Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen

Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility to these sequences within the context of the natural allergens. Strong IgE-binding epitopes of Lol p 5 have been identified down to single critical amino acid residues and are shown to occur as linear or continuous domains in the natural conformation of natural Lol p 5 and other group 5 grass pollen allergens. The fact that such an allergenic synthetic epitope has the capacity to strongly inhibit IgE-binding to natural allergens highlight its potential for use as a candidate in future therapeutics to treat pollen-associated allergies.     

Analysis of the IgE-epitope of Der f 2, a major mite allergen, by in vitro mutagenesis

Der f 2 is a major mite allergen composed of 129 amino acid residues. To determine the major epitopes on Der f 2 recognized by human IgE antibodies, artificial mutations were introduced to Der f 2 protein. The IgE-binding activity of Der f 2 was significantly decreased by deletion of 10 amino acids at the N-terminus or nine amino acids at the C-terminus. Site-directed mutagenesis with a single amino acid replacement by Ala or Leu in both N- and C-terminal regions as well as a central portion was performed to generate 42 single-site mutations. Amino acid replacement around a disulfide bond of Cys8-Cys119 caused a marked decrease in IgE-binding activity. Furthermore, a distinct decrease in IgE-binding was also caused by Ala-substitution close to a disulfide bond of Cys73-Cys78 and by mutations of a few charged residues. From these results, it was concluded that the two disulfide-forming regions of Der f 2 and several charged residues are important for forming major epitope structures recognized by human IgE antibodies.     

In vitro viability of cryopreserved equine embryos following different freezing protocols

Aspergillus fumigatus grown in submerged and surface cultures was extracted, and the extracts were analyzed separately. The submerged extract contained 31.9% protein and 8.3% carbohydrate, while the corresponding values were 17.0% and 33.3% for the surface material. With individual sera from patients with allergic asthma, SDS-PAGE combined with immunoblotting revealed that the submerged extract contained at least six strong IgE-binding components (20, 30, 38, 50, 68, and 90 kDa) in addition to several weak to medium IgE-binding components. The surface extract contained about the same number of IgE-binding components, but only one gave a strong reaction (20 kDa). The allergens present were shown to have pI between 4.5 and 5.6 as demonstrated by isoelectric focusing (IEF) combined with immunoblotting. For identification of A. fumigatus glycoprotein allergens, both extracts were treated with periodate under mild conditions. Two allergens of the submerged extract (90 and 38 kDa) partly lost their IgE-binding ability by this treatment, indicating that these components are glycoproteins and that the carbohydrate moiety is involved in the IgE binding. The IgE-binding ability of the 20-kDa allergen was not influenced by periodate. For assessment of the stability of the two allergen extracts, aqueous solutions were kept at 4 degrees C for 2, 7, and 21 d and then analyzed by SDS-PAGE and immunoblotting. The results showed that most allergens of the submerged extract were partly inactivated after 2 d. After 21 d, only the 20-kDa and 30-kDa components were still able to bind IgE. Similar results were obtained by analyzing the surface extract.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of an immunodominant IgE epitope of the Parietaria judaica major allergen

Par j 1.0101 is one of the two major allergens of the Parietaria judaica (Pj) pollen, and its three-dimensional structure was built by three-dimensional structural homology modeling. The resultant model was used to identify putative IgE binding regions. Western blot analysis of gene fragmentation products showed that the 1 to 30 region was capable of binding specific IgE from a pool of sera (n = 30) of patients allergic to Pj pollen. Using the structural model as a guide, deletion and site-directed mutagenesis of the 1 to 30 region was performed, and the amino acids involved in IgE binding were identified. In addition, a synthetic peptide covering the 1 to 30 region was capable of binding human IgE without triggering histamine release from basophils of Pj allergic patients (n = 6) and thus represents a haptenic molecule with potential use as an immunotolerant agent. This epitope is also present on the Par j 2.0101 major allergen representing a common IgE epitope. It is an immunodominant epitope, since it was capable of inhibiting 30% of all specific IgE against the Pj major allergens, and therefore, it might be a candidate for the future development of immunotherapeutics.     

