Identification of peroxidase-1 and beta-glucosidase as cross-reactive wheat allergens in grass pollen-related wheat allergy.

BACKGROUND: Some patients with wheat-dependent exercise-induced anaphylaxis (WDEIA) or wheat allergy showed negative ω-5 gliadin-specific IgE test and high level of grass pollen-specific IgE. It was presumed that these patients developed allergic reaction upon cross-reaction of their IgE antibodies raised against grass pollen allergens to wheat allergens. This study aimed to clarify clinical characteristics and wheat allergens of this phenotype of WDEIA/wheat allergy, which were tentatively diagnosed as grass pollen-related wheat allergy (GPWA). METHODS: A total of six patients with GPWA were enrolled, and controls were 17 patients with grass pollen allergy but no episode of wheat allergy, and 29 patients with other wheat allergies: 18 with conventional WDEIA and 11 with hydrolyzed wheat protein allergy. Sensitization to wheat proteins was determined by basophil activation test (BAT). IgE-binding proteins in wheat flour were identified by immunoblotting followed by mass spectrometry. Wheat allergen-specific IgE tests were established by CAP-FEIA system. RESULTS: All the six patients with GPWA were sensitized to water-soluble wheat proteins in BAT and IgE-immunoblotting, and peroxidase-1 (35 kDa) and beta-glucosidase (60 kDa) were identified as specific IgE-binding wheat proteins. The binding of patient IgE to these proteins was inhibited by pre-incubation of patient sera with grass pollen. The peroxidase-1- and beta-glucosidase-specific IgE tests identified three and four of six patients with GPWA, respectively, but only two of 29 controls, indicating high specificity of these tests. CONCLUSIONS: Peroxidase-1 and beta-glucosidase are specific wheat allergens for GPWA among grass pollen allergy and other types of wheat-induced food allergies.

ß-1,3-glucanase rOle e 9 and MnSOD rAsp f 6 IgE reactivity are the signature of atopic dermatitis in the Mediterranean area.

BACKGROUND: Atopic dermatitis (AD) represents a chronic skin disorder seriously affecting patients' QoL and is often associated with immunological imbalance, disorders of the skin barrier function and environmental factors. OBJECTIVE: We extensively studied the proteomic IgE sensitization profile in a large AD Mediterranean cohort. METHODS: A total of 588 individuals with moderate-severe (70.6%) or mild and/or history of (29.4%) AD were evaluated in comparison to 1285 unselected atopic controls (AC) with a history of adverse reactions to foods, allergic rhinitis and/or bronchial asthma by means of ImmunoCAP ISAC112 ® and Allergy Explorer-ALEX® microarray analysis. RESULTS: The olive tree pollen ß-1,3-glucanase rOle e 9 and the manganese superoxide dismutase from Aspergillus rAsp f 6 were the molecules most significantly associated with AD occurrence and allowed to discriminate among the moderate and severe forms of disease. An IgE hyper-reactivity to cypress, grasses, olive tree, house dust mites (including rDer p 11), and to all cross-reactive components except profilin and polcalcin was observed. About 60% of adults with severe AD were sensitized to nsLTPs. Cross-reactive carbohydrate determinants (CCDs) IgE was found in about one-third of AD participants. Hen eggs nGal d 1 IgE sensitization was more prevalent in the paediatric population, whilst rAsp f 6 and rOle e 9 reactivity was found particularly in older patients. Despite the status of widespread IgE sensitization to both environmental and food allergens, a reduced frequency of patient-reported severe reactions to food or of asthma was observed in AD patients compared to AC, particularly in case of concomitant Ole e 9 reactivity. CONCLUSION AND CLINICAL RELEVANCE: Testing IgE reactivity to a large panel of molecular components unveils important associations between IgE reactivity profiles and AD clinical presentation, highlights the allergens useful for a precise AD signature and allows the detection of interesting sensitisations patterns.

Exoproteome Analysis of Human Pathogenic Dermatophyte Species and Identification of Immunoreactive Proteins.

