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Allergenic Cross-Reactivity Between Cashew and Pistachio Nuts Pallavi D

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Allergenic Cross-Reactivity Between Cashew and Pistachio Nuts Pallavi D Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2004 Allergenic Cross-Reactivity Between Cashew and Pistachio Nuts Pallavi D. Tawde Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES ALLERGENIC CROSS-REACTIVITY BETWEEN CASHEW AND PISTACHIO NUTS By PALLAVI D TAWDE A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science Degree Awarded: Fall Semester, 2004 The members of the Committee approve the Thesis of Pallavi D Tawde defended on Nov 15, 2004. Kenneth Roux Professor Directing Thesis Shridhar Sathe Outside Committee Member Tom Keller Committee Member Approved: Timothy Moerland, Chair, Department of Biological Science The Office of Graduate Studies has verified and approved the above named committee members. ii For my parents iii ACKNOWLEDGEMENTS First and foremost, I would like to thank Florida State University and the Department of Biology for providing me with the opportunity to conduct the research of my interest. I would like to offer my gratitude to my committee members, Dr. Shridhar Sathe, and Dr. Tom Keller for their consistent support and advice on this project. Dr. Sathe and his lab provided us with valuable tree nut protein samples, which formed an essential component of this research. Most importantly, I would like to thank my committee members for being highly accommodative and having agreed to read my submitted thesis in less than a week’s time. For having provided me with tree nut allergic patients sera, I would like to thank Dr. Suzanne Teuber of University of California, Davis. I want to take this opportunity to specially thank Rani Dhanrajan, and Margaret Seavy for offering their technical expertise on issues related to this project. A special thanks to all my lab-mates, Jason Robotham, Henry Grise, Lauren Porter, Vanessa Seamon, Dillon Fritz and Richard Penny, for their support and encouragement. I would like to thank each one of them personally for making it such a fun experience even in the most trying times. My heartfelt thanks to Manacy Pai for her unwavering support and help. I thank my parents and family for all their encouragement. Finally, I would like to tender my sincere appreciation to my advisor, Dr. Kenneth Roux for having invested his time, resources and more importantly his unbiased criticism on every step of the way. He always provided me with complete freedom in any and every endeavour I wished to pursue. This has contributed to my overall learning experience, not merely as a researcher but also as a writer, and a thinker. iv TABLE OF CONTENTS List of Tables ................................................................................................ Page vii List of Figures ................................................................................................ Page viii Abstract ....................................................................................... Page xi 1. INTRODUCTION .......................................................................................... Page 1 Food allergy ................................................................................................ Page 1 Peanut and tree nut allergy.......................................................................... Page 4 Allergic cross-reactivity.............................................................................. Page 4 Tree nut allergens........................................................................................ Page 5 The cupin superfamily ................................................................................ Page 5 11S globulins (Legumin) ............................................................................ Page 6 2S albumins ................................................................................................ Page 7 Allergenic cross-reactivity between peanut and tree nuts .......................... Page 8 Allergenic cross-reactivity between tree nuts and coconut ........................ Page 8 Cross-reactivity between tree nuts.............................................................. Page 8 Cross-reactivity between pistachio nut and other members of the ............. Page 9 family, Anacardiaceae Aims and findings of the thesis work ......................................................... Page 9 2. MATERIALS AND METHODS.................................................................... Page 11 Total cashew and pistachio protein extracts and cashew major ................. Page 11 protein (CMP) preparation Production of rabbit anti-cashew globulin polyclonal antiserum............... Page 11 Human allergic sera ................................................................................... Page 11 v One dimensional electrophoresis (SDS-PAGE) and immunoblot.............. Page 12 Two dimensional gel electrophoresis ......................................................... Page 13 Staining of the 2-DE gel with colloidal Coomassie blue ........................... Page 14 Immunoblot of 2-DE separated proteins .................................................... Page 14 Transblot for N-terminal amino acid sequencing ...................................... Page 15 Inhibition immunoblot with patients #38 and #270 sera ............................ Page 15 ELISA ........... Page 16 Inhibition ELISA ........................................................................................ Page 17 Construction and IgE immunoscreening of cashew cDNA library ............ Page 17 and 2S albumin (Ana o 3) preparation 3. RESULTS ................................................................................................ Page 19 Identification of immuno-reactive antigens and allergens in total ............. Page 19 cashew and purified cashew globulin extracts SDS-PAGE and Immunoblotting................................................................ Page 19 Two-dimensional electrophoresis of cashew proteins................................ Page 20 2-DE immunoblotting of total cashew extract proteins with rabbit anti- ... Page 21 cashew polyclonal antisera and pooled human cashew and tree nut allergic sera Homology searches of Ana o 2 using NCBI BLAST Program .................. Page 22 Identification of immuno-recative antigens and allergens in total pistachio Page 23 extract SDS-PAGE and immunoblotting................................................................ Page 23 2-DE and immunoblotting using rabbit anti-cashew globulin antisera ...... Page 24 and pooled sera from cashew and tree nut allergic patients vi Detection of specific IgE antibodies to cashew and pistachio proteins...... Page 27 by ELISA Cross-reactivity of cashew and pistachio, Inhibition ELISA ..................... Page 28 Inhibition of IgE reactivity of pre-adsorbed sera to cashew....................... Page 28 Inhibition of IgE reactivity of pre-adsorbed sera to CMP .......................... Page 29 Inhibition of IgE reactivity of pre-adsorbed sera to pistachio .................... Page 30 Inhibition of IgE reactivity of pre-adsorbed sera to Ana o 3...................... Page 30 Cross-inhibition immunoblot to detect cross-reactivity between ............... Page #32 cashew and pistachio proteins IgE reactivity of pre-adsorbed sera from patient #38 towards.................... Page 33 cashew and pistachio extract IgE reactivity of pre-adsorbed patient #270 sera towards cashew ............. Page 34 and pistachio extract 4. DISCUSSION ................................................................................................ Page 36 REFERENCES ................................................................................................ Page 42 BIOGRAPHICAL SKETCH ............................................................................... Page 49 vii LIST OF TABLES Table 1: Clinical characteristics of cashew and tree nut allergic patients ........... Page 12 Table 2: NCBI-BLAST – proteins showing homology to Ana o 2 ..................... Page 22 Table 3: Comparison of the N-terminal amino acid sequences of pistachio ...... Page 25 protein spots 1, 2, 3, and 4 with those of various 11S globulins Table 4: Alignment of N-terminal amino acid sequences of pistachio................ Page 26 protein spots A, B, C, and D with basic subunits from various 11S globulins Table 5: Alignment and comparison of the N-terminal amino acid sequence..... Page 26 of spot E with various 2S albumins from the NCBI database viii LIST OF FIGURES Figure 1: IgE binding to the mast cell via Fcε receptors .................................... Page 2 Figure 2: Allergen cross-linking IgE antibodies leading to activation of mast cells Page 3 Figure 3: Sequence of events and symptoms of mast /basophil degranulation .. Page 3 Figure 4: Comparative SDS-PAGE analysis and immunoblotting of proteins .. Page 19 from total cashew extract (T) and cashew globulin (A) Figure 5: Two dimensional gel electrophoresis (2-DE) of water and salt soluble Page 20 proteins from total cashew and cashew globulin extracts stained with colloidal Coomassie blue Figure 6A: 2-DE immunoblot analysis of cashew globulin with rabbit anti-cashew Page 21 globulin antisera Figure 6B: Cashew globulin 2-DE immunoblot probed with pooled cashew and Page 21 tree nut allergic human sera Figure 7: Total cashew extract 2-DE immunoblot probed with pooled human .. Page 22 cashew allergic sera
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