Chapter CHAPTER 5: DISCUSSION, CONCLUSION AND FUTURE PROSPECTS OF THE RESEARCH WORK 189 Chapter 5.1 DISCUSSION AND CONCLUSION Not many allergenic fungi are studied well and very few of them have been characterized for the allergens in detail. Characterization of the fungus and its constituent allergens is vital towards proper understanding of the fungus and the patterns of disease elicited by it. This study highlighted the importance of a clinically known but less studied (for allergenicity) tropical fungal species, Curvularia lunata. The fungus is prevalent in the tropics and is found both indoors and outdoors. Being an important fungus of the Singapore environment, it is very important to understand the effects of this fungus on the sensitive patients. This study aided in the better understanding of the IgE binding patterns; geographically (on the local as well as international population) as well as on patients with varied atopy. To date, around 100 fungal allergen sequences are present in various databases. Apart from the allergens of Aspergillus fumigatus and Alternaria alternata, very few have been well characterized for: IgE binding frequency/intensity over a population, crossreactivity, structure and critical residues for allergenicity, levels in the environment, function, etc. Here, we report various allergens from Curvularia lunata. For the rapid isolation of the allergenic proteins from Curvularia lunata, combinatorial strategy involving expressed sequence tagging (EST) approach as well as proteomics approach was utilized. EST approach utilizes sequence similarity as a means of identification whilst proteomics approach is based on identification of IgE binding proteins by mass spectrometry. Hence, the known allergens would have been identified by ESTs while the novel unknown allergens (if any) would have been discovered by proteomics. EST based 190 Chapter identification is a faster and high throughput way of identifying allergens based on sequential similarity. A total of 1683 ESTs were sequences and catalogued from a nonnormalized C.lunata library. More than 50% of the ESTs matched a known protein available in the GenBank protein database. Most of these proteins were classified under the general house keeping proteins such as metabolism, nucleotide biosynthesis, gene expression and protein synthesis. ESTs serve as a basis for studying various aspects like allergen component, identification of allergen variants, phylogenetic studies and protein identification by proteomics. About 2% of the ESTs encoded for 14 different (fungal as well as non-fungal) allergen types. To identify novel IgE binding components (if any) present in Curvularia lunata, 1D western blots of various fungi (including C.lunata) were developed. The IgE binding bands present in C.lunata were excised and subjected to tandem mass spectrometric identification. different IgE binding components (Alcohol dehydrogenase, Manganese superoxide dismutase, Thioredoxin and Cyclophilin) were identified. These allergens were already identified by ESTs though. To identify the proteome of C.lunata for standardization of the allergen extracts as well as to study the levels/forms of various allergen transcripts, 2D SDS PAGE were done. Further, around 150 spots were identified by tandem mass spectrometry. Spots for different allergen types (Manganese superoxide dismutase, Cyclophilin, heat shock protein 70, Calcium binding protein and Enolase) were identified suggesting these proteins to be expressed in the proteome. All of these allergens (except) enolase were already found in the ESTs. 191 Chapter Out of the 14 different putative allergen types isolated, recombinant proteins for 11 different types were generated using the pET32Ek/LIC expression system. Also, the recombinant homologues for various (fungal as well as human) homologs of these proteins were generated for cross-comparison. For the confirmation of allergenicity of these identified putative allergens as well as characterization of the allergens (along with the homologs) further studies were performed. IgE binding patterns over patients` sera from populations (fungal atopic Singaporean population, atopic Italian population, asthmatic Colombian population and fungal atopic Indian population) were studied for better understanding of the allergenicity of these proteins. The populations were selected based on differential geography as well as reactivity to various allergens. C.lunata recombinant proteins were found to bind patients IgE from all the populations confirming the allergenicity. Further, out of the 11 different C.lunata recombinant proteins tested, allergens [Cur l (manganese superoxide dismutase), Cur l (thioredoxin), Cur l 10 (alcohol dehydrogenase) and Cur l 12)] were considered immunologically important allergens showing IgE binding to more than 50% of the patients suggesting them to be major allergens of C.lunata. Comparing the IgE binding amongst various homologous allergen groups, differential IgE binding patterns were observed suggesting some allergen groups to bind patients IgEs with higher frequency than others. Within the homologous allergen groups, it was observed that reaction pattern for one allergen type correlated strongly with that of its homolog suggesting possible cross-reactivity/co-sensitization. 192 Chapter Confirmation of cross-reactivity by IgE inhibition studies suggested that most of the homologous allergen groups are cross-reactive within themselves making them Pan allergens. A possible explanation for this can be due to the fact that the isolated allergens were generally from the house keeping genes, known to be conserved across phylogeny giving rise to cross-reactivity due to the conserved domains. Also, the human homologs of the fungal allergens also showed IgE binding; suggesting that the cross-reactivity is prevalent not only amongst the fungal proteins but is also extended to mammalian proteins. This might be due to the formation of autoantibodies to human proteins (due to cross-reactivity with fungal allergens) after the initial exposure from fungal allergens (Valenta et al., 2000). This phenomenon is termed as Autoallergy. This phenomenon was first reported by Storm van Leeuwen and Keller who independently reported reactions to aqueous human dander extracts in severely atopic patients (Keller, 1924; Storm van Leeuwen et al., 1926). Moreover, some studies showed that the individual exhibiting cross-reactivity between environmental and endogenous human proteins (e.g. profilins and manganese superoxide dismutases) showed stronger IgE binding to the environmental allergens that to the endogenous counterparts suggesting the environmental allergens to be the primary sensitizers (Valenta et al., 1991; Crameri et al., 1996). Our data hence confirms the fact that autoallergy exist among fungal atopic patients and the primary sensitizers are the fungal allergen counterparts. This phenomenon is alarming and need to be dealt with by studying in detail as it might involve severe disease symptoms, especially in the patients with atopic dermatitis who are constantly being exposed to the environmental as well as skin (self) antigens. 