Cloning of the minor allergen Api g 4 profilin from celery (Apium graveolens) and its cross-reactivity with birch pollen profilin Bet v 2

BACKGROUND: Profilin is a panallergen that is recognized by IgE from about 20% of birch pollen- and plant food-allergic patients. A subgroup of celery-allergic patients shows IgE-reactivity with this minor allergen. To investigate the IgE-binding potential and cross-reactivity of celery profilin at the molecular level, this study was aimed at the cloning and immunological characterization of this allergen. OBJECTIVES: Cloning, expression and purification of profilin from celery tuber to characterize its immunological properties and its cross-reactivity with birch pollen profilin. METHODS: Cloning of celery profilin was performed by polymerase chain reaction using degenerated primers and a 5'RACE method for the identification of the unknown 5'-end of the cDNA. Expression was carried out in Escherichia coli BL21 (DE3) using a modified vector pET-30a. The recombinant profilin was purified by affinity chromatography on poly L-proline coupled to sepharose. Immunological characterization was performed by immunoblotting, EAST and IgE-inhibition experiments. RESULTS: The coding region of the cDNA of celery profilin was identified as a 399-bp open reading frame, coding for a protein of 133 amino acids with a calculated molecular weight of 14.3 kDa. The deduced amino acid sequence of the corresponding protein showed high identity with other plant profilins (71-82%) recently described as allergens. Celery profilin was isolated as highly pure nonfusion protein. The IgE-reactivity of celery profilin was similar to that of natural protein. Seven of 17 celery-allergic patients tested presented specific IgE-antibodies to the recombinant protein tested by immunoblotting. Inhibition experiments showed high cross-reactivity of IgE with both profilins from celery and birch pollen. Moreover, the biological activity of recombinant celery profilin was demonstrated by a histamine release assay. CONCLUSIONS: Celery profilin is an important allergenic compound in celery and shows high homology to birch pollen profilin, Bet v 2. According to the revised IUIS allergen nomenclature, we suggest naming the celery profilin Api g 4. In addition to the cross-reacting major allergens Api g 1 and Bet v 1, birch pollinosis and associated allergies to celery can therefore additionally be explained by the cross-reactivity between homologous profilins. Moreover, recombinant Api g 4 may be used for target-specific diagnosis and structural analyses.     

IgE reactivity to profilin in pollen-sensitized subjects with adverse reactions to banana and pineapple

BACKGROUND: The so-called 'latex-fruit syndrome' is a well-documented phenomenon in cross-reactive allergies. By contrast, there is a lack of information about allergy to exotic fruits in patients with a predominant pollen sensitization. Since the ubiquitous protein profilin has been identified as an allergen in natural rubber latex as well as in pollen-related foods, the aim of this study was to investigate the role of profilin in allergy to certain exotic fruits. METHODS: Recombinant profilins from banana and pineapple were cloned by a PCR technique after isolation of total RNA using degenerated profilin-specific primers. The unknown 5' ends of copy DNA (cDNA) were identified by rapid amplification of 5'cDNA ends (5'-RACE) and expression in Escherichia coli BL21(DE3) cells. The recombinant profilins were purified by affinity chromatography using poly-(L)-proline as the solid phase. IgE-binding capabilities were characterized by means of immunoblot and Enzyme Allergosorbent Test (EAST). The cross-reactivity to birch pollen profilin and latex profilin was studied by EAST as well as by immunoblot inhibition experiments. RESULTS: Both banana and pineapple profilin were found to consist of 131 amino acid residues with high amino acid sequence identity to known allergenic pollen and food profilins (71-84%). IgE binding to the recombinant profilins was observed in 7/16 sera from subjects with suspected banana allergy (44%) and in 8/19 sera from subjects with suspected pineapple allergy (42%). Inhibition experiments indicated similar IgE reactivity of natural and recombinant allergens. In addition, high cross-reactivity to birch pollen profilin Bet v 2 and latex profilin Hev b 8 was demonstrated by immunoblot inhibition as well as EAST inhibition experiments. CONCLUSIONS: Since a high IgE-binding prevalence of about 40% was obtained in both banana and pineapple allergy, we conclude that profilin is an important mediator of IgE cross-reactivity between pollen and exotic fruits.     

Cross-reactivity within the profilin panallergen family investigated by comparison of recombinant profilins from pear (Pyr c 4), cherry (Pru av 4) and celery (Api g 4) with birch pollen profilin Bet v 2.

Profilin is a panallergen which is recognised by IgE from about 20% of birch pollen- and plant food-allergic patients. Little is known about epitope diversity among these homologous proteins, and about the correlation between IgE-cross-reactivity and allergenic reactivity. Plant food profilins from pear (Pyr c 4) and cherry (Pru av 4) were cloned by polymerase chain reaction and produced in Escherichia coli BL21. The profilins were purified as non-fusion proteins by affinity chromatography on poly-(L-proline)-Sepharose and characterized by immunoblotting, IgE-inhibition experiments and histamine release assays. The coding regions of the cDNA of pear and cherry profilin were identified as a 393 bp open reading frame. The deduced amino acid sequences showed high identities with birch pollen profilin Bet v 2 (76-83%) and other allergenic plant profilins. Pyr c 4 and Pru av 4 were investigated for their immunological properties in comparison with profilins from celery (Api g 4) and birch pollen (Bet v 2). Fourty-three of 49 patients (88%), preselected for an IgE-reactivity with Bet v 2 showed specific IgE-antibodies to the recombinant pear protein, 92% of the sera were positive with the recombinant cherry allergen and 80% of the sera were reactive with the celery protein. Inhibition experiments showed a strong cross-reactivity of IgE with profilins from plant food and birch pollen. However, IgE binding profiles also indicated the presence of epitope differences among related profilins. All investigated profilins, Pyr c 4, Pru av 4, Api g 4 and Bet v 2, presented almost identical allergenic properties in cellular mediator release tests. Therefore, cross-reactivities between related profilins may explain pollen-related allergy to food in a minority of patients. The nucleotide sequences reported have been submitted to the Genbank database under accession numbers AF129424 (Pyr c 4) and AF129425 (Pru av 4).     

Profilins: mimickers of allergy or relevant allergens?

Profilins are ubiquitous proteins, present in all eukaryotic cells and identified as allergens in pollen, latex and plant foods. The highly conserved structure justifies the cross-reactive nature of IgE antibodies against plant profilins and their designation as pan-allergens. Primary sensitization to profilin seems to arise from pollen sensitization with later development of cross-reactive IgE antibodies against plant food (and possibly latex) profilins. The role of profilin in inducing allergic symptoms needs to be evaluated and raises important issues in allergy diagnosis due to cross-reactivity. IgE cross-reactivity among profilins is associated with multiple pollen sensitization and with various pollen-food syndromes. In respiratory allergy, sensitization to pollen to which the patient has virtually no environmental exposure has been identified as a manifestation of profilin sensitization. As a food allergen, profilin usually elicits mild reactions, such as oral allergy syndrome, is not modified by processing and is especially important in allergy to some fruits, such as melon, watermelon, banana, tomato, citrus fruit and persimmon. Purified natural and recombinant profilins for in vitro and in vivo allergy tests are helpful in the diagnostic work-up. Herein we review the current state of knowledge about the allergen profilin and its implications in the diagnosis and treatment of allergic diseases. We conclude that, although its role in triggering allergic symptoms is still controversial, profilin is undoubtedly a relevant allergen. As a pan-allergen, profilin is associated with multiple pollen sensitization and pollen-food-latex syndromes that the allergist has to be aware of in order to accomplish an accurate diagnosis and successful treatment of allergic diseases.     

