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Cloning and expression of Aca f 1: a new allergen of Acacia farnesiana pollen
Gholam Reza Khosravi, Mohammad-Ali Assarehzadegan, Payam Morakabati, Bahareh Akbari, Fatemeh Dousti
(Cent Eur J Immunol 2016; (3): 273-281)
- Cloning.pdf [0.32 MB]
IntroductionAcacia farnesiana (Vachellia farnesiana), a member of the Fabaceae family, is a widespread tree throughout the tropical and subtropical regions of the United States, Asia, Africa, and Australia [1, 2]. Pollens from Acacia have been reported as a main source of pollinosis in subtropical countries such as Iran, Saudi Arabia, and the United Arab Emirates, where the frequency of sensitisation ranges from 25% to 48% [3-8].
Immunochemical analysis of the protein profile of Acacia pollen indicated several components ranging from 12 to 105 kDa. Moreover, the 15, 45, 50, 66, and 85 kDa proteins are recognised as dominant IgE-binding components of Acacia pollen [1, 9, 10]. A significant IgE cross-reactivity between the Acacia tree and other plants, particularly Prosopis juliflora (mesquite) and Lolium perenne (rye grass) pollen allergens, has also been described [1, 11, 12]. In a recent study, Aca f 2, the first allergen from A. farnesiana pollen, was identified as belonging to the family of profilins .
Despite a high prevalence of sensitisation to Acacia pollen in semiarid countries, there is little information about the molecular characterisation of A. farnesiana pollen allergens. Here we report Aca f 1, a new allergen from Acacia pollen, which is a member of the Ole e 1-like protein family. The first allergenic member of this family was isolated from Olea europaea pollen as the major allergen of olive pollen (Ole e 1) . In previous studies, similar allergens were also identified in other plants such as Fraxinus excelsior (Fra e 1) , Ligustrum vulgare (Lig v 1)  Syringa vulgaris (Syr v 1) , Salsola kali (Sal k 5) , and Chenopodium album (Che a 1) .
In this study, we expressed and purified Aca f 1 in Escherichia coli, and subsequently evaluated the immunoreactivity of this recombinant allergen. Moreover, the nucleotide sequence homology of Aca f 1 was investigated with the most common allergenic Ole e 1-like proteins in tropical regions.
Material and methodsPollen collection and crude extract preparation
Pollen was collected from the plants during pollination season (February-May) from different areas in and around Ahvaz city, a tropical region in southwest Iran. Collection and processing of pollen materials were performed with care by trained pollen collectors according to previous studies [19, 20]. Pollen materials were defatted using repeated changes of diethyl ether. Pollen was extracted as defined previously . In brief, two grams of pollen were mixed with 10 ml of phosphate-buffered saline (PBS) 0.01 M (pH 7.4) by continuous stirring for 18 hours at 4oC. The supernatant was separated by centrifugation at 14,000 × γ for 30 minutes, filtered, and the supernatant collected. The extract was then freeze-dried. The protein content of the extract was measured using the method of Bradford . Finally, the extract was freeze-dried and stored at –20°C for later use in the current study.
Study population and skin prick tests (SPTs)Twenty-five patients with SPT positive for Acacia pollen and respiratory allergy history participated in this study. Five additional subjects who showed negative SPT responses and no specific IgE to A. farnesiana pollen extract were included as negative controls. All patients and controls provided informed consent. SPTs were performed by an experienced nurse under a physician’s supervision, and patients with a positive SPT donated a serum sample. Afterwards, patients’ serum samples were immediately stored at 20°C before use.
