Association Between the Seed Storage Proteins 2S Albumin and 11S Globulin and Severe Allergic Reaction After Flaxseed Intake.

BACKGROUND: Given the increased popularity of flaxseed in meals, several cases of allergy to these seeds have been reported. Little is known about the allergens implicated in hypersensitivity reactions to flaxseed. The present study aimed to identify the allergens involved in IgE-mediated reactions in 5 patients with a clinical history of severe systemic symptoms after flaxseed consumption. METHODS: Proteins that were potential allergens with IgE-binding capacity were purified from flaxseed extract using chromatography and identified via MALDI-TOF mass spectrometry. Immunoassays were performed using the 5 allergic patients' sera tested individually and as a pool. RESULTS: Immunoblotting of the flaxseed extract revealed a low-molecular-mass protein (around 13 kDa) in 4 of the 5 patients, while a protein of approximately 55 kDa was detected in 2 patients. The proteins were identified by mass spectrometry as flaxseed 2S albumin, which is included in the WHO/IUIS allergen nomenclature as Lin u 1, and 11S globulin. Inhibition assays revealed in vitro IgE-mediated cross-reactivity between Lin u 1 and peanut and cashew nut proteins, while IgE-mediated recognition of 11S globulin by patients' sera was partially inhibited by several plant-derived sources. CONCLUSIONS: Seed storage proteins from flaxseed were involved in the development of severe symptoms in the 5 patients studied and exhibited cross-reactivity with other allergenic sources. Besides the severity of flaxseed allergy in patients sensitized to 2S albumin, this is the first time that 11S globulin has been identified as a potential allergen. Taking these data into account should ensure a more accurate diagnosis.

Effects of ole e 1 on human bronchial epithelial cells cultured at the air-liquid interface

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Amaranthaceae pollens: review of an emerging allergy in the mediterranean area

The Amaranthaceae family is composed of about 180 genera and 2500 species. These common weeds have become increasingly relevant as triggers of allergy in the last few years, as they are able to rapidly colonize salty and arid soils in extensive desert areas. The genera Chenopodium, Salsola, and Amaranthus are the major sources of pollinosis from the Amaranthaceae family in southern Europe, western United States, and semidesert areas of Saudi Arabia, Kuwait, and Iran. In Spain, Salsola kali is one of the most relevant causes of pollinosis, together with olive and grasses. To date, 9 Amaranthaceae pollen allergens from Chenopodium album, Salsola kali, and Amaranthus retroflexus have been described and are listed in the International Union of Immunological Societies allergen nomenclature database. The major allergens of Amaranthaceae pollen belong to the pectin methylesterase, Ole e 1-like, and profilin panallergen families, whereas the minor allergens belong to the cobalaminindependent methionine synthase and polcalcin panallergen families. These relevant allergens have been characterized physicochemically, and immunologically at different levels. Recombinant forms, allergenic fusion recombinant proteins, and hypoallergenic derivatives of these allergens have been expressed in bacteria and yeast and compared with their natural proteins from pollen. In this review, we provide an extensive overview of Amaranthaceae pollen allergens, focusing on their physicochemical, and immunological properties and on their clinical significance in allergic patients. We also review studies where these recombinant allergens and their hypoallergenic derivatives have been used in clinical diagnosis and their potential use in personalized therapy (AU)