PURPOSE: Increasing incidence of onychomycosis and tinea pedis in humans of industrialized countries together with deep tissue infections are a therapeutic challenge in clinical mycology. For a better understanding of the pathology and immunology of infection, the authors analyze the exoproteomes of three reference strains of the most common clinical dermatophyte species (Trichophyton rubrum, Trichophyton interdigitale, Arthroderma benhamiae) and of Trichophyton strains isolated from affected patients. EXPERIMENTAL DESIGN: Extracellular proteins of those in vitro grown strains are separated via 2D High Performance Electrophoresis and identified by mass spectrometry to find proteins with provoked host immune reactivity. RESULTS: More than 80 secreted proteins including virulence factors such as peptidases and other hydrolases are identified. By Western blotting with respective patient sera, up to 31 proteins with significant antigen-antibody reactions are detected in comparison with control sera, for example, peptidases as well as several oxidoreductases. One protein, beta-glucosidase F2SZI9 seems to be a commonly processed antigen in all Trichophyton infections. CONCLUSIONS AND CLINICAL RELEVANCE: These first global exoproteome data of three dermatophyte species can be a stepping stone on the way to further study the molecular mechanisms of Trichophyton pathogenicity-associated traits. Possible candidates for potential new diagnostic methods or vaccination have to be validated in further investigations.

An Enzymatically Active ß-1,3-Glucanase from Ash Pollen with Allergenic Properties: A Particular Member in the Oleaceae Family.

Endo-ß-1,3-glucanases are widespread enzymes with glycosyl hydrolitic activity involved in carbohydrate remodelling during the germination and pollen tube growth. Although members of this protein family with allergenic activity have been reported, their effective contribution to allergy is little known. In this work, we identified Fra e 9 as a novel allergenic ß-1,3-glucanase from ash pollen. We produced the catalytic and carbohydrate-binding domains as two independent recombinant proteins and characterized them from structural, biochemical and immunological point of view in comparison to their counterparts from olive pollen. We showed that despite having significant differences in biochemical activity Fra e 9 and Ole e 9 display similar IgE-binding capacity, suggesting that ß-1,3-glucanases represent an heterogeneous family that could display intrinsic allergenic capacity. Specific cDNA encoding Fra e 9 was cloned and sequenced. The full-length cDNA encoded a polypeptide chain of 461 amino acids containing a signal peptide of 29 residues, leading to a mature protein of 47760.2 Da and a pI of 8.66. An N-terminal catalytic domain and a C-terminal carbohydrate-binding module are the components of this enzyme. Despite the phylogenetic proximity to the olive pollen ß-1,3-glucanase, Ole e 9, there is only a 39% identity between both sequences. The N- and C-terminal domains have been produced as independent recombinant proteins in Escherichia coli and Pichia pastoris, respectively. Although a low or null enzymatic activity has been associated to long ß-1,3-glucanases, the recombinant N-terminal domain has 200-fold higher hydrolytic activity on laminarin than reported for Ole e 9. The C-terminal domain of Fra e 9, a cysteine-rich compact structure, is able to bind laminarin. Both molecules retain comparable IgE-binding capacity when assayed with allergic sera. In summary, the structural and functional comparison between these two closely phylogenetic related enzymes provides novel insights into the complexity of ß-1,3-glucanases, representing a heterogeneous protein family with intrinsic allergenic capacity.

The C-terminal domains of two homologous Oleaceae ß-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR.

Ole e 9 and Fra e 9 are two allergenic ß-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9.

The non-lysosomal ß-glucosidase GBA2 is a non-integral membrane-associated protein at the endoplasmic reticulum (ER) and Golgi.