Cloning of the panallergen profilin from lychee fruit and its cross-reactivity with birch pollen profilin Bet v 2

Lychee fruits can cause severe anaphylactic reactions and has cross-reactivity with other allergens. Panallergen profilin is partly responsible for the cross-reactivity between allergens. In the present study, the cDNA of lychee profilin was performed by RT-PCR and 3'RACE from total RNA. The lychee profilin cDNA includes an open reading frame coding for 131 amino acids. The deduced amino acid sequence of the corresponding protein show high identity with other allergenic profilins. Expression of the recombinant lychee profilin was carried out in Escherichia coli BL21 (DE3) using vector PET-28a, and the purification of the recombinant protein was performed via affinity chromatography with Ni⁺ coupled to sepharose. IgE reactivity of recombinant lychee profilin was investigated by immunoblot, and five of 15 lychee-allergic patients tested presented specific IgE antibodies to recombinant lychee profilin. IgE inhibition and ELISA inhibition experiments were performed to analyze lychee profilin cross-reactivity with profilins from birch pollen, and a high cross-reactivity between lychee profilin and birch profilin has been found in sera from lychee allergic patients.     

Cloning and characterization of profilin (Pru du 4), a cross-reactive almond (Prunus dulcis) allergen

BACKGROUND: The identity of allergenic almond proteins is incomplete. OBJECTIVE: Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen. METHODS: An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin. RESULTS: The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins. CONCLUSION: Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients. CLINICAL IMPLICATIONS: Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.     

Pho d 2, a major allergen from date palm pollen, is a profilin: cloning, sequencing, and immunoglobulin E cross-reactivity with other profilins

BACKGROUND: Up to now, some date palm pollen (DPP) allergens have been described but very few data are available about their molecular nature. The aim of this study was to identify and characterize Pho d 2, a major allergen from this pollen. METHODS: Sera from 25 patients allergic to DPP were analysed by immunoblotting. Purification of DPP profilin was performed by poly-l-proline affinity chromatography. Profilin-encoding cDNA from DPP was cloned by using a RT-PCR strategy and recombinant allergen was expressed as a non-fusion protein in Escherichia coli. Natural and recombinant Pho d 2 were investigated by means of enzyme allergosorbent test to compare the immunologic properties of both allergens and to analyse cross-reactivity with other profilins. RESULTS: A 14.4 kDa protein was identified as a major allergen in DPP extract. Purification, cloning, heterologous expression, and inhibition experiments identified it as profilin (Pho d 2). Pho d 2 comprises 131 amino acids and has high sequence identity with other allergenic food and pollen profilins. The prevalence of specific IgE antibody reactivity to natural Pho d 2 by ELISA was 56% and 64% by skin prick test (SPT). Pho d 2 is an important allergen as it is responsible for more than 70% of the IgE reactivity to the pollen extract. IgE directed against Pho d 2 showed a strong cross-reactivity with other profilins such as those from olive tree and grass pollens. CONCLUSION: Pho d 2, a 14.4 kDa protein identified as profilin, is a major and relevant allergen in DPP, as confirmed by SPT and thereby may elicit clinical symptoms in sensitized patients.     

Cloning and molecular and immunological characterisation of two new food allergens,Cap a 2 and Lyc e 1, profilins from bell pepper (Capsicum annuum) and Tomato (Lycopersicon esculentum)

BACKGROUND: Profilins are recognised by IgE of about 20% of patients allergic to birch pollen and plant foods. They are ubiquitous intracellular proteins highly cross-reactive among plant species. Therefore, they were called panallergens and are made responsible for cross-sensitisation between plant pollen and food. OBJECTIVES: The aim of the present study was to clone the cDNAs encoding profilins from bell pepper and tomato, to produce and purify the recombinant proteins and to compare their IgE-binding capacities to those of the natural proteins. METHODS: cDNA clones coding for profilin were obtained by RT-PCR from total RNA of tomato and bell pepper fruits, sequenced and expressed as non-fusion proteins in ESCHERICHIA COLI. The recombinant profilins were subsequently purified and tested for IgE-binding and inhibition capacity with sera from 34 food-allergic patients. Possible oligomerisation of recombinant profilins was investigated by HPLC analysis and its influence on IgE binding assayed by ELISA. RESULTS: The open reading frame from both profilins encompasses 393 bp with a predicted molecular mass of 14,184 kD and a pI of 4.44 for bell pepper profilin (Cap a 2) and 14,257 kD and a pI of 4.46 for the profilin from tomato (Lyc e 1). The two protein sequences display 91% identity, whereas tomato profilin from pollen shares only 75% identity with tomato fruit profilin. Eleven out of 34 food-allergic patients (32%) display IgE binding to both purified profilins. Preincubation of a serum pool with either purified rCap a 2 or rLyc e 1 nearly abolished IgE binding to natural Cap a 2 and Lyc e 1, respectively. In addition, purified recombinant Cap a 2 was able to inhibit IgE-binding to rLyc e 1 by approximately 50%, whereas rLyc e 1 completely blocked IgE-binding to rCap a 2 in cross-inhibition assays. HPLC analysis showed that in solution Cap a 2 and Lyc e 1 can be found predominantly as dimers, which can be partially reduced to monomers by addition of dithiothreitol (DTT). In ELISA DTT-treated Lyc e 1 displayed a clearly lower IgE-binding capacity than untreated profilin. CONCLUSIONS: Purified rCap a 2 and rLyc e 1 proved to be valuable tools for studying cross-reactivity to profilins in patients allergic to pollen and food.     

