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Identification of homologues of manganese superoxidase dimutase, thioredoxin, cyclophilin, profilin, a calcium-binding protein, and a fibrinogen-binding protein as IgE binding components in Curvularia lunata
J ALLERGY CLIN IMMUNOL VOLUME 11 I, NUMBER 2
Abstracts
$327
pnea when exposed to birds were studied after informed consent was obtained. Prick tests (prick by prick) were performed with raw and boiled egg white and yolk and chicken meat. Other extracts included feather mix (duck and chicken), livetins, chicken serum, chicken albumin, ovalbumin and ovomucoid. Other tests included specific IgE determinations and bronchial provocation with livetins in one patient. SDS-PAGE immunoblot with livetins was performed. Patients were also skin tested with 3 air filter extracts collected in a home housing 6 canary birds. RESULTS: All patients had a positive skin prick test with raw yolk, livetin, feathers and air filter extracts. They showed specific lgE against yolk and livetin. Bronchial provocation with livetins was positive. SDSPAGE immunoblot with livetins showed several lgE binding bands including one of 66.2 KDa (Gal d 5). CONCLUSIONS: We confirm that livetins induce positive bronchial challenges in patients with bird-egg syndrome and that these allergens seem to be airborne in homes with birds.
50 kD) together with their isoforms. Six components that matched ESTs were further studied. RACE and gene-specific primers were generated to amplify these sequences, after which the coding regions were cloned into a pET32a expression vector and expressed in Escherichia coil Recombinants homologous to manganese superoxidase dimntase (Asp f 6), thioredoxin (Cop c 2), cyclophilin (Mal f 6), profilin, calcium-binding protein (Juno 4), and fibrinogeu-binding protein (Asp f 2) were generated, lgEbinding frequencies to these recombinants among patients' sera tested ranged from 13 to 50%. CONCLUSIONS: We have thus identified the primary structure of six different IgE-binding components from C. lunata, which showed homology to several pan-allergens.
Funding: C.B.E LETI, S.A.
A. M. K a h n j, C. C. Castro I, E. G. Wolff 1, M. J, Gregorio I, M. Ordofiez~, M. D. Romero-Piffeguer I, D. Beltramo 2, J. C. Muifiol; IAIlergy & Immunology Seccirn, Internal Medicine Service, Hospital Misericordia, Crrdoba, ARGENTINA, 2Immunology Departament, Ceprocor, Crrdoba, ARGENTINA. RATIONALE: Although rat allergen is known to cause occupational asthma and rhinitis in laboratory workers, its potential significance in home environments and indoor allergens have been previously reported. However the responsible antigens have been not identified yet. The purpose of this study is to identify the rat allergens that bind to lgE by immunoblot. METHODS: We isolate sera from 25 subjects with asthma and rhinitis from C6rdoba, Argentina, who have positive skin prick test to Urine Rat Allergen Extract (URAE). RESULTS: Sera from 22 of 25 patients presented IgE that binds to one or more allergens of URAE. We could identify at least 4 different bands according to their molecular weight as follows: 14 kd, 18 kd, 28 kd, and 45 kd, The non allergic control group (n: 10) was negative for all the different allergens of URAE. CONCLUSIONS: In the present study we were able to show specific IgE to rat allergens in sera of allergic patients who were positive in prick test to rat.
