Biochemical characterization and surfactant properties of horse allergens.

A new allergen from horse dander, Equ c 5 has been purified. Its biochemical and biophysical properties have been characterized and compared with those of Equ c 1, Equ c 2 and Equ c 4. Their molecular masses, determined by mass spectrometry, were 22 kDa for Equ c 1, 16 kDa for Equ c 2, 18.7 kDa for Equ c 4 and 16.7 kDa for Equ c 5. Their pI values were between 3.8 and 5.25. Equ c 2 and Equ c 5 are not glycosylated, while Equ c 4 contains a tri-antennary tri-sialylated N-linked glycan. Linkages of terminal N-acetylneuraminic acid to galactose were: alpha-(2-->6) in Equ c 4, and both alpha-(2-->3) and alpha-(2-->6) in Equ c 1. Oligosaccharide portions of Equ c 1 or Equ c 4 were barely involved in IgE-immunoreactivity. Partial N-terminal sequence of Equ c 4 shares a significant sequence homology with the rat submandibular gland protein A. No matching was found for two internal peptides of Equ c 5. Surfactant properties of horse allergens as well as other proteins were investigated. In contrast to Equ c 2 and Equ c 3, solutions of Equ c 1, Equ c 4 and Equ c 5 significantly lowered the surface tension. Relationship between a property such as this, involving oriented hydrophobic patches of a molecule and allergenicity, is addressed.

Crystal structure of the allergen Equ c 1. A dimeric lipocalin with restricted IgE-reactive epitopes.

The three-dimensional structure of the major horse allergen Equ c 1 has been determined at 2.3 A resolution by x-ray crystallography. Equ c 1 displays the typical fold of lipocalins, a beta-barrel flanked by a C-terminal alpha-helix. The space between the two beta-sheets of the barrel defines an internal cavity that could serve, as in other lipocalins, for the binding and transport of small hydrophobic ligands. Equ c 1 crystallizes in a novel dimeric form, which is distinct from that observed in other lipocalin dimers and corresponds to the functional form of the allergen. Binding studies of point mutants of the allergen with specific monoclonal antibodies raised in mouse and IgE serum from horse allergic patients allowed to identify putative B cell antigenic determinants. In addition, total inhibition of IgE serum recognition by a single specific monoclonal antibody revealed the restricted nature of the IgE binding target on the molecular surface of Equ c 1.

Thiophilic adsorption chromatography: purification of Equ c2 and Equ c3, two horse allergens from horse sweat.

Purification of two allergens from horse (Equus caballus) sweat, Equ c2 and Equ c3, by means of salt-promoted chromatography on a "thiophilic" (T-gel) adsorbent is described. Immobilization of these proteins was found to be dependent on the presence of water-structure-forming salts where the ammonium sulphate concentration in the equilibration buffer was 2 M. Equ c2 showed higher affinity towards the thiophilic matrix than Equ c3. Their molecular mass (Mr) values established by SDS-polyacrylamide gel electrophoresis were for Equ c2 approximately 17,000 and for Equ c3 approximately 16,000, and both proteins showed a low isoelectric point of approximately 3.8. Their allergenic properties were also investigated using sera from horse-sensitized patients, where it was demonstrated that these proteins exhibited an IgE antibody binding capacity. In this report we show the broad potential applications of thiophilic adsorption chromatography for the efficient purification of allergens.

Simultaneous quantitation of specific IgE against 20 purified allergens in allergic patients sera by checkerboard immunoblotting (CBIB).

A simple technique, checkerboard immunoblotting (CBIB), is described for simultaneous quantitation of specific IgE antibodies against several allergens in human sera. Using as little as 50 microliters of each of the 20 sera examined against 20 different allergens, it was possible in a single run to achieve 400 tests. To guarantee high specificity and sensitivity of the assay, this new application of CBIB employs purified allergens, cyanogen bromide-activated nitrocellulose membrane, and Phosphorimager technology. Results are expressed both qualitatively (five classes) and quantitatively in kilo units per liter equilibrated against the World Health Organization (WHO) standard for IgE. There was excellent agreement between the results of CBIB and the results of Pharmacia Cap System, an alternative method widely used for measuring serum-specific IgE. The CBIB method certainly could be useful in any laboratory interested in allergy clinical research for easy screening and relative quantitation of allergen-specific human IgE.

cDNA cloning and sequencing reveal the major horse allergen Equ c1 to be a glycoprotein member of the lipocalin superfamily.

