A recombinant functional variant of the olive pollen allergen Ole e 10 expressed in baculovirus system

Pollens have been reported as important sources of antigens causing type-I allergy and, among them, olive pollen has high clinical relevance in Mediterranean countries. The most recently described olive allergen, Ole e 10, is involved in cross-reactivity phenomena and related to asthma induction in allergic patients. These immunologic features make this allergen a good candidate to be included in diagnosis and therapy of protocols of allergic diseases. Since the availability of Ole e 10 from the olive pollen is limited, the allergen has been efficiently expressed in the baculovirus/insect cell system. The Ole e 10-cDNA inserted into the transfer vector pBacPAK8 allowed the expression of the recombinant protein in cultured Sf21 cells. Recombinant Ole e 10 (rOle e 10) was purified from the culture after dialysis and three chromatographic steps. Mass spectrometry, Edman degradation, IgE- and IgG-binding analyses were employed to characterize the recombinant allergen, which showed molecular and immunological equivalence with the natural protein. Affinity gel electrophoresis in presence of laminarin (1,3-beta-glucan) revealed that rOle e 10 retains identical carbohydrate-binding capacity than the natural allergen. In conclusion, the recombinant expression of Ole e 10 in baculovirus/insect cell system produces a homogeneous and biologically active allergen that could be useful for clinical and scientific purposes.     

Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR

Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen allergen, is a small (5.8 kDa) and acidic protein (pI 4.2) and no homologous proteins have been isolated or characterized so far. Ole e 6 has been efficiently expressed in the methylotrophic yeast Pichia pastoris. The cDNA encoding Ole e 6 was inserted into the plasmid vector pPIC9 and overexpressed in GS115 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reverse-phase HPLC. N-terminal sequencing, amino acid composition analysis, CD, NMR, and IgG-binding experiments were employed to characterize the purified protein. NMR data revealed the oxidation of the methionine at position 28 in approximately 50% of the recombinant protein but, although this alters its electrophoretic behavior, it did not affect folding or IgG-binding properties of rOle e 6. The recombinant form of Ole e 6 expressed in P. pastoris can be employed for structural and biochemical studies.     

Production and detailed characterization of biologically active olive pollen allergen Ole e 1 secreted by the yeast Pichia pastoris.

The glycoprotein Ole e 1 is a significant aeroallergen from the olive tree (Olea europaea) pollen, with great clinical relevance in the Mediterranean area. To produce a biologically active form of recombinant Ole e 1, heterologous expression in the methylotrophic yeast Pichia pastoris was carried out. A cDNA encoding Ole e 1, fused to a Saccharomyces cerevisiae alpha-mating factor prepropeptide using the pPIC9 vector, was inserted into the yeast genome under the control of the AOX1 promoter. After induction with methanol, the protein secreted into the extracellular medium was purified by ion-exchange and size-exclusion chromatography. The structure of the isolated recombinant Ole e 1 was determined by chemical and spectroscopic techniques, and its immunological properties analysed by blotting and ELISA inhibition with Ole e 1-specific monoclonal antibodies and IgE from sera of allergic patients. The allergen was produced at a yield of 60 mg per litre of culture as a homogeneous glycosylated protein of around 18.5 kDa. Recombinant Ole e 1 appears to be properly folded, as it displays spectroscopic properties (CD and fluorescence) and immunological reactivities (IgG binding to monoclonal antibodies sensitive to denaturation and IgE from sera of allergic patients) indistinguishable from those of the natural protein. This approach gives high-yield production of homogeneous and biologically active allergen, which should be useful for scientific and clinical purposes.     

A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies [correction of interspecie] cross-reactivity

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.     

Isolation of three allergenic fractions of the major allergen from Olea europea pollen and N-terminal amino acid sequence

A method to isolate the major allergen from olive pollen (Ole e I) in high yield is described. The allergenic fraction has been separated into 3 subfractions by reverse-phase HPLC. All these fractions were reactive to allergic sera from olive-sensitized patients, giving similar responses. No significant differences were observed between the amino acid compositions of these three proteins. The amino acid sequence of the first 27 amino acid residues from the N-terminal end is given. No homologies have been detected between Ole e I and other known allergens obtained from pollen.     