Purification and immunochemical characterization of Pla l 2, the profilin from Plantago lanceolata

Profilins are small actin-binding proteins found in eukaryotes and involved in cell development, cytokinesis, membrane trafficking, and cell motility. From an allergenic point of view, profilins are panallergens usually involved in allergic polysensitization, although they are generally recognized as minor allergens.The objectives of this study were to identify and characterize the profilin from Plantago lanceolata pollen and to investigate the cross-reactivity between profilins from different pollen allergenic sources.Profilins from P. lancelolata (Pla l 2) and palm tree pollen (Pho d 2) were purified by affinity chromatography, deeply characterized and identified by mass spectrometry. Pla l 2 allergenicity was confirmed by immunoblot with serum samples from a patient population sensitized to profilin. Immunoblot inhibition was performed to study IgG reactivity between different pollen profilins. IgE cross-reactivity was demonstrated by ImmunoCAP inhibition.Pla l 2 is the second P. lanceolata allergen included in the IUIS Allergen Nomenclature database. Four peptides from purified Pla l 2 were identified with percentages of homology with other pollen profilins between 73 and 86%. Eighty-six percent (21/24) of the patient population recognized Pla l 2. The allergenic relatedness between Pla l 2, Pho d 2 and six pollen profilins was confirmed, and IgE cross-reactivity of Pla l 2 with rBet v 2 and rPhl p 12 was demonstrated.Pla l 2 is the profilin from P. lanceolata. The demonstrated allergenicity of this protein and its cross-reactivity with other pollen profilins support its use in profilin diagnostic assays.     

Ara h 8, a Bet v 1-homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy

BACKGROUND: We recently described patients with soybean allergy mainly mediated by cross-reactivity to birch pollen allergens. A majority of those patients were reported to have peanut allergy. OBJECTIVE: We sought to study the occurrence of peanut allergy in patients allergic to birch pollen and characterized the Bet v 1-homologous peanut allergen Ara h 8. METHODS: Recombinant Ara h 8 was cloned with degenerated primers and expressed in Escherichia coli. Nine Swiss and 11 Dutch patients with peanut and birch pollen allergy and a positive double-blind, placebo-controlled food challenge result to peanut were investigated for IgE reactivity to birch pollen and purified peanut allergens and cross-reactivity between birch and peanut. Ara h 8 stability against digestion and roasting was assessed by means of RAST inhibition. The IgE cross-linking potency of Ara h 8 was tested on the basis of basophil histamine release. RESULTS: During double-blind, placebo-controlled food challenge, all patients experienced symptoms in the oral cavity, progressing to more severe symptoms in 40% of patients. CAP-FEIA detected recombinant (r) Ara h 8-specific IgE in 85%. IgE binding to Ara h 8 was inhibited by Bet v 1 in peanut extract immunoblotting and in RAST inhibition. In EAST inhibition recombinant rAra h 8 inhibited IgE binding to peanut in 4 of 7 tested patient sera. Antipeanut response was dominated by Ara h 8 in 12 of 17 tested patients. Furthermore, our results demonstrate a low stability of Ara h 8 to roasting and no stability to gastric digestion. Basophil histamine release with rAra h 8 was more than 20% in 5 of 7 tested sera. CONCLUSIONS: Peanut allergy might be mediated in a subgroup of our patients by means of cross-reaction of Bet v 1 with the homologous peanut allergen Ara h 8.     