1034 Hevein-like Domain IV in Wheat Germ Agglutinin (WGA) is Responsible for the Majority of ]gE Cross-reactivity between WGA and Hevein (Hey b 6.02) P. Karisola j , H. Alenius2, K. Turjanmaa3, T. Reunala 3, N. Kalkkinen 4, T. Palosuo 5, M. Kulomaa6; JBiological and Environmental Sciences, Molecular Biology, University of Jyvaskyla, Jyvaskyla, FINLAND, 2Finnish Institute of Occupational Health, Helsinki, FINLAND, 3Tampere University Hospital, Tampere, FINLAND, 4University of Helsinki, Helsinki, FINLAND, 5National Public Health Institute, Helsinki. FINLAND, 6University of Jyvaskyla, Jyvaskyla, FINLAND. R A T I O N A L E : Crystal structure of wheat germ agglutinin (WGA) reveals four covalently connected homologous domains resembling hevein (Hev b6.02), a major latex allergen, lgE from up to half of latexallergic patients recognize WGA in ELISA or immunoblotting. The detailed molecular basis of this alleged cross-reactivity was examined. METHODS: The 4 hevein-like domains of WGA were separately produced in insect cell system and their immunogenicity was tested with sera from herein-allergic patients. RESULTS: In ELISA no detectable binding of IgE was seen to heveinlike domains I and II of WGA. In contrast, domain IV and, to a some extent, domain Ill bound IgE from hevein-allergic patients. CONCLUSIONS: Majority of the IgE-binding ability of WGA appears to reside in its hevein-like domain IV. To assess the clinical significance of this cross-reactivity in vivo studies are needed.
Funding: Academy of Finland
1035
Identification of Homologues to Manganese Superoxidase Dimutase, Thioredoxin. Cyclophi]in, Profilin. a CalciumBinding Protein, and a Fibrinogen-Binding Protein as IgE Binding Components in Curvularia lunata
F. L. Wong, S. S. Joshi, B. W. Lee, T. K. Tan, F. T. Chew; National University of Singapore, Singapore, SINGAPORE. RATIONALE: Curvularia spp. is an important fungal aeroallergen in tropical regions. Our studies showed that it exist as a heterogeneous mix of species in the environment with C. lunata being the most prevalent and allergenic. This study aims to identify, sequence and express its allergenic component for further characterization. METHODS: C. lunata protein was isolated from mats separated by 2-D gel electrophoresis, and immunoblotted with fungal allergic patients' sera. Selected lgE-binding spots were identified via MALD1-TOF MS and QTOF MS-MS. These peptide sequence information were compared to both public databases and in-house fungi expressed sequence tag (EST) collections to identify the proteins of interest. Recombinant proteins to several of these were generated, and further characterized for IgE binding using 16 patients' and 2 negative sera. RESULTS: From our initial IgE western blotting and MS-MS sequencing, we identified 14 different IgE-binding components (ranging from 13-
Funding: Biomedical Research Council, Singapore
1036 Immunoblot Analysis of the Sera of Patients Allergic to Rat in Cordoba. Argentina
Funding: Self-funded
1037 ,.isAoprene e t Increases i v i t GST Y AProduction l t e r nanda Allergenic r i a Altertlata
J. S h a n k a r =, S. Sridhara 2. B. R Singh 2, N. Arora2; ZAllergy and Immunology Section, Center for Biochemical Technology, New Delhi, INDIA, 2Center for Biochemical Technology, New Delhi, INDIA. RATIONALE: The glutathione-s-transferases (GST) are a family of enzymes that catalyze the neucleophilic addition of the thiol of reduced glutathione to a variety of electrophiles and thus detoxify drugs. GST from fungi had been shown as pan allergen, and hence it becomes important to study their allergenic property. Since the production of GST is about 1-2% of the total protein synthesized, it is difficult to purify GST in bulk from native sources. In this study, attempts were made to enhance the production of the GST in Ahernaria alternata using isoprene (2-methyl 1,3 butadiene). METHODS: Sabouraud's medium (SB) containing varying concentration of isoprene (lmM, 2mM, 5raM, 20mM, 100mM, IM) was used to grow the fungus. Day 9 culture (surface growth) extract was prepared in 0.1MPBS containing 5mM EDTA and lmM PMSF. GST activity was quantitated using CDNB and ELISA. The allergenic activity was measured using pooled allergic patient's sera. RESULTS: Increasing concentration of isoprene upto 100raM resulted in four fold increase in enzymatic activity (0. 538/min/ml to 2.067/min/ml). A concomitant increase in allergenic activity (O.D at A490n m 0.7 tO 2.4) was also observed. CONCLUSIONS: These observations suggest that isoprene can be used to enhance the production of GST and GST-like proteins in fungi to enable their purification in bulk for use in diagnosis and treatment.
