Thioredoxin from the Indianmeal moth Plodia interpunctella: cloning and test of the allergenic potential in mice.

BACKGROUND/OBJECTIVE: The Indianmeal moth Plodia interpunctella is a highly prevalent food pest in human dwellings, and has been shown to contain a number of allergens. So far, only one of these, the arginine kinase (Plo i 1) has been identified. OBJECTIVE: The aim of this study was to identify further allergens and characterise these in comparison to Plo i 1. METHOD: A cDNA library from whole adult P. interpunctella was screened with the serum of a patient with indoor allergy and IgE to moths, and thioredoxin was identified as an IgE-binding protein. Recombinant thioredoxin was generated in E. coli, and tested together with Plo i 1 and whole moth extracts in IgE immunoblots against a large panel of indoor allergic patients' sera. BALB/c mice were immunised with recombinant thioredoxin and Plo i 1, and antibody production, mediator release from RBL cells, T-cell proliferation and cytokine production were measured. RESULT: For the first time a thioredoxin from an animal species was identified as allergen. About 8% of the sera from patients with IgE against moth extracts reacted with recombinant P. interpunctella thioredoxin, compared to 25% reacting with recombinant Plo i 1. In immunised BALB/c mice, the recombinant allergens both induced classical Th2-biased immune responses such as induction IgE and IgG1 antibodies, upregulation of IL-5 and IL-4 and basophil degranulation. CONCLUSION: Thioredoxin from moths like Plo i 1 acts like a classical Type I allergen as do the thioredoxins from wheat or corn. This clearly supports the pan-allergen nature of thioredoxin. The designation Plo i 2 is suggested for the new P. interpunctella allergen.

The vacuolar serine protease, a cross-reactive allergen from Cladosporium herbarum.

Subtilisin-like serine proteases make up one of the most important allergen-families regarding the number of individual allergens. Previously, fungal subtilisin-like serine proteases have been identified from Aspergillus-, Penicillium-, and Trichophyton-species having a prevalence of IgE-reactivity between 33% and 80%. Since IgE-cross-reactivity is a common phenomenon within fungal species we wanted to know whether this protein also represents an allergen in Cladosporium herbarum. Hence, a screening of a C. herbarum cDNA library was performed using the coding sequence of the Penicillium oxalicum vacuolar serine protease (Pen o 18) as hybridization probe, ending up with a full-length clone. Biochemical and immunological characterization of this clone revealed that C. herbarum vacuolar serine protease most likely is synthesized as a precursor with an N-terminal pro-enzyme sequence and represents a minor allergen (Cla h 9) with a prevalence of IgE-reactivity of 15.5%. Furthermore Cla h 9 specifically reacted with the two monoclonal antibodies FUM20 and PCM39, as do the vacuolar serine proteases from Aspergillus fumigatus and Penicillium species. Investigation of IgE-cross-reactivity between Cla h 9 and other fungal serine proteases revealed that cross-reactivity is higher between vacuolar than alkaline serine proteases. IgE-epitope mapping of Cla h 9 was done in order to test whether four Cla h 9-peptides having a high sequence homology to previously determined Pen ch 18-IgE-epitopes also harbour IgE-epitopes. Three-dimensional models of the vacuolar serine proteases from C. herbarum and Penicillium chrysogenum were generated for the three-dimensional localization of the Cla h 9- and Pen ch 18- IgE-reactive and -non-reactive peptides. Taken together a new C. herbarum allergen has been identified, which may be useful in a molecule-based approach of C. herbarum allergy-diagnosis and -therapy. Moreover, Cla h 9 represents a further member of the subtilisin-like serine protease allergen-family, which stresses the importance of these proteins with respect to fungal IgE-cross-reactivity.

Vacuolar serine protease is a major allergen of Cladosporium cladosporioides.

BACKGROUND: Cladosporium is an important allergenic fungus worldwide. We report here a major allergen of C. cladosporioides. METHODS: Major C. cladosporioides allergens were characterized by immunoblotting, N-terminal amino acid sequencing, protein purification and cDNA cloning. RESULTS: Seventy-four sera (38%) from 197 bronchial asthmatic patients demonstrated IgE binding against C. cladosporioides extracts. Among these 74 sera, 41 (55%) and 38 (51%) showed IgE binding against a 36- and a 20-kDa protein of C. cladosporioides, respectively. Both IgE-reacting components reacted with FUM20, a monoclonal antibody against fungal serine proteases. N-terminal amino acid sequencing results suggest that they are vacuolar serine proteases, and the 20-kDa component is possibly a degraded product of the 36-kDa allergen. A corresponding 5'-truncated 1,425-bp cDNA fragment was isolated. The mature protein after N-terminal processing starts with an N-terminal serine that is the ninth residue encoded by the 5'-truncated cDNA. The protein sequence deduced shares 69-72% sequence identity with Penicillium vacuolar serine proteases and was designated as Cla c 9. The purified 36-kDa Cla c 9 allergen showed proteolytic activity with peptide Z-Ala-Ala-Leu-pNA as substrate. IgE cross-reactivity was detected between the purified Cla c 9 and serine protease allergens from Aspergillusfumigatus and Penicillium chrysogenum. CONCLUSION: We identified a vacuolar serine protease as a major allergen of C. cladosporioides (Cla c 9) and a major pan-allergen of prevalent airborne fungi. IgE cross-reactivity among these highly conserved serine protease pan-fungal allergens was also detectable.

