Swati Sharma1, Bharti Arora1, S N Gaur2, Naveen Arora3. 1. Allergy and Immunology Section, CSIR-Institute of Genomics and Integrative Biology, New Delhi, 110007, India; Academy of Scientific and Innovative Research, Ghaziabad, U.P., 201002, India. 2. Department of Pulmonary Medicine, V.P. Chest Institute, University of Delhi, New Delhi, 110007, India. 3. Allergy and Immunology Section, CSIR-Institute of Genomics and Integrative Biology, New Delhi, 110007, India; Academy of Scientific and Innovative Research, Ghaziabad, U.P., 201002, India. Electronic address: naveen@igib.res.in.
Abstract
INTRODUCTION: GSTs are multifunctional enzymes involved in cellular detoxification and present as potent allergens in several sources. Present study investigates allergenic relevance of GST from P. americana and determine its cross reactive potential with other indoor allergen sources. METHODS: Computational analysis with FASTA and ConSurf webserver was performed to determine potentially cross reactive allergens. Further, Per a 5 gene was cloned in pET 22b+ vector and expressed in E.coli BL21 cells and the rPer a 5 protein was purified using Ni-NTA affinity chromatography. Enzymatic activity of rPer a 5 was assessed using CDNB and cumene hydroperoxide. ELISA and immunoblot were performed using cockroach hypersensitive patient's sera. Functional activity of rPer a 5 was evaluated by basophil activation test. Inhibition studies were carried out with D. pteronyssinus, A. alternata and C. lunata extracts. RESULTS: Per a 5 demonstrates highest sequence similarity with delta class GST of Blattella germanica (94.9%). It also exhibits significant sequence similarity (50-58%) with mite, fungal and helminth allergenic GSTs. ConSurf analysis reveals high degree of evolutionary similarity in N terminal region of Per a 5, especially at GST dimerization interface. The purified rPer a 5 protein resolved at 27 kDa on SDS-PAGE. The rPer a 5 protein exhibits GST activity and possess upto 65% immunoreactivity with cockroach hypersensitive patient's sera in ELISA and immunoblot. It upregulates expression of CD203c on basophils signifying its biological ability to activate effector cells. rPer a 5 significantly inhibits corresponding GSTs in P. americana, D. pteronyssinus, A. alternata and C. lunata with EC50 values of 15.5 ng. 38.38 ng, 41.4 ng and 61.66 ng, respectively. CONCLUSION: Recombinant delta class GST of P. americana is a clinically relevant allergen showing upto 65% immunoreactivity with hypersensitive patient's sera. Per a 5 GST allergen showed phylogenetic similarity with dust mite, fungal and birch allergens thereby demonstrating allergen cross reactivity.
INTRODUCTION: GSTs are multifunctional enzymes involved in cellular detoxification and present as potent allergens in several sources. Present study investigates allergenic relevance of GST from P. americana and determine its cross reactive potential with other indoor allergen sources. METHODS: Computational analysis with FASTA and ConSurf webserver was performed to determine potentially cross reactive allergens. Further, Per a 5 gene was cloned in pET 22b+ vector and expressed in E.coli BL21 cells and the rPer a 5 protein was purified using Ni-NTA affinity chromatography. Enzymatic activity of rPer a 5 was assessed using CDNB and cumene hydroperoxide. ELISA and immunoblot were performed using cockroach hypersensitivepatient's sera. Functional activity of rPer a 5 was evaluated by basophil activation test. Inhibition studies were carried out with D. pteronyssinus, A. alternata and C. lunata extracts. RESULTS: Per a 5 demonstrates highest sequence similarity with delta class GST of Blattella germanica (94.9%). It also exhibits significant sequence similarity (50-58%) with mite, fungal and helminth allergenic GSTs. ConSurf analysis reveals high degree of evolutionary similarity in N terminal region of Per a 5, especially at GST dimerization interface. The purified rPer a 5 protein resolved at 27 kDa on SDS-PAGE. The rPer a 5 protein exhibits GST activity and possess upto 65% immunoreactivity with cockroach hypersensitivepatient's sera in ELISA and immunoblot. It upregulates expression of CD203c on basophils signifying its biological ability to activate effector cells. rPer a 5 significantly inhibits corresponding GSTs in P. americana, D. pteronyssinus, A. alternata and C. lunata with EC50 values of 15.5 ng. 38.38 ng, 41.4 ng and 61.66 ng, respectively. CONCLUSION: Recombinant delta class GST of P. americana is a clinically relevant allergen showing upto 65% immunoreactivity with hypersensitivepatient's sera. Per a 5 GST allergen showed phylogenetic similarity with dust mite, fungal and birch allergens thereby demonstrating allergen cross reactivity.