Molecular cloning and characterization of Cup a 4, a new allergen from Cupressus arizonica

Sensitization to Cupressaceae pollen has become one of the most important causes of pollinosis in Western countries during winter and early spring. However, the characterization of the extracts, the allergens involved and the cross-reactivity with other pollen sources still remain poorly studied; in the case of Cupressus arizonica only two allergens have been described so far. A new allergen from C. arizonica pollen, Cup a 4, was cloned and expressed in Escherichia coli as an N-terminally His-tag recombinant protein that was characterized biochemically, immunologically and by circular dichroism spectroscopy. The new allergen has high sequence identity with Prickly Juniper allergen Jun o 4 and contains four EF-hand domains. The recombinant protein has structural similarities with other calcium binding allergens such as Ole e 3, Ole e 8 and Phl p 7. Cup a 4 is expressed in mature pollen grains and shares antigenic properties with the recombinant form. Sera from 9.6% C. arizonica allergic patients contain specific IgE antibodies against recombinant Cup a 4.     

Vegetable proteomics: the detection of Ole e 1 isoallergens by peptide matching of MALDI MS/MS spectra of underivatized and dansylated glycopeptides

Ole e 1 (NCBI entry gi|14424429) is the major allergen of Oleaceae family. Multiple isoforms and variants are present in varying degrees of distribution. In this report, we present a new approach to the resolution of multiple forms of Ole e 1 from whole antigen extracts, based on a preliminary chemical fractionation procedure followed by MALDI MS and MS/MS measurements. The characterization of Ole e 1 isoallergens was accomplished through the identification of the amino acid sequence including the glycosylation site and the structure of the glycan moieties. The structure feature of the identified Ole e 1.0102 (gi|2465127), main olive allergen [Olea europaea] (gi|13195753), Major pollen allergen Ole e 1 (gi|33329740) and Ole e 1c (gi|1362131) is represented by the point mutation K(106) --> I and by the presence of a glycan moiety. Two other variants Major pollen allergen (Allergen Ole e1) (Ole e I) (gi|14424429) and Ole e 1.0103 protein [Olea europea] (gi|2465129) were identified as nonglycosylated species. These results, partially in disagreement with Swiss-Prot annotation, were validated by matching the MALDI MS/MS spectra of the natural tryptic mixture with those obtained after deglycosylation.     

Analysis of the pollen allergen content of twelve olive cultivars grown in Portugal

Olive pollen presents intercultivar variability as regards to its antigenic and allergenic composition. In this study, we report the presence of differences among the SDS-PAGE pollen protein profiles of twelve Portuguese olive cultivars. Though most soluble proteins from these extracts seemed similar, three bands of about 18, 20 and 22 kDa presented sharp differences in intensity among the cultivars analyzed. The dissimilarity of patient’s sera reactivity to these protein extracts and the presence of several allergens already characterized (Ole e 1, Ole e 2, Ole e 5 and Ole e 9) in the extracts were also investigated. Epidemiological data indicated that 53.3 % out of the 428 patients analyzed with reactivity to pollen extracts, presented specific IgE levels to Olea europaea. A representative number of these sera were assayed in immunoblotting experiments. The cultivars ‘Galega’ and ‘Conserva de Elvas’ displayed low reactivity to the sera of atopic patients, whereas the extracts corresponding to the cultivars ‘Cobrançosa’, ‘Ascolana’ and ‘Verdeal de Serpa’ led to higher IgE reactivity. The use of antibodies to the allergens Ole e 1, Ole e 2, Ole e 5 and Ole e 9 in immunoblotting experiments also allowed cultivar discrimination. The cultivar ‘Verdeal de Serpa’ presented the highest Ole e 1, Ole e 5 and Ole e 9 allergen loads but the lowest Ole e 2. ‘Carrasquenha’ was the second cultivar in terms of the higher allergen content. Oppositely, the lowest allergen loads were those of the cultivars ‘Galega’ and ‘Conserva de Elvas’ coincidentally with their low IgE reactivity. These data may help interpret physiological differences in pollen performance for successful olive fertilization and, moreover, to better define future strategies for allergy diagnosis and treatment by specific immunotherapy.     

