Type of Document	Dissertation
Author	Sharma, Girdhari M
Author's Email Address	gms06@fsu.edu
URN	etd-02262010-140241
Title	Identification and Characterization of Select Allergens in Pecan [Carya illinoinensis (Wangenh.) K. Koch] and Brazil Nut (Bertholletia excelsa L.) Seeds
Degree	Doctor of Philosophy
Department	Nutrition, Food, and Exercise Science, Department of
Advisory Committee	Advisor Name Title Shridhar K. Sathe Committee Chair Bahram H. Arjmandi Committee Member Thomas C.S. Keller III Committee Member Kenneth H. Roux University Representative
Keywords	11S Legumin 2S Albumin Allergy Brazil Nut Pecan
Date of Defense	2010-02-03
Availability	unrestricted
Abstract Tree nut allergies affect up to 0.2% young children and 0.5% adults in the US. The current investigation focused on two tree nuts (pecan and Brazil nut), with four specific aims: (i) to clone and characterize 2S albumin, a major allergen in pecan, (ii) to clone and characterize 11S legumin, a major allergen in pecan, (iii) to develop a sensitive and robust competitive ELISA for Brazil nut detection, and (iv) to purify and characterize BN seed allergens. Pecan cDNA expression library was constructed to meet specific aims (i) and (ii). The genes corresponding to 2S albumin and 11S legumin in pecan was amplified and expressed as fusion proteins. The fusion proteins were screened for IgE-binding with pecan allergic human sera. The corresponding native protein in pecan was identified using proteomic tools and inhibition immunoblots. The cross-reactivity of fusion protein with corresponding walnut allergen was also assessed. Overlapping synthetic peptides were used to determine the linear epitopes. Homology modeling of pecan allergens was done to obtain the structural insight and compare with known epitopes of corresponding allergens in other tree nuts. Of the 28 patients’ serum IgE tested by dotblot, 22 (79%) bound to 2S albumin (Car i 1) and 16 (57%) bound to 11S legumin (Car i 4). The native pecan 2S albumin is ~16 kDa composed of a large subunit (~12 kDa) linked to small subunit (~4 kDa) by disulfide bond. The native pecan 11S legumin is a hexameric protein, each monomer composed of ~33 kDa acidic subunit linked via disulfide bond to ~20-22 kDa basic subunit. IgE inhibition immunoblots suggested Car i 1 and Car i 4 to be cross-reactive with corresponding walnut allergens Jug r 1 and Jug r 4, respectively. Linear epitope mapping of Car i 1 indicated weak, moderate, and strong reactivity of serum pools against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all 3 serum pools, 5 peptides were strongly reactive. These strongly reactive polypeptides were located in 3 discrete regions of the Car i 1 sequence (residues 43-57, 67-78, and 106-120). Epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all 3 sera pools, of which 2 were strongly reactive. The strongly reactive peptides were located in 3 discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 1 and Car i 4 revealed significant overlapping regions shared in common with other tree nuts. Rabbit anti-Brazil nut polyclonal antibodies were used to develop the inhibition ELISA for specific aim (iii). The assay was evaluated for cross-reactivity and robustness. The developed ELISA was sensitive (IC50 = 23.2 ± 9 ng/ml, n=76). Among the 66 tested foods/ingredients, only cinnamon exhibited detectable interference (1.36%). The ELISA could detect Brazil nut seed proteins over a pH range of 5-12, with optimal pH range of 7-10. Exposing Brazil nut seeds to processing did not adversely affect the nut seed protein detection using the assay. Brazil nut seed protein recovery from 100 mg of foods spiked with 10 and 1 μg of soluble Brazil nut proteins or 100 and 10 μg of defatted Brazil nut flour exhibited a wide recovery range, 63-315%, indicating protein-food matrix interaction. Brazil nut storage proteins were purified using column chromatography to meet specific aim (iv). Analytical ultracentrifugation of the purified Brazil nut albumin, vicilin and legumin proteins registered sedimentation coefficients of 1.8S, 7.1S and 11.8S, respectively. Under reducing conditions, the major polypeptide bands in 2S albumin were observed at 6.4, 10-11, and 15.2 kDa. The 7S vicilin was composed of one 12.6 kDa, two ~38-42 kDa, and two ~54-57 kDa polypeptides, while 11S globulin contained two major classes of polypeptides: ~30-32 kDa and ~20-21 kDa. The 7S vicilin is a glycoprotein. The estimated molecular mass and Stokes’ radius for 2S albumin and 7S and 11S globulins were (19.2 kDa, 20.1 Å), (114.8 kDa, 41.1 Å), and (289.4 kDa, 56.6 Å), respectively. Circular dichroism spectroscopic analysis indicated the secondary structure of the 3 proteins to be mainly -sheets and turns. Emission fluorescence spectra of the native proteins registered a λmax at 337, 345, and 328 nm for 2S albumin and 7S and 11S globulin, respectively. When probed with rabbit anti-Brazil nut polyclonal antibodies, 7S vicilin exhibited higher immunoreactivity than 2S albumin and 11S globulin.
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