GBA1 and GBA2 are both ß-glucosidases, which cleave glucosylceramide (GlcCer) to glucose and ceramide. GlcCer is a main precursor for higher order glycosphingolipids but might also serve as intracellular messenger. Mutations in the lysosomal GBA1 underlie Gaucher disease, the most common lysosomal storage disease in humans. Knocking out the non-lysosomal GBA2 in mice results in accumulation of GlcCer outside the lysosomes in various tissues (e.g. testis and liver) and impairs sperm development and liver regeneration. However, the underlying mechanisms are not well understood. To reveal the physiological function of GBA2 and, thereby, of the non-lysosomal GlcCer pool, it is important to characterize the localization of GBA2 and its activity in different tissues. Thus, we generated GBA2-specific antibodies and developed an assay that discriminates between GBA1 and GBA2 without the use of detergent. We show that GBA2 is not, as previously thought, an integral membrane protein but rather a cytosolic protein that tightly associates with cellular membranes. The interaction with the membrane, in particular with phospholipids, is important for its activity. GBA2 is localized at the ER and Golgi, which puts GBA2 in a key position for a lysosome-independent route of GlcCer-dependent signaling. Furthermore, our results suggest that GBA2 might affect the phenotype of Gaucher disease, because GBA2 activity is reduced in Gba1 knock-out fibroblasts and fibroblasts from a Gaucher patient. Our results provide the basis to understand the mechanism for GBA2 function in vivo and might help to unravel the role of GBA2 during pathogenesis of Gaucher disease.

Prunasin hydrolases during fruit development in sweet and bitter almonds.

Amygdalin is a cyanogenic diglucoside and constitutes the bitter component in bitter almond (Prunus dulcis). Amygdalin concentration increases in the course of fruit formation. The monoglucoside prunasin is the precursor of amygdalin. Prunasin may be degraded to hydrogen cyanide, glucose, and benzaldehyde by the action of the ß-glucosidase prunasin hydrolase (PH) and mandelonitirile lyase or be glucosylated to form amygdalin. The tissue and cellular localization of PHs was determined during fruit development in two sweet and two bitter almond cultivars using a specific antibody toward PHs. Confocal studies on sections of tegument, nucellus, endosperm, and embryo showed that the localization of the PH proteins is dependent on the stage of fruit development, shifting between apoplast and symplast in opposite patterns in sweet and bitter cultivars. Two different PH genes, Ph691 and Ph692, have been identified in a sweet and a bitter almond cultivar. Both cDNAs are 86% identical on the nucleotide level, and their encoded proteins are 79% identical to each other. In addition, Ph691 and Ph692 display 92% and 86% nucleotide identity to Ph1 from black cherry (Prunus serotina). Both proteins were predicted to contain an amino-terminal signal peptide, with the size of 26 amino acid residues for PH691 and 22 residues for PH692. The PH activity and the localization of the respective proteins in vivo differ between cultivars. This implies that there might be different concentrations of prunasin available in the seed for amygdalin synthesis and that these differences may determine whether the mature almond develops into bitter or sweet.

Feasibility of using cryopreserved lymphoblastoid cells to diagnose some lysosomal storage diseases.

OBJECTIVE: The Epstein-Barr virus (EBV) is utilized as a tool in the study of cellular biology because of its capacity to transform B-lymphocytes. For this reason, EBV is used in conservation of human B-lymphocytes for long periods for subsequent evaluation of lysosomal hydrolase activity. Lymphoblastoid cell lines have several advantages for use over other cell types, such as prompt availability and possibility to develop, characterize and standardize cell banks, to test effects of promising pharmaceutical reagents. The study below presents biochemical data that demonstrate validity of lymphoblastoid cell lines for diagnosis of GM1-gangliosidosis, Gaucher, Fabry and Pompe diseases and mucopolysaccharidosis type I. MATERIALS AND METHODS: Cultures were prepared from peripheral blood, collected from 25 normal subjects and 13 affected individuals. Enzyme activities and immunohistochemistry (IHC) were measured. Activities of enzymes beta-galactosidase, beta-glucosidase, alpha-iduronidase, alpha-galactosidase and alpha-glucosidase were measured before and after cryopreservation for 180 days. Enzymatic activity was measured when transformation was confirmed by IHC. RESULTS: We observed some significant alterations in enzymatic activity of non-cultured cells when compared to others that had been cultured for 12 days and kept frozen for 180 days. CONCLUSIONS: However, these alterations did not invalidate use of the technology of transformation of lymphoblastoid cell lines with EBV, to diagnose the diseases mentioned above, in view of the fact that the cultured cells, before and after freezing, demonstrated similar enzymatic activities.