The gene encoding the major horse allergen, designated Equus caballus allergen 1 (Equ c1), was cloned from total cDNA of sublingual salivary glands by reverse transcription-polymerase chain reaction using synthetic degenerate oligonucleotides deduced from N-terminal and internal peptide sequences of the glycosylated hair dandruff protein. A recombinant form of the protein, with a polyhistidine tail, was expressed in Escherichia coli and purified by immobilized metal affinity chromatography. The recombinant protein is able to induce a passive cutaneous anaphylaxis reaction in rat, and it behaves similarly to the native Equ c1 in several immunological tests with allergic patients' IgE antibodies, mouse monoclonal antibodies, or rabbit polyclonal IgG antibodies. Amino acid sequence identity of 49-51% with rodent urinary proteins from mice and rats suggests that Equ c1 is a new member of the lipocalin superfamily of hydrophobic ligand-binding proteins that includes several other major allergens. An RNA blot analysis demonstrates the expression of mRNA Equ c1 in liver and in sublingual and submaxillary salivary glands.

Cross-antigenicity of horse serum albumin with dog and cat albumins: study of three short peptides with significant inhibitory activity towards specific human IgE and IgG antibodies.

Horse serum albumin is present in the near vicinity of the animal, while dog and cat serum albumins are very common allergens present in house dust. Human patients clinically defined as allergic to horse could react with horse serum albumin by means of IgE or IgG antibodies. Studies regarding the specificities of these antibodies by inhibition enzyme-linked immunosorbent assay (ELISA) and depletion experiments have demonstrated that they are directed against dog serum albumin and cross-react not only with horse serum albumin but with other serum albumins from different origins. To investigate these observations further, we isolated and characterized three tryptic peptides (P1, P2 and P3) from horse serum albumin. The peptide P1 contains loops 1 and 2 of the first domain, P2 is derived from loop 4 of the second domain, and P3 contains the disulphide loop 9 of the third domain. These were able to inhibit the binding of the patients' IgE and IgG antibodies to horse albumin as well as to dog and cat serum albumins. This indicates that these peptides are involved in the observed cross-reactions. They also shared common epitopes, as revealed by human IgE antibodies. After reduction and alkylation, they totally lost their inhibitory capacity, suggesting that the intra-chain disulphide bridges, essential for the preservation of the loop structure, probably maintain their allergenic/antigenic reactivity.

Hydrophobic interaction chromatography for isolation and purification of Equ.cl, the horse major allergen.

Equ.cl, the horse (Equus caballus) major allergen, was identified in a partially purified extract obtained from a crude aqueous horse dander extract, by acetonic precipitation and a salting-out process. It was isolated and purified by size-exclusion chromatography followed by hydrophobic interaction chromatography. Equ.cl appeared as an almost pure protein in a fraction eluted at 1.2 M ammonium sulphate from a phenyl Superose column. It is a single peptide with a relative molecular mass of 20,000 and a pI of ca. 3.9.

H2 genotype and IgE immune response to Fel dI, the cat major allergen, in mice.

Mice of 6 strains were immunized with a highly purified Fel dI allergen adsorbed to alum. Their ability to display a significant IgE response was detected via passive cutaneous anaphylaxis (PCA) tests, performed in rats. Linkage of the responsiveness to the H2 genotype is not totally obvious. IgE response can be delayed, particularly in the SJL strain (H2-s histocompatibility allele), and lacking in the C57 B1/6 strain (H2-b). Mice with H2-k, H2-d alleles and the B6D2F1 hybrid H2-b/d are good responders, leading to the hypothesis that the IgE response to Fel dI may be related to the H2-d allele. For all good responders, individual variations are rather important. All our observations show that mice perfectly mimic human hypersensitization to the cat major allergen Fel dI, at least where the IgE response is concerned.

Isolation of Der pI, the Dermatophagoides pteronyssinus major mite allergen, from a crude mite culture extract, purification by ion-chromatography, and comparison between the material obtained and a cDNA-coded Der pI.

A high degree of purity is a prerequisite for an allergen preparation to be suitable for clinical diagnosis and therapy. A pure allergen can easily be obtained from a crude mite culture extract by using an immunosorbent prepared with highly specific monoclonal antibodies or from a cDNA-coded material. However, up to now none of these methods has been performed on a process scale. Here large-scale purification is defined as a process in which a crude Dermatophagoides pteronyssinus mite culture extract is essentially fractionated by acetone and ammonium sulphate precipitations followed by anion-exchange high-performance liquid chromatography. A high yield of a very pure Der pI allergen is obtained during the first isocratic run, as shown by sodium dodecylsulphate-polyacrylamide gel electrophoresis, capillary electrophoresis, chromatofocusing and a two site monoclonal antibody enzyme-linked immunosorbent assay. Microsequencing revealed that the 25-residue sequence obtained is entirely in agreement with the sequence derived from the cDNA of Der pI.

Isolation and purification of cat albumin from cat serum by copper ion affinity chromatography: further analysis of its primary structure.

