Allergenic Cross-Reactivity Between Cashew and Pistachio Nuts Pallavi D

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2004 Allergenic Cross-Reactivity Between Cashew and Pistachio Nuts Pallavi D. Tawde

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THE FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS AND SCIENCES

ALLERGENIC CROSS-REACTIVITY BETWEEN CASHEW AND PISTACHIO NUTS

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.

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.

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

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

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

Figure 8: Comparative SDS-PAGE and immunoblotting profile of pistachio and Page 23 cashew extracts

Figure 9: 2-DE and immunoblotting of total pistachio extract proteins ...... Page 24

Figure 10: 2-DE immunoblot of pistachio extract proteins probed with pooled Page 25 cashew and tree nut allergic human sera and N-terminal sequencing of the immuno-reactive spots

Figure 11: IgE binding to total cashew and pistachio extracts of ten patients ... Page 27 sera with cashew and tree nut allergy

Figure 12: Comparison of IgE reactivity to total cashew extract, CMP, and Ana o 3 Page 28

Figure 13: Inhibition ELISA of total cashew extract ...... Page 29

Figure 14: Inhibition ELISA of CMP ...... Page 29

Figure 15: Inhibition ELISA of total pistachio extract ...... Page 30

Figure 16: Inhibition ELISA of r Ana o 3 ...... Page 31

Figure 17: Dose-related curves of inhibition of IgE binding to cashew and ...... Page 32 pistachio with sera from patients #38 and #270, preadsorbed with cashew and pistachio extracts

Figure 18: Cashew and pistachio 1-DE immunoblot with patient #38 sera ...... Page 33 preadsorbed with pistachio or cashew extracts

Figure 19: Cashew and pistachio 2-DE inhibition immunoblot probed with...... Page 34 patient #38 sera preadsorbed with pistachio or cashew extracts

Figure 20: Cashew and pistachio 1-DE immunoblot and preadsorption of...... Page 35 patient #270 sera with pistachio or cashew extracts

Figure 21: The 2-DE inhibition immunoblot of cashew and pistachio extract and Page 35 preadsorbed patient #270 sera with cashew or pistachio extracts

ABSTRACT

Rationale: Cashew and pistachio belong to Anacardiaceae family and strong allergenic cross- reactivity between nuts of these two species has been reported. The aim of our study was to identify the cross-reactive allergenic proteins from cashew and pistachio nuts.

Methods: Extracted cashew and pistachio nut proteins were separated by means of 1- and 2- dimensional PAGE. Pooled human sera from cashew-allergic patients was tested for reactivity to soluble cashew and pistachio proteins by IgE immunoblotting after one-dimensional (1-D) and 2-D electrophoresis. The identities of the IgE-reactive bands from the pistachio immunoblot were further analyzed by means of N-terminal amino acid sequencing and comparison to previously published data from the cashew. ELISAs were performed using individual sera from subjects with cashew and tree nut allergy to assess the degree of IgE reactivity to cashew and pistachio nut extracts. Inhibition ELISA studies were conducted to assess the degree of allergenic cross-reactivity between cashew and pistachio nuts.

Results: IgE immunoblots of cashew and pistachio extract probed with cashew-allergic sera identified proteins of 35kDa, 22kDa, and 7-9kDa. N-terminal amino acid sequencing of the IgE- reactive spots from pistachio immunoblot identified them as the acidic subunit, basic subunit of 11S globulin and 2S albumin seed storage proteins respectively. Seed storage proteins are known food allergens in cashew and have been designated as Ana o 1 (7S globulin), Ana o 2 (11S globulin) and Ana o 3 (2S albumin). ELISA results with ten individual cashew-allergic sera (two out of the ten patients have pistachio allergy, and the remaining eight patients had never eaten pistachio) showed IgE reactivity to both cashew and pistachio nut. Inhibition ELISA demonstrated that pre-incubation of sera with pistachio extract resulted in a marked decrease in IgE binding to cashew extract, and vice versa indicating allergenic cross-reactivity.

Conclusion: The results demonstrate the presence of cross-reactive B cell epitopes on cashew and pistachio nut allergens. The plant taxonomic classification of cashew and pistachio nuts does appear to predict allergenic cross-reactivity.

CHAPTER 1

INTRODUCTION

Food Allergy

Food allergy is defined as an adverse immunological (hypersensitivity) response to food and encompasses a range of disorders. This group of conditions includes acute, potentially fatal reactions, and a host of chronic diseases that mainly affect the skin and gastrointestinal tract. The Type I allergy/hypersensitivity represents a relevant health problem in industrialized countries and affects almost 500 million people worldwide 1.

Most type I food allergies appears in the first 2 years of life and occurs in 6–8% of infants 2. As their immune systems mature (by 5 years or so), 80% of allergic infants will lose their food allergies 3. Food allergy appears to be less common in adults. Recent epidemiologic studies suggest that nearly 4% of Americans are afflicted with IgE- mediated food allergies, a prevalence much higher than appreciated in the past 4. Food allergens range from fruits and vegetables to meats; the big 8 foods have been identified as the most frequent human food allergens and account for 90% of food allergies. They are milk, egg, peanut, tree nut (walnut, cashew, almonds etc), fish, shellfish, soy and wheat. The typical allergens of infancy and early childhood are egg, milk, peanut, wheat and soy, whereas allergens responsible for causing severe reactions in older children and adults are mainly by peanuts, tree nuts and seafood 1;5.

While fruits mainly cause oral symptoms, legume seeds and nuts are likely to provoke acute generalized symptoms and even anaphylactic reaction. Symptoms range from mild rashes to life-threatening systemic anaphylaxis and are of four main types: dermatological (hives, local swelling, dermatitis, and eczema), gastrointestinal (nausea, vomiting, diarrhea, and abdominal pain), respiratory (runny nose, asthma, and tightening of the throat), and systemic (anaphylactic shock, organ failure, cardiac arrhythmia, and death).

Food allergic disorders can be broadly divided into those that are mediated by IgE antibodies and those that are not. The acute onset of symptoms, following ingestion of offending food mediated by IgE antibodies, tissue mast cells and blood basophils, results in a state termed sensitization. Upon re-exposure, the causal food protein(s) cross-links the effector cell (mast cell, basophil) – bound specific IgE molecules and triggers the release of inflammatory mediators (histamine, serotonin, leukotrienes), causing the symptoms of allergy. The non-IgE mediated reactions to food are not as clearly defined.

The process of mast cell activation and degranulation is schematically outlined in the following figures. Allergic patients are characterized by their intrinsic tendency to form IgE antibodies to otherwise harmless antigens and allergen specific IgE are produced upon re-exposure to a food allergen. The IgE antibodies bind to the Fcε receptors present on the surface of mast cells and basophils. Upon subsequent exposure or ingestion of the food allergen, the specific effector cell bound IgE molecules are cross-linked by the food

1 allergens on the surface, which activate and trigger the mast cells and basophils to release inflammatory mediators. Both mast cells and basophils contain special cytoplasmic granules that store mediators of inflammation. There are two categories of inflammatory (anaphylactic) mediators in mast cells and basophils. Preformed mediators, stored in secretory granules and secreted upon cell activation, typically include histamine, proteoglycans, either heparin, chondroitin sulphates or both, and a spectrum of neutral proteases. Newly generated mediators, often absent in the resting mast cells, are typically produced during activation and consist of arachidonic acid metabolites, principally leukotriene C4 (LTC4) and prostaglandin D2 and cytokines. Of particular interest in humans is the production of (TNF-α), IL-4, IL-5 and IL-6. Allergic reactions can occur within minutes to a few hours after eating the offending food.

Mast cells and IgE antibodies

Mast cells express FcεRI and Fc RII receptors that ε Fcε receptor I specifically recognize IgE (FcεRI) antibodies IgE is bound to Fcε receptors Antigen-binding to IgE induces clustering of Fcε receptors and IgE antibody thereby activates mast cell

Fig. 1. IgE binding to the mast cell via Fcε receptors.

Activation of mast cells

Resting mast cell Activated mast cell

Fcε receptor I (FcεRI)

IgE antibody

Fig. 2. Allergen cross-linking IgE antibodies leading to activation of mast cells.

Effects of mast cell activation

Mast cell activation and granule release

Gastrointestinal tract Airways Blood vessels

Increased fluid secretion, Decreased diameter, Increased blood flow, incresased peristalsis increased mucus secretion Increased permeability

Congestion and blockage of increased fluid in tissues Expulsion of gastrointestinal airways (wheezing, coughing, causing increased flow of tract contents plegm) lymph to lymph nodes, increased cells and protein (diarrhea, vomiting) Swelling and mucus secretion in tissue in nasal passages increased effector response

Fig. 3. Sequence of events and symptoms of mast /basophil degranulation

3 Peanut and tree nut allergy

Peanut and tree nut allergy is characterized by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence 1. There appears to be a strong clinical association between peanut and tree nut allergy. Allergies to peanuts and tree nuts are severe, common, and long lasting 1;5 and there is a strong clinical impression that allergies to these foods have increased. Peanut and tree nut allergies are particularly severe, common, and rarely outgrown.

Tree nuts encompass a group that includes walnut, cashew, almond, pistachio, hazelnut, pecan, macademia nut, pine nut, and Brazil nuts. Patients allergic to tree nuts do not outgrow or lose their sensitivity during their lifetime, akin to peanut allergy. Approximately 0.5% of the US population is considered to be allergic to tree nuts 5. Data from a voluntary registry of peanut and tree nut allergic US patients reveals that 62% of those reporting allergy to tree nuts record sensitivity to walnut, 44% report sensitivity to cashew, and 29% to hazelnut 1;5.

Allergic cross-reactivity

Patterns of allergic cross-reactivities, are important because they may reflect (a) the pattern of clinical sensitivities, (b) regulation of allergic sensitization via B- and T-cell epitope cross-reactivity, and (c) the need for screening of novel foods for allergenicity 6. A large proportion of patients with pollen allergies show hypersensitivity after eating fresh fruit or vegetables. For example, the cross-reactivity of the major allergens of birch pollen (Bet v 1), sweet cherry (Pru a 1), apple (Mal d 1), pear (Pyr c 1), celery tuber (Api g 1), and carrot (Dau c 1) were traced to high structural and amino acid sequence similarities 7. The cross-reactivity of IgE epitopes can be traced to a single amino acid and mutation studies illustrate that small changes in amino acid sequence can result in a significant reduction of allergenicity. Similar results have been reported in the study of cross-reactivity of profilins from birch and celery 8. In contrast, studies with the latex allergen Hev b 5 indicate that amino acid substitutions in multiple epitopes are necessary for reduction in IgE binding 9. In some cases high sequence homology is related to pan- allergenicity as in lipid transfer proteins 10, while in other cases high homology does not always result in cross-reactivity as in birch and carrot cyclophylins 11.

The FAO/WHO expert recommendations provide guidelines for the assessment of potential cross-reactive allergenicity of food proteins (FAO/WHO 2001). These include sequence comparison and several steps of homology search against protein databases and multiple sequence analysis. According to the recommendations, cross-reactivity has to be considered if there is >35% homology with any known allergen across any 80-amino acid window of a query protein, or if there is an identity of six or more contiguous amino acids. However, similarity in short stretches of a linear amino acid sequence is unlikely to result in cross-reactivity between two proteins unless they share similar protein folding. Identification of common structural motifs 12;13 is likely to improve the quality of assessment of cross-reactivity and allergenicity. More advanced methods of

4 establishing the structure-activity relationship will provide further insight into the molecular basis of allergenicity.

Tree nut allergens

Most tree nut allergens identified to date are seed storage proteins and belong to major protein superfamilies such as the vicilins (7S trimeric globulins composed of ~50 kDa subunits), legumins (11-13S hexameric globulins with subunits composed of 30 to 40 kDa acidic and 17 to 20 kDa basic peptides), and 2S albumins (~15 kDa), with ~9 and ~5 kDa subunits 14-16. Seed storage proteins have been traditionally characterized according to their solubility as albumins (water soluble) and globulins (soluble in low-salt buffers), and by their sedimentation constants (e.g. 7S, 11S globulins and 2S albumins). Albumins and globulins are the major seed storage proteins of angiosperms and important cross- reactive food allergens 17. Some aspects of protein structure are known to be relevant for allergenicity, such as solubility, compactness, stability, and possibly an ability to interact with lipids in the membrane. In this regard the structures of 11S globulins and 2S albumins are highly compact, thereby conferring stability to thermal denaturation and to digestion by proteolytic enzymes.

The cupin superfamily

Cupins are very stable proteins and comprise a large super-family of proteins that are considered to have originated by divergent evolution from a common ancestor. The globular storage proteins called legumins (11S) and vicilins (7S) are two-domain cupins. They share a common architecture, which has been described as 'double-stranded α-helix' or 'jelly-roll' barrel-like structure.

Vicilins appear to have defense-related properties as well. The vicilins and legumin-like proteins are members of the cupin superfamily characterized structurally in plants by their β-barrel conformation and acidic and basic chain subunit polypeptides. The 7S globulins or vicilins are homo-trimeric proteins and were identified as allergens in peanuts, tree nuts and legumes. These allergens have been designated as Ara h 1 from peanuts 18, Ana o 1 from cashew 19, Jug r 2 from walnut 20, conglycinin from soybean 21, and Len c 1 from lentils 22. Linear epitope mapping by means of overlapping peptides have been used to identify the IgE-binding epitopes of Ara h 1 18. At least 23 different linear IgE-binding epitopes, located throughout the length of Ara h 1 were identified. Four of the peptides were immunodominant IgE-binding epitopes being recognized by more than 80% of the patients tested. Cashew Ana o 1 and peanut Ara h 1, share 27% identity and 45% similarity in amino acid sequence and presumably are similar in overall structure, however there was no significant sequence conservation between epitopes of the two allergens 19. Although it is still possible that conformational (discontinuous) epitopes could be shared in common, these observations may explain the lack of cross- reactivity between tree nut-reactive and peanut-reactive patients sera.

