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Comparative Analysis of IgE Binding Proteins in GM and Non-GM Rice Varieties Using Atopic Patients Sera
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A Method of Reducing Allergenicity of Legume Proteins Naveen Arora1, Ramkrashan Kasera2, Anand Singh, PhD3, Shakuntala Lavasa4, Komerla Nagendra5; 1CSIR- IGIB, Delhi, India, 2 CSIR-Institute of Genomics and Integrative Biology, India, 3Institute of Genomics and Integrative Biology, DELHI, India, 4USLavasa Medical & Research Center, India, 5Bengaluru Allergy Centre, India. RATIONALE: Legumes are implicated in IgE mediated food allergy in different countries. The present study aimed to investigate the effect of enzymatic hydrolysis on allergenicity of kidney bean, black gram and peanut. METHODS: The extracts were subjected to sequential enzymatic treatment by Alcalase and flavorzyme. The allergenicity of hydrolysates were determined by ELISA and SPT. Allergenic potential of legume proteins were assessed by ELISA inhibition and basophil histamine release. RESULTS: Hydrolysis reduced the number of IgE binding fractions of the three legumes- kidney bean, black gram and peanut. Specific IgE binding was reduced 62%, 87% and 92% in the hyrolysates of kidney bean, black gram and peanut, respectively after 8 h of hydrolysis (p<0.01). Alcalase treatment alone increased the IC50 value of kidney bean, black gram and peanut to 4, 8.5 and 123 folds respectively. Sequential hydrolysis using both alcalase and flavourzyme increased the IC50 of the kidney bean, black gram and peanut to 25, 368 and 393 folds respectively further as compared to single enzyme treatment. Basophil histamine release showed reduced allergenicity of the hydrolyzed proteins. Significant reduction in the biopotency of hydrolyzed protein of kidney bean, black gram and peanut was observed in SPT where 6/13 kidney bean sensitive individuals, 8/13 black gram sensitive individuals and 9/15 peanut sensitive individuals were found SPT negative to their respective hydrolysates. CONCLUSIONS: Sequential hydrolysis by alcalase and flavourzyme reduced the allergenicity of legume proteins. The method has potential to reduce allergenicity of food extracts.
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Comparative Analysis of IgE Binding Proteins in GM and NonGM Rice Varieties Using Atopic Patients Sera Chandni Mathur1, P. C Kathuria2, Shakuntala Lavasa3, Pushpa Dahiya4, Anand Singh, PhD5; 1Institute of Genomics & Integrative Biology, Delhi, India, 2National Allergy Centre, India, 3USLavasa Medical & Research Center, India, 4M.D University, India, 5Institute of Genomics and Integrative Biology, Delhi, India. RATIONALE: Food allergy is recognized as IgE-mediated immune response. Safety issues regarding foods derived from genetically modified (GM) plants are central to their acceptance into the food supply and its commercial production. METHODS: Three GM rice varieties containing cry protein Cry 1ab, Cry 1ac and Cry 1c along with their native variety were selected for the present investigation. All the seed powders were processed for protein extraction and estimation. Extracts were subjected to SDS-PAGE for protein profiling. Simulated Gastric Fluid Digestion (SGF) was also done for all seed powders. IgE binding protein bands in rice were observed through Immunoblot with sera of allergic patients. Bioinformatic analysis of above transgenic proteins across databases was also performed. RESULTS: Protein content in non-GM rice was 0.9 mg/ml while in GM rice varieties it varied from 0.95 – 1.2 mg/ml. Proteins fractionated in 11 -14 bands in all varieties within a range of 9-81 kD. Native rice and GM rice with Cry 1Ab subjected to SGF showed a very faint appearance of smear around 9kD. Rice variety with Cry 1Ab had faint proteins around 27 and 47 kD. IgE binding protein bands observed in rice varieties were aggregating around 37-47 kD. Comparative analysis across different databases yielded that cry proteins used in GM Rice showed sequence identity of < 27 % . CONCLUSIONS: It is suspected that no major detectable difference was observed in GM and non-GM rice varieties.