Proteins, regardless of their origin, have to be highly purified, particularly from the immunochemical point of view, if they are to be used to study their allergenicity. It is shown that cat albumin, a highly potent allergen for cat-sensitive humans, can be isolated and purified from cat serum using immobilized metal ion affinity chromatography (copper ions) instead of a salting-out process or precipitation with alcohol, techniques generally used for the preparation of serum proteins. During the process described, immunoglobulins are concomitantly isolated in a relatively pure form. Cat albumin amino acid composition and sequence were analysed after an ultimate purification by ion-exchange chromatography. The highest homology (greater than 80%) was found with the rat serum albumin.

Relationship between specific circulating IgE, basophil cell-bound IgE and histamine release induced by purified allergens of Dermatophagoides farinae.

In this study we analysed the presence and proportions of IgE specific for Df 6 and Df 11, two purified allergens of Dermatophagoides farinae. The characterisation of serum IgE and cell-bound IgE for 3 D. farinae-sensitive patients were performed by CRIE, RAST and PRIST assays. Furthermore the basophils from these same patients were studied by histamine release assays in the presence of Df 6 and Df 11. All the individual patient's cell and serum IgE samples displayed the presence of IgE antibodies specific for Df 6 and Df 11, but the relative quantities of the IgE for these two specificities were characteristic of each patient. The ratios (Specific IgE for Df 11) : (Specific IgE for Df 6) (ratio 11 : 6) were similar in the serum and on the cells for an individual patient. As judged by histamine release assays, the basophil sensitivities towards Df 6 and Df 11 were very different from one atopic patient to another. Moreover cell sensitivities reflected the proportion of Df 6- and Df 11-specific IgE antibodies found in the serum and in the cell eluate.

Comparison of antigenic and allergenic composition of two partially purified extracts from Dermatophagoïdes farinae and Dermatophagoïdes pteronyssinus mite cultures.

Dermatophagoïdes farinae (Df 80d) and Dermatophagoïdes pteronyssinus (Dp 80d) extracts were analyzed for their antigenic and allergenic composition by means of crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE). By CIE, 11 antigens could be numbered in Df 80d (Df 1 to Df 11) and seven antigens in Dp 80d (Dp 1 to Dp 7). This technique allowed us also to define antigens with common as well as specific parts for the two mite species. Among the antigens of D. farinae and D. pteronyssinus, only the antigen corresponding to Df 5 and Dp 5 seems to bear common epitopes to the two mite species, whereas Df 6 and Dp 4 appear to bear, respectively, specific epitopes of each species. Moreover, Df 11 appears to bear specific epitopes of D. farinae, although it shows a partial identity with Dp 7. By CRIE, on 20 mite-sensitive patients' sera, we identified, for each mite extract, the allergens responsive to human specific IgE. The allergograms show that the majority of mite-sensitive patients react with Df 11 and Df 6 and with Dp 7 and Dp 4. Thus, these antigens can be considered as major allergens. The minor allergens were also identified. None of these antigens was recognized by the control sera. Moreover, we observed that for one antigen (antigen 5) there exist antigenic determinants common to the two species of mites toward the rabbit serum and specific allergenic determinants to the human IgE response. A significant correlation was found between the specific IgE binding in CRIE and in RAST (Spearman coefficient: "rs" = 0.61 p less than 0.01 for Df; "rs" = 0.78 p less than 0.01 for Dp).

Antigens and allergens in Dermatophagoïdes farinae mite. I. Immunochemical and physicochemical study of two allergenic fractions from a partially-purified Dermatophagoïdes farinae mite extract.

The fractionation of a partially-purified extract of Dermatophagoïdes farinae mite culture has been undertaken by gel filtration. Two fractions were isolated. One, P25, is a protein-rich fraction with mol. wt about 25,000. The other, GP8, is a polysaccharide-rich fraction with mol. wt around 8,000. By crossed immunoelectrophoresis, we detected eleven antigens in the partially-purified dialysed D. farinae extract (Df 80d) as well as in P25 fraction. One of them, ag 11, seems the most important allergen since in crossed-radio immunoelectrophoresis experiments it displays the faster radiostaining, implying that it binds the greatest part of the mite-specific IgE present in a pool of sera from mite-sensitive patients. By crossed-line immunoelectrophoresis, we demonstrated the absence of ag 11 in GP8, in which only ag 5 and ag 6 were identifiable. By radioalloergosorbent tests (RAST), it was found that P25- and GP8- coated paper discs can fix specific IgE induced in the majority of D. farinae sensitive patients. Defining a 'major allergen' as an allergen to which the majority of sensitive patients develop specific IgE, both P25 and GP8 do appear to contain at least one major allergen. By RAST inhibition method, using Df 80d as a solid phase, the allergenic activity of P25 appeared as slightly higher than that of Df 80d, whereas GP8 displayed a very weak inhibitor capacity. Thus, the allergic specificity of GP8 differs from that of Df 80d or P25.