Allergy 2002: 57: Suppl. 71: 17–23Printed in UK. All rights reserved

17

Copyright © Blackwell Munksgaard 2002

ALLERGYISSN 0108-1675

Blackwell Science Ltd

The allergenic relevance of profilin (Ole e 2) from Olea europaea pollen

A. Martínez1, J. A. Asturias1, J. Monteseirín2, V. Moreno2, A. García-Cubillana2, M. Hernández 2, A. de la Calle2, C. Sánchez-Hernández2, J. L. Pérez-Formoso2, J. Conde2

1Bial-Arístegui, R&D Department, Bilbao; 2Hospital Virgen Macarena, Servicio de Alergia, Seville, Spain

Alberto Martínez Gárate Bial-Aristegui Departamento de I+D Alda. Urquijo, 27 48008 Bilbao Spain

Many works have dealt with the study of the allergenic relevanceof profilin from allergenic extracts, mainly derived from pollens andvegetable foods. Olive pollen extracts also contain a profilin allergen(Ole e 2). This protein has been characterized in detail, so the amino-acid sequence of three isoforms and the structural model of one ofthem are already known. The prevalence of Ole e 2 for olive allergenicpatients has been evaluated by different in vivo and in vitro methods,and the results compared with those obtained for another pollenprofilins.

Profilins are ubiquitous proteins found in mam-mals, animal cells, plants, and even in viruses (1).Profilins sequester actin monomers in a 1:1complex and inhibit actin polymerization, so areinvolved in the organization of the cytoskeleton ineukaryotic cells. they also affect the two ends ofthe actin filament (pointed and barbed) retardingpolymerization (2). For many years, sequesteringwas considered to be profilin’s primary function.However, more recent data indicate that profilinhas more complex effects on actin dynamics (3).

Because of its affinity for phosphatidylinositol4,5-biphosphate, profilin also functions as a regu-lator of the signal transduction pathway throughphospholipase C (4). Profilin can bind not onlyactin and polyphosphoinositides, but also polyl-proline (PLP) sequences. The functionality ofthe affinity for this ligand has not been elucidated,but it could allow the anchorage of profilin tospecific cell regions. The ability to bind to PLP(8–10 sequential prolines are required to bindprofilin), a nearly unique feature of profilins, hasbeen exploited for the purification of the protein (5).

Profilins were first described by Carlsson et al.(6) as low-molecular-weight proteins in calf spleen,which inhibited the growth of actin filaments in vitro.As a result of the formation of ‘profilamentous’complexes with actin, they were named ‘Profilins’.

The presence of profilin as an allergen was firstreported by Valenta et al. in 1991 (7), during their

research on birch pollen allergenic extracts. Sincethen, studies have demonstrated that profilin isindeed an allergen present in a large variety ofvegetable foods as well as in pollen (8). Its ubiquityand high sequence conservation characterize thisallergen with a broad spectrum of cross-reactivityamong taxonomically related and unrelated species.This has led to the term of ‘pan-allergen’ for thisprotein (9).

Profilin in plant allergenic extracts

In a wide range of allergenic extracts from plantsources, the presence of profilins has been ascer-tained by direct protein purification or cDNAcloning, or by indirect immunochemical detectionin Western blots using animal polyclonal mono-specific antisera, monoclonal antibodies, or evenhuman sera from profilin-sensitized patients. Thebetter characterized profilins are those describedas allergens according to the current allergennomenclature list of the IUIS shown in Table 1.

Allergenic relevance of profilins

Profilin was identified as a minor allergen in birch(Betula verrucosa), timothy grass (Phleum pra-tense), and mugwort (Artemisia vulgaris) pollens

Martínez et al.

18

(9). In this study, about 20% of all pollen-allergicpatients tested (patients were from CentralEurope; n = 65) displayed IgE reactivity to recom-binant birch profilin (rBet v 2) in SDS-PAGEimmunoblotting inhibition experiments. The samesensitization frequency was observed in anotherstudy by IgE immunoadsorption of sera from30 timothy pollen allergen patients, also usingrBet v 2 (10).

