Partial characterization of red gram (Cajanus cajan L. Millsp) polypeptides recognized by patients exhibiting rhinitis and bronchial asthma

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Abstract

We sought to assess the allergenic potential of red gram by identifying and characterizing the responsible proteins. Immunoblotting was performed to detect IgE binding proteins. Identities of these proteins were confirmed by mass spectrometry. To evaluate allergenic potential, BALB/c mice were sensitized with red gram proteins and levels of specific immunoglobulins, histamine, Th2 cytokines were measured. Allergenic response was evident by significant increase in specific IgE, IgG1, histamine and Th2 cytokine levels. Prominent anaphylactic symptoms, discernible histopathological responses and down regulation of IFN-γ levels give strong support towards allergenicity of red gram proteins. IgE immunoblot detected five proteins; one of 66 kDa, three of 45 kDa (pI of ∼5.3, 5.9 and 6.6) and one of 30 kDa. All these proteins showed homology to known allergens of soybean (different subunits of β-conglycinin), lentil (Len c1 and Len c2), peanut (Ara h1) and pea (vicilin). In conclusion, five novel IgE binding proteins (namely Caj c1, Caj c2, Caj c3, Caj c4 and Caj c5) were identified as putative clinically relevant allergens.

Introduction

Red gram (Cajanus cajan L. Millsp), commonly known as “pigeon pea” or “Arhar” is a perennial plant that belongs to family Leguminosae. It is now cultivated throughout the tropical and subtropical areas of the world. Red gram is nutritionally very important, as it contains high levels of protein (about 22%) with presence of important essential amino acids like methionine, lysine and tryptophan (Salunkhe et al., 1986).

Red gram is consumed as whole/split pulse and in form of green vegetable to supplement cereal-based diet. Apart from its medicinal role in jaundice, diabetes, bronchitis, coughs, genital irritation, pneumonia and skin problems; utilization of red gram for human nutrition may be constrained by the presence of protease inhibitors (Ambekar et al., 1996, Akande and Balogun, 2009) which are known to reduce the digestibility of proteins in general (Nielsen, 1991, Alonso et al., 2000). Stability to digestion against gastric enzymes is thought to be an important feature of many of the allergenic food proteins (Astwood et al., 1996, Thomas et al., 2004). High consumption of any proteinaceous food may increase the chances of sensitization against the potentially allergenic proteins in susceptible individuals (Dalal et al., 2002) Allergens of peanut and soybean have been isolated, identified, cloned and well defined (Helm et al., 2000, Koppelman et al., 2003). Some data exist on adverse reaction to pea (Sanchez-Monge et al., 2004), chickpea (Mercedes et al., 2000) and lentil (Sanchez-Monge et al., 2000). Therefore, the present study was undertaken to assess the allergenic potential of red gram in Balb/c mice and then further extended to human population. Identification and characterization of allergens is an important step for immunotherapy and clinical screening. Moreover, identification of allergens is also helpful for the development of transgenic non-allergenic variety. The objective of the current investigation was to identify and partly characterize the seed proteins identified by the sera IgE from red gram-sensitive patients exhibiting rhinitis and bronchial asthma symptoms.

Section snippets

Test material and reagents

All the chemicals were of highest grade purity available. RPMI-1640, antibiotic–antimycotic solution, fetal bovine serum (FBS), pepsin from porcine gastric mucosa (Cat #P-6887), O-phenylenediamine (OPD), bovine serum albumin (BSA), reagents for sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and goat anti-human IgE peroxidase conjugate(cat #A9667) were purchased from Sigma Chemical Company, St. Louis, USA. The anti-human IgE antibody preparation is specific for human IgE

SGF digestibility of red gram CPE

As pepsin stability of a protein is one of the distinguishing features of allergens, SGF assay was carried out to detect the pepsin resistant proteins of red gram. SDS–PAGE of red gram CPE resulted in nine proteins of mol wts 18–130 kDa (Fig. 2, lane 2). Out of nine, two proteins of 45 and 30 kDa were stable up to 60 min (Fig. 2, lane 11), whereas 66 kDa protein got digested after 4 min in SGF (Fig. 2, lane 8).

Antigen challenge of sensitized mice resulted in anaphylactic symptoms and increased histamine levels

To measure the extent of anaphylactic responses induced by red gram proteins, allergic

Discussion

Allergy to legumes is well known, with peanut being the most prominent and soybean as the next most important allergenic legume. In the current study, we have reported the allergenic potential of red gram and identified five proteins that could be regarded as candidate allergens of red gram. Stability to digestion against gastric enzymes is thought to be an important feature of allergenic food proteins (Astwood et al., 1996, Besler et al., 2001, Thomas et al., 2004). The theoretical basis for

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

We are grateful to the Director of the Institute for his keen interest in this study. Thanks are due to SIP-08 of Council of Scientific and industrial Research (CSIR), New Delhi for financial support. Amita Misra, Rahul Kumar and Vivek Mishra are thankful to Indian Council of Medical Research (ICMR), CSIR and University Grant Commission (UGC) New Delhi, respectively, for the award of their Senior Research Fellowships. Authors thank Mr. B.D. Bhattacharji for his help in editing of the

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