Solution structure of the C-terminal domain of Ole e 9, a major allergen of olive pollen.

Ole e 9 is an olive pollen allergen belonging to group 2 of pathogenesis-related proteins. The protein is composed of two immunological independent domains: an N-terminal domain (NtD) with 1,3-beta-glucanase activity, and a C-terminal domain (CtD) that binds 1,3-beta-glucans. We have determined the three-dimensional structure of CtD-Ole e 9 (101 amino acids), which consists of two parallel alpha-helices forming an angle of approximately 55 degrees , a small antiparallel beta-sheet with two short strands, and a 3-10 helix turn, all connected by long coil segments, resembling a novel type of folding among allergens. Two regions surrounded by aromatic residues (F49, Y60, F96, Y91 and Y31, H68, Y65, F78) have been localized on the protein surface, and a role for sugar binding is suggested. The epitope mapping of CtD-Ole e 9 shows that B-cell epitopes are mainly located on loops, although some of them are contained in secondary structural elements. Interestingly, the IgG and IgE epitopes are contiguous or overlapped, rather than coincident. The three-dimensional structure of CtD-Ole e 9 might help to understand the underlying mechanism of its biochemical function and to determine possible structure-allergenicity relationships.

Allergenic contribution of the IgE-reactive domains of the 1,3-beta-glucanase Ole e 9: diagnostic value in olive pollen allergy.

BACKGROUND: Designing of methods for an accurate diagnosis is a main goal of allergy research. Olive pollen allergy is currently diagnosed using commercially available pollen extracts that do not allow identification of the molecules that elicit the disease. OBJECTIVE: To analyze the suitability of using the N- and C-terminal domains (NtD and CtD, respectively) of the 1,3-beta-glucanase Ole e 9, a major allergen from olive pollen, for in vitro diagnosis. METHODS: Serum samples from 55 olive-allergic patients were assayed using enzyme-linked immunosorbent assay to study hypersensitive patients with IgE reactivity to Ole e 9. The specific IgEs to NtD and CtD, obtained by recombinant technology, were determined by means of immunoblotting, enzyme-linked immunosorbent assay, and inhibition assays. RESULTS: Thirty-one of 33 serum samples from Ole e 9-allergic patients were IgE reactive to recombinant NtD (rNtD) (n = 26 [79%]), recombinant CtD (rCtD) (n = 22 [67%]), or both (n = 17 [52%]). Nine patients (27%) were exclusively reactive to rNtD and 5 (15%) to rCtD. Inhibition assays of IgE binding to Ole e 9 with a mixture of both domains abolished 90% of the binding, whereas 44% and 45% were abolished when rNtD and rCtD were used, respectively. CONCLUSIONS: Because sensitization to NtD or CtD of Ole e 9 could be correlated to vegetable food-latex-pollen cross-reactivity processes or to the exacerbation and persistence of asthma, respectively, these molecules could be used in vitro as markers of disease to classify patients and to design a patient-tailored immunotherapy approach.

The role of major olive pollen allergens Ole e 1, Ole e 9, and Ole e 10 on mice sensitization.

BACKGROUND: Olive pollen is an important cause of allergy in Mediterranean countries. To date, 10 allergens (Ole e 1 to Ole e 10) have been isolated and characterized. Animal models of olive pollen allergy are suitable tools for testing the efficacy and safety of new forms of immunotherapy. OBJECTIVES: To characterize the immune response in mice sensitized with olive pollen extract and to compare it with that of allergic patients. METHODS: BALB/c mice were sensitized by 4 intraperitoneal injections of olive pollen extract in aluminum hydroxide. The allergic state was proved by measuring serum specific IgG1 and total IgE antibody levels. The IgG1 responses to olive pollen allergens were assayed by immunoblotting and enzyme-linked immunosorbent assay. Competition experiments between human IgE and mouse IgG1 binding to olive pollen allergens were performed. RESULTS: Sensitization with olive pollen extract induced high levels of specific IgG1 and total IgE in all tested animals. Immunoblotting experiments showed that the mouse IgG1 binding pattern to pollen extract was complex and heterogeneous, as occurs with human IgE. High IgG1 antibody levels to the major olive pollen allergens described for humans were detected in serum samples from sensitized mice, whereas minor olive pollen allergens induced no significant IgG1 response. Coincubation of mouse serum samples with a cocktail of Ole e 1, Ole e 9, and Ole e 10 resulted in a significant decrease (60%) in IgG1 binding to olive pollen extract. Specific mouse IgG1 strongly inhibited human IgE binding to olive pollen allergens. CONCLUSIONS: This mouse model of olive pollen sensitization mimics immunologic features of human pollinosis and could be a useful tool for designing novel forms of immunotherapy for olive pollen allergy based on allergen cocktails.

