Characterisation of oyster allergens for improved diagnosis of mollusc allergy

• Main Author: Nugraha, Roni
• Format: Thesis
• Language: unknown
• Published: 2018
• Subjects: Pacific; Pacific oyster; Mite
• Online Access: https://researchonline.jcu.edu.au/60529/1/JCU_nugraha_2018_thesis.pdf

Discovery and characterisation of allergens in various food and inhalant sources is central to our understanding of the molecular mechanisms of allergic reactions. Allergen characterization is the most important underlying factor for better patient management with improved diagnostics, and the design and development of novel immunotherapeutics. Of the 'Big eight' allergen food groups, shellfish presents a unique challenge in terms of allergen discovery due to the large number and diversity of consumed species, leading to heterogeneity of allergen structure and cross-reactivity among various sources. The group of 'shellfish' comprises of two invertebrate phyla; arthropods and molluscs. Although all shellfish are invertebrate animals, these two groups are very distinct in evolutionary terms and subsequently contain different molecular repertoires of allergenic proteins. Co-sensitisation of patients with crustacean and mollusc allergy is often described, however, the current diagnostic approaches to manage patients is not based on sufficient molecular knowledge of these shellfish allergens. Consequently, mollusc allergy is clinically underreported and allergens are ill-defined. To date, only five mollusc allergens are listed in the WHO/International Union of Immunological Societies (IUIS) Allergen Nomenclature SubCommittee database, all of which are different tropomyosin's (http://www.allergen.org/index.php). Additional mollusc allergens have been reported, but not yet fully characterised. A detailed review of the current status and the diagnosis of mollusc allergy is provided in Chapter 1. Current strategies for allergen identification are time- and resource-consuming, which are highly prone to missing hidden allergens present in low concentrations. Allergenic proteins are traditionally identified based on their serum-specific IgE antibody recognition. Soluble proteins derived from whole protein preparations of a suspected allergen source are screened for IgE antibody binding proteins using sera from individuals with clinically confirmed allergy. Although this approach is the current standard for allergen identification, there are three major drawbacks. Firstly, this approach often does not detect allergenic proteins present in low abundance. Secondly, cross-reactive allergens are not easily identified due to their possible presence in unrelated allergen sources. Thirdly, the IgE recognition patterns are highly dependent on the demographics of the particular allergic patient cohort under investigation. This PhD thesis presents a comprehensive study on the improvement of allergen discovery from Pacific oyster, the most widely consumed mollusc species and immunological characterisation of the major allergen, tropomyosin, using a cohort of mollusc-sensitised patients in Australia. Furthermore, novel ways to diagnose cross-reactivity between crustacean and mollusc species were developed. Chapter 2 describes a comprehensive discovery pipeline, for allergenic proteins, that accounts for biological and molecular variability using allergenomics, high-throughput screening of genomic databases and high-resolution mass spectrometry. This methodological approach was successful in identifying 24 previously unreported allergens from over 25,000 proteins from the Pacific oyster. This is the first study to demonstrate the presence of 24 hidden allergens, also found in very different allergen sources from animals, including fish and mites, as well as plant allergens from pollen, latex and fungi. Importantly all of these allergenic proteins identified are reactive with shellfish allergic patients' IgE antibodies. However, it was demonstrated in chapter 2 that not all allergens present in the genome and transcriptome of oyster are also detected in the extracted proteome. Allergens are often overlooked during the extraction process due to the use of inappropriate buffers which might affect the in vitro diagnostic methods that use whole proteins extracts. In Chapter 3 various buffer compositions covering a wide range of pH were evaluated to improve the detection of the unreported allergens described in chapter 2. The IgE-reactivity of protein extracts from each buffer was determined against a pool of serum from five shellfish allergic patients. In addition, the protein composition of the Pacific oyster was analysed using high-resolution mass spectrometry. High concentrations of protein were recovered after extraction using high salt content or high pH buffers, subsequently revealing more IgE-reactive bands on the immunoblotting. Low pH buffers, however, resulted in poor protein recovery and affected negatively patient IgE-reactivity. Mass spectrometry analysis discovered that the novel IgE-reactive proteins, particularly of high molecular weight, emerged due to an increased abundance in the allergen extract. Overall, increasing the ionic strength and pH of the extraction buffers improves the solubility of allergenic proteins. In Chapter 4, a detailed analysis of immunological characteristics of the Pacific oyster extracts and the major allergen, tropomyosin were conducted. Twentyone oyster-sensitised patients were analysed to determine the prevalence of each allergen in the patient cohort. Eighteen out of 21 patients showed reactivity to tropomyosin although the binding intensity varied between patients. Patients who lacked IgE-binding to tropomyosin were shown to be sensitised to other oyster allergens. These allergenic proteins were preliminary abundant in the raw extract of the Pacific oyster. Further investigation was carried out with the Pacific oyster tropomyosin. The natural wild type and recombinant tropomyosin were successfully purified, and their structural properties observed. Both purified natural and recombinant tropomyosin had very similar structural and immunological properties. Cross-reactivity analysis using ELISA demonstrated patients who demonstrated IgE reactivity to the Pacific oyster tropomyosin were also reactive to other tropomyosin's from Black tiger prawn as well as the House dust mite. The degree of crossreactivity correlated well with the tropomyosin amino acid sequence identity. Sequence alignment of tropomyosin from those three species revealed five protein regions containing predicted IgE-binding epitopes responsible for the strong cross-reactivity observed. Finally, in Chapter 5 to improve the prediction of clinical cross-reactivity between crustaceans and molluscs as well as other invertebrate species, conservation analysis of IgE-binding epitopes of four shrimp allergens were carried out. The results demonstrated that within a large directory of shrimp IgE-binding epitopes there are a substantial fraction of epitopes that are highly conserved across various invertebrate species. Shrimp TM and AK shared a higher number of conserved epitopes compared to shrimp SCP and MLC; in fact, no conserved epitope could be found for SCP, while MLC only shared two epitopes in one region with cockroach MLC. These results suggest that TM and AK are the major contributing proteins in immunological and clinical cross-reactivity between crustacean and other invertebrate groups. Furthermore, comparative evaluation of the number of conserved epitopes in TM and AK revealed a clear cross-reactive hierarchy where cockroach has the highest number followed by mite, and molluscs are on the bottom of this hierarchy. In conclusion, the outcomes of this thesis have demonstrated that many unreported allergens have been overlooked due to the limitations of the current allergen discovery methodology. The utilisation of transcriptome data and proteomic techniques in addition to the well-established allergenomic approach improves discovery of unreported allergens. While tropomyosin seems to be a clinically relevant cross-reactive major allergen, the presence of additional allergens that has never been reported in mollusc species, suggests mollusc species contain different molecular repertoires of allergens. Supported by the findings of the in-depth bioinformatics analysis of IgE-binding epitopes, component-resolved diagnostics for mollusc allergy could be developed, enabling precise identification of patients sensitised to a specific mollusc group and distinguish from patients with extensive cross-reactivity to ingested and inhaled allergens from other invertebrate sources.