Birch pollen profilin was found to be recognizedby 20% of birch-allergic patients in another twostudies; one of them by means of skin tests withrBet v 2 (n = 51) (11), and the other in a serasample from 34 patients using recombinant birchprofilin coupled to CAP solid phase (12).

However, different profiles in specific IgE torBet v 2 have been described depending on thegeographic localization of subjects. Thus, in a studythat involved 293 patients (from six countries)who were allergic to birch pollen, it was foundthat, in the Swedish and Finish groups, 5–7%were reactive to rBet v 2, by CAP, comparedwith 20–38% in the Central /Southern Europeangroups (13).

A 20% level of profilin reactivity was alsoestablished for a Spanish sample population ofBermuda grass (Cynodon dactylon) allergic patientsby SDS-PAGE immunoblotting using a recom-binant form (rCyn d 12), because six out of 30sera tested exhibited IgE-binding reactivity to theisolated protein (14).

Some higher sensitization frequencies for pollenprofilin have been reported in another study byour group (15): profilin from Helianthus annuus(sunflower) was shown to be reactive to 30% ofsera from a sample of patients (n = 121) allergic topollen from this Compositae plant, when assayedby Western blot using H. annuus recombinantprofilin (rHel a 2).

The same character of minor allergen has beenencountered for profilin in some reports of foodhypersensitivitiy. This is the case with peanutextracts. Sera (n = 40) of peanut-allergic individualswere tested by immunoblotting with several peanutrecombinant allergens; 13% of them showed IgEbinding to rAra h 5 (profilin) (16). In a technicallysimilar study using recombinant profilin fromcelery (rApi g 4), a 42% frequency of sensitization(n = 17) was determined (17).

In some specific systems, profilin has beendescribed as a major allergen, because SDS-PAGEimmunoblotting of whole pollen extracts revealedIgE binding at the level of profilin bands (previ-ously identified with monospecific polyclonalor monoclonal antibodies) in more than the 50%of tested sera from patients allergic to pollens ofMercurialis annua (18), Croccus sativus (19), andPhoenix dactylifera (20). However, these data shouldbe further corroborated with assays using isolatedprofilins.

Soybean profilin has also been demonstratedto be a major allergen for patients showing aller-gic manifestations after soybean ingestion. In animmunoblot analysis, the recombinant soybeanprofilin (rGly m 3) was recognized by IgE in nine(69%) of the 13 sera tested (21).

The frequency of sensitization to profilin hasalso been investigated in latex-allergic individuals,but some controversial results have been found.Thus, in a study by Nieto et al. (22), natural andrecombinant latex profilins (Hev b 8) were testedin 17 spina bifida (SB) patients and 14 adultsallergic to latex (AL), giving positive results in 15and 14 of them, respectively; only 39% of the seraexhibited IgE binding when studied using SDS-PAGE immunoblotting with Hev b 8. On thecontrary, Rihs et al. (23) recently reported a muchlower prevalence of IgE antibodies to latex profilin:12% (n = 17) in SB patients and 20% (n = 25) inhealth care workers allergic to latex, by CAP andWestern blot of rHev b 8.

The documented relevance of profilin as apan-allergen in plant sources is evidenced by itspreponderance, in terms of frequency of sensitiza-tion, when considering only groups of patients withpollinosis and associated food hypersensitivity.Thus, in areas without birch trees, such as Spain,profilins may represent important food allergens inpatients with combined grass pollen/food allergy.Several reports illustrate this conclusion. At leastfour studies have been performed with patientsallergic to grass pollen and Rosaceae fruits suchas apple and peach, revealing profilin sensitizationfrequencies of 75% (n = 16 and 22, respectively) by

Table 1. Profilins described as allergens according to the current IUIS (March2001) nomenclature. All of them have been cloned

Profilin source Allergen Mol. mass (kDa)

Betula verrucosa pollen Bet v 2 15Cynodon dactylon pollen Cyn d 12 14Helianthus annuus pollen Hel a 2 15.7Mercurialis annua pollen Mer a 1 14Olea europaea pollen Ole e 2 15–18Phleum pratense pollen Phl p 12 14Celery Api g 4 14Cherry Pru av 4 15Latex Hev b 8 14Peanut Ara h 5 15Pear Pyr c 4 14Soybean Gly m 3 14

Relevance of profilin from Olea europaea pollen

19

RAST to purified profilin (24,25), 90% (n = 10),by CAP to rBet v 2 (26), and 52% (n = 29) by thesame technical procedure (27).

