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Natural rubber latex allergy is an IgE-mediated disease that is caused by proteins that elute from commercial latex products. A complementary DNA (cDNA) coding for Hev b 9, an enolase (2-phospho- d-glycerate hydrolyase) and allergen from latex of the rubber tree Hevea brasiliensis, was amplified by PCR. The PCR primers were designed according to conserved regions of enolases from plants. The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino-acid residues with a calculated molecular mass of 47.6 kDa. Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as important allergens. In addition, the crucial amino-acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolases were also conserved. Hevea latex enolase was produced as a recombinant protein in Escherichia coli with an N-terminal hexahistidyl tag, and purified by affinity chromatography. The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture. The recombinant allergen bound IgE from latex, as well as mold-allergic patients, in immunoblot and ELISA experiments. The natural enolase was isolated from Hevea latex by (NH4)2SO4 precipitation and ion exchange chromatography. The natural and the recombinant (r)Hev b 9 showed equivalent enzymatic activity. Patients’ IgE-antibodies preincubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B-cell epitopes on both molecules. Cross-reactivity with two enolases from Cladosporium herbarum and Alternaria alternata was determined by inhibition of IgE-binding to these enolases by rHev b 9. Therefore, enolases may represent another class of highly conserved enzymes with allergenic potentials.
IgE-mediated type 1 allergy to natural rubber latex (NRL) is caused by proteins present in diverse latex products. Latex is the milky sap produced by the laticiferous cells of the tropical rubber tree Hevea brasiliensis of the family Euphorbiaceae. Sensitization occurs via the skin, mucosal or wound contact, or inhalation of airborne allergens released from powdered latex gloves . NRL-allergy has been acknowledged to impose serious health problems, especially among occupationally exposed individuals, e.g. health care workers and individuals who have to undergo repeated surgeries such as patients with spina bifida [2,3].
In NRL a variety of IgE-binding proteins with molecular masses ranging from 5 to 110 kDa were detected by two-dimensional immunoblotting [4–6]. Since 1993, a total of 10 NRL allergens have received an international nomenclature designation. With the exception of Hev b 4, full coding sequences are known for all other designated latex allergens [7–9] (S. Wagner & E. Ganglberger, unpublished data) . Some of these latex allergens have been identified in latex only, while others cross-react with homologous proteins present in fruits . These cross-reactivities have been termed the ‘latex–fruit syndrome’. Immunological cross-reactivity has been reported between allergens from latex and banana, avocado, chestnut, peanut, papaya, passion fruit, fig, melon, pineapple, kiwi, and tomato . In addition, cross-reactivities between allergens from NRL and Ficus benjamina, sweet pepper, grass, and weed pollen have also been described [12–14].
Prohevein from Hevea latex (Hev b 6.01) and its homologous proteins in vegetables and fruits have been implicated in cross-reactivity of latex with sweet pepper, chestnut, banana, peach, and kiwi . Prohevein belongs to the group of chitin-binding proteins with a hevein domain. This group also includes plant class I chitinases . Hevein (Hev b 6.02), the N-terminal 4.7-kDa domain of prohevein, shares epitopes with class I chitinases from avocado, chestnut, and banana, and represents one cause for their cross-reactivity [16–18]. Another protein that may play a role in the cross-reactivity of latex with pollen and fruits is Hev b 8, the latex profilin. Profilin was the first allergen to be named a ‘panallergen’ because of its high cross-reactivity . Latex profilin was detected in a nonammoniated latex extract  and a certain amount of cross-reactivity to pollen proteins was shown by inhibition studies . Hev b 2, a β-1,3-glucanase, was separated from nonammoniated latex and described as a latex-allergen. Hev b 2 was recognized by several atopic patients, indicating a possible cross-reactivity .
Based on data obtained by immunoprobing two-dimensional Hevea latex protein blots with sera from latex allergic patients and by protein microsequencing, several proteins from NRL have been described as putative allergens . Two IgE-reactive protein spots shared over 90% sequence identity with the N-terminus of Ricinus communis enolase (SWISS-PROT P42896). Enolase (2-phospho- d-glycerate hydrolyase) is a highly expressed key enzyme of glycolysis and gluconeogenesis that is needed for the conversion of glycerate 2-phosphate to phosphoenolpyruvate and vice versa. Enolase occurs as a homodimer in all eukaryotic and many prokaryotic cells. Owing to its essential role as a cytoplasmic household enzyme, enolase is a highly conserved enzyme. It has been identified and characterized from diverse sources ranging from bacteria to higher vertebrates. Enolases are described as important allergens in several molds. An enolase was first identified as an allergen in Saccharomyces cerevisiae, but IgE cross-reactivity exists between enolases of Cladosporium herbarum (Cla h 6), Alternaria alternata (Alt a 5), Candida albicans, Aspergillus fumigatus, and Fusarium solani[22–25].
Here, we report the nucleotide and deduced amino-acid sequence of the new latex allergen Hev b 9, which is an enolase. Further, we describe the cloning and expression of the recombinant molecule in E. coli and its biological activity. Characterization as an 48-kDa latex allergen was shown by IgE-immunoblot and IgE-inhibition experiments with the natural Hevea enolase isolated from latex C-serum. The results from IgE-immunoblots and ELISA-inhibition experiments performed with Hevea latex enolase and the mold allergens Alt a 5 and Cla h 6 indicate a new area of cross-reactivity of latex allergens.
