain caused a portion of the activated 4S glucocorticoid receptor to sediment as 7 or 9S in sucrose gradients containing 0.4 M KCl, but did not alter the sedimentation of the nontransformed 8S receptor. Specificity of the glucocorticoid receptor-antibody interaction was demonstrated by loss of reactivity after preabsorption with peptide antigens. Antisera that interacted specifically with the glucocorticoid receptor inhibited DNA binding of the activated receptor by as much as 80%. Thus, antibody probes directed against DNA-binding domain sequences provide immunological evidence that glucocorticoid receptor activation exposes the DNA-binding region of the receptor.     

Monoclonal antibodies to the rat liver glucocorticoid receptor.

Monoclonal antibodies against the 90 000 mol. wt. form of the activated rat liver glucocorticoid receptor were generated from mice immunized with a partially purified receptor preparation. The screening assay was based on the precipitation of liver cytosol, labelled with [3H]triamcinolone acetonide, with monoclonal antibodies bound to immobilized rabbit anti-mouse IgG. Out of 102 hybridomas obtained, 76 produced immunoglobulin and eight of them were found to react with the receptor molecule. Only one of the positive clones secreted IgG whereas the other seven produced IgM. The complexes of receptor and antibodies were identified by sucrose density gradient centrifugation. All seven monoclonal antibodies tested reacted with the 90 000 mol. wt. form of the receptor but not with the 40 000 mol. wt. form that contains the steroid and DNA binding domains. None of the monoclonal antibodies interfered with the binding of the receptor to DNA cellulose, thus suggesting that the antigenic determinants are located in a region of the receptor that is not directly implicated in either steroid binding or DNA binding. These antigenic determinants were common to glucocorticoid receptors from several tissues of the rat, whereas glucocorticoid receptors from other species react only with some of the antibodies.     

Studies with antibodies against the conserved cysteine region of progesterone receptor

Polyclonal antibodies were generated against two synthetic peptides corresponding to sequences from the DNA-binding domain of steroid receptors. The sequence for peptide 1 (13 amino acids) lies between the two putative metal-binding loops of the conserved cysteine region while the sequence for peptide 2 (12 amino acids) lies within one loop. Peptide antibodies were generated by injecting rabbits with peptide conjugated to bovine serum albumin. By Western blot analysis, antibodies to peptide 2 recognized chick and human progesterone receptor and human glucocorticoid receptor, but peptide 1 antibodies did not. No cross-reactivity with native chick progesterone receptor was detected with either anti-peptide. These findings suggest that the epitopes for peptide 2 antibodies, and possibly for peptide 1 antibodies, are inaccessible to antibody in the native receptor.     

The glycine-alanine repeating region is the major epitope of the Epstein-Barr nuclear antigen-1 (EBNA-1)

The Epstein-Barr nuclear antigen-1 (EBNA-1) is a protein containing a large glycine-alanine repeat that has been shown to be antigenic. Antibodies to EBNA-1 can be detected by means of immunoblotting. Preincubation of antisera with purified EBNA-1 protein inhibits the binding of IgG antibodies in this system, indicating that those epitopes detected by immunoblots are also accessible on the native molecule. A number of synthetic peptides the sequences of which were derived from the glycine-alanine repeating region of EBNA-1 and from regions adjacent to it also inhibited antibody binding to EBNA-1. These showed, however, a 1000-fold variation in their inhibitory activities. Peptides containing only glycine and alanine were the most effective inhibitors. The anti-EBNA-1 antibodies did not react with several other peptides representing sequences from unrelated proteins. At saturating concentrations of peptide 85 to 100% of anti-EBNA-1, antibody binding was inhibited in all sera tested with one exception. Similar results are obtained when antibody binding is assayed by an enzyme immunosorbent assay by using partially purified EBNA-1 to coat the plates. Thus the glycine-alanine region, either through its primary structure or through conformations assumed by this region, forms the major epitope(s) of the EBNA-1 molecule.     