IgE to Bet v 1 and profilin: cross-reactivity patterns and clinical relevance

BACKGROUND: Individuals with pollen allergy often have IgE against plant-derived foods. This can be due to cross-reactive IgE against Bet v 1 and homologues, profilins, and/or cross-reactive carbohydrate determinants. OBJECTIVE: The aim of this study was to correlate sensitization to Bet v 1 and profilin with individual recognition patterns to plant foods and clinical relevance. METHODS: Fifty-two patients with pollen allergy and IgE against at least one plant-derived food were included in the study. Adverse reactions to plant-derived foods were documented by using standardized interviews. Skin prick tests were performed for pollen (grass, birch, and mugwort) and 14 plant-derived foods. In addition, recombinant (r) Bet v 1 and rBet v 2 (profilin) were tested intracutaneously. Specific IgE against the abovementioned allergens were determined by means of RAST. Cross-reactivity was studied by means of RAST inhibition. RESULTS: Eighty-five percent of patients were sensitized to Bet v 1, and 71% were sensitized to profilin. Profilin was associated with a higher number of positive RAST results to plant-derived foods than Bet v 1. In contrast, Bet v 1 was associated with more positive skin prick test responses and more food-related symptoms. Sensitization to Bet v 1 was associated with IgE against apple, hazelnut, and peach, whereas sensitization to profilin was associated with positive RAST results to all investigated plant-derived foods except apple, peach, and melon. CONCLUSIONS: IgE antibodies against Bet v 1 have a more limited spectrum of cross-reactivity than those against profilin, but they frequently give rise to clinically relevant cross-reactivities to food. In analogy to anticarbohydrate IgE, cross-reactive IgE against food profilins have no or very limited clinical relevance.     

Sensitization to cross-reactive carbohydrate determinants and the ubiquitous protein profilin: mimickers of allergy

BACKGROUND: During the last decade, evidence has been provided for profilins and cross-reactive carbohydrate determinants (CCDs) to be capable of inducing cross-reactive IgE antibodies with little clinical relevance. OBJECTIVE: To investigate the prevalence of sensitization to CCD and profilin in isolated allergies (birch, timothy grass, house dust mite, pets (cat and/or dog), natural rubber latex (NRL) and hymenoptera venom). To study the contribution of anti-CCD and anti-profilin IgE antibodies as a cause of clinically irrelevant IgE for NRL and apple. METHODS: For the first part of the study, 100 patients with inhalant allergy, 17 patients with NRL allergy and 40 patients with venom anaphylaxis were enrolled. Diagnosis was based on a questionnaire and a positive IgE determination and skin test for relevant allergen. Patients were identified as sensitized to CCD if they had a negative prick test and positive IgE for the glycoprotein bromelain. Sensitization to profilin was assessed by IgE for rBet v 2 (recombinant birch profilin). For the second part of the study, sera containing IgE against apple (n=82) or NRL (n=38) were classified as true-negative or false-positive according to the presence or absence of an oral allergy syndrome (OAS) or NRL-induced anaphylaxis. In these patients, sensitization to CCD and profilin was evaluated as described above. RESULTS: No sensitization to bromelain-type CCD and profilin was found in isolated birch pollen or NRL allergy. In contrast, sensitization to bromelain-type CCD was found in 4/17 patients with isolated grass pollinosis, 5/24 patients with combined pollinosis (birch, timothy, mugwort) and 7/33 patients with venom anaphylaxis. Sensitization to profilin was almost restricted to patients with combined pollen allergy (5/24). In pollen-allergic individuals with a false-positive IgE against NRL the prevalence of sensitization to bromelain-type CCD and profilin IgE was higher than in NRL-allergic patients (P<0.00001 and P=0.0006, respectively). In pollen-allergic individuals with a false-positive IgE to apple, the frequency of sensitization to bromelain-type CCD was higher than in OAS patients (P=0.004). Clinically irrelevant NRL and apple were also found in four and five out of the seven patients sensitized to venom CCD, respectively. In pollinosis, clinically irrelevant NRL and apple IgE antibodies were inhibited by bromelain and recombinant birch profilin, whereas in isolated venom anaphylaxis these antibodies were inhibited by bromelain. CONCLUSIONS: Patients monoallergic to NRL or birch pollen showed no sensitization to bromelain-type CCD or profilin. Sensitization to profilin and/or bromelain-type CCD, caused by pollen (timothy grass, mugwort) or hymenoptera venom allergens, can elicit false-positive IgE antibodies against NRL and apple.     

Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins.

BACKGROUND: Birch pollen is a major cause of pollinosis and is responsible for cross-reactive oral allergies to fruits, nuts, and vegetables. The major allergen, Bet v 1, has been extensively characterized, and 3 minor allergens, Bet v 2, Bet v 3, and Bet v 4, have been cloned and sequenced. Recently, another birch pollen protein with an apparent mass of 35 kd was described as a new IgE-binding protein in birch pollen with cross-reacting homologues in plant foods. OBJECTIVE: The aim of this study was to determine the primary structure of the 35-kd birch pollen allergen and to investigate its immunologic properties. METHODS: On the basis of a known complementary DNA fragment, a PCR strategy was applied to obtain the full-length nucleotide sequence of the coding region. The protein was expressed as His-Tag fusion protein in Escherichia coli and purified by Ni-chelate affinity chromatography. Nonfusion protein was obtained by cyanogen bromide treatment of the fusion protein. IgE-binding characteristics and potential allergenicity were investigated by immunoblot, immunoblot inhibition analysis, rat basophil leukemia-cell mediator release assay, and basophil histamine release and compared with those of natural (n) Bet v 5, recombinant (r)Bet v 1, and rBet v 2. RESULTS: Recombinant Bet v 5 has a mass of 33 kd, an isoelectric point of 9.0, and sequence identity of 60% to 80% to isoflavone reductase homologue proteins from various plants. On immunoblots the recombinant Bet v 5 bound IgE from 9 (32%) of 28 sera from patients allergic to birch pollen with a CAP class of at least 3; Bet v 1 was detected by 89% of these patients. IgE immunoblot and inhibition experiments showed that nBet v 5 and rBet v 5 shared identical epitopes. A rabbit antiserum raised against pea isoflavone reductase and patients' IgE reacted with Bet v 5 and proteins of similar size in several vegetable foods, including exotic fruits. A similar reaction pattern was obtained with 2 Bet v 5-specific mAbs. Furthermore, Bet v 5 triggered a dose-dependent mediator release from rat basophil leukemia 2H3 cells passively sensitized with murine anti-birch pollen IgE and from basophils of a Bet v 5-reactive subject with birch pollen allergy. In contrast, no mediator release could be induced from basophils of a subject who was monosensitized to Bet v 1. CONCLUSIONS: This 33-kd protein, designated as Bet v 5, is a new minor allergen in birch pollen and may be responsible for pollen-related oral allergy to specific foods in a minority of patients with birch pollen allergy. Amino acid sequence comparison and immunoreactivity to anti-isoflavone reductase serum indicate that Bet v 5 is related to isoflavone reductase, a protein family that is involved in plant defense reactions.     

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.     