5 Legumins or 11S globulins are hexameric proteins. Each subunit is made up of an acidic and a basic chain linked by disulphide bonds. Among others, allergenic 11S globulins were characterized in peanut (Ara h 3, 4), hazelnut (Cor a 9), cashew nut (Ana o 2), and soybean (glycinin G1 and G2) 21;23-25.

The 2S albumins (members of the prolamin superfamily) are 10-14 kDa in size and are structurally related to the lipid transfer proteins (LTPs, 7-9 kDa) are also considered to be defense-related proteins. The 2S albumins are heterodimeric proteins with the two subunits linked by disulphide bonds. Allergenic 2S albumins were identified in mustard seeds (Sin a 1, Bra j 1), oilseed rape seed (Bra n 1), Brazil nut (Ber e 1), walnut (Jug r 1), peanut (Ara h 2, 6, 7), cashew (Ana o 3), and sesame seed 14;26-28[Robotham et al. submitted]. Although one immunodominant linear epitope was identified in Jug r 1, strong evidence for the existence of conformational epitopes was also obtained 26.

The LTPs, as the name implies, are believed to be involved in lipid exchange between cellular membranes and are also involved in cutin biosynthesis. Other tree nut allergens include profilins and hevein-related proteins. Profilins, heveins, and LTPs are considered panallergens because of their contribution to the allergenicity of a wide variety of pollens, nuts, seeds, fruits, and vegetables and their propensity for exhibiting a significant degree of IgE-mediated cross-reactivity 17.

Much of the recent work on tree nut allergens is derived from the study of recombinant proteins. The cDNA libraries of walnut, hazelnut, and cashew nut have been described 15;20;23;24, and are an excellent tool to study various tree nut allergens.

11S Globulins (Legumin)

The 11S globulins are hexameric heteroligomeric proteins of 360 kDa MW, with each subunit comprising an acidic polypeptide 30–40 kDa in size that is disulfide-linked to a 20 kDa basic polypeptide. The 11S globulins are synthesized, assembled, and sequestered into protein bodies in a complex process that includes several posttranslational modifications. Each polypeptide pair is initially synthesized as a single precursor and is cleaved post-translationally at an Asn-Gly bond by an asparaginyl endopeptidase 29.

A number of 11S globulins have been identified as major plant food allergens, and are found in abundance in tree nuts and are also allergenic in legumes like peanut and soybean 15;21;30. Several legumins identified as food allergens include Ana o 2 from cashew 24, Ara h 3 30, and Ara h 4 31 from the peanut; soybean G1 and G2 glycinins 21;32, Cor a 9 from hazelnut 23 and possibly a legumin from buckwheat 33;34, sesame seed 11S globulin 35 and also coconut and walnut 36. Furthermore, almond 11S globulin has also been identified as allergenic 37.

Soybean 11S globulin is synthesized as a single polypeptide precursor (preproglycinin) containing a signal peptide that gets removed co-translationally in the endoplasmic

6 reticulum 38. The resultant proglycinin assembles into trimers, which are then sorted to protein storage vacuoles. Finally, most, but not all, of the proglycinin is post- translationally cleaved by asparaginyl endopeptidase 39 resulting in a mature hetero- trimer consisting of disulfide bonded acidic and basic polypeptides 29. Following cleavage, the hetero-trimers pair up to form (300-450 kDa) hexamers of ~ 11S. The 11S globulins are non-glycosylated members of a complex family of seed storage proteins that can account for up to 50% of the total seed protein content in tree nuts 40. To date, five genes have been identified that encode glycinin subunits 41 leading to several isoforms of the proteins.

Linear epitope mapping of allergenic 11S globulins has been reported 24;30; Helm et al., 2000; Beardslee et al., 2000). The alignment of six available 11S globulin maps highlight five allergenic hot spots. Remarkably the biochemical characteristics and positional overlap of these epitope-spanning regions is unique. Several Ana o 2 epitopes were shown to overlap those of the peanut legumin group allergen (Ara h 3), in position but shared little sequence similarity. Considerably greater positional overlap and identity was observed between Ana o 2 epitopes and those of soybean glycinin 21;24;30;42. The majority of Ana o 2 linear epitopes (68% overall and 83% of the most strongly reactive) are on the acidic chain of the protein as detected with reactivity with human cashew allergic sera 24.

2S Albumins

The 2S albumins belong to a family of seed storage proteins that show the presence of a conserved skeleton of eight cysteine residues within the protein sequences 43. In addition these proteins have similar three-dimensional (3D) structures that are rich in α-helices and are held together by four disulfide bonds. They are typically hetero-dimeric proteins with large and small subunits having molecular weights of about 4 and 9 kDa, and are encoded on one message as part of a large precursor polypeptide. After removal of the signal peptide, the polypeptide is cleaved, and thereby producing two subunits linked by disulfide bonds.

The 2S albumins are characterized by their solubility in water and high contents of proline and glutamine 44. The 2S albumins have been identified as allergens from tree nuts and seeds. The identified tree nut allergenic 2S albumins include Ber e 1 from Brazil nut 45;46, Jug r 1 from the English walnut 47, Ana o 3 from cashew nuts 14[Robotham et al. submitted]. Ses i 2, is the clinically most important allergen of sesame seeds 48 and the allergenicity of sunflower seed 2S albumin SFA-8 is still under investigation 49. Mustard seeds have been reported to provoke strong allergic reactions sometimes leading to anaphylactic shock and the allergens responsible for these reactions have been characterized as Sin a 1 50, and Bra j 1 from oriental mustard seeds 51;52. A 10 kDa MW buckwheat 2S albumin, designated as BW10 kDa protein has been identified as an allergen 53. A 2S albumin from almonds has also been identified as an allergen 54. The 2S albumin seed storage proteins are major food allergen and can be viewed as

7 universal allergens among seeds in which they occur although they are not necessarily cross-reactive.

The linear epitope mapping of Sin a 1 from yellow mustard led to identification of two immunodominant regions on the protein. One is located very close to the hypervariable region of the 2S albumins, which forms a flexible loop between helices 3 and 4 43. The four amino acid residue epitope of Jug r 1, Arg-Gly-Glu-Glu, is also located in the hypervariable region 26 detected using overlapping peptide approach.

Allergenic cross-reactivity between peanut and tree nuts

In a study, the prevalence of allergenic cross-reactivity between peanut and tree nuts (almonds, Brazil nut, hazelnut) was reported 55. As a result multiple peanut and tree nut sensitivities observed in allergic patients may be due to cross-reactive IgE-targeted epitopes present in different peanut and tree nut allergens 17;55.

Allergenic cross-reactivity between tree nuts and coconut

Very few patients are allergic to coconut 1;56;57. In a study, substantial IgE cross- reactivity between the walnut and coconut was witnessed with sera from two patients with reported primary walnut allergy and secondary coconut allergy 36. IgE-reactive coconut extract bands were detected at 35, 36.5 and 55 kDa on a reducing immunoblot. Adsorption of the patients' sera with walnut, almond and peanut extracts inhibited IgE binding to these bands. Conversely, coconut only partially inhibited IgE reactivity to 35 and 36 kDa bands on a walnut immunoblot, suggesting that IgE resulting from primary walnut exposure was responsible for a secondary cross-reaction to coconut. Indirect evidence suggests that the coconut 35 and 36.5 kDa bands represent the 11S legumin-like subunits 20;58;59. The coconut 35 kDa protein is known to share similar physical and biochemical characteristics with other globulins of the legumin group, including soybean glycinin, pea legumin, and peanut arachin 58. These two studies support the incidence of cross-reactivity between coconut and legumin-like seed storage proteins.

Cross-reactivity between tree nuts

It has been estimated that 30% of patients who are allergic to at least one food in the nut group are allergic to several tree nuts 60. Considerable research has been conducted in recent years in an attempt to characterize the allergens that are most responsible for allergy sensitization and triggering. Both native and recombinant nut allergens have been identified and characterized and, for some, the IgE-reactive epitopes described. The major seed storage protein constituents of the nuts are legumins, vicilins, and 2S albumins. Allergens such as lipid transfer proteins, profilins, and members of the Bet v 1-related family, represent minor constituents in tree nuts. These allergens are frequently

8 cross-reactive with other food and pollen homologues, and are considered panallergens 15;17.

Cross-reactivity between pistachio nut and other members of the family, Anacardiaceae

Cashew and pistachio are members of the Anacardiaceae family and are implicated in tree nut allergies 15;61-65. Adverse reactions to other members of the Anacardiaceae family, e.g., mango fruit, have also been reported 64;66;67. As observed in earlier studies, significant serologic cross-reactivity among cashew and pistachio, members of the Anacardiaceae family has been reported. However pistachio allergy is not extensively investigated, resulting in limited information on pistachio allergens.

In one study of 42 patients with cashew allergy, seven of the 42 (17%) reported both cashew and pistachio allergies and all the children had positive cashew SPTs., twenty- nine children had positive SPTs for other tree nuts: pistachio (28 cases, 67%), almond (10 cases, 23.8%), hazelnut (6 cases, 14.3%), walnut (4 cases, 9.5%), pine nut (3 cases, 7%), pecan (2 cases, 4.8%) and Brazil nut (2 cases, 4.8%) 68.

Studies suggest that there may be cross-reactive allergens between pistachio, peanut, walnut, chestnut, almond and cashew nuts as well as pine nut and almond 15. There is a strong in vitro cross-reactivity between cashew and pistachio, both of which belong to the same botanical family 62-64.

Cross-reactivity between cashew and pistachio has been reported using the CAP- inhibition studies 62 and the presence of specific IgE antibodies to both cashew and pistachio nuts were demonstrated by CAP and immunoblotting 64. The strongest IgE- binding bands had similar molecular weights (15, 30 and 60 kDa) in cashew and pistachio nuts 64. The detection of allergens in pistachio nut and cross-reactive antigens in other members of the same plant family, specifically cashew and mango has also been observed by RAST inhibition 63. SDS-PAGE analysis followed by immunoblotting of cashew and pistachio nut extracts, demonstrated that serum from both patients recognized several pistachio and cashew allergens with a molecular weight ranging from 14-70 kDa. Cross-reactivity was detected between pistachio nut and mango seed but not with mango pulp 63.

Aims and findings of the thesis work

Cashew and pistachio nut belong to Anacardiaceae family, and have been reported to demonstrate allergenic cross-reactivity. There have been reports suggesting serologic as well as clinical cross-reactivity 62-64. Reports of cashew and pistachio allergy linked to the greater use of tree nuts in the food industry raises concerns for tree nut allergy and its impact on people with known or latent allergies to peanuts and other tree nuts. It is evident that we need careful biochemical and immunological studies to evaluate IgE- binding proteins in both nut species, with a goal of identifying homologous proteins that

9 can potentially elicit serious allergic cross-reactions. However none of the previous reports identify the cross-reactive allergens at the protein level. Our aim was to identity these cross-reactive allergens.

Of the known cashew allergens, the 11S globulins and 2S albumins are major allergens involved in cashew allergy 14;24[Robotham et al. submitted]. The 11S globulins represent the most abundant seed storage protein. It is the most widely expressed allergenic protein in cashew, walnut, Brazil nut, hazelnut, peanut and soybean The 11S globulins have been observed to be the most immunogenic of the seed storage proteins in cashew 24. Our studies have focused on the 11S globulins because of their abundance (>50% of seed protein) in cashew and their high sequence similarity with other tree nut and legume 11S globulin family of seed storage proteins 14;15;24.

In this thesis work, I have focused on detecting IgE-immunoreactivity to cashew and pistachio nut proteins. The sera from cashew and tree allergic patients, containing IgE reacting with both cashew and pistachio proteins, were used for detection and identification of cross-reactive proteins in these two nuts.

Two dimensional gel electrophoresis and immunoblotting, of cashew and pistachio proteins using sera from cashew and tree nut allergic patients has been applied to detect the IgE immunoreactive proteins. Several of these immunoreactive protein spots from cashew and pistachio have been analyzed by N-terminal amino acid sequencing. The N- terminal sequences have been identified as seed storage proteins belonging to the family of 11S globulins and 2S albumins, by means of the NCBI BLASTp homology program.

Inhibition ELISA and inhibition immunoblot studies have revealed allergenic cross- reactivity between cashew and pistachio proteins. Thus, biochemical and immunological studies with cashew and pistachio nut extracts have been significant in pinpointing the potential key players in allergenic cross-reactivity between these tree nuts belonging to the Anacardiaceae family.

CHAPTER 2

MATERIALS AND METHODS

Total cashew and pistachio protein extracts and cashew major protein (CMP) preparation

Soluble protein extract was prepared from defatted cashew and pistachio nut flour (provided by Dr. Shridhar Sathe), as previously described41. Whole cashew or pistachio flour extract was prepared by mixing 100 mg of defatted cashew flour with 1 ml borate buffered saline (BSB). The mixture was further mixed for 1 hr at room temperature (RT) on a rotator. The soluble aqueous layer was separated by centrifugation at 11,100 g for 10 min. Protein concentrations were determined by using the Bradford protein assay (BioRad Laboratories, Inc., Hercules, CA) using bovine serum albumin as the standard protein. Approximately 40-45% of cashew and pistachio defatted flour protein is solubilized by this method. The purification and biochemical analysis of CMP has been previously described in detail74. The purified CMP was dissolved in 0.1M Tris-HCL, pH 8.1, at 5mg/ml and stored at –20oC. CMP accounts for about 50% of the total soluble protein.