Pollen Ole e 1 content variations in olive cultivars of different Portugal regions

Olive is recognized as a crop with great impact in agricultural, socioeconomic, environmental and public health sectors. The last is becoming more important during recent years as consequence of the increase of the pollen allergy in south Europe prompted by the widespread Olea pollen allergic reactions. The aim of the study was to quantify, for the first time, the variations of the Ole e 1 allergen amount in Olea pollen grains from four cultivars in three regions of Portugal. How weather parameters can affect the allergen production was also assessed. The study was conducted in three olive producer areas of Portugal from 2010 to 2013, Santarém (Central), Elvas (Southeast) and Mirandela (Trás-os-Montes region, Northeast). Mature pollen of four different cultivars (Cobrançosa, Arbequina, Picual and Verdeal) was collected during the olive flowering season. Ole e 1 was quantified using specific 2-site antibody ELISA. Pollen of the olive groves at the boundary Olea bioclimatic distribution in the Mirandela registered the higher allergen content for all varieties in each study year. Arbequina was the variety that showed the lower Ole e 1 allergen concentration, whereas the higher content was registered for Cobrançosa. The main meteorological parameters that influenced the allergen Ole e 1 concentration in the pollen grains were the rainfall and temperatures related variables. The knowledge of the allergenicity in different olive cultivars is an important tool in the selection of the most adequate for planting as ornamental crop and to adjust the pollen extracts used for diagnosis or even immunotherapy.

     

Recombinant expression, purification and cross-reactivity of chenopod profilin: rChe a 2 as a good marker for profilin sensitization

Chenopod pollen is one of the major sources of allergens in some locations in the US, southern Europe and desert countries, and pollen profilin (Che a 2) is a major allergen. Recombinant Che a 2 (rChe a 2) has been produced in Escherichia coil cells with a final yield of 25 mg/l of cell culture. The expressed protein was isolated and structurally characterized by means of mass spectrometry, Edman degradation and circular dichroism. rChe a 2 displayed a molecular mass of 13 959 Da, which agrees with that of the amino acid sequence. The N-terminal amino acid sequence indicated the correct processing of the recombinant product. The immunological analysis of rChe a 2 showed IgG- and IgE-binding capabilities equivalent to those of its natural counterpart, Che a 2, isolated from the pollen. Inhibition experiments showed high cross-reactivity degrees with different allergenic sources. Inhibition degrees of >95% and >80% were obtained for chenopod profilin and, respectively, latex and pollen extracts, whereas 10-95% of inhibition was observed for different plant-derived foods. Due to its close relation to other allergenic profilins from pollens, plant-derived foods and latex, rChe a 2 could be a useful tool in clinical trials to detect profilin-allergic patients and perhaps, depending on its clinical relevance, in specific immunotherapy of these hypersensitive individuals.     

Purification strategy for recombinant Phl p 6 is applicable to the natural allergen and yields biochemically and immunologically comparable preparations

The recombinant major grass pollen allergen Phl p 6 has been expressed with a N-terminal 6 x His-tag sequence and subsequently purified using nickel-chelating Sepharose. After cleavage of the tag-sequence, a second pass over the affinity chromatography revealed that even untagged rPhl p 6 bound tightly. In order to determine if that property is typical for Phl p 6, the natural allergen was purified in the same way starting with a grass pollen extract. Indeed, nPhl p 6 could be highly enriched in one step using nickel-chelating Sepharose. In addition to this new powerful purification method, the results provide further information in that the recombinant and natural allergens share a lot of properties, since biochemical characteristics are reflected in the purification strategies. The preparations of natural and recombinant Phl p 6 were used for comparative electrophoretic, chromatographic and immunological analysis which demonstrated high similarity.     

Expression and purification of the mitochondrial serine protease LACTB as an N-terminal GST fusion protein in Escherichia coli

LACTB is a mammalian mitochondrial protein sharing sequence similarity to the beta-lactamase/penicillin-binding protein family of serine proteases that are involved in bacterial cell wall metabolism. The physiological role of LACTB is unclear. In this study we have subcloned the cDNA of mouse LACTB (mLACTB) and produced recombinant mLACTB protein in Escherichia coli. When mLACTB was expressed as an N-terminal GST fusion protein (GST-mLACTB), full-length GST-mLACTB protein was recovered by glutathione-agarose affinity chromatography as determined by MALDI-TOF mass spectrometry and immunoblotting. Expression of mLACTB as a C-terminal GST fusion protein or with either an N- or C-terminal His6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length mLACTB. Analysis of GST-mLACTB by Fourier transform infrared spectrometry revealed the presence of alpha-helices, beta-sheets and turns, consistent with a well-defined secondary structure. These results show that mLACTB can be expressed as a GST fusion protein in E. coli and suggest that GST-mLACTB was properly folded.     

