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ALLERGY |ISSN 0105-4538 t

Olive-pollen profilin. Molecular andimmunologic properties

Ledesma A, Rodriguez R, Villalba M. Olive-pollen profilin. Molecular andimmunologic properties.Allergy 1998: 53: 520-526. © Munksgaard 1998.

Olive-pollen profilin has been isolated and characterized as a significantallergen. Its molecular properties, such as a molecular mass of 15 kDa;amino-acid composition; and secondary repetitive structure percentagesof 15% a-helix, 33% P-strand, 20% P-tum, and 32% random coil, have beendetermined. Its allergenic capability, a recognition frequency estimated at24% of olive-hypersensitive patients, and high cross-reactivity with all thepollen used have been found. The presence of conformational epitopes inthe olive profilin, as well as a high structural and immunologic similarityto other pollen sources such as birch and ash, can be established from thesestudies.

A. Ledesma, R. Rodriguez,M. VillalbaDepto. Bloquimica y Biologia Molecular,

Facultad de Qufmica, UCM, Madrid, Spain

Key words: allergen; allergenic cross-reactivity;

olive-tree pollen, profilin,

Dr Mayte Villalba

Depto Bioquimica y Biologfa Molecular

Facultad de Quimica

Unlversidad Complutense

28040 Madrid

Spain

Accepted for publication 20 January 1998

Profilin is a ubiquitous protein that has beendetected in various animal and plant tissues.Profilins interact with skeleton proteins of the cell,being involved in polymerization of actin filaments(1, 2) and signal transduction events through phos-phoinositide pathways (3). Profilin has been identi-fied as a panallergen because of its allergenicity; itcan induce type-I hypersensitivity as a constituentof both pollens (4) and fruits (5). Sera from hyper-sensitive patients have been shown to contain IgEantibodies which recognize profilin from birch,wheat, celery, or Phleum (6).

Although profilins are not major allergens, theycause significant cross-reactivity among related andunrelated sources of allergens (7). About 20% ofpollen-allergic patients are sensitized to profilins(8), but these proteins seem to have greater rele-vance in pollen-food cross-sensitizations (9). Thecross-reactivity could be due to the similaritybetween their amino-acid sequences, which reachesup to 80% identity among pollen profilins (10).

Allergy to olive {Olea europaea) pollen is aclinical disorder of great incidence in Mediterraneancountries (11, 12). Most of the allergic patients(about 70% in the studied populations) are sensi-tized to Ole e 1, the major allergen described inolive pollen (13, 14). However, many other IgE-reactive bands have been detected in SDS-PAGEof the saline extract of this pollen (15, 16), someof them exhibiting a molecular mass under 16 kDathat could be attributed to profilin. In fact, profilin

has been already detected in peach and apple (17)and its cDNA cloned and sequenced (18). In thiswork, we report the isolation of olive-pollenprofilin. The analysis of its physicochemical andimmunologic properties allowed us to assess itssimilarity to other known profilins and, thus, toestablish the cross-sensitivity of olive pollen withother pollen sources.

Material and methodsPollen extracts

Pollens from O. europaea, Betula verrucosa, Zeamays, Cynodon dactylon, and Fraxinus excelsiorwere obtained from Allergon AB. Pollen (5% w/v)was extracted with 50 mM ammonium bicarbonate,pH 8.0, containing 1 mM phenylmethylsulfonylfluoride. The pellet was twice re-extracted in thesame solution. Total protein determinations ofsaline extracts were carried out according to Lowryet al. (19).

Human sera and rabbit antiprofilin antibodies

Sera from donors allergic to olive pollen and notundergoing immunotherapy were used. The serawere selected by their recognition of 14-16-kDaprotein bands of the olive-pollen extract. A serumfrom a patient allergic to Der p 1 was used ascontrol.

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A polyclonal rabbit antiserum obtained againstrecombinant birch profilin, kindly donated byDr R. Valenta, was used in the purification stepsof the antigen. On the other hand, a specific rabbitpolyclonal antiserum was raised against olive-pollenprofilin by the intra-Iymph-node technique (20),An amount of 50 ig of the purified olive profilinwas used for immunization. One month later, therabbit was boosted with the same dose of antigen.