Production of rabbit anti-cashew globulin polyclonal antiserum

A rabbit was immunized with 5 mg cashew globulin extract along with Freund’s complete adjuvant (Sigma Chemical Co., St. Louis, MO). A booster dose was administered 4 weeks later with 5 mg of cashew extract in incomplete Freund’s adjuvant (Sigma Chemical Co.). The rabbit was subsequently bled and the serum was stored at - 20°C. Guidelines for animal care and welfare as set down in the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources (National Research Council, National Academy Press, revised 1996) were followed.

Human allergic sera

Blood samples were drawn after informed consent from patients with life-threatening systemic reactions to cashew nut, and the sera (provided by Dr. Suzanne Teuber) were frozen at –20oC until use. The human subjects review committee, at the University of California, Davis, approved the study with human allergic sera. The subjects had clinical histories of severe reactions to cashew and the presence of cashew-reactive IgE was confirmed either by ImmunoCAP assay (Pharmacia Diagnostics, Uppsala, Sweden) or immunoblotting. Some patients reported a history of anaphylaxis to peanuts and tree nuts including walnut, pecan, pistachio, almonds, and hazelnut. Of the ten patients sera used, two of them reported allergy to pistachio, and the remaining eight patients had never

11 eaten the nut. One of the ten patients reported allergy reaction to sunflower. The clinical characteristics of the ten patients used in this thesis study are listed in Table 1. The negative control serum used was from patients reporting pollenosis, but no sensitivity to tree nuts.

Table 1. Clinical characteristics of cashew and tree nut allergic patients

Patient Sex Age Age of Other Other ImmunoCAP No. onset of atopy food score cashew allergy allergy

4 F 30 10 AD1, AR2 peanut, walnut, hazelnut 4.04

32 F 26 2 AD, AR, Asthma peanut, walnut, pistachio 9.51

38 F 35 2 AD, AR, Asthma walnut, pecan, almonds 35.1

68 F 43 1 AD, AR, Asthma peanut, tree nuts 41.9

98 M 25 3 Asthma walnut, pecan, hazelnut 5.66

41 F 31 2 AR, Asthma walnut, sunflower 4.42

157 F 26 3 AR, Asthma peanut, tree nuts 2.41

237 F 39 1 AD, AR, Asthma peanut, tree nuts 9.53

196 F 39 5 Asthma pistachio, tree nuts 94.7

270 ? ? ? ? ? ?

AD1 = Atopic dermatitis AR2 = Allergic rhinitis (Patient clinical information provided by Dr. S Teuber) (? – Clinical history of patient #270 was not available)

One dimensional (1-DE) electrophoresis (SDS-PAGE) and immunoblot.

Cashew extract (300µg) was mixed in the ratio of 1:2 with reducing sample buffer (70 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.05% bromophenol blue) and boiled for 5 min before it was loaded into 12% SDS-PAGE gels. Electrophoresis was conducted (Bio-Rad Mini Protean 3 Electrophoresis Cell System) at 130 V on a Power Pac300 (Bio-Rad Laboratories). Proteins from the gels were

12 transferred onto 0.45 µm Protran® Pure nitrocellulose (NC) transfer membrane (Shleicher & Schuell, Keen, NH) for 3 h at 80 mA or overnight (o/n) at 30 mA, using an electrophoresis power supply (Northeastern Science Company, Boston, MA) and a Mini Trans-Blot Transfer Cell (Bio-Rad Laboratories).

The NC membranes were cut into 4 mm strips, blocked with 2% bovine serum albumin (BSA, Sigma) in TBS-T (20 mM Tris, 137 mM NaCl, pH 7.6, with 0.2% Tween 20) for 1 h at RT, and washed once with TBS-T. The strips were then incubated with either a pool of cashew allergic patients sera (diluted 1:20 in TBS-T, overnight at 4°C), goat anti-total cashew protein extract IgG (diluted 1:250, 1 hr at RT), or rabbit anti-CMP sera (diluted 1:50,000, 1 h RT). Strips probed with human cashew allergic antisera were washed two times in TBS-T and a third time in PBS (pH 7.3) for 20 min each wash and incubated o/n at 4°C with 125I-anti-human IgE (Hycor Biomedical Inc, Garden Grove, CA) diluted 1:10 in a mixture of phosphate buffered saline (PBS), 5% nonfat dry milk, and 0.05% Tween- 20. Three final washes in TBS-T were performed, and the strips were exposed to Kodak Biomax X-ray film (Rochester, NY) for 24 h for up to 2 weeks at –80oC. The HRP- labeled rabbit anti-goat IgG or goat anti-rabbit IgG were added to the strips containing the corresponding primary antibodies and incubated for 1 h at RT. The strips were washed once for 15 min and three times for 5 min each in TBS-T after antiserum and secondary antibody incubation. The reactive bands were identified using Enhanced Chemiluminescence Plus kit (ECL+, Amersham Pharmacia) and subsequent exposure to Kodak X-OMAT X-ray film.

Two dimensional (2-DE) gel electrophoresis

Two-dimensional gel electrophoresis was performed using ZOOM® IPGRunnerTM System (Invitrogen Life Technologies). The total cashew, pistachio extract protein, or CMP (400 µg in a total volume of 10 µl) were solublized in 155 µl sample rehydration buffer (8M urea, 2% CHAPS, 0.5% (v/v) ZOOM® Carrier Ampholytes (pH 3-10) non- linear, 0.002% bromophenol blue, 20mM DTT). This rehydration buffer containing the protein samples were loaded into the individual channels of the ZOOM® IPGRunnerTMCassette, and was rehydrated o/n at RT with immobilized pH gradient gel strip pH 3-10 NL (ZOOM®IPG Strip, pH 3-10, non-linear, 7 cm, Invitrogen).

Following rehydration of the IPG strips, isoelectric focusing (IEF) was performed on the entire cassette assembly using the ZOOM® IPGRunnerTM Mini-cell (Invitrogen). IEF of the proteins was performed by applying step voltage for 90 min as follows: 200 V for 20 min, 450 V for 15 min, 750 V for 15 min, 2000 V for 30 min, using an electrophoresis power supply (Northeastern Science Company, Boston, MA).

Thereafter the gel strips were subjected to SDS-PAGE as the second dimension of 2D electrophoresis. Prior to SDS-PAGE, the ZOOM® IPG strips were equilibrated in NuPAGE®LDS sample reducing buffer (Invitrogen) for 15 min at RT. The equilibrated ZOOM® Strips were then loaded onto Novex® 4 – 20%Tris-Glycine gel (Invitrogen). The second dimension, SDS-PAGE was performed using a Xcell SureLockTM Mini-Cell

13 (Invitrogen life technologies) by applying a constant current of 125 V for 90 min at RT using an electrophoresis power supply (Northeastern Science Company, Boston, MA).

The 2-DE gel was either stained with colloidal Coomassie blue stain (Brilliant Blue G, Sigma-Aldrich Co, St. Louis, MO) or transferred to nitrocellulose or PVDF membranes for further analysis. A molecular weight standard marker set, KPS (Kaleidoscope Prestained Standards, Bio-Rad, Hercules, CA) was used in all experiments.

Staining of the 2-DE gel with colloidal Coomassie blue

The 2-DE gel electrophoresis of soluble total cashew, pistachio proteins and CMP were performed with the above protocol and the gels thus obtained were stained with colloidal Coomassie blue. The gel was first placed in a fixative solution (40% methanol, 7% acetic acid and 53% ddH2O) for 30 min at RT with agitation. The excess fixative solution was then discarded, and replaced with colloidal Coomassie blue solution and incubated o/n at RT with agitation. The gel was then destained with a destaining solution (25% methanol, 10% acetic acid and 65% ddH2O).

Immunoblot of 2-DE separated proteins

The second dimension gels (cashew, pistachio extract and CMP) from 2-DE gel electrophoresis were transferred either to 0.2 µm Protran® Pure nitrocellulose immobilization membrane (Shleicher & Schuell, Keen, NH), 0.2 µM PVDF Problott® membrane (Applied Biosystems) or Immobilon-P or Immobilon-PSQ transfer membrane (Millipore Inc). The transfer protocol used was the same as that mentioned above in the 1-DE transfer immunoblot. The transferred membranes were stored dry in a desiccator at 4oC until used. The PVDF Problott® immobilization membranes were used for transfer blot, as they were preferred for N-terminal amino acid sequencing of the IgE immuno- reactive spots. For immunoblot analysis, cashew and pistachio proteins were transferred to nitrocellulose membranes, Immobilon-P and Immobilon-PSQ membranes. The later membranes were preferred to nitrocellulose and hence were used for immunoblot analysis of cashew and pistachio proteins with human allergic (cashew and tree nut allergy) sera. Immobilon-PSQ membrane served as an ideal transfer membrane for proteins less than 20 kDa molecular weight and was used to detect IgE binding to low molecular weigh proteins in soluble cashew and pistachio proteins.

The 2-DE immunoblots of cashew, pistachio proteins and CMP were analyzed for immuno-reactivity with rabbit anti-cashew globulin antiserum, using Enhanced Chemiluminescence Plus kit and subsequent exposure to Kodak X-OMAT X-ray film as described above in the immunoblot protocol.

Additionally, the 2-DE immunoblot of cashew and pistachio proteins were examined for immuno-reactivity with pooled human cashew and tree nut allergic sera using the 125I- labeled anti-human IgE secondary antibody. The 2-DE immublotting was performed as

14 described above and were exposed to Kodak Biomax X-ray film for 24 h to up to 2 weeks at –80oC.

Transblot for N-terminal amino acid sequencing

The 2D gel electrophoresis of cashew and pistachio proteins were transferred to 0.2µM PVDF Problott® membrane (Applied Biosystems, Foster City, CA) using a transfer buffer suitable for PVDF membrane (CAPS, 100mL methanol, 800mLcold ddH2O) at 4oC applying a current of 80V for 3 h or 30V overnight using an electrophoresis power supply (Northeastern Science Company, Boston, MA). When transfer was complete, the PVDF membrane was removed from the transfer assembly and washed with water for 5- 10 min with shaking on the rocker and was followed by placing the membrane in a glass petridish containing pure methanol for not more than 1 min. The membrane was stained with 0.1% Coomassie Blue R in 50% methanol for 5-10 min and then destained with several changes, 5-10 min each, of destain solution (50% methanol, 10% acetic acid). The membrane will remain a faint blue color, and was destained until the protein spots were faintly visible. Finally the membrane was air-dried thoroughly and placed in a plastic petridish and stored dry at -20oC. They were then submitted for N-terminal amino acid sequence analysis, at the Sequencing Facility (FSU). The immuno-reactive protein spots from 2D blot of cashew and pistachio were detected by immunoblotting. The selected immuno-reactive spots were excised and subjected to N-terminal amino acid sequencing with the aid of ABI 477A sequencer (Applied Biosystems Inc, Foster City, CA)

The amino acid sequence data were collected with ABI Procise software (Applied Biosystems Inc, Foster City, CA). This analysis led to the identification of 10-12 amino acids at the N-terminal and further analyzed using the NCBI protein-protein BLAST (BLASTp) program. The input amino acid was entered in the space provided and searched for short, nearly exact matches (http://www.ncbi.nlm.nih.gov/BLAST).

BLAST stands for Basic Local Alignment Search Tool and is a set of programs designed to perform similarity searches on all available sequence data. The BLASTp compares an amino acid query sequence with others stored in protein sequence databases. BLASTp uses an algorithm developed by NCBI that seeks out local alignment (the alignment of some portion of two amino acid sequences) as opposed to global alignment (the alignment of two sequences over their entire length). By searching for local alignments, BLAST is able to identify regions of similarity within two protein sequences yielding functional and evolutionary clues about the structure and function of the input amino acid sequence.

Inhibition immunoblot with patients #38 and #270 sera

The cashew and pistachio 2D immunoblot containing 300 µg total cashew or pistachio extract protein was blocked for 1 hr at RT in 2% BSA made in TBS-T. Patients #38 and

15 #270 sera were diluted 1:20 in 1% BSA made in PBS and 0.2% TX-100, and were each incubated o/n at RT with either 400 µg/ml of pistachio extract or cashew extract. The pre-incubated serum were then added to the respective blots and incubated overnight at 4oC on a rocker. The patient sera pre-incubated with cashew protein were used to probe IgE binding to the pistachio 2-DE immunoblot, and similarly the pistachio absorbed sera were used to probe IgE inhibition to the cashew 2-DE immunoblot. The blots were washed for 20 min each, 3 times at RT with TBS-T and incubated with 125I-anti-human IgE o/n at 4oC as described earlier in the immunoblot procedure. The blots were washed and exposed were exposed to Kodak Biomax X-ray film for 24 h to up to 10 days at – 80oC.

ELISA

ELISA was used to determine the IgE reactivity of ten individual cashew and tree nut allergic patients sera towards cashew and pistachio proteins.

ELISA was carried out using 40 µg/ml of total cashew, pistachio protein extract, and CMP and 20 µg/ml of rAna o 3 diluted in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and coated (50 µl/well) onto the wells of 96-microwell round bottom polyvinyl microtiter ELISA plates (Seracluster “U” Vinyl, no. 2797, Costar, Cambridge, MA) either o/n at 4oC or for 1 h at 37°C. The plates were rinsed three times with phosphate buffered saline containing 0.1% Tween®20 (PBS-T), and slapping each time on paper towels to shake off the excess buffer solution. Plates were blocked with 100 µl/well of blocking buffer (5% nonfat dried milk powder, PBS-T) for 1 h at 37°C. The emptied plates could either be sealed with Parafilm (American National Can., Chicago, IL) and stored at -20°C for several months or washed with PBS-T and used immediately. Human patients sera allergic to cashew and other tree nuts listed in Table 1 were used. The ten individual cashew and tree nut allergic sera were diluted 1:10 in incubation buffer (1% BSA in PBS-T), and 50 µl/well was added to the plates and incubated for 3 h at 37°C or o/n at 4°C.