193 Chapter The three dimensional structures are of great importance as they open new avenues to reduce the allergenic activity of allergens by genetic engineering. The structural similarity of the allergens (as observed in our work) can be correlated to the allergenicity of the allergens since it’s the three dimensional structure of the allergen which determines the interaction of the allergen to the IgE antibodies. From the isolated C.lunata allergens, some of them belonged to the stress response pathway. For example, the alcohol metabolism pathway involves biological conversion of alcohol to aldehyde, which is further converted to carboxylic acids. The two enzymes responsible for these bioconversions are alcohol dehydrogenase and aldehyde dehydrogenase; both of them are identified as allergens in C.lunata. Similarly, for the elimination of toxic oxygen free radical (O2-), superoxide dismutase is required which converts it to less harmful hydrogen peroxide (H2O2) which can be further converted to water. This superoxide dismutase protein was also showed to be allergenic. Hence, this observation suggests that the stress response proteins form an important cohort of the fungal allergens. As most of the fungi are parasitic or saprophytic, they need to overcome stress (chemical or biochemical) present in the host environment for their survival. For this purpose, they have various stress response proteins for their defense in the unfavorable environments. These proteins being allergenic suggest that the stress response drive of the fungi gives rise to this cohort of allergens. Further characterization of the allergen groups showed that these allergens are present in the quantifiable amounts in the environment and may cause possible sensitization. Last but not the least, this work helps in identifying the allergens which are to be used for immunotherapy. In immunotherapy, increasing doses of allergens are administered 194 Chapter to allergic patients which induces the IgE (blocking antibodies) inhibiting the allergic response to IgE. Traditionally, allergen immunotherapy involved crude allergen extracts. The problem with the use of crude allergen extract is that it’s a heterogeneous mix of proteins and has varying amounts of antigens. The level of allergens may vary from batch to batch. Also, the extracts might also be contaminated by allergens from other sources and may also possess toxins. Hence, vaccination with crude extracts at times gives heterogeneous immune response. Hence, for this reason, specific immunotherapy based on specific allergen is proposed. Recombinant allergens, mimicking structures and immunogenicity represent potential candidates for specific allergen immunotherapy. Hence, our work helps in providing with a platform for generating allergenic protein candidates for specific immunotherapy. Moreover, instead of diagnosing the patients by using crude extracts, we suggest using recombinant proteins or homologous allergen groups to identify the exact protein which is causing sensitization in the individual. Specific allergen immunotherapy should then be tailored as per patient’s sensitivity for effective administration. In conclusion, we have successfully identified the allergens of C.lunata, crosscompared it with other fungal/non-fungal homologs for allergenicity, cross-reactivity, structure and allergen levels in the environment 195 Chapter 5.2 FUTURE PROSPECTS In relation to the work reported, future studies were suggested to enhance and build up on the current available knowledge about the C.lunata allergens and fungal allergens in general; which are enlisted below: 1) Further study in detail the levels of exposure of various fungi in the indoor as well as outdoor environments. Also, identify various fungal species which are prevalent but unknown in the environment and test them for possible allergenicity. 2) To identify allergens from other immunologically important allergenic fungi using expressed sequence tagging approach in order to understand allergenicity, allergen composition and variability in the allergens of these fungi. 3) Novel allergen identification (using western blots followed by proteomics) of various important fungi using sera from different populations in order to study the complete allergen repertoire of an organism. 4) Details regarding the effect of biological function, localization of the protein, biochemical composition (stability, activity, composition) on allergenicity are to be evaluated in order to further understand allergenicity and its evolution. 196 Chapter 5) Confirmation of reactivity of human allergens and autoallergy by using mouse models in order to understand the underlying autoallergy mechanisms. 6) Comparison of IgE binding patterns of recombinant proteins against their native counterparts in order to study the effect of post-translational modifications (if any) on allergenicity. 7) Determination of the three dimensional structures of the studied allergens to validate the homology-model as well as to understand the binding of allergen to IgE in the three dimensional space. 8) Epitope mapping (B-cell as well as T-cell) of the allergens to define the critical residues for IgE binding as well as the ones involved in activation. 9) Generation of hypoallergenic fragments (lacking B-cell epitopes as identified from the epitope mapping) for specific allergen based immunotherapy. 197 . well and very few of them have been characterized for the allergens in detail. Characterization of the fungus and its constituent allergens is vital towards proper understanding of the fungus and. further understand allergenicity and its evolution. Chapter 5 197 5) Confirmation of reactivity of human allergens and autoallergy by using mouse models in order to understand the underlying. cross- reactivity, structure and critical residues for allergenicity, levels in the environment, function, etc. Here, we report various allergens from Curvularia lunata. For the rapid isolation of the allergenic