Isolation and characterization of allergens from curvularia lunata 4

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CHAPTER 4:
CROSS-COMPARISON OF VARIOUS HOMOLOGOUS
ALLERGEN GROUPS







131

4.1 INTRODUCTION
It has been found that allergens can be classified into a small number of structural
protein families, regardless of their biological source (Ferreira et al., 2004). Also, as
evident from the previous chapter, there exists possible cross-reactivity amongst fungal
allergens across the fungal phylogeny. Cross-reactivity is the ability of an antigen (in
this case allergen) to bind with an antibody that was raised to a different antigen. The
phenomenon of cross-reactivity can be explained by two mechanisms: Firstly, because
of the shared epitopes amongst various orthologues, or secondly because of the
conformational similarity of the epitopic regions amongst phylogenetically related

proteins. The extent of binding (affinity) may vary depending on where the proteins
with shared epitopes may show stronger binding affinity compared to the
conformational epitopes which may bind with a lower affinity (Richard and Weber,
2000). Conformational dynamics of the epitopes play an important role towards
allergenicity (Rossjohn J and McCluskey J, 2007). Phylogenetically-related species
show high degree of homology in the primary structure of the proteins, which results
in homologous 3-D structures and thus potentially, in cross-reactivity (Aalberse et al.,
2001). Also, at times, for non-related spcies, there might be cases where two proteins
might share low homology in primary structures but higher homology in 3-D
structures (Kvansakul et al., 2007).
The cross-reactivity of IgE antibodies is of interest among allergologists for various
reasons. Cross-reactivity studies are important as they impact diagnosis and therapy of
allergies as they mask the correct source of sensitization (because of the reactions to
the shared epitopes) and make it difficult to identify the primary sensitizer. From the
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clinical point of view, it is crucial to know the patterns of cross-reactivity as it reflects
the pattern of clinical sensitivities (Aalberse et al., 2001), which could be helpful in the
diagnosis and treatment of fungal allergy. Moreover, fungal sensitization can
contribute to auto-reactivity against self-antigens in humans due to shared epitopes
with homologous fungal allergens (Crameri et al., 2006).
Furthermore, cross-reactivity can additionally be useful in standardization of allergen
extracts by aiding in proper quantitation of allergens by comparing the strength of
reactivity to the source, in order to administer proper amount of allergen. Hence, by
recognizing the correct source of allergen by cross-reactivity studies, one can
implement effective therapy to suit to individual atopic patient with differential
reactivity to various allergens. Hence, immunological cross-comparison of allergens
across phylogeny is important and can help answer various basic questions related to
cross-reactivity.
In the present chapter, the generated Curvularia lunata recombinant allergens were

cross-compared with other fungal as well as mammalian (human) homologs. Also, the
characterization of allergens was done on the basis of homologous allergen families
(classifying all the allergens with their homologs into respective unique cohort) rather
than just considering individual allergens.

4.2 MATERIALS AND METHODS
4.2.1 Cloning, expression and purification of the C. lunata allergens homologs
from various fungal as well as non-fungal sources
4.2.1.1 Fungal cultures and raw materials
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Aspergillus fumigatus (ATCC42202), Penicilium citrinum (ATCC16040), Alternaria
alternata (ATCC6663) and Cladosporium herbarum (ATCC38810) were bought from
American Type Culture Collection (ATCC) and cultured in the laboratory as per the
ATCC (www.atcc.org). Briefly potato dextrose broth (as per ATCC protocol) was
used to culture A. fumigatus, P. citrinum and A. alternata, while malt extract agar (as
per ATCC protocol) was used to culture Cl. herbarum. The Saccharomyces cerevisiae
(w303) was previously ordered and cultured in-house in the YEPD (1% Yeast extract
w/v, 2% Bacto-peptone w/v and 5% Glucose w/v) medium with 48 h shaking (200
rpm) at 26°C and harvested by centrifugation (10,000g). The fungi were grown as per
the ATCC protocol and harvested mat was stored at -80°C till further use.
4.2.1.2 mRNA extraction and cDNA synthesis
One gm of the dried fungal mat was powdered with liquid nitrogen. RNA extraction
was performed using RNeasy mini kit (QIAGEN) as per manufacturer’s protocol.
cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad).
4.2.1.3 Amplification of the C. lunata allergen homologs from other species
Various homologs of C. lunata were amplified from A. fumigatus, P.citrinum, A.
alternata, Cl. herbarum, S. cerevisiae cDNA. For designing the primers, the sequence
of the corresponding allergen homolog was taken from the NCBI sequence database.
As described earlier, the primers had LIC overhangs to make it compatible for cloning

