Purification, characterization and allergenicity assessment of 26 kDa protein, a major allergen from Cicer arietinum

Highlights

• A 26 kDa protein from chickpea was purified and characterized as plant albumin.

• Chickpea 26 kDa protein administration in BALB/c mice induces allergic responses.

• In vitro study on RBL-2H3 cells showed an enhanced release of allergic mediators when sensitized with the sera of treated mice and challenged with the same 26 kDa chickpea protein.

• Upregulated expressions of mast cell signaling molecules were observed.

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 ∼26 kDa protein from chickpea crude protein extract (CP-CPE). Purification of CP 26 kDa 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 25.8 kDa by Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis. Further, allergenic potential of purified 25.8 kDa 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 ∼26 kDa 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–3 K, PKC) were also found increased at mRNA and protein levels in the intestines of mice treated with CP ∼26 kDa protein. In addition, enhanced release of β-hexosaminidase, histamine, cysteinyl leukotriene and prostaglandin D2 were observed in RBL2H3 cell line when treated (125 μg) with CP 26 kDa protein. Conclusively, in vivo and in vitro studies revealed the allergenic potential of purified CP 26 kDa 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.

Introduction

Allergic reactions are consequences of abnormal immune response to common and harmless substances, present in the environment. A substance that causes allergic reaction is known as allergen. In last two decades, the prevalence and severity of allergic diseases are increasing worldwide and food allergy constitutes a major part of this increase (Sicherer and Sampson, 2010). Generally, the food allergy may be defined as an adverse health effect that arises from a specific immune response on exposure to a given food (Boyce et al., 2010). Moreover, food induced allergy is characterized by induction of Th2 cells and production of IL-4 cytokine that further acts upon naive T-cells and causes their differentiation into allergen specific Th2 cells to secrete Th2 cytokines including IL-4, IL-5, IL-13 which assist B-cell responses and induce the production of allergen specific IgE (McKenzie et al., 1998). Further, various transcription factors like GATA-3, STAT-6, SOCS-3, C-MAF also play an important role in skewing of immune response towards Th2 reactions thereby provoking allergic manifestations by enhancing the production of Th2 cytokines (Kumar et al., 2012).

The most common allergenic foods include milk and dairy products, egg, gluten containing cereals (e.g., wheat, rye, barley), fish, crustaceans, shellfish and legumes (e.g., peanut, soybean) (Śpičák, 2010). Legumes are essential constituent of human diets as they are rich source of proteins and oils but unfortunately, legumes are also important source of allergens. The legume allergens are mainly associated with four protein families (i) Cupins (ii) Prolamins (both included in seed storage proteins), (iii) Profilins and (iv) Pathogen related proteins. In recent years, the major allergens from different legumes have been identified and characterized including peanut (Ara h1–Ara h7), soybean (Gly mBD), green gram (Vig r2-Vig5), red gram (Caj c1–Caj c6) and green bean (Pha v3) that are responsible for IgE- mediated allergic reactions in susceptible individuals (Verma et al., 2013). Among leguminous crops, chickpea (CP) is also one of the most commonly consumed legume, particularly in the populations of developing nations, and also in mediterranean areas as it is a good source of protein, soluble and insoluble fibres. Mature chickpea grains contain 60–65% carbohydrate, 6% fat, and 17% protein (Gopalan et al., 1989). However, allergenic potential of CP has not been explored well. Therefore, it is important to purify and explore the allergenic potential of IgE-binding CP proteins to understand their distinct role in allergy.

In the earlier study, it was reported that CP is able to cause IgE-mediated allergic reactions in nasobronchial allergic patients and sensitized BALB/c mice, as CP-CPE contains seven IgE- binding proteins in the range of 15–95 kDa (Verma et al., 2012). However, purification and characterization of major CP allergens are necessary to explore the extent of allergenicity caused by purified allergens. Therefore, the aim of the present study was to purify the IgE- binding protein from CP-CPE by using column chromatography and further characterization by Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis. In the current study, purification of CP 26 kDa protein was done as this protein showed the IgE-binding property with all screened CP sensitive patients. So, we decided to purify this 26 kDa major allergenic protein from CP and assessed the allergenic potential of this protein in vitro as well as in vivo situations.