Molekulare und immunologische Charakterisierung von Profilin aus Weizen (Triticum aestivum)

Krankheiten die nach dem Verzehr von Weizenprodukten bzw. nach der Inhalation von Weizenmehl auftreten sind Zöliakie, Weizenallergie und Bäckerasthma. Eine weitere, jedoch selten auftretende Krankheit, ist die durch den Verzehr von Weizen mit nachfolgender körperlicher Betätigung ausgelöste Anaphylaxie (WDEIA) Ein potentieller Auslöser für Weizenallergie, WDEIA und Bäckerasthma ist Profilin aus Weizen. Ziel dieser Arbeit war es, Profilin (Tri a 12), ein Protein aus Weizen (Triticum aestivum) zu klonieren, als rekombinantes nicht-Fusionsprotein in E. coli zu exprimieren, zu reinigen und seine Eigenschaften zu untersuchen. Gesamt-RNA wurde aus Weizen (Cultivar Edison) isoliert und mittels PCR konnte die Tri a 12 cDNA kloniert und sequenziert werden. Die kodierende Sequenz besteht aus 396 Basenpaaren (inklusive Start/Stoppcodon) und wurde in den Expressionsvektor pET17b kloniert. Für die Expression bei 30°C wurden E. coli BL21(DE3) Zellen verwendet. Rekombinantes Tri a 12 wurde aus der löslichen Zellfraktion isoliert und mittels Affinitätschromatographie gereinigt. Tri a 12 besteht aus 131 Aminosäuren (inklusive Start-Methionin) und hat eine intakte molekulare Masse von 14,031 Da und einen isoelektrischen Punkt von 5,04. N-terminales Sequenzieren der ersten fünf Aminosäuren und Massenspektroskopie verifizierten die Sequenz von Weizenprofilin. Zirkuläre Dichroismus Spektroskopie zeigte, dass rTri a 12 nach Reinigung als gefaltetes Protein vorliegt und eine gemischte α/β-Sekundärstruktur besitzt. IgE Bindung wurde mittels IgE Immunoblot und IgE ELISA untersucht. Hierfür wurden Seren von Patienten mit diagnostizierten allergischen Symptomen nach dem Verzehr von Weizenprodukten bzw. Weizenmehlinhalation verwendet. Tri a 12, ein Mitglied der Profilin-Familie, wurde identifiziert und charakterisiert.There are three major diseases caused by either wheat consumption or wheat flour inhalation: celiac disease, wheat allergy and bakers’ asthma. A fourth disease is the very rare wheat-dependant exercise induced anaphylaxis (WDEIA). These four diseases display different symptoms and are caused by different allergens. One potential causative agent is wheat profilin. Total RNA from wheat (cultivar Edison) was isolated and PCR with gene specific primers was performed. cDNA encoding for Tri a 12 was cloned and sequenced. The coding sequence consisting of 396 bp (including start- and stopcodon) was cloned into the expression-vector pET17b. E.coli BL21(DE3) was used as host strain for expression at 30°C. Recombinant Tri a 12 was isolated from the soluble cell fraction and purified by affinity chromatography. Tri a 12 has a deduced amino acid sequence of 131 amino acid residues (including initial methionine) and an intact molecular mass of 14, 031 kDa and an isoelectric point of 5.04. N-terminal sequencing of the first five amino acid residues and mass spectroscopy confirmed the expected wheat profilin sequence. Circular dichroism spectroscopy showed that rTri a 12 was expressed and purified as a folded protein since it displayed a mixed α/β-secondary structure. IgE binding properties were investigated by IgE immunoblotting and IgE ELISA. Sera from diagnosed wheat allergic patients were used for the investigation of IgE binding properties. Tri a 12, a member of the profilin protein family, was identified and characterised. It represents another molecule suitable for component-resolved diagnosis and for defining cross reactivity among profilins

Expression, Purification and Crystallization of Wheat Profilin (Tri a 12)

Profilin from wheat (Triticum aestivum) has been identified as an allergen (Tri a 12). The recombinant 14 kDa protein was produced in Escherichia coli, purified and crystallized using the hangingdrop vapour-diffusion method. A diffraction-data set was collected in-house from a single crystal to a resolution of 3.3 Å. The crystals belonged to space group P3221, with unit-cell parameters a = b = 58.9 Å, c = 82.5 Å, α = β = 90° and γ = 120°. (doi: 10.5562/cca1790

Comparison of two multiplex arrays in the diagnostics of allergy

Abstract The objective of this analysis was to compare the multiplex ImmunoCAP ISAC (Thermo Fisher Scientific, Sweden) and the multiplex Alex Allergy Explorer (Macro Array Diagnostics GmbH, Austria) platform on specific IgE to grass pollen (Phl p 1, Phl p 5), tree pollen (Bet v 1), house dust mites (Der p 1, Der p 2) and cat (Fel d 1) allergens in allergic patients. Our findings demonstrate a good correlation of presently used methods to detect serum sIgE. Multiplex testing of allergen-specific IgE can be the method of choice for a prospective component-resolved diagnosis of type I allergy, and the basis for the design and monitoring of a patient-tailored specific therapy

Identification of Potentially Tolerated Fish Species by Multiplex IgE Testing of a Multinational Fish-Allergic Patient Cohort

Background: Although recent studies indicated that many fish-allergic patients may safely consume certain fish species, no clinical guidelines are available for identification of the exact species tolerated by specific patients. Objective: To investigate whether multiplex immunoglobulin E (IgE) testing reveals potentially tolerated fish through absence of IgE to parvalbumin (PV) and extracts from specific species. Methods: Sera from 263 clinically well-defined fish-allergic patients from Austria, China, Denmark, Luxembourg, Norway, and Spain were used in a research version of the ALEX 2 multiplex IgE quantification assay. Specific IgE to PVs from 10 fish species (9 bony and 1 cartilaginous), and to extracts from 7 species was quantified. The IgE signatures of individual patients and patient groups were analyzed using SPSS and R. Results: Up to 38% of the patients were negative to cod PV, the most commonly used molecule in fish allergy diagnosis. Forty-five patients (17%) tested negative to PVs but positive to the respective fish extracts, underlining the requirement for extracts for accurate diagnosis. Between 60% (Spain) and 90% (Luxembourg) of the patients were negative to PV and extracts from ray, a cartilaginous fish, indicating its potential tolerance. Up to 21% of the patients were negative to at least 1 bony fish species. Of the species analyzed, negativity to mackerel emerged as the best predictive marker of negativity to additional bony fish, such as herring and swordfish. Conclusions: Parvalbumins and extracts from multiple fish species relevant for consumption should be used in fish-allergy diagnosis, which may help identify potentially tolerated species for individual patients