Biomedical subjects

Oscar Palomares

Publications and source records attributed to Oscar Palomares.

13 recordsLinked to original sources

PubMed2006

Emerging pollen allergens.

Numerous pollen allergens have been reported over the last few years. Most of them belong to well-known families of proteins but some others constitute the first member of new allergenic families. Some of the factors that can contribute to the detection and identification of new pollen allergens are: a) advances in the technology tools for molecular analysis; and b) the deep knowledge of many allergenic sources. The combination of these factors has provided vast information on the olive pollen allergogram and the identification of minor allergens that become major ones for a significant population. The close taxonomical relationship between olive tree and ash -both Oleaceae- has permitted to identify Fra e 1 (the Ole e 1-like allergen) in ash pollen and to detect the presence of protein homologues of Ole e 3 and Ole e 6. In the other hand, extensive areas of south Europe are suffering an increasing desertification. As a consequence of this, new botanical species are spontaneously growing in these areas or being used in greening ground programs: Chenopodium album and Salsola kali are some examples recently recognized as allergenic woods. The identification of the complete panel of allergens from the hypersensitizing sources might help to develop more accurate diagnosis, and efficient and safer therapy tools for Type-I allergic diseases.

Allergens↗

PubMed2006

NMR assignment of the C-terminal domain of Ole e 9, a major allergen from the olive tree pollen.

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Allergens↗

PubMed2006

The major allergen of olive pollen Ole e 1 is a diagnostic marker for sensitization to Oleaceae.

BACKGROUND: Trees of the family Oleaceae are important allergen sources, with a strongly varying geographic distribution. For example, olive pollen is an important allergen source in Mediterranean countries, whereas ash pollen dominates in Northern and Central Europe and North America. The aim of this study was to compare the profiles of olive and ash pollen allergens and to study the degree of cross-reactivity using populations of allergic patients selectively exposed to olive or ash pollen. METHODS: Olive and ash pollen extracts were analyzed by IgE immunoblotting using sera from Spanish patients highly exposed to olive pollen and Austrian patients without olive but ash pollen exposure. IgE cross-reactivity was studied by qualitative immunoblot inhibition assays and semiquantitative ELISA inhibitions using olive, ash, birch, mugwort, timothy grass pollen extracts and the major olive pollen allergen, Ole e 1. RESULTS: Spanish and Austrian patients exhibited an almost identical IgE-binding profile to olive and ash pollen allergens, with major reactivity directed against Ole e 1, and its homologous ash counterpart, Fra e 1. IgE inhibition experiments demonstrated extensive cross-reactivity between olive and ash pollen allergens. However, whereas cross-reactions between profilins and calcium-binding allergens also occurred between unrelated plant species, cross-reactivity to Ole e 1 was confined to plants belonging to the Oleaceae. CONCLUSIONS: Ole e 1 is a marker allergen for the diagnosis of olive and ash pollen allergy.

Allergens↗

PubMed2006

Variability of Ole e 9 allergen in olive pollen extracts: relevance of minor allergens in immunotherapy treatments.

BACKGROUND: Clustered severe adverse reactions to immunotherapy with olive pollen extracts have been occasionally reported in areas where olive trees are extensively grown. Allergic patients from these areas, in addition to the major olive pollen allergen Ole e 1, frequently recognize a recently described allergen, Ole e 9. OBJECTIVE: We aimed to develop an immunoassay to measure Ole e 9 concentration and to study the variability of this allergen in olive pollen extracts. METHODS: Monoclonal antibodies (mAb) to Ole e 9 were produced from mice immunized with the pure allergen. One of these mAbs was used to develop a sandwich ELISA with an anti-olive pollen extract rabbit serum as the tracer. Olive pollen batches from several suppliers were analyzed using this method. These batches were also analyzed for Ole e 1 content and biological activity. RESULTS: A 10-fold variation between the extreme values was found for the biological activity of the batches analyzed. Ole e 1 concentration showed a 25-fold variation. Variability of Ole e 9 concentration was extremely high, up to 161 times. The ratio Ole e 1/Ole e 9 varied in a range from 0.6 to 390.4. CONCLUSION: The availability of a mAb-based ELISA for Ole e 9 made it possible for us to detect an important source of variability in olive pollen batches. This variability may be the cause of outbreaks of adverse reactions in the course of immunotherapy treatments, which have sometimes been observed among olive-allergic patients living in areas with very high levels of airborne olive pollen.

Allergens↗

PubMed2006

Allergenic contribution of the IgE-reactive domains of the 1,3-beta-glucanase Ole e 9: diagnostic value in olive pollen allergy.