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Analysis of the Association Between Food Allergy and Sensitization to Ltp and Profilin Felicia Berroa1, M Jose Goikoetxea, PhD, MD2, Marta M. Ferrer, MD, PhD, FAAAAI2, Paula Cabrera-Freitag, MD, PhD3, Gracia Javaloyes, MD, PhD1, Maria L Sanz, MD, PhD1, Gabriel Gastaminza, MD, PhD1; 1Department of Allergy, Clinica Universidad de Navarra, Spain, 2Department of Allergy, Clinica Universidad de Navarra, Pamplona, Spain, 3Clinica Universidad de Navarra, Pamplona, Spain. RATIONALE: LTP and profilin panallergens are associated with food allergy (FA). The aim of our study was to evaluate the association of suffering oral allergy syndrome (OAS) and systemic symptoms (SS) according to sensitization to LTP or profilin. METHODS: Retrospective analysis of 238 patients in whom skin prick test (SPT) to palm profilin and peach LTP(ALK-Abello, Madrid, Spain) was performed. Specific IgE rPhl p 12 and rPru p 3 were analyzed by ImmunoCAP (Phadia, Sweden) from serum obtained on the day of consultation. The association to present OAS or SS after ingestion of plant-food was studied by Chi-square and Odds Ratio(OR) calculation. RESULTS: Out of 238 patients, 72 (30%) had FA: 36 presented OAS, 20 patients SS and 16 OAS and SS. Eighty-three patients had positve SPT to profilin and/or LTP (42 profilin, 29 LTP and 12 both panallergens).Skin sensitization to LTP was associated with: OAS (OR: 3.279 [1.595-6.741]; p50.001) and SS (OR: 5.664[2.598-12.349]; p<0.001). Neither LTP-SPT nor rPru p 3 were associated with OAS without SS. A positive result to rPrup3 by ImmunoCAP was associated with OAS, only in those patients suffering also from SS. ImmunoCAP (OR: 2,568 [1,254-5,258]; p50.008) but not SPT (OR: 2.206[1.121-4.342]; p50.020) to profilin were associated with OAS without SS. CONCLUSIONS: Having positive SPT and/or ImmunoCAP is associated with having systemic symptoms with or without OAS. When OAS is present without systemic symptoms is associated only with ImmunoCAP to rPhl p 12.
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Structure and Function of the Peanut Panallergen Ara h 8 Barry K. Hurlburt, PhD1, Lesa Celeste, PhD2, Karolina A. Majorek3, Jane McBride4, Soheila J. Maleki, PhD1, Wladek Minor, PhD3, Maksymilian Chruszcz, PhD5; 1USDA-ARS-SRRC, New Orleans, LA, 2university of south carolina, columbia, SC, 3University of Virginia, Charlottesville, VA, 4usda-ars-srrc, new orleans, LA, 5University of South Carolina, Columbia, SC. RATIONALE: Ara h 8 is hypothesized to be the panallergen responsible for oral allergy syndrome between birch pollen and peanut. In addition, it may also be a signaling molecule carrier. We sought to investigate these notions with structural, and biochemical characterization. METHODS: Recombinant Ara h 8 was expressed in E. coli and purified. The structure was determined using X-ray crystallography. Ligand binding was tested by displacement of bound fluorescent 8-anilo-1-naphthalenesulfonic acid. RESULTS: Purification by a combination of heat treatment, ammonium sulfate precipitation and hydrophobic interaction chromatography yielded > 95% pure Ara h 8. The crystal structure was solved by molecular replacement and refined at 1.7 A resolution. The overall fold of Ara h 8 was very similar to Bet v 1, as well as Bet v 1 homologs Api g 1 (celery), Gly m 4 (soy), and Pru av 1 (cherry). Physiologically-relevant ligands have been identified. CONCLUSIONS: Our results support both the idea that Ara h 8 could be contributing to oral allergy syndrome between birch pollen and peanut, and the hypothesis that Bet v 1-like proteins could be transporters of signaling molecules important for plant development.
