Original article Cross-reactivities between date palm (Phoenix dactylifera L.) polypeptides and foods implicated in the oral allergy syndrome Background: Date fruit and pollen antigens share a number of cross-reactive epitopes. Date pollen has been shown to cross-react with antigens from Artemisia, cultivated rye (Secale cereale), Timothy grass (Phleum pratense), Sydney golden wattle (Acacia longifolia) and Bermuda grass (Cynodon dactylon) pollen. The present study was carried out to examine any cross-reactivities between date palm polypeptides and antigens of some common foods and vegetables that have been implicated in the oral allergy syndrome (OAS). Because most of such cross-reactivities in other allergens are attributable to the presence of carbohydrate chains and profilin, their role was also investigated. Methods: Fresh extracts of 20 common fruits and vegetables were prepared. Putative date profilins were isolated by affinity chromatography using a poly L- proline column. Date fruit extracts were digested by various endoglycosidases and the immunoglobulin (Ig)E binding of the postdigest products was assessed in immunoblots. Rabbit antisera to whole date fruit extracts, Timothy grass profilin and putative date profilins, as well as human sera from date sensitive individuals were used in immunoblotting, ELISA and in inhibition experiments. Results: IgG ELISA and immunoblot results with the different rabbit antisera and date-sensitive atopic sera showed several antigenic cross-reactivities and similar cross-reactivities were seen with birch, date and timothy grass profilins. IgE ELISA and immunoblot experiments with pooled date sensitive human sera showed a range of cross-reactivities with some food extracts. A number of the IgE cross-reactivities could be inhibited after preabsorption of pooled sera with date extracts. Sixty-six percent of individual date hypersensitive human sera bound IgE in putative date fruit profilin and their pooled sera bound IgE in birch pollen profilin. IgE-binding of the endoglycosidase digested date fruit extracts to atopic serum pool was restricted to only a very low molecular weight band of 6.5–8 kDa. Conclusion: These results indicate that date palm polypeptides share cross- reactive IgG and IgE epitopes with a number of foods implicated in the oral allergy syndrome, bind to birch and Timothy grass profilins and bind IgE through glycosyl residues. The clinical relevance of these cross-reactivities needs to be further elucidated. A. A. A. Kwaasi 1 , H. A. Harfi 2 , R. S. Parhar 1 , S. Saleh 1 , K. S. Collison 1 , R. C. Panzani 3 , S. T. Al-Sedairy 1 , F. A. Al-Mohanna 1 1 Department of Biological and Medical Research; 2 Department of Medicine/Paediatric, Section of Allergy and Clinical Immunology, King Faisal Specialist Hospital and Research, Center, Riyadh, Saudi Arabia; 3 Laboratoire de Recherches, Marseille, France A. A. A. Kwaasi PhD CBiol FIBiol MBC-03 Biological & Medical Research Department King Faisal Specialist Hospital and Research Center PO Box 3354, Riyadh 11211 Saudi Arabia Accepted for publication 15 February 2002 Patients sensitized to allergens from one source often react clinically to other allergens, and some patients with type I allergies to pollens frequently produce IgE to a wide range of food and vegetable allergens with the same specificity (1). This IgE binding occurs if proteins from the different source materials are homologous and contain identical, or nearly identical epitopes. Three explanations have been given for these cross-reactiv- ities: The first is related to taxonomic relationship of closely related organisms which often have great similarities and share a number of antigens as is the situation with pollen from different species of the same genus and family (2). The second type of cross- reactivities is thought to be caused by evolutionarily conserved protein structures. The ubiquitous pan- allergen, profilin, is a conserved protein in eukaryotes and is known to be responsible for most of the cross- reactivities between a birch pollen allergen (birch pollen profilin, Bet v 2) and extracts of some ‘vegetal’ foods (3). A third type of cross-reactivity which is responsible for reactions between even less phylogenetically related species is of glycoprotein origin and is as a result of the carbohydrate chains of these allergens (4–10). The unfortunate consequence of these cross-reactiv- Allergy 2002: 57: 508–518 Printed in UK. All rights reserved Copyright # 2002 Blackwell Munksgaard ALLERGY ISSN 0105-4538 508
Transcript
Original article
Cross-reactivities between date palm (Phoenix dactylifera L.)
