Fusarium solani major allergen peptide IV-1 binds IgE but does not release histamine

BACKGROUND: Fusarium solani (FS) is an important allergen source afflicting 4% of the nasobronchial allergy patients. Fus s I3596*, a 65 kDa major glycoprotein allergen of FS reacts with 95% fungus sensitive patients. OBJECTIVES: To purify and characterize a potent peptide from Fus s I3596* which may be useful for therapeutic purposes. METHODS: The 65 kDa protein was sequentially cleaved with trypsin and cyanogen bromide (CNBr). The cleaved products were purified on reverse phase high performance liquid chromatography (rpHPLC) column and functionally characterized by in vitro and in vivo methods for its IgE binding and histamine release. RESULTS: The protein on cleavage showed 11 peaks (I to XI). Of these, peaks I, III, IV and V were highly allergenic as determined by IgE ELISA. These peaks were further purified and peptide IV-1 was most potent in comparison to other peptides by ELISA-inhibition. This peptide showed IgE binding but could not evoke intradermal response in Fusarium-sensitive patients. Heparinized blood challenged with peptide IV-1 does not release histamine. Preincubation of heparinized blood with peptide IV-1 and challenging with crude extract blocked histamine release in a dose dependent manner. CONCLUSION: Peptide IV-1 binds to IgE but does not release histamine, demonstrating its potential use in therapy of Fusarium-allergic patients.     

Construction of the PG-deficient mutant of Fusarium equiseti by CRISPR/Cas9 and its pathogenicity of pitaya

Fusarium is an important plant pathogen and many cell wall-degrading enzymes (CWDEs) are produced in Fusarium-infected plant tissues. To investigate the role of CWDEs in the pathogenicity of pitaya pathogen, we isolated a Fusarium equiseti strain from the diseased pitaya fruit and the activities of CWDEs were determined. The higher polygalacturonase (PG) activity was confirmed both in vitro and vivo. Aiming at the PG gene, the CRISPR/Cas9 system of F. equiseti was constructed and optimized for the first time. Through the process of microhomology-mediated end joining, the flanking region containing 30 bp was used to mediate the homologous recombination of Cas9 double-strand breaks, and the PG gene knockout mutants were obtained by protoplast transformation. Through the phenotypic and pathogenicity experiments of the wild-type strain and mutant strain, the results showed that the colony growth rate and spore production of the strain without the PG gene decreased to some extent, and the lesion diameter and the degree of pericarp cell damage decreased, which showed that the CRISPR/Cas9 system could be used in F. equiseti and PG enzyme and can play a significant role in the interaction between F. equiseti and pitaya fruit.     

Isolation and characterization of the 18-kDa major apple allergen and comparison with the major birch pollen allergen (Bet v I)

The major allergen from birch pollen, Bet v I, and the cross-reacting 18-kDa major allergen from Golden Delicious and Granny Smith apples were isolated by micropreparative SDS-PAGE followed by electroelution. In the case of apples, highly active, low-temperature extracts were used. The purity of the allergens was checked by analytic SDS-PAGE and immunoblotting with allergic patients’ sera, as well as by N-terminal amino acid microsequencing, and the allergens were found to be very pure. The strong immunologic activity of the isolates was determined by the enzyme allergosorbent test (EAST) and EAST inhibition assays; this activity was, in the case of Bet v I, similar to that of a preparation obtained by monoclonal antibody affinity chromatography. The allergenic potency of Bet v I and of the cross-reactive apple allergen was determined by EAST inhibition and dose-related histamine release. With both assay systems, the allergenic reactivity of Bet v I was considerably higher than that of the major apple allergen. Fürthermore, skin prick tests with the purified allergens and with whole allergenic extracts were performed on a group of 33 patients suffering from birch-pollen and apple hypersensitivity, and on a control group of 10 patients. The frequency of positive prick test results in the allergic patient group ranged from 73% for the major allergen from Golden Delicious apples to 97% with Bet v I and whole birch pollen extract, respectively. In contrast to our low-temperature extracts, commercial prick test solutions of four different manufacturers were found to be unreliable for the diagnosis of apple allergy. The skin test results again indicated the strong immunologic activity of the allergen isolates and the predominance of the major allergens in context with birch-pollen and apple hypersensitivity. Taken together, the results support the view that the 18-kDa major allergen represents most of the allergenicity of the the apple fruit, and that all allergenic epitopes of the apple proteins are present on Bet v I.     

