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Divisions of
*
Specific Prophylaxis and Tropical Medicine and
Immunopathology, Department of Pathophysiology, and Division of
Hematology and Hemostaseology, Department of Internal Medicine I, University of Vienna, Vienna, Austria; and
Institute for Stored-Product Protection, Biological Research Center for Agriculture and Forestry, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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That moths could be the causative agent of inhalant allergies had been mentioned as early as 1928 by Vaughan (7). Over the years, there have been scattered case reports on bronchial asthma caused by the clothes moth Tineola bisselliella (8) and wax moth Galleria mellonella (9). Baldo and Panzani (10) and more recently Komase et al. (11) characterized various insect extracts by IgE immunoblotting and demonstrated several IgE-Ags in the clothes moth or silkworm moth. In studies conducted in Japan, a high proportion of patients with asthma bronchiale (12, 13) or allergic rhinitis (14) were found to react with silkworm moth allergens.
In recent years the Indianmeal moth, Plodia interpunctella, has become a widely spread household and stored product pest throughout the United States and Europe. Its larvae feed on dry foodstuffs such as nuts, grains, dried fruit, and chocolate (15). Although it was mentioned as a possible cause of allergies in a review on allergens in mills (16), no detailed studies have been performed whether the Indianmeal moth represents an indoor allergen source. We examined a panel of 102 sera from indoor allergic patients and found a high prevalence of IgE reactivity against Indianmeal moth Ags. One of these IgE-reactive Ags was characterized on the molecular level, and was identified as an arginine kinase by cDNA cloning, demonstration of sequence homology, and enzymatic activity of the recombinant protein. Finally, we demonstrate that this allergen has IgE cross-reactive homologs in several invertebrate species such as mite, cockroach, lobster, king prawn, and mussel.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sera from the following groups of patients were tested for the presence of IgE Abs against moth allergens: 1) patients with type I allergic symptoms (rhinitis/conjunctivitis, allergic asthma bronchiale) indoors (n = 90, patients H1–H90, ages ranging from 17 to 60 years, average age 32 years); 2) patients with type I allergic symptoms indoors as above plus atopic dermatitis (n = 12, patients AH1–AH12, ages from 11 to 47 years, average age 28 years); 3) control individuals without type I allergies or atopic dermatitis (n = 10, individuals N1–N10, ages from 26 to 35 years, average age 31 years).
The diagnosis of type I allergy was based on case history, skin prick testing, and CAP-RAST (radioallergosorbent test; Pharmacia, Uppsala, Sweden) testing using a panel of extracts from indoor (house dust mite, cat dander) and outdoor (birch pollen, grass pollen) allergen sources. The diagnosis of atopic dermatitis was based on the criteria of Hanifin and Rajka (17). The cDNA library was screened with the serum from patient AH11. Skin prick tests were performed in patients AH11 and H60.
IgE reactivity of natural moth, mite, and cockroach extracts as well as the purified recombinant arginine kinase
Preparations from two moth species, house dust mite, and cockroach were used to detect specific IgE in patients’ sera. Extracts from the Indianmeal moth P. interpunctella were obtained by homogenizing 25 late-stage larvae per 1 ml of PBS. Reducing gel loading buffer was added 1/1, samples were denatured for 10 min at 95°C, and debris was removed by centrifugation in a microcentrifuge (5 min, room temperature, 10,000 x g). In the same way, extracts were prepared from commercial preparations from adult Mediterranean flour moth (Ephestia kuehniella), house dust mite (Dermatophagoides pteronyssinus), and cockroach (Blattella germanica) obtained from Allergon Pharmacia (Uppsala, Sweden). The extracts or the purified arginine kinase were electrophoresed on preparative 12.5% SDS-polyacrylamide gels with an approximate protein concentration of 20 µg cm-1 (extracts) or 10 µg cm-1 (purified recombinant arginine kinase) as estimated by Coomassie blue-stained test gels. Proteins were blotted onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany), and 5-mm strips were cut from the sheets after the transfer. The nitrocellulose membranes were blocked 2 x 5 min and 1 x 30 min at room temperature with three changes of buffer G (42 mM Na2HPO4, 6.4 mM NaH2PO4, 0.5% (v/v) Tween 20, 0.5% (w/v) BSA, 0.05% (w/v) NaN3, pH 7.5) and incubated with a 1/10 dilution of patients’ sera in buffer G overnight at 4°C. After washing 2 x 5 min and 1 x 30 min in buffer G, bound IgE was detected by overnight incubation at room temperature with 125I-labeled anti-IgE Abs (Pharmacia), washing as above, and autoradiography.