polypeptides and foods implicated in the oral allergy syndrome
Background: Date fruit and pollen antigens share a number of cross-reactiveepitopes. Date pollen has been shown to cross-react with antigens fromArtemisia, cultivated rye (Secale cereale), Timothy grass (Phleum pratense),Sydney golden wattle (Acacia longifolia) and Bermuda grass (Cynodon dactylon)pollen. The present study was carried out to examine any cross-reactivitiesbetween date palm polypeptides and antigens of some common foods andvegetables that have been implicated in the oral allergy syndrome (OAS).Because most of such cross-reactivities in other allergens are attributable to thepresence of carbohydrate chains and profilin, their role was also investigated.Methods: Fresh extracts of 20 common fruits and vegetables were prepared.Putative date profilins were isolated by affinity chromatography using a poly L-proline column. Date fruit extracts were digested by various endoglycosidasesand the immunoglobulin (Ig)E binding of the postdigest products was assessed inimmunoblots. Rabbit antisera to whole date fruit extracts, Timothy grass profilinand putative date profilins, as well as human sera from date sensitive individualswere used in immunoblotting, ELISA and in inhibition experiments.Results: IgG ELISA and immunoblot results with the different rabbit antiseraand date-sensitive atopic sera showed several antigenic cross-reactivities andsimilar cross-reactivities were seen with birch, date and timothy grass profilins.IgE ELISA and immunoblot experiments with pooled date sensitive human serashowed a range of cross-reactivities with some food extracts. A number of theIgE cross-reactivities could be inhibited after preabsorption of pooled sera withdate extracts. Sixty-six percent of individual date hypersensitive human serabound IgE in putative date fruit profilin and their pooled sera bound IgE in birchpollen profilin. IgE-binding of the endoglycosidase digested date fruit extracts toatopic serum pool was restricted to only a very low molecular weight band of6.5–8 kDa.Conclusion: These results indicate that date palm polypeptides share cross-reactive IgG and IgE epitopes with a number of foods implicated in the oralallergy syndrome, bind to birch and Timothy grass profilins and bind IgEthrough glycosyl residues. The clinical relevance of these cross-reactivities needsto be further elucidated.
A. A. A. Kwaasi1, H. A. Harfi2,R. S. Parhar1, S. Saleh1,K. S. Collison1, R. C. Panzani3,S. T. Al-Sedairy1, F. A. Al-Mohanna11Department of Biological and Medical Research;2Department of Medicine/Paediatric, Section of
Allergy and Clinical Immunology, King Faisal Specialist
Hospital and Research, Center, Riyadh, Saudi Arabia;3Laboratoire de Recherches, Marseille, France
A. A. A. Kwaasi PhD CBiol FIBiol
MBC-03 Biological & Medical
Research Department
King Faisal Specialist Hospital
and Research Center
PO Box 3354, Riyadh 11211
Saudi Arabia
Accepted for publication 15 February 2002
Patients sensitized to allergens from one source oftenreact clinically to other allergens, and some patientswith type I allergies to pollens frequently produce IgEto a wide range of food and vegetable allergens with thesame specificity (1). This IgE binding occurs if proteinsfrom the different source materials are homologous andcontain identical, or nearly identical epitopes. Threeexplanations have been given for these cross-reactiv-ities: The first is related to taxonomic relationship ofclosely related organisms which often have greatsimilarities and share a number of antigens as is thesituation with pollen from different species of the same
genus and family (2). The second type of cross-reactivities is thought to be caused by evolutionarilyconserved protein structures. The ubiquitous pan-allergen, profilin, is a conserved protein in eukaryotesand is known to be responsible for most of the cross-reactivities between a birch pollen allergen (birch pollenprofilin, Bet v 2) and extracts of some ‘vegetal’ foods(3). A third type of cross-reactivity which is responsiblefor reactions between even less phylogenetically relatedspecies is of glycoprotein origin and is as a result of thecarbohydrate chains of these allergens (4–10).The unfortunate consequence of these cross-reactiv-
Allergy 2002: 57: 508–518Printed in UK. All rights reserved
Copyright # 2002 Blackwell Munksgaard
ALLERGYISSN 0105-4538
508
ities is that individuals can be sensitized to someallergens without exposure and so can they suffersevere, or at times fatal and detrimental, effects of suchallergens (1, 11). Birch pollen allergic patients oftenexhibit oral allergy syndrome after ingestion of variousfruits of the Family Rosaceae (apple, pear, cherry,peach), nuts and some vegetables such as carrots, celeryand potato. Other forms of pollenosis, now referred toin the revised EAACI nomenclature for allergy asallergic hypersensitivity (12) have been associated withcross sensitivities to various groups of food; allergy to
ragweed pollen has been noted to cause intolerance tobanana and melon (13). Hazelnut and hazel pollenallergic patients produce IgE to birch pollen, potato andapple (14) and children allergic to birch pollen are alsosensitized to apple, carrot and potato (15). Even thoughallergy to celery has been known for almost a century(1), the immunological relationship or cross-reactivityof celery to other allergens was not evident. It had longbeen presumed that in the groups of allergenic sourcesthat contribute to the OAS celery, whether cooked, rawor as a spice can cause a range of symptoms including
A
B
CD
Figure 1. (a) Poly L-proline affinity chromatograph elution profile monitored at A279. Peak A is the flow-through peak of unboundextract components during washing with matrix buffer. After the trace had returned to baseline, it was washed through with the samebuffer as detailed in the text and the poly L-proline binding components were eluted using matrix buffer containing 30% DMSO (peakB). (b) SDS-PAGE of date fruit components eluted from the poly L-proline column. Lane M = Amersham rainbow molecular weightmarker M (14.3–220 kDaMr). Lanes 1–8 are the proteins contained in the various fractions. Lanes 1 and 2 contain residual unboundhigh molecular weight material as shown by bands of j 14.3 kDa. Lanes 3–8 contain the poly L-proline binding putative profilin asseen. (c) SDS-PAGE of fractions of date pollen components eluted from the poly L-proline column. These fractions were collected inthe same way as in Fig. 1(b). The material eluted in lanes 1 and 2 as in 1c contain some unbound material as well as some undesired highmolecular weight proteins. Lanes 3–7 contain poly L-proline binding material, which are of a much lower molecular weight(<14.3 kDa) than that from date fruit extract. (d) Sephadex G-75 fractionation of pooled proteins from poly L-proline column wasused to further separate and purify the contaminating high molecular weight proteins and nonpoly L-proline binding proteins from thedesired low molecular weight proteins. Peak ‘‘A’’ contained all the high molecular weight material and peak ‘B’ was only made up of thedesired low molecular weight proteins.