Heterogeneity of grass pollen allergens (Dactylis glomerata) recognized by IgE antibodies in human patients sera by a new nitrocellulose immunoprint technique

A new nitrocellulose immunoprint technique has been developed to detect specific antigens or/and allergens present among a heterogeneous solution such as a water-soluble crude extract of a grass pollen (Dactylis glomerata). The antigens are separated by isoelectric focusing (IEF) in an agarose gel and characterized by their isoelectric point (pI). These antigens are transferred and immobilized on a nitrocellulose sheet. They are recognized by the binding of specific antibodies contained in an unfractionated serum to be studied. Finally, the binding of these antibodies is visualized by species- or/and class-specific antibodies themselves labeled by an enzyme or by radioactivity. So one can detect the allergens recognized by the specific serum IgE antibodies and also the other antigens recognized by specific IgG, IgA or IgM antibodies.     

Isolation and partial characterization of a major peanut allergen

We isolated and partially characterized a major peanut allergen (Peanut-I) using the radioallergosorbent test (RAST) to monitor the allergenicity of various fractions. Raw peanuts were pulverized, defatted, and extracted; the resulting crude extract (CPE) was fractionated by diethylaminoethyl (DEAE) cellulose anion-exchange chromatography using a linear salt gradient. Peak allergenic fractions were further purified by a second DEAE cellulose chromatography step, followed by a preparative polyacrylamide gel electrophoresis (PAGE) step. The final product, Peanut-I, contained 11% nitrogen and 8.7% carbohydrate and was homogeneous by both analytical PAGE and immunoelectrophoresis against rabbit anti-CPE. Peanut-I was heterogeneous by thin-layer electrofocusing and by PAGE in 1% sodium dodecyl sulfate. Biologic activity of Peanut-I was demonstrated by positive skin tests and leukocyte histamine release assays in patients with peanut allergy. By RAST inhibition assay Peanut-I did not account for all of the allergenic activity of CPE.     

Purification and immunobiochemical characterization of folding variants of the recombinant major wasp allergen Ves v 5 (antigen 5)

Background: Antigen 5 is one of three major allergens in wasp venoms, but unlike phospholipase A(1) and hyaluronidase, both of which are enzymes, its biological function is unknown. The cDNA coding for this allergen has been isolated and used for recombinant expression. Thorough analysis of the expression product is essential in order to evaluate the usefulness for in vivo or in vitro application. Objective: In this study, folding variants of the recombinant major allergen Ves v 5 from Vespula vulgaris were immunologically and biochemically investigated in order to determine their possible applicability for diagnostic or therapeutic purposes. Method: The cDNA encoding Ves v 5 was cloned into the expression vector pSE420 which generates recombinant products lacking a tag sequence. After expression, inclusion bodies were purified, subsequently denatured and dialyzed against different solutions. The structural properties of soluble proteins were analyzed by size exclusion chromatography, non-reducing SDS-PAGE, native PAGE, N-terminal sequencing, proteolytic digestion and ion exchange chromatography. Immunological investigations were performed by using different monoclonal antibodies (mAbs) specific for Ves v 5 and IgE from patients allergic to wasp venom allergens. Results: After dialysis, soluble monomeric recombinant Ves v 5 was more than 95% pure in each case. Using different dialysis solutions, clearly distinguishable folding variants were obtained. In one case, the recombinant allergen was comparable with the natural counterpart in respect of migration in non-reducing SDS-PAGE, native PAGE and IgE reactivity. This variant reacted with two different Ves v 5-specific mAbs and produced a stable fragment after proteolytic digestion. Elution from a cation exchange chromatography column was achieved with 320 mM NaCl. In two other cases, folding variants exhibited a different migration behavior in SDS-PAGE and native PAGE compared with the natural allergen. Also, the mAb 1E11 recognized none of these variants since it presumably detected a conformational epitope. Moreover, the IgE reactivity was clearly reduced and proteolytic digestion effected almost complete degradation. These variants eluted from the cation exchange column with 400 mM NaCl. Conclusion: Defined folding strategies resulted in both soluble misfolded variants with reduced IgE reactivity, potentially suitable for immunotherapy, and natural-like folded variants for diagnosis.     

Isolation and partial characterization of allergens from Helianthus annuus (sunflower) pollen

We have purified four allergens from Helianthus annuus (sunflower) pollen, hereafter named as allergens a, b, c , and d. Under native conditions, allergen a has a mol. mass of 32, allergen b has one of 24, and allergens c and d each have one of 55 kDa. At the least, allergens b , c, and d demonstrate charge heterogeneity, and the electrophoretic mobility of allergens c and d increases when these allergens are deglycosylated with trifluoromethanesulfonic acid. Cross-reactivity among the four allergens and with the whole extract is very high, and each allergen recognizes IgE in a high proportion of patients sensitized to sunflower pollen.     

Identification of the soybean hull allergens involved in sensitization to soybean dust in a rural population from Argentina and N-terminal sequence of a major 50 KD allergen

BACKGROUND: Sensitization to soybean hull (SH) allergens occurs in subjects from Argentina, a soybean producer country. However, the causative allergens have not been identified. The purposes of this study are to: (i) identify the SH allergens using sera of 29 subjects with asthma and/or allergic rhinitis from Argentina exposed to soybean dust who have a positive (weal with SH/weal with histamine > or = 0.5) skin prick test to SH; and (ii) determine the N-terminal amino acid sequence of a major 50 K SH allergen that sensitizes this population. METHODS: All sera were assayed for specific IgE (RIA), IgG4 (ELISA), and IgE and IgG4-Western blots. A sera pool from 10 healthy subjects was a negative control. N-terminal amino acid sequencing was performed by the Edman degradation method. RESULTS: Positive specific IgE only was found in 12/29 (41.4%), IgG4 in 3/29 (10.3%), and both IgE and IgG4 in 14/29 (48.3%) sera. IgE-Western blot demonstrates: (i) an allergen, MW 50 K (51.7% binding); (ii) one or two distinct allergens, MW < 20.2 K (72.4% binding), depending on the sera; and (iii) 1-5 additional IgE binding proteins, MW > 20.2 to < 46.9 K (41.4% binding), depending on the sera. IgG4-Western blot demonstrates: (i) a band, MW 70K (31% binding); (ii) a band, MW 50 K (17.2% binding); (iii) one or two additional bands, MW < 20.2 K (51.7% binding), depending on the sera; and (iv) a band, MW > 20.2 to < 28.5 K (20.7% binding). The 50 K allergen N-terminal amino acid sequence of the first 17 amino acids indicates a significant homology with chlorophyll A-B binding protein precursors from tomato, spinach, and petunia. CONCLUSIONS: Specific IgE and IgG4 to SH are common in sera from allergic individuals living in rural areas in Argentina. SH contain an IgE binding protein, MW about 50 K, not previously described. Sensitization to this allergen is common in subjects who are repeatedly exposed to soybean dust inhalation.     

Isolation and characterization of a major allergen from oriental mustard seeds, BrajI.

A 2 S albumin from oriental mustard (Brassica juncea) seeds has been isolated and characterized as an allergen. This protein, BrajI, was recognized by human IgE from mustard-sensitive individuals, as proved by using two different enzyme immunoassays. BrajI was found to be closely related to Sin a I, the major allergen from yellow mustard seeds. Many fractions with molecular weights ranging from 16,000 to 16,400 and with differences in charge were separated by ion-exchange chromatography. They exhibited small but significant amino acid composition differences for Glx, Val, Ile, Lys, and Arg contents. The heterogeneity of BrajI can be explained by size and charge differences of its heavy and light chains. All of the isoallergenic forms of BrajI gave a single precipitation band in double diffusion immunoassays when using a Sin a-I-specific rabbit polyclonal serum.     