Variability of Ole e 9 allergen in olive pollen extracts: relevance of minor allergens in immunotherapy treatments.

BACKGROUND: Clustered severe adverse reactions to immunotherapy with olive pollen extracts have been occasionally reported in areas where olive trees are extensively grown. Allergic patients from these areas, in addition to the major olive pollen allergen Ole e 1, frequently recognize a recently described allergen, Ole e 9. OBJECTIVE: We aimed to develop an immunoassay to measure Ole e 9 concentration and to study the variability of this allergen in olive pollen extracts. METHODS: Monoclonal antibodies (mAb) to Ole e 9 were produced from mice immunized with the pure allergen. One of these mAbs was used to develop a sandwich ELISA with an anti-olive pollen extract rabbit serum as the tracer. Olive pollen batches from several suppliers were analyzed using this method. These batches were also analyzed for Ole e 1 content and biological activity. RESULTS: A 10-fold variation between the extreme values was found for the biological activity of the batches analyzed. Ole e 1 concentration showed a 25-fold variation. Variability of Ole e 9 concentration was extremely high, up to 161 times. The ratio Ole e 1/Ole e 9 varied in a range from 0.6 to 390.4. CONCLUSION: The availability of a mAb-based ELISA for Ole e 9 made it possible for us to detect an important source of variability in olive pollen batches. This variability may be the cause of outbreaks of adverse reactions in the course of immunotherapy treatments, which have sometimes been observed among olive-allergic patients living in areas with very high levels of airborne olive pollen.

Prophylactic intranasal treatment with fragments of 1,3-beta-glucanase olive pollen allergen prevents airway inflammation in a murine model of type I allergy.

BACKGROUND: Olive pollen is an important cause of allergy in Mediterranean countries. More than 50% of olive-pollen-allergic patients are sensitized against the 1,3-beta-glucanase Ole e 9. To date, prophylactic and therapeutic treatments using purified recombinant allergens have not been studied in animal models of olive pollen allergy. METHODS: BALB/c mice were immunized against Ole e 9 combining intraperitoneal injections of the allergen in Al(OH)3 with airway allergen challenges. A prophylactic treatment was performed by intranasal administration of a mixture of the recombinant fragments of the allergen prior to Ole e 9 sensitization. Serum levels of specific IgE, IgG1, IgG2a and IgG2b were measured by ELISA, and total IgE levels by sandwich ELISA. Bronchoalveolar lavage and lungs from mice were collected to study airway inflammation by light microscopy. RESULTS: BALB/c mice immunized against Ole e 9 developed a predominantly Th2-like immune response with allergen-specific immunoglobulin induction and airway inflammation accompanied by the infiltration of eosinophils, lymphocytes, and neutrophils in the lung. Prophylactic treatment by intranasal application of the recombinant fragments of Ole e 9 avoids airway inflammation induced by sensitization with this allergen although the levels of Ole e 9-specific antibodies remain unchanged. CONCLUSIONS: Prophylactic intranasal treatment with recombinant fragments of Ole e 9 prevents airway inflammation triggered by immunization to this allergen in a murine model of type I allergy.

1,3-beta-glucanases as candidates in latex-pollen-vegetable food cross-reactivity.