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anaphylactic shock (16–18). However, recent work byBreitender et al. (19) and Luttkopf et al. (20) haveunequivocally clarified this anomaly. Breiteneder et al.have cloned and characterized Api g 1, the majorallergen of celery (Apium graveolens) and established itsimmunological and structural relationships to a groupof 17-kDa tree pollen allergens. Luttkopf et al. haveproved for the first time using double-blind placebo-controlled food challenge (DBPCFC) Api g 1, Api g 4,cross-reactive carbohydrate determinants (CCD), and a60-kDa allergen as celery allergens (19, 20).These multiplicity of syndromes make the diagnosis
and treatment of allergic diseases difficult, and accuraterecognition of the relationship between allergen expo-sure, sensitization and disease is paramount. Most dateallergic (hypersensitive) patients react to a number ofpollen and other allergens. Some of these patients whoare skin prick test (SPT) positive to date pollen and fruitextracts are individuals who do not eat dates, have
eaten dates on special occasions only, or have not hadany earlier exposure or contact with date pollen andcould therefore not have been previously sensitized (21).In a previous study of date pollen, IgG cross-reactivitieswere observed between date pollen and pollen extractsof Bermuda grass, Artemisia, rye, Timothy grass andAcacia longifolia (20, 21).Date fruits are eaten in varying quantities around the
world and are an important component in hundreds offoods sold in supermarkets (21). The major date pollenand fruit allergens and antigens are cross-reactive and50–100% of pollen and fruit allergic patients’ sera bindIgE to three main fruit-specific major allergen bandswith relative molecular weights of 6.5 to 12–14, 28–33and 54–58 kDa, provisionally assigned the notationsPho d.f. 1, 2 and 3 (22). Some of these molecular weightranges are similar to those reported for some majorallergens of many fruits and vegetables that cross-reactwith birch pollen profilin and have been implicated in
Figure 2. ELISA scores histogram of the food extracts with antisera to: (a) date pollen, (b) date fruit and (c) a mixture of the putativedate fruit and pollen profilin. Figure 1 (d) is IgE ELISA of individual date allergic sera with the putative date profilin as used in Fig. 2(c). It is clearly demonstrated in Fig. 1 (a–c) that irrespective of the extract there is a high degree of antigenic recognition betweenconstituents of some of the foods tested, birch pollen and the various date antisera sources. (d) IgE ELISA of date allergic human serawith the putative date profilin also shows that out of 24 sera tested 16 (66%) were positive to date fruit profilin. The cut-off level forpositivity has been denoted by lines. In order to determine positive from negative ELISA titres a Dynatech MR 600 automaticMicroplate reader (Dynatech Laboratories Inc.) interphased with an IBM computer and specifically programmed to calculate the meanand SD was used. Results were considered positive only if an ELISA score was 2 SD or higher than mean of all controls. Note that: RB= reagent blank control and NS = normal serum used as blank control, PIS=preimmune serum.
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OAS (5, 13, 14, 16, 17). Ingestion of dates is known tocause itching of the mouth and throat in someindividuals (21) and even anaphylactic shock has beenreported (11).There is however, no published reference to cross-
reactivities of date fruit polypeptides with any of thefoods involved in OAS. The aims of the present studywere: 1) to investigate any antigenic and allergeniccross-reactivities that may exist between date palmproteins and some of the foods to which our dateallergic patients react, and 2) to identify the cross-reactive components and to assess how specific they arein relation to date allergens and 3) find out whether dateallergenic molecules demonstrate any immunologicalrelatedness to any of the molecules known to beresponsible for IgE cross-reactivities in pollen andvegetable foods such as the profilins and carbohydratemoieties.