Evaluation of total and specific IgETotal serum IgE levels were measured using a commercially available ELISA kit according to the manufacturer’s instructions (Euroimmun, Lübeck, Germany). For the detection of specific IgE against A. farnesiana pollen proteins in allergic patients, an indirect ELISA was developed. For this purpose, 3 µg/well of A. farnesiana pollen extract in 100 µl carbonate buffer (15 mM Na2CO3 and 35 mM NaHCO3, pH 9.6) was incubated at 4°C overnight in a 96-well ELISA microplate (Nunc A/S, Roskilde, Denmark). The plates were blocked with 150 µl of PBS-2% bovine serum albumin (BSA) solution (1 hour at 37°C) and then incubated with 100 µl of patients’ sera for three hours at room temperature with shaking. Each well was then incubated for two hours at room temperature with a 1 : 500 dilution of biotinylated goat anti-human IgE antibody (Nordic-MUbio, Susteren, Netherlands) in 1% BSA. Following five gentle washes with T-PBS (PBS containing 0.05% Tween 20) 100 µl of a 1 : 10,000 dilution of horseradish peroxidase-conjugated streptavidin (Bio-Rad laboratories, Hercules, CA, USA) (diluted in PBS – 1% BSA) and incubated for one hour at room temperature. After an additional washing step, 100 µl of a tetramethylbenzidine (TMB-H2O2; Sigma-Aldrich, St. Louis, MO) substrate solution was used as the HRP-substrate. Following a 20-minute incubation in the dark, the reaction was stopped by the addition of 100 µl of 3 M HCl. Then, the optical density (OD) in each well was determined at 450 nm using an ELISA reader. OD three times greater than the mean values of three determinations of pooled sera from negative controls (i.e. > 0.16 OD units) was considered to be positive.
Amplification of Aca f 1 gene and sequencingFollowing the isolation of total RNA and synthesis of cDNA, the coding sequence of Aca f 1 was cloned using a degenerate forward primer and reverse primer used for cDNA amplification, designed according to consensus nucleotide sequences for reported allergens from the Ole e 1-like protein family, which had a high degree of amino acid sequence identity [14, 17, 18, 23-25]. These include the forward and reversed primers 5-ACKATKTTYCCMAACCTCCA-3 and 5-TTAATTAGCTTTAACATCATAAAGATCC-3, respectively.
The amplified fragment was ligated into a pTZ57R/T TA cloning vector using the InsTAcloneTM PCR Cloning Kit (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Escherichia coli TOP10 cells (Invitrogen, Carlsbad, CA, USA) were transformed with the recombinant pTZ57R/T vector using the manufacturer’s protocol. Recombinant plasmid was subsequently purified from the gel using a Plasmid Extraction Kit (GeNet Bio, Chungnam, Korea) and sequenced using the dideoxy chain termination method at Bioneer Inc. (Daejeon, Korea). The DNA sequence of Aca f 1 was submitted to the NCBI GenBank database of (http://www.ncbi.nlm.nih.gov/) under accession number KR870435.
Production and purification of recombinant Aca f 1 (rAca f 1) allergenSpecific primers for the amplification of Aca f 1 cDNA were designed using the obtained sequence of Aca f 1. These contained overhangs with Not I and Xho I restriction sites for direct cloning into expression plasmid pET-21b(+) (Novagen, Gibbstown, NJ, USA) as follows: the sense primer (5-TCCGCGGCCGCACKATKTTYCCMAACCTCCA-3, Not I restriction site is bolded) and the antisense primer (5-CCCTCGAGTTAATTAGCTTTAACATCATAAAGAT-3, Xho I restriction site is bolded). After PCR amplification, the resulting product was digested with Not I and Xho I restriction enzymes according to the manufacturer’s protocol (Thermo Scientific, Waltham, MA, USA). After digestion of the PCR product and ligation into the digested pET-21b (+) plasmid with the same enzymes, correct constructs were transformed into competent E. coli BL21 (DE3) cells (Novagen, Gibbstown, NJ, USA). Subsequently, a clone of recombinant plasmid pET-21b(+)/Aca f 1 was inoculated into 2 ml of Lysogeny broth (LB) medium containing 100 µg/ml of ampicillin and incubated at 37°C. Expression of rAca f 1 was induced by isopropyl -D thiogalactopyranoside (IPTG) to a concentration of 0.5 mM. Subsequently, the cells were isolated by centrifugation (3500 × g, 20 minutes, 4°C), resuspended in lysis buffer (50 mM Tris–HCl pH 6.8, 15 mM imidazole, 100 mM NaCl, 10% glycerol, and 0.5% Triton X-100) and then disrupted by sonication. Purification of rAca f 1 was performed using Ni-NTA affinity chromatography (Invitrogen, Carlsbad, CA, USA) from the soluble phase of the lysate, following the manufacturer’s instructions.