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We report the cloning and sequencing of Hev b 9, a new and cross-reactive Hevea latex allergen. The Hev b 9 cDNA encodes a cytoplasmic latex protein of 445 amino-acid residues with a predicted molecular mass of 47.6 kDa and a calculated pI of 5.6. The protein displays 62% identity with Cla h 6, the enolase of the mold C. herbarum, and 60% identity with Alt a 5, the enolase of the mold A. alternata ( Fig. 1). We have produced Hev b 9 as a recombinant His-tagged protein in E. coli and demonstrated that the molecule possesses IgE reactivity equivalent to nHev b 9 ( Fig. 3). Furthermore, cross-reactivity with enolases from molds was tested using IgE-inhibition ELISA.
Enolase is a highly expressed key enzyme of glycolysis and gluconeogenesis and a highly conserved enzyme in all eukaryotic and most prokaryotic cells. Total latex RNA was used to amplify the cDNA coding for the Hevea enolase. The deduced amino-acid sequence of the enolase from H. brasiliensis shows an identity in the range of 60% to enolases from several molds. The important amino-acid residues responsible for the active site of enolases have been described for the enolase of S. cerevisiae. Interestingly, sequence alignments of enolases showed that these amino-acid residues are located in conserved regions , which are also present in the Hev b 9 sequence.
Enolases are described as allergens in several molds. Alt a 5, the enolase from A. alternata, is the second most important allergen of this mold, and recognized by 22% of all A. alternata allergic patients . The same has been reported from the enolase of C. herbarum, which is also the second most important allergen of this mold, with 22% of the patients reacting positively [25,33]. In addition, a 46- to 48-kDa enolase is also a main allergen and cross-reacting component of C. albicans, S. cerevisiae, and C. utilis[22,24,34]. Comparing several mold protein extracts, a 46-kDa band appeared as the only dominant band present in all the protein extracts . Therefore, enolases are not only important allergens but also predominant proteins in mold extracts.
Two-dimensional electrophoresis and N-terminal sequences lacking the N-terminal methionine provided the first evidence that enolase is an allergen of Hevea latex . We have produced Hev b 9 as a recombinant His-tagged protein in E. coli and were able to purify rHev b 9 from E. coli cells at a yield of 110 mg per litre culture medium.
The biological activity of rHev b 9 was tested with biochemical and immunological assays to confirm that the recombinant protein was equivalent to its natural counterpart and correctly folded. Therefore, in a first step the enzymatic activity of rHev b 9 was tested in an enolase assay. In this assay, the enzyme catalyzed the conversion of glycerate 2-phosphate to phosphoenolpyruvate and the extinction coefficient of the produced phosphoenolpyruvate was measured. Recombinant Hev b 9 showed an activity of 22 U·mg−1 enolase in this enzyme assay. Thus, the recombinant protein possessed an active site because of its correct folding. Furthermore, enolases are described to be enzymatically active only as dimers with identical subunits . Our rHev b 9 was enzymatically active, indicating its ability to form homodimers.
In latex C-serum natural Hev b 9 is a minor component. Detection of IgE binding to Hev b 9 in latex C-serum is difficult because a high percentage of IgE reactivity is directed to proteins in the 43–50 kDa range [5,6]. Therefore, we isolated natural Hev b 9 from C-serum. The protein showed enzymatic activity of 20 U·mg−1 protein, which is comparable with the activity found for the recombinant protein. Both natural and recombinant Hev b 9 bound IgE from a serum-pool of latex allergic individuals with comparable intensities in immunoblots. Furthermore, rHev b 9 was able to abolish IgE binding to nHev b 9 when the serum-pool was preincubated with the recombinant protein ( Fig. 3). Consequently, the highly purified and standardized recombinant protein is a superior tool compared to the natural protein.
We tested the IgE binding capacity of the recombinant Hev b 9 by IgE immunoblots using 110 individual serum samples from latex-allergic patients. Sixteen patients (14.5%) revealed IgE-binding to the recombinant Hev b 9. Four out of these 16 patients were also allergic to molds. Three out of 12 mold allergic patients showed IgE-binding to rHev b 9. All rHev b 9 positive patients were also tested for their IgE-binding to rAlt a 5 and rCla h 6. Except one patient ( Fig. 4, lane 8), all others showed IgE reactivity to the mold enolases ( Fig. 4), but IgE binding intensities to the three recombinant enolases were different when tested in IgE immunoblots. The cross-inhibition ELISA studies confirmed these results, that rHev b 9 was only able to diminish IgE binding to rAlt a 5 and rCla h 6 in the range of 19 and 24%, respectively. From these data we conclude that the three enolases share a number of epitopes. It has been described that enolases from molds contain several common IgE binding epitopes, but inhibition occurs only partially . This implies that the mold enolases also have unique epitopes. Therefore, it becomes evident that sensitization to Hev b 9 may occur from Hevea latex and does not represent an epiphenomenon of sensitization to the homologous mold allergens.
Some Hevea latex allergens seem to have a high potential for cross-reactivity, including well-conserved proteins such as manganese superoxide dismutases, chitinases, profilins, and enolases. Sequence comparisons, three-dimensional structure determinations and cross-inhibition assays could help to predict immunogenic regions of the individual proteins. These cross-reactivities often occur with proteins from several fruits and were described as the ‘latex–fruit syndrome’. The latex allergens involved in this syndrome are often defence-related enzymes such as Hev b 2 and class I chitinases . In contrast, enolases are house-keeping enzymes that have been described as important allergens of several molds. Another important cross-reactive allergen from molds is a manganese superoxide dismutase [35,36]. MnSOD from Hevea latex is also a described allergen and has been designated Hev b 10 (IUIS Allergen Nomenclature Subcommittee: Official list of allergens available at ftp://biobase.dk/pub/who-iuis/allergen.list). Although Hev b 9 is a minor latex allergen, its cross-reactivities and the possible cross-reactivities of Hev b 10 to mold allergens indicate the possible existence of a ‘latex–mold-syndrome’ that will have to be confirmed in the future by the use of the relevant recombinant allergens.