Identification of oilseed rape (Brassica napus) pollen profilin as a cross-reactive allergen

BACKGROUND: Major allergens of oilseed rape (OSR) pollen with molecular weights of 6/8, 14 and between 27 and 69 kD have been described. The aim of the present study was to further characterize the 14-kD allergen. METHODS: The 14-kD protein was purified from OSR pollen extracts by poly-(L-proline) (PLP)-Sepharose affinity chromatography and characterized immunologically by means of allergic patients' IgE antibodies, profilin-specific rabbit antisera, Western blot and ELISA inhibition using recombinant birch profilin (rBet v 2), and skin prick testing. RESULTS: By PLP affinity chromatography, OSR pollen profilin was purified as a single protein of 14.5 kD and further identified as a profilin by three polyclonal rabbit antisera raised against ragweed and tobacco pollen profilin and the C-terminus of birch profilin. IgE binding of a human serum pool (n = 15) and four profilin-reactive sera to nitrocellulose-blotted OSR profilin was completely inhibited by 1 microg/ml rBet v 2 (birch profilin). Reciprocal ELISA inhibition using increasing concentrations of rBet v 2 and purified OSR profilin, respectively, showed that rBet v 2 strongly inhibits antibody binding to OSR profilin, whereas almost 100 times the amount of OSR profilin was needed to inhibit IgE binding to rBet v 2. Skin prick tests were positive (wheal >/=3 mm) with 5 microg/ml rBet v 2 in all three patients tested, and with OSR profilin in two patients at a concentration of 50 microg/ml. CONCLUSIONS: OSR pollen profilin shares IgE and IgG epitopes with Bet v 2 and other plant profilins and may represent a potentially relevant allergen for profilin-sensitized patients.     

Cross-reactive and species-specific immunoglobulin E epitopes of plant profilins: an experimental and structure-based analysis

BACKGROUND: Profilins are cross-reactive plant allergens responsible for multiple pollen sensitization and pollen-associated food allergy. While it is assumed that profilins from different species are immunologically equivalent, some studies suggest partial or even lacking IgE cross-reactivity between certain profilins. OBJECTIVE: We aimed to obtain a semi-quantitative assessment of the contributions of conserved and species-specific epitopes to IgE binding of plant profilins. METHODS: We compared model structures of profilins from timothy, mugwort, celery and bell pepper with crystal structures of birch and latex profilins. We predicted potential conformational epitopes that consisted of contiguous patches of at least 20% surface-exposed residues. Celery and timothy profilins were purified from their natural sources, and profilins from birch, mugwort, bell pepper and latex were expressed in Escherichia coli. The structural integrity of all purified proteins was confirmed by circular dichroism spectroscopy. IgE ELISAs and ELISA inhibitions using sera from 22 profilin-sensitized allergic patients were carried out. RESULTS: Peptide backbone conformations of all six profilins were highly similar. Nine variable epitopes and two containing high proportions of conserved residues were predicted. IgE from all sera bound to all tested profilins and the amounts were highly correlated. However, IgE inhibition experiments revealed that up to 60% of total IgE binding was mediated by species-specific epitopes. The extent of cross-reactivity among profilins from timothy, birch, latex and celery was greater than cross-reactivity to mugwort and bell pepper profilins. This pattern was mirrored by sequence similarities among one of the predicted variable epitopes. Patients with IgE to cross-reactive epitopes displayed allergic reactions to a greater number of plant foods than patients having IgE directed to species-specific epitopes. CONCLUSION: The large extent of cross-reactivity among plant profilins justifies using a single profilin for diagnosis. However, the fine specificity of IgE directed to variable epitopes may influence the clinical manifestation of profilin sensitization.     

Hev b 8, the Hevea brasiliensis latex profilin, is a cross-reactive allergen of latex, plant foods and pollen