Site of binding of IgG2b and IgG2a by mouse macrophage Fc receptors by using cyanogen bromide fragments

Cyanogen bromide fragments of murine IgG2b and IgG2a immunoglobulins were used to localize the sequences that are bound by specific IgG2b and IgG2a Fc receptors on murine macrophages. One fragment from the CH2 domain of IgG2b bound to the gamma 2b Fc receptor. Two fragments from IgG2a--one one from the CH2 domain, differing by only four amino acids from the homologous IgG2b fragment, and the other from the CH3 domain--specifically bound to the gamma 2a Fc receptor. In both a rosetting assay and a radioactive binding assay, these two fragments from IgG2a competed with intact IgG2a: however, they did not compete with each other. Rather, binding of the fragment from the CH3 domain of IgG2a augmented the binding of the fragment from the CH2 domain of IgG2a but not that of the homologous fragment from IgG2b. The binding of both IgG2a fragments was abolished by trypsin treatment of macrophages. These data suggest that 1) a sequence in the CH2 domain of IgG2b is sufficient for binding to the gamma 2b Fc receptor, 2) sequences from both the CH2 and CH3 domains of IgG2a bind to the gamma 2a Fc receptor, and 3) the binding of sequences from the CH3 domain of IgG2a may induce a conformational change in the gamma 2a Fc receptor that leads to enhanced binding of sequences from the CH2 domain.     

Topographic analysis of human epidermal growth factor by monospecific antibodies and synthetic peptides

The mode of interaction between human epidermal growth factor (hEGF) and its receptor has been investigated by immunochemical studies and a synthetic peptide approach. Two types of monoclonal and five different monospecific polyclonal antibodies against hEGF have been prepared, whose epitopes are regions 1-13, 13-32, 33-53, 33-43, 22-32, and discontinuous sequences of hEGF. Antibody against 22-32 (Type I) and antibody against 33-53 (PRE 4) inhibited the binding of 125I-hEGF to membrane receptor on A 431 cells more markedly than the other antibodies. When hEGF was bound to the receptor, only antibody against 13-32 (PRE 2) could bind to hEGF-receptor complex whereas antibody against 22-32 (Type I) could not. These data suggest that region 13-20 is exposed outside during receptor-binding and both region 22-32 and region 33-53 contact the hEGF receptor. The activity of synthetic peptides corresponding to the amino acid residues 1-13, 13-32, 33-53, 13-20, 22-32, and 33-43 of hEGF was also examined. Out of the six peptides, only 13-32 stimulated DNA synthesis of BALB 3T3 cells. The activity was approximately 1/10(6) of that of intact hEGF. All of these data suggest that region 22-32 is responsible for binding to the receptor for signal transduction and region 33-53 binds to the receptor to stabilize the ligand-receptor interaction. This dual binding model fits in well with the three-dimensional hEGF structure deduced from NMR spectra.     

Human epidermal growth factor (EGF) receptor sequence recognized by EGF competitive monoclonal antibodies. Evidence for the localization of the EGF-binding site

Epitopes recognized by three epidermal growth factor (EGF) competitive monoclonal antibodies, LA22, LA58, and LA90, have been localized to a 14-amino acid region in the extracellular domain of the human EGF receptor. The binding of each of these mutually competitive antibodies to A431 epidermoid carcinoma cells was inhibited up to 87% by EGF. Furthermore, binding to A431 cells was inhibited 100% by the EGF competitive monoclonal antibody 528 IgG. The EGF receptor monoclonal antibody 455 IgG, which recognizes a blood group A-related carbohydrate modification of A431 receptors and does not inhibit EGF binding, did not inhibit the binding of these three antibodies to A431 cells. Antibodies LA22, LA58, and LA90 were unusual in that they bound to recognized denatured and endoglycosidase F-treated antigenic determinants in Western blots. This suggested that the antibodies recognized continuous peptide epitopes. The epitopes for these antibodies were first localized in cyanogen bromide- and V8 protease-generated fragments of a truncated form of the EGF receptor secreted by A431 cells. In experiments with synthetic peptides, all three antibodies were found to bind to the 14 amino acids from Ala-351 to Asp-364 of the mature human EGF receptor. These amino acids are located between the two Cys-rich regions of the extracellular domain of the receptor, and they include an Arg-Gly-Asp-Ser recognition site for adhesion molecule receptors. The homologous sequence in the chicken EGF receptor, which binds mouse EGF with a 100-fold lower affinity than the human EGF receptor, contains four amino acid differences including two in the Arg-Gly-Asp-Ser tetramer. The mutually competitive binding of EGF and antibodies LA22, LA58, and LA90 implied that the amino acids between Ala-351 and Asp-364 participated in the formation of the EGF-binding site of the human EGF receptor.     