After washings, bound specific IgE were detected by adding HRP conjugated mouse anti- human IgE (Zymed Laboratories Inc) at a dilution of 1:1000 made in the incubation buffer and was incubated for 1 h at 37°C. After subsequent washings with PBS-T, the IgE reactivity was detected by colorimetric reaction using o-phenylenediamine (OPD, Zymed Laboratories Inc.) and H2O2 as substrate. One tablet of OPD was dissolved in 12 ml of 0.1M citrate phosphate buffer, pH 5.0, containing 0.03% H2O2. The substrate solution was added to the plate (50 µl/well) and incubated for 15-20 min. The plates were covered with aluminium foil as the chromogen is light sensitive.

To stop the reactions, 50 µl of 2N H2SO4 was added to each well. Optical density (OD) was measured in a KC4 v2.5 ELISA reader (Bio-Tek Instruments Inc, Winooski, VT) at wavelength of 495 nm. All samples were assayed in duplicate, and the assays were repeated 3-4 times on different days. Sera from ten cashew and tree nut allergic patients and normal healthy non-cashew allergic donor were used in this assay.

Inhibition ELISA

ELISA inhibition studies were carried out on sera from ten cashew and tree allergic patients and showed IgE reactivity with both cashew and pistachio proteins. In these experiments, IgE reactivity to plate bound (40 µg/ml) cashew and pistachio extracts, CMP (40 µg/ml) and r Ana o 3 (20 µg/ml), was measured before and after pre-incubation of allergic sera with inhibitors (400 µg/ml) namely, the total cashew, pistachio extract or CMP, overnight at 4oC.

The inhibition ELISA and IgE binding was detected using HRP labeled anti-human IgE (1:1,000; Zymed Laboratories Inc) and performed as the ELISA protocol described above. The negative control used was 1% BSA in PBS-T.

Percent inhibition was calculated using the following formula:

% Inhibition = OD of test sample – OD of inhibited sample X 100 OD of test sample

Construction and IgE immunoscreening of cashew cDNA library and 2S albumin (Ana o 3) preparation

The construction of cashew cDNA library has previously been described in detail (Wang et al., 2003) and was performed by F. Wang in our lab. Briefly, cashew nuts in late maturation were chopped, frozen in liquid nitrogen, and ground to a fine powder. Total RNA from cashew was extracted and mRNA was isolated with a Poly-ATtract kit (Promega, Madison, Wis), according to the manufacturer’s instructions. The construction of the cDNA library was performed with the Uni-ZAP XR Gigapack Cloning Kit (Stratagene Inc, Cedar Creek, Tex) and cloned into the lambda Uni-ZAP XR expression vector. The library was amplified using Escherichia coli strain XL1-Blue. The amplified library was initially screened with rabbit anti-cashew serum and the clones were subsequently screened with antiserum from patients with cashew allergy. The immuno- positive clones were picked, plaque purified, and stored in SM buffer supplemented with 2% chloroform at 4°C for further use.

A 2S albumin gene from the cashew cDNA library was amplified by PCR with the aid of degenerate primers designed as per the identified cashew 2S albumin N-terminal amino acid sequence data (Robotham et al. submitted). This was then amplified and ligated to the maltose-binding protein (MBP) fusion expression vector pMAL-c2 (New England Biolabs Inc., Beverly, MA) with an engineered thrombin cleavage site. The cashew 2S albumin has been designated as Ana o 3 (Teuber et al., 2002; Robotham et al. in prep). For expression of Ana o 3, competent E.coli BL21 (DE3) cells (Novagen Inc., Madison, WI) were transformed (with the above mentioned plasmid containing the Ana o 3 coding

17 insert) and induced with isopropyl-D-thiogalactopyranoside (IPTG). The cells were harvested and lysed by sonication and then centrifuged at 12,000g for 20 min. The lysate supernatant was passed over an amylase affinity column and the fusion protein was eluted with 10 mM maltose and concentrated using centricon filter (YM-3). A yield of 1- 3 mg of soluble fusion protein per liter of cultured cells was routinely recovered after column purification and stored at –20oC for long term storage or at 4oC for brief storage.

CHAPTER 3

RESULTS

IDENTIFICATION OF IMMUNO-REACTIVE ANTIGENS AND ALLERGENS IN TOTAL CASHEW AND PURIFIED CASHEW GLOBULIN EXTRACTS

The separation of water and salt soluble proteins in total cashew extract and cashew globulin (CMP) was achieved by means of SDS-PAGE and two-dimensional electrophoresis (2-DE). Total protein detection was accomplished by colloidal Coomassie blue staining of the gels and the immuno-detection of antigenic or allergenic cashew proteins was carried out with either rabbit anti-cashew globulin, polyclonal antisera, or pooled human sera from cashew and tree nut allergic patients. All of the cashew allergic patients used in this study have previously documented systemic allergic reactions to cashew following consumption (Table 1).

SDS-PAGE and immunoblotting

SDS-PAGE (one-dimensional gel electrophoresis, 1-DE) analysis of total cashew extract depicts proteins having molecular weights (MW) ranging from 6-70 kDa (Fig. 4A, lane T). Similar analysis of HPLC purified cashew globulin revealed a protein profile with bands corresponding to those of the total cashew extract at 22, 35, 45, and 55 kDa MW (Fig 4A, lane G). It is apparent that the total extract contains more low-molecular-weight proteins or polypeptides than in the cashew globulin preparation, but the patterns are otherwise very similar.

A B RT HT RG HG T G MW (kDa) MW (kDa) 66 66 Vicilin-like protein 45 45 31 Legumin group acidic subunits 31

Legumin group 22 22 basic subunits

6.5 6.5 Fig.4. Comparative SDS PAGE analysis and immunoblotting of proteins from total cashew extract (T) and cashew globulin (A) SDS-PAGE gel stained with colloidal Coomassie blue. Bands at 22, 35, 45, and 55 kDa are seen in both cashew preparations (lane T and G). (B) The rabbit anti-cashew antisera used to probe both the whole cashew extract (lane RT) and cashew globulin (lane RG) fractions as was the human cashew and tree nut allergic sera (lane HT and RG, respectively). Both sera recognize protein bands at 22, 30-35, and 45 kDa. However, the 55 and 66 kDa proteins were reactive solely with rabbit sera (lane RT) and the ~9 kDa protein with human sera lane HT).

19 The immunoblot analysis of total cashew extract and cashew globulin with rabbit anti- cashew globulin antisera demonstrated that the polypeptide bands at 20-22, 30-35, 45, 55, and 66 kDa MW were strongly immunoreactive. Fig. 4B compares the immuno- reactivity of the rabbit antisera and cashew and tree nut allergic patient sera with total cashew and cashew globulin extracts. Both sera react with proteins at 22-25, 30-35, 45 kDa MW from total cashew and CMP extracts (Fig. 4B). The rabbit sera demonstrated reactivity to the 55 and 66 kDa in total cashew extract (lane RT), which was not detected by human sera (lane HT).

Two-dimensional electrophoresis of cashew proteins

2-DE and subsequent immunoblotting and N-terminal sequencing of allergens have contributed to the identification of the major allergens 23;70, their multiple isoforms, and the conserved IgE-binding epitopes in food allergens 23. Thus, using these tools enabled us to separate, identify, and characterize IgE-binding proteins in cashew. A typical 2-DE map obtained of the BSB soluble total cashew extract proteins and pure cashew globulin (which predominantly consists of native 11S globulins, termed as legumins) stained with colloidal Coomassie blue is seen in Fig 5.

Cashew globulin Total cashew extract MW (kDa) 4% 200 116 S 97 D 66 S | 45 P A 31 G E 21

6.5 20% 3 10 3 10 pI pI

Fig. 5. Two-dimensional gel electrophoresis (2-DE) of water and salt soluble proteins from total cashew and cashew globulin extracts stained with colloidal Coomassie blue. Distinct spots at 31-35 kDa and 22-25 kDa were observed at a pI in the range 5-6 and 8-9, respectively. Low molecular weight protein spots at 6-9 kDa at an acidic pI range of 4-5 were observed in total cashew extract.

Several distinct spots at ~35 and 55 kDa with pI of 5-6 and 25 kDa with pI of 8-9 were observed in both the cashew protein preparations and spots having the same molecular weight but slightly different pIs, suggest the presence of isoforms for the cashew seed storage proteins (Fig. 5).

20 2-DE immunoblotting of total cashew extract proteins with rabbit anti-cashew polyclonal antisera and pooled human cashew and tree nut allergic sera

The 2-DE cashew globulin immunoblot probed with pooled cashew and tree nut allergic sera (Fig. 6B) demonstrated similar immuno-reactivity pattern as that observed with rabbit anti-cashew sera (Fig. 6A). The 30-35 kDa MW spot, corresponds to the acidic subunit and 20-25 kDa MW spot, to the basic subunit of 11S globulin (Fig. 6) and are compatible to the corresponding MW bands observed in the 1-DE gel of cashew globulin depicted in Fig. 4.

Cashew globulin MW (kDa) 4% 200 116 S Fig. 6A. 2-DE immunoblot analysis 97 D of cashew globulin with rabbit anti- S 66 cashew globulin antisera. Two major | immuno-reactive spots were detected P 45 P A at MWs 20-22 kDa, pI 8-9, and ~30- 31 G 35 kDa, pI 5-6. E

6.5 20% 3 10 pI

4% 200 Fig. 6B. Cashew globulin 2-DE 116 S immunoblot probed with pooled 97 D cashew and tree nut allergic human S 66 sera. The immunoreactive spot at 30- | 35 kDa MW, pI 5-6 could correspond P 45 A to the acidic subunit and the spot at 31 G 20-25 kDa MW, pI 8-9, could E correspond to the basic subunits of 21 11S globulins. 20% 6.5 3 10 pI

The prominent IgE-binding spots on the cashew extract 2-DE immunoblot were identified at MW of 6-9, 20-25, 30-40, 45, and 55 kDa (Fig. 7). Several immuno-reactive spots in cashew having the same molecular weight but expressing different pIs were detected, thereby suggesting the existence of isoforms. The 11S globulins seed storage proteins are products of multi-gene family and have been shown to exhibit isoforms in other plant species 71.

21 Total cashew extract Fig. 7. Total cashew extract 2-DE MW (kDa) 4% immunoblot probed with pooled 200 human cashew allergic sera. Proteins 116 S in the spots A (30-35 kDa MW, pI 5- 97 A D 6) and B (22-25 kDa MW, pI 8-9) S 66 were identified based upon N-terminal | amino acid sequencing of 10-12 P 45 residues and comparison with known A proteins in databases. 31 G E 21 N-terminal amino acid sequences: B 6.5 20% Spot A - SRQEWQQQDECQI Spot B - GIEETICTMRLK 3 10 pI

The N-terminal sequences of spots A and B (Fig. 7) appeared to be identical to the earlier published sequences of acidic and basic subunits of Ana o 2, the cashew 11S globulin 14. The Ana o 2 allergen has been cloned from a cashew cDNA library and has been identified earlier as a major allergen in cashew (Wang et al., 2003).

Homology searches of Ana o 2 using NCBI BLAST Program

The identified N-terminal amino acid sequences of cashew 11S globulin (Ana o 2) were shown to possess significant homology (45-58% identity and 63-74% similarity) to the 11S globulins from diverse tree nuts, seeds, and legumes and are listed in Table 2.

24 Table 2: NCBI-BLAST – Proteins showing homology to Ana o 2 (Data from ) ______Protein Organism Accession AA1 % Identity %Simlarity Publication Description Number Overlap2 Legumin-like protein Ricinus communis (castor bean) AAF73007 1-446 58 74 Sherarer et al (unpubl. results) 11S globulin Corylus avellana (hazelnut) AF449424 2-444 55 71 Beyer et al., 2002 Legumin precursor Quercus robur (English Oak) CAA67879 2-445 55 72 Fischer (unpubl. results) 11S globulin Sesamum indicum (sesame) AAK15087 20-451 51 69 Tai and Tzen (unpubl. results) Legumin precursor Magnolia salicifolia S54206 1-454 50 68 Fischer (unpubl.results) 11S globulin Amaranthus hypochondriacus S49422 1-454 48 68 Barba de la Rose et al. (grain amaranth) (unpubl.results) Glycinin G1 subunit Glycine max (soybean) AAB23209 2-439 47 66 Nielsen et al., 1989 11S Globulin Coffea arabica (coffee) AAC61881 1-457 46 64 Acuna et al., 1999 Legumin A precursor Vicia sativa (spring vetch) S44294 1-439 45 65 Nong et al., (unpubl. results) 11S globulin Cucurbita pepo (winter squash) AAA33110 3-455 45 64 Hayashi et al., 1988 Glucinin Ara h 3 Arachis hypogaea (peanut) AF093541 21-439 43 59 Rabjohn et al., 1999 Glucinin Ara h 4 Arachis hypogaea (peanut) AF086821 4-439 42 58 Kleber-Jankeet et al., 1999 1AA= Amino acid 2Overlap = Ana o 2 residue numbers ______

22 IDENTIFICATION OF IMMUNO-REACTIVE ANTIGENS AND ALLERGENS IN TOTAL PISTACHIO EXTRACT

Cashew and pistachio nuts belong to Anacardiaceae family and in order to investigate antigenic and allergenic cross-reactivity between cashew and pistachio, SDS-PAGE, 2- DE and immunoblotting of total pistachio extract with rabbit anti-cashew globulin antisera and pooled cashew and tree nut allergic patient sera was performed.