Material and methods
Fruits and vegetables
Owing to the large number of food and vegetables that have beenreported as cross-reactive only those that evoke allergic symptoms inat least one of the date sensitive patients seen in our clinic wereselected for this study. Fresh apple, apricot, asparagus, avocado,banana, beans, bell pepper, celery, fig, kiwi, mango, melon, olives,parsley, peach, peas, potato, spinach, strawberry and tomato werepurchased from local supermarkets and extracted, dialyzed,lyophilized and stored as previously described (21, 24). Purifiedbirch profilin was as a kind gift from Dr Irene Mitterman (AKH,Vienna, Austria).
Isolation of poly L-proline binding proteins from date fruit and pollen
Coupling of cyanogen bromide (CNBr)-activated sepharose 4B topoly L-proline. Cyanogen bromide activated Sepharose waspurchased from Pharmacia Diagnostics AB (Uppsala, Sweden).Five g of powder was suspended in 20 ml of 1 mM HCl (pH 2–3)and allowed to swell. This low pH was required to wash awayadditives and also preserve the activity of the reactive groups (25,26). The swollen gel was immediately washed for 15 min with1.5 L of 1 mM HCl on a sintered glass filter. The method used inthe preparation of poly L-proline-sepharose was essentially thatreported by Lindberg et al. (27). Poly L-proline (Sigma ChemicalCo, Poole, UK) was dissolved in coupling buffer (0.1 MNaHCO3 pH 3 containing 0.5 M NaCl) at a ratio of 1 : 5 (w/v).The coupling solution containing the ligand was mixed with thegel in a stoppered container and allowed to rotate end-over-endfor 1 h at room temperature or overnight at 4uC. After this step,the excess ligand was washed away with 10 gel volumes ofcoupling buffer. In order to block any remaining reactive sites, thegel was transferred into either of 0.1 M Tris HCl, pH 8.0 or 1 Methanolamine, pH 8.0 and allowed to stand for 2 h. The finalproduct was then washed with three cycles of alternating pH (acidand alkali) consisting of 0.1 M acetate buffer pH 4.0 containing0.5 M NaCl followed by a wash with 0.1 M Tris-HCl buffer pH 8,containing 0.5 M NaCl.
Affinity isolation of putative profilin by poly L-proline) sepharose 4B
chromatography
The poly L-proline column was equilibrated with Tris-Glycine
Figure 3. Immunoblots of rabbit antiserum to Timothy grassprofilin with purified birch pollen in lanes 1 and 2 (50 mg and200 mg, respectively). Lane 3 is 200 mg of putative date fruitprofilin. In the right panel is the flow-through of unbound pollenextract from poly L-proline column (lane 1) and lane 2 is 200 mgof date pollen profilin.
Figure 4. ECL developed IgG immunoblots of date fruit extractson nitrocellulose strips, demonstrating the cross-reactivity of datefruit and pollen antigens. Strips in lanes 1–3 were immunoreactedwith equal amounts of pooled antisera that have been preab-sorbed with 0.1, 1 and 10 mg/ml of pooled pollen extract. Theinitial absorption was enough to remove all fruit immunostainingbands except those at j 14.3 and 30 kDa. Progressive loss ofbands at j 14.3 and 30 kDa in a dose dependent manner fromlanes 1–3 is clearly demonstrated.
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Figure 5. (a) IgG immunoblots of fruit and vegetable extracts immunoreacted with hyper-immune rabbit antiserum to date fruit andpollen mix using enhanced chemiluminescence (ECL) horseradish peroxidase kit (Amersham Life Sciences, Buckinghamshire, UK);Lane HM is high molecular weight rainbow marker (14.3–220 kDaMr). Lanes 1–20 are the different extracts as indicated. Lane LM isof a lower molecular range rainbow marker (2.5–45 kDa). All Extracts displayed some degree of cross antigenicity. (b) is immunoblotof IgE–binding components of various fruit and vegetables extracts exhibiting cross-allergenicity with date fruit and pollen allergichuman serum pool. The markers are exactly as in 5a. Apart from Capsicum, kiwi, parsley and peas which did not resolve any bandssome IgE reactivities are seen with all extracts. (c) is an immunoblot of a similar gel which has been immunoreacted with atopic humanserum pool as in Fig. 5(b) but had been preabsorbed with pooled date allergenic fruit extract.
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‘matrix buffer A’ (0.1 M KCl, 0.1 M Glycine, 10mMm Tris-HCl,pH 7.8) containing 0.5 mM dithiothreitol (DTT) at room tempera-ture (27). Date pollen was defatted as previously described (28–30),and the date fruits were washed and the seeds removed (21, 22).Defatted pollen or fruits were homogenized in 5 volumes of ‘matrixbuffer A’ containing 0.5 mM DTT and 0.5% Triton X-100. Afterultracentrifugation (105 000rg, for 30 min at 4uC), the supernatantwas applied to the poly L-proline column and left overnight at 4uC.The column was then attached to a fraction collector (LKBPharmacia, Turku, Finland) and washed with matrix buffer only(25). Elution was monitored (A279) until the trace returned tobaseline. The elution buffer was at this stage changed and replacedby buffer A containing 30% DMSO, and fractions were collectedevery 2 min. Care was taken to avoid contact between DTT andDMSO because their reaction product; dimethyl sulfide is hazardousto health. Fractions were dialyzed freeze-dried and aliquots ofrepresentative samples were electrophoresed by SDS-PAGE to checkthe presence of proteins. These fractions were further purified by sizeexclusion chromatography using Sepahdex G-75 beads. Fractionsrich in the putative profilin were pooled and precipitated by dialysisagainst 10 volumes of saturated ammonium sulfate (pH 7.6) to
which had been added 5 mMmercaptoethanol. Protein samples wereeither lyophilized or stored as precipitate in ammonium sulfatesolution without loss of activity.