Quantification of Specific IgE to rAca f 1 by ELISAAn indirect ELISA was developed for the determination of levels of specific IgE against rAca f 1 using Acacia allergic patients’ sera, as previously reported . The wells of the ELISA microplate were coated with 100 µl/well of purified recombinant rAca f 1 (3 µg/ml).
IgE-binding reactivity of rAca f 1 by immunoblotting assaySodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of Acacia pollen extract was performed using 12.5% acrylamide separation gels as described previously . The protein bands from the electrophoresis of Acacia pollen extract or purified rAca f 1 were electro-transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, UK), and after blocking and washing, membranes were incubated with a serum pool or individual sera from patients with Acacia allergy or with control sera (1 : 5 dilutions) for three hours. Biotinylated goat anti-human IgE (Nordic-Mubio, Susteren, Netherlands) (1 : 1000 v/v in PBS) was added to detect specific IgE, and the blotted membrane strips were then incubated with 1 : 12,000 v/v in TPBS-HRP-linked streptavidin (Sigma Aldrich, St. Louis, Mo, USA). Finally, strips were incubated with Super signal West Pico Chemiluminescent Substrate Kit (Thermo Scientific, Waltham, MA, USA) for five minutes, and proteins were then visualised by chemiluminescence using ChemiDoc XRS+ system (Bio-Rad).The molecular masses of protein bands were estimated using Image Lab Analysis Software (Bio-Rad) by comparison with protein markers of known molecular weights (Amersham Low molecular weight Calibration Kit for SDS electrophoresis, GE Healthcare, UK).
Evaluation of cross-reactivity using in vitro inhibition assaysELISA inhibition assay were performed as reported previously [19, 20]. In this method a pooled serum (1 : 2 v/v) from hyper-reactive Acacia allergic patients (No. 1, 3, 8, and 9) was pre-incubated for overnight at 4°C with either 1000, 100, 10, 1, 0.1, or 0.01 µg of rAca f 1 as inhibitors or with BSA as a negative control before use. The percentage inhibition was calculated using the following formula: (OD of sample without inhibitor-OD of sample with inhibitor/OD of sample without inhibitor) × 100.
To study cross-inhibition between natural and recombinant Aca f 1, a mixture of 100 µl of pooled serum (1 : 5 v/v) was incubated with natural Acacia pollen extract (15 µg/ml, as inhibitor), rAca f 1 (8 µg/ml, as inhibitor), or BSA (as negative control) overnight at 4°C with shaking. Preincubated sera were used to assess the reactivity of a PVDF membrane blotted with natural Acacia pollen extract and rAca f 1.
ResultsPatients and SPTs
Twenty-five patients – 11 males and 14 females (mean age: 28.56 ±5.93 years; age range 19-40 years) – were included in present study. All patients suffered from respiratory allergies and seasonal rhinitis during pollination season of Acacia. The patients were all positive by SPT with Acacia pollen extracts (Table 1). A serum pool of five nonallergic subjects who showed negative SPT responses and no specific IgE against Acacia pollen extract were considered as negative control.
Total and specific IgE levelsThe mean total IgE of patients’ sera was determined as 256.63 IU/ml. In Aca f 1 reactive patients, the mean of total IgE was 202.90 IU/ml (Table 1). Serum from 25 allergic patients were assessed for specific IgE binding to proteins from Acacia pollen extract. All of these patients had significantly elevated specific IgE levels to the extract of Acacia pollen (mean: OD450, 1.15 ±0.46; range: 0.70-2.3). The mean OD450 for specific IgE in rAca f 1 reactive patients was 1.36 ±0.45; range: 0.70-1.93) (Table 1).
Analysis of nucleotide and deduced amino acid sequences of Aca f 1The sequence analysis of Aca f 1 allergen showed an open reading frame of 453-bp coding 150 amino acid residues with a predicted molecular mass of 16.525 kDa and a calculated isoelectric point (pI) of 4.62. The obtained nucleotide sequence was submitted to the NCBI GenBank (Accession Number: KR870435).