Likewise, Petersen et al. (28) reported that, withina group of eight patients allergic to grass pollen andtomato, sera predominantly presented IgE specificto profilin (63%), as evaluated by Western blot.

Olive profilin, Ole e 2

The purification and characterization of oliveprofilin from its natural source (olive tree pollen)were reported by Ledesma et al. (29). Isolationwas achieved by gel filtration (Sephadex G-75column) followed by affinity (PLP-Sepharose 4B)and additional gel filtration fast protein liquidchromatography, a yield of 100 µg/g of dried olivepollen was obtained. An average molecular mass ofabout 15 kDa was estimated. A circular-dichroismspectrum revealed a secondary structure with per-centages of 15% α-helix, 33% β-strand, 20% β-turn,and 32% random coil.

In a previous study by our group (30), oliveprofilin cDNA was amplified by PCR with theuse of degenerate primers, producing a 400-bpfragment which was subcloned in the expressionvector pKN172. Three full-length cDNAs, i.e.OePRO1, OePRO2 and OEPRO3, were obtained.All of them had a length of 405 bp and encodeda polypeptide of 134 amino acids with a predictedaverage molecular mass of 14.4 kDa and an averageisoelectric point value of 5.1. A high degree ofidentity (>98%) was found between the sequences(Fig. 1). Heterogeneity appears at four positionson the primary structure: Asp-20 → Gly, Val-122→ Ala, Val-130 → Leu and Met-134 → Leu. Pairwise,alignments indicated 73–86% identical aminoacidscompared with other cloned plant profilins, withthe greatest identity being with profilin from theBetula verrucosa tree, and to a lesser extent withthose of grass species. Much lesser identities werefound with non-plant profilins, like the humanprofilin (32%).

A structural model of OePRO1 was constructedfrom the X-ray coordinates of profilin (3nul) from

OePRO1 -MSWQAYVDDHLMCDIEGHEDHRLTAAAIVGHDG--SVWAQSAT--FPQFKPEEMNGIMT 55

OePRO2 -...................G.............--........--.............. 55

OePRO3 -...................G.............--........--.............. 55

Cynodon -.............E..---G.H..S...I....--T......A--..A......AN..K 52

Phleum -....T...E....E...---.H.AS...L....--T......D--........IT...K 52

Hordeum -....T......C.E.D.---QH..S...L....--R..V..PN--........IA..IK 52

Betula -....T...E......D.QASN-SL.S.......--......SS--......Q.IT...K 54

Parietaria -..............VGD--GNT.AS...I....--.......N--...L....VT...N 53

humanI MAG.N..I.NLMADG-------TCQD.....YKDSP....AVPGKT.VNITPA.VGVLVG 53

OePRO1 DFNEPGHLAPTGLHLGGTKYMVI------QGEAGAVIRGKKGSGG----ITIKKTGQALV 105

OePRO2 ......................------................----............ 105

OePRO3 ......................------................----............ 105

Cynodon ..D...F......F..P......------...P............----V.V........ 102

Phleum ..D.........MFVA.A.....------...P.........A..----........... 102

Hordeum ..D..........F.........------...P.V.......T..----.......MP.I 102

Betula ..E..............I.....------................----........... 104

Parietaria ....G.F......F.........------...S....-.......----A.L......I. 102

humanI --KDRSSFYVN..T...Q.CS..RDSLLQD..FSMDL.T.STG.APTFNV.VT..DKT.. 111

OePRO1 FGIYEEPVTPGQCNMVVERLGDYLVEQGM 134

OePRO2 ................A.......L...L 134

OePRO3 ........................L...L 134

Cynodon I...D..M........I.K.....I.... 131

Phleum V...D..M..................... 131

Hordeum L...D..M......L.............F 131

Betula ........................ID..L 133

Parietaria I...D..M......L.........L.... 131

humanI LLMGK.G.HG.LI.KKCY.MASH.RRSQY 140

Figure 1. Amino acid deduced sequences of olive profilins (OePRO1, OePRO2 and OePRO3), comparison with differentplant profilins: Cynodon dactylon (Y08390), Phleum pratense (X77583), Horden vulgare (U49505), Betula verrucosa (M65179),Parietaria judaica (Y15208209) and with respect to human profilin I (BC002475). Points indicate identity of residues with respectto the upper sequence.

Martínez et al.

20

Arabidopsis thaliana (V43325) using Swissmodelsoftware (31) (Fig. 2). As other plant profilins,olive profilin is built around a central six strandedantiparallel β-sheet. Both termini are α-helicaland packed against the same side of the centralsheet, connected by two short loops. Residues onthe opposing face of the central sheet form twoα-helices and a small two-stranded β-sheet. When

superimposed to Arabidopsis profilin (77.1% iden-tity to OePRO1), a characteristic loop in oliveprofilin corresponding to residues 18–21 can beobserved. Apart of that, the rest of secondarystructure looks fairly coincidental.

Olive profilin was expressed as a non-fusion pro-tein and purified to homogeneity after a single stepof affinity chromatography with a poly l-prolineSepharose column (30). The yield of recombinantprofilin was about 70 mg/l of all culture, withmore than 95% in soluble form. In the same study,natural profilin was isolated from olive pollenextracts by PLP-Sepharose chromatography. Anelectrophoretic pattern composed of four bandswas obtained; all of them were immunoreactiveand showed apparent molecular masses of 17.8,17.0, 16.0 and 15.2 kDa. The identity of these bandsas profilin isoforms was later corroborated byWestern blot inhibition using rOle e 2 (32). ThisSDS-PAGE pattern of natural isolated profilin isnot commonly reported for other pollen profilins,which usually exhibit single electrophoretic bands(7,14,15) (Fig. 3).

Further experiments (30) using olive profilinand the olive major allergen Ole e 1 revealed thatpreadsorption of a serum pool of O. europaea aller-gic patients to rOle e 1 could inhibit up to 80%of the binding of IgE antibodies to olive pollenextract, while preadsorption to rOle e 2 showedlow reduction of IgE binding capacity, but nearlyreaching 20%. This suggests that a cocktail con-taining rOle e 1 and rOle e 2 allergens could havean equivalent specificity and sensitivity – from adiagnostic point of view – to the whole olive pollen

Figure 2. Molecular model of O. europaea profilin (OePRO1)(dark ribbon) and A. thaliana (light ribbon). The Swiss modelprogram was used for graphic visualization and manipulations.

Figure 3. Comparative SDS-PAGE pattern obtained by analysis of affinity chromatography purification of natural olive(A) and Bermuda grass (B) pollen profilins. (1): retained profilin fraction (2); unretained fraction; (3) whole crude extracts;(M): molecular mass markers.

Relevance of profilin from Olea europaea pollen

21

extract and a potential therapeutic effect. Thispossibility, which would resemble the Bet v 1/Bet v 2 model for birch pollen allergic patientsdescribed by van Ree et al. (33), is worth furtherinvestigation.

Ole e 2 prevalence

A first estimation of IgE binding frequency toprofilin in patients allergic to olive pollen wasreported by Ledesma et al. (29). They found a24% recognition frequency by Western blot of olivepollen crude extract, using 191 sera from olive-hypersensitive patients.

In a study by our group (32), also using SDS-PAGE immunoblotting with whole O. europaeapollen extract, after identification of profilin bandsby blot inhibition with rOle e 2, a prevalence of27% was found within a sample of 120 sera fromolive-allergic subjects (Fig. 4). An almost identicalpercentage (25%) was encountered using the samesera and Western blot technique with isolatedOle e 2 (34).

