Immunological characterization of a ... - Wiley Online Library

Clin Exp Allergy 2005; 35:483–489

doi:10.1111/j.1365-2222.2005.02214.x

Immunological characterization of a recombinant tropomyosin from a new indoor source, Lepisma saccharina B. Barletta*, C. Butteroni*, E. M. R. Puggioni*, P. Iacovacci*, C. Afferni*, R. Tinghino*, R. Arianow, R. C. Panzaniz, C. Pini* and G. Di Felice* *Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanita`, Rome, Italy, wUSL 1 Imperiese, Department

Allergology, Bordighera (IM), Italy and zCentre de Recherche en Allergologie, Marseille, France

Summary Background The presence of specific IgE antibodies to invertebrates is common among patients with rhinitis and asthma. Tropomyosin has been described as an invertebrate cross-reactive allergen. We have recently characterized an allergenic extract from silverfish (Lepisma saccharina). Since this insect could be a new source of tropomyosin in the indoor environment, we have thought important to clone and characterize the tropomyosin from it. Methods Recombinant tropomyosin was cloned and characterized by means of immunoblotting with tropomyosin-specific monoclonal antibodies, rabbit polyclonal antibodies and IgE from allergic patients. Its allergenic activity was investigated in histamine release assays. Immunoblotting and ELISA inhibition were carried out to identify the natural tropomyosin in the silverfish extract and to study the cross-reactivity among other arthropod tropomyosins. Results Tropomyosin-specific antibodies recognized in immunoblotting the natural tropomyosin in the insoluble fraction of silverfish extract. The silverfish tropomyosin (Lep s 1) was cloned and fully expressed. It shared high homology with other arthropod tropomyosins. rLep s 1 was recognized by tropomyosin-specific monoclonal and polyclonal antibodies and by IgE of allergic patients. It was able to inhibit the IgE binding to the insoluble fraction of silverfish extract, and to induce histamine release by an arthropod-allergic serum. Inhibition experiments revealed IgE cross-reactivity between rLep s 1 and other arthropod tropomyosins. Conclusion rLep s 1 is the first allergen cloned and characterized from silverfish extract. It enabled us to identify the natural counterpart in the insoluble fraction of silverfish extract, suggesting that the tropomyosin is not readily extractable with a classic aqueous extraction procedure. rLep s 1 displayed biological activity, suggesting that it could be regarded as a useful tool to study the role of silverfish tropomyosin in the sensitization to invertebrate allergic sources. Keywords insect allergy, silverfish, tropomyosin Submitted 3 May 2004; revised 26 July 2004; accepted 20 December 2004

Introduction Insects represent about 80% of all animal kingdom, since more than one million of species are known and this number increases day by day with the progress of the entomological research. Many groups of insects have been identified as environmental triggers for asthma [1, 2]. Exposure can occur indoor or outdoor and in occupational or nonoccupational settings. During the last decades, insects other than those already known as allergenic have been investigated for their potential role in inducing and triggering IgE immune response [3]. Among these, the silverfish, an insect belonging to the Thysanura order, appeared of particular interest [4]. In Correspondence: Bianca Barletta, Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanita`, Rome, Italy. Viale Regina Elena, 299-00161-Rome, Italy. E-mail: [email protected]

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spite of its antiquity, silverfish has succeeded in exploiting the new opportunity created by man. In southern Europe and in some regions of Asia these insects live out in the open, under stones and in crevices, but elsewhere they are almost exclusively associated with human houses, stables, outhouses and so on. Silverfish is not easily seen by dwellers because it is nocturnal and can run very swiftly. However, it is considered a pest, or at least a nuisance, by homeowners. These insects prefer vegetable matter with a high carbohydrate and protein content. However, when living indoors they will feed on almost anything. A partial list includes flour, starch, paper, gum, glue, cotton, linen, rayon, silk, sugar, moulds, dried beef and breakfast cereals. Recently, we have prepared and characterized a silverfish extract and our findings indicated that classic aqueous extraction procedure could be not completely satisfactory, since important allergenic components are lost at a pH close to neutrality. In fact, we found an allergenic component with 483