La familia Amaranthaceae se compone de alrededor de 180 géneros y 2500 especies vegetales. En los últimos años, el polen de estas malezas está adquiriendo una relevancia cada vez mayor como inductor de alergia, ya que estas plantas son capaces de colonizar rápidamente los suelos salinos y áridos de zonas desertificadas. El polen de los géneros Chenopodium, Salsola y Amaranthus es el causante del mayor número de casos de polinosis asociados a la familia Amaranthaceae en países del sur de Europa, oeste de Estados Unidos, y en las zonas semi-desérticas de Arabia Saudí, Kuwait o Irán. En España, el polen de Salsola kali es una de las causas más relevantes de polinosis junto con los pólenes de olivo y gramíneas. Hasta la fecha, se han descrito un total de nueve alérgenos del polen de Chenopodium album, Salsola kali y Amaranthus retroflexus, los cuales se han depositado en la base de datos de nomenclatura de alérgenos IUIS. Los alérgenos principales del polen de la familia Amaranthaceae pertenecen a las familias pectin metilesterasa, Ole e 1, o a la familia de panalérgenos -profilina-, mientras que los alérgenos secundarios descritos pertenecen a la familia de panálergenos -polcalcina-, o bien, corresponden a la metionina sintasa independiente de cobalamina. Estos relevantes alérgenos se han caracterizado fisicoquímica e inmunológicamente en mayor o menor profundidad. Las formas recombinantes, y sus variantes recombinantes o derivados hipoalergénicos fusionados a un tag, se han expresado en bacteria o levadura y se ha comparado su funcionalidad con sus correspondientes homólogos naturales presentes en el polen. En esta revisión, ofrecemos una extensa descripción de los alérgenos del polen de la familia Amaranthaceae, centrándonos en sus propiedades físico-químicas e inmunológicas, y en su importancia clínica en los pacientes alérgicos. Por otra parte, también hemos revisado aquellos estudios en donde se han utilizado estos alérgenos recombinantes y sus derivados hipoalergénicos en el diagnóstico clínico, o bien, en donde se describe su potencial uso en la terapia personalizada (AU)

Characterization of profilin and polcalcin panallergens from ash pollen

Background: Ash ( Fraxinus excelsior ) is an important source of allergenic pollen in temperate areas of Europe. Profilin and polcalcin are 2 important panallergens involved in cross-reactivity between different sources. Objective: To clone and produce Fra e 2 (profilin) and Fra e 3 (polcalcin) as recombinant proteins and evaluate their immunological properties using the natural forms obtained from ash pollen. Methods: Total RNA from ash pollen was used as a template to obtain the specific complementary DNA (cDNA) sequences of the 2 panallergens. The cDNA-encoding sequences were cloned into the pET11b expression vector and used to transform BL21 (DE3) Escherichia coli cells. Proteins were expressed, purified by chromatography, and characterized structurally by circular dichroism, mass spectrometry, and immunologically by western blot and ELISA using profilin and polcalcin polyclonal antibodies and human sera from ash pollen-sensitized patients. Results: Profilin and polcalcin amino acid sequences from ash pollen showed a high degree of identity with homologous allergens from different sources. The cDNA-encoding allergen sequences were expressed as nonfusion recombinant proteins and purified to homogeneity. Secondary structure values were similar to those obtained from other members of these families. Allergenic properties of the recombinant allergens were observed to be equivalent to those of the natural counterparts of F excelsior pollen. Conclusions: Fra e 2 and Fra e 3 recombinant allergens might be used in clinical diagnosis to determine profilin- and polcalcin-specific IgE levels present in the sera of ash pollen-sensitized patients, thus facilitating the finding of the sensitizing source in areas with complex sensitization profiles (AU)

Antecedentes: El polen de fresno (Fraxinus excelsior ) es una importante fuente alergénica en zonas cálidas de Europa. La profilina y polcalcina son 2 panalérgenos implicados en reactividad cruzada. Objetivos: Clonar y producir Fra e 2 (profilina) y Fra e 3 (polcalcina) como alérgenos recombinantes. Comparar sus propiedades inmunológicas con sus formas naturales del polen de fresno. Métodos: El RNA total de polen de fresno se utilizó como molde para obtener los cDNAs específicos de ambos panalérgenos. Dichos cDNAs se clonaron en el vector de expresión pET11b y se transformaron células de Escherichia coli BL21(DE3). Las proteínas se caracterizaron mediante dicroísmo circular, espectrometría de masas, inmunodetección en membrana y ELISA utilizando anticuerpos policlonales frente a profilina y polcalcina y sueros de pacientes alérgicos al polen de fresno. Resultados: Las secuencias de aminoácidos de la profilina y polcalcina de polen de fresno presentaban una identidad de secuencia elevada con alérgenos homólogos. Dichos alérgenos se expresaron como proteínas recombinantes independientes y se purificaron a homogeneidad. Los valores de estructura secundaria fueron similares a los de otros miembros de estas familias. Las propiedades alergénicas de los alérgenos recombinantes resultaron ser equivalentes a los de sus homólogos naturales del polen. Conclusiones: Los alérgenos recombinantes Fra e 2 y Fra e 3 podrían usarse en diagnóstico clínico para determinar los niveles de IgE específicos para profilina y polcalcina en los sueros de los pacientes sensibilizados al polen de fresno, facilitando así la identificación de la fuente sensibilizante en áreas donde los pacientes presentan perfiles alergénicos complejos (AU)

Amaranthaceae pollens: review of an emerging allergy in the mediterranean area.