SDS-PAGE and immunoblotting

Comparison of pistachio and cashew extracts by SDS-PAGE is illustrated in Fig. 8A. Bands at 6-9, 25-30, 35, 45, 55, and 66 kDa MW are seen in both pistachio and cashew (Fig. 8A, lanes P and C). The immuno-reactivity of pistachio proteins analyzed with rabbit anti-cashew globulin antisera (Fig. 8B, lane R) demonstrated antigenic cross- reactivity between cashew and pistachio protein bands at 31-35 and 55 kDa MW (Fig. 8B, lane R). The human cashew and tree nut allergic serum (Fig. 8B, lane H) showed IgE reactivity to pistachio protein bands at 9-12, 25, 31-35, and 45 kDa MW. No reactivity was detected by the rabbit sera to the basic subunit of 11S globulins (22-27 kDa MW) in Fig. 8B, lane R. The human IgE reactivity pattern with pistachio (Fig. 8B, lane H) was similar to that of cashew (Fig. 4B, lane HT). Lane H demonstrated patient IgE reactivity to low MW bands at 9-12 kDa.

A B P R H P C MW (kDa) MW (kDa) 66 66 Vicilin-like protein 45 45

31 Legumin group 31 acidic subunits

Legumin group 22 22 basic subunits

6.5 6.5

Fig 8. Comparative SDS-PAGE and immunoblotting profile of pistachio and cashew extracts. (A) Cashew (lane C) and pistachio (lane P) 22-27, and 30-40 kDa MW proteins correspond to the acidic and basic subunits of 11S globulins, respectively. (B) Lane R represents an immunoblot of pistachio extract probed with rabbit anti-cashew globulin antisera, and lane H probed with human cashew and tree nut allergic sera. Both the sera demonstrated immuno-reactivity with the bands at 31- 35 and 55 kDa (lane R). Lane H demonstrated IgE reactivity to low MW bands at 9-12 kDa.

23 2-DE and immunoblotting using rabbit anti-cashew globulin antisera and pooled sera from cashew and tree nut allergic patients

To further investigate antigenic and allergenic cross-reactivity between cashew and pistachio nuts, 2-DE immunoblot of total pistachio extract was probed with rabbit anti- cashew globulin antisera (Fig. 9B). The 2-DE gel profile of soluble proteins from pistachio extract depicts several distinct protein spots at 30-40 kDa and 7-12 kDa in the acidic pI range and basic pI proteins having 20-27 kDa and 7-9 kDa MW (Fig. 9A).

AB Total pistachio extract Total pistachio extract MW (kDa) MW (kDa) 4% 200 4% 200 116 S 116 97 30-40 kDa, pI 4-6 S D 97 S 2 D 66 3 S | 66 P | 45 P A 45 A 31 G E 31 G E 21 22-27 kDa, pI 8-9 21 4 1 6.5 20% 6.5 20% 3 10 3 10 pI pI C

N-terminal amino acids Spot 1 – SQQRQLQQQRF; Spot 2 – SRQQPQQNE; Spot 3 – SPQQGXQQNE; Spot 4 - GPGQQQNE Q G

Fig. 9. 2-DE gel electrophoresis and immunoblotting of total pistachio extract proteins. (A) 2-DE of pistachio extract stained with colloidal Coomassie blue. Pistachio proteins were detected in the molecular weight range of 6-12 and 30-40 kDa in the acidic pI range and basic pI proteins having 6 and 22-27 kDa MW, respectively. (B) 2-DE immunoblot probed with rabbit anti-cashew globulin antisera, and (C) N-terminal sequencing of the immuno-reactive spots 1, 2, 3, and 4. The pistachio antigens at 30- 40 kDa MW and acidic pI were immunoreactive and identified as acidic subunits of 11S globulins, based upon the N-terminal amino acid sequencing and comparison with known proteins in the database

The 2-DE immunoblot of pistachio extract probed with rabbit anti-cashew globulin antisera demonstrated reactivity with several protein spots, chiefly in the acidic pI range (5-6) at 35-40 kDa (Fig. 9B). The N-terminal amino acid sequences of the four spots show sequence similarities with each other and suggest the existence of isoforms. The N-terminal amino acids from cashew and pistachio share high homology, and provided preliminary data indicative of a shared structural motif and potential cross-reactivity between these two nuts. The N-terminal amino acid sequences of the reactive spots from pistachio (Fig. 9C) analyzed by the NCBI- BLASTp program, detected high homology (87-98% identity; 97-100% similarity) to the acidic subunits of 11S globulins from cashew (Ana o 2), soybean glycinin (G1 and G3), pea legumin A, and chick pea legumin (Table 3).

24 Table 3. Comparison of the N-terminal amino acid sequences of pistachio protein spots 1, 2, 3, and 4 with _ those of various 11S globulins

______Protein Organism Access no. Overlap % % Sequence3 AA2 Identity Similar Spot 21 SRQQP-QQ-NE Spot 3 .Q...... Spot 4 --G..G..Q.. Ana o 2 Anacardium occidentale (cashew) AAN76862 15-25 98 100 ...EW....D. Glycinin G1 Glycine max (soybean) AAB23209 22-30 88 100 ---.E...... Legumin A Pisum sativum (pea) CAA26720 23-30 87 100 --L.E...... Glycinin G3 Glycine max (soybean) AAB23211 23-30 87 100 --FREQPQQNE Legumin Cicer arietinum (chick pea) CAB60140 23-30 87 97 --L.D......

1Spot 2 = Spots in Fig. 9B 2AA = Amino acid 3Sequence = N-terminal amino acid sequences The highlighted amino acids in green represent identity and are indicated with a dot (·), a dash (-) indicates where a space was added in the sequence to maximize alignment.

The pistachio 2-DE immunoblot probed with cashew and tree nut allergic patients sera (Fig. 10) demonstrated a similar reactivity profile as that seen with rabbit anti-cashew antisera (Fig. 9B). Several spots in the acidic pI range of 4-6 and MW of 30-40, and 45 kDa show immuno-reactivity with both sera (Fig. 9 and 10). However, exclusive IgE reactive spots were detected with human sera at MW of 22-27 kDa and basic I, 8-9. N-terminal sequences of these IgE immuno-reactive spots are included in Fig.10 and are labeled as A, B, C, and D. They were identified as the basic subunits of 11S globulins (Fig. 10) and revealed significant sequence similarity with the 11S globulins from several nuts, seeds, and legumes including cashew Ana o 2, sesame seed, soybean glycinin, almond prunin, etc., as listed in Table 4.

Total pistachio extract

MW( kDa) 4% 200 N – terminal amino acids: S 116 D B Spot A – GLEETIXTMK 97 S C | 66 P Spot B – GLEETICTMKLKENI A 45 G Spot C – GLEETFCTMTLK E 31 E A 21 Spot D – GLEETFCTMTLKLNI

Spot E – RQESFRQCCQELQE 6.5 D 20% Spot E – RQESFRQCCQELQE

3 10 pI Fig. 10. 2-DE immunoblot of pistachio extract proteins probed with pooled cashew and tree nut allergic human sera and N-terminal sequencing of the immuno-reactive spots. Several pistachio protein spots at MW 6-9 (pI 4-5), 20-27 (pI 8-9), and 30-40 kDa (pI 5-6) were detected. The N- terminal sequences of spots (A, B, C, D, and E) were compared with known proteins in the databases.

Table 4. Alignment of N-terminal amino acid sequences of pistachio protein spots A, B, C, and D with basic subunits from various 11S globulins

______Protein Organism Access no. AA1 % % Sequence3 Overlap Identity Similar SpotB2 GLEETICTMKLKENI SpotD .....F...T..L.. SpotC ...... F...T..-- Spot A ...... X...----- Ana o 2 Anacardium occidentale(cashew) AAN76862 272-286 96 100 .I...... R..... 11S globulin Sesamum indicum (sesame seed) AAD42944 278-289 90 93 ...... KFR... Glycinin Glycine max (soybean) AAA33964 345-359 80 86 .V..N...... H... 11S globulin Coffea arabica (coffee) AAC61881 302-316 80 93 .....L..V..S... Citrin Sweet orange AAB52963 300-314 80 86 .F...... RH.. Cruciferin Brassica napus (rape seed) CAA40980 225-235 88 88 .....L...RCT--- Prunin Prunus dulcis (almond) CAA55009 368-382 73 93 .....F.SLR..... Legumin B Vicia faba (fava bean) CAA27313 304-318 73 100 ...... SL.IR... Legumin like Ricinus communis(castor bean) AAF73007 290-304 73 93 .V...F...RM.... Legumin Pisum sativum (garden pea) CAA35056 336-350 73 80 .....V..AK.R--- 13S Globulin Fagopyrum esculentum P83004 1-15 66 93 .ID.NV.....R..- B chain (common buckwheat) ______1AA = Amino acid 2Spot B = Spots labeled in Fig. 10 3Sequence = N-terminal amino acid sequences The highlighted amino acids in green represent identity and are indicated with a dot (·), a dash (-) indicates where a space was added in the sequence to maximize alignment.

The N-terminal amino acid sequence of spot E (Fig. 10) revealed remarkably high homology (87- 94%) to 2S albumins from cashew (Ana o 3), walnut, sunflower and sesame seed (Table 5). The 2S albumins have been identified as allergenic in a variety of tree nuts and seeds 14;48[Robotham et al. submitted]38;49. The cashew 2S albumin, designated as Ana o 3, has been cloned from a cashew cDNA library, and has been identified as an important cashew allergen 14[Robotham et al. submitted].

Table 5. Alignment and comparison of the N-terminal amino acid sequence of spot E with various 2S albumins from the NCBI database. ______Protein Organism Access no. AA1 % % Sequence3 Overlap Identity Similar ______Spot E2 RQESFRQCCQELQE Ana 0 3 Anacardium occidentale(cashew) AAL91665 69- 82 87 94 ....L.E...... 2S albumin Juglans regia (English walnut) AAM54365 93-103 73 83 ..H-...... Q... 2S albumin Sesamum indicumc (sesame) AAD42943 81- 90 70 80 .H..E..N..---- 2S albumin Helianthus annus(sunflower) CAC81359 77-83 100 100 ------...... basic 2S albumin chain 1AA = Amino acid 2Spot E = Spots labeled in Fig. 10 3Sequence = N-terminal amino acid sequences The highlighted amino acids in green represent identity and are indicated with a dot (·), a dash (-) indicates where a space was added in the sequence to maximize alignment.

26 DETECTION OF SPECIFIC IGE ANTIBODIES TO CASHEW AND PISTACHIO PROTEINS BY ELISA

The data obtained from (1-DE and 2-DE) immunoblotting with pooled sera from cashew and tree nut allergic patients resulted in immuno-detection of IgE reactive proteins (11S globulins and 2S albumins) in both cashew and pistachio nuts. To quantify the specific IgE to cashew and pistachio proteins in individual sera from ten patients, direct ELISA studies were performed. All of the ten patients displayed life threatening systemic reactions to cashew nut were analyzed and their clinical histories are tabulated in Table 1. Two of the ten patients had reported allergy to pistachio nuts and the remaining eight patients reported having never eaten pistachio.

The total cashew, CMP, and pistachio extract proteins and rAna o3 were separately coated onto ELISA plates to detect IgE reactivity to the antigens in the solid phase. The results are the mean of two experiments performed in duplicate. The resultant OD (optical density) values were measured and represent the intensity of IgE binding and are denoted as % IgE binding; the higher the value, stronger the IgE reactivity (Fig. 11 and 12). The OD readings of patient #38 to cashew and patient #237 to pistachio were the highest and were assigned a value of 100%. Each of the ten patients’ sera demonstrated specific IgE reactivity to both cashew and pistachio extracts, in varying intensities (Fig. 11). In patients #38, #68 and #270, the IgE binding to cashew was higher as compared to that of pistachio and patients # 41, #32, #98, #237, #196, and #4 demonstrated higher IgE binding to pistachio as compared to cashew. The IgE binding of patient #157 to both cashew and pistachio was equivalent. However, it is inappropriate to directly compare the IgE binding values to cashew and pistachio with each other, due to the potential variability of the antigen concentration that is actually coated in the well (both cashew and pistachio coated at 40µg/ml, though the amount actually bound is unknown). The ImmunoCAP score of the patients listed in Table 1 does not correlate to the intensity of IgE binding to cashew as recorded in ELISA (Fig. 11).

ELISA and ImmunoCAP

Cashew Pistachio ImmunoCAP Fig. 11. IgE binding to total 100 100 cashew and pistachio extracts of ten individual 80 80 patients with cashew and tree nut allergy. Each of the ten sera displayed 60 60 unique IgE reactivities to cashew and pistachio. The 40 40 highest OD reading (i.e., most binding) for each assay % IgE Binding 20 20 was assigned a value of ImmunoCAP score score ImmunoCAP ImmunoCAP 100%. ImmunoCAP score for cashew in range of 0-100. 0 0 ( ) P41 P32 P38 P98 P237 P157 P68 P196 P4 P270* * – ImmunCAP score for patient #270 not available Patient number

27 The studies with 1-DE and 2-DE immunoblotting with cashew (Fig. 4 and 7) and pistachio extracts (Fig. 8 and 10) resulted in detection of IgE reactive proteins, which were subsequently identified as the 11S globulins and 2S albumins. Therefore, ELISA studies were performed to evaluate the IgE reactivity of each of the ten patients to total cashew extract, CMP (native cashew 11S globulin), and rAna o 3 (cashew 2S albumin) (Fig. 12). The OD readings of patient # 38 to total cashew extract and CMP, and patient #270 to pistachio were the highest and were assigned a value of 100%. Patients #270 and #38 showed significantly high IgE reactivity to all the three cashew protein preparations. Patients #32, #237, #68, and #4 exhibited high IgE reactivity to CMP and total cashew extract as that compared to rAna o 3 (Fig. 12). Patient #98 reacted equally to CMP and rAna o 3. Patient #157 demonstrated IgE reactivity to CMP and total cashew extract but negligible reactivity observed with rAna o 3 (Fig. 12) implying that this patient is allergic to the cashew 11S globulin but not to 2S albumin.