Purification of the antigen

The lyophilized saline extract was dissolved in 0.2M ammonium bicarbonate, pH 8.0, and subjectedto gel filtration on a Sephadex G-75 column. Theabsorbance at 280 nm was measured. The fractionswere assessed with the anti-birch-profilin polyclonalantibody in immunoblotting analysis. Fractionscontaining positive bands in the range of 14-16kDa were pooled, loaded in a poly-(L-proline)column (21), and eluted with TBS-ATP containing6 M urea. The eluted material was dialyzed againstdistilled water at 4°C overnight. This material waslyophilized and dissolved in 20 mM sodium phos-phate, containing 20 mM NaCl, pH 7,6, and loadedonto a Superdex-75, HR 10/30 gel filtration column.The elution was done on an FPLC system(Pharmacia). Absorbance was measured at280 nm.

The amino-acid composition of the isolated pro-tein (1-2 nmol) was determined by acid hydrolysisand amino-acid analysis as described (13). Theprotein concentration of the purified antigen wasdetermined by amino-acid analysis. NHj-terminalEdman degradations of native protein were per-formed on an Applied Biosystems model 477Asequencer (Applied Biosystems), as previouslydescribed (13).

Polyacrylamide gel electrophoresis (PAGE)

SDS-PAGE was performed according to Laemmli(22) in 15% polyacrylamide gels, Profilin sampleswere reduced by using P-mercaptoethanol 5% (v/v)and heating at 80°C for 20 min. Protein was stainedwith silver, as previously described (23), Molecularweight determination was done according to Weber& Osborn (24), by using protein markers providedby Sigma. In immunoblotting assays, we usedprestained molecular weight markers provided byBio-Rad, and their molecular masses are indicatedin Fig. 1.

Circular-dichroism (CD) analysis

Olive-pollen profilin

1 2 3 4 5 6 7 8 9 10 C

84"-53»-35»-29 • -20 •-

CD spectrum was obtained on a Jasco J-715dichrograph fitted with a 150-W xenon lamp. The

Fig. 1. a) IgE recognition of olive-pollen extract (50 ng) by 10individual sera from patients allergic to olive pollen (lanes 1-10). Scrum of Der p 1-aIlergic patient (c) was used as control,b) Detection of profilin in crude extract with polyclonalantibody obtained against recombinant birch profilin. Mol-ecular mass markers are indicated by arrowheads.

sample was analyzed in 0.1-cm optical-path cells,in 20 mM sodium phosphate and 20 mM sodiumchloride, pH 7,6. The spectra in the far-ultravioletregion were recorded at 50 nm/min scanning speed,and the accumulation of four spectra was achieved.The protein concentration was in the 0.05-0.1 mg/ml range. Mean residue mass ellipticities werecalculated on the basis of 111 as the averagemolecular mass/residue and expressed in terms of0 (degreexcm^xdmol"'). Secondary structure esti-mations were performed by computer fit by themethod of Perczel et al. (25).

Immunoblotting

Protein bands in SDS-PAGE were transferred tonitrocellulose membranes (Bio-Rad) with an LKBNovaBlot 2117 apparatus (Pharmacia LKB), asdescribed by Batanero et al, (26). After transfer-ence, the nitrocellulose sheet was saturated in140 mM sodium chloride and 10 mM sodium phos-phate (PBS), pH 7,2. containing 3% nonfat milkand 0.1% Tween 20 (blocking buffer), for 1 h. Themembrane was incubated with patient sera 1:10diluted, for 2 h at 25°C. After washing, the stripswere incubated with mouse antihuman IgE (1:5000diluted) in blocking buffer for 1 h. Blots werewashed, incubated with horseradish-peroxidase(HRP)-labeled goat antimouse IgG, washed again,and developed with the ECL^'^Western-blottingreagent (Amersham, UK), over Hyperfilm sheets(Amersham).

The immunostainings with polyclonal antibodiesraised against recombinant birch profilin (1:20000diluted) and against olive-pollen profilin (1:50000diluted) were followed by incubation with HRP-labeled goat antirabbit IgG.

521

Ledesma et al.

