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American Cockroach Allergen rPer a 3 Expressed in Pichia pastoris
AB146 Abstracts
572
Identification of T-Cell Epitopes of Phl p 5a as a Basic Requirement for Construction of Hypoallergenic Mutants H. Kahlert, A. Nandy, M. Wald, O. Cromwell, H. Fiebig; ALLERGOPHARMA Joachim Ganzer KG, Reinbek, GERMANY. RATIONALE: Recombinant Phl p 5a derivatives are promising candidates for an allergen cocktail for specific immunotherapy based on hypoallergenic Timothy grass major allergens. An important prerequisite for construction of such mutants is identification of T-cell epitopes on Phl p 5a. METHODS: T-cell lines were raised from PBMC of grass pollen allergic subjects with natural or recombinant Phl p 5a. T-cell epitopes were analyzed with synthetic dodeca peptides spanning the whole sequence of 288 amino acids of rPhl p 5a overlapping by 3 residues. T-cell reactivity was measured by proliferation assays using 3H-Thymidine. RESULTS: Results from 30 TCL of 16 different grass pollen allergics are included. Two dominant regions of T-cell reactivity are found in the N-terminal part of Phl p 5a at positions 13-27 and 40-54. Four further dominant regions are located in the C-terminal half of the molecule at positions 160174, 172-186, 196-228 and 229-252. Further peptides with lower frequencies of T-cell reactivity were found between positions 55-162. The T-cell reactivity of a mutant with deletions at positions 94-113 and 175-190 coincides with these findings. CONCLUSIONS: Five regions of frequent T-cell reactivity could be determined on Phl p 5a, together with one region with T-cell reactivity of lower frequency and 5 - 6 regions with no T-cell reactivity. The latter segments of the molecule are those where genetic engineering might produce promising mutants with a changed conformation and reduced IgEreactivity.
573
MONDAY
Comparative Analysis Of Enzyme Activities From Dust Mites And Cockroaches K. Jeong, C. Kim, T. Yong; Yonsei University College of Medicine, Seoul, REPUBLIC OF KOREA. RATIONALE: Enzyme activity is believed to influence on the allergenicity. The objectives of this study was to evaluate the enzymatic activity of allergen extracts from dust mites and cockroaches commonly found in Korean homes. METHODS: The allergen extracts were prepared from 3 dust mites (Dermatophagoides farinae, D. pteronyssinus and Tyrophagus putrescentiae) and 3 cockroaches (Blattella germanica, Periplaneta americana and P. fuliginosa) which have been maintained in the Korea National Arthropods of Medical Importance Resource Bank. Proteins were extracted in phosphate buffered saline buffer after homogenization under liquid nitrogen. The various enzyme activity was investigated by API Zym system. Protease activity was examined by the gelatin zymography. RESULTS: T. putrescentiae extract showed the highest protease activity, followed by cockroaches extracts. D. farinae and D. pteronyssinus showed only weak protease activity. Gelatinolytic activity was mainly detected in proteins with 30 kDa in D. farinae, 28 kDa in D. pteronyssinus, > 26 kDa in T. putrescentiae, > 20 kDa in B. germanica, and > 23 kDa in P. americana and P. fuliginosa. No significant difference of phosphatase, lipase, and glycosidase activity was observed among 6 allergen extracts. CONCLUSIONS: The strong protease activity of cockroach could play an important role in the sensitization to cockroach allergens which is known to be associated with development of asthma.
574
Identification of Cockroach Allergen using Proteomic Analysis L. K. Arruda1, M. C. R. Barbosa1, A. B. R. Santos1, L. D. Santos2, M. S. Palma2, F. Ferreira-Briza3, P. Briza3, A. Erler3, M. D. Chapman4, A. Pome´s4; 1School of Medicine of Ribeirao Preto, Ribeirao Preto, BRAZIL, 2 State University of Sao Paulo, Rio Claro, BRAZIL, 3University of Salzburg, Salzburg, AUSTRIA, 4Indoor Biotechnologies Inc, Charlottesville, VA. RATIONALE: Recombinant allergens offer the possibility of preparing well defined mixtures of allergens for component-resolved diagnosis and treatment of allergy. We have demonstrated that a panel of 5 recombinant
J ALLERGY CLIN IMMUNOL FEBRUARY 2010
allergens (rPer a 1, rPer a 7, rBla g 2, rBla g 4, rBla g 5) was sufficient to identify 50% of patients with asthma and/or rhinitis, with positive skin tests to cockroach. METHODS: 2-D electrophoresis was used to detect proteins in natural cockroach extracts. Immunoblotting was performed using undiluted serum pool constructed with sera from 27 patients with asthma and/or rhinitis allergic to P. americana who presented negative skin tests and serum IgE to Per a 7 and Per a 1 allergens. The membrane was incubated with biotinconjugated anti-human IgE, followed by Streptavidin Horseradish Peroxidase. Detection was carried out by Enhanced Chemiluminescence. Spots of interest were digested with trypsin and peptide sequencing was determined by mass spectrometry. RESULTS: Approximately 100 spots were identified in cockroach extract, and 5 showed IgE reactivity using the cockroach-allergic serum pool. These IgE-binding proteins comprised peptides showing homology to hexamerins from Blaberus discoidalis (97% identity) and P. americana (67% identity), based on analysis of 10% of the sequence of the molecule. Hexamerin from P. americana had been identified as allergen Per a 3 among patients with asthma in Taiwan, therefore our results suggested identification of an isoform of Per a 3. CONCLUSIONS: Proteomic analysis P. americana extract could provide novel information on cockroach allergens and related isoforms, which could contribute to better diagnosis and treatment of cockroach allergy.