The Amaranthaceae family is composed of about 180 genera and 2500 species. These common weeds have become increasingly relevant as triggers of allergy in the last few years, as they are able to rapidly colonize salty and arid soils in extensive desert areas. The genera Chenopodium, Salsola, and Amaranthus are the major sources of pollinosis from the Amaranthaceae family in southern Europe, western United States, and semidesert areas of Saudi Arabia, Kuwait, and Iran. In Spain, Salsola kali is one of the most relevant causes of pollinosis, together with olive and grasses. To date, 9Amaranthaceae pollen allergens from Chenopodium album, Salsola kali, and Amaranthus retroflexus have been described and are listed in the International Union of Immunological Societies allergen nomenclature database.The major allergens ofAmaranthaceae pollen belong to the pectin methylesterase, Ole e 1-like, and profilin panallergen families, whereas the minor allergens belong to the cobalamin- independent methionine synthase and polcalcin panallergen families. These relevant allergens have been characterized physicochemically, and immunologically at different levels. Recombinant forms, allergenic fusion recombinant proteins, and hypoallergenic derivatives of these allergens have been expressed in bacteria and yeast and compared with their natural proteins from pollen. In this review, we provide an extensive overview ofAmaranthaceae pollen allergens, focusing on their physicochemical, and immunological properties and on their clinical significance in allergic patients. We also review studies where these recombinant allergens and their hypoallergenic derivatives have been used in clinical diagnosis and their potential use in personalized therapy.

Olive pollen recombinant allergens: value in diagnosis and immunotherapy.

Olive pollen has a complex allergenic profile, from which more than 10 allergens have been identified and characterized. Some of these belong to well-known protein families and others cannot be included in reported biochemical types. Most of these allergens have been produced by recombinant technology, mainly in Escherichia coli or in Pichia pastoris, and they are good candidates for diagnostic and therapeutic purposes. Diagnosis and immunotherapy of allergy currently use extracts prepared from homogenates of natural sources, which only allow us to detect sensitivity to the complete source. These extracts can be successfully replaced by mixtures with controlled amounts of specific allergenic proteins obtained by recombinant technology in order to define the sensitization profile of individual patients. Recombinant Ole e 1 can be used as a marker for sensitization to Oleaceae. Recombinants Ole e 2 (profilin) and Ole e 3 (polcalcin) can serve as markers of polysensitivity. Finally, recombinant forms of Ole e 6, Ole e 10, and the carboxy-terminal and amino-terminal domains of Ole e 9 would help to detect sensitization to these minority allergens that could be overlooked in the complete olive pollen extract. These recombinant molecules can help provide an accurate diagnosis of sensitivity to individual allergens and, therefore, improve the design of more efficacious allergen-based immunotherapy strategies.

Allergenic diversity of the olive pollen.

A great number of allergenic proteins have been detected in olive pollen extracts. To date, nine allergens have been isolated and characterized, which have been called Ole e 1 to Ole e 9. The most prevalent olive allergen is Ole e 1, which affects more than 70% of patients hypersensitive to olive pollen, but others, such as Ole e 2, Ole e 8, and Ole e 9, have been demonstrated to be major allergens, and Ole e 6 or Ole e 7 reach high values of clinical incidence. Many of these allergens, such as Ole e 2 (profilin) and Ole e 3 (polcalcin), are involved in cross-reactivities, which agrees with their adscription to panallergenic families. Among the many olive allergens of high molecular mass, only Ole e 9 (46 kDa) has been characterized. The allergen is a polymorphic and glycosylated beta-1,3-glucanase, which belongs to a pathogenesis-related (PR-2) protein family. In addition to the polypeptide epitopes, Ole e 1 also exhibits IgE-binding determinants in the carbohydrate, which are recognized by more than 60% of the sera from patients sensitive to the whole allergen, although the level of such glycan-specific IgE seems not to be clinically relevant in the overall content of the sera. Recent advances in the elucidation of the structure of the Ole e 1-oligosaccharide component allows us to explain the antigenicity of the molecule. Finally, the recombinant production of several allergens from olive pollen in both bacterial and eukaryotic cells has allowed us to resolve problems derived from the polymorphism and scarcity of the natural forms of these allergens. The biological equivalence between the natural and recombinant forms lets us initiate studies on the design of mixtures for clinical purposes, in which hypoallergenic derivatives of these allergens could play a definitive role.