Purification and characterization of a cross-reactive 45-kD major allergen of Fusarium solani

BACKGROUND: Fusarium solani (FS) is an important source of fungal allergen. A 45-kD major allergen of FS showed reactivity with patients' sera sensitive to many fungi. OBJECTIVES: To purify and characterize a 45-kD common allergenic protein from FS, which may be useful for the diagnosis of and therapy for fungal allergy. METHODS: FS culture filtrate extract was seperated on SDS-PAGE; 45-kD protein was electroeluted and purified on C18 column using reverse-phase high-pressure liquid chromatography (rpHPLC). The purified protein was functionally and biochemically characterized by in vitro and in vivo methods. RESULTS: The 45-kD protein showed a single peak on rpHPLC. The N-terminal amino acid sequence of this protein did not show homology to enolase or known fungal proteins. It showed cross-reactivity with Epicoccum nigrum, Curvularia lunata, Cladosporium herbarum and Alternaria alternata by ELISA and ELISA inhibition using rabbit antibodies raised against these fungi. IgE ELISA inhibition with patients' sera positive to different fungi demonstrated allergenic cross-reactivity of the 45-kD protein with other fungal extracts. This 45-kD protein released a significant amount of histamine in FS-allergenic patients. CONCLUSION: A cross-reactive 45-kD allergenic/antigenic protein was purified to homogeneity and characterized. It has prospects for use in allergen therapy.     

Isolation and immunochemical characterization of the major allergen of birch pollen (Betula verrucosa)

The classification of some of the extractable birch pollen antigens as allergens was established by crossed radioimmunoelectrophoresis (CRIE). In CRIE the major allergen (antigen 23) exhibited the strongest “radiostaining” and only a few other components of birch pollen extract were visibly radiostained. The major allergen and a preparation containing mainly the minor allergens, antigens 25 and 19, were isolated from a crude aqueous birch pollen extract by a combination of anion-exchange, size-exclusion, and chelate chromatography. Antigen 23 was purified to near homogeneity. The molecular weights and the pIs of antigens 23, 25, and 19 were determined to be 17,000 daltons, pI 5.25 (5.5, 5.0); 25,000 daltons, pI 5.0 (4.9, 5.4); and 29,000 daltons, pI 6.2 (5.4), respectively. The classification of antigen 23 as the major allergen in birch pollen was supported by results of RAST inhibition experiments, RAST screening, and skin prick testing.     

IgE-mediated allergy to corn: a 50 kDa protein, belonging to the Reduced Soluble Proteins, is a major allergen

BACKGROUND: Although corn is often cited as an allergenic food, very few studies have been devoted to the identification of corn allergens and corn allergy has been rarely confirmed by double-blind, placebo-controlled food challenge (DBPCFC). Recently, Pastorello et al. (1) identified some salt-soluble IgE-binding proteins of corn flour as potential allergens. One of these, corresponding to corn Lipid Transfer Protein (LTP), appeared to be the major one. The aim of this study was to verify the clinical significance of the skin prick test (SPT) and CAP-FEIA CAP-System IgE fluozoenzyme immunosorbent assay (Pharmacia Diagnostic, Uppsala, Sweden) positivities to corn and to identify the presence of IgE-binding proteins in the corn flour salt-insoluble protein fractions (comprising up to 96% of the total protein) using sera of patients with DBPCFC-documented food allergy to corn. In addition the effect of cooking and proteolytic digestion on the corn allergens was investigated. METHODS: Sixteen subjects with SPT and CAP-FEIA positivities to corn flour were examined. Only six of them complained of suffering from urticaria and/or other symptoms after ingestion of corn-based foods. The patients were food challenged with cooked corn flour (polenta). IgE-binding proteins were detected by immunoblotting. The digestibility of the IgE-binding proteins was examined during a pepsin attack followed by a pancreatin digestion performed on a cooked corn flour sample. RESULTS: Oral challenge was positive only for six patients with symptoms after ingestion of corn. A 50 kDa protein, belonging to the corn Reduced Soluble Protein (RSP) fraction was recognized by the serum IgE of all the DBPCFC-positive subjects and resulted to be resistant to both heating and peptic/pancreatic digestion. SPT with the purified RSP fraction gave positive results for all of the DBPCFC-positive patients examined. CONCLUSIONS: SPT and CAP-FEIA positivities to corn flour had no clinical significance for most of the patients and food allergy to corn has to be proved by DBPCFC. A salt-unextractable protein of 50 kDa, belonging to the RSP fraction, represents a potential allergen in food hypersensitivity to corn because of its stability to cooking and digestion.     