575
American Cockroach Allergen rPer a 3 Expressed in Pichia pastoris J. Glesner1, L. K. Arruda2, M. D. Chapman1, A. Pome´s1; 1Indoor Biotechnologies, Inc., Charlottesville, VA, 2School of Medicine of Ribeirao Preto, Ribeirao Preto, BRAZIL. RATIONALE: Per a 3, an American cockroach (Periplaneta americana) allergen shows homology to hemocyanin superfamily proteins and is reported to show skin test reactivity in ~80% of cockroach allergic patients. Since neither the natural or recombinant allergen is available, we expressed rPer a 3 in Pichia pastoris for allergenicity studies. METHODS: DNA encoding for a mature Per a 3 isoallergen that shares 92.8% amino acid identity with Per a 3.01 was mutated to eliminate a Xho I site. The mutated DNA was PCR-amplified and sub-cloned into the Pichia pastoris expression vector pGAPZaB using Xho I and Not I restriction sites. A Histidine-tag was added to the C-terminus for purification purposes. Monoclonal antibodies raised against natural Per a 3 (kindly provided by Dr. C-H Wu) were used to develop a standard and chimeric ELISA for allergen and IgE quantification, respectively. RESULTS: Linearized plasmid encoding for rPer a 3 was electroporated into Pichia pastoris KM71 cells. The allergen was constitutively expressed from a selected colony at up to 24 mg/L and purified from the media by affinity nickel agarose chromatography. The 670 amino acid allergen has two N-glycosylation sites and ran as a ~80kDa protein in a silver stained SDSPAGE gel. ELISA using the monoclonal antibodies 2A2-A4 as capture and biotinylated-E4 or IgE from sera of cockroach allergic patients showed that rPer a 3 appears to fold correctly. CONCLUSIONS: rPer a 3 expressed in Pichia pastoris shows immunoreactivity and appears to be a good recombinant allergen for structure-function studies related to allergenicity to P. americana.
572
Identification of T-Cell Epitopes of Phl p 5a as a Basic Requirement for Construction of Hypoallergenic Mutants H. Kahlert, A. Nandy, M. Wald, O. Cromwell, H. Fiebig; ALLERGOPHARMA Joachim Ganzer KG, Reinbek, GERMANY. RATIONALE: Recombinant Phl p 5a derivatives are promising candidates for an allergen cocktail for specific immunotherapy based on hypoallergenic Timothy grass major allergens. An important prerequisite for construction of such mutants is identification of T-cell epitopes on Phl p 5a. METHODS: T-cell lines were raised from PBMC of grass pollen allergic subjects with natural or recombinant Phl p 5a. T-cell epitopes were analyzed with synthetic dodeca peptides spanning the whole sequence of 288 amino acids of rPhl p 5a overlapping by 3 residues. T-cell reactivity was measured by proliferation assays using 3H-Thymidine. RESULTS: Results from 30 TCL of 16 different grass pollen allergics are included. Two dominant regions of T-cell reactivity are found in the N-terminal part of Phl p 5a at positions 13-27 and 40-54. Four further dominant regions are located in the C-terminal half of the molecule at positions 160174, 172-186, 196-228 and 229-252. Further peptides with lower frequencies of T-cell reactivity were found between positions 55-162. The T-cell reactivity of a mutant with deletions at positions 94-113 and 175-190 coincides with these findings. CONCLUSIONS: Five regions of frequent T-cell reactivity could be determined on Phl p 5a, together with one region with T-cell reactivity of lower frequency and 5 - 6 regions with no T-cell reactivity. The latter segments of the molecule are those where genetic engineering might produce promising mutants with a changed conformation and reduced IgEreactivity.
573
MONDAY
Comparative Analysis Of Enzyme Activities From Dust Mites And Cockroaches K. Jeong, C. Kim, T. Yong; Yonsei University College of Medicine, Seoul, REPUBLIC OF KOREA. RATIONALE: Enzyme activity is believed to influence on the allergenicity. The objectives of this study was to evaluate the enzymatic activity of allergen extracts from dust mites and cockroaches commonly found in Korean homes. METHODS: The allergen extracts were prepared from 3 dust mites (Dermatophagoides farinae, D. pteronyssinus and Tyrophagus putrescentiae) and 3 cockroaches (Blattella germanica, Periplaneta americana and P. fuliginosa) which have been maintained in the Korea National Arthropods of Medical Importance Resource Bank. Proteins were extracted in phosphate buffered saline buffer after homogenization under liquid nitrogen. The various enzyme activity was investigated by API Zym system. Protease activity was examined by the gelatin zymography. RESULTS: T. putrescentiae extract showed the highest protease activity, followed by cockroaches extracts. D. farinae and D. pteronyssinus showed only weak protease activity. Gelatinolytic activity was mainly detected in proteins with 30 kDa in D. farinae, 28 kDa in D. pteronyssinus, > 26 kDa in T. putrescentiae, > 20 kDa in B. germanica, and > 23 kDa in P. americana and P. fuliginosa. No significant difference of phosphatase, lipase, and glycosidase activity was observed among 6 allergen extracts. CONCLUSIONS: The strong protease activity of cockroach could play an important role in the sensitization to cockroach allergens which is known to be associated with development of asthma.
574
Identification of Cockroach Allergen using Proteomic Analysis L. K. Arruda1, M. C. R. Barbosa1, A. B. R. Santos1, L. D. Santos2, M. S. Palma2, F. Ferreira-Briza3, P. Briza3, A. Erler3, M. D. Chapman4, A. Pome´s4; 1School of Medicine of Ribeirao Preto, Ribeirao Preto, BRAZIL, 2 State University of Sao Paulo, Rio Claro, BRAZIL, 3University of Salzburg, Salzburg, AUSTRIA, 4Indoor Biotechnologies Inc, Charlottesville, VA. RATIONALE: Recombinant allergens offer the possibility of preparing well defined mixtures of allergens for component-resolved diagnosis and treatment of allergy. We have demonstrated that a panel of 5 recombinant
J ALLERGY CLIN IMMUNOL FEBRUARY 2010
allergens (rPer a 1, rPer a 7, rBla g 2, rBla g 4, rBla g 5) was sufficient to identify 50% of patients with asthma and/or rhinitis, with positive skin tests to cockroach. METHODS: 2-D electrophoresis was used to detect proteins in natural cockroach extracts. Immunoblotting was performed using undiluted serum pool constructed with sera from 27 patients with asthma and/or rhinitis allergic to P. americana who presented negative skin tests and serum IgE to Per a 7 and Per a 1 allergens. The membrane was incubated with biotinconjugated anti-human IgE, followed by Streptavidin Horseradish Peroxidase. Detection was carried out by Enhanced Chemiluminescence. Spots of interest were digested with trypsin and peptide sequencing was determined by mass spectrometry. RESULTS: Approximately 100 spots were identified in cockroach extract, and 5 showed IgE reactivity using the cockroach-allergic serum pool. These IgE-binding proteins comprised peptides showing homology to hexamerins from Blaberus discoidalis (97% identity) and P. americana (67% identity), based on analysis of 10% of the sequence of the molecule. Hexamerin from P. americana had been identified as allergen Per a 3 among patients with asthma in Taiwan, therefore our results suggested identification of an isoform of Per a 3. CONCLUSIONS: Proteomic analysis P. americana extract could provide novel information on cockroach allergens and related isoforms, which could contribute to better diagnosis and treatment of cockroach allergy.
575
American Cockroach Allergen rPer a 3 Expressed in Pichia pastoris J. Glesner1, L. K. Arruda2, M. D. Chapman1, A. Pome´s1; 1Indoor Biotechnologies, Inc., Charlottesville, VA, 2School of Medicine of Ribeirao Preto, Ribeirao Preto, BRAZIL. RATIONALE: Per a 3, an American cockroach (Periplaneta americana) allergen shows homology to hemocyanin superfamily proteins and is reported to show skin test reactivity in ~80% of cockroach allergic patients. Since neither the natural or recombinant allergen is available, we expressed rPer a 3 in Pichia pastoris for allergenicity studies. METHODS: DNA encoding for a mature Per a 3 isoallergen that shares 92.8% amino acid identity with Per a 3.01 was mutated to eliminate a Xho I site. The mutated DNA was PCR-amplified and sub-cloned into the Pichia pastoris expression vector pGAPZaB using Xho I and Not I restriction sites. A Histidine-tag was added to the C-terminus for purification purposes. Monoclonal antibodies raised against natural Per a 3 (kindly provided by Dr. C-H Wu) were used to develop a standard and chimeric ELISA for allergen and IgE quantification, respectively. RESULTS: Linearized plasmid encoding for rPer a 3 was electroporated into Pichia pastoris KM71 cells. The allergen was constitutively expressed from a selected colony at up to 24 mg/L and purified from the media by affinity nickel agarose chromatography. The 670 amino acid allergen has two N-glycosylation sites and ran as a ~80kDa protein in a silver stained SDSPAGE gel. ELISA using the monoclonal antibodies 2A2-A4 as capture and biotinylated-E4 or IgE from sera of cockroach allergic patients showed that rPer a 3 appears to fold correctly. CONCLUSIONS: rPer a 3 expressed in Pichia pastoris shows immunoreactivity and appears to be a good recombinant allergen for structure-function studies related to allergenicity to P. americana.