Construction and IgE immunoscreening of a cDNA library from P. interpunctella larvae
The insect larvae were grown on oats. One hundred eighty larvae (2.4 g) in the prepupal stage were homogenized in 30 ml of TRIzol reagent (Life Technologies, Frederick, MD), and total RNA was prepared. Poly(A)+ RNA was prepared with the Poly(A)Ttract system (Promega, Madison, WI). The cDNA library was prepared in the Uni-ZAP system (Stratagene, La Jolla, CA) according to the supplier’s protocol. The primary library from 5 µg of poly(A)+ RNA contained 3 x 106 clones and was amplified with standard methods.
ZAP phages (360,000 in total) were used to infect Escherichia
coli XL1-Blue (Stratagene) in 24 140-mm petri dishes. Synthesis of
recombinant proteins was induced by adding nitrocellulose filters
soaked in 10 mM isopropylthio 
-D-galactoside.
The filters were blocked and probed with the serum from patient AH11 as
described above.
Sequence analysis of the IgE-reactive clones
The cDNA-containing plasmids were obtained from the 31 isolated IgE-reactive phages by in vivo excision (18). The DNAs were sequenced using Thermosequenase (Amersham Pharmacia Biotech, Piscataway, NJ) and IRD800-labeled primers (MWG Biotech, Ebersberg, Germany) on a LI-COR sequencer (LI-COR, Lincoln, NE).
The deduced protein sequences were compared with sequences deposited in the SwissProt database using the FastA program (19). Clones were aligned with each other and homologous protein sequences with the GAP program from the University of Wisconsin Genetics Computer Group package (Madison, WI) (20). Further protein sequence analysis was performed by software provided at the ExPASy molecular biology server (http://www.expasy.ch/tools/) such as NetPhos (21), PROSITE (22), and NetOglyc (23) for the predictions of putative phosphorylation and N- and O-linked glycosylation sites.
Expression and purification of the recombinant moth allergen in E. coli
One full-length cDNA, coding for a 40-kDa protein with
end-to-end sequence similarity with arginine kinases, was inserted in
two steps between the EcoRI and XhoI sites of the
plasmid pET23+ (Novagen, Madison, WI). The
ribosome binding site was inserted by oligonucleotide-directed
mutagenesis (24) using the oligonucleotide 5'-GGT AGC GGC
GTC CAC CAT GGT ATA TCT CCT TCT AGA GGG AAA CCG-3' giving the vector
pETAK1. A second mutagenesis with the oligonucleotide 5'-ATC TCA GTG
GTG GTG GTG GTG GTG CAG GGA TTT CTC GAT TTT GAT-3' inserted the coding
sequence for a hexahistidine tag for purification by nickel chelate
affinity chromatography giving plasmid pETHisAK1. This plasmid was
checked by DNA sequencing and transformed into E. coli BL21
(DE3) for protein expression. The cells were grown at 37°C to an
optical density at 600 nm of 0.8. Recombinant protein synthesis was
induced for 3 h by adding isopropylthio
-D-galactoside to a final concentration of 0.4
mM. The cells were pelleted and lysed by 30-min treatment in buffer L
(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 1
mg ml-1 (w/v) lysozyme, pH 8). Undissolved material was
pelleted by 30-min centrifugation at 2000 x g and
4°C. The recombinant protein was then purified by nickel chelate
affinity (25) under native conditions using small spin columns (Qiagen,
Hilden, Germany).
Measurement of the arginine kinase activity of the recombinant allergen
Arginine kinase (Enzyme Commission number: EC 2.7.3.3) activity was measured by determining the rate of formation of ADP. The ADP is converted back to ATP by pyruvate kinase, and the pyruvate formed is reduced to lactate by lactate dehydrogenase. The rate of consumption of NADH in this reaction is measured photometrically (26). Protein concentration was estimated from the optical density at 280 nm and the extinction coefficient calculated from the deduced protein sequence of the recombinant allergen (27). The reaction was performed at 30°C in 1-ml volumes containing 2 mM L-arginine and 4 mM ATP, 50 mM Tris/acetate, 5 mM Mg-acetate, 0.75 mM phosphoenolpyruvate, 0.2 mM NADH, 10 µg ml-1 pyruvate kinase, and 12.5 µg ml-1 lactate dehydrogenase at pH 6.8.
