Isolation and characterization of a 28 kDa major allergen from blackgram (Phaseolus mungo).
Legumes are the major elicitors of IgE-mediated food allergy in many countries of the world. Purified major allergens are prerequisite for component resolved diagnosis of allergy. The present study was aimed to isolate and characterize a major allergenic protein from blackgram (Phaseolus mungo). Respiratory allergy patients with history of blackgram allergy were skin prick tested (SPT) and sera were collected from SPT positive patients. The blackgram extract was fractionated using a combination of anion exchange and hydrophobic interaction chromatography. The purified protein was characterized by indirect ELISA, immunoblot, ELISA inhibition, SPTs, stripped basophil histamine release, lymphoproliferation assay and digestibility assay. The purified protein separated at 28 kDa on 12% gel and showed IgE binding with 81% of blackgram hypersensitive patients' sera on immunoblot indicating it to be a major allergen. Periodic Acid Schiff's and meta-periodate treatment staining detected it to be a glycoprotein. The 28 kDa protein recognized 7/9 (77.8%) of blackgram positive patients by SPT, where as all 9 patients showed significant histamine release on stimulation with protein as compared to controls. The 28 kDa protein remained stable up to 15 min on incubation with SGF. Bands of 14-16 kDa appeared after 15 min of pepsin digestion that remained stable up to 60 min of incubation. However, purified protein degraded within 5 min after incubation with SIF. The N-terminus-12 residues sequence of 28 kDa protein was GRREDDYDNLQL. A stretch of residues 'DDYDNLQL' showed homology with Rho-specific inhibitor of transcription termination (E=0.42, Identity=87%) and NBS-LRR type disease resistant protein from peanut (Arachis hypogaea) (E=2, Identity=77%). In conclusion, the purified 28 kDa protein is a potent major allergen that may have implication in diagnosis of blackgram allergy.
Allergy and Immunology Lab, CSIR-Institute of Genomics and Integrative Biology, Delhi 110007, India.
Dolly
D
Kumari
Naveen
N
Arora
Ramkrashan
R
Kasera
Susheela
S
Sridhara
Raj
R
Kumar
Bhanu Pratap
BP
Singh
D000485:Allergens / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D001491:Basophils / Q000276:immunology; D006636:Histamine Release / Q000276:immunology; D006801:Humans; D006967:Hypersensitivity / Q000175:diagnosis / Q000276:immunology; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D008970:Molecular Weight; D027805:Phaseolus / Q000737:chemistry* / Q000276:immunology*; D010936:Plant Extracts / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D012882:Skin Tests
D000485:Allergens; D052179:Antigens, Plant; D010936:Plant Extracts; D007073:Immunoglobulin E