Monoclonal antibodies to different sites on human transcobalamin II

Two IgG1K monoclonal antibodies to human transcobalamin II (TC II) were generated. These antibodies, 16.1 and 16.6, did not cross-react with the other two types of human cobalamin-binding proteins, intrinsic factor and R binder (TC I). Both antibodies cross-reacted with orangutan and simiang TC II but not with TC II from cynomolgus and howler monkeys, who are less closely related to humans. This finding suggests close structural similarity of human to ape TC II. The antibodies also did not react with TC II of lower mammals which included the horse, dog, guinea pig, and mouse; in particular, reaction did not occur with rabbit TC II, which has been considered structurally close to human TC II. Neither of the two antibodies was directed at the cobalamin-binding site of TC II. However, antibody 16.6 hindered TC II binding to cell receptor. This reactivity with the receptor-binding site should prove particularly useful in studies of that region of the TC II molecule.     

Immunological studies of the toxic site in ammodytoxin A

Two monoclonal antibodies against the native ammodytoxin A and four site-directed polyclonal antibodies against synthetic peptides derived from the primary structure of the toxin were prepared in order to estimate the localization of its toxic site. Some of the antibodies neutralized the lethal toxicity of the toxin, thus indicating an approximate position of the toxic or receptor binding site on the molecule that is different from those predicted by comparison with a number of known sequences.     

Characterization of an antiserum against the glucocorticoid receptor

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [³H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [³H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [³H]TA-GR-antibody complex compared to that of the [³H]TA-GR complex, and (III) an increased molecular size of the [³H]TA-GR-antibody complex when compared to that of the [³H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (Stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptor, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physiocochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.     

Generation of an auto-anti-idiotypic antibody that binds to glucocorticoid receptor

A monoclonal antibody (8G11-C6) that is directed to a region near the ligand-binding site of the glucocorticoid receptor was obtained by an auto-anti-idiotypic route, using a derivative of triamcinolone coupled to thyroglobulin to immunize a mouse. The resulting hybridomas were screened for anti-idiotypic antibody (anti-antisteroid) with Fab fragments of affinity-purified polyclonal rabbit anti-triamcinolone antibody. The anti-idiotypes were then screened for binding to rat cytosol glucocorticoid receptor by a depletion procedure, yielding a clone, 8G11-C6, whose specificity for receptor was verified by sucrose density and Western blot analyses. Depletion was not significantly reduced by prelabeling the cytosol with [3H]triamcinolone acetonide. The anti-idiotype (8G11-C6) bound to Fab fragments of antisteroid and to partially purified receptor in a concentration-dependent manner. Both binding reactions were inhibited only by rabbit serum albumin conjugates of steroids known to bind to the glucocorticoid receptors. Triamcinolone derivatives of lysine and of oligopeptides containing up to six amino acids inhibited the binding of the anti-idiotype to the Fab fragments but not to the receptor, implying that the target epitope of the antisteroid antibody may be closer to its glucocorticoid-binding site than the cross-reacting epitope of the receptor. Our findings demonstrate further the versatility of the auto-anti-idiotypic route for the preparation of anti-receptor antibodies.     

Two MSA 2 peptides that bind to human red blood cells are relevant to Plasmodium falciparum merozoite invasion.

Plasmodium falciparum merozoite membrane surface antigen 2 (MSA2) has been associated with the development of protective immunity against malaria. MSA2 antibodies were able to inhibit in vitro merozoite invasion. In our search for experimental evidence concerning the participation of MSA2 in merozoite invasion, 40 peptides were synthesized according to sequences reported for the CAMP and FC27 prototype Plasmodium strains. These peptides were purified, 125I-radiolabeled and tested for their ability to bind to erythrocytes. Two MSA2 synthetic peptides with high specific binding to human erythrocytes were found. The peptide coded 4044 (KNESKYSNTFINNAYNMSIR), located in the MSA2 N-terminal conserved region, has an affinity coefficient of 72 nM and showed a positive cooperativity for the receptor-ligand interaction. The other peptide, coded 4053 (NPNHKNAETNPKGKGEVQKP) and located in the central variable region of MSA2, has an affinity coefficient of 49nM and also showed a positive cooperativity for the receptor-ligand interaction. The binding capacity of these peptides is affected by erythrocytes treated with neuraminidase and trypsin, but it is not affected by chymotrypsin. Both of these sequences inhibit in vitro erythrocyte parasite invasion by up to 95% suggesting that they have an important role in the parasite's invasion process. Furthermore, as published previously [A. Saul et al. (1992) J. Immunol., 148, 208-211], a protective B epitope is included in the 4044 peptide sequence.     