To evaluate sequence homology between Aca f 1 and the known allergenic Ole e 1-like protein in the protein database, the deduced amino acid sequences of these allergenic proteins were compared (Fig. 1). A high degree of sequence identity (82%) was detected between Aca f 1 and Che a 1 and Cro s 1 (allergen from Crocus sativus pollen) (Table 2).
SDS-PAGE and IgE-binding components of Acacia pollen extractThe SDS-PAGE separation of the pollen extract showed several protein bands in the Acacia pollen extract with molecular weights ranging from approximately 10 to 85 kDa (Fig. 2). IgE-binding reactivity of the separated protein bands from the electrophoresis of the Acacia pollen extract was assessed by conducting immunoblotting experiments. The results indicated several IgE-reactive bands ranging from around 12 to 85 kDa.
Expression and purification of Aca f 1A pET-21b (+)/Aca f 1 clone was created and confirmed by digestion with NotI and XhoI restriction enzymes. This recombinant plasmid was expressed in E. coli strain BL21 (DE3) pLysS as a fusion protein with His6-tag in the C-terminus.
The rAca f 1 was present in a soluble form in the supernatant, where it was further purified by Ni2+ affinity chromatography to yield purified protein. The purified rAca f 1 was quantified using Bradford’s protein assay, which indicated that nearly 14 mg of recombinant protein had been purified from one litre of the bacterial expression medium. SDS-PAGE revealed that the apparent molecular weight of the fusion protein was about 17.5 kDa (Fig. 3). The allergenic Ole e 1-like protein from Acacia pollen, as a new allergen, was designated Aca f 1 by the WHO/IUIS Allergen Nomenclature Subcommittee.
Analysis of IgE-binding potential of rAca f 1The levels of specific IgE to the purified rAca f 1 were determined using 25 individual patients’ sera. Eleven patients (44%) had significant specific IgE levels to rAca f 1 (Table 1). Serum samples from patients allergic to Acacia pollen were subsequently tested for IgE reactivity to rAca f 1 in immunoblotting experiments. The results showed that the recombinant form of Aca f 1 was reactive with 11 individuals’ sera (Fig. 3). These results were consistent with those obtained from specific IgE ELISA (Table 1).
In vitro inhibition assaysELISA inhibition experiments were performed to investigate the IgE-binding potential of the purified rAca f 1 compared to its natural form in Acacia pollen crude extract. ELISA inhibition results revealed a dose-dependent inhibition of the IgE directed to rAca f 1 in patients’ sera positive to Acacia pollen extract. Pre-incubation of pooled serum with 1 mg/ml of rAca f 1 and Acacia pollen extract revealed significant inhibition (92% and 85%, respectively) of IgE binding to rAca f 1 in microplate wells (Fig. 4).
Immunoblot inhibition assays showed that pre-incubation of serum samples with rAca f 1 nearly completely inhibited the IgE binding to a protein band with an apparent molecular weight of 17 kDa (Fig. 5, line 3). Altogether, in vitro inhibition assays revealed a similar IgE reactivity for rAca f 1 and its natural counterpart in Acacia pollen extract. In addition, results indicated that pre-incubation of serum samples with native crude extract of Acacia pollen inhibited IgE binding to natural Aca f 1 in Acacia pollen extract and other reactive proteins (Fig. 5, line 2). However, pre-incubation of the pooled serum with BSA did not affect the IgE-reactivity to rAca f 1 (Fig. 5, line 1).