Identification of grass pollen allergens by two-dimensional gel electrophoresis and serological screening

Approximately 50% of allergic patients are sensitized against grass pollen allergens. The characterization of specific immunoglobulin E (IgE) reactivity to allergen components in pollen-allergic patients is fundamental for clinical diagnosis and for immunotherapy. Complex allergen extracts are commonly used in diagnostic tests as well as in immunotherapy preparations, but their composition in single allergenic molecules is only partially known. Diagnostic tests which utilize recombinant or immuno-purified allergens have been made available in clinical practice. They allow to obtain specific profiles of IgE reactivity, but the panel of available molecules is far from complete. Here, we used a proteomic approach in order to detect grass allergens from a natural protein extract. A five-grass pollen extract used for diagnosis and immunotherapy was resolved by two dimensional gel electrophoresis (2-DE), and assayed with 9 sera from pollen-allergic patients whose sensitization profile was dissected by using IgE reactivity to recombinant allergens. 2-DE immunoreactivity patterns were matched with IgE reactivity to identify protein spots as candidate allergens. Identity was confirmed by mass spectrometry analysis. We identified 6 out of 8 expected clinically relevant allergens in the natural grass extract. Moreover, we identified different molecular isoforms of single allergens, thus obtaining a more detailed profile of IgE reactivity. Some discrepancies in protein isoform profile and sera immunoreactivity between recombinant and native allergen 5 from Phleum pratense were observed and a new putative allergen was described. The proteomic approach applied to the analysis of a natural allergen allows the comprehensive evaluation of the sensitization profile of allergic patients and the identification of new allergens.     

Identification of the N-terminal residue of the heavy chain of both native and recombinant human hepatocyte growth factor.

The N-terminal amino acid of the heavy chain of native (purified from human plasma) and recombinant human hepatocyte growth factor (hHGF) was determined by analyses of amino acid composition and sequence of peptide fragments derived by enzymatic cleavage, peptide mapping, and fast atom bombardment mass spectrometry. Our results indicate that the N-terminal amino acid of the heavy chain of hHGF, both native and recombinant, is pyroglutamate, derived from glutamine at the 32nd residue from the initiation methionine.     

Solution structure of the C-terminal domain of Ole e 9, a major allergen of olive pollen

Ole e 9 is an olive pollen allergen belonging to group 2 of pathogenesis-related proteins. The protein is composed of two immunological independent domains: an N-terminal domain (NtD) with 1,3-beta-glucanase activity, and a C-terminal domain (CtD) that binds 1,3-beta-glucans. We have determined the three-dimensional structure of CtD-Ole e 9 (101 amino acids), which consists of two parallel alpha-helices forming an angle of approximately 55 degrees , a small antiparallel beta-sheet with two short strands, and a 3-10 helix turn, all connected by long coil segments, resembling a novel type of folding among allergens. Two regions surrounded by aromatic residues (F49, Y60, F96, Y91 and Y31, H68, Y65, F78) have been localized on the protein surface, and a role for sugar binding is suggested. The epitope mapping of CtD-Ole e 9 shows that B-cell epitopes are mainly located on loops, although some of them are contained in secondary structural elements. Interestingly, the IgG and IgE epitopes are contiguous or overlapped, rather than coincident. The three-dimensional structure of CtD-Ole e 9 might help to understand the underlying mechanism of its biochemical function and to determine possible structure-allergenicity relationships.     