IgE binding by individual allergic sera

Relative affinities of the individual allergic sera tothe isolated allergen were estimated by IgE bindingof solid-phase-fixed antigen, as described byBatanero et al. (26). A volume of 100 |il of theantigen (1.25 |ig/ml) in PBS, pH 7.2, was added toindividual wells and incubated at 4°C overnight.After treatment with the blocking solution, plateswere incubated with individual sera 1:10 diluted.Incubations with mouse antihuman IgE (1:5000diluted) and HRP-labeled goat antimouse IgGwere carried out. The peroxidase reaction wasdeveloped with fresh enzyme substrate (0.03%H2O2, and 0,63 mg/ml of o-phenylenediamine in0.1 M sodium citrate, pH 5.0), and the absorbancewas measured at 492 nm.

ResultsEvidence of profilin presence in the olive-pollenextract

Immunoblotting analyses of olive-pollen crudeextract were carried out with 191 IgE-specific serafrom patients allergic to olive pollen, in order todetect a profilin-like protein (Fig. la). Twenty-fourpercent of the sera (46/191 tested) recognizedprotein bands of 14-16 kDa, which is the molecularmass of profilin (4, 27). A specific rabbit polyclonalantibody raised against the birch recombinant profilinwas also used to check blots with transferred olive-pollen extract. Fig. lb shows the reaction of theantibody to the 14-16-kDa protein band. Theseresults support the presence of profilin in the olive-pollen extract.

Isolation and purification of the olive profilin

The isolation and purification of the olive profilinwas performed in three chromatographic steps. Gelfiltration chromatography on a Sephadex G-75column was carried out to allow the enrichment ofthe profilin and remove polypeptides of highermolecular mass and pigment components. Thepooled fractions that contained the allergen wereloaded into a poly-(L-proline) Sepharose 4B affinitycolumn, where profilin was specifically retained.The eluted fraction has an abnormally high contentof proline, indicating a coelution besides profilin ofpoiy-L-proline peptides from the matrix. Thesecontaminants were eliminated through additionalgel filtration FPLC chromatography. After eachpurification step, the presence of profilin in theeluted fractions was tested by SDS-PAGE andWestern blot analysis, with both a pool of allergicsera and the polyclonal antibody specific for birch

recombinant profilin. The final yield of the purifiedprotein was around 100 p,g/g of dried pollen.

Molecular characterization

Two main close bands of around 15 kDa molecularmass were observed by silver staining of the puri-fied protein after SDS-PAGE. Both bands couldbind the antiprofilin polyclonal antibody, as well asthe IgE from a pool of sera from five subjectsallergic to olive pollen (Fig. 2). Olive-pollen profi-lin was blocked at the NH2-terminal end, as theattempt to obtain the amino-acid sequence of theallergen by automatic Edman degradation failed torelease any phenylthiodantoin amino-acid deriva-tive after five cycles of degradation.

The quantitative amino-acid composition of thisallergen was determined after standard acidhydrolysis. The total number of residues given inTable 1 was calculated according to the totalcontent of glutamic acid plus glutamine residuespresent in the other profilins. In Table 1, theamino-acid composition of the olive profilin iscompared with those of the other pollen profilins.

The CD spectrum of the purified protein wasdetermined as a three-dimensional property of itsstructure (Fig. 3). The results obtained for theregular secondary structure, 15% a-helix, 33% p-strand, 20% P-turn, and 32% random confor-mation, agree with the molecular data of manymembers of the profilin family, such as Acanth-amoeba (28) and human profilins (29).

IgE binding of purified profilin with individual sera

The allergenic reactivity of the purified protein wasassayed by analyzing the IgE-binding capacity ofthe protein by immunoblotting (Fig. 4A) and

a p 8

6 6 -4 5 -3 6 -2 9 -2 4 -

2 0 -

14— I

Fig. 2. Sodium dodecyl sulfate - polyacrylamide gel electro-phoresis (SDS-PAGE) analysis of purified profilin. a) Silverstaining; p) imtnunostaining of protein transferred to nitro-cellulose membranes with rabbit polyclonal antiserum raisedagainst birch profilin; s) immunostaining with pool of sera frompatients allergic to olive pollen.