into pET32Ek/LIC vector. Clones of the putative human and mouse homologs were
purchased from Open Biosystems. These were then amplified with LIC primers. The
NCBI/Open Biosystems accession ids for individual amplified allergens are mentioned
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along with the discussion on the respective proteins in the results and discussion
section of this chapter.

4.2.1.4 Cloning and expression of the C. lunata allergen homologs
After amplification with LIC overhangs, the amplicons were cloned into pET32Ek/LIC
expression vector as discussed in chapter 3. Further, the recombinant proteins of the
homologs were expressed (with the pET fusion protein) and purified as explained in
chapter 3. The purified proteins were quantified and were used for further experiments.

4.2.2 IgE binding studies of the homologous allergen groups
4.2.2.1 IgE binding studies of the homologous allergen groups over various
populations using IgE immunodot-blot assays
Sera from four populations viz. the Singaporean fungal atopic population, the Italian
atopic population, the Colombian asthmatic population and the Indian atopic
population as discussed in chapter 3 were used for IgE immuno-dotblot screening of
the homologs.
The total fungal extraction and quantification of the recombinants and the
standardization of the proteins for dot-blot was done as mentioned earlier. Protein
samples along with the positive (human IgE in dilutions from 150IU to 9.375IU) and
negative controls (pET32 fusion protein) as well as blanks (PBS for fungal extracts
and elution buffer for recombinants) were dotted onto the membranes and were
screened over the sera from the above mentioned populations. Each sample, control
and blanks were dotted in duplicates.
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The developed membranes were blotted, scanned and the color intensity was analyzed.
The Spearman correlation coefficient within homologous protein group was computed
by SPSS.
4.2.2.2 IgE binding studies of the homologous allergen groups by skin prick
testing
To test the in vivo IgE binding of the allergens, skin prick testing was carried out.
Equimolar mixtures of various allergens in the allergen groups were used. A drop of
0.05mg/ml of the equimolar mixture of homologous recombinant group was each
placed on the inner forearm of the patient. Histamine was used as positive control
while saline was used as negative control. After 20 min, the results were analyzed.
Results were considered as positive when weal diameter was greater than or equal to
3mm×3mm. A total of 34 fungal atopic and 10 non-atopic adult Singaporean patients
were tested. Of the atopic fungal patients, 20 (58.8%) were males while 14 (41.2%)
were females. Out of the 34 fungal atopic patients, 18 (52.9%) were previously
diagnosed with atopic eczema while 13 (38.2%) patients were asthmatic.

4.2.3 Raising polyclonal antibodies against the C. lunata allergens in rabbit
Antibodies (serum IgGs) with allergen-specific binding capacity were raised for
individual C. lunata allergens. Polyclonal antibodies were raised instead of
monoclonal antibodies as reactivity to multiple epitope sites on the allergens was
desired. New Zealand White female rabbits were chosen as the host for immunization
in order to get large volumes (~80ml) of highly-specific sera and also because it could
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be harvested from the rabbits after the initial immunization and 3 to 4 booster
injections.
Individual recombinant allergens were diluted to 0.6mg/ml in up to 700µl of PBS.
Each diluted allergen preparation was subsequently mixed with an equal volume of
complete or incomplete Freund’s complete (F5881) as well as incomplete (F5506)
adjuvant (Sigma) respectively for the first immunization and subsequent boosters. The

adjuvant was added for the non-specific activation of the macrophages, T-cells, and B-
cells to amplify the humoral antibody response, for prolonging antigen exposure to
host and also for protecting the antigen from degradation (Stills and Bailey, 1991).
This allergenic protein-adjuvant mixture was emulsified and injected subcutaneously
into the rabbit. Ketamine-xylazine solution was injected subcutaneously to sedate the
rabbits before injecting them. Pre-immune blood was collected before the whole
immunization process and blood was drawn immediately after every immunization
booster (every three weeks). The sera were harvested by ensanguining the rabbits. Sera
was collected after centrifuging the clotted blood, aliquoted and stored at -20°C till
future use.
The antibody titre (lowest dilution of sera that binds significantly to the specific
antigen) was quantified using Enzyme Linked Immuno-Sorbant Assay (ELISA). The
specific recombinant allergen was diluted 10µg/ml and coated on the absorbent surface
of the ELISA plate (NUNC) and was absorbed overnight at 4°C. Simultaneously, sera
obtained from the various boosters as well as the pre-immune sera were serially-
diluted in PBS. As the proteins were expressed with the pET32 fusion protein, the sera
were pre-absorbed with pET32 protein (100µg/ml) to reduce any background binding
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with the fusion protein. Later, the plates were washed thrice with 200µl 0.05% PBS-T
to remove any excess allergen. The plates were then blocked using 200µl 0.1% PBS-T
for 30mins. After three washes of the ELISA plate, diluted rabbit polyclonal sera was
appropriately added to each well and incubated for 2.5 h. Anti-rabbit alkaline
phosphatase-conjugated secondary antibody diluted 1:2000 was added to each well
and allowed to incubate for 2 h. A final wash was carried out to remove excess of the
secondary antibody before the reaction was developed using p-nitrophenyl phosphate
(Sigma). The reaction was read at 405nm. The generated rabbit polyclonal antibodies
were then used for the immunological detection and characterization of the allergens
as discussed later.

4.2.4 Competitive inhibition studies to establish cross-reactivity
To confirm the cross-reactivity of homologous allergens, inhibition studies were
carried out by measuring the ability to inhibit binding of the antigen to the specific
antibody by homologous antigen was measured. The inhibition of a specific antibody
by its homologous antigen was compared to that of self-inhibition by the antigen
against which the antibody was raised as well as against a heterologous protein against
which the antibody would have no reaction. The inhibition studies were done using
ELISA. As explained previously, 1µg of the C. lunata specific recombinant protein
was coated on to a MaxiSorp ELISA plate. Dilution of the protein inhibitors (the
homologous antigen and BSA heterologous inhibitor) of volume 50µl was prepared
and individually mixed with an equal volume of the appropriate dilution factor of the
primary antibody (as determined during antibody titering). This pre-absorption was
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allowed to occur at 4°C overnight. The ELISA was subsequently completed following
the previously described protocol. The degree of cross-reactivity was expressed in
terms of percentage inhibition based on the formula: [(Iu-Ii) / (Iu) x 100], where Iu
represents the uninhibited dot intensity, Ii is the intensity at i µg inhibitor.
Both the antigenic as well as allergenic cross-reactivity studies were done for the
homologous allergen groups. For the antigenic cross-reactivity, rabbit polyclonal
antibodies generated against specific C. lunata allergens were used. For the allergenic
cross-reactivity, sera from reactive patients (individually or in pool) were used.

4.2.5 Structural comparison of various homologs within an allergen group
Various homologs of C. lunata allergens were compared for their primary structure
using multiple sequence alignments, hydropathy plots and homology trees. All the
sequence alignments, hydropathy plots as well as the trees were generated using
DNAMAN v4.15 (Lynnon BioSoft).
For the comparison of the three dimensional (3D) structures, the 3D structure of the
known allergen as well as human/mouse protein homologs were downloaded from the

Protein Data Bank (PDB) database (www.
The 3D structures for C. lunata as well as other fungal proteins (for which there was
no structure available) were modeled using DeepView3.7 freeware from the ExPASy
server ( Briefly, the protein sequence of the target
molecule was loaded to find the possible matching template from the PDB database.
Further, it was checked whether the protein was monomeric or oligomeric. Based on
the state (monomeric or oligomeric) of the template, the corