Detection, isolation and complete amino acid sequence of an aeroallergenic protein from rapeseed flour.

BACKGROUND: Seed proteins have been found to cause hypersensitivity by ingestion or inhalation. Rapeseed flour was responsible for allergic symptoms in a patient, who develops into allergy to mustard spice. OBJECTIVE: To determine the presence of allergenic proteins in rapeseed flour, and analyse the structure of the main component and its crossreactivity with the mustard allergen. METHODS: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and subsequent immunoblotting with a serum from a rapeseed allergic patient were performed to detect IgE-binding proteins. Proteolytic digestions and high performance liquid chromatography were used to obtain the peptides from the allergenic BnIII napin from rapeseed flour. Automatic Edman degradations were carried out to determine their amino acid sequences, which were compared with other sequences in nucleotide and amino acid sequence databases. Crossreactivity assays were carried out by ELISA inhibition using sera from a rapeseed allergic patient and from patients allergic to mustard. RESULTS: The 2S albumins of rapeseed were recognized by the serum from a patient allergic to this seed. The most abundant isoform of the allergenic napins, BnIII, was used for structural and immunological analysis. The protein consists of two different chains of 9.5 and 4.5 kDa. Their complete amino acid sequences were determined. The protein exhibited structural relationships with other napin-like storage proteins from seeds. IgE and IgG crossreactivity between rapeseed and mustard allergens was also demonstrated. Considering the structural and immunological data, certain polypeptide regions are suggested to be involved in the allergenicity of these proteins. CONCLUSIONS: Rapeseed contains 2S storage proteins which may cause allergy in hypersensitive individuals. These proteins exhibit great sequence similarity with 2S albumins from different seeds. Crossreactivity between mustard and rapeseed flours can be explained by sequence homology.

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.

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.

The amino acid sequence of Ole e I, the major allergen from olive tree (Olea europaea) pollen.

The complete primary structure of the major allergen from Olea europaea (olive tree) pollen, Ole e I (IUIS nomenclature), has been determined. The amino acid sequence was established by automated Edman degradation of the reduced and alkylated molecule as well as of selected fragments obtained by proteolytic digestions. Ole e I contains a single polypeptide chain of 145 amino acid residues with a calculated molecular mass of 16331 Da. No free sulfhydryl groups have been detected in the native protein. The molecule contains a putative glycosylation site. A high degree of microheterogeneity has been observed, mainly centered in the first 33% of the molecule. Comparison of Ole e I sequence with protein sequence databases showed no similarity with other known allergens. However, it has a 36% and 38% sequence identity with the putative polypeptide structures, deduced, respectively, from nucleotide sequences of genes isolated from tomato anthers and corn pollen, which have been suggested to be involved in the growing of the pollen tube. Therefore, the olive tree allergen may be a constitutive protein of the pollen involved in reproductive functions.

Characterization of a new oriental-mustard (Brassica juncea) allergen, Bra j IE: detection of an allergenic epitope.

Bra j IE, a major allergen from oriental-mustard (Brassica juncea) seeds, has been isolated and characterized. Its primary structure has been elucidated. This protein is composed of two chains (37 and 92 amino acids) linked by disulphide bridges. The amino acid sequence obtained is closely related to that previously determined for Sin a I, an allergen isolated from yellow mustard (Sinapis alba). A common epitope has been detected in the large chain of both Bra j IE and Sin a I by means of electroblotting and immunodetection with 2B3, which is a monoclonal antibody raised against the yellow-mustard allergen. A histidine residue of the large chain of both mustard allergens has been found to be essential for the recognition by 2B3 antibody. A synthetic multiantigenic peptide containing this His was recognized by 2B3 as well as by sera of mustard-hypersensitive individuals. Therefore this antigenic determinant must be involved in the allergenicity of these proteins.

Cloning and expression of the major allergen from yellow mustard seeds, Sin a I.

The cloning and bacterial expression of the major allergen from yellow mustard (Sin a I) is reported. Cloning has been carried out by means of the polymerase chain reaction using non-degenerate oligo primers encoding the N- and C-terminal regions of the mature protein. Two nucleotide sequences (SA2S1 and SA2S2) have been found and analysed. The observed polymorphism suggests the existence of multiple isoforms for Sin a I allergen. SA2S2 has been expressed as a fusion protein, linked to the choline-binding domain of the Streptococcus pneumoniae murein hydrolase. The resulted fusion protein is recognized by Sin-a-I-specific antibodies.

Isolation and characterization of a major allergen from oriental mustard seeds, BrajI.

A 2 S albumin from oriental mustard (Brassica juncea) seeds has been isolated and characterized as an allergen. This protein, BrajI, was recognized by human IgE from mustard-sensitive individuals, as proved by using two different enzyme immunoassays. BrajI was found to be closely related to Sin a I, the major allergen from yellow mustard seeds. Many fractions with molecular weights ranging from 16,000 to 16,400 and with differences in charge were separated by ion-exchange chromatography. They exhibited small but significant amino acid composition differences for Glx, Val, Ile, Lys, and Arg contents. The heterogeneity of BrajI can be explained by size and charge differences of its heavy and light chains. All of the isoallergenic forms of BrajI gave a single precipitation band in double diffusion immunoassays when using a Sin a-I-specific rabbit polyclonal serum.