Allergy to bumblebee venom. II. IgE cross-reactivity between bumblebee and honeybee venom

To obtain more information on IgE cross-reactivity between bumblebee venom and honeybee venom, we tested sera from venom-sensitized patients for specific IgE against venoms from the European bumblebee (Bombus terrestris), the North American bumblebee (Megabombus pennsylvanicus), and the honeybee (Apis mellifera). RAST, RAST-inhibition, and immunoblotting experiments indicate that bumblebee venom and honeybee venom contain venom-specific IgE-binding epitopes. These results suggest that immunotherapy using honeybee venom may not be effective in all bumblebee venom-allergic patients. Our experiments also revealed differences in IgE binding for venom from European and American bumblebees.     

Purification and characterization of an allergenic monomeric hemoglobin from a chironomid distributed worldwide, Polypedium nubifer

The Pol n component MV, a potent experimental allergen for mice, was purified to homogeneity from extracts of a chironomid distributed worldwide, Polypedium nubifer (PN). The Pol n I component MV was shown to have cross-reactivity to hemoglobins (Hb) derived from all species of chironomids tested. Determination of the amino acid sequence of the first 37 N-terminal residues revealed that it had 30-59% homology to Hb of an European chironomid, Chironomus thummi thummi, which had been known as an important allergen for humans. By Western blot analysis, we showed that sera from asthmatic patients, which had positively reacted to the extract of the adult PN midge, bound to the purified Pol n I component MV. Furthermore, using rabbit polyclonal antibodies raised against synthetic polypeptides corresponding to the N-terminal residues, it was demonstrated that the N-terminal amino acid sequence between position 15 and 35 contained antigenic epitope(s) for human IgE. The results indicate that the Pol n I component MV is an allergen for human beings as well as for mice, and useful as a diagnostic tool for chironomid allergy.     

Replacement of lysine 45 by uncharged residues modulates the redox-Bohr effect in tetraheme cytochrome c3 of Desulfovibrio vulgaris (Hildenborough)

The structural basis for the pH dependence of the redox potential in the tetrahemic Desulfovibrio vulgaris (Hildenborough) cytochrome c3 was investigated by site-directed mutagenesis of charged residues in the vicinity of heme I. Mutation of lysine 45, located in the neighborhood of the propionates of heme I, by uncharged residues, namely threonine, glutamine and leucine, was performed. The replacement of a conserved charged residue, aspartate 7, present in the N-terminal region and near heme I was also attempted. The analysis of the redox interactions as well as the redox-Bohr behavior of the mutated cytochromes c3 allowed the conclusion that residue 45 has a functional role in the control of the pKa of the propionate groups of heme I and confirms the involvement of this residue in the redox-Bohr effect.     

Determinant analysis of IgE and IgG4 antibodies and T cells specific for bovine alpha(s)1-casein from the same patients allergic to cow's milk: existence of alpha(s)1-casein-specific B cells and T cells characteristic in cow's-milk allergy

In an effort to clarify the etiology of milk allergy from the standpoint of allergen-specific immune reactions, we investigated the determinants of IgE, IgG4, and T cells specific for bovine alpha(s)1-casein from the same individual patients by using its synthetic peptides and cyanogen bromide-digested fragments. Alpha(s)1-casein is a major allergen in cow's milk, and its unique conformation enabled us to investigate the determinants of antibodies without consideration about missing the reactivities because of conformational changes. Nine patients were selected as subjects from among 129 milk-sensitive infants screened by ELISA to assess the anti-alpha(s)1-casein IgE levels in their sera. By using ELISA for epitope mapping, a C-terminal region of alpha(s)1-casein was identified as a common binding site for IgE from all of these patients, whereas those for anti-alpha(s)1-casein IgG4 were located in multiple regions of alpha(s)1-casein. We determined the specificities of seven alpha(s)1-casein-specific T-cell lines established from peripheral blood mononuclear cells of two of the patients. These T cells have been shown to secrete IL-4. All of the T-cell lines had different specificities to alpha(s)1-casein. However, a common amino acid residue use was found among the determinants of various T-cell lines from each patient. The results suggest that patients allergic to cow's milk have characteristic B cells recognizing a limited region of alpha(s)1-casein and secreting alpha(s)1-casein-specific IgE. These B cells may interact particularly with T cells recognizing determinants with a common structure.     