BACKGROUND: Designing of methods for an accurate diagnosis is a main goal of allergy research. Olive pollen allergy is currently diagnosed using commercially available pollen extracts that do not allow identification of the molecules that elicit the disease. OBJECTIVE: To analyze the suitability of using the N- and C-terminal domains (NtD and CtD, respectively) of the 1,3-beta-glucanase Ole e 9, a major allergen from olive pollen, for in vitro diagnosis. METHODS: Serum samples from 55 olive-allergic patients were assayed using enzyme-linked immunosorbent assay to study hypersensitive patients with IgE reactivity to Ole e 9. The specific IgEs to NtD and CtD, obtained by recombinant technology, were determined by means of immunoblotting, enzyme-linked immunosorbent assay, and inhibition assays. RESULTS: Thirty-one of 33 serum samples from Ole e 9-allergic patients were IgE reactive to recombinant NtD (rNtD) (n = 26 [79%]), recombinant CtD (rCtD) (n = 22 [67%]), or both (n = 17 [52%]). Nine patients (27%) were exclusively reactive to rNtD and 5 (15%) to rCtD. Inhibition assays of IgE binding to Ole e 9 with a mixture of both domains abolished 90% of the binding, whereas 44% and 45% were abolished when rNtD and rCtD were used, respectively. CONCLUSIONS: Because sensitization to NtD or CtD of Ole e 9 could be correlated to vegetable food-latex-pollen cross-reactivity processes or to the exacerbation and persistence of asthma, respectively, these molecules could be used in vitro as markers of disease to classify patients and to design a patient-tailored immunotherapy approach.

Adolescent↗

PubMed2006

Hypersensitivity to black locust (Robinia pseudoacacia) pollen: "allergy mirages".

BACKGROUND: The allergenicity of the ornamental tree Robinia pseudoacacia, or black locust, is unknown. OBJECTIVE: To evaluate the prevalence of sensitization to R. pseudoacacia pollen, its possible allergenic cross-reactivity with other common pollens, and the potential implication of pollen panallergens (profilin, polcalcin, and 1,3-beta-glucanase) as a cause of sensitization to R. pseudoacacia pollen. METHODS: Skin prick testing with R. pseudoacacia pollen was performed in 149 patients with pollinosis. Nasal challenge with R. pseudoacacia pollen was performed in 10 patients. The prevalence of sensitization to the recombinant forms of profilin (rChe a 2), polcalcin (rChe a 3), and the N-terminal of the 1,3-beta-glucanase (rNtD of Ole e 9) was investigated. Immunoblotting, enzyme-linked immunosorbent assay, and competitive inhibition assays were performed with R. pseudoacacia pollen and recombinant pollen allergens. RESULTS: Sixty-four patients (43%) had positive skin prick test reactions to R. pseudoacacia pollen. Nasal challenge results were positive in 5 sensitized patients and negative in 4 controls and 1 sensitized patient. The allergenic profile of R. pseudoacacia pollen comprises at least the panallergen profilin, a calcium-binding protein, and a 1,3-beta-glucanase. The prevalence of sensitization to rChe a 2, rChe a 3, and rNtD of Ole e 9 was 60%, 33%, and 87%, respectively, among patients sensitized to R. pseudoacacia pollen. Binding of IgE to R. pseudoacacia extract was completely inhibited by Robinia, Chenopodium, Olea, Cupressus, and Lolium extracts. CONCLUSIONS: The high prevalence of R. pseudoacacia pollen sensitization in patients with pollinosis is likely to be due to cross-sensitization to panallergens (profilin, polcalcin, and 1,3-beta-glucanase) from other common pollens. This phenomenon may lead to a diagnosis of "allergy mirages."

Allergens↗

PubMed2005

A recombinant precursor of the mustard allergen Sin a 1 retains the biochemical and immunological features of the heterodimeric native protein.

BACKGROUND: Mustard has been an important cause of food allergy of increasing incidence in the last years. Sin a 1, a storage 2S albumin, is the most relevant allergen from this spice. METHODS: Pichia pastoris has been used as host for the recombinant production of the precursor form of Sin a 1 (rproSin a 1). rproSin a 1 was purified to homogeneity by three chromatographic steps: gel filtration, anion exchange and reverse-phase HPLC. Molecular characterization was performed using Edman degradation, mass spectrometry, amino acid composition, and circular dichroism. Immunological properties were analyzed by immunoblotting, ELISA, and ELISA inhibition experiments. RESULTS: We overexpressed rproSin a 1 as a single polypeptide with both large and small chains linked by an internal processed fragment at high yield. The purified rproSin a 1 (>95%) was obtained as a monomeric and soluble protein. rproSin a 1 showed equivalent structural and immunological properties to natural heterodimeric Sin a 1 allergen. rproSin a 1 was recognized by 75% of the patients allergic to mustard. The inhibitory capacity of rproSin a 1 to the total allergenicity of mustard extracts varied from 13 to 83% in different patients, with a mean value of 54%. CONCLUSIONS: rproSin a 1 is a good candidate to replace natural allergen in diagnosis protocols of mustard allergy. P. pastoris has been demonstrated to be a suitable expression system for the production of allergenic derivates of Sin a 1 that could be used for immunotherapy purposes in future.