484 B. Barletta et al.

MW of 31–35 kDa, presents only in the insoluble material, which is normally discarded. This component was recognized by out out of the 19 sera (21%) reactive in IgE immunoblotting to silverfish extract [4]. Some evidences suggested that such component could be silverfish tropomyosin [4]. Tropomyosin has been recognized as a cross-reactive inhalant/food allergen, and its pivotal role in cross-reactivity between foods and aeroallergens of animal origin has been described [5–11]. The aim of the present paper was to identify and characterize an important allergenic component represented in the insoluble fraction of silverfish extract, starting from the finding that tropomyosin specific antibodies recognized a component of 31–35 kDa in this fraction. This issue has been addressed by the molecular cloning and the characterization of tropomyosin from silverfish. In fact, we have identified its natural counterpart in the insoluble fraction of the extract. Moreover, we evaluated the biological activity of recombinant silverfish tropomyosin by means of histamine release assay and carried out studies on the cross-reactivity among invertebrate tropomyosins.

Materials and Methods Antisera Human sera Since a diagnostic preparation of Lepisma saccharina was not available, 133 patients allergic to arthropods were selected on the basis of clinical history, skin prick test (SPT) and RAST reactivity. The panel of allergens used in SPT included shrimp, house fly, household insects, mosquito, cockroach, spider and mites (Dermatophagoides pteronyssinus or D. farinae), whereas RAST analysis was performed by using shrimp, mosquito, cockroach and mites (D. pteronyssinus or D. farinae). Nine out of these 133 sera were selected for their IgE reactivity in immunoblotting to a diffuse component with a MW of 31–35 kD present in the insoluble fraction of silverfish extract, identified in our previous paper [4] as natural silverfish tropomyosin. Clinical features of these nine patients are reported in Table 1. The three sera showing the highest immunoblotting reactivity (sera n. 1–3) were chosen for the characterization of recombinant silverfish tropomyosin. Serum from one nonatopic subject was used as control. Informed consent was obtained from all subjects. Rabbit antisera One New Zealand rabbit was immunized three times at 2-week intervals with shrimp tropomyosin, purified according to the method of Smillie [12], by intramuscular injection of 100–200 mg of protein dissolved in 0.5 mL phosphate-buffered saline (PBS) and mixed with an equal volume of Freund’s adjuvant. Serum was aliquoted and stored at 20 1C until use. The animal was maintained in the Animal Care Unit of the Istituto Superiore di Sanita` according to the local guidelines for animal care. Monoclonal antibody Monoclonal antibody 1A/6 specific to D. pteronyssinus tropomyosin, and able to recognize also other arthropod tropomyosins [5, 8], was kindly supplied by Prof. Aalberse.

Sodium dodecylsulfate-polyacrilamide gel electrophoresis and immunoblotting The silverfish extract was prepared according to Barletta et al. [4], and two fractions were obtained and named Ppt and Sup. SDS-PAGE and immunoblotting were carried out as previously described [13, 14] in 15% poliacrylamide gels. The blotted nitrocellulose strips were incubated at room temperature overnight (o. n.) with either individual human sera diluted 1 : 100 in PBS-Tween, or 1 h with the monoclonal antibody 1A/6 diluted 1 : 2000 or 1 h with the rabbit polyclonal antibody diluted 1 : 10 000. The strips were developed with either 125I-labelled anti-human IgE (Bioallergy, Rome, Italy), or peroxidase-labelled goat anti-mouse IgG (Bio-Rad, Richmond, CA) or peroxidase-labelled goat anti-rabbit IgG (Bio-Rad).

Molecular cloning of tropomyosin Total RNA was isolated from adult insect bodies by using RNAzolTM B (BiothechItalia, Rome, Italy), in a ratio of 20 mL/mg of insect according to the Manufacturer’s instructions. cDNA was amplified from 5 mg of total RNA by using the Takara ‘PCR synthesis kit’ (Takara Biochemical Inc., Berkeley, CA). Degenerated oligodeoxynucleotide primers for cDNA amplification were designed as already described by Asturias et al. [9]. Trop 1: 5 0 -CTGGATCCATGGA(G/T) GC(C/T)ATCAAGAA(A/G)AA (sense primer) and trop 2: 5 0 -GGCCGAATTCT(T/C)A(A/G)TA(G/A/T)ACCAG(T/ A/C)(A/C)A(G/A)(T/C)TCGG (antisense primer), restriction sites are underlined. The PCR was carried out in the following conditions: after denaturation at 94 1C for 7 min, the sample was subjected to five cycles at 94 1C for 1 min, 42 1C for 1 min, 72 1C for 1 min, followed by 30 cycles at 94 1C for 1 min, 58 1C for 1 min, 72 1C 1 min, and a final step at 72 1C for 10 min. A product of about 850 bp was obtained and cloned in pBSII KS (Stratagene, La Jolla, CA). Several clones were obtained and some of the positive ones underwent sequence analysis.