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Table 1 Amino-acid composition of olive-pollen profilin compared witfi birch(Betula) and maize isoform 1 profilins

Aminoacid

CysAsxThrSerGlxProGlyAlaValMetHeLeuTyrPheHisLysArgTrpTotal

Amount inolive Drofilin -

(%)

1,4"9,95.95.6

12,7

6313,2

8,64,83,35.56.03,1U2,56.2US

Not done

Number of residues in profilin

Olive

21387

169

1711758843383

Not done132

Betula

2969

166

20895

119444722

133

Maize 1

2795

177

18977

1010435722

131

' Determined by oxidation with performic acid,

ELISA (Fig. 4B) with 14 individual sera frompatients allergic to olive pollen which recognizedprotein bands of around 15 kDa with differentintensities in the saline extract of the olive pollen.As shown in Fig. 4, several sera were positive to aband around 32 kDa, which could correspond tothe dimer form of the protein. This band is veryoften observed in immunoblotting assays with thepolyclonal antibody. These data indicated thestrong tendency to aggregation of this protein.

Olive-pollen profilin

Serum from a patient sensitized to Der p 1, and anonallergic serum, were included as negative con-trols. Each of the sera showed IgE reactivity inELISA analysis, whereas only 11 of them reactedto nitrocellulose-blotted protein. Moreover, severalsera exhibited intense response in ELISA, while avery weak or no signal was observed in immuno-blotting. These results indicate the existence ofconformational IgE epitopes in the olive profilin,since denaturing conditions during SDS-PAGEmight affect the protein folding.

Cross-reactivity studies

The presence of this allergen was checked in blotsof pollen extracts of olive, ash, birch, Bermudagrass, and maize, by using a specific antibody againstolive-pollen profilin. In all cases, a positive band ofaround 15 kDa was bound by the antiserum (datanot shown). Preincubation of the antiserum withthe same amounts (600 ng) of crude extracts inhi-bited binding to purified profilin to a varyingdegree (Fig. 5).

Cross-reactivity analysis was carried out inimmunoblotting by using a pool of five sera frompatients whose IgE recognized this protein. Completeinhibition was observed when amounts of 50 g ofthe above pollen extracts were electrophoresed andtransferred to nitrocellulose membranes, and theIgE-binding assay was performed with a previous

-1000

= -3000

-4000

-5000

-6000

200 220 230 240

nmFig- 3. Circular-dichroism spectrum of olive-pollen profilin infar-ultraviolet region (200-250 nm). Molar ellipticity isexpressed as 0 (degreesxcm^xdmol"').

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 C N

SERUM NUMBER

Fig. 4. IgE recognition of purified profilin by individual serafrom patients hypersensitive to olive pollen. A) Immunoblot-ting assay responses for sera of 14 patients (lanes 1-14), B)ELISA responses with same sera are presented. Serum ofpatient allergic to Der p 1 (C) and serum of nonallergic subject(N). instead of allergic serum, were used as controls. Molecularmass markers are indicated by arrowheads, as in Fig. 1.

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Ledesma et al.

a o g m b e

Fig. 5. Effect of preineubation with different extracts on rabbitantiprofilin antiserum immunodeteetion of purified profilin inimmunoblotting analysis, Polyclonal antibody was preincubatedwitb 600 Hg of following extracts: a) ash, o) olive, g) Bermudagrass, m) maize, b) birch, and c) PBS as control.

incubation with 10 |a.g of the purified profilin(Fig, 6A). The same results were observed whenthe profilin (1 |ig) was transferred to the mem-branes, and 400-ng amounts of the selected pollenswere used as inhibitors (Fig. 6B).

Discussionl\vo of the most significant allergic inducers are theinhalant and food allergens. These allergenicsources appear to be unrelated, but, as shown byEbner et al. (30), they can exhibit cross-reactivitiessince they share common allergens. For particularfood sources (apple and nuts), the presence ofallergens cross-reacting with those in birch (Bet v1 and Bet v 2) has been demonstrated (9). Theidentification of plant profilins as relevant allergensin a wide variety of sources such as grasses, weeds,and trees (4, 25), and the great ubiquity andsimilarity of these actin-binding proteins indicatedthat they are probably responsible for the cross-reactivities between different biologic sources.

Olive pollen is a main cause of seasonal hyper-sensitivity in the Mediterranean area (11, 12).Besides Ole e 1, other low-molecular-mass proteincomponents are detected when crude extract blotsare immunostained with sera of patients allergic toolive pollen. The identification of the olive profilinin the crude extract could be done with rabbitantiserum specific for birch recombinant profilin.Therefore, our study focused on the characteriza-tion of the molecular and immunologic propertiesof this allergen,

Profilin can be purified from a wide variety ofanimal and plant sources by taking advantage ofits affinity for poly-L-proline (31). Like profilinsfrom other sources, olive-pollen profilin bindsspecifically to this matrix. The yield of the purifi-cation (100|ig/g of dried olive pollen) is higher

than that obtained for celery profilin (325 Hg/kg ofcrude extract).