The beta-amyloid epitope masking activity in human brain is identified as albumin

Human brain homogenate proteins were analyzed for binding and processing activity in relation to brain beta-amyloid precursor protein (APP). The homogenate was purified by arginine-Sepharose 4B affinity chromatography, which traps proteins with affinity to certain groups of arginine residue, such as serine proteases and zymogens. A 69 kDa protein that masks epitope(s) of brain APP was found in a weakly bound fraction. The nature of the 69 kDa brain protein was identified as albumin by N-terminal amino acid sequencing and Western blot analysis using anti-human albumin antibody. Western blot analysis with domain-specific anti-APP antibodies revealed that the masking activity is complete for beta-amyloid epitope(s), but incomplete for cytoplasmic and extracellular domain epitopes, suggesting that the interaction site of the albumin is beta-amyloid itself. Therefore, it seems that brain albumin is not merely a carrier protein for beta-amyloid in cerebrospinal fluid, but also a modulator which interferes with processing of beta-amyloid precursor protein and its peptides.     

Allergenic and antigenic activity of peptide fragments in a whey hydrolysate formula

BACKGROUND: Milk hydrolysates, although frequently used as substitutes in cases of cow's milk allergy, show a reduced but never a complete abolishment of antigenicity and allergenicity. OBJECTIVE: Our purpose was to determine the lower molecular weight limit of peptides to elicit skin reactions and to bind IgE antibodies in vitro. METHODS: Using FPLC, an ultrafiltrated whey hydrolysate, was fractionated in different molecular weight fractions. Skin-prick tests were performed with the hydrolysate and its fractions in five cow's milk allergic children, and RAST inhibition tests were done using the serum of these children. RESULTS: On the basis of the lowest extinction values between two peaks of the chromatogram, seven fractions with molecular weights between 15000 and 125 Da were obtained. Peptides of > 2600 Da elicited a clearly positive skin reaction and inhibited IgE-binding, while peptides of < 1400 Da did not give any positive skin reaction but were still able to inhibit to a small extent IgE-binding to the hydrolysate. CONCLUSION: Our findings suggest that for skin reactivity peptides of > 1400 Da are needed. The minimal molecular mass for IgE binding in vitro appears to be situated between 1400 and 970 Da. Such peptides might be used to develop a safe formula for patients reacting to milk hydrolysates or even for tolerance induction.     

The MHC class I-restricted immune response to HIV-gag in BALB/c mice selects a single epitope that does not have a predictable MHC-binding motif and binds to Kd through interactions between a glutamine at P3 and pocket D

Using a strain of Listeria monocytogenes that stably expresses and secretes HIV gag to deliver this Ag to the MHC class I pathway of Ag processing, we have identified the immunodominant CTL epitope to gag in the BALB/c mouse and shown that it is Kd restricted. The specific motif for the peptides that bind the MHC class I molecule H-2 Kd is believed to be a nonamer with residues tyrosine or phenylalanine in the second amino acid position and leucine or isoleucine in the carboxyl-terminal or ninth amino acid position as dominant anchoring positions. Surprisingly, the identified gag peptide, AMQMLKETI, does not contain an anchoring aromatic residue in position two although competition assays with other Kd-restricted epitopes indicated that it binds to Kd with comparable affinity. Using a theoretical molecular dynamics approach to probe the stability of peptide binding to MHC class I molecules, we show that the absence of an appropriate anchor residue at P2 in AMQMLKETI is compensated by favorable interactions of the glutamine at P3 with pocket D of Kd. These findings were verified experimentally, demonstrating the predictive power of this theoretical approach in analyzing MHC class I/peptide interactions. These studies also indicate that CTL epitope prediction that relies on dominant peptide motifs may not always identify the correct epitope.     