Recently, data have been obtained by in vivomethods using isolated olive pollen profilin. Resultsare shown in Fig. 5. Olive-pollen-allergic patientssuffering from rhinoconjunctivitis and/or asthma,and not undergoing immunotherapy were selected.On average, in vivo frequencies of profilin recog-nition were in the same range as those reportedfor in vitro techniques. It should be remarked thatmost patients reacting to olive profilin did not givea positive response to Bermuda grass pollen pro-filin used as the control, indicating a certain degreeof epitopic difference between them, despite theirhigh sequence homology. None of the patients

Figure 4. SDS-PAGE immunoblotting of O. europaea pollen extracts incubated with representative of 20 sera out of 120 olive-allergic patients involved in study. (M) molecular mass markers. Arrowhead indicates IgE-binding bands corresponding toprofilin.

26%(12/45)

20%

(9/45)

19%(3/16)

0 20 40 60 80 100

NPT

CPT

Prick

Sensitization frequency (%)

37%

(7/19)

28.5%

(6/21)

30%

(3/10)

0 20 40 60 80 100

RAST/Blot

NPT

Prick

Sensitization frequency (%)

47%(17/36)

33%(10/30)

20%(7/35)

28%(10/36)

0 20 40 60 80 100

RAST/Blot

NPT

CPT

Prick

Sensitization frequency (%)

Olea europaea

Artemisia vulgaris

Cynodon dactylon

Figure 5. Histogram showing prevalence of positive responsesby in vivo and in vitro methods to different isolated naturalprofilins. (CPT): Conjunctival Provocation Test; (NPT): NasalProvocation Test. (Positive patients/tested patients).

Martínez et al.

22

studied showed associated food allergic mani-festation, so no conclusions could be drawn at thispoint. Therefore, a study on the profilin preval-ence in patients suffering from olive pollinosis andfood hypersensitivity could represent an attractivetopic for further work.

Comparatively, two other natural purified pro-filins from pollens were studied (Fig. 5). Whileolive profilin was overall positive, in around 20%of allergic patients evaluated by three in vivotechniques, natural isolated profilin from mugwort(Artemisia vulgaris) reacted in vivo (prick andnasal provocation test) in an average of 30% ofpatients, specific IgE being detected in 37% oftheir sera. This coincides with the in vitro preval-ence values (30%) found in (another Compositae)sunflower profilin by immunoblotting with Hel a 2(15). However, natural isolated profilin fromBermuda grass pollen (Cynodon dactylon) alsorevealed, by in vivo and in vitro tests, a minorallergenic character, with sensitization frequenciesranging from 20% to 47% in conjunctival provoca-tion and cutaneous prick tests respectively.

Finally, a collaborative preliminary study withmono- and polysensitized patients to O. europaeapollen has been carried out to estimate by the pricktest the in vivo reactivity of recombinant Ole e 2.Cutaneous tests were considered positive wheneliciting of a wheal with diameter 3 mm greaterthan the negative (glycerinated saline) control.Recombinant protein was applied at 0.1 mg/ml.Only one of six monosensitized patients reactedpositively. However, a positive wheal was detectedin 18 out of 83 (21.6%) patients polysensitized toolive pollen and other inhalants, but without foodhypersensitivity. Natural isolated olive profilin testedas the control within the same sample populationrevealed higher prevalence percentages (66% forpolysensitized and 50% for monosensitized patients).These results are not in accordance with previousstudies using other group of patients and neitherwith the in vitro results. A tentative explanation,

which should be investigated further, could be theloss of conformational profilin epitopes occurringas a consequence of the SDS-PAGE treatment ofthe samples. The possibility of some contaminanttraces in isolated natural profilins should not beruled out as an alternative explanation for thediscordant prick values.

Despite the small group of monosensitizedpatients, the results tend to corroborate the datareported by Dubost et al. (35), according to whichthe incidence of sensitization to profilin is higherin polysensitized patients than in those who aremonosensitized.