Besides the specific binding to the poly-(L-proline)Sepharose, it is possible to find other featurescharacteristic of every member of this family. Theabsence of the NH^-terminal sequence has beenreported for profilins such as those from Acanth-amoeba (32) and calf spleen (33). In the silver-staining of the purified protein after SDS-PAGE,two bands appeared, the main one having a molec-ular weight of 15.6, which is in the range of birch(14.0), Bermuda grass (15,0), and maize (14.0 and14.8) profilins. The presence of two bands indicatesthe existence of isoforms, as determined in celery(27) and maize (34), in which three profilin isoformshave been detected, two of them of differentlength. Like other members of this plant-proteinfamily, olive-pollen profilin is an acidic protein,while in other genera, such as Acanthamoeba,profilins exist in both acidic (pi 5.5) and basic (pi9.2) forms (35) that differ in their ability to bindthe polyphosphoinositides.

The reaction of IgE-containing sera to olive-pollen allergens with the purified profilin confirmsthat this protein is an allergen, for which wepropose the designation Ole e 2, according to therules of the IUIS Allergen Nomenclature Committee(36), Of sera arbitrarily selected from patientsallergic to olive pollen, 24% contained specific IgE

g m- + _ +

b- +

* m

CO a b g m s

IB

Fig. 6. Inhibition studies with pollens from different species byimmunoblotting analysis. A) Pollen extracts (50 ng) from olive(o), ash (a), Bermuda grass (g), maize (m), and birch (b) weretransferred to nitrocellulose membranes, and IgE binding bypool of allergic sera was inhibited by purified profilin (tOng).- ) Without inhibitor: +) inhibited by profilin from olive pollen.B) IgE binding with same pool of sera to purified profilintransferred to nitrocellulose, as inhibited by 400 ng of sameextracts of pollens used in A. (c) Incubation with PBS and (s)with Sinapis alba seed extract as controls.

524

for olive profilin, as detected after SDS-PAGE ofthe purified protein and immunoblotting. This fre-quency was increased when the binding assay wascarried out under nondenaturing conditions. There-fore, the IgE of a population of allergic sera shouldbe directed against conformational epitopes of theprofilin.

One of the purposes of this study was to deter-mine the cross-reactivities between profilins fromdiverse plant origins. In fact, there is a significantimmunologic similarity among homologous pro-filins. The specific recognition of the profilin bandsin different pollen extracts by a rabbit polyclonalantibody raised against purified olive profilin con-firms a narrow structural relationship among theseproteins. The inhibition assays using this polyclonalantibody lead to the conclusion that the relativeaffinity is higher in birch and ash (unrelated andrelated species, respectively), and lower in Bermudagrass, a fact which may be due to a lower immuno-logic relationship or to less abundancy in the pollen.Allergenic cross-reactivity can be observed in inhibi-tion studies using a pool of sera allergic to olivepollen and the purified profilin as inhibitor. Thepresence of proteins cross-reacting with the purifiedprofilin has been demonstrated for all the pollensources tested, indicating the existence of sharedIgE epitopes. The fact that less cross-reactivity wasobserved with the rabbit antiserum than withserum IgE could be due to a higher IgG titeragainst IgE or even a different route of sensitiza-tion to profilins from other pollens. These analysesconfirm the similar immunologic behavior of pollenprofilins, and this should be taken into considera-tion in the diagnosis of olive allergy, given the highprevalence of hypersensitivity to olive profilin.

Our study confirms that olive-pollen profilin is arelevant allergen which could play an importantrole in the patients' hypersensitivities to differentallergenic sources, since it is shared by a highnumber of animal and plant species, mainly inpollens and foods.

AcknowledgmentsWe thank Dr R, Valenta for kindly donating a polyclonalantibody specific for recombinant birch profilin. We also thankthe Direcci(3n General de Investigacion Cientffica y T6cnica,M. E. C, Spain, for financial support (grants PB92/0195 andPM95/0074),

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