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

Isolation and Functional Characterization of the Novel Clostridium botulinum Neurotoxin A8 Subtype

Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT). Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

The detection of botulinum neurotoxins (BoNT) is extremely challenging due to their high toxicity and the multiple BoNT variants. To date, seven serotypes with more than 30 subtypes have been described, and even more subtypes are expected to be discovered. The fact that the BoNT molecules are released as large complexes of different size and composition adds further complexity to the issue. Currently, in the diagnostics of botulism, the mouse bioassay (MBA) is still considered as gold standard for the detection of BoNT in complex sample materials. Over the years, different functional, immunological, and spectrometric assays or combinations thereof have been developed, supplemented by DNA-based assays for the detection of the organism. In this review, advantages and limitations of the current technologies will be discussed, highlighting some of the intricacies of real sample analysis

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

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

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

Multiplex detection of microbial and plant toxins by immunoaffinity enrichment and matrix-assisted laser desorption/ionization mass spectrometry

Plant and microbial toxins such as ricin, staphylococcal enterotoxin B (SEB), and the botulinum neurotoxins (BoNT) are considered as potential biological warfare agents. Specific screening methods are, therefore, required that enable unambiguous and sensitive identification of these biohazards, particularly for the occurrence of the toxins in complex sample matrixes. The present study describes a combination of a multiplex-immunoaffinity purification approach, followed by matrix-assisted laser desorption/ionization (MALDI)-based detection for the simultaneous identification of ricin, SEB, BoNT/A, and BoNT/B. The method comprises an affinity enrichment step, using specific monoclonal antibodies for each of the four toxins which have been selected from a pool of antibodies. The selected antibodies allow for specific and simultaneous capture of ricin, SEB, BoNT/A, BoNT/B, and the corresponding BoNT complexes. These were subsequently identified by MALDI time-of-flight (TOF) mass spectrometry (MS), following tryptic digest. The sensitivity of the technique was approximately 500 fmol for each of the toxins. These toxins were detectable within 8 h, even when present in complex matrixes such as milk or juice. Furthermore, the MALDI-based multiplex assay allowed for the discrimination of closely related BoNT sero- and subtypes, including a real case of food-borne botulism in Germany