The spectrum of olive pollen allergens.

Olive pollen is one of the most important causes of seasonal respiratory allergy in Mediterranean countries, where this tree is intensely cultivated. Among the high number of protein allergens detected in this pollen, 8 - Ole e 1 to Ole e 8 - have been isolated and characterized. Ole e 1 is the most frequent sensitizing agent, affecting more than 70% of the patients suffering of olive pollinosis, although others, such as Ole e 4 and Ole e 7, have also been shown to be major allergens. In this context, the prevalence of many olive pollen allergens seems to be dependent on the geographical area where the sensitized patients live. Some of the olive allergens have been revealed as members of known protein families: profilin (Ole e 2), Ca(2+)-binding proteins (Ole e 3 and Ole e 8), superoxide dismutase (Ole e 5) and lipid transfer protein (Ole e 7). No biological function has been demonstrated for Ole e 1, whereas Ole e 4 and Ole e 6 are new proteins without homology to known sequences from databases. cDNAs encoding for Ole e 1, Ole e 3 and Ole e 8 have been overproduced in heterologous systems. The recombinant products were correctly folded and exhibited the functional activities of the natural allergens. In addition to the Oleaceae family, other species, such as Gramineae or Betulaceae, contain pollen allergens structurally or immunologically related to those of the olive tree. This fact allows to detect and evaluate antigenic cross-reactivities involving olive allergens. The aim of this research is the development of new diagnostic tools for olive pollinosis and new approaches to improve the classical immunotherapy.

Identification, isolation, and characterization of Ole e 7, a new allergen of olive tree pollen.

BACKGROUND: Olive tree (Olea europaea) pollen is an important cause of pollinosis in countries of the Mediterranean area and California. OBJECTIVE: The aim of this study was to identify and purify a new allergen of olive tree pollen. METHODS: Detection of a pollen allergen was done with individual allergic sera by immunoblotting and ELISA tests. Two allergenic fractions were isolated from olive pollen extract by using gel filtration and reverse-phase HPLC. Molecular characterization was achieved by acid hydrolysis and amino acid analysis, as well as by mass spectrometry. Sequencing of the N-terminal end of the allergen was carried out by Edman degradation of the polypeptide chain. Allergenic characterization was performed with sera from subjects with olive allergy by means of ELISA and immunoblotting after SDS-PAGE. RESULTS: The new allergen Ole e 7 exhibits a high degree of polymorphism. Its molecular mass is in the range of 9875 d to 10,297 d. Twenty-one amino acid residues from the N-terminal end of 2 isoforms of the allergen have been sequenced revealing no homology with proteins contained in database banks. Ole e 7 has an average frequency of about 47% in patients with olive allergy. The strategy of purification of Ole e 7 can be useful on the isolation of new allergens. CONCLUSIONS: A new olive pollen allergen of clinical significance has been purified and characterized, contributing to the study of the complete allergogram of the olive tree pollen.

Direct effects on antigen-presenting cells and T lymphocytes explain the adjuvanticity of a nontoxic cholera toxin mutant.