Expression and purification of recombinant tropomyosin One of the tropomyosin-encoding cDNA was cloned into p-GEX-6P to obtain a recombinant Lep s 1 (rLep s 1) as a Table 1. Clinical symptoms, positive skin prick test (SPT) and positive RAST in patients recognizing natural and recombinant silverfish tropomyosin Clinical symptoms

Positive SPT*

Serum n. 1

Asthma

SH, HF, MO, MI

SH, CO, MO, MI

Serum n. 2 Serum n. 3

Asthma Allergic rhinitis

CO, HF SH

SH, CO, MO, MI SH, CO, MO, MI


Asthma Asthma

HF, CO, SP HF, HI

ND ND


Asthma Allergic rhinitis

SH, MO, MI SH, MO

SH, MO, MI SH, MO


Allergic rhinitis Allergic rhinitis

SH, MO, MI SH, MO, MI

SH, MO, MI SH, MO, MI

Positive RASTw

*Panel of allergens used in SPT: SH, shrimp; HF, house fly; HI, household insects; MO, mosquito; CO, cockroach; MI, mites (Dermatophagoides pteronyssinus or D. farinae); SP, spider. wPanel of allergens used in RAST: SH, shrimp; MO, mosquito; CO, cockroach; MI, mites (D. pteronyssinus or D. farinae); ND, not determined.

r 2005 Blackwell Publishing Ltd, Clinical and Experimental Allergy, 35:483–489

Recombinant silverfish tropomyosin

fusion protein to GST. The expression of rLeps 1 was obtained in E. coli BL 21 bacterial strain. Bacteria were grown in Luria–Bertami media with 200 mg/mL of ampicillin at 37 1C up to OD600 0.6 when 0.5 mM IPTG was added and the cells were harvested after 2 h by centrifugation. The pellet was resuspended in phosphate buffer (pH 8) containing 100 mg/mL lysozyme, 50 mL Triton X-100, 10 U/mL DNAse, 10 mg/mL RNAse, incubated for 10 min on ice and lysed by sonication. The recombinant molecule was purified on a glutathione Sepharose 4B column (Amersham Pharmacia Biotec, Uppsala, Sweden) according to the Manufacturer’s instructions and was recovered from the resin after an oncolumn cleavage with 80 U of PreScission Protease (Amersham Pharmacia Biotec) for mL of washed glutathione Sepharose bed volume, o. n. at 4 1C. The purity of the protein was tested by SDS-PAGE gel and the protein concentration by method of Bradford [15]. rLeps 1 was aliquoted and stored to 20 1C until use.

Nucleotide and protein sequencing analysis Nucleotide sequences were determined using ABI PRISM Dye Terminator Cycle Sequencing Ready reaction kit and run on ABI 310 DNA sequencer. The analysis of the sequences was achieved using the BLAST program. The deduced protein sequence and its analysis were performed by PROSITE database [16]. The alignment of Lep s 1 sequence and homologous protein sequences from other species was obtained by Clustal W (1.74 and 1.81) program.

Immunoblotting inhibition Inhibition was carried out as previously described [14]. Inhibition of specific IgE binding on blotted insoluble fraction of silverfish extract (Ppt) was performed by preincubating serum n. 1 diluted 1 : 100 with increasing concentrations (20 and 50 mg/mL) of recombinant rLep s 1.

were used as inhibitors. Amount of inhibitors ranged from 10 to 0.00064 mg protein/mL.

