Isolation and characterization of allergens from curvularia lunata 5

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CHAPTER 5:
DISCUSSION, CONCLUSION AND FUTURE
PROSPECTS OF THE RESEARCH WORK






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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, 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 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
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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 non-
normalized 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. 4 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 5 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.
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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 4 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 4 populations confirming the allergenicity. Further, out of the
11 different C.lunata recombinant proteins tested, 4 allergens [Cur l 3 (manganese
superoxide dismutase), Cur l 4 (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.
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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 do 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.
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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 (O
2
-

), superoxide dismutase is required which
converts it to less harmful hydrogen peroxide (H
2
O
2
) 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
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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, cross-
compared it with other fungal/non-fungal homologs for allergenicity, cross-reactivity,
structure and allergen levels in the environment



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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.

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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.