Investigation of the involvement of IgE-binding cross-reactivecarbohydrate determinants (CCDs)
Date fruit extracts from the most allergenic cultivars were pooledand after extensive dialysis, lyophilized and reconstituted in de-ionized distilled water to a concentration of 1 mg/ml. Aliquots werefurther purified on Detoxi-Gel columns (Pierce, Rockford, USA) toremove any toxic substances that may interfere with enzymaticdigestion. Solutions of endoglycosidases D, F and H (BoehringerMannheim GmbH, Mannheim, Germany) were prepared in theirrespective buffers (25, 31, 32) according to manufacturer’s instruc-tions. Endoglycosidase F (N-glycosidase F-free) was prepared in100 mM citrate buffer pH 4.5 (32), endoglycosidase D was preparedin 0.1 mM citrate-phosphate buffer (pH 5.0 33), and endoglycosi-dase H was prepared in 50 mM sodium acetate buffer of pH 5.5 (35).Solutions containing the recommended unit (s) of the enzymes wereadded to 0.1 mg date fruit or pollen extract and then incubated at
A B
Figure 6. (a) Effects of undigested, and allergenic samples digested with endoglycosidases D, F and H are shown. Lane M=molecularweight markers, Lane 1=CBB stained undigested date fruit allergenic extract pooled from the 5 most allergenic cultivars. Lanes 2–4=isSDS-PAGE of the same extract digested overnight as for 1 but digested with endoglycosidases D, F and H, respectively. In order tocheck that these enzymes did not possess proteinase activity a bovine serum albumin (BSA) control was included to see the effect ofthese enzymes. Results (not shown) indicated that the enzymes did not have any effect on the SDS-PAGE banding pattern of BSA. (b)Enhanced chemiluminescence developed autoradiograph of IgE immunoblot of endoglycosidase treated allergenic date extracts. Lane 1was incubated as others but without any enzyme, instead a mixture of the three buffers that were used in the enzyme digests lanes 2,3 and4 were added as controls.
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37uC overnight. In all cases 1 mM phenylmethyl-sulfonyl fluoride(PMFS) was added to inhibit proteolysis. Aliquots of these enzymeswere also incubated with different concentrations of bovine serumalbumin (BSA) as further controls to establish the absence ofnonspecific proteolytic activity by the endolycosidases. Afterincubation, the reaction was stopped and the digested extractswere electrophoresed and used in immunoblotting.
Antisera
Hyperimmune antisera to date pollen, date fruits and putative dateprofilins were produced in rabbits as previously described (21, 22,27–30). Briefly New Zealand White rabbits were weaned, bled forpreimmune serum and then immunized with either whole dateextracts or purified poly L-proline binding fractions from date fruitor pollen. Antiserum production in test bleeds was monitored byOuchterlony’s double immunodiffusion (21). Antisera were collectedand pooled after test bleeds from good responders, and the Igfractions were isolated (21, 28–30). Polyclonal antibody againstTimothy grass profilin was a kind gift from Dr Ronal van Ree (CLB,Amsterdam, Netherlands).
Human sera and patient symptoms
Normal human sera were collected as previously described (21, 22,29, 30). The patient serum pool used in the present study werecollected from patients who were SPT and RAST positive for datepalm allergens. These sera also gave positive IgE-immunoblotreactions with all the major date fruit and pollen allergens (22, 29,30). Additionally, these date-sensitive patients were polysensitive andreacted to some indoor and outdoor allergens as well as at least oneof the food and vegetables under investigation.Apart from reporting of various symptoms of allergic hypersensi-
tivity (12), the majority of the patients had reported at least oneepisode of throat itching, constant sneezing, mouth swelling, andlacrimation after eating dates or some of these foods. Some of thesesubjects complained of these symptoms after ingestion of certain datefruit cultivars only.
ELISA and ELISA inhibition
In order to assess the antigenic and allergenic relatedness of datepalm extracts to structures in these foods, in birch pollen and othercross-reactive molecules, indirect ELISAs were carried out. Rabbitantisera to whole date fruit and pollen, putative date fruit and pollenprofilins and Timothy grass profilin as well as human sera wereinvestigated. Indirect and ELISA inhibition experiments wereperformed with various foods as follows. Aliquots of antisera orhuman serum were preincubated with each of the food extracts withcontinuous mixing (4uC, overnight) and centrifuged to removeimmune precipitate before the supernatant was added to the coatedwells. Wells were washed after incubation and secondary antibodiesadded. Further washing and chromogen development steps werecarried out as described previously and results analyzed (28–30).