Funding: Council for Scientific and hzdustrial Research
Abstracts
$327
pnea when exposed to birds were studied after informed consent was obtained. Prick tests (prick by prick) were performed with raw and boiled egg white and yolk and chicken meat. Other extracts included feather mix (duck and chicken), livetins, chicken serum, chicken albumin, ovalbumin and ovomucoid. Other tests included specific IgE determinations and bronchial provocation with livetins in one patient. SDS-PAGE immunoblot with livetins was performed. Patients were also skin tested with 3 air filter extracts collected in a home housing 6 canary birds. RESULTS: All patients had a positive skin prick test with raw yolk, livetin, feathers and air filter extracts. They showed specific lgE against yolk and livetin. Bronchial provocation with livetins was positive. SDSPAGE immunoblot with livetins showed several lgE binding bands including one of 66.2 KDa (Gal d 5). CONCLUSIONS: We confirm that livetins induce positive bronchial challenges in patients with bird-egg syndrome and that these allergens seem to be airborne in homes with birds.
50 kD) together with their isoforms. Six components that matched ESTs were further studied. RACE and gene-specific primers were generated to amplify these sequences, after which the coding regions were cloned into a pET32a expression vector and expressed in Escherichia coil Recombinants homologous to manganese superoxidase dimntase (Asp f 6), thioredoxin (Cop c 2), cyclophilin (Mal f 6), profilin, calcium-binding protein (Juno 4), and fibrinogeu-binding protein (Asp f 2) were generated, lgEbinding frequencies to these recombinants among patients' sera tested ranged from 13 to 50%. CONCLUSIONS: We have thus identified the primary structure of six different IgE-binding components from C. lunata, which showed homology to several pan-allergens.
Funding: C.B.E LETI, S.A.
A. M. K a h n j, C. C. Castro I, E. G. Wolff 1, M. J, Gregorio I, M. Ordofiez~, M. D. Romero-Piffeguer I, D. Beltramo 2, J. C. Muifiol; IAIlergy & Immunology Seccirn, Internal Medicine Service, Hospital Misericordia, Crrdoba, ARGENTINA, 2Immunology Departament, Ceprocor, Crrdoba, ARGENTINA. RATIONALE: Although rat allergen is known to cause occupational asthma and rhinitis in laboratory workers, its potential significance in home environments and indoor allergens have been previously reported. However the responsible antigens have been not identified yet. The purpose of this study is to identify the rat allergens that bind to lgE by immunoblot. METHODS: We isolate sera from 25 subjects with asthma and rhinitis from C6rdoba, Argentina, who have positive skin prick test to Urine Rat Allergen Extract (URAE). RESULTS: Sera from 22 of 25 patients presented IgE that binds to one or more allergens of URAE. We could identify at least 4 different bands according to their molecular weight as follows: 14 kd, 18 kd, 28 kd, and 45 kd, The non allergic control group (n: 10) was negative for all the different allergens of URAE. CONCLUSIONS: In the present study we were able to show specific IgE to rat allergens in sera of allergic patients who were positive in prick test to rat.
1034 Hevein-like Domain IV in Wheat Germ Agglutinin (WGA) is Responsible for the Majority of ]gE Cross-reactivity between WGA and Hevein (Hey b 6.02) P. Karisola j , H. Alenius2, K. Turjanmaa3, T. Reunala 3, N. Kalkkinen 4, T. Palosuo 5, M. Kulomaa6; JBiological and Environmental Sciences, Molecular Biology, University of Jyvaskyla, Jyvaskyla, FINLAND, 2Finnish Institute of Occupational Health, Helsinki, FINLAND, 3Tampere University Hospital, Tampere, FINLAND, 4University of Helsinki, Helsinki, FINLAND, 5National Public Health Institute, Helsinki. FINLAND, 6University of Jyvaskyla, Jyvaskyla, FINLAND. R A T I O N A L E : Crystal structure of wheat germ agglutinin (WGA) reveals four covalently connected homologous domains resembling hevein (Hev b6.02), a major latex allergen, lgE from up to half of latexallergic patients recognize WGA in ELISA or immunoblotting. The detailed molecular basis of this alleged cross-reactivity was examined. METHODS: The 4 hevein-like domains of WGA were separately produced in insect cell system and their immunogenicity was tested with sera from herein-allergic patients. RESULTS: In ELISA no detectable binding of IgE was seen to heveinlike domains I and II of WGA. In contrast, domain IV and, to a some extent, domain Ill bound IgE from hevein-allergic patients. CONCLUSIONS: Majority of the IgE-binding ability of WGA appears to reside in its hevein-like domain IV. To assess the clinical significance of this cross-reactivity in vivo studies are needed.