The measured activity was 18.5 U mg-1, which corresponded to a turnover number kcat of 12.3 s-1. When either arginine or ATP was omitted from the reaction mixture, no activity was observed.
Histamine release assay
Heparinized blood samples were obtained from the patients H20 and AH11 with IgE-reactivity to arginine kinase, and granulocytes were prepared by dextran sedimentation (28). Cells were resuspended in histamine release buffer and incubated with increasing concentrations of recombinant moth allergen or, for positive control, with anti-IgE mAb E124-2-8 (Immunotech, Marseille, France) at 37°C for 30 min. Then cells were sedimented by centrifugation at 4°C and the cell-free supernatants were recovered. Liberated histamine expressed as percentage of total histamine was measured in the cell-free supernatants by radioimmunoassay (Immunotech) (28). Triplicate determinations of histamine release by the recombinant moth allergen were conducted.
Skin prick test
After written informed consent was obtained, skin prick tests were performed in two atopic individuals, one with (AH11) and one without (H60) IgE, against the recombinant moth arginine kinase. Purified recombinant allergens (recombinant arginine kinase (this study) and, for control purposes, recombinant birch pollen allergen Bet v 1 (Biomay, Linz, Austria)) were diluted in sterile 0.9% NaCl to five different concentrations: 50 ng µl-1 allergen, 25 ng µl-1, 12.5 ng µl-1, 6.25 ng µl-1, and 3.12 ng µl-1. NaCl (0.9%) and histamine dihydrochloride (Allergopharma, Reinbek, Germany) in a concentration of 1 mg ml-1 were used for negative or positive controls.
IgE immunoblot inhibition experiments
The cross-reactivity of the moth arginine kinase with allergens from mite (D. pteronyssinus), cockroach (B. germanica), king prawn (Penaeus monodon), lobster (Homarus gammarus), mussel (Mytilus edulis), and cod (Gadus morhua) was tested in this experiment. Fresh, uncooked seafood was purchased from a local market, and white meat was prepared. The different samples (1–5 g) were frozen in liquid nitrogen and crushed to a powder in a mortar. Ice-cold H2O containing 5 mM PMSF was added and Ags were extracted by stirring for 1 h at 4°C. After addition of 1 volume of gel loading buffer, samples were denatured for 10 min at 95°C and insoluble particles were removed by centrifugation. The protein concentration of the extracts was estimated on a Coomassie blue-stained SDS-PAGE gel. Preparative 12.5% gels containing 20 µg cm-1 protein were run and blotted onto nitrocellulose which was cut into strips. The sera from patients AH11, H89, and H32 were diluted 1/10 in buffer G and preincubated in buffer G with or without 10 µg ml-1 recombinant moth arginine kinase overnight at 4°C, and then exposed to the nitrocellulose-blotted extracts from the different species. Bound IgE was detected as described for IgE immunoblotting.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the prevalence of IgE recognition and the allergen
profile of the Indianmeal moth, we used sera from patients with type I
allergic symptoms indoors (n = 90, H1–H90, average age
32), sera from patients with type I allergic symptoms and atopic
dermatitis in addition (n = 12, AH1–AH12, average age
28), and sera from nonallergic individuals (n = 10,
N1–N10, average age 31) to detect nitrocellulose-blotted IgE-reactive
Ags (Figs. 1
and 2, and Table I). Forty-two of 90 indoor allergic
patients (47%) and 10 of the indoor allergic patients with atopic
dermatitis (83%) had IgE Abs against P. interpunctella
larval Ags. The m.w. profile of the IgE-binding components varied
significantly for the different patients’ sera, and a number of Ags of
various m.w. were recognized. More than one-third of the sera revealed
significant IgE reactivity to high molecular mass components above 70
kDa. None of the nonallergic control individuals exhibited IgE
reactivity to the P. interpunctella extract. Reactivity of
the 125I-labeled anti-human IgE Abs with a
component of around 66 kDa was noted in one set of experiments
(Fig. 1).
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). The results were
comparable with those obtained for the Indianmeal moth larval extracts.
Thirty-four (38%) from the indoor allergic group without atopic
dermatitis and six (50%) from the indoor allergic group with atopic
dermatitis showed specific IgE reactivity.
When we tested those sera containing IgE-Abs against Indianmeal moth
larval Ags with commercial extracts of house dust mite (D.
pteronyssinus) and cockroach (B. germanica), we found
that many of the sera did not cross-react with mite or cockroach Ags.