SDS-PAGE, immunoblotting and absorption experiments
Each of the extracts was adjusted to the same protein concentrationand electrophoresed on either 13% or 5–15% gradient gels using thediscontinuous system of Laemmli’s (34). The protein components ofextracts were electrotransferred onto nitrocellulose membranes(0.45 mm pore size, Bio-Rad, Richmond, CA, USA) according tothe method of Towbin et al. (35). After transfer, the membranes weretreated with 3% BSA to block non-specific binding sites. Membraneswere allowed to react with appropriate dilutions of the various seraand antisera followed after washes by the addition of secondaryantibodies and all further steps were carried out as described (21, 29,30).
In order to ascertain the specificity of these extracts absorptionexperiments were carried out by preincubation of pooled human serawith date fruit and date pollen extracts as previously described (21,28–30).
Results
Isolation of putative profilin from date fruit and pollen
Putative profilins from date fruits and pollen, birchpollen profilin and antiserum to Timothy grass profilinswere used in experiments in order to assess the role ofprofilins in the cross-reactivity of date proteins.Figure 1a is a typical affinity purification chromato-
graphy elution profile obtained when date fruit orpollen extracts are applied to the poly L-prolinecolumn. The two distinct peaks indicate fractionscontaining unbound (peak A) and bound (peak B)proteins.Figures 1b and c are SDS-PAGE gels of fractions
collected from poly L-proline chromatography of datefruit and pollen extracts, respectively.Figures 1b and c are results of further purification of
poly L-proline binding material on Sephadex G-75column. Through repeated fractionation steps thismethod separated high molecular weight contaminatingproteins from the desired low molecular weight material(putative profilins). Figure 1d is a typical separationprofile.
Cross antigenicity of date fruit and pollen components with antigens
in other foods
Figure 2a–c are histograms of ELISA scores of the fruitand vegetable extracts with various antisera.Figure 2d shows histograms of the reactivity of
individual sera of patients who are hypersensitive toputative date fruit profilin.Figure 3 demonstrates the cross-reactivity of
Timothy grass profilin with date and birch profilins.These results reveal the allergenic cross-reactivities thatare evident between date antigens, birch profilin andTimothy grass profilin.Figure 4 demonstrates the antigenic cross-reactivities
between date fruit and date pollen.Pre-absorption was effective in the complete abroga-
tion of all date fruit immunoreactive bands and onlyfaintly staining residual bands can be resolved at j12–14 and 28–33 kDa in a dose-dependent manner andall other bands were totally removed. In a previousstudy it was shown that there was complete absence ofinhibitable cross-reactivity between date proteins andpollen extracts from two genera of the Palmaceae familynamely, Washingtonia spp. and coconut (Cocos nuci-fera) (22).Figure 5a is IgG immunoblots of the various fruit
and vegetable extracts immunostained with hyperim-mune rabbit antiserum to date extract.
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Figure 5b is a similar blot to that in Fig. 5a that hasbeen immunostained with pooled atopic human serum.Figure 5c is a parallel blot to Fig. 5b but the polled
serum was preabsorbed sequentially with date fruitextract followed by date pollen extracts.The cross-antigenicity of date proteins with extracts
of apple, apricot, asparagus, avocado, banana, beans,bell pepper, celery, fig, kiwi, mango, melon, olives,peach, peas, potato, spinach, strawberry and tomatoare evident in IgG immunoblots.No inhibitable cross-reactivities were seen with any of
