Girdhari M Sharma

Florida State University, Tallahassee, Florida, United States

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Publications (12)32.29 Total impact

  • Shridhar K Sathe · Harshal H Kshirsagar · Girdhari M Sharma
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    ABSTRACT: Effects of different solvents, ionic strength, and pH on Inca peanut seed protein solubility were assessed by quantitatively analyzing solubilized proteins using Lowry and Bradford methods. Soluble proteins were fractionated using Osborne procedure and the polypeptide composition of solubilized proteins was determined by one dimensional 25 % monomer acrylamide linear gradient SDS-PAGE. Osborne protein fractions were analyzed by the 2D gel electrophoresis. Total seed proteins were efficiently solubilized by 2 M NaCl among the tested solvents. The soluble seed proteins registered a minimum solubility at pH ~4.0. Osborne protein fractions, albumins, globulins, prolamins, and glutelins accounted for 43.7, 27.3, 3.0, and 31.9 %, respectively, of the total aqueous soluble proteins. Soluble seed flour proteins are mainly composed of polypeptides in the MW range of 6-70 kDa of which the predominant polypeptides were in the 20-40 kDa range. Prolamin fraction was mainly composed of four polypeptides (MW < 15 kDa). Glycoprotein staining indicated 32-35 and <14 kDa peptides to be positive.
    No preview · Article · Aug 2012 · Plant Foods for Human Nutrition
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    ABSTRACT: Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential 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 three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.
    No preview · Article · Jun 2011 · Journal of Agricultural and Food Chemistry
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    ABSTRACT: Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain to be identified and characterized. The 2S albumin gene was amplified from the pecan cDNA library. Dot-blots were used to screen the recombinant protein with pecan allergic patients' serum. The affinity purified native protein was analyzed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS) analysis. Cross-reactivity with walnut was determined by inhibition enzyme-linked immunosorbent assay (ELISA). Sequential epitopes were determined by probing the overlapping peptides with three different patients' serum pool. The 3-dimensional homology model was generated, and the locations of the pecan epitopes were compared with those of known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot-blot, 22 (79%) bound to 2S albumin, designated as Car i 1. Edman sequencing and the MS/MS sequencing of native 2S albumin confirmed the identity of recombinant (r) Car i 1. Both pecan and walnut protein extracts inhibited the IgE-binding to rCar i 1. Sequential epitope mapping indicated weak, moderate, and strong reactivity against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all serum pools, 5 peptides were strongly reactive and located in 3 discrete regions of the Car i 1 (amino acids 43-57, 67-78, and 106-120). Three-dimensional modeling revealed IgE-reactive epitopes to be solvent accessible and share significant homology with other tree nuts providing a possible basis for previously observed cross-reactivity.
    No preview · Article · Mar 2011 · Journal of Agricultural and Food Chemistry
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    ABSTRACT: Native, undenatured amandin and anacardein secondary structures were estimated to be, respectively, 56.4 and 49% β-sheet, 14 and 23.7% α-helix, and 29.6 and 27.4% random coil. Circular dichroic (CD) and fluorescence spectroscopy were used to assess structural changes in amandin and anacardein subjected to denaturing treatments that included heat (100 °C, 5 min), guanidium HCl (GuHCl), urea, sodium dodecyl sulfate (SDS), and reducing agent, 2% v/v β-mercaptoethanol (βME) + heat. Mouse monoclonal antibodies (mAbs) 4C10 and 4F10 directed against amandin and 1F5 and 4C3 directed against anacardein were used to assess the influence of denaturing treatments on the immunoreactivity of amandin and anacardein. Among the denaturing treatments investigated, SDS and β-ME caused a significant reduction in the immunoreactivity of amandin and anacardein when probed with mAb 4C10 and 4C3, respectively.
    No preview · Article · Jan 2011 · Journal of Agricultural and Food Chemistry
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    ABSTRACT: Brazil nut storage proteins, 2S albumin, 7S vicilin, and an 11S legumin, were purified using column chromatography. Analytical ultracentrifugation of the purified albumin, vicilin, and legumin proteins, respectively, registered sedimentation coefficients of 1.8, 7.1, and 11.8 S. Under reducing conditions, the major polypeptide bands in 2S albumin were observed at 6.4, 10-11, and 15.2 kDa. The 7S globulin was composed of one 12.6 kDa, two approximately 38-42 kDa, and two approximately 54-57 kDa polypeptides, whereas the 11S globulin contained two major classes of polypeptides: approximately 30-32 and approximately 20-21 kDa. The 7S globulin stained positive when reacted with Schiff reagent, indicating that it is a glycoprotein. The estimated molecular mass and Stokes radius for 2S albumin and 7S and 11S globulins were 19.2 kDa and 20.1 A, 114.8 kDa and 41.1 A, and 289.4 kDa and 56.6 A, respectively. Circular dichroism spectroscopic analysis indicated the secondary structure of the three proteins to be mainly beta-sheets and turns. Emission fluorescence spectra of the native proteins registered a lambda(max) at 337, 345, and 328 nm for 2S albumin and 7S and 11S globulins, respectively. When probed with anti-Brazil nut seed protein rabbit polyclonal antibodies, 7S globulin exhibited higher immunoreactivity than 2S albumin and 11S globulin.
    Preview · Article · May 2010 · Journal of Agricultural and Food Chemistry