Allergens↗

PubMed2005

Isolation and identification of an 11S globulin as a new major allergen in mustard seeds.

BACKGROUND: Although mustard seed allergy has been largely reported during the preceding 20 years, currently only 2 allergens, Sin a 1 and Bra j 1, have been identified. OBJECTIVE: To improve the characterization of the allergenic profile of yellow mustard seeds by reporting the identification and biochemical characterization of an 11S globulin as a new major allergen. METHODS: Mustard seed proteins were separated using size exclusion and ion-exchange chromatographic columns, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 2-dimensional polyacrylamide gel electrophoresis. Separation of different polypeptide chains was achieved by reverse-phase high-performance liquid chromatography. Mass spectrometry after tryptic digestion and Edman degradation were used to determine amino acid sequences of peptides. IgE binding assays were performed with 13 serum samples from mustard allergic patients in immunoblotting and enzyme-linked immunosorbent inhibition assays. RESULTS: A protein of 51 kDa was recognized as a major allergen by patients allergic to mustard and called Sin a 2. The allergen was dissociated in 2 chains of 36 and 23 kDa, which also bound IgE. N-terminal end and internal amino acid sequences allowed identification of the new allergen as a seed storage 11S globulin belonging to the Cupin super family. Purified allergen was able to inhibit the IgE binding of sera from allergic patients to mustard seeds extract in up to 55% of the responses. CONCLUSIONS: An 11S globulin storage protein has been isolated and identified as a novel major allergen of mustard seeds.

Adolescent↗

PubMed2004

Solution structure and stability against digestion of rproBnIb, a recombinant 2S albumin from rapeseed: relationship to its allergenic properties.

NMR spectroscopy has been used to determine the solution structure of the precursor form of the recombinant napin BnIb, rproBnIb, a 2S albumin, 109-residue protein from the seeds of Brassica napus. More than 90% of the side-chain proton resonances were unambiguously assigned from the analysis of two-dimensional correlation (COSY), total correlation (TOCSY), and nuclear Overhauser effect (NOESY) spectra. The final structures were computed by using restrained molecular dynamics on the basis of 1316 upper-limit distance constraints derived from NOE cross-correlation intensities. The computed structures exhibited a root-mean-square deviation (RMSD) radius of 0.66 A for the backbone and 1.16 A for the side-chain heavy atoms of the structural core. The resulting structure consists of five amphipathic helices arranged in a right-handed super helix, a folding motif found in other proteins of the prolamin superfamily. As in the case of the mature protein, the recombinant precursor behaves as a plant food allergen. To trace out the origin and characteristics of its allergenic properties, rproBnIb was assayed against simulated gastric fluid and found to be very resistant to proteolysis. Also, heat treatment of the protein followed up to 85 degrees C by circular dichroism showed a very limited unfolding, which was recovered after cooling to 20 degrees C, indicating a high thermal stability. These results suggest that rproBnIb, as other 2S albumins, may be able to reach the gut immune system intact. A comparison of the putative epitopes against IgE antibodies of the three members of the prolamine family [2S albumins, nonspecific lipid transfer proteins (nsLTPs), and alpha-amylase/trypsin inhibitors] indicates that there are not common surfaces of interaction with IgE. Though the epitopes appear to be located in different regions of the proteins, they do comply with the requirements of being solvent-exposed and flexible.

2S Albumins, Plant↗

PubMed2004

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.

Adult↗

PubMed2004

Characterization of recombinant forms of the yeast Gas1 protein and identification of residues essential for glucanosyltransferase activity and folding.