A quantitative comparison of machined commercially pure titanium and titanium-aluminum-vanadium implants in rabbit bone

BACKGROUND: Grass group I consists of very potent allergenic components which are found in the pollen of all temperate grasses. Several post-translational modifications are predicted from the cDNA data. OBJECTIVE: The aim of this study was to identify sequential IgE-binding sites on the allergen Phl p 1 and to determine their influence on IgE reactivity. METHODS: Based on cDNA data and microsequencing results we synthesized overlapping decapeptides covering the complete Phl p 1 molecule and tested them for immunological reactivity by means of the PEPSCAN technique. In a dot test we determined the frequency of IgE reactivities to post-translationally modified structures (hydroxylated proline residues, carbohydrate structure, and disulphide formations). RESULTS: Screening by overlapping peptides demonstrated an IgE binding site on the 10 N-terminal amino acids. Comprehensive studies showed that the two hydroxyproline residues of the native Phl p 1 allergen (at positions 5 and 8) and the N-glycan (at position 9) can result in an increased IgE reactivity; 3.3% of the sera exclusively bound to the hydroxyproline bearing peptide, while only 0.4% bound to the proline containing peptide. With regard to glycosylation, we estimated that 20% of sera recognized protein and carbohydrate epitopes, while one serum exclusively bound to the glycan. The formation of disulphide bonds has no detectable effect on the IgE reactivity to Phl p 1. CONCLUSION: Our results indicate that the post-translational modifications, the carbohydrate structure and the hydroxylation of proline residues, can enhance the IgE reactivity of Phl p 1.     

A male-specific nuclease-resistant chromatin fraction in the mealybug Planococcus lilacinus

BACKGROUND: The domestic mites Euroglyphus maynei and Blomia tropicalis frequently co-inhabit homes in subtropical/tropical regions around the world. Both species are the source of substances that cause allergic reactions in patients. OBJECTIVE: The purpose of this study was to examine the immunologic cross-reactivity between E. maynei and B. tropicalis. METHODS: Sera of 19 mite-sensitive patients who were skin test positive to B. tropicalis and/or RAST positive to E. maynei were used to probe immunoblots or crossed radioimmunoelectrophoresis (CRIE) gels. RESULTS: Western blotting showed that individual sera had IgE that bound to 0 to 17 and 2 to 15 proteins in E. maynei and B. tropicalis extracts, respectively. Corresponding IgE-binding proteins of 105, 75, 57, 18, and 14 kD were detected in both E. maynei and B. tropicalis extracts. The majority of IgE-binding proteins did not show corresponding bands in both extracts. Heterologous CRIE showed IgE binding to six of the nine E. maynei antigens precipitated by anti-B. tropicalis serum with individual sera recognizing 0 to 4 of the six allergens. In the reciprocal reaction, 10 of the 12 proteins of B. tropicalis that were precipitated by anti-E. maynei serum bound IgE with individual sera binding to 0 to 5 proteins. CONCLUSION: This study indicated that E. maynei and B. tropicalis are the source of both species-specific and cross-reactive allergens, but most allergens in each extract were species-specific.     