Conclusions

The pan-allergen profilin has been detected inO. europaea pollen. The frequency of sensitizationto this allergen for olive tree pollen allergic patientsby in vitro and most in vivo techniques is around20%.

Although rOle e 2 is a minor allergen, its presenceshould be ascertained and quantified in O. europaeaallergenic extracts intended for diagnostic and ther-apeutic use. The potential diagnostic and therapeuticusefulness of the Ole e 1/Ole e 2 combination incomparison with the whole allergenic extractremains an attractive topic to be investigated.

Acknowledgments

This work was supported in part by BIAL-Aristeguiand Grants No. 53–07 from the Plan Nacional deI+D (Programa PROFIT. Ministerio de Cienciay Tecnología, Spain), and No. TEI-0166–2000/2from the Programa INTEK (Departamento deIndustria, Agricultura y Pesca, Gobierno Vasco).The authors thank the Protein Design Group fromCNB-CSIC (Spain) for helping in the molecularmodelling.

References

1. Witke W, Sutherland JD, Sharpe A, et al. Profilin I is essential for cell survival and cell division in early mouse development. Proc Natl Acad Sci USA 2001;98:3832–3836.

2. Haarer BK, Brown SS. Structure and function of profilin. Cell Motility Cytoskel 1990;17:71–74.

3. Ayscough KR. In vivo functions of actin-binding proteins. Curr Opin Cell Biol 1998;10:102–111.

4. Goldschmidt-Clermont PJ, Machesky LM, Baldassare JJ, et al. The actin-binding profilin binds to PIP2 and inhibits its hydrolysis by phospholipase C. Science 1990;247:1575–1577.

5. Lindberg U, Schutt CE, Hellsten E, et al. The use of poly(l-proline)-Sepharose in the isolation of profilin and profilactin complexes. Biochim Biophys 1988;967:391–400.

6. Carlsson L, Nystön LE, Sunkvist I, et al. Profilin a low

molecular weight protein controlling actin polymerisability. In: Perry SV, et al., editors. Contractile Systems in Non-Muscle Tissues. Amsterdam: Elsevier, 1976:39–49.

7. Valenta R, Duchene M, Pettenburger K, et al. Identification of profilin as a novel pollen allergen; IgE autoreactivity in sensitised individuals. Science 1991;253:557–560.

8. van Ree R, Voitenko V, van Leeuwen WA, et al. Profilin

Relevance of profilin from Olea europaea pollen

23

is a cross-reactive allergen in pollen and vegetable food. Int Arch Allergy Immunol 1992;98:97–104.

9. Valenta R, Duchene M, Ebner C, et al. Profilins constitute a novel family of functional plant pan-allergen. J Exp Med 1992;175:377–385.

10. Laffer S, Vrtala S, Duchene M, et al. IgE-binding capacity of recombinant timothy grass (Phleum pratense) pollen allergens. J Allergy Clin Immunol 1994;94:88–94.

11. Pauli G, Oster JP, Deviller P, et al. Skin testing with recombinant allergens rBet v 1 and birch profilin, rBetv2: diagnostic value for birch pollen and associated allergies. J Allergy Clin Immunol 1996;97:1100–1109.

12. Scheiner O, Aberer W, Ebner C, et al. Cross-reacting allergens in tree pollen and pollen-related food allergy: implications for diagnosis of specific IgE. Int Arch Allergy Immunol 1997;113:105–108.

13. Elfman L, Svensson M, Lidholm J, et al. Different profiles in specific IgE to rBetv1 and rBet v 2 in patients allergic to birch pollen from six countries. Int Arch Allergy Immunol 1997;113:249–251.

14. Asturias JA, Arilla MC, Gómez-Bayón N, et al. Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen. Clin Exp Allergy 1997;27:1307–1313.

15. Asturias JA, Arilla MC, Gómez-Bayón N, et al. Cloning and immunological characterization of the allergen Hel a 2 (profilin) from sunflower pollen. Mol Immunol 1998;35:469–478.

