Supplementary material for the article: Nikolić, J.; Nešić, A.; Kull, S.; Schocker, F.; Jappe, U.; Gavrović-Jankulović, M. Employment of Proteomic and Immunological Based Methods for the Identification of Catalase as Novel Allergen from Banana. Journal of Proteomics 2018, 175, 87–94. https://doi.org/10.1016/j.jprot.2018.01.007

Supplementary material for: [https://doi.org/10.1016/j.jprot.2018.01.007]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2117

Supplementary material for the article: Nikolić, J.; Nešić, A.; Kull, S.; Schocker, F.; Jappe, U.; Gavrović-Jankulović, M. Employment of Proteomic and Immunological Based Methods for the Identification of Catalase as Novel Allergen from Banana. Journal of Proteomics 2018, 175, 87–94. https://doi.org/10.1016/j.jprot.2018.01.007

Supplementary material for: [https://doi.org/10.1016/j.jprot.2018.01.007]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2117

Maternal transfer of the most potent peanut allergen Ara h 2 into human breast milk in a German cohort

Background Peanut allergy is known to be one of the most severe food allergies. The aim of our breast milk study was to investigate the transfer of the most potent peanut allergen Ara h 2 into human breast milk in a German cohort. Therefore, the time courses of appearance after ingestion, the potential risk of sensitization to peanuts via breast milk in Germany and, if possible, the way Ara h 2 will be processed in vivo after secretion into breast milk was studied. Methods Of 32 lacating, non-peanut allergic women, breast milk samples were collected at different time points after ingestion of 100 g dry roasted peanuts (approved by the local ethics committee). Breast milk samples were analysed for peanut protein in SDS-PAGE, Western blot and ELISA (Neogen Veratox ELISA® and an ELISA against digestion resistant Ara h 2 (DRP-Ara h 2)). Natural Ara h 2 was digested by Enzynorm f® and Kreon® to mimic the effect of the combined gastric and duodenal digestion in vivo and, subsequently, analysed by N-terminal sequencing and MALDI TOF MS. Results Ara h 2 was undetectable using Western blot. Performing the Neogen Veratox ELISA® against crude peanut extract peanut proteins still remained undetectable. However, Ara h 2 was identified by an ELISA against DRP-Ara h 2 in 8/32 women (25 %) at different concentrations and time points of appearance. To assess the way Ara h 2 is processed in vivo, natural Ara h 2 was digested into several digestion resistant immunoreactive peptides \u3c15 kDa after treatment with Enzynorm f® and Kreon®, and a 12 kDa fragment was identified by N-terminal sequencing and mass spectrometry corresponding to the middle part of Ara h 2. Conclusions After maternal ingestion Ara h 2 is secreted into breast milk in our German cohort in 25 % of the volunteers, individually either rapidly (after 1h, 2h or 4 h) or delayed (after 8h or 12h) and in different concentrations. To study Ara h 2 or Ara h 2 peptides that survive digestion and pass into human breast milk antibodies against the 12 kDa fragment are now raised for enrichment strategies to characterize these sensitizing or tolerogenic peanut structures in our breast milk samples

Employment of proteomic and immunological based methods for the identification of catalase as novel allergen from banana

Diagnostic reagents based on food allergen extracts often lack sufficient sensitivity. The introduction of well characterized food allergens in molecular allergy diagnosis has been recognized as valid approach to circumvent unstandardized allergen extracts. Banana fruit (Musa acuminate) is a well-established allergen source which besides six characterized allergens, contains unidentified IgE reactive proteins whose clinical relevance remains undefined. By employment of a combinatorial peptide ligand library (CPLL) methodology with 2-D PAGE, mass spectrometric and 2-D immunoblot analysis, a novel allergen from banana fruit was detected in banana as catalase. A recombinant homologue of natural catalase was produced, isolated and biochemically characterized. The recombinant protein showed IgE reactivity in 7 out of 13 tested patients with suspected allergy to banana in immunoblot. Novel banana fruit allergens should be added as components to allergen-microarrays for the diagnosis and the monitoring of banana allergy. Significance: By employment of CPLL methodology with 2-D PAGE, mass spectrometric and 2-D immunoblot analysis catalase from banana fruit is identified as a novel allergen, with proposed designation as Mus a 7. IgE reactive recombinant Mus a 7 was produced and should be included in a component-resolved allergy diagnosis.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3203

Development of a novel strategy to isolate lipophilic allergens (oleosins) from peanuts.