As many as 23% of all moth-allergic patients had IgE reactive with
neither mite nor cockroach extracts (Table II).
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Thirty-one IgE-reactive clones were isolated from a ZAP cDNA
library prepared from P. interpunctella larvae using the
serum of patient AH11. Although the clones obtained in the first
screening experiment had different insert sizes, all of them were
derived from the same cDNA. Two more screening experiments using
another three patients’ sera identified three more P.
interpunctella IgE-binding Ags (B. Hayek, unpublished data). The
longest cDNA clone coded for a polypeptide of 39.9 kDa including an
initiator methionine (Fig. 3). The
untranslated regions were 24 bp at the 5'-end and 195 bp at the 3'-end
upstream of the poly(A) tail. Comparison with the databases showed an
end-to-end similarity of the deduced amino acid sequence with arginine
kinases from various arthropod species. The closest homologs of the
moth enzyme were arginine kinases from the grasshopper
(Schistocerca americanus) (29) and the honeybee
(Apis mellifera) (30), with 86 and 85% amino
acid sequence identity, respectively (Fig. 3). Fig. 3 shows that there
was also a very high degree of sequence identity (82%) with the enzyme
from lobster (H. gammarus) (31). Even an
arginine kinase from a protozoan, Trypanosoma cruzi
(32), had 70% identical residues.
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(Ser20,
Ser44, Ser156,
Ser281, Thr333,
Tyr74, Tyr133, and
Tyr144). All except Ser20
were conserved in all three species. No O-glycosylation
sites were identified, but there was one possible
N-glycosylation site at Asn214 and an
actinin-type actin-binding site (residues Glu213
to Asn222, marked in Fig. 3). In this context it
is noteworthy that scallop muscle arginine kinase AK1 binds to
filamentous actin, leading to noncompetitive inhibition of the enzyme
(33). Recently, the crystal structure of arginine kinase
from the horseshoe crab (Limulus polyphemus) has
been reported (34). All the residues putatively
interacting with the substrates (Arg126,
Glu225, Arg229,
Cys271, Thr273,
Arg280, Arg309, and
Glu314) are also present in the corresponding
positions in the moth, honeybee, and lobster arginine kinases
(double-underlined in Fig. 3). Purification of recombinant enzymatically active P. interpunctella arginine kinase
The pET23+-derived expression plasmid
pETHisAK1 was constructed, and recombinant P. interpunctella
arginine kinase was expressed and purified as shown in Fig. 4. The induced cultures produced the
recombinant protein as a major, soluble protein which could be purified
under native conditions to high purity by a single step of nickel
chelate affinity chromatography. The yield was around 5 mg of purified
protein per 1000 ml of E. coli culture. A standard coupled
assay for arginine kinase activity was performed using the recombinant
enzyme, and an activity of 18.5 U/mg of protein was measured,
corresponding to a turnover number of 12.3 molecules per second.
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All the sera from the patients and control individuals were tested
for specific IgE against the nitrocellulose-blotted recombinant
arginine kinase. Ten of the 90 house dust allergic patients and three
of 12 patients with atopic dermatitis and indoor allergy, but none of
the nonallergic individuals, had IgE Abs to recombinant arginine
kinase. This corresponded to 13% of all patients and 25% of the
patients who were IgE-reactive with moth larval extract (Figs. 1 and 2,
and Table I).
The allergenic activity of recombinant arginine kinase was demonstrated
by histamine release assay using basophils from the two sensitized
patients H20 and AH11 (Fig. 5, A and B). In both patients, the recombinant
allergen induced a dose-dependent histamine release with a maximum
between 1 and 10 ng ml-1 and a significant
release with as low as 10 pg ml-1 allergen for
patient H20 and 100 pg ml-1 for patient
AH11.
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). Recombinant Bet v 1 induced skin
reactions in both patients having specific IgE Abs. After 24 h
patient AH11 developed papules within the marked borders of the
previous immediate reactions to the moth arginine kinase and to Bet v
1, being indicative of a late-phase reaction (not shown). No reactions
in either patient were induced with physiological NaCl alone.
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The moth arginine kinase displayed high sequence similarity with
homologs from other species (Fig. 3). To investigate whether cockroach
(B. germanica), mite (D. pteronyssinus), king
prawn (P. monodon), lobster (H. gammarus),
mussels (M. edulis) and cod (Gadus morhua), a
vertebrate, contained allergens cross-reactive with moth arginine
kinase, IgE immunoblot inhibition experiments were performed.