Coating antigen Cashew CMP Ana o 3 100 Fig. 12. Comparison of IgE reactivity to total

80 cashew extract, CMP, and rAna o 3. The highest OD

ng reading (i.e., most binding)

di 60

n for each assay was assigned bi

E a value of 100%. Highest g

I 40 IgE binding to total cashew % extract and CMP seen with 20 patient #38, and highest IgE binding with pistachio 0 recorded with patient #270. P41 P32 P38 P98 P237 P157 P68 P196 P4 270P Patient number

CROSS-REACTIVITY OF CASHEW AND PISTACHIO PROTEINS STUDIED BY INHIBITION ELISA

Specific IgE binding determined by ELISA (Fig.11) with sera from cashew and tree nut allergic patients detected IgE reactivity with both cashew and pistachio proteins. Therefore to verify the IgE reactivity whether this was due to independent responses or because of cross- reactivity between cashew and pistachio nuts, cross-inhibition ELISA studies were performed. Each of the ten cashew and tree nut allergic sera were pre-incubated with either CMP, total cashew or pistachio extract (400 µg/ml), and subsequently used to determine the relative IgE inhibition to antigens (CMP, total cashew, pistachio extracts and rAna o 3) coated on to the solid phase.

Inhibition of IgE reactivity of preadsorbed sera towards total cashew extract

Adsorption of sera with cashew extract completely abolished IgE binding to cashew extract in all the ten patients (Fig. 13). CMP displayed moderate to almost total IgE inhibition (77-97%) with each of the ten patients sera. Adsorption of sera with pistachio extract completely

28 removed IgE binding to cashew extract in eight of the ten patients (Fig. 13) thus indicating extensive cross-reactivity between the IgE reactive epitopes of these two nuts, for these patients. Partial IgE inhibition (40-53%) was recorded in two of the ten patients thus indicating, that these patients (patients #38 and #68) recognized some unique epitopes in cashew that were not present in pistachio.

Inhibitor CMP Cashew Pistachio Fig. 13. Inhibition ELISA of 100% total cashew extract. Adsorption with cashew 80% extract completely removed IgE binding to total cashew 60% extract. CMP displayed moderate to almost total 40% inhibition (77-97%) of IgE

% IgE Inhibition reactivity. Pistachio extract 20% completely inhibited IgE reactivity to cashew extract, in 0% six of the ten patients sera P41 P32 P38 P98 P237 P157 P68 P196 P4 P270 tested. Patient number

Inhibition of IgE reactivity of preadsorbed sera towards CMP

To investigate cross-reactivity between CMP (the native cashew 11S globulin) and the homologous protein in pistachio, inhibition ELISA was performed. Adsorption of sera with CMP and total cashew extract completely abolished IgE binding to CMP in all the ten patients as expected (Fig. 14). Adsorption with total pistachio extract, substantially inhibited IgE binding to CMP in eight patients and partially in two (patients #38 and #270) of the ten patients sera (Fig. 14). The inhibition studies thereby confirmed the presence of in vitro allergenic cross-reactivity between cashew and pistachio proteins, the 11S globulin family. The 11S globulins are major seed storage proteins in cashew accounting for approximately 50% of the total seed protein 14;41 thus resulted in significantly high IgE inhibition to total cashew extract following CMP adsorption of sera seen in Fig. 13.

Inhibitor CMP Cashew Pistachio 100% Fig. 14. Inhibition ELISA of CMP. Adsorption of 80% each of the ten patients sera with CMP and total cashew 60% extract completely removed IgE binding to CMP. Strong 40% IgE inhibition (70-98%) to % IgE Inhibition CMP was observed in seven 20% of the ten sera with adsorption with pistachio

0% extract. P41 P32 P38 P98 P237 P157 P68 P196 P4 P270 Patient number

Inhibition of IgE reactivity of pre-adsorbed sera towards total pistachio extract

In Figs. 13 and 14, substantial inhibition to cashew extract and CMP was observed with pistachio pre-adsorption. Further cross-inhibition ELISA studies confirmed the presence of cross- reactivity between cashew and pistachio (Fig. 15). When CMP or total cashew extract were used as inhibitor, significantly high inhibition to pistachio was seen in eight of the ten patients, implying that all of the IgE binding epitopes of cashew 11S globulins are present in pistachio. Interestingly, patients #38 and #68 which were previously shown to totally inhibit IgE binding to pistachio with cashew preadsorption, demonstrated the converse of the IgE inhibition to cashew with pistachio adsorption Figs. 13 and 14). The IgE binding of patients #41and #32 to total cashew extract and CMP was almost totally inhibited by pre-adsorption with pistachio (Figs. 13 and 14), in contrast to partial inhibition observed to pistachio as a consequence of pre-adsorption with either total cashew extract or CMP (Fig. 15). Patient #32 had reported incidence of pistachio allergy and patient #41 had reported not eaten the nut.

Inhibitor CMP Cashew Pi st achi o Fig. 15. Inhibition ELISA of 100% total pistachio extract. Adsorption of each of the ten 80% patients sera with pistachio extract totally abolished IgE binding to pistachio. 60% Adsorption with cashew extract completely inhibited IgE

E inhibition 40%

g binding to pistachio extract in eight of the ten sera validating % I 20% the presence of cross-reactivity.

0% P41 P32 P38 P98 P237 P157 P68 P196 P4 P270 Patient number

Inhibition of IgE reactivity of pre-adsorbed sera towards rAna o 3

The 2S albumin detected in the pistachio 2-DE IgE immunoblot (Fig. 10) revealed significantly high sequence homology with the cashew 2S albumin (Ana o 3). Inhibition ELISA was used to confirm in vitro allergenic cross-reactivity (Fig. 16). These data are expressed as the mean of duplicates. Five sera displaying strong IgE reactivity with rAna o 3 (Fig. 12) were used in this assay. Adsorption with total cashew extract and rAna o 3 totally inhibited IgE binding to rAna o 3 in each of the five patients. Adsorption with pistachio extract substantially reduced IgE binding to Ana o 3 in four of the five patients, thereby confirming that pistachio and cashew 2S albumins share IgE cross-reactivity. However, partial inhibition of patient #38 IgE binding to Ana o 3 with pistachio adsorption was observed suggesting that this patient recognized unique IgE epitopes on the cashew 2S albumin (Fig. 16). The IgE binding to cashew extract was partially

30 inhibited following pistachio absorption as was shown previously (Fig. 13) implying the presence of unique cashew IgE reactive epitopes.

Inhibitor rAna o 3 Cashew Pistachio 100% Fig. 16. Inhibition ELISA using rAna o 3. Total inhibition of IgE 80% binding rAna o 3 was observed with pre-incubation with rAna o 60% 3 and total cashew extract. Pre- incubation of the sera with total 40% pistachio extract significantly

% IgE Inhibition % IgE decreased IgE binding to rAna o 20% 3 in four of the five patients.

0% P41 P32 P38 P196 P270 Patient number

Dose-dependent inhibition assays was performed to determine the degree of IgE cross-reactivity between cashew and pistachio using patients #38 and #270. Both of these patients displayed high specific IgE (anti-cashew and pistachio) reactivity (Figs. 11 and 12) and unique inhibition profiles (Figs. 13, 14, and 15); hence were selected for further investigation.

Adsorption of patients #38 and #270 sera with increasing concentration of the inhibitor (cashew or pistachio extracts) resulted in a pattern co-relating the relative degree of IgE inhibition (Fig. 17). ELISA inhibition assays revealed almost full inhibition of the IgE-binding of patient #270 to cashew when the pistachio extract was used as an inhibitor, and vice a versa, whereas the IgE- binding of patient #38 to cashew is only partially inhibited by pistachio (Fig. 17). The pistachio extract was able to inhibit 42% of the IgE binding of patient #38 to cashew at 400 µg/ml of protein (Fig. 17A, whereas cashew totally inhibited IgE binding to pistachio at the same protein concentration (Fig. 17B). Adsorption of patient #38 sera with pistachio at the highest concentration of (3200 µg/ml) resulted in 68% IgE inhibition to cashew (Fig. 17A). These observations suggest that patient #38 sera contain IgE that react with cross-reactive cashew and pistachio proteins; and additionally to unique cashew IgE epitopes. Patient #270 sera preadsorbed with pistachio showed a maximum inhibition of 93% of IgE binding to cashew (Fig. 17C), and pre-adsorption with cashew showed 97.7% inhibition to pistachio, at inhibitor concentrations of 800 µg/ml (Fig. 17D). The observed cross-reactivity suggests that the cashew and pistachio proteins share similar IgE-binding epitopes. Both the cashew and pistachio extracts, inhibited the IgE binding of patient #270 sera to cashew or pistachio extracts in a similar fashion (Figs. 17A and B) and indicating that this particular patient reacted to the cross-reactive IgE epitopes on cashew and pistachio.

31 Inhibition ELISA with Patient #38 sera A Inhibition ELISA with Patient #38 sera B Antigen coated – Total cashew extract Antigen coated – Total pistachio extract 100% 100% Inhibitor Inhibitor Cashew extract Cashew extract 80% 80% Pistachio extract Pistachio extract on on 60% 60% hibiti hibiti n n I I

E Inhibition E Inhibition 40% 40% g g IgE IgE

% % % I % I 20% 20%

0% 0% 12.5 25 50 100 200 400 800 1600 3200 12.5 25 50 100 200 400 800 Inhibitor concentration µg/ml Inhibitor concentration µg/ml

C Inhibition ELISA with Patient #270 sera D Inhibition ELISA with Patient #270 sera Antigen coated – Total cashew extract Antigen coated – Total pistachio extract 100% 100% Inhibitor Inhibitor Cashew extract Cashew extract 80% 80% Pistachio extract Pistachio extract

60% 60% E Inhibition E Inhibition E Inhibition E Inhibition 40% 40% g g g g % I % I % I % I 20% 20%

0% 0% 12.5 25 50 100 200 400 800 12.5 25 50 100 200 400 800

Inhibitor concentration Inhibitor concentration µg/ml µg/ml Fig. 17. Dose-related curves of inhibition of IgE binding to cashew and pistachio with sera from patients #38 and #270, preadsorbed with cashew or pistachio extracts. (A) Patient #38 sera, inhibition of IgE binding to cashew; (B) inhibition of IgE binding to pistachio, with pistachio and cashew used as inhibitors; (C) Patient #270 sera, inhibition of IgE binding to cashew; (D) inhibition of IgE binding to pistachio, with pistachio and cashew used as inhibitors

CROSS-INHIBITION IMMUNOBLOT TO DETECT CROSS-REACTIVITY BETWEEN CASHEW AND PISTACHIO PROTEINS

Substantial IgE reactivity to total cashew, pistachio and CMP extracts, and rAna o 3 were detected with sera from two patients #38 and #270 as shown in Figs. 11 and 12. Both the sera displayed distinct IgE reactivity in inhibition ELISAs studies (Figs. 13, 14; and 15) hence used for further analysis in inhibition immunoblot to visualize the proteins involved in inhibition correlating to invitro allergenic cross-reactivity.

32 IgE reactivity of pre-adsorbed sera from patient #38 towards total cashew and pistachio extract

The IgE binding pattern of patient #38 serum to cashew extract can be seen in lane 1 (Fig.18A), and to pistachio extract in lane 4 of Fig. 18B. Adsorption with pistachio extract eliminated IgE binding proteins at 22-25kDa (basic subunit of 11S globulins), 45 and 55 kDa MWs. The IgE binding to the 31-40 kDa (acidic subunit) wide band has been considerably inhibited, however the IgE reactivity has not been totally eliminated. The same is true for the low MW 9-12 kDa band (lane 2). The IgE binding pattern in the pistachio blot demonstrated reactivity with 9-12, 22-27, 31-40, 45 and 55 kDa MW bands (Fig. 18B). Adsorption with cashew extract totally abolished IgE reactivity to pistachio proteins (lane 5). These data validate the results observed in cross-inhibition ELISA, indicating that IgE binding of patient #38 to total pistachio extract was totally inhibited by pre-adsorption with cashew extract (Figs. 13 and 15), in contrast to partial inhibition observed to cashew extract as a consequence of pre-adsorption with total pistachio extract. Similarly, patient #38 demonstrated partial inhibition of IgE binding to Ana o 3 with pistachio adsorption, suggesting that this patient recognized unique IgE epitopes on the cashew 2S albumin (Fig. 16). Therefore the inhibition immunoblot data with patient #38 sera (Fig. 18) revalidates the presence of cross-reactivity between cashew and pistachio and additionally indicates the presence some unique epitopes in cashew (acidic subunit of 11S globulin and the 2S albumin) that were not present in pistachio.

1 2 3 4 5 6 A MW (kDa) B

66 66 É 45 45 É

31 31

21 É 21

6.5 6.5

Fig 18. Cashew and pistachio 1-DE inhibition immunoblot with patient #38 sera preadsorbed with pistachio or cashew extract. (A) Cashew inhibition immunoblot are shown as indicated. Lane 1, standard IgE immunoblot against cashew; Lane 2, sera preadsorbed with pistachio extract; Lane 3, sera preadsorbed with cashew extract (the arrow indicates the absence of a band at 22-25 kDa). Pistachio preadsorption (lane 2) removed almost all IgE binding to 22-25, 45, 55 and 66 kDa and bands at 30-35 and 6-9 kDa were partially inhibited. (B) Pistachio I-DE inhibition immunoblot probed with patient #38 sera preadsorbed with pistachio or cashew extract. Lane 4, patient #38 serum IgE binding to pistachio; cashew adsorption totally abolished IgE binding to pistachio (lane 5). Lane 6, pre-adsorption of patient #270 sera with pistachio, and resulted in complete IgE inhibition.

The 2-DE immunoblot with cashew (Fig. 7) and pistachio extracts (Figs. 9 and 10) demonstrated the possible existence of isoforms for the 11S globulins and 2S albumins.

33 Therefore, 2-DE cross-inhibition immunoblot was performed to better visualize the absence of IgE binding to protein spots (isoforms) following inhibition. Adsorption of patient #38 sera with pistachio, and probed with cashe 2-DE immunoblot demonstrates inhibition of IgE binding to the basic subunit of 11S globulins and is marked with a red arrow (Fig, 19A). The IgE binding at spots with MW of 6-9, 30-35, 55 kDa appear to be un-inhibited (Fig. 19A). Total inhibition of IgE binding to pistachio 2-DE immunoblot was seen with preadsorption with cashew (Fig. 19B). These data with patient #38 revalidate the presence of cross-reactivity between cashew and pistachio and also the presence of unique cashew IgE epitopes on the 2S albumins and the acidic subunit of 11S globulins.

A B MW (kDa) 200 4% 4% 116 97 S S 66 D D S S 45 - | P P 31 A A G G 21 É E E 6.5 20% 20% 3 10 3 10

Fig. 19. Cashew and Pistachio 2-DE inhibition immunoblot probed with patient #38 sera preadsorbed with pistachio or cashew extracts. (A) The absence of IgE binding to spot at 22-25 kDa (basic subunit of 11S globulins) indicated by an arrow in the cashew immunoblot. (B) Pistachio 2-DE immunoblot and preadsorption of patient #38 sera with cashew extract. Cashew preadsorption resulted in total inhibition of IgE binding to pistachio.

IgE reactivity of preadsorbed patient #270 sera towards total cashew and pistachio extract

In the cross-inhibition ELISA, patient #270 had demonstrated total inhibition to total cashew extract as a consequence of preadsorption with total pistachio extract and vice a versa (Figs. 13 and 15), thereby indicating the presence of cross-reactivity between cashew and pistachio proteins. Futher, the 1-DE cross-inhibition immunoblot with patient #270 validated these observations (Fig. 20) and confirm cross-reactivity between these two nuts. The IgE binding pattern of patient #270 sera to cashew (lane 1) and pistachio (lane 4) is illustrated in Fig. 20A and B. Adsorption with pistachio totally inhibited IgE binding to cashew proteins at 6-9,22- 25, 35, 45, and 55kDa bands and the same was true for the reverse immunoblot (Fig. 20A,lane 2 and Fig. 20B, lane 5). The IgE reactive epitopes on cashew and pistachio (11S globulins and 2S albumins) seem to be highly cross-reactive.

AB 1 2 3 MW (kDa) 4 5 6 Fig. 20 Cashew and pistachio 1D 66 immunoblot and preadsorption of patient # 270 sera with pistachio or cashew 45 extracts. (A) Lane 1, standard IgE immunoblot against cashew); Lane 2, sera preadsorbed with pistachio extract; Lane 3, 31 sera preadsorbed with cashew extract. (B) Pistachio inhibition immunoblot, Lane 4, 21 standard IgE immunoblot; Lane 5, sera preadsorbed with pistachio; Lane 6, sera 6.5 preadsorbed with cashew extract. Total IgE inhibition seen in lane 5 and 6

The 2-DE cross-inhibition immunoblot (Fig. 21) performed to visualize cross-reactivity between cashew and pistachio proteins validate the observations of the 1-DE inhibition immunoblot (Fig. 20). Pre-adsorption of patient #270 sera with pistachio extract resulted in total inhibition of IgE reactivity to both the acidic and basic subunits of 11S globulin from cashew extract (Fig 21A). The IgE reactivity to low molecular weight proteins with acidic pI identified as 2S albumin has also been significantly inhibited (Fig. 21A). Adsorption with cashew completely abolished IgE binding to pistachio blot. These data with patient #270 indicate significantly high in vitro allergenic cross-reactivity between cashew and pistachio proteins, the 11S globulins and 2S albumins.

AB MW (kDa) 200 4% 116 97 S D 66 S 45 | P 31 A G 21 E

6.5 20% 3 10 3 10 pI pI

Fig. 21. The 2-DE inhibition immunoblot of cashew and pistachio extract and preadsorbed patient # 270 sera with cashew or pistachio extracts. (A) Cashew immunoblot probed with patient # 270 sera preadsorbed with pistachio extract. (B) Pistachio immunoblot probed with patient #270 sera preadsorbed with cashew.

CHAPTER 4

DISCUSSION

Tree nut allergies are common, potentially life-threatening food allergies and typically show lifelong persistence. The voluntary registry of peanut and tree nut allergic US patients (FAAN), reported that 40% of tree nut allergy patients exhibit sensitivity to cashew, the second highest percentage for any tree nut following walnut. This registry also lists sensitivities to other tree nuts, including almond (15%), pecan (9%) and pistachio (7%) 1.

Cashew nut (Anacardium occidentale) and Pistachio (Pistacia vera) are members of the Anacardiaceae family, which also includes mango, Asian sumac, poison ivy and others. Allergy to the cashew nut and other plants belonging to the same taxonomic family has been reported 56;65;68;69. Several studies have reported clinical allergic reactions to pistachio but there is relatively little information regarding the identity of the relevant allergens 1;69;72;73. Additionally, clinical and in vitro cross-reactivity anticipated between cashew, pistachio and mango has been reported previously 62-64;73.

Three allergens from cashew nut have been identified so far, namely Ana o 1 (vicilin), Ana o 2 (legumin), and Ana o 3 (2S albumin), all of which are seed storage proteins 14;19;24[Robotham, unreported data]. The seed storage proteins, which includes the 2S albumins, legumin-group proteins (11S globulins), and vicilins (7S globulins) have been identified as major allergens is various tree nuts, seeds and legumes and are implicated in many cases of severe, anaphylactic tree nut allergy 15.

The objective of this thesis work was to detect and identify the IgE reactive pistachio proteins by immunological and biochemical means. This current study, also aims to determine in vitro IgE cross-reactivity between the allergenic proteins from cashew (11S globulins and 2S albumins) and the homologous proteins present in pistachio, using sera from a group of patients sensitized to cashew and other tree nuts (Table 1).

The antigenic and allergenic profile of cashew and pistachio was analyzed by SDS-PAGE, 2-DE followed by immunoblotting, and subsequently by N-terminal amino acid sequencing. The comparative SDS-PAGE analysis of cashew and pistachio extracts show a similar profiles, detecting proteins at MW ranging from 6-66 kDa (Fig. 8A). Our extraction technique was expected to solubilize both albumins and globulins, and indeed our 1-DE Coomassie-stained gels show that the albumins and globulins described by others are represented 14;41;63-65;74. The immunoreactivity pattern of both cashew and pistachio extracts with rabbit anti-cashew globulin antisera was demonstrated to be identical and detected proteins at 30-35 and 45 kDa MW (Fig. 4B, Lane RT and Fig. 8B, Lane R). However no reactivity was observed with the 22 kDa protein from pistachio as opposed to that observed in cashew extract (Fig. 4B, Lane RT). Additionally, the cashew and pistachio immunoblots probed with sera from cashew and tree nut allergic patients show a similar IgE reactivity pattern, recognizing proteins at 6-9, 22-25, 30-35, and 45

36 kDa MW (Fig. 4B, Lane HT and Fig. 8B, Lane H). In a study reported earlier, the screening of 15 cashew allergic patients by immunoblot with cashew extract resulted in dominant IgE reactions to the 20-27 and 31-35 kDa MW polypeptides 14; N-terminal and partial amino acid sequencing characterized these proteins as the basic subunit and acidic subunit of the 11S globulins, respectively 14;41. The 11S globulin is a major protein and allergen in the cashew nut, ~ 50% of the aqueous-extractable proteins are represented by this fraction 14;41;74. The IgE binding pistachio and cashew proteins at corresponding MW have been reported previously and could be equivalent to the proteins that were detected in this thesis study 63-65.

Apart from the high MW proteins, a low MW band at 6.5-12 kDa was detected in the IgE immunoblot of both cashew (Fig. 4B, Lane HT) and pistachio extracts (Fig. 8B, Lane H). These data correspond to the observations reported previously, whereby the IgE binding to the low MW cashew protein was detected in eleven of the 15 sera from cashew allergic patients tested (73%); and was characterized as the 2S albumin by N-terminal amino acid sequencing 14. In another study, a similar protein band in pistachio was reported to be detected by IgE immunoblot and was speculated to be a 2S albumin 65. These reports are analogous to the observations made in the current study.

The 11S globulins and 2S albumins are seed storage proteins (2S albumins also have metabolic functions e.g., enzyme inhibitors) and both belong to families of proteins that are known to exist as isoforms 71. Characterization of the legumin-group (11S globulins) and 2S albumin allergens is complex because multiple genes with substantial variation in posttranslational processing of the encoded legumin-group and 2S albumin precursors have been described in some plant species 42,36. The 2-DE and subsequent immunoblotting and N-terminal sequencing of allergens have contributed to the identification of the major allergens, their multiple isoforms, and the conserved IgE-binding epitopes in food allergens 23;70. The 2-DE is an ideal method of choice to separate proteins first by their isoelectric point and then by their MW under denaturing conditions. The high resolution of 2-DE detected several distinct spots in both the cashew (Fig. 5) and pistachio (Fig. 9A) extracts. Identification of the acidic and basic subunits of cashew 11S globulins on the cashew 2-DE immunoblot was accomplished with pooled sera from cashew and tree nut allergic patients (Fig. 7) followed by N-terminal amino sequence analysis. The corresponding immuno-reactive spots in 2-DE immunoblot cashew were also detected with rabbit anti-cashew globulin antisera (Fig. 6). These sequences appeared to be identical to the earlier published sequences of acidic and basic subunits of cloned Ana o 2, a cashew 11S globulin 14. Several 11S globulins have been identified as food allergens including Ana o 2 from cashew, Ara h 3 31;75 and Ara h 4 32 from peanut, the G1 and G2 glycinins from soybean 21;33;76, Cor a 9 from hazelnut 23, legumin from buckwheat 34;35, 11S globulins from sesame seeds 77, legumin like protein in coconut and walnut 37.

Antigenic cross-reactivity between cashew and pistachio proteins, detected by rabbit anti-cashew globulin sera on the pistachio 2-DE immunoblot; designated as spots 1, 2, 3, 4(30-35 kDa MW and pI 5-6) (Fig. 9B), were identified to be the acidic subunits of 11S globulins based on the N- terminal amino acid sequence analysis. The rabbit anti-cashew globulin sera reacted intensely with the acidic subunits of 11S globulin of pistachio however, no reactivity was detected with the basic subunits (Fig. 9B) suggesting that the presence of cross-reactive epitopes are limited to the acidic subunit of the 11S globulins. The human cashew and tree nut allergic sera displayed IgE

37 immuno-reactivity with several spots (A, B, C, D as well as those detected with rabbit polyclonal sera) on the pistachio 2-DE immunoblot (Fig. 10). Both the acidic and basic subunits of 11S globulins displayed strong reactivity to IgE antibodies (Fig. 10). In an earlier study, IgE binding to pistachio peptides at 34, 41, 52, and 60 kDa were described, and was strongest at 34 kDa 63-65; and could possibly be equivalent to the corresponding IgE binding profile of pistachio observed in the current study (Fig. B, Lane H and Fig. 10).

N-terminal amino acid sequences of the pistachio protein spots 1, 2, 3, and 4 (Fig. 9) and spots A, B, C, and D (Fig. 10) displayed significantly high sequence homology with the 11S globulins from cashew, almond, soybean, sesame, coffee, rape seed, to name a few, and the alignment has been listed in Table 3 and 4. The N-terminal amino acid sequences of pistachio protein spots 1, 2, 3 and 4 share amino acid sequence homology with each other and could represent the isoforms of the acidic subunit of 11S globulins (Fig. 9C). The N-terminal amino acid sequences of spots A, B, C and D also share sequence homology with each other and could represent the isoforms of the basic subunit of 11S globulins. Protein spots having the same molecular weight but slightly different pIs, possibly due to posttranslational modifications and multi-gene complexity, suggests the presence of isoforms, which is a characteristic of the seed storage proteins and also a common feature of several seed, legume, and tree nut allergens 15;22;52;77. In a previous study, the charge heterogeneity of cashew 11S globulins was demonstrated by isoelectric focusing, and the presence of at least seven bands within the narrow pI range of 6.5-7 2 was reported 41;74.

In addition to the detection of 11S globulins, the 2S albumin on the pistachio 2-DE immunoblot was detected with the pooled sera from cashew and tree nut allergic patients, as a low MW protein at 6-9 kDa at the acidic end (pI 4-6) and is designated as Spot E in Fig. 10. The 2S albumins are a group of seed storage proteins, typically heterodimeric that consist of two polypeptide chains of approximately 4 and 9 kDa that are linked together by four disulfide bonds. On the 2-DE pistachio immunoblot, three adjacent protein spots at MW (6-9 kDa) and displaying slight differences in the pIs (4-6) were observed (Fig. 10) suggesting the presence of isoforms of the pistachio 2S albumins. Such charge heterogeneity has been reported earlier with other 2S albumins [Moreno et al., 2004] such as cashew Ana o 3 14 sesame seed Ses i 2 23, and hazelnut Cor a 1 78. The 2S albumins from cashew were reported previously to have three isoforms, represented by bands showing slight differences in electrophoretic mobility and sequence differences in the N-terminal sequences in the large chain 14. Studies in our lab with rAna o3 demonstrate that 2S albumin is an important allergen in cashew along with rAna o 2, the 11S globulin 14;24[Robotham et al. submitted]. Significantly high amino acid sequence homology at the N-terminus of pistachio 2S albumin was detected with the 2S albumins from cashew (Ana o 3), English walnut, sesame and sunflower (Table 5). Interestingly, the N-terminal sequence of one of the cashew 2S albumin isoforms displays 100% homology with the pistachio 2S albumin N-terminal amino acid sequence.