Results Identification of natural tropomyosin in the silverfish extract To better identify the IgE reactivity of silverfish extract components not readily extractable in classical aqueous buffer, three human sera reactive to the 31–35 kDa component only in IgE-immunoblotting were selected. Figure 1a shows the IgE reactivity of one representative serum to the two fractions of silverfish extract. A restricted reactivity to the 31–35 kDa component of the insoluble fraction was detected and no bands of reactivity were found in aqueous fraction. The monoclonal antibody 1A6, specific for mite tropomyosin, reacted with the insoluble fraction only, where it was able to identify a component of 31–35 kDa (Fig. 1b). The reactivity of rabbit antibody specific for the natural purified shrimp tropomyosin, confirmed the presence of tropomyosin in the insoluble fraction of the silverfish extract (Fig. 1c).

Molecular cloning, expression, purification and characterization of tropomyosin Using two degenerated primers, already employed for the cloning of American Cockroach tropomyosin [9], the cDNA of Lep s 1 was obtained. The cDNA nucleotide sequence corresponds to a 855 bp open reading frame (EMBL accession number: AJ309202). The analysis of the sequence by BLAST showed the higher homology with tropomyosins from Homarus americanus (69%). The cDNA encoded for a protein of 284 aa designed rLep s 1 with an estimated molecular mass of 32.4 kDa and a pI of 4.88 The analysis of the protein sequence showed one potential N-linked glycosy-

Histamine release assay from passively sensitized basophils

(a)

The histamine release assay was performed as reported by Iacovacci et al. [17]. One representative serum out of tropomyosin-positive patients (serum n. 2) and one control serum, both diluted 1 : 2, were used in this test. Histamine release from the passively sensitized basophils was measured by using the Immunotech Pharmaceuticals kit (San Diego, CA), according to the Manufacturer’s instructions. The rLep s 1 was used in the test at the final concentrations of 10, 1, 0.1 and 0.01 mg/mL.

97.4 66.2 45.0

ELISA inhibition

14.4

ELISA inhibition experiments were carried out essentially as reported in Barletta et al. [14]. The amount of rLep s 1 coated on the ELISA plates (polystyrene microtitre, Greiner, Frickenhausen, Germany) was 0.1 mg/mL. Human sera were diluted 1 : 100 or 1 : 50 in PBS-Tween 20% and 1% wt/vol gelatin. Recombinant tropomyosins from D. pteronyssinus and Periplaneta americana, rDer p 10 and rPer a 7, respectively, purchased from BIAL (Bilbao, Spain) and natural shrimp tropomyosin, purified according to the method of Smillie [12],

485

(b)

(c)

KDa

31.0 21.5

Ppt

Sup

Ppt

Sup

Ppt

Sup

Fig. 1. Two fractions of silverfish, aqueous (Sup) and insoluble (Ppt), were probed in immunoblotting with a representative allergic serum (a), monoclonal antibody 1A6 (b) and anti shrimp tropomyosin rabbit antiserum (c).



Lep s 1 D. mel. Hom a 1 Per a 7 Der p 10

MEAIKKKMQAMKLEKDNAMDKADALEAQARDANRKADKILEEVQDLKKKPSQVETDFTTT *D****************I****TC*N**K***SR***LN***R**E**FV*****LV*A *D******************R**T**Q*NKE**IR*E*TE**IRITH**MQ***NELDQV *D******************C*LLC*Q******LR*E*AE**ARS*Q**IQ*I*N*LDQ* *E***N************I*R*EIA*QK*****LR*E*SE***RA*Q**IQ*I*NELDQV

Lep s 1 D. mel Hom a 1 Per a 7 Der p 10

KENLATANKNLEDKEKTLTNTESEVASLNRKVQMIEENLERSEERLGTALTKLGEASHAA **Q*EK**TE**E***L**A******TQ*****Q***D**K****ST**QQ**L**TQS* Q*Q*SL**TK**E***A*Q*A*G***A***RI*LL**D********N**T***A***QA* M*Q*MQV*AK*DE*D*A*Q*A*****A***RI*LL**D********A**TA**A***QAV Q*Q*SA**TK**E***A*QTA*GD**A***RI*LI**D********KI*TA**E***QS* E3

E6

E2


DEASRMCKVLENRSQQDEERMDQLTNQLKEARMLAEDADGKSDEVSRKMAQVEDDLEVAE **NN***********************************T********L*F***E***** **SE**R*******LS******A*E*******F***E**R*Y***A**L*M**A***R** **SE*AR*I**SKGLA******A*E*******FM**E**K*Y***A**L*M**A***R** **SE**R*M**H**IT*****EG*E********M*****R*Y***A**L*M**A***R**