the major apple allergens of 9, 18 and 31 kDa but IgEcross-reactive bands at 55 and 75 kDa (Fig. 5b) wereremoved after absorption with date fruit extracts.Apricot, asparagus and avocado displayed a widerange of antigenic and allergenic cross-reactivities,most of which were either completely removed ortheir intensities substantially reduced after absorptionwith date extracts. IgE cross-reactivities were seen inbanana at 20, 35, 37, 57 and above 75 kDa, however,the only bands that could be removed postabsorptionwere the 35 and 37 kDa bands. Beans were cross-reactive with bands of about 2–25 and 37–75 kDa, andall of them could be absorbed with date extracts.Capsicum antigens were difficult to resolve and nodistinct antigenic or allergenic cross-reactive bands wereevident. A number of cross-reactive celery-specificbands were seen in immunoblots with rabbit antiserumfrom as low as 3 kDa to over 100 kDa. Similarly IgEimmunoblots of celery extracts with human atopicserum pool showed distinct immunostaining bandsfrom j6-i100 kDa, which were inhibited post-absorption. However, no clear IgE-immunostainingbands were seen with the major celery band Api g 1 of14.3 kDa.The bands in fig distinctly exhibited IgG and IgE
cross-reactivities, which could be inhibited with dateextracts at 47 and 67 kDa. Kiwi fruit showed a fewimmunoreactive IgG bands with rabbit antiserum butapart from a smear of 2.5–20 kDa no IgE reactivitieswere evident with atopic human sera. Protein bands inmango were cross-reactive with rabbit antiserum above20 kDa but were not discernible owing to smearing andhigh background. Several mango-specific IgE cross-reactive bands of between 20 and 100 kDa were seenand all of them could be inhibited by preabsorptionexcept two bands of 22–25 kDa.All protein bands in melon were antigenically cross-
reactive as indicated by IgG blots but only bands ofbetween 30 and 62 kDa bound IgE in atopic sera. Apartfrom a distinct band at 66 kDa, olive extract did notresolve well and only showed smearing at molecularweights of between 2 and 6 kDa. No bands could beresolved with parsley. Peach extract possessed onedistinct IgG- and IgE-immunostaining band of about8–9 kDa, which could be totally abrogated afterabsorption with date extract. Potato gave a distinct
antigenic and allergenic reactive band at about 45 kDaand a faint band at 23 kDa. Both bands were totallyinhibited by preabsorption with date extracts. Spinachdisplayed a number of IgG and IgE cross-reactivebands. All the IgE reactive bands were inhibited post-absorption except a sharp band of about 21 kDa.Strawberry was cross-reactive giving a distinct IgE-staining band at 4.5–7 kDa and weaker bands at 42 and58 kDa. The 4.5–7 kDa band was unaffected inimmunoblot inhibition experiments but the highermolecular weight bands were completely removed.Weak IgG immunostains of 5–17 kDa and some 3IgE cross-reactive bands were seen with tomato extract.Only the bands of 42 and 66 kDa were inhibited afterpreabsorption.In general cross-reactive IgE bands can be seen in the
different fruit and vegetable extracts at differentmolecular weight ranges with the unabsorbed datehypersensitive (12) serum pool (Fig. 5b), whilst anumber of these IgE reactive bands, particularly atlow molecular weights of between 6 and 30 kDa areeither much reduced in intensity or missing with thepostabsorption serum pool. These are particularlyevident in apricot, asparagus, avocado, celery, fig,peach, peas, potato, strawberry and tomato extracts.
Involvement of carbohydrate epitopes in allergenicity of datepolypeptides
Figure. 6a and b are results of experiments withendoglycosidases digests to assess the degree and effectsof glycosylation on the IgE binding of date fruit extractsand results are presented.Figure 6a is SDS-PAGE of undigested allergenic
samples and Fig. 6b is the post digestion productsimmunoblotted with date-sensitive atopic serum todetect IgE binding proteins. Figure 6b shows clearlythat, the only postdeglycosylation IgE-binding is seen ata very low molecular weight of c. 6.5–8 kDa.
Discussion
Cross-reactivity occurs when specific antibodies formedin response to one epitope reacts with another similar oridentical epitope on another antigen. Because naturehas a habit of conserving successful protein structures,and using common metabolic pathways, cross-reactiveallergens are distributed widely throughout the animaland plant kingdoms. The most broadly distributedcross-reactive allergens are the pan-allergens. Examplesof these are proteins like profilin (3, 23), lipid transferproteins (37, 38) and cross-reactive carbohydratedeterminants (5–10, 39). Most of these cross-reactiv-ities could be of clinical importance so it is essential forallergist and patients to be aware of them especially forpreviously unknown allergens such as date palmallergens.