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    ABSTRACT: Amandin, the primary storage protein in almonds, contains key polypeptides recognized by almond-allergic patients. A variety of food matrices representing diverse categories of foods were analyzed to assess the effect of food matrix on amandin recognition and recovery using rabbit polyclonal antibody based immunoassays. Food matrices from dairy, nuts, and vegetables typically resulted in over-estimation of amandin. Some foods representing legumes and cereals resulted in over-estimation while others in under-estimation of amandin. The amandin recovery range was 116–198 μg/100μg (dairy) 110–292 μg/100μg (tree nuts), 43–304 μg/100μg (legumes), 106–183 μg/100μg (most cereals- with the exception of barley, whole-wheat flour, wild rice and raisin bran whole mix). Amandin recovery from spices was typically low (2–85 μg/100μg) with a few exceptions where higher recoveries were observed (121–334 μg/100μg). Salt (black and white), tea, confectionery (sugar, cocoa, dark chocolate), and fruits (1–83 μg/100μg) generally resulted in lower recoveries. Tested food matrices did not adversely affect amandin immunorecognition in Western blots. The pH and the extraction buffer type affected amandin recovery. The results suggest that food matrix effects as well as extraction conditions need to be carefully evaluated when developing immunoassays for amandin detection and quantification.
    No preview · Article · May 2010 · Lebensmittel-Wissenschaft und-Technologie
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    ABSTRACT: Borate saline buffer (0.1 M, pH 8.45) solubilized proteins from almond, Brazil nut, cashew nut, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean seeds were prepared from the corresponding defatted flour. The yield was in the range from 10.6% (macadamia) to 27.4% (almond). The protein content, on a dry weight basis, of the lyophilized preparations ranged from 69.23% (pine nut) to 94.80% (soybean). Isolated proteins from Brazil nut had the lightest and hazelnut the darkest color. Isolated proteins exhibited good solubility in aqueous media. Foaming capacity (<40% overrun) and stability (<1 h) of the isolated proteins were poor to fair. Almond proteins had the highest viscosity among the tested proteins. Oil-holding capacity of the isolated proteins ranged from 2.8 (macadamia) to 7 (soybean) g of oil/g of protein. Least gelation concentrations (% w/v) for almond, Brazil nut, cashew, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean were, respectively, 6, 8, 8, 12, 20, 12, 10, 14, 14, and 16.
    Full-text · Article · Mar 2010 · Journal of Agricultural and Food Chemistry
  • Shridhar K Sathe · Girdhari M Sharma
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    ABSTRACT: Food allergies are on the rise in Western countries. With the food allergen labeling requirements in the US and EU, there is an interest in learning how food processing affects food allergens. Numerous foods are processed in different ways at home, in institutional settings, and in industry. Depending on the processing method and the food, partial or complete removal of the offending allergen may be possible as illustrated by reduction of peanut allergen in vitro IgE immunoreactivity upon soaking and blanching treatments. When the allergen is discretely located in a food, one may physically separate and remove it from the food. For example, lye peeling has been reported to produce hypoallergenic peach nectar. Protein denaturation and/or hydrolysis during food processing can be used to produce hypoallergenic products. This paper provides a short overview of basic principles of food processing followed by examples of their effects on food allergen stability. Reviewed literature suggests assessment of processing effects on clinically relevant reactivity of food allergens is warranted.
    No preview · Article · Aug 2009 · Molecular Nutrition & Food Research
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    ABSTRACT: The solubility of almond, Brazil nut, cashew nut, hazelnut, macadamia, pecan, pine nut, pistachio, walnut, and peanut proteins in several aqueous solvents was qualitatively and quantitatively assessed. In addition, the effects of extraction time and ionic strength on protein solubility were also investigated. Electrophoresis and protein determination (Lowry, Bradford, and micro-Kjeldahl) methods were used for qualitative and quantitative assessment of proteins, respectively. Depending on the seed, buffer type and ionic strength significantly affected protein solubility. The results suggest that buffered sodium borate (BSB; 0.1 M H(3)BO(3), 0.025 M Na(2)B(4)O(7), 0.075 M NaCl, pH 8.45) optimally solubilizes nut seed proteins. Qualitative differences in seed protein electrophoretic profiles were revealed. For a specific seed type, these differences were dependent on the solvent(s) used to solubilize the seed proteins. SDS-PAGE results suggest the polypeptide molecular mass range for the tree nut seed proteins to be 3-100 kDa. The results of native IEF suggested that the proteins were mainly acidic, with a pI range from >4.5 to <7.0. Western immunoblotting experiments indicated that rabbit polyclonal antibodies recognized substantially the same polypeptides as those recognized by the corresponding pooled patient sera IgE.
    No preview · Article · Aug 2009 · Journal of Agricultural and Food Chemistry
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    ABSTRACT: Soybean flours stored for 20 years at -20 degrees C retained protein polypeptide profile integrity. Proteins in stored soybean flours retained their immunoreactivity. Long-term frozen storage of seed flours at -20 degrees C did not adversely affect seed protein in vitro pepsin digestibility.
    No preview · Article · Mar 2009 · Journal of Agricultural and Food Chemistry
  • Girdhari M Sharma · Kenneth H Roux · Shridhar K Sathe
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    ABSTRACT: Undeclared Brazil nut residue in food products is of great concern because it can trigger life-threatening allergic reactions in sensitive patients. A rabbit polyclonal antibody-based competitive ELISA (IC(50) = 23.2 +/- 9 ng/mL, n = 76) with good sensitivity, detection range of 10-90 ng/mL, was developed. The ELISA could detect Brazil nut seed proteins over a pH range of 5-12. The optimal pH range for the detection assay was 7-10. Among the 66 tested foods/ingredients, only cinnamon exhibited statistically significant interference (1.36%, p = 0.05). 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 microg of soluble Brazil nut proteins or 100 and 10 microg of defatted Brazil nut flour exhibited a wide recovery range, 63-315%, indicating protein-food matrix interaction.
    No preview · Article · Jan 2009 · Journal of Agricultural and Food Chemistry
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    Girdhari M. Sharma
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    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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