Identification and Characterization of B-Cell Epitopes in the DBL4ε Domain of VAR2CSA

Malaria during pregnancy in Plasmodium falciparum endemic regions is a major cause of mortality and severe morbidity. VAR2CSA is the parasite ligand responsible for sequestration of Plasmodium falciparum infected erythrocytes to the receptor chondroitin sulfate A (CSA) in the placenta and is the leading candidate for a placental malaria vaccine. Antibodies induced in rats against the recombinant DBL4ε domain of VAR2CSA inhibit the binding of a number of laboratory and field parasite isolates to CSA. In this study, we used a DBL4ε peptide-array to identify epitopes targeted by DBL4ε-specific antibodies that inhibit CSA-binding of infected erythrocytes. We identified three regions of overlapping peptides which were highly antigenic. One peptide region distinguished itself particularly by showing a clear difference in the binding profile of highly parasite blocking IgG compared to the IgG with low capacity to inhibit parasite adhesion to CSA. This region was further characterized and together these results suggest that even though antibodies against the synthetic peptides which cover this region did not recognize native protein, the results using the mutant domain suggest that this linear epitope might be involved in the induction of inhibitory antibodies induced by the recombinant DBL4ε domain.     

Sequences in the promoter region of the chicken lysozyme gene required for steroid regulation and receptor binding

We have constructed a series of deletion mutants in the lysozyme promoter region fused to the SV40 T-antigen coding region. Regulated expression was tested after microinjection of the lysozyme deletion mutants into primary cultures of chicken oviduct cells using fluorescent antibodies against T antigen. Deletion of lysozyme gene sequences upstream of position - 164 was accompanied by loss of both progesterone- and glucocorticoid-induced expression. Using the rat liver glucocorticoid receptor for binding studies, two separate binding sites have been identified: a strong binding site that is destroyed by deletion of lysozyme sequences between positions -74 and -39 and a weaker binding site contained between positions -208 and -161 upstream of the lysozyme cap site.     

Identification of dexamethasone-binding sites on male-rat liver plasma membranes by affinity labelling.

Binding studies with [3H]dexamethasone identified two binding sites on plasma membranes prepared from the male rat liver, a low-capacity site with a KD of 7.0 nM and a higher-capacity site with a KD of 90.1 nM. Both sites exhibited glucocorticoid responsiveness and specificity for glucocorticoids and progestins. Triamcinolone acetonide, which competes well for the binding of dexamethasone to the cytosolic glucocorticoid receptor, did not compete well for the binding of [3H]dexamethasone to the plasma-membrane binding sites. The binding sites were sensitive to protease and neuraminidase treatment, and resistant to extraction with NaCl, but were extracted with the detergent Triton X-100. As these experiments indicated the presence of plasma-membrane protein components which bind glucocorticoids at physiological concentrations, affinity-labelling experiments with dexamethasone mesylate were conducted. Two peptides were specifically labelled, one at approx. Mr 66,000 and one at Mr 45,000. The Mr-66,000 peptide was not sensitive to glucocorticoids, and was extracted by NaCl, and so did not correspond to either of the sites identified in the dexamethasone-binding studies. The Mr-45,000 entity, on the other hand, resembled the dexamethasone-binding sites in its response to glucocorticoid manipulation of the animal and in its resistance to salt extraction. This peptide was not present in rat serum. Thus we have identified a plasma-membrane peptide which binds dexamethasone. Whether this peptide is involved in transport of the glucocorticoid across the plasma membrane remains to be determined.     

The main immunogenic region of the nicotinic acetylcholine receptor: interaction of monoclonal antibodies with synthetic peptides

Monoclonal antibodies to the main immunogenic region of the nicotinic acetylcholine receptor have been studied with regard to their binding to synthetic peptides. It was found that monoclonal antibody 210 to the main immunogenic region binds to the synthetic fragment spanning residues 66 to 76 of the alpha subunits of the acetylcholine receptor from human muscle, but not to the homologous sequence from Xenopus. This parallels the reactivities of antibodies to the main immunogenic region with intact receptors from two species, and confirms the biological significance of the weak interactions observed between antibodies to this region and synthetic peptides. It also suggests that N alpha 68 and D alpha 71 are critical contact residues.