16. Kleber-Janke T, Crameri R, Appenzeller U, et al. Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology. Int Arch Allergy Immunol 1999;119:265–274.

17. Scheurer S, Wangorsch A, Haustein A, et al. Cloning of the minor allergen Api g 4 profilin from celery (Apium graveolens) and its cross-reactivity with birch pollen profilin Bet v 2. Clin Exp Allergy 2000;30:962–971.

18. Vallverdú A, García-Ortega P, Martínez J, et al. Mercurialis annua: characterization of main allergens and cross-reactivity with other species. Int Arch Allergy Immunol 1997;112:356–364.

19. Feo F, Martínez J, Martínez A, et al. Occupational allergy in saffron workers. Allergy 1997;52:633–641.

20. Fernández J, Asturias JA, Martínez A. Biological standardization and allergen characterization of date palm pollen (Phoenix dactylifera). Allergy 2000;55(Suppl. 63):140 (Abstract).

21. Rihs HP, Chen Z, Rueff F, et al. IgE binding of the recombinant allergen soybean profilin (rGly m 3) is mediated by conformational epitopes. J Allergy Clin Immunol 1999;104:1293–1301.

22. Nieto A, Mazón A, Estornell F, et al. Profilin, a relevant allergen in latex allergy. J Allergy Clin Immunol 1998;101:S207 (Abstract).

23. Rihs HP, Chen Z, Rozynek P, et al. PCR-based cloning, isolation, and IgE-binding properties of recombinant latex profilin (rHevb8). Allergy 2000;55:712–717.

24. van Ree R, Fernández-Rivas F, Cuevas M, et al. Pollen-related allergy to peach and apple: an important role for profilin. J Allergy Clin Immunol 1995;95:726–734.

25. Fernández-Rivas M, van Ree R, Cuevas M. Allergy to Rosaceae fruits without related pollinosis. J Allergy Clin Immunol 1997;100:728–733.

26. Daschner A, Crespo JF, Pascual CY, et al. Allergens in pepper and paprika. Immunologic investigation of the celery–birch–mugwort–spice syndrome. Allergy 1998;53:36–41.

27. Díez-Gómez ML, Quirce S, Cuevas M, et al. Fruit-pollen–latex cross-reactivity: implication of profilin (Bet v 2). Allergy 1999;54:951–961.

28. Petersen A, Vieths S, Aulepp H, et al. Ubiquitous structures responsible for IgE cross-reactivity between tomato fruit and grass pollen allergens. J Allergy Clin Immunol 1996;98:805–815.

29. Ledesma A, Rodriguez R, Villalba M, et al. Olive-pollen profilin. Molecular and immunologic properties. Allergy 1998;53:520–526.

30. Asturias JA, Arilla MC, Gómez-Bayón N, et al. Cloning and expression of the panallergen profilin and the major allergen (Ole e 1) from olive tree pollen. J Allergy Clin Immunol 1997;100:365–372.

31. Guex N, Peitsch MC. Swiss-model and the Swiss-PdbViewer: an enviroment for comparative protein modelling. Electrophoresis 1997;18:2714–2723.

32. Martínez A, Asturias JA, Palacios R, et al. Identification of a 36-kDa olive-pollen allergen by in vitro and in vivo studies. Allergy 1999;54:584–592.

33. van Ree R, van Leeuwen WA, Akkerdaas JH, et al. How far can we simplify in vitro diagnostics for Fagales tree pollen allergy?. A study with three whole pollen extracts and purified natural and recombinant allergens. Clin Exp Allergy 1999;29:848–855.

34. Asturias JA, Martínez A, Palacios R, et al. Profilinas recombinantes: Capacidad y frecuencia de fijación de IgE. Rev Esp Alergol Inmunol Clin 1998;13:251 (Abstract).

35. Dubost R, Ruet N, Deviller P. Incidence of sensitisation to profilin in a population allergic to pollen: responsibility of profilin in pollen polysensitizations in patients with a normal level of total IgE. Allerg Immunol (Paris) 2000;32:199–206.