Purification and structural analysis of the novel glycoprotein allergen Cyn d 24, a pathogenesis-related protein PR-1, from Bermuda grass pollen

Bermuda grass pollen (BGP) contains a very complex mixture of allergens, but only a few have been characterized. One of the allergens, with an apparent molecular mass of 21 kDa, has been shown to bind serum IgE from 29% of patients with BGP allergy. A combination of chromatographic techniques (ion exchange and reverse phase HPLC) was used to purify the 21 kDa allergen. Immunoblotting was performed to investigate its IgE binding and lectin-binding activities, and the Lysyl-C endopeptidase digested peptides were determined by N-terminal sequencing. The cDNA sequence was analyzed by RACE PCR-based cloning. The protein mass and the putative glycan structure were further elucidated using MALDI-TOF mass spectrometry. The purified 21 kDa allergen was designated Cyn d 24 according to the protocol of International Union of Immunological Societies (IUIS). It has a molecular mass of 18,411 Da by MALDI-TOF analysis and a pI of 5.9. The cDNA encoding Cyn d 24 was predicted to produce a 153 amino acid mature protein containing tow conserved sequences seen in the pathogen-related protein family. Carbohydrate analysis showed that the most abundant N-linked glycan is a alpha(3)-fucosylated pauci-mannose (Man3GlcNAc2) structure, without a Xyl beta-(1,2)-linked to the branching beta-Man. Thus, Cyn d 24 is a glycoprotein and the results of the sequence alignment indicate that this novel allergen is a pathogenesis-related protein 1. To the best of our knowledge, this is the first study to identify any grass pollen allergen as a pathogenesis-related protein 1.     

Exosomes from bronchoalveolar fluid of tolerized mice prevent allergic reaction

Exosomes are nanovesicles originating from multivesicular bodies that are secreted by a variety of cell types. The dual capability of exosomes to promote immunity or to induce tolerance has prompted their clinical use as vehicles for vaccination against different human diseases. In the present study, the effect of allergen-specific exosomes from tolerized mice on the development of allergen-induced allergic response was determined using a mouse model. Mice were tolerized by respiratory exposure to the olive pollen allergen Ole e 1. Exosome-like vesicles were isolated from bronchoalveolar lavage fluid of the animals by the well-established filtration and ultracentrifugation procedure, characterized by electron microscopy, Western blot, and FACS analyses, and assessed in a prophylactic protocol. To this end, BALB/c mice were intranasally treated with tolerogenic exosomes or naive exosomes as control, 1 wk before sensitization/challenge to Ole e 1. Blood, lungs, and spleen were collected and analyzed for immune responses. Intranasal administration of tolerogenic exosomes inhibited the development of IgE response, Th2 cytokine production, and airway inflammation--cardinal features of allergy--and maintained specific long-term protection in vivo. This protective effect was associated with a concomitant increase in the expression of the regulatory cytokine TGF-beta. These observations demonstrate that exosomes can induce tolerance and protection against allergic sensitization in mice. Thus, exosome-based vaccines could represent an alternative to conventional therapy for allergic diseases in humans.     

NMR solution structure of Ole e 6, a major allergen from olive tree pollen

Ole e 6 is a pollen protein from the olive tree (Olea europaea) that exhibits allergenic activity with a high prevalence among olive-allergic individuals. The three-dimensional structure of Ole e 6 has been determined in solution by NMR methods. This is the first experimentally determined structure of an olive tree pollen allergen. The structure of this 50-residue protein is based on 486 upper limit distance constraints derived from nuclear Overhauser effects and 24 torsion angle restraints. The global fold of Ole e 6 consists of two nearly antiparallel alpha-helices, spanning residues 3-19 and 23-33, that are connected by a short loop and followed by a long, unstructured C-terminal tail. Viewed edge-on, the structured N terminus has a dumbbell-like shape with the two helices on the outside and with the hydrophobic core, mainly composed of 3 aromatic and 6 cysteine residues, on the inside. All the aromatic rings lie on top of and pack against the three disulfide bonds. The lack of thermal unfolding, even at 85 degrees C, indicates a high conformational stability. Based on the analysis of the molecular surface, we propose five plausible epitopes for IgE recognition. The results presented here provide the structural foundation for future experiments to verify the antigenicity of the proposed epitopes, as well as to design novel hypoallergenic forms of the protein suitable for diagnosis and treatment of type-I allergies. In addition, three-dimensional structure features of Ole e 6 are discussed to provide a basis for future functional studies.