IgE–Mediated Reactions to Autoantigens in Allergic Diseases

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Background: Complex allergenic sources such as moulds, foods and mites contain complex panels of IgE-binding molecules which need to be cloned, produced and characterized in order to mimic the entire allergenicity of whole extracts reconstituted by mixing single standardized recombinant allergens. Methods: Phage surface display of cDNA libraries selectively enriched for allergen-expressing clones using IgE from allergic patients allows rapid isolation of large panels of allergens. For the characterization of all different clones present in enriched cDNA libraries in a fast and cost-effective way, high-throughput screening technology is required. Results: The combination of selective enrichment of cDNA libraries based on biopanning against serum IgE from sensitized patients and automated robot technology for picking and high-density gridding of clones onto filter membranes, followed by hybridization, enables fast identification of all the different clones present in an enriched library. The consequent application of selective enrichment and robotic-based screening allows, within weeks, cloning and characterization of the whole allergenic repertoire of any organisms. Conclusions: Robotic-based high-throughput screening of clones selected for IgE-binding capacity from phage surface-displayed cDNA libraries of Aspergillus fumigatus, Cladosporium herbarum, Coprinus comatus, Malassezia furfur, peanut and human lung tissue allowed rapid characterization of 81, 28, 37, 27, 8 and 151 different sequences, respectively. All these cDNAs bear a high probability to encode allergens derived from the respective allergenic source.

Section: Resultsmentioning

confidence: 99%

Tapping Allergen Repertoires by Advanced Cloning Technologies

Crameri¹,

Kodzius

Konthur

et al. 2001

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“…So far, Mn-dependent SODs have been identified as allergen and extensively studied in different allergenic sources ( Aspergillus fumigatus [31], Hevea brasiliensis latex [32]). Mn-SOD allergen from A. fumigatus shows a high homology with the human correspondent enzyme and seems to be involved in the IgE recognition of autoallergens [31, 33, 34]. The Mn-SOD from latex (Hev b 10) could also be involved in IgE cross-reactivity with SODs from related or unrelated species [32].…”

Section: Discussionmentioning

confidence: 99%

Cloning and Expression of the <i>Olea europaea</i> Allergen Ole e 5, the Pollen Cu/Zn Superoxide Dismutase

Butteroni

Afferni²,

Barletta³

et al. 2005

Background: Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity. Methods: cDNA of Ole e 5 was amplified by nested 3′-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients’ IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition). Results: The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein. Conclusions: Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens.

“…The presence of autoantibodies against the ribosomal P proteins in autoimmune diseases [38] and their association in IgE-mediated allergic diseases [39] confirm the allergenicity of Pen b 26 protein of P. brevicompactum . All P proteins seem to contain an important epitope structure in their C-terminus that is recognized by the antibodies in the sera of individuals with autoimmune diseases, such as systemic lupus erythematosus [40], protozoan infections like leishmaniosis [41], and Chagas’ heart disease [42].…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of a cDNA Clone Encoding One IgE-Binding Fragment of <i>Penicillium brevicompactum</i>

Sevinc

Kumar

Abebe

et al. 2005

Background:The abundance of allergenic Penicillium species has been associated with an increased incidence of childhood asthma and pulmonary bleeding. Penicillium brevicompactum has been identified as the most prevalent indoorspecies of this genus. However, detailed studies on the allergens of the ubiquitous Penicillium species are still lacking. For the characterization of allergens of prevalent Penicillium species, molecular cloning of the allergen genes of P. brevicompactum was performed in the present study. Methods:A phage cDNA library of P. brevicompactum was constructed in Uni-ZAP^® XR vector using mRNA isolated from the organism. The cDNA library of P.brevicompactum was screened using pooled atopic sera. Results: Screening of P. brevicompactum cDNA library resulted in one positive clone encoding an estimated molecular weight of 11 kDa polypeptide, rich in acidic residues (>20%), with a pI of 3.87. This clone was designated as Pen b 26 and found to be reactive only against the atopic sera obtained from individuals sensitive to P. brevicompactum. The amino acid sequence analysis of Pen b 26 revealed that it had strong homology to the 60S acidic ribosomal protein P1 family from different eukaryotic sources, predominantly fungal aero-allergens. Other features of Pen b 26 including having high alpha-helical content (>50%), alanine-rich residues (>20%), and a well-conserved C-terminal epitope region fits well into the common properties of 60S acidic ribosomal proteins. Conclusions:The results obtained suggest that the allergenic clone, Pen b 26 is a 60S acidic ribosomal protein P1 of P. brevicompactum and shows strong similarity to other P1 family proteins.