Predicting unbound phenytoin concentrations in the critically ill neurosurgical patient

BACKGROUND: This study was carried out to compare two techniques used to determine the food specific IgE antibody. METHODS: Thirty-four allergic patients were evaluated for most common food IgE antibodies by multiple allergosorbent chemiluminescent assay (MAST-CLA). IgE antibodies to eight of these food allergens were also measured by Pharmacia CAP (CAP) test. RESULTS: The food specific IgE showed good agreement between MAST-CLA and CAP (kappa = 0.3-0.77). The sensitivity of MAST-CLA assay for food specific IgE antibody was variable comparing with that of CAP system. The accuracy ranged from 0.76 to 0.97. The agreement between the results of MAST-CLA and skin test was variable (kappa = 0.03-0.58). The agreement was poor in wheat, peanut and soybean (kappa = 0.03-0.12). Similar result between CAP and skin test was also obtained (kappa = 0.06-0.82). The agreement was poor in wheat (kappa = 0.06) and milk (kappa = 0.15). CONCLUSIONS: Different results might be related to quality of the extract, how they are performed in vitro test and difference of correspondent allergens employed in the tests.     

Tropomyosin implicated in host protective responses to microfilariae in onchocerciasis

A cDNA from adult female Onchocerca volvulus encoding the C-terminal portion of a tropomyosin isoform (termed MOv-14) has been shown previously to confer protective immunity in rodent models of onchocerciasis. The full-length sequence (designated Ov-tmy-1) obtained by PCR amplification, codes for a protein of 33 kDa and shares 91% identity with tropomyosins from other nematodes, falling to 57% identity with human alpha-tropomyosin. Ov-TMY-1 migrates with an apparent molecular mass of 42 kDa on SDS/PAGE and is present in all life-cycle stages, as determined by immunoblotting. Immunogold electron microscopy identified antigenic sites within muscle blocks and the cuticle of microfilariae and infective larvae. Anti-MOv14 antibodies were abundant in mice exhibiting serum-transferable protection against microfilariae conferred by vaccination with a PBS-soluble parasite extract. In contrast, little or no MOv14-specific antibody was present in mice inoculated with live microfilariae, in which resistance is mediated by antibody-independent mechanisms. In human infections, there was an inverse correlation between anti-tropomyosin IgG levels and densities of microfilariae in the skin. Seropositivity varied with the relative endemicity of infection. An immunodominant B cell epitope within Ov-TMY-1 (AQLLAEEADRKYD) was mapped to the N terminus of the MOv14 protein by using sera from protectively vaccinated mice. Intriguingly, the sequence coincides with an IgE-binding epitope within shrimp tropomyosin, believed to be responsible for hypersensitivity in individuals exhibiting allergy to shellfish. IgG and IgE antibodies reacting with the O. volvulus epitope were detected in human infections. It is concluded that antibody responses to tropomyosin may be important in limiting microfilarial densities in a proportion of individuals with onchocerciasis and have the potential to mediate hypersensitivity reactions to dead microfilariae, raising the possibility of a link with the immunopathology of infection.     

Ubiquitous structures responsible for IgE cross-reactivity between tomato fruit and grass pollen allergens

The simultaneous presence of IgE reactivity to tomato fruit and grass pollen allergens is evident in many patients with allergy and may be caused by cross-reactivity. Using sera from polysensitized patients with a positive enzyme allergosorbent test (EAST) result (score > 2), we tested reactivity to both allergen sources. IgE reactivity against both extracts was demonstrated in eight serum samples, and cross-reactivity was confirmed by the EAST inhibition assay. The structures responsible for this cross-reactivity were identified by Western blotting: five of the eight sera demonstrated a 16 kd protein in both extracts, which was identified as profilin. Additionally, seven of the eight sera showed IgE binding to epitopes on carbohydrate moieties, which contained alpha 1, 3 fucosylations. To determine the allergens of tomato fruit extract, we performed two-dimensional polyacrylamide gel electrophoresis blotting. We were able to demonstrate one highly concentrated and about 20 weaker proteins possessing terminal fucose residues. These are similarly found in grass pollen extracts. It is therefore postulated that the cross-reactivity is affected by profilins and similar carbohydrate determinants. If carbohydrate structures can provoke IgE cross-reactivity between phylogenetically distant species, such structures may play an important role in sensitization and mediator release. The ubiquitous nature of the IgE-binding determinants was studied by additional EAST inhibition tests with tomato allergen disks and extract from birch pollen, mugwort pollen, apple, and celery, leading to significant inhibitions among all these allergen sources. Epitopes exclusive to grass pollen and tomato have not been detected.     