DRVKSGDSKIMELEEELKVVGNSLKSLEVSEEKANQRVEEYKRQIKTLTVKLKEAEARAE ******E*********************************F**EM***SI******Q*** E*AET*E***V******R****N**************E*A**E*****AN***A****** E*AE**E***V******R****N************L*E****Q******TR********* E*AET*E***V******R****N***********Q**E*AHEQ**RIM*T********** E4


YAEKYVKKLQKEVDRLEDELGINKDRYRALADEMDQTFAELSGY ***Q**R**********R*FNE*EK*K*IC*DL*******T** F**RS*Q*************VNE*EK*KSIT**L****S***** F**RS*Q*************VHE*EK*KFIC*DL*M**T**A** F**RS*Q******G******VHE*EK*KSIS**L*******T**

[100%] [ 76%] [ 67%] [ 65%] [ 64%]

Fig. 2. Sequence alignment of rLep s 1 with other tropomyosins: Lep s 1 (Lepisma saccharina, CAC84590), Drosophila melanogaster (P09491), Hom a 1 (Homarus americanus, O44119), Per a 7 (Periplaneta americana, Q9UB83), Der p 10 (Dermatophagoides pteronissinus, O18416). The percentage of each sequence homology is given in parentheses. Regions corresponding to the four IgE-binding epitopes identified on Penaeus aztecus tropomyosin (E2, E3, E4, E6) are marked in grey.

KDa

KDa

KDa

97.2 66.2 97.2 66.2

45.0

45.0

31.0

31.0 21.5

21.5

97.2 66.2

14.4

45.0

14.4

31.0

21.5 1

2

3

4

5

6

7

Fig. 3. SDS-PAGE and Immunoblotting analysis of rLep s 1 detected with

14.4

Coomassie Brilliant Blue staining (lane 1) and incubated with three allergic human sera selected for following experiments (lanes 2–4), a nonatopic human serum (lane 5), mouse monoclonal antibody 1A6 (lane 6), and antishrimp tropomyosin rabbit serum (lane 7).

lation site (NRSQ 132–135). Both the EAIKKK N-terminal motif and the LKEAExRAE signature sequences, highly conserved in tropomyosin molecules, were present in rLep s 1. Search of protein sequence similarity revealed the highest degree of homology with tropomyosin from Drosophila melanogaster (74%) (Fig. 2). The sequences of the IgE reactive epitopes E2 (RKLAMVEADLERA 157–169), E3

1

2

3

Fig. 4. Immunoblotting inhibition pattern of IgE binding to insoluble fraction of silverfish extract (Ppt) inhibited with 50 and 20 mg/mL of recombinant tropomyosin, rLep s 1 (lanes 1–2), no inhibitor added (lane 3).

(SDEERMDALENQ 126–137), E4 (NEKEKYKSITDELDQTFSELS 252–272), and E6 (EADRKYDEVARKL



147–159), obtained from Penaeus aztecus tropomyosin [18], showed a degree of identity of 69%, 76%, 52% and 61%, respectively, to the corresponding regions identified on the rLep s 1 sequence. The cDNA obtained was cloned into p-GEX-6P expression vector. The MW of the purified recombinant protein was about 36 kDa, as calculated by Rf analysis after SDS-PAGE (Fig. 3 lane 1). The yield of the purified protein was 2 mg/L of bacterial culture, as measured by the Bradford assay [15]. To evaluate the IgE reactivity of the recombinant molecule, the three human sera recognising the above described component

Histamine release (%)

of 31–35 kDa were tested in immunoblotting (Fig. 3, lanes 2–4). All sera recognized the recombinant tropomyosin. The recombinant molecule did not react with a nonallergic human sera used as negative control (Fig. 3, lane 5). Specific IgE did not recognize notinduced cell lysate (data not shown). The monoclonal antibody specific to mite tropomyosin and the rabbit serum, raised against shrimp tropomyosin, recognized rLep s 1 (Fig. 3, lanes 6 and 7).