Identification of italian cypress (Cupressus sempervirens) pollen allergen Cup s 3 using homology and cross-reactivity.

BACKGROUND: The prevalence of seasonal allergic diseases of the upper airways is increasing in industrialized countries. The Cupressaceae are important causes of pollinosis, particularly in Europe. OBJECTIVE: To determine whether the pollen from Cupressus sempervirens (Italian cypress) contains a pathogenesis-related group 5 (PR-5) protein, similar to that found in other allergenic Cupressaceae pollens. METHODS: Messenger RNA was purified from Italian cypress pollen, and complementary DNA (cDNA) was synthesized. cDNAs for PR-5 proteins were amplified by polymerase chain reaction and extended by rapid amplification of cDNA ends methods. Recombinant Cup s 3 was expressed in Escherichia coli as a fusion protein. Inhibition enzyme-linked immunosorbent assays were used to test the allergenicity of Cup s 3. RESULTS: Three cDNAs were cloned. These clones had approximately 95% identity to Jun a 3 and Cup a 3. Recombinant Cup s 3.0102 maltose-binding protein inhibited the IgE from most patients from binding to an extract of Italian cypress. The extent of inhibition suggested that antibodies to Cup s 3 were a prominent component of the IgE response to Italian cypress pollen. CONCLUSION: Cup s 3, an allergen of Italian cypress pollen, was identified based on cross-reactivity and homology with other pollen PR-5 proteins, despite an apparently low level of protein expression. Variations in the content of Cup s 3 in the pollen from different regions or trees should be considered in the choice of extracts for diagnosis and specific immunotherapy for Italian cypress pollen hypersensitivity.

Allergic memory of patients sensitized to castor bean after a long stimulation-free period.

We have taken advantage of the temporary exposure of Marseilles population to castor bean seed proteins to follow 26 allergic patients more than 20 years after sensitization. Skin tests, specific immunoglobulin E (IgE) antibody assays, and specific immunoblots were performed. Skin test reactivity to Ricinus Communis and specific IgE concentrations decreased progressively and almost completely disappeared after 20 years. Specific IgE concentration displayed a fairly exponential decrease, with a half-life of 4.7 years. Thus, in the absence of any antigenic stimulation, directly by castor bean, or indirectly by cross-reactivity to other Euphorbiaceae, especially latex, IgE sensitization is bound to disappear.

Cloning and Expression of the Olea europaea allergen Ole e 5, the pollen Cu/Zn superoxide dismutase.

BACKGROUND: Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity. METHODS: cDNA of Ole e 5 was amplified by nested 3'-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients' IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition). RESULTS: The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein. CONCLUSIONS: Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens.

Allergen cross-reactivity between proteins of the latex from Hevea brasiliensis, seeds and pollen of Ricinus communis, and pollen of Mercurialis annua, members of the Euphorbiaceae family.

Allergen cross-reactions among three strongly sensitizing Euphorbiaceae species, i.e., the rubber tree (Hevea brasiliensis), castor bean (Ricinus communis), and the Mediterranean weed Mercurialis annua were studied in Finnish patients (n = 25) allergic to natural rubber latex (NRL), but with no known exposure to castor bean or M. annua, and French patients allergic to castor bean (n = 26) or to M. annua (n = 9), but not to NRL. In immunoglobulin E (IgE)-immunoblotting, 28% of NRL-allergic patient sera recognized castor bean seed and 48% reacted to castor bean pollen proteins. Likewise, 35% of the NRL-allergic patient sera bound to M. annua pollen allergens. Nineteen percent of castor bean-allergic patients showed IgE to NRL and 8% to M. annua proteins. Sera from patients allergic to M. annua reacted in 44% to NRL, in 56% to castor bean seed, and in 78% to castor bean pollen proteins. In immunoblotting, castor bean seed extract inhibited the binding of NRL-reactive IgE to 20 kDa, 30 kDa of NRL, and 55 kDa of proteins; NRL extract, in turn, inhibited the binding of castor bean-reactive IgE to 14, 21-22, 29, and 32-34 kDa of castor bean proteins. In ELISA inhibition, NRL extract inhibited 33% of the binding of M. annua--reactive IgE of pooled sera to M. annua pollen. In conclusion, allergen cross-reactivity in vitro was observed among three botanically related Euphorbiaceae members, H. brasiliensis, R. communis, and M. annua, but the molecular specificity of the observed cross-reactions as well as their clinical significance remains to be elucidated. Allergen cross-reactivity should be taken into account in diagnostic work.