BACKGROUND: Plant profilins are important pan-allergens. They are responsible for a significant percentage of pollen-related allergies. Limited information is available about their involvement in the latex-fruit syndrome and the cross-reactivities between latex and pollen. We aimed to clone and express the Hevea brasiliensis latex profilin to investigate its allergological significance and serological cross-reactivities to profilins from plant foods and pollens. METHODS: A DNA complementary to messenger RNA (cDNA) coding for the Hevea latex profilin, Hev b 8, was amplified by polymerase chain reaction from latex RNA. Recombinant (r)Hev b 8 was produced in Escherichia coli and used to screen sera from 50 latex- allergic health care workers (HCWs) with well-documented histories of food and pollen allergy and 34 latex-allergic spina bifida (SB) patients. The cross-reactivity of natural Hev b 8 and rHev b 8 with other plant profilins was determined by ELISA inhibition assays. A three-dimensional homology model of Hev b 8 was constructed based on known profilin structures. RESULTS: The cDNA of Hev b 8 encoded a protein of 131 amino acids with a predicted molecular mass of 14 kD. Twelve of the 50 HCWs and 2 of the 34 SB patients were sensitized to Hev b 8. All Hev b 8-sensitized patients showed allergic symptoms to pollen or plant foods. Cross-reactivities between profilins of latex, pollen and plant food were illustrated by their ability to inhibit IgE binding to rHev b 8. Homology modeling of Hev b 8 yielded a structure highly similar to Bet v 2, the birch pollen profilin, with the most distinct differences located at the N-terminus. CONCLUSIONS: We conclude that primary sensitization to latex profilin in the majority of cases takes place via pollen or food profilins. Additionally, pollinosis and food-allergic patients with profilin-specific IgE can be at risk of developing latex allergy.     

A mimotope defined by phage display inhibits IgE binding to the plant panallergen profilin

Birch pollen and mugwort pollen allergies are often associated with hypersensitivity to plant foods. This clinical and serological cross-reactivity is mediated by IgE antibodies reacting with homologous proteins in pollen and food. Cross-reacting homologs of the important birch pollen allergen Bet v 2 (profilin) could be detected in other pollen, fruits, nuts, and vegetables, such as celery tuber. We purified IgG/IgE antibodies from the serum of an exclusively profilin-allergic patient using affinity columns either coupled with protein extracts from mugwort pollen, birch pollen, or celery tuber. Constrained and unconstrained random nonapeptide libraries were pooled and screened with the anti-profilin antibody preparations to define cross-reactive ligands. Specific ligands were enriched by successive panning rounds using the profilin-specific antibodies in series. After the last panning round enriched phage clones were screened with purified profilin-specific antibodies and IgE-binding clones were sequenced. Five out of eight positive clones (62.5 %) displayed the same circular peptide CAISGGYPVC. This peptide was synthesized and examined for its ability to inhibit IgE binding to blotted mugwort pollen, birch pollen, or celery tuber profilin. Inhibition studies showed reduction of IgE binding to profilins in all three protein extracts. As the sequence of the mimotope did not show any homology to the known birch profilin sequence this peptide is considered to mimic a common conformational IgE epitope for these examined profilins.     

Allergy to Rosaceae fruits without related pollinosis

Background: Rosaceae fruit allergy is frequently associated with birch pollinosis in Central and Northern Europe and with grass pollen allergy in Central Spain. The main cross-reactive structures involved for birch pollinosis are Bet v 1 and profilin, and for grass pollinosis they are profilin and carbohydrate determinants. Rosaceae fruit allergy can occasionally be observed in patients without pollinosis. Objective: We investigated the clinical presentation and the allergens involved in allergy to Rosaceae fruit without pollinosis. Methods: Eleven patients from Central Spain allergic to apples, peaches, and/or pears but not to pollens were compared with 22 control subjects with combined grass pollen and fruit allergy. Skin prick tests and RASTs to apple, peach, and pear were performed. Cross-allergenicity was studied by RAST inhibition. Bet v 1 was tested with an indirect RAST, and profilin was tested in skin prick tests, histamine release, and RAST. Results: Rosaceae fruit allergy without pollinosis is severe with 82% of patients reporting systemic symptoms, mainly anaphylaxis (73%), whereas oral symptoms are less frequent (64%). Anaphylactic shock was observed in 36% of patients. The fruit allergens involved showed cross-reactivity among Rosaceae species but were not related to profilin or Bet v 1. Ninety-one percent of patients with combined grass pollinosis and fruit allergy reported oral allergy, 45% reported systemic symptoms, 18% reported anaphylaxis, and 9% reported anaphylactic shock. Conclusion: Allergy to Rosaceae fruits in patients without a related pollen allergy is a severe clinical entity. Profilin- and Bet v 1-related structures are not involved in Rosaceae fruit allergy without pollinosis.(J Allergy Clin Immunol 97;100;728-33)