Gas1p is a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae and is a representative of Family GH72 of glycosidases/transglycosidases, which also includes proteins from human fungal pathogens. Gas1p, Phr1-2p from Candida albicans and Gel1p from Aspergillus fumigatus have been shown to be beta-(1,3)-glucanosyltransferases required for proper cell wall assembly and morphogenesis. Gas1p is organized into three modules: a catalytic domain; a cys-rich domain; and a highly O-glycosylated serine-rich region. In order to provide an experimental system for the biochemical and structural analysis of Gas1p, we expressed soluble forms in the methylotrophic yeast Pichia pastoris. Here we report that 48 h after induction with methanol, soluble Gas1p was produced at a yield of approximately 10 mg x L(-1) of medium, and this value was unaffected by the further removal of the serine-rich region or by fusion to a 6 x His tag. Purified soluble Gas1 protein showed beta-(1,3)-glucanosyltransferase activity that was abolished by replacement of the putative catalytic residues, E161 and E262, with glutamine. Spectral studies confirmed that the recombinant soluble Gas1 protein assumed a stable conformation in P. pastoris. Interestingly, thermal denaturation studies demonstrated that Gas1p is highly resistant to heat denaturation, and a complete refolding of the protein following heat treatment was observed. We also showed that Gas1p contains five intrachain disulphide bonds. The effects of the C74S, C103S and C265S substitutions in the membrane-bound Gas1p were analyzed in S. cerevisiae. The Gas1-C74S protein was totally unable to complement the phenotype of the gas1 null mutant. We found that C74 is an essential residue for the proper folding and maturation of Gas1p.

Amino Acid Sequence↗

PubMed2003

The C-terminal segment of the 1,3-beta-glucanase Ole e 9 from olive (Olea europaea) pollen is an independent domain with allergenic activity: expression in Pichia pastoris and characterization.

Several allergenic proteins, such as the 1,3-beta-glucanases, have been associated with plant defence responses. Ole e 9 (46 kDa) is a 1,3-beta-glucanase and major allergen from olive pollen, which is a principal cause of allergy in Mediterranean countries. Its C-terminal segment (101 amino acid residues) has been produced as a recombinant polypeptide in the yeast Pichia pastoris. The cDNA encoding the polypeptide was inserted into the plasmid vector pPICZalpha-A and overexpressed in KM71 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reversed-phase HPLC. Edman degradation, MS and CD were used to determine molecular properties of the recombinant polypeptide, which exhibited 16% alpha-helix and 30% beta-sheet as regular elements of secondary structure. Disulphide bridges of the molecule were determined at positions Cys-14-Cys-76, Cys-33-Cys-94 and Cys-39-Cys-48. The high IgE-binding capability of the recombinant C-terminal segment of Ole e 9 against sera from Ole e 9-sensitive individuals, which was determined by immunoblotting and ELISA inhibition, supported the proper folding of the polypeptide and the maintenance of antigenic properties that it exhibits as a part of the whole allergen. These data indicated that this portion of Ole e 9 constitutes an independent domain, which could be used to study its three-dimensional structure and function, as well as for clinical purposes such as diagnosis and specific immunotherapy. Since it shows sequence similarity with portions of 1,3-beta-glucanases from plant tissues and the Gas/Phr/Epd protein families involved in yeast morphogenesis, we suggest that this domain could play an equivalent functional role within these enzymes.

Allergens↗

PubMed2002

Recombinant pronapin precursor produced in Pichia pastoris displays structural and immunologic equivalent properties to its mature product isolated from rapeseed.

2S albumin storage proteins from rapeseed (Brassica napus), called napins, consist of two different polypeptide chains linked by disulphide bridges, which are derived by proteolytic cleavage from a single precursor. The precursor form of the napin BnIb (proBnIb) has been cloned using a PCR strategy and sequenced. The amino-acid sequence deduced from the clone includes 31 residues of the small chain and 75 of the large chain, which are connected by the peptide Ser-Glu-Asn. Expression of the cDNA encoding proBnIb has been carried out in the methylotrophic yeast Pichia pastoris. The induced protein was secreted to the extracellular medium at a yield of 80 mg.L(-1) of culture and was purified by means of size-exclusion chromatography and reverse phase-HPLC. Recombinant proBnIb appeared properly folded as its molecular and spectroscopic properties were equivalent to those of the mature heterodimeric protein. As 2S albumin storage proteins from Brassicaceae have been shown to be type I allergy inducers, the immunological activity of the recombinant proBnIb was analysed as a measure of its structural integrity. The immunological properties of the recombinant precursor and the natural napin were indistinguishable by immunoblotting and ELISA inhibition using polyclonal antisera and sera of patients allergic to mustard and rapeseed. In conclusion, the recombinant expression of napin precursors in P. pastoris has been shown to be a successful method for high yield production of homogeneous and properly folded proteins whose polymorphism and complex maturation process limited hitherto their availability.

2S Albumins, Plant↗