SATURDAY
Abstracts AB19
J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2
A Method of Reducing Allergenicity of Legume Proteins Naveen Arora1, Ramkrashan Kasera2, Anand Singh, PhD3, Shakuntala Lavasa4, Komerla Nagendra5; 1CSIR- IGIB, Delhi, India, 2 CSIR-Institute of Genomics and Integrative Biology, India, 3Institute of Genomics and Integrative Biology, DELHI, India, 4USLavasa Medical & Research Center, India, 5Bengaluru Allergy Centre, India. RATIONALE: Legumes are implicated in IgE mediated food allergy in different countries. The present study aimed to investigate the effect of enzymatic hydrolysis on allergenicity of kidney bean, black gram and peanut. METHODS: The extracts were subjected to sequential enzymatic treatment by Alcalase and flavorzyme. The allergenicity of hydrolysates were determined by ELISA and SPT. Allergenic potential of legume proteins were assessed by ELISA inhibition and basophil histamine release. RESULTS: Hydrolysis reduced the number of IgE binding fractions of the three legumes- kidney bean, black gram and peanut. Specific IgE binding was reduced 62%, 87% and 92% in the hyrolysates of kidney bean, black gram and peanut, respectively after 8 h of hydrolysis (p<0.01). Alcalase treatment alone increased the IC50 value of kidney bean, black gram and peanut to 4, 8.5 and 123 folds respectively. Sequential hydrolysis using both alcalase and flavourzyme increased the IC50 of the kidney bean, black gram and peanut to 25, 368 and 393 folds respectively further as compared to single enzyme treatment. Basophil histamine release showed reduced allergenicity of the hydrolyzed proteins. Significant reduction in the biopotency of hydrolyzed protein of kidney bean, black gram and peanut was observed in SPT where 6/13 kidney bean sensitive individuals, 8/13 black gram sensitive individuals and 9/15 peanut sensitive individuals were found SPT negative to their respective hydrolysates. CONCLUSIONS: Sequential hydrolysis by alcalase and flavourzyme reduced the allergenicity of legume proteins. The method has potential to reduce allergenicity of food extracts.
68
Comparative Analysis of IgE Binding Proteins in GM and NonGM Rice Varieties Using Atopic Patients Sera Chandni Mathur1, P. C Kathuria2, Shakuntala Lavasa3, Pushpa Dahiya4, Anand Singh, PhD5; 1Institute of Genomics & Integrative Biology, Delhi, India, 2National Allergy Centre, India, 3USLavasa Medical & Research Center, India, 4M.D University, India, 5Institute of Genomics and Integrative Biology, Delhi, India. RATIONALE: Food allergy is recognized as IgE-mediated immune response. Safety issues regarding foods derived from genetically modified (GM) plants are central to their acceptance into the food supply and its commercial production. METHODS: Three GM rice varieties containing cry protein Cry 1ab, Cry 1ac and Cry 1c along with their native variety were selected for the present investigation. All the seed powders were processed for protein extraction and estimation. Extracts were subjected to SDS-PAGE for protein profiling. Simulated Gastric Fluid Digestion (SGF) was also done for all seed powders. IgE binding protein bands in rice were observed through Immunoblot with sera of allergic patients. Bioinformatic analysis of above transgenic proteins across databases was also performed. RESULTS: Protein content in non-GM rice was 0.9 mg/ml while in GM rice varieties it varied from 0.95 – 1.2 mg/ml. Proteins fractionated in 11 -14 bands in all varieties within a range of 9-81 kD. Native rice and GM rice with Cry 1Ab subjected to SGF showed a very faint appearance of smear around 9kD. Rice variety with Cry 1Ab had faint proteins around 27 and 47 kD. IgE binding protein bands observed in rice varieties were aggregating around 37-47 kD. Comparative analysis across different databases yielded that cry proteins used in GM Rice showed sequence identity of < 27 % . CONCLUSIONS: It is suspected that no major detectable difference was observed in GM and non-GM rice varieties.