Identification of IgE-binding egg white proteins: comparison of results obtained by different methods

The binding of IgE to egg white proteins was investigated for 34 sera from adults with a positive case history and/or positive RAST towards egg, and the impact of experimental conditions on IgE binding in commonly used methods was studied. Radioimmunoblotting after SDS-PAGE of both reduced and unreduced egg white extracts showed complex reaction patterns. The results were confirmed by crossed radioimmunoelectrophoresis (CRIE). Radio dot immunobinding was used to investigate the effect of treatment of allergens for SDS-PAGE and to evaluate the other methods. As a conclusion, the use of combinations of at least two methods is recommended for the identification of IgE-binding egg white proteins. Of the 34 sera, 18 reacted with ovotransferrin, 13 with ovomucoid, 11 with ovalbumin and 5 with lysozyme. The amounts of IgE bound to ovalbumin and lysozyme were generally lower than the amounts bound to ovotransferrin and ovomucoid.     

Expandable metal stents for the treatment of colonic obstruction: techniques and outcomes

Modulation of CD8(+) T-cell responses specific for an exogenous antigen by epitope variants would be advantageous to develop a novel means of antigen-specific immune regulation. We have analyzed CD8(+) T-cell responses to single amino acid-substituted variants of a peptide corresponding to residues 142-149 (p142-149; LAYFYPEL) of alphas1-casein, a major milk allergen, which is a dominant determinant restricted by H-2Kb. An analog peptide L142I with a substitution of Ile for Leu at the nonanchor N-terminal residue induced more IFN-gamma secretion than p142-149 from specific CD8(+) T cells. Furthermore, L142I could prime CD8(+) T cells more efficiently in vivo, and these L142I-primed cells secreted more IFN-gamma than p142-149-primed CD8(+) T cells upon stimulation with p142-149 in vitro. These findings are mainly explained by the greater ability of L142I to form stable Kb-peptide complexes. These findings indicate that appropriate analog peptides may be useful as efficient inducers of CD8(+) T cells which recognize the parent peptide and secrete IFN-gamma, a potent inhibitor of Th2-dependent events, including IgE production.     

IgE from latex-allergic patients binds to cloned and expressed B cell epitopes of prohevein

Prohevein is one of the major allergens associated with latex allergy. In the present study, we identified IgE binding regions of prohevein, and expressed multiple IgE binding epitopes by selective cloning. These truncated polypeptides were then used to demonstrate IgE in the sera of patients. Decapeptides of prohevein were synthesized on derivatized cellulose membrane with an offset of one amino acid. The IgE reactivity of these linear peptides was evaluated separately using pooled sera from latex-allergic health care workers (HCW) and spina bifida (SB) patients. A total of 10 IgE binding epitopes representing unique as well as shared epitopes from both the N- and C-domains of the prohevein were identified. Recombinant polypeptides were constructed based on the identified epitopes, and clones carrying DNA fragments were overexpressed. These recombinant peptides were evaluated for IgE binding with sera from HCW, SB, and normal individuals. Recombinant prohevein, hevein, and the C-domain exhibited IgE binding in 84, 88, and 40% of HCW sera, respectively, as against reactivity of 84% with crude latex allergens. However, only 48% of the sera from SB patients showed IgE binding with recombinant prohevein, while 56 and 28% had reactivity with recombinant N- and C-domains, respectively. Among the three remaining recombinant peptides of the C-domain, only CA44-103 showed IgE binding with SB patients. The results of the present study suggest that linear IgE epitope analysis and construction of recombinant peptides increase the sensitivity and specificity of the immunodiagnosis of latex allergy and provide more information on the immunopathogenesis of hypersensitivity reaction mediated by type I allergy.