Inhibition of specific IgE binding to insoluble fraction of silverfish extract To further elucidate whether the natural counterpart of rLep s 1 was really present in the insoluble fraction of silverfish extract, IgE immunoblotting inhibition experiments were carried out with serum n. 1 (Fig. 4). rLep s 1 was able to inhibit the IgE binding to the blotted insoluble fraction at a concentration of 50 mg/mL, confirming that the natural tropomyosin corresponds to the allergenic component with MW of 31–35 kDa identified in the insoluble fraction.

Allergen: rLep s 1

100

Serum n. 2

80

487

60 40

Histamine release from basophils 20 0 0.001

0.01

0.1

1

The allergenic activity of recombinant tropomyosin was demonstrated by the histamine release assay, using basophils from a nonatopic donor passively sensitized with IgE from one representative allergic patient (serum n. 2). The doserelated release curve obtained after stimulation with rLep s 1 is shown in Fig. 5. Specific IgE induced a histamine release up to 54% at the highest amount of allergen. Serum from one nonatopic subject, used as negative control, did not induce

10

Amount of allergen (µg/mL) Fig. 5. Basophil histamine release tests performed with IgE from serum n. 2 Dose-related release curves obtained after stimulation with rLep s 1. Allergen was used at concentration of 10, 1, 0.1 and 0.01 mg/mL.

ELISA inhibition Antigen: rLep s 1 Serum n. 2 Serum n. 3 Serum n. 1

% of inhibition

(a)

Inhibitor: rPer a 7

(b)

100

100

75

75

50

50

25

25

0 0.0001 0.001 0.01

(c)

0.1

1

10

100

Inhibitor: shrimp tropomyosin

0 0.0001 0.001 0.01

(d)

100

100

75

75

50

50

25

25

0 0.0001 0.001 0.01

0.1

1

10

100

Inhibitor: rDer p 10

0.1

1

10

100

10

100

Inhibitor: rLep s 1

0 0.0001 0.001 0.01

0.1

1

Amount of inhibitor (µg/mL) Fig. 6. ELISA inhibition of serum n. 1 (diamond), serum n. 2 (circle) and serum n. 3 (triangle) against rLep s 1 with rPer a 7 (panel a), rDer p 10 (panel b), purified shrimp tropomyosin (panel c) and rLep s 1 (panel d) as inhibitors.



histamine release when tested with the highest allergen amount (data not shown).

Cross-reactivity among tropomyosins from different allergenic sources The presence on the recombinant tropomyosin of crossreactive IgE epitopes with tropomyosins from different sources was studied by ELISA inhibition assays, performed with the three sera positive to silverfish tropomyosin (Fig. 6). The results were expressed as percent inhibition of the total specific binding. As shown in Fig. 6, recombinant tropomyosins from P. Americana (Per a 7) (Fig. 6a), and D. pteronissinus (Der p 10) (Fig. 6b) as well as natural shrimp tropomyosin (Fig. 6c), were able to inhibit IgE binding to recombinant silverfish tropomyosin up to 75%, 89% and 99%, respectively, at the highest inhibitor concentration tested. IgE reactivity of the three patients shows similar inhibition curves.

Discussion Although previous studies demonstrated that house dust contains significant silverfish antigens [19], and reported that silverfish could be regarded as a significant source of many allergenic components [4], a commercial silverfish extract is not available so far. In a previous study, we have prepared and characterized a silverfish extract and investigated the IgE reactivity of its components [4]. An evaluation of IgE reactivity indicated a proportion of sera (21%) recognising a diffuse and intense component with MW of 31–35 kDa, present in the insoluble fraction of silverfish extract only. Results obtained suggested that such component could be the silverfish tropomyosin that could be missed with a classic aqueous extraction. This finding suggests that a dedicated extraction procedure may be needed for some insect sources, since tropomyosin does not appear to be readily extractable in classic aqueous buffer. Because of common features between silverfish and other insects, this last issue could be extended to all allergenic extract preparations from insect sources, since lack of important allergenic components in an extract could affect several aspects, such as exposure assessment, diagnosis sensitivity and therapy efficacy. To further investigate this important aspect, in the present paper we have analysed the two fractions of the silverfish extract in immunoblotting developed with monoclonal and polyclonal antibodies raised against mite and shrimp tropomyosin, respectively. The use of these specific antibodies allowed us to identify the tropomyosin in a new allergenic source present in indoor environment that could give a possible contribution to increase tropomyosin indoor levels. This is in line with that previously evidenced by other authors reporting that some dust samples have too high concentration of tropomyosin if compared with mite presence, indicating the contribution of additional tropomyosin sources other than mites [19]. Tropomyosin belongs to a family of highly conserved proteins, some of which have allergenic activity. In fact, in contrast to vertebrate tropomyosins, invertebrate tropomyo-