Preincubation of sera from patients (AH11, H89, and H32) containing
specific IgE against the recombinant moth arginine kinase inhibited IgE
binding to a 40-kDa allergen in cockroach, house dust mite, lobster,
king prawn, and mussel, but not in cod extracts (Fig. 7). IgE reactivity to the 40-kDa protein
was blocked completely after preincubation with moth arginine kinase in
the cockroach, house dust mite, and mussel extracts. In lobster and
king prawn strong IgE binding was observed to the 40-kDa component,
which was blocked only partially by recombinant moth arginine kinase.
No inhibition of IgE binding to cod fish allergens by the allergen was
observed (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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So far, moth allergens have not been characterized on the molecular level. We therefore constructed a moth cDNA library and used the serum from a moth-sensitized patient to identify IgE-reactive clones. A set of overlapping cDNA clones all coded for a 40-kDa Ag with high end-to-end similarity with arginine kinases from various invertebrate species. This is probably due to a very strong IgE reactivity of the patient to this Ag and, in addition, to a high level of expression of arginine kinase in the moth larvae. Further screening experiments using other patients’ sera led to the identification of several other moth allergens (B. Hayek, unpublished data).
Arginine kinases (EC 2.7.3.3) catalyze the reversible transfer of a high-energy phosphate from ATP to L-arginine yielding ADP and N-phospho L-arginine. In various invertebrate species, excess energy can thus be stored as arginine phosphate (31, 35), whereas vertebrate species use creatine phosphate to store energy and therefore possess creatine kinases. To our knowledge, arginine kinases have not been described as allergens; however, Lin et al. (36) obtained partial peptide sequences from a shrimp (Parapenaeus fissurus) allergen, which shared similarity with the arginine kinase sequence published soon afterward by Dumas and Camonis (31).
We produced and purified the recombinant arginine kinase and demonstrated its enzymatic activity. IgE immunoblot experiments showed that 13% of all indoor allergic patients and 25% of the moth-allergic patients had specific IgE to the recombinant allergen.
Previous studies by Baldo and Panzani (10) and by Komase et al. (11) had demonstrated strong IgE-binding to a 40-kDa component in immunoblots of various insects or silkworm moth, but this component was not identified. In the present study, the allergenic activity of the recombinant moth arginine kinase was demonstrated by its ability to induce the specific and dose-dependent release of histamine from basophils down to a concentration of 10 pg ml-1, and to give specific skin reactions. We therefore suggest a tentative designation of Plo i 1 for this first identified allergen from the Indianmeal moth.
Arginine kinases with highly similar amino acid sequences have been identified in various invertebrate species. The results from our IgE immunoblot inhibition experiments identify arginine kinase-related allergens of 40 kDa in mite (D. pteronyssinus), cockroach (B. germanica), king prawn (P. monodon), lobster (H. gammarus), and mussel (M. edulis). No homologous cross-reactive component was observed in the cod, a vertebrate species. These data suggest that arginine kinases represent a novel class of invertebrate cross-reactive pan-allergens which may be implicated in the induction and maintenance of respiratory and food allergy in polysensitized patients (10, 36).
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Dermatology, University of Erlangen-Nuremberg, Erlangen, Germany.
3 Current address: Division of Allergy, Immunology, and Infectious Diseases, Department of Dermatology, University of Vienna, Vienna, Austria.
4 Current address: Biologische Beratung bei Insektenproblemen (BIp), Berlin, Germany.
5 Address correspondence and reprint requests to Dr. Michael Duchêne, Division of Specific Prophylaxis and Tropical Medicine, Department of Pathophysiology, University of Vienna, AKH, Währinger Gürtel 18-20, A-1090 Vienna, Austria. E-mail address: michael.duchene{at}univie.ac.at
Received for publication April 9, 2001. Accepted for publication August 31, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ZAP: a bacteriophage expression vector with in vivo excision properties. Nucleic Acids Res. 16:7583.This article has been cited by other articles:
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  C.-J. Yu, Y.-F. Lin, B.-L. Chiang, and L.-P. Chow Proteomics and Immunological Analysis of a Novel Shrimp Allergen, Pen m 2 J. Immunol., January 1, 2003; 170(1): 445 - 453. [Abstract] [Full Text] [PDF]
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