Funding: Academy of Finland
1035
Identification of Homologues to Manganese Superoxidase Dimutase, Thioredoxin. Cyclophi]in, Profilin. a CalciumBinding Protein, and a Fibrinogen-Binding Protein as IgE Binding Components in Curvularia lunata
F. L. Wong, S. S. Joshi, B. W. Lee, T. K. Tan, F. T. Chew; National University of Singapore, Singapore, SINGAPORE. RATIONALE: Curvularia spp. is an important fungal aeroallergen in tropical regions. Our studies showed that it exist as a heterogeneous mix of species in the environment with C. lunata being the most prevalent and allergenic. This study aims to identify, sequence and express its allergenic component for further characterization. METHODS: C. lunata protein was isolated from mats separated by 2-D gel electrophoresis, and immunoblotted with fungal allergic patients' sera. Selected lgE-binding spots were identified via MALD1-TOF MS and QTOF MS-MS. These peptide sequence information were compared to both public databases and in-house fungi expressed sequence tag (EST) collections to identify the proteins of interest. Recombinant proteins to several of these were generated, and further characterized for IgE binding using 16 patients' and 2 negative sera. RESULTS: From our initial IgE western blotting and MS-MS sequencing, we identified 14 different IgE-binding components (ranging from 13-
Funding: Biomedical Research Council, Singapore
1036 Immunoblot Analysis of the Sera of Patients Allergic to Rat in Cordoba. Argentina
Funding: Self-funded
1037 ,.isAoprene e t Increases i v i t GST Y AProduction l t e r nanda Allergenic r i a Altertlata
J. S h a n k a r =, S. Sridhara 2. B. R Singh 2, N. Arora2; ZAllergy and Immunology Section, Center for Biochemical Technology, New Delhi, INDIA, 2Center for Biochemical Technology, New Delhi, INDIA. RATIONALE: The glutathione-s-transferases (GST) are a family of enzymes that catalyze the neucleophilic addition of the thiol of reduced glutathione to a variety of electrophiles and thus detoxify drugs. GST from fungi had been shown as pan allergen, and hence it becomes important to study their allergenic property. Since the production of GST is about 1-2% of the total protein synthesized, it is difficult to purify GST in bulk from native sources. In this study, attempts were made to enhance the production of the GST in Ahernaria alternata using isoprene (2-methyl 1,3 butadiene). METHODS: Sabouraud's medium (SB) containing varying concentration of isoprene (lmM, 2mM, 5raM, 20mM, 100mM, IM) was used to grow the fungus. Day 9 culture (surface growth) extract was prepared in 0.1MPBS containing 5mM EDTA and lmM PMSF. GST activity was quantitated using CDNB and ELISA. The allergenic activity was measured using pooled allergic patient's sera. RESULTS: Increasing concentration of isoprene upto 100raM resulted in four fold increase in enzymatic activity (0. 538/min/ml to 2.067/min/ml). A concomitant increase in allergenic activity (O.D at A490n m 0.7 tO 2.4) was also observed. CONCLUSIONS: These observations suggest that isoprene can be used to enhance the production of GST and GST-like proteins in fungi to enable their purification in bulk for use in diagnosis and treatment.
Funding: Council for Scientific and hzdustrial Research