The present study has elucidated two distinct mechanisms that may explain how a mutant of cholera toxin (mCT), E112K, retains adjuvant effects though it lacks ADP-ribosyltransferase activity and associated toxicity. In the first mechanism, we show that mCT E112K, like native cholera toxin (nCT), enhances B7-2 expression, but, to some extent, also enhances B7-1 on Peyer's patch B cells and macrophages. Cocultivation of CD4+ T cells with E112K- or nCT-treated B cells and macrophages in the presence of anti-CD3 stimulation resulted in the induction of T cell-proliferative responses. Further, the responses were blocked by mAbs to B7-1 and/or B7-2; however, the effect of anti-B7-1 was minimal. In the second mechanism, addition of mCT E112K or nCT to anti-CD3 mAb-stimulated Peyer's patch CD4+ T cells inhibited proliferative responses, while recombinant CT-B subunit (rCT-B) did not. Analysis of cytokine responses showed that both mCT E112K and nCT preferentially inhibited IFN-gamma production. Interestingly, however, nCT, but not mCT E112K, induced apoptosis in CD4+ T cells activated via the TCR-CD3 complex. These results indicate that CT uses at least two pathways for inhibition of Th1 responses and that, while nCT induces cAMP accumulation that in turn leads to apoptosis in Th1-type cells, mCT E112K, which lacks ADP-ribosyltransferase activity, inhibits IFN-gamma synthesis by a separate mechanism. Thus, mCT E112K, like nCT, induces adjuvant responses via up-regulation of mainly B7-2 on APCs and through preferential inhibition of Th1-type CD4+ T cell responses in the absence of ADP-ribosyltransferase activity.

Molecular cloning and expression of active Ole e 3, a major allergen from olive-tree pollen and member of a novel family of Ca2+-binding proteins (polcalcins) involved in allergy.

A cDNA encoding Ole e 3, a major allergen from olive-tree pollen, has been cloned and sequenced. A strategy based on two-step PCR amplification towards the 5' end and 3' end, with an internal specific primer, has been used. The isolated cDNA contains an open reading frame coding for a polypeptide of 84 amino acids, which is in agreement with the composition and molecular mass of the natural allergen, exhibiting two 12-residue segments homologous to Ca2+-binding sites of EF-hand type. The cDNA was inserted into the pET-11b expression vector and over-expressed in Escherichia coli. The purified recombinant protein shows identical secondary structure to that of the natural allergen and is able to bind both IgE from sera of patients allergic to olive pollen and polyclonal antibodies raised against olive-pollen Ole e 3. The capacity of binding Ca2+ has been demonstrated for both natural and recombinant allergens. RNA transcripts of Ole e 3 were only detected in pollen tissue. Northern-blot and Western-blot analyses of poly(A)+ RNA and protein extracts, respectively, obtained from a variety of olive-tree-related and nonrelated mature pollens demonstrated the presence of Ole e 3 homologous proteins. This indicates a sequence conservation and widespread distribution for this family of Ca2+-binding proteins that can be responsible for allergenic cross-reactivity. We suggest the tentative generic name of polcalcins for the members of this family of Ca2+-binding proteins from pollen.

Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine-enriched allergen from olive tree pollen.

The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse-phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH2-terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH2-terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys-X3-Cys-X3-Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides.

Isolation, cDNA cloning and expression of Lig v 1, the major allergen from privet pollen.

BACKGROUND: An olive allergen-like protein has been detected in privet pollen. This protein could be involved in the allergenic cross-reactivity described for privet and olive tree pollen extracts. OBJECTIVE: Isolation and characterization of natural Lig v 1. Cloning and expression of its cDNA in order to assess its structural similarity with the olive allergen. METHODS: Current chromatographic methods were used to isolate the privet counterpart of Ole e 1. A pool of sera from subjects allergic to olive tree pollen was used to immunodetect the protein in the elution profiles. Ole e 1-specific polyclonal antibody and allergic sera were used in immunoblotting assays of the isolated protein. Polymerase chain reaction amplification of the first strand cDNA synthesized from the privet pollen total RNA was carried out to prepare a full-length fragment encoding Lig v 1. After nucleotide sequencing, expression of one clone was performed in Escherichia coli, under the form of a fusion protein with glutathione S-transferase. The IgE binding capability of the recombinant protein was also analysed. RESULTS: The major allergen from privet pollen. Lig v 1, was purified to homogeneity by two gel filtration chromatographies and one reverse-phase high-performance liquid chromatography. Its amino acid composition and N-terminal amino acid sequence were determined. Two different clones encoding Lig v 1 were sequenced. Strong sequence similarity between Lig v 1 and Ole e 1 was observed, the identity being 85 and 96%. One of the sequenced clones was expressed and the recombinant product exhibited IgG and IgE binding activities against both anti-Ole e 1 polyclonal antibodies and olive-allergic sera. CONCLUSION: Privet pollen contains a protein structurally and immunologically related to the major allergen of olive pollen. The similarity exhibited by these proteins could explain the cross-reactivity observed between the two pollen extracts. Since these allergens are highly polymorphic, the expression of an immunologically active recombinant Lig v 1 will permit the preparation of well defined molecules for both research and clinical purposes.