BACKGROUND: 1,3-beta-glucanases (group 2 of pathogenesis-related proteins) are enzymes widely distributed among higher plants and have been recently proven to be significant allergens. OBJECTIVE: The aim of this work was to study the potential implication of 1,3-beta-glucanases in cross-reactivities among latex, pollen and vegetable foods. METHODS: The cDNA encoding the N-terminal domain (NtD) of Ole e 9, a major allergenic 1,3-beta-glucanase from olive pollen, was amplified by polymerase chain reaction and produced as a recombinant protein in Pichia pastoris (recombinant N-terminal domain, rNtD). Circular dichroism, ELISA, immunoblotting and immunoblotting inhibition experiments were carried out. Sera from olive pollen allergic patients and a rNtD-specific polyclonal antiserum were used. RESULTS: The NtD of Ole e 9 has been produced at high yield in the yeast P. pastoris and possesses 1,3-beta-glucanase activity. The expressed polypeptide conserves IgE and IgG immunodominant epitopes of the whole Ole e 9. A rNtD-specific polyclonal antiserum and sera from olive pollen allergic patients allowed detection of IgG and IgE reactive peptidic epitopes common to 1,3-beta-glucanase Ole e 9 in extracts from ash and birch pollen, tomato, potato, bell-pepper, banana and latex. CONCLUSION: rNtD and homologous glucanases are new molecules to be used in diagnostic protocols as they could help to identify allergic pollen patients who are at risk for developing allergic symptoms to fruits, vegetables and latex.

Beta-glucosidase enzymatic activity of crystal polypeptide of the Bacillus thuringiensis strain 1.1.

The crystals of Bacillus thuringiensis strain 1.1 consist of the 140 kDa delta-endotoxin, which exhibits beta-glucosidase enzymatic activity, based on the following data. (i) Purified crystals exhibit beta-glucosidase enzymatic activity. When the crystals are reacted with specific antibodies directed either against the commercial (almond purified) beta-glucosidase or against the 140 kDa polypeptide, then considerable reduction of enzymatic activity is observed almost at the same level with both antibodies. (ii) Commercial beta-glucosidase and the 140 kDa crystal polypeptide share antigenic similarities; in Western immunoblots, the 140 kDa crystal polypeptide is recognized by anti-beta-glucosidase antibodies, and commercial beta-glucosidase is recognized by anti-140-kDa antibodies. (iii) The enzymatic properties of commercial beta-glucosidase and that resident in the crystals of B. thuringiensis strain 1.1 are very similar. Thus, both enzymes hydrolyze a wide range of substrates (aryl-beta-glucosides, disaccharides with alpha- or beta-linkage polysaccharides) and have an optimum activity at 40 degrees C and pH 5. Both enzymes are relatively thermostable and are resistant to end-product inhibition by glucose. Additionally, they show the same pattern of inhibition or activation by several chemical compounds. (iv) The crystals and commercial beta-glucosidase show almost equivalent levels of insecticidal activity against Drosophila melanogaster larvae and, furthermore, cause reduction in adult flies that emerge from larvae surviving treatment.

Significance of carbohydrate epitopes in a latex allergen with beta-1,3-glucanase activity.

BACKGROUND: One of the latex allergens, Hev b 2, has beta-1,3-glucanase activity. The entire sequence of this allergen is already known. There is one potential N-glycosylation site in this molecule ((27)Asn). Heterogeneous glycosylation of this Asn residue could be a source of the multiplicity of natural Hev b 2. Possible participation of the carbohydrate epitopes of latex beta-1,3-glucanase isoenzymes in their IgE-binding capacity and cross-reactivity was investigated in this study. METHODS: beta-1,3-Glucanase isoenzymes were separated based on their affinities for concanavalin A. IgE-binding capacity and cross-reactivity were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Sequence heterogeneity among the isoenzymes was probed by peptide mass mapping after lysyl endopeptidase digestion. To clarify the relation to Hev b 2, N-terminal sequencing was performed on a fragmented peptide common to the separated isoenzymes. RESULTS: Basic beta-1,3-glucanase was subdivided into two glycosylated isoenzymes (GI and GII) and one non-glycosylated isoenzyme (GIII). IgE antibodies in latex-positive sera chiefly recognized the glycosylated isoenzymes. Inhibition ELISA supported the significance of the carbohydrate epitopes for the IgE recognition and cross-reactivity. However, non-glycosylated GIII, as well as GI and GII, produced positive results in a skin prick test. The three beta-1,3-glucanase isoenzymes shared a partial sequence in common with Hev b 2. CONCLUSIONS: Our results suggest that the carbohydrate epitopes in Hev b 2 homologues are relevant to an in vitro diagnosis of latex allergy and the accompanying cross-reactivity. Carbohydrate epitopes do not necessarily provoke allergic symptoms. Therefore, the actual allergenicity of Hev b 2 and its homologues should be carefully evaluated not only by in vitro IgE tests but also by in vivo tests.

