title: Purification, characterization and allergenicity assessment of 26kDa protein, a major allergen from Cicer arietinum.
abstract: Chickpea (CP), a legume of the family Fabaceae, is an important nutrient-rich food providing protein, essential amino acids, vitamins, dietary fibre, and minerals. Unfortunately, several IgE-binding proteins in CP have been detected that are responsible for allergic manifestations in sensitized population. Therefore, the prevalence of CP induced allergy prompted us towards purification, characterization and allergenicity assessment of a major ∼26kDa protein from chickpea crude protein extract (CP-CPE). Purification of CP 26kDa protein was done using a combination of fractionation and anion exchange chromatography. This protein was further characterized as "Chain A, crystal structure of a plant albumin" from Cicer arietinum with Mol wt 25.8kDa by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Further, allergenic potential of purified 25.8kDa protein was assessed using in vivo and in vitro model. Purified protein showed IgE-binding capacity with sensitized BALB/c mice and CP allergic patient's sera. Enhanced levels of specific and total IgE, MCP-1, MCPT-1, myeloperoxidase, histamine, prostaglandin D2, and cysteinyl leukotriene were found in sera of mice treated with CP ∼26kDa protein. Further, expressions of Th2 cytokines (i.e. IL-4, IL-5, IL-13), transcription factors (i.e. GATA-3, STAT-6, SOCS-3) and mast cell signaling proteins (Lyn, cFgr, Syk, PLC-γ2, PI-3K, PKC) were also found increased at mRNA and protein levels in the intestines of mice treated with CP ∼26kDa protein. In addition, enhanced release of β-hexosaminidase, histamine, cysteinyl leukotriene and prostaglandin D2 were observed in RBL2H3 cell line when treated (125μg) with CP 26kDa protein. Conclusively, in vivo and in vitro studies revealed the allergenic potential of purified CP 26kDa protein. Being a potential allergen, plant albumin may play a pivotal role in CP induced allergenicity. Current study will be helpful for better development of therapeutic approaches to prevent the allergenicity in CP sensitized individuals.
authors.affiliation: Food Toxicology Laboratory, Food, Drug and Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research, Vishvigyan Bhavan, 31, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), CSIR- Indian Institute of Toxicology Research, Lucknow 226001, Uttar Pradesh, India.
authors.forename: Alok Kumar
authors.initials: AK
authors.lastname: Verma
authors.affiliation: Food Toxicology Laboratory, Food, Drug and Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research, Vishvigyan Bhavan, 31, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), CSIR- Indian Institute of Toxicology Research, Lucknow 226001, Uttar Pradesh, India.
authors.forename: Akanksha
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Food Toxicology Laboratory, Food, Drug and Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research, Vishvigyan Bhavan, 31, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India; Department of Pharmacology and Therapeutics, BLSC-Centre for Genetics and Pharmacology L4-113, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263, USA.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Food Toxicology Laboratory, Food, Drug and Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research, Vishvigyan Bhavan, 31, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India.
authors.forename: Rinkesh Kumar
authors.initials: RK
authors.lastname: Gupta
authors.affiliation: Food Toxicology Laboratory, Food, Drug and Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research, Vishvigyan Bhavan, 31, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India.
authors.forename: Dinesh
authors.initials: D
authors.lastname: Kumar
authors.affiliation: Food Toxicology Laboratory, Food, Drug and Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research, Vishvigyan Bhavan, 31, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India.
authors.forename: Kriti
authors.initials: K
authors.lastname: Gupta
authors.affiliation: Department of Pulmonary Medicine, KS Hegde Medical Academy, Mangalore 575018, India.
authors.forename: B H
authors.initials: BH
authors.lastname: Giridhar
authors.affiliation: Food Toxicology Laboratory, Food, Drug and Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research, Vishvigyan Bhavan, 31, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), CSIR- Indian Institute of Toxicology Research, Lucknow 226001, Uttar Pradesh, India.
authors.forename: Mukul
authors.initials: M
authors.lastname: Das
authors.affiliation: Food Toxicology Laboratory, Food, Drug and Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research, Vishvigyan Bhavan, 31, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), CSIR- Indian Institute of Toxicology Research, Lucknow 226001, Uttar Pradesh, India. Electronic address: pddwivedi@yahoo.com.
authors.forename: Premendra D
authors.initials: PD
authors.lastname: Dwivedi
mesh_terms: D000485:Allergens / Q000276:immunology* / Q000302:isolation & purification*; D000818:Animals; D002853:Chromatography, Liquid; D029863:Cicer / Q000737:chemistry / Q000276:immunology*; D005512:Food Hypersensitivity / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010940:Plant Proteins / Q000276:immunology* / Q000302:isolation & purification*; D053719:Tandem Mass Spectrometry
chemical_list: D000485:Allergens; D010940:Plant Proteins
keywords: Allergy; Chickpea; Mast cell; Plant albumin; Transcription factors