BACKGROUND:Peanut allergy is one of the most severe class I food allergies with increasing prevalence. Especially lipophilic allergens, such as oleosins, were found to be associated with severe symptoms, but are usually underrepresented in diagnostic extracts. Therefore, this study focused on isolation, molecular characterization and assessment of the allergenicity of peanut oleosins. METHODS AND RESULTS:A comprehensive method adapted for the isolation of peanut oil bodies of high purity was developed comprising a stepwise removal of seed storage proteins from oil bodies. Further separation of the oil body constituents, including the allergens Ara h 10, Ara h 11, the presumed allergen oleosin 3 and additional oleosin variants was achieved by a single run on a preparative electrophoresis cell. Protein identification realized by N-terminal sequencing, peptide mass fingerprinting and homology search revealed the presence of oleosins, steroleosins and a caleosin. Immunoblot analysis with sera of peanut-allergic individuals illustrated the IgE-binding capacity of peanut-derived oleosins. CONCLUSION:Our method is a novel way to isolate all known immunologically distinct peanut oleosins simultaneously. Moreover, we were able to provide evidence for the allergenicity of oleosins and thus identified peanut oleosins as probable candidates for component-resolved allergy diagnosis

Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library

The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA library of hazelnut was constructed. The library was screened with serum of six hazelnut allergic patients displaying different IgE-binding patterns on hazelnut immunoblot. Rapid amplification of cDNA ends (RACE) protocols were applied to obtain full-length clones. Expression experiments were carried out in Eschericchia coli. Expression was monitored by SDS-PAGE, protein staining and immunoblotting. A hazelnut cDNA library was constructed. IgE screening resulted in the cloning of two isoforms of a novel putative hazelnut allergen. The clones were identified as oleosins, with theoretical molecular masses of 16.7 and 14.7 kDa and pI of 10.5 and 10.0, respectively. The isoforms demonstrated only 37% amino acid sequence identity but contained the typical hydrophobic stretch in the middle of the protein (53% identity) with the characteristic oleosin proline knot region (11/12 amino acids identical). Expression in E. coli of the longer isoform resulted in a clear band on SDS-PAGE. The expressed protein was recognized on an immunodot blot by IgE from serum that was used for screening the cDNA library. Hazelnut contains multiple isoforms of oleosin. IgE binding of a hazelnut-allergic patient to a recombinant version suggest that hazelnut oleosin is an allergen, as has been described for peanut and sesam

Identification of peanut oleosins using peptide mass fingerprinting and N-terminal sequencing.

<p>Peptides resulting from tryptic digestion were searched against the NCBInr database using the Mascot search engine. Bold letters indicate identified sequences by MALDI-TOF-MS, whereas underlined letters mark sequences obtained after N-terminal sequencing. The hydrophobic domains, not accessible to tryptic cleavage, are written in italics. The methionine residue, highlighted in grey, was not observed.</p

Western blot of oleosin prototypes isolated from roasted peanuts and purified by preparative electrophoresis.

<p>Fractions obtained by preparative electrophoresis containing the prototypes 1/2 (A) and prototypes 3/4 (B) were pooled, subjected to SDS-PAGE and subsequent immunoblotting. M, molecular mass marker; G, Gold staining; CS, Coomassie staining; A, polyclonal anti-oleosin antibody; P1–P4, allergic patients’ sera; P°, allergic individual (not to peanut) with respiratory symptoms; P*, healthy control; C1, secondary antibody control (mouse anti-human IgE antibody); C2, secondary antibody control (goat anti-rabbit IgG antibody).</p

Recombinant lipid transfer protein Cor a 8 from hazelnut: a new tool for in vitro diagnosis of potentially severe hazelnut allergy

BACKGROUND: Cor a 1.04 has been identified as the major hazelnut allergen in 65 European patients with positive double-blind, placebo-controlled food challenge results to hazelnut. Recently, the 11S globulin Cor a 9 was shown to be a pollen-independent hazelnut allergen in the United States, whereas preliminary data suggest the lipid transfer protein (LTP) as an important birch pollen-unrelated hazelnut allergen in Europe. OBJECTIVE: We sought to recruit a group of European patients allergic to hazelnut without birch pollen allergy and to identify and clone the major food allergen(s) in this study population. METHODS: We recruited 26 such Spanish patients, including 10 patients with anaphylaxis. IgE immunoblotting was performed with hazelnut extract. Hazelnut LTP Cor a 8 was cloned by using a PCR strategy, purified, and subjected to IgE immunoblotting. Recombinant Cor a 8, rCor a 1.0401, and rCor a 2 (profilin) were further investigated by means of enzyme allergosorbent test. Immunoblot inhibition experiments were used to compare the immunologic properties of natural and recombinant LTP. RESULTS: A 9-kd major allergen was identified in hazelnut extract. Cloning, sequencing, heterologous expression, and inhibition experiments identified it as an LTP. The prevalence of specific IgE antibody reactivity to LTP was 62% in hazelnut extract and 77% when recombinant LTP was tested by means of immunoblotting. IgE immunoblot inhibition with hazelnut extract showed that natural Cor a 8 and rCor a 8 shared identical epitopes. Only one patient had positive reactivity to Cor a 1.04, and no patients had positive reactivity to Cor a 2. Two sera bound to high-molecular-weight allergens. The LTP was denominated as Cor a 8 and submitted to the allergen database of the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Subcommittee. CONCLUSIONS: Cor a 8 is a relevant allergen for a majority of Spanish patients with hazelnut allergy that can cause severe allergic reaction