sins have been demonstrated to be allergenic [11]. Molecular cloning of tropomyosin from different sources could provide useful tools to investigate their allergenicity. To this aim, we have cloned and characterized a silverfish tropomyosin, named rLep s 1, by means of immunoblotting with specific tropomyosin antibodies and IgE from crustacean-and house insect-allergic patients. The nucleotide sequence shows 69% of homology with tropomyosin from Homarus americanus, and the deduced amino-acid sequence shows the highest degree of homology with tropomyosin from Drosophila melanogaster. Moreover, a significant homology with the IgE-binding epitopes E2, E3, E4, E6 described on P. aztecus tropomyosin by Reese et al. [18] has been found in the corresponding regions identified in the Lep s 1 sequence. These significant homologies found within regions identified as critical for IgE-binding could contribute to the wide crossreactivity among tropomyosins from different sources. rLep s 1 is able to inhibit the IgE binding to the insoluble fraction of silverfish extract suggesting that the IgE epitopes are shared by recombinant and natural tropomyosin. A strong IgE crossreactivity among inhalant and edible invertebrates allergenic sources, because of tropomyosin molecule, has been extensively investigated also at level of IgE epitopes [20]. In order to investigate whether silverfish tropomyosin shared IgE epitopes with tropomyosins from other sources, ELISA inhibitions were performed. Tropomyosins from three arthropod sources were used as inhibitors and three human sera from patients allergic to arthropods were selected as IgE sources. The highest inhibiting capacity is displayed by natural shrimp tropomyosin; this result could be because of the fact that the purified preparation contains a wide range of isoforms. However, significant inhibition of IgE binding to rLep s 1 was also obtained with rPer a 7 and rDer p 10, suggesting that IgE crossreactive epitopes shared by tropomyosins from different sources were represented on the rLep s 1 molecule. Interestingly, when we used the homologous inhibitor rLep s 1 we found different behaviours by the three sera. These differences could reflect different causes of primary sensitization. In fact, serum n. 3, which is unable to reach plateau inhibition, was selected on the basis of SPT reactivity to shrimp allergens only (Table 1). This widespread cross-reactivity may result in clinical consequences. In fact, several studies indicate that the exposure and sensitization to a particular food allergen may ultimately lead to sensitization to certain aeroallergens [5] and vice versa [21, 22]. Several authors have attempted to identify the primary source and consequently the initial route of sensitization (ingestion or inhalation) by means of inhibition experiments. Recently, an interesting study has indirectly suggested that sensitization to shrimp tropomyosin could occur by inhalation route, on the basis of the observation of a group of Ortodox Jews who strictly observe Kosher dietary laws that prohibit eating shellfish, so excluding the exposure by ingestion route [23]. It was also suggested that immunotherapy for house dust mite allergy may lead to sensitization to cross-reacting seafood tropomyosin [21, 24], adding a possible third sensitization route involved in tropomyosin sensitization. We have evaluated the capability of rLep s 1 to trigger histamine release from basophils passively sensitized with a



representative insect allergic patient. The results obtained demonstrated that rLep s 1 was able to induce histamine release up to 54%, confirming its allergen activity. In conclusion, Lep s 1 represents the first allergen identified in silverfish extract or rather in its insoluble fraction that, in a standard extraction procedure, is normally discarded. Recombinant silverfish tropomyosin, rLep s 1, can be regarded as an allergenic molecule cross-reactive with tropomyosins from inhalant and food sources. Moreover, since tropomyosin molecules can elicits hypersensitivity reactions by sensitising through three different routes [inhalation, ingestion and parenteral administration], rLep s 1 could be an useful tool to evaluate the role that the sensitization route plays in the development of allergy disease.

Acknowledgements We thank Dr A. Orlandi for her helpful assistance and Prof. R.C. Aalberse of Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, for providing monoclonal antibody 1A6 specific to D. pteronyssinus tropomyosin.

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