Ole e 3, an olive-tree allergen, belongs to a widespread family of pollen proteins.

An allergen has been isolated from a saline extract of olive tree (Olea europaea) pollen. The protein consists of a single polypeptide chain of 9.2-kDa, as determined by mass spectrometry. It contains neither tryptophan nor tyrosine residues, and displays an acidic isoelectric point. The secondary structure of the protein, estimated from the analysis of the circular-dichroism spectrum in the peptide-bond region, is composed of 52% alpha-helix, 10% beta-strand, 29% beta-turn and 9% non-regular conformation. The N-terminal end of the protein is blocked. Amino-acid-sequence data have been obtained from peptides produced by CNBr treatment of the native allergen. A partial sequence of 36 amino acids has thus been elucidated. The protein exhibits sequence similarity with pollen allergens from Brassica species and contains a Ca(2+)-binding motif. The isolated protein displays IgE-binding activity against sera of patients allergic to olive-tree pollen. It has been named Ole e 3, according to the recommendations of the IUIS Nomenclature Committee. IgG ELISA inhibition assays with polyclonal antibodies specific for Ole e 3 reveal the presence of proteins similar to Ole e 3 in the pollen from non-related plant species, which may explain allergic cross-reactivity processes.

Cross-reactivity between the major allergen from olive pollen and unrelated glycoproteins: evidence of an epitope in the glycan moiety of the allergen.

Ole e 1, the major allergen from olive pollen, is a glycoprotein containing a single Asn-linked glycan moiety. Rabbit antiserum against this protein has been obtained; and its immunologic cross-reactivities in Western blotting with ascorbate oxidase, horseradish peroxidase, bromelain, ovalbumin, and honeybee venom phospholipase A2 have been studied. Ascorbate oxidase, peroxidase, and bromelain are recognized by the Ole e 1 antiserum. When these three proteins are deglycosylated by periodate treatment, such an immunologic reaction does not occur. The relative affinities of these proteins have been analyzed by direct and inhibition ELISA experiments. A commercially available antibody against horseradish peroxidase has also been considered in these studies. This antibody reacts with Ole e 1 but not with the periodate-deglycosylated allergen. Horseradish peroxidase, bromelain, and ascorbate oxidase are recognized by the IgE of sera from patients who are hypersensitive to olive tree pollen. This binding is also abolished by periodate treatment. The results are interpreted in terms of the presence of an epitope in the carbohydrate moiety of Ole e 1, which would contain a xylose involved in recognition by both IgE and IgG antibodies.

Expression in Escherichia coli of Sin a 1, the major allergen from mustard.

Sin a 1, the major yellow mustard allergen, is a seed storage protein that belongs to the 2S albumin family. It is composed of two disulfide-bonded polypeptide chains. The cloning of this allergen has been carried out by means of the polymerase chain reaction using non-degenerate oligonucleotides encoding the N-terminal and C-terminal regions of the mature protein as primers. Five genomic nucleotide sequences have been analyzed, encoding both mature polypeptide chains linked by the internal processed fragment. The sequence data show the existence of microheterogeneities at ten positions, demonstrating the polymorphism exhibited by the natural protein. One of the genomic clones was expressed in Escherichia coli by fusion to glutathione S-transferase from Schistosoma japonicum. The resulting chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B matrix, and digested with thrombin to release the recombinant allergen. The recombinant Sin a 1 is recognized by rabbit polyclonal and mouse monoclonal antisera raised against natural Sin a 1, as well as by the IgE of mustard-sensitive human sera. In addition, recombinant Sin a 1 possesses a high resistance to trypsin digestion, like the native mustard allergen.