DiscussionIn recent years, the pollen of Acacia species such as A. farnesiana has been recognised as a cause of respiratory allergy in various parts of the Southern hemisphere [1, 4, 6, 7, 27-31]. Up to now, only one allergen (Aca f 2) of Acacia pollen has ever been identified as a member of the profilin family . In this study a cDNA encoding another major allergen from Acacia pollen has been cloned, expressed, and identified as a member of the Ole e 1-like protein family, and its importance has been analysed with a population of patients living Ahvaz city, Southwest Iran, a tropical region in which Acacia pollen induces nearly 48% of the total sensitisation of patients with allergic rhinitis . This allergenic member of the Ole e 1-like protein family of A. farnesiana was named Aca f 1 in accordance with the International Union of Immunological Societies (IUIS) Allergen Nomenclature Subcommittee (http://www.allergen.org/). The IgE-reactivity to some allergenic members of Ole e 1-like protein family has been demonstrated between 30% and 77% in allergic patients in different regions where it was considered as a major allergen in pollen of the allergenic plants such as O. europaea (Ole e 1), F. excelsior (Fra e 1), C. album (Che a 1), S. kali (Sal k 5), and Ligustrum vulgare (Lig v 1) [15, 17, 18, 23, 24].
The open reading frame contains 453 bases encoding a mature Aca f 1 polypeptide of 150 amino acids, with six cysteine residues. Encoded within this open reading frame is a 16.5 kDa protein that exhibits molecular characteristics similar to known members of the plant Ole e 1-like protein family (ranging from 16 to 19 kDa) [14, 17, 24]. Moreover, Aca f 1, like Che a 1, Cro s 1, and Sal k 5 has a conserved sequence for potential N-glycosylation in the same position of the polypeptide chain (Asn-Ile/Leu-Thr-Ala), which is actually occupied by a glycan in these proteins.
The results of immunoblotting assay on Acacia pollen extract using pooled sera from patients also revealed an IgE-binding protein band with an estimated MW of 17 kDa (Fig. 1).
The IgE-binding capacity of the purified rAca f 1 to sera from A. farnesiana allergic patients was evaluated using specific ELISA and immunoblotting assays in order to confirm that rAca f 1 was correctly folded and bound to IgE as the natural counterpart in Acacia extract. The immunoblot analysis with individual patients’ sera demonstrated diverse IgE reactivity with six proteins of 15, 17, 28, 39, 45, and 50 kDa as major IgE binding components. The results of immunoblotting assays for natural Aca f 1 with an apparent molecular weight of 17 kDa were consistent with those obtained from rAca f 1. A nearly complete inhibition of IgE-binding to natural Aca f 1 was also obtained after pre-incubation of pooled sera with purified rAca f 1. It seems that rAca f 1 is comprised of IgE-epitopes similar to those of its natural counterpart.
Cross-reactivity among Acacia pollen components with other allergenic plants has been described [1, 11, 12]. The importance of the Acacia and the most allergenic members of the Fabaceae (Prosopis juliflora) and the Amaranthaceae (Salsola kali, Chenopodium album) families’ pollens have been described as main causes of respiratory allergy [3-5, 7]. This study was performed to elucidate the amino acid sequence homology of Ole e 1-like proteins to prevalent allergenic regional plants. The results of amino acid sequence identity analyses revealed that Aca f 1 has a significant degree of identity with selected members of the allergenic Ole e 1-like protein family from the most common allergenic regional plants, particularly C. album (Che a 1), Crocus sativus (Cro s 1), and S. kali (Sal k 5) (82%, 82% and 77%, respectively). Identification of the Aca f 1 sequence will allow further studies on the basis of in vitro assays to evaluate the molecular basis of crossreactivity between these important allergenic pollens.
In conclusion, Aca f 1, with a detectably specific IgE in 44% of Acacia allergic patients and the second allergen from the A. farnesiana pollen was identified as a member of the Ole e 1-like protein family. Furthermore, the results indicate that E. coli is a relevant expression system for the production of rAca f 1 with immunoreactivity similar to that of the allergen’s natural form. Analysis of the amino acid sequences of Aca f 1 and several allergenic members of the Ole e 1-like protein family from other plants also showed a possibility of cross-reactivity among plants belonging to unrelated families, which may be predicted by the degree of amino acid sequence identity of potential conformational epitopes. However, this hypothesis needs to be confirmed in further studies where cross-reactivity among these allergens are evaluated using in vitro assays.
AcknowledgmentsThis article is issued from the thesis of Mr. Khosravi, and financial support was provided by Ahvaz Jundishapur University of Medical Sciences (Grant No. U-93180).
The authors declare no conflict of interest.
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