Similar sequence homology shared among proteins can erroneously lead to an assumption that potential cross-reactivity is likely. However, findings from a few earlier studies have proven otherwise positing that simply comparing the amino-acid sequences of the two peptides remains inadequate in determining if the two are cross-reactive 79. Despite the presence of homologous proteins in peanuts and tree nuts, those allergic to one are unlikely to be allergic to the other on the basis of cross-reactivity. This was evident in a study of patients allergic to both peanuts (Ara

38 h 1) and walnut (Jug r 2); although IgE reactivity to vicilin from both sources was demonstrated, cross-reactivity was not detected between the allergens 29.

It is reasonable to assume that extensive cross-reactivity can exist among the different members of the same taxonomic family 80. Anticipated here is the presence of in vitro allergenic cross- reactivity between cashew and pistachio and to address this issue, cross-inhibition ELISA and inhibition immunoblot were perfomed. Detection of specific IgE by ELISA using sera of ten cashew and tree nut allergic patients to whole cashew and pistachio extracts, CMP (native cashew 11S globulin), and rAna o 3 (cashew 2S albumin) (Fig. 11 and 12) was performed. Each of the ten sera displayed IgE reactivity to CMP, and to total cashew and pistachio extracts (Fig. 10 and 12), and six of these ten patients exhibited strong IgE binding to rAna o 3 (Fig 12).

Cross-reactivity between pistachio and cashew (11S globulins and 2S albumins) was studied by inhibition ELISA; the degree of cross-reactivity between these two nuts was found to be very high in the patients studied. Significant cross-reactivity was observed in eight of the ten patients sera as evidenced by the ability of pistachio to inhibit most of the IgE binding to cashew extract and CMP (Figs. 13 and 14) and partial inhibition was detected in two of the ten patients #38 and #68 sera (Figs. 13 and 14). Conversely, complete inhibition of IgE binding to pistachio was noted following pre-adsorption with cashew in these two patients. (Fig. 15). These data thus suggest that while cross-reactivity is detected, certain cashew-unique epitopes are also present. Interestingly, these particular patients had reported having never consumed pistachio.

The reactivity of sera from patients #32 and #41 to pistachio was not significantly inhibited by pre-incubation with cashew proteins. These patients also recognize cross-reactive IgE epitopes in cashew and pistachio (Fig. 15). Patient #32 had reported pistachio allergy, which is consistent with the above finding. Patient #41, on the other hand had reported not having consumed pistachio. The plausible explanation for this could be that the patient #41 might have been inadvertently exposed to pistachio, which was not recorded in the clinical history.

Substantial cross-reactivity was observed between cashew 2S albumin (Ana o 3) and the homologous protein in pistachio as demonstrated in four of the five patients sera tested by inhibition ELISA (Fig. 16). Interestingly, partial IgE inhibition to rAna o 3 was observed when pistachio extract was used as an inhibitor, in patient #38 thereby demonstrating the presence of certain IgE epitopes that are distinctive to cashew.

Patients #38 and #270 were studied further in 1-DE, and 2-DE inhibition immunoblot because while one displayed partial inhibition, the other showed complete inhibition of IgE binding to cashew resulting from pre-adsorption with pistachio extract. These data validate the presence of cross-reactivity between cashew and pistachio as revealed by the inhibition ELISA and immunoblot studies (Figs. 18, 19, 20 and 21), and also demonstrate the presence of unique cashew epitopes as observed with patient #38 (Figs. 18 and 19). Extensive cross-reactivity is evident with the basic subunit of 11S globulins from cashew and pistachio, and the presence of cashew-unique IgE epitopes on the 2S albumins and the acidic subunit of 11S globulins (Figs. 18 and 19) is highlighted.

39 Cross-reactivity between almond, walnut and hazelnut 2S albumins has been documented 55 and a related finding from another study also attests the same [Teuber et al. in prep]. The later study demonstrates complete inhibition of IgE binding to cashew 2S albumin resulting from pre-adsorption with walnut extract and partial inhibition in regards to pre-adsorption with hazelnut extract. These reports further substantiate the presence of cross-reactivity between 2S albumins from cashew, walnut and hazelnut [Teuber et al.in prep]. Of the eight linear IgE binding epitopes identified in Ana o 3, one exhibits positional overlap and sequential homology (81% similarity) with the sole linear epitope of the English walnut (allergenic 2S albumin) Jug r 1 [Robotham et al. submitted]. This epitope similarity between cashew and walnut 2S albumins could perhaps explain why the cashew allergic patients employed in the present study are also allergic to walnut.

Facets of protein structure tend to be highly pertinent to the understanding of allergenicity and cross-reactivity. Past structural studies of 11S globulins and 2S albumins indicate their potential as allergens 39;44. The 11S globulins belong to bicupin family possessing a common β-barrel structure that appears to be a remarkably stable structural motif, resisting both thermal denaturation and proteolysis16. The 2S albumins are structurally homologous, compact globular proteins with a conserved skeleton of cysteine residues 44. This compactness and rigidity probably plays an important role, and could be responsible, in part at least, for the thermostable nature of the allergenic activity of these proteins. Such properties, coupled with the abundance in the cashew and pistachio nuts, may contribute to their being able to act as potent allergens.

Cross-reactivity is largely contingent upon structural aspects, as has been established by several previous studies 16;81. Interestingly, most of the proteins displaying IgE mediated cross-reactions have shared attributes on both the primary sequence and tertiary structural level. It is important to note here is that while all cross-reactive proteins exhibit an analogous fold, the reverse fails to hold true; proteins with a similar fold are not necessarily cross-reactive. The outer protein surface as well as protein folding is greatly influenced by differences in critical amino acid residues consequently resulting in a reduction in antibody reactivity to the specific epitope involved. Worth noting is that while cross-reactivity is found to be a rare outcome where identity is below 50%, similar protein folds are found with as little as 25% amino acid identity. In most circumstances, cross-reactivity entails 70% or greater sequence identity 81. High structural homology results from the sequence similarities of 2S albumins and, in particular, the common and conserved pattern of eight cysteine residues, suggesting that tertiary structure constraints imposed by disulfide bonds dominate sequence conservation. The 3 dimensional modeling of cashew and pistachio 11S globulins and 2S albumins, based on homology modeling with other homologous proteins and allergens (whose structures are resolved) could provide additional insights of their role as allergens.

The overall results of the current study posit that cross-reactivity between cashew and pistachio proteins, especially the 11S globulins and 2S albumins could be attributable to structural similarity. However, the in vitro allergenic cross-reactivity suggesting IgE binding and thereby identification of putative allergens cannot be considered as tantamount to its actual allergenicity under clinical circumstances. The clinical relevance of cross-reactivity seems to be influenced by a myriad of factors including the host immune response against the particular allergen,

40 exposure, and the intrinsic qualities of the allergen itself (Ferreira et al., 2004). In addition, the levels of specific IgE antibodies and their relative affinities are key mediators for allergic cross- reactions. Although cross-reactivity among tree nuts is proven to be clinically significant, it fails to inform us why some individuals exhibit clinical reactions to only one nut or seed while others reacting to several.

In summary, findings from the current study have supported the claim of existence of cross- reactive allergens in cashew and pistachio. This may explain the incidence of pistachio nut sensitivity among cashew allergic individuals. It would therefore be highly advisable for the cashew allergic patients to refrain from consuming pistachio nuts and vice versa. Inhibition studies (ELISA and immunoblot) and N-terminal amino acid sequence comparisons of allergens from cashew and pistachio nuts suggest that the 11S globulins and the 2S albumins seed storage proteins may perhaps prove to play a key role in clinical cross-reactivity. Differences in the amounts of specific allergens, within and between tree nuts, and the variations of affinities of the IgE antibodies may form the basis for the different degrees of cross-reactivity observed in this study.

Directions for future study involve identifying B-cell epitopes on 11S globulins and 2S albumins from pistachio. Doing so may facilitate the comparison of epitope maps of cashew and pistachio, perhaps even revealing locations on the proteins that confers cross-reactivity. Studies performing double-blind placebo-controlled food challenges with tree nut allergens and provocation tests are likely to be valuable in assessing the clinical relevance of in vitro cross- reactivity and the risk of a subject developing multiple allergies to various tree nuts. Further studies allowing the molecular identification and characterization of other potential cross- reactive allergens as well as the corresponding IgE-binding epitopes are expected to make a major contribution for improved diagnosis and treatment of cashew and pistachio nut allergy.

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48 BIOGRAPHICAL SKETCH Pallavi Tawde

Mailing address: Tel: 850-222-6067 (Home) 322 Conradi St, Apt#8 850-321-2279 (Cell) Tallahassee, FL 32304 E-mail: [email protected]

Education M. S., Biology (2004), Florida State University (FSU) in process. M. S. in Microbiology (1993), University of Bombay, India B. S., Microbiology (1989), University of Bombay, India

Qualifications Summaries • Comprehensive background and solid knowledge in immunology, biochemistry, molecular biology and microbiology • Experience in Site directed Mutagenesis, Bioinformatics, ELISA, Western blot, Electrophoresis, Chromatography, PCR, and many other laboratory techniques. • Quick study with ability easily assimilate job requirements and aggressively employ new ideas, concepts, methods and technologies. • Good communication and leadership skills. Attention to detail and concern for quality. • Thrive in work situations requiring an ability to manage multiple and concurrent responsibilities.

Experience Highlights Molecular biology experience

• Performed molecular biology techniques such as DNA/RNA extraction and purification, PCR, RT-PCR and sequencing. • Worked on cashew and almond cDNA library construction, allergen screening, cloning and recombinant protein expression, purification and characterization. • Sequencing, primer design, agarose and polyacrylamide gel electrophoresis • Site-directed mutagenesis of Ana o2, to produce hypoallergenic mutants.

Immunology and biochemistry • Site-Directed Mutagenesis • Two dimensional gel electrophoresis • Isoelectric focusing • Blue Native gel electrophoresis • Proficient in SDS-PAGE, gel staining, and immunoblotting • Western blot, immunodot blot to evaluate the activity of antigen and antibody. • Proficient in techniques of affinity chromatography, size exclusion chromatography and protein G/A chromatography. • Performed bacterial cell culture for the expression of recombinant protein. • Immunohistochemistry • Molecular, cellular, and experimental immunology techniques

49 Immunoassay • Immunoassays (Sandwich ELISA, inhibition ELISA) for the detection of tree nut allergen in foods. • Expertise in ELISA techniques and background knowledge. • Extensive experience in optimizing and evaluating the effective of the immunoassays. • Proficient in using KC4 v2.5 ELISA reader, KC4 software and multichannel pipettes.

Tissue culture

• Production and characterization of monoclonal antibodies using the hybridoma technology. • Experience in handling human peripheral blood mononuclear cells • Culturing of mouse and rat cell lines • Working on rat basophilic cell line (RBL) expressing human Fc epsilon receptors • Histamine release assay and β-Hexosaminidase assay – to study the mechanism of degranulation of basophils and mast cells upon specific IgE cross-linking by native, recombinant and hypoallergenic mutant allergens. • T cell proliferation assay

Microbiology

• Culturing Bacteria – Mycobacterium tuberculosis and other bacterial species

Other Experience • Expertise in the designing of experiments and analyzing data. Be capable of work independently on projects. • Ability to exercise independent judgment and excellent problem-solving skills. • Quick study with ability to learn and master new techniques/skills. • Prepare manuscript, thesis and protocols. Presented research findings and reviewed relevant scientific literatures in weekly meetings and seminars.

Skills Summarization • PCR thermocyclers • Plasmid extraction and purification • UV spectrophotometer • DNA electrophoresis • Public databases (GenBank etc.) • SDS-PAGE and protein transfer • Sequencer Analysis software • Immunoblotting, Western blot, Dot • ELISA reader blotting • Centrifuge • Sepharose 4B active beads protein • Electrophoresis and transfer coupling equipment • Affinity chromatography • Phospho scan • HPLC • PCR • Protein extraction and purification • DNA purification 50 • Recombinant protein expression • Tissue culture and purification • Site-directed mutagenesis of • Enzyme-linked immunosorbant allergens assay (ELISA) • Structural Analysis of proteins • Lowry protein assay • Handling of laboratory animals- • Bio-Rad protein assay mice and rabbits

Employment History • Research Assistant (2001- 2003), Florida State University, FL. • Teaching Assistant (2000 – 2004), Florida State University, FL. 2000-2004 Teaching assistant for Immunology lab 2001 Teaching assistant for Freshmen Biology lab 2002 and 2004 Teaching assistant Prokaryotic Biology (Microbiology) lab

• Research Fellow (1994 – 1999), Jaslok Hospital & Research Centre, Mumbai, India

Computer Proficiency • Windows 98/2000 & NT, UNIX. • Microsoft Word, Excel, Power Point, Outlook, Photo Editor. • Adobe PhotoShop, Paint Shop Pro 5 and other image-manipulation software. • Statistical packages: JMP, Minitab. • Public databases (GenBank etc.), Sequencer Analysis software (GCG). • Highly familiar with accessing the Internet for research purposes. • Quantitative image analysis. • Structural studies

Publications

Ana o 1, a cashew (Anacardium occidental) allergen of the vicilin seed storage protein family. Wang F, Robotham JM, Teuber SS, Tawde P, Sathe SK, Roux KH. J Allergy Clin Immunol 2002 Jul; 110(1):160-6

Ana o2, a major cashew nut allergen of the legumin family. Robotham JM, Wang F, Teuber SS, Tawde P, Sathe SK, Roux KH. Manuscript in progress.

Abstracts and Presentations Mutational Analysis of the Cashew Vicilin Allergen, Ana o 2. American Academy of Allergy, Asthma and Immunology, 60th Annual Meeting, San Francisco, CA. 2004.

Hypoallergenic Variants of the Legumin, Ana o 2, A Major Cashew Allergen 12th International Congress of Immunology and 4Th Annual Conference of FOCIS. July 18-23, 2004, Montreal Canada 51