Cloning and expression of Ole e I, the major allergen from olive tree pollen. Polymorphism analysis and tissue specificity.

Ole e I, the major allergen from the olive tree (Olea europaea), is one of the main causes of allergy in Mediterranean countries and some areas of North America. The cloning and sequencing of several cDNAs coding for the olive allergen have been achieved. cDNA has been synthesized from total pollen RNA and amplified by using the polymerase chain reaction. The nucleotide sequence data demonstrate the existence of microheterogeneities in at least 37 positions out of the 145 amino acids of Ole e I, thus explaining the high degree of polymorphism exhibited by the natural protein. One of the sequenced cDNAs encoding a full-length isoform was inserted into the plasmid vector pGEX-2T and overexpressed. The recombinant Ole e I has been produced in Escherichia coli as a fusion protein with glutathione S-transferase of Schistosoma japonicum. This chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B column and digested with thrombin to release the recombinant allergen. Both the fusion protein and the recombinant Ole e I were recognized in Western blot analysis by rabbit polyclonal and mouse monoclonal antisera raised against native Ole e I as well as by the IgE of olive pollen-sensitive human sera. This indicates that the recombinant production of individual isoforms may be useful for the improvement of reagents to be used in diagnosis and therapy of IgE-mediated disorders. In addition, Ole e I mRNA has been observed to be pollen-specific as shown in a Northern blot analysis.

Isolation and characterization of an olive allergen-like protein from lilac pollen. Sequence analysis of three cDNA encoding protein isoforms.

An olive allergen-like protein has been isolated from lilac (Syringa vulgaris) pollen extract. The protein can be considered as an allergen since is recognized by IgE from olive hypersensitive human sera, and has been called Syr v I (IUIS nomenclature). This protein consists of a glycosylated polypeptide of 20 kDa, which has an amino acid composition, spectroscopic properties, and an N-terminal sequence similar to the major allergen from olive pollen, Ole e I. The lilac allergen is recognized by rabbit polyclonal antisera raised against olive allergen as well as by an Ole e I-specific monoclonal antibody. Using a polymerase chain reaction strategy, based on the similarities observed between these olive and lilac proteins, three cDNA clones encoding Syr v I have been isolated and sequenced. These clones code for a polymorphic protein of 145 residues with a derived molecular mass of about 16,400Da, which contains a potential N-glycosylation site. Comparison of the deduced amino acid sequences of these Syr v I isoforms to each other revealed identities of 90-97%. Moreover, these sequences showed a high degree of similarity (85.5-89.6% identity) with Ole e I. The structural and immunological characterization of Syr v I justify the cross-reactions observed between olive and lilac pollen extracts. The molecular cloning of Syr v I is relevant for the epitope mapping in Oleaceae allergens, and may contribute to an improvement in the design of reagents for diagnosis and therapy of IgE-dependent allergic reactions.

Glycosylation site of the major allergen from olive tree pollen. Allergenic implications of the carbohydrate moiety.

The electrophoretic analysis of purified Ole e I, the major allergen from Olea europaea pollen, reveals the presence of two main variants, glycosylated (20.0 kDa) and non-glycosylated (18.5 kDa) components. The glycosylated variant has been identified as a concanavalin A-binding glycoprotein. Its carbohydrate moiety has a molecular mass of about 1.3 kDa (5% weight of the glycosylated allergen), based on mass spectrometry analysis. Enzymatic treatment of native Ole e I with the specific glycosidase PNGase F accounts for an oligosaccharide N-linked to the polypeptide chain. This treatment does not sensibly modify the secondary structure of the protein but diminishes the affinity of the allergen for specific IgE antibodies. Tryptic digestion of Ole e I reveals the presence of a single carbohydrate-containing peptide. This peptide was recognized by the sera of hypersensitive individuals. The amino acid sequence of this peptide is Phe-Lys-Leu-Asn-Thr-Val-Asn-Gly-Thr-Thr-Arg, asparagine at the seventh being the carbohydrate attaching site. The obtained data are discussed in terms of the potential role of the sugar moiety in the allergenic activity of Ole e I.