Production, purification, and characterization of a novel galactose oxidase from Fusarium acuminatum

Extra-cellular production of a novel galactose oxidase from Fusarium acuminatum using submerged fermentation was studied. Glucose (1.0% w/v) was used as the sole carbon source. Maximum galactose oxidase production (approximately 4.0 U/ml) was obtained when fermentation was carried out at 25 degrees C, with orbital shaking (100 rpm) and an initial medium of pH 7.0, for 96 h, using a 2% (v/v) inoculum made from a homogenized four-day-old liquid culture, in the presence of copper, manganese, and magnesium. The enzyme was purified by one-step affinity chromatography, with a recovery of 42% of the initial activity. The purified enzyme ran as a single band of 66 kDa in SDS-PAGE. Optimal pH and temperature for the enzyme activity were 8.0 and 30 degrees C, respectively. The enzyme was thermoinactivated at temperatures above 60 degrees C. The purified enzyme was active toward various substrates, including galactose, dihydroxyacetone, guar gum, lactose, melibiose, methyl-galactopyranoside, and raffinose. SDS was an inhibitor but EDTA, Tween 80, NH(4)(+), Na(+), Mg(2+), K(+), and glycerol were not. The Michaelis-Menten constant (K(m)) for galactose was estimated to be 16.2 mM, while maximal velocity (V(max)) was 0.27 micromol of H(2)O(2) . ml(-1) . min(-1).     

Optimization and characterization of freeze-dried multilamellar liposomes incorporating different standardized allergen extracts

Desensitization therapy for type I allergy is now current practice. Liposomes have been proposed as a support for allergens to improve safety and effectiveness. The aim of this work was to optimize liposomal formulations of three different standardized allergen extracts and to test their allergenicity in vitro and in a preclinical trial. Allergen extracts ( Dactylis glomerata, Dermatophagoides pteronyssinus , and cat hair and dander) were associated with multilamellar liposomes of varying compositions at different pH. Liposome-bound allergens were quantified by RAST inhibition after ultracentrifugation, and analyzed qualitatively by SDS-PAGE followed by immunoblotting. Their allergenicity was assessed by basophil degranulation in vitro , as compared with the allergenicity of an aqueous extract, and by skin tests in allergic subjects. The best association, about 50% of the added allergen, was obtained with negatively charged liposomes, when the pH of the allergen solution was adjusted so as to impart a net positive charge to the proteins. One-third of the liposome-associated allergens was located on the surface of the liposomes and was free to interact with antibody, as shown by RAST inhibition assays and the basophil degranulation test; the remaining two-thirds was encapsulated within the liposomes. AH the major immunoreactive proteins in the extract were included. These liposomes could be readily freeze-dried and reconstituted without changing their properties. This study reveals the allergenic characteristics of liposomes and suggests their potential use in the treatment of allergic patients.     

Identification and characterization of the major allergen of the Humulus japonicus pollen

BACKGROUND: Pollen of Humulus japonicus has been known as one of the important causes of pollinosis in Korea and China. To date, the major allergen of H. japonicus has not been determined. OBJECTIVE: To identify the major allergen of H. japonicus pollen and characterize its biochemical properties. METHODS: With the sera of 29 patients reactive to H. japonicus, the major allergen of H. japonicus was determined from the results of IgE immunoblotting and ELISA inhibition. The biochemical properties of the major allergen of H. japonicus were evaluated by lectin blotting assay and 2-dimensional PAGE blot. N-terminal amino acid sequences were determined by the Edman degradation method. The suggested major allergen was purified by DEAE anion exchange and gel filtration chromatography. RESULTS: Twenty-nine sera contained IgE bound to the 10, 16, 20, 29 and 42 kDa proteins of H. japonicus in immunoblot analysis. A protein of 10 kDa was the most prevalent allergen in the sera of H. japonicus-reactive patients (72%). The ELISA optical density of H. japonicus-specific IgE was not inhibited by pollen extracts of birch, oak, rye grass and mugwort. The 10-kDa allergen was neither stained with PAS nor bound with ConA and five other lectins. The isoelectric point of the 10-kDa allergen was approximately pH 5.1. We sequenced the N-terminal amino acids of the 10-kDa allergen, which was not homologous with any previously characterized allergen. The 10-kDa allergen could be purified with DEAE anion exchange and gel filtration chromatography. Maximum inhibitions of H. japonicus-specific IgE ELISA by whole extract of H. japonicus and purified 10-kDa allergen were more than 97 and 88%, respectively, while the 50% inhibitory concentration of the whole extract of H. japonicus and purified 10 kDa were 38 and 20 ng/mL, respectively. CONCLUSION: The 10-kDa peptide could be a major allergen of H. japonicus. Its isoelectric point was 5.1 and it did not bind with lectins. The N-terminal amino acid sequence of the 10-kDa major allergen was also determined.     