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In this study hyperimmune rabbit antisera to datepollen, date fruit and putative profilin from date fruitsand pollen have been used to demonstrate the range ofcross-antigenicity that exists between date polypeptidesand antigens from taxonomically different fruit andvegetables. The use of purified birch pollen profilin andantibody to Timothy grass profilin, also made itpossible to show that poly L-proline binding proteins(the putative date pollen and fruit profilins) possessantigenic components that cross-react with birch andTimothy grass profilins making date allergens apotential player in the OAS.IgE to food allergens are known to be highly
prevalent in patients allergic to pollens, with andwithout symptoms of food allergy and in many of thesepollen allergic patients the presence of food specific IgEdoes not always correspond to clinical symptoms offood allergy (40). The allergenic capability of date palmpollen and fruit proteins has been known for a relativelyshort time (21, 22, 28–30). To date, nothing is knownabout their cross-reactivities apart from the fact thatdate pollen and fruit polypeptides cross-react with eachother and date pollen cross-reacts with pollen extractsof Bermuda grass, Artemisia, rye, Timothy grass andAcacia longifolia (29).Even though the patient population under study has
not been screened for the true presence of oral allergy todates and the various foods, most of the patientspresent with symptoms that are very much like or mimicthose reported for OAS to other foods. The fact thatdate-allergic sera bind IgE to birch profilin is a pointerto the fact that oral allergy syndrome may exist in thedate-allergic population and only needs thorough andwell controlled clinical investigations to establish thisfact.Immunoblot inhibition results hint at the probability
that low molecular weight proteins like lipid transferproteins (LTPs) may be involved in the allergenicity ofdate polypeptides. The reason for this surmise is that,most of the low molecular weight bands at c. 6–15 kDain these cross-reactive fruit and vegetables (apple,peach, kiwi, melon, strawberry and celery) that areknown to contain lipid transfer proteins (37, 38) gavediffering intensities of IgG bands with rabbit antiserumto dates. Most of them were inhibited (complete orpartial abrogation of bands) in IgE immunoblots bypreabsorption of pooled atopic human positive serumwith date fruit extract.Lipid transfer proteins are highly conserved and are
resilient to enzymatic digests (40). Their exact functionis not yet completely clear, but they have beenimplicated in the transport of fatty acids, bothintracellularly and extracellularly (41, 42). All livingplants possess natural protection from invading patho-gens and lipid transfer proteins have been reported toact as plant defence proteins against bacterial andfungal infections (38, 44–46). LPTs have been detected
in many botanically unrelated plants such as peach,apple, tomato, rape, carrot, barley, corn, wheat, rice,sorghum, broccoli, onion and grapevine (37, 46, 47). Arole in the transport of cutin monomers to the cuticularlayer of leaves and fruits could also explain theirabundance in peels of allergenic fruits (36) and in thewaxy layer of some leaves like broccoli leaves. All theseattributes are necessary for a plant like the date palmthat thrives in harsh arid environments. Furthermore,some of the foods mentioned above share antigeniccross-reactivity with date structures making furtherstudies of these low molecular weight date proteinsessential for further understanding of allergy to datesand whether dates in fact, possess LTPs.The importance of carbohydrate epitopes in plant
allergy has been widely emphasized in recent literatureand cross-reactivity between pollen and vegetable foodsowing to allergen-specific IgE that recognizes carbohy-drate epitopes is a phenomenon that is gaining attention(5–10, 48). Results of experiments with endoglycosi-dases were interesting: the shift in most of the bands andthe virtual disappearance of most IgE binding compo-nents in lanes 2–4 in Fig. 6a especially at 8–14 kDa isobvious. Some of the fused IgE bands at 30 kDa andj14.3 kDa were replaced by discrete bands withseeming shifts in their molecular weights, indicatingthat glycans, that are susceptible to these enzymes areinvolved in the IgE binding to date allergens. This alsoimplies that carbohydrates and glycopeptides in datesmay play a key role in their IgE-binding capability.Such cross-reacting carbohydrate determinants are
present not only in a wide variety of plant extracts (48),but also in material of invertebrate origin, includinginsect venom, caddis fly and molluscs, leading to a widerange of unexpected cross-reactivities (48, 49). Datescontain a high amount of sugars and fresh date fruitscontain between 60 and 70% of carbohydrate dependingon the variety or cultivar (50), which also endow dateswith high glycoprotein and probable CCD content; andcould influence or be related to its allergenicity. Resultsof SDS-PAGE using endoglycosidase treated extractsshowed distinct shifts in some IgE-binding peptidebands in date fruits and pollen. Similarly, IgEimmunoblots with the post-digest extracts alsoshowed that IgE binding in date fruit extract wasrestricted to a low molecular weight band compared tothe rather large and diffused IgE bands seen inimmunoblots with date cultivars (51). This indicatesclearly that sugar (or glycosyl) residues play a key rolein date palm allergy.Even though these cross-reactive date palm polypep-
tides are yet to be identified and their role in date palmallergy elucidated, this maiden study provides strongevidence that there is significant amount of antigenicand allergenic cross-reactivity between date fruitcomponents and those of some of the foods studied.Date palm proteins cross-react with birch and Timothy
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grass profilins as well as with polypeptides in a numberof fruit and vegetables that have been proven to causeor have been associated with OAS. Like some of thefoods investigated here date polypeptides appear tobind IgE through glycosyl residues and some also cross-react with some LTP-like structures in some of thesefoods. It can be concluded that: firstly, date palmproteins exhibit a broad range of cross-reactivity withstructures in most of the foods studied; secondly, someof these antigenic and allergenic relatedness are specificand can be inhibited in date reactive atopic serum poolby preabsorption with date extracts and thirdly, datepolypeptides cross-react with Timothy grass and birchpollen profilins.Dates possess some epitopes, which bind IgE through
carbohydrate or glycosyl moieties, and some of these
proteins are highly cross-reactive with structure infoods that are known to possess LTPs and can induceOAS.A detailed study of these cross-reactive structures in
dates, and their pathophysiologic role in patients whoare hypersensitive to dates and the individual foods isneeded to explain some of the unresolved observations.
Acknowledgments
We would like to thank Delia Bonifaco and Rita Sison for secretarialassistance. Staff of the Photographics and the Ground Maintenancedepartments are acknowledged for their respective contributionstowards the study.
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