Allergens and natural rubber proteins.

BACKGROUND: Allergy to natural rubber latex (NRL) results from exposure to proteins derived from Hevea brasiliensis. Type I latex hypersensitivity is observed in certain occupational and other high-risk groups with frequent exposure to NRL products. This includes health care workers (HCWs), workers in the latex industry, children with spina bifida, and atopic individuals. OBJECTIVES: Early reliable diagnosis and avoidance are required for better patient care. Standardized reagents are not presently available for in vitro and in vivo testing and treatment of patients with latex allergy. However, a number of allergens have been isolated and characterized from Hevea latex and NRL products. Currently, a total of 11 major and minor allergens are designated by the International Allergen Nomenclature Committee. This article reviews the structural and functional characteristics of these latex allergenic proteins. RESULTS: NRL-allergenic proteins include those involved in the biosynthesis of polyisoprene and coagulation of latex rubber elongation factor, small rubber particle protein, prohevein, and patatin. Pathogenesis-related proteins include beta-1,3-glucanases, chitinases, and hevamine; and the structural proteins include microhelix protein complex, proline-rich protein, profilins, enolases, and manganese superoxide dismutase. Recombinant allergens demonstrated skin test reactivity in patients with latex allergy. The minimal level of skin test reactivity was about 70 pg/mL for NRL and 1 ng/mL for recombinant allergens. The use of selected recombinant latex allergens (Hev b 5, Hev b 6, and Hev b 7) in skin prick tests identified 93% of allergic individuals, mainly health care workers. CONCLUSIONS: Recombinant latex allergens are clinically reactive and can be produced in a standardized manner, which could potentially provide safe and sensitive reagents for the diagnosis and treatment of type I latex allergy.

Cloning and expression of the gene which encodes a tube precipitin antigen and wall-associated beta-glucosidase of Coccidioides immitis.

We report the structure and expression of the Coccidioides immitis BGL2 gene which encodes a previously characterized 120-kDa glycoprotein of this fungal respiratory pathogen. The glycoprotein is recognized by immunoglobulin M tube precipitin (TP) antibody present in sera of patients with coccidioidomycosis, a reaction which has been used for serodiagnosis of early coccidioidal infection. The deduced amino acid sequence of BGL2 shows 12 potential N glycosylation sites and numerous serine-threonine-rich regions which could function as sites for O glycosylation. In addition, the protein sequence includes a domain which is characteristic of family 3 glycosyl hydrolases. Earlier biochemical studies of the purified 120-kDa TP antigen revealed that it functions as a beta-glucosidase (EC 3.2.1.21). Its amino acid sequence shows high homology to several other reported fungal beta-glucosidases which are members of the family 3 glycosyl hydrolases. Results of previous studies have also suggested that the 120-kDa beta-glucosidase participates in wall modification during differentiation of the parasitic cells (spherules) of C. immitis. In this study we showed that expression of the BGL2 gene is elevated during isotropic growth of spherules and the peak of wall-associated BGL2 enzyme activity correlates with this same phase of parasitic cell differentiation. These data support our hypothesis that the 120-kDa beta-glucosidase plays a morphogenetic role in the parasitic cycle of C. immitis.