Peanut allergen Ara h 3: isolation from peanuts and biochemical characterization

BACKGROUND: Peanut allergen Ara h 3 has been the subject of investigation for the last few years. The reported data strongly depend on recombinant Ara h 3, since a purification protocol for Ara h 3 from peanuts was not available. METHODS: Peanut allergen Ara h 3 (glycinin), was purified and its posttranslational processing was investigated. Its allergenic properties were determined by studying IgE binding characteristics of the purified protein. RESULTS: Ara h 3 consists of a series of polypeptides ranging from approximately 14 to 45 kDa that can be classified as acidic and basic subunits, similar to the subunit organization of soy glycinin. N-terminal sequences of the individual polypeptides were determined, and using the cDNA deduced amino-acid sequence, the organization into subunits was explained by revealing posttranslational processing of the different polypeptides. IgE-binding properties of Ara h 3 were investigated using direct elisa and Western blotting with sera from peanut-allergic individuals. The basic subunits, and to a lesser extent the acidic subunits, bind IgE and may act as allergenic peptides. CONCLUSIONS: We conclude that peanut-derived Ara h 3, in contrast to earlier reported recombinant Ara h 3, resembles, to a large extent, the molecular organization typical for proteins from the glycinin family. Furthermore, posttranslational processing of Ara h 3 affects the IgE-binding properties and is therefore an essential subject of study for research on the allergenicity of Ara h 3.     

The allergen profile of ash (Fraxinus excelsior) pollen: cross-reactivity with allergens from various plant species

BACKGROUND: Ash, a wind-pollinated tree belonging to the family Oleaceae, is distributed world-wide and has been suggested as a potent allergen source in spring time. OBJECTIVE: The aim of this study was to determine the profile of allergen components in ash pollen in order to refine diagnosis and therapy for patients with sensitivity to ash pollen METHODS: The IgE reactivity profile of 40 ash pollen-allergic patients was determined by immunoblotting. Antibodies raised to purified pollen allergens from tree and grass pollens were used to identify cross-reactive structures in ash pollen extract. IgE immunoblot inhibition studies were performed with recombinant and natural pollen allergens to characterize ash pollen allergens and to determine the degree of cross-reactivity between pollen allergens from ash, olive, birch, grasses and weeds. RESULTS: The allergen profile of ash pollen comprises Fra e 1, a major allergen related to the major olive allergen, Ole e 1, and to group 11 grass pollen allergens, the panallergen profilin, a two EF-hand calcium-binding protein, a pectinesterase-like molecule and an allergen sharing epitopes with group 4 grass pollen allergens. Thus, the relevant allergens of ash are primarily allergens that share epitopes with pollen allergens from other tree, grass and weed species. CONCLUSIONS: Allergic symptoms to ash pollen can be the consequence of sensitization to cross-reactive allergens from other sources. The fact that ash pollen-allergic patients can be discriminated on the basis of their specific IgE reactivity profile to highly or moderately cross-reactive allergens has implications for the selection of appropriate forms of treatment.     

Isolation and Partial Characterization of Three Allergens of Timothy Pollen

Three allergens, antigen Nos. 3, 25, and 30, were isolated from freeze-dried aqueous extract of timothy pollen by various combinations of anionic and cationic exchange and gel chromatography. The allergens were all of protein nature and contained less than 2% (w/w) immunochem;cally detectable impurities. Molecular weights and pI's were determined to less than 10 X 10(3), 15 X 10(3) and 3.9, 4.5, 9.4, respectively, and antigen 25 was determined as the major component of the earlier isolated antigen B. amino acid analyses performed an antigens 3 and 30 revealed large variation s in the amino acid composition. The allergenic activities were verified by means of RAST inhibition and pick tests and proved antigen 25 to be quantitatively the most important allergen of timothy pollen.