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Analysis of the Association Between Food Allergy and Sensitization to Ltp and Profilin Felicia Berroa1, M Jose Goikoetxea, PhD, MD2, Marta M. Ferrer, MD, PhD, FAAAAI2, Paula Cabrera-Freitag, MD, PhD3, Gracia Javaloyes, MD, PhD1, Maria L Sanz, MD, PhD1, Gabriel Gastaminza, MD, PhD1; 1Department of Allergy, Clinica Universidad de Navarra, Spain, 2Department of Allergy, Clinica Universidad de Navarra, Pamplona, Spain, 3Clinica Universidad de Navarra, Pamplona, Spain. RATIONALE: LTP and profilin panallergens are associated with food allergy (FA). The aim of our study was to evaluate the association of suffering oral allergy syndrome (OAS) and systemic symptoms (SS) according to sensitization to LTP or profilin. METHODS: Retrospective analysis of 238 patients in whom skin prick test (SPT) to palm profilin and peach LTP(ALK-Abello, Madrid, Spain) was performed. Specific IgE rPhl p 12 and rPru p 3 were analyzed by ImmunoCAP (Phadia, Sweden) from serum obtained on the day of consultation. The association to present OAS or SS after ingestion of plant-food was studied by Chi-square and Odds Ratio(OR) calculation. RESULTS: Out of 238 patients, 72 (30%) had FA: 36 presented OAS, 20 patients SS and 16 OAS and SS. Eighty-three patients had positve SPT to profilin and/or LTP (42 profilin, 29 LTP and 12 both panallergens).Skin sensitization to LTP was associated with: OAS (OR: 3.279 [1.595-6.741]; p50.001) and SS (OR: 5.664[2.598-12.349]; p<0.001). Neither LTP-SPT nor rPru p 3 were associated with OAS without SS. A positive result to rPrup3 by ImmunoCAP was associated with OAS, only in those patients suffering also from SS. ImmunoCAP (OR: 2,568 [1,254-5,258]; p50.008) but not SPT (OR: 2.206[1.121-4.342]; p50.020) to profilin were associated with OAS without SS. CONCLUSIONS: Having positive SPT and/or ImmunoCAP is associated with having systemic symptoms with or without OAS. When OAS is present without systemic symptoms is associated only with ImmunoCAP to rPhl p 12.
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Structure and Function of the Peanut Panallergen Ara h 8 Barry K. Hurlburt, PhD1, Lesa Celeste, PhD2, Karolina A. Majorek3, Jane McBride4, Soheila J. Maleki, PhD1, Wladek Minor, PhD3, Maksymilian Chruszcz, PhD5; 1USDA-ARS-SRRC, New Orleans, LA, 2university of south carolina, columbia, SC, 3University of Virginia, Charlottesville, VA, 4usda-ars-srrc, new orleans, LA, 5University of South Carolina, Columbia, SC. RATIONALE: Ara h 8 is hypothesized to be the panallergen responsible for oral allergy syndrome between birch pollen and peanut. In addition, it may also be a signaling molecule carrier. We sought to investigate these notions with structural, and biochemical characterization. METHODS: Recombinant Ara h 8 was expressed in E. coli and purified. The structure was determined using X-ray crystallography. Ligand binding was tested by displacement of bound fluorescent 8-anilo-1-naphthalenesulfonic acid. RESULTS: Purification by a combination of heat treatment, ammonium sulfate precipitation and hydrophobic interaction chromatography yielded > 95% pure Ara h 8. The crystal structure was solved by molecular replacement and refined at 1.7 A resolution. The overall fold of Ara h 8 was very similar to Bet v 1, as well as Bet v 1 homologs Api g 1 (celery), Gly m 4 (soy), and Pru av 1 (cherry). Physiologically-relevant ligands have been identified. CONCLUSIONS: Our results support both the idea that Ara h 8 could be contributing to oral allergy syndrome between birch pollen and peanut, and the hypothesis that Bet v 1-like proteins could be transporters of signaling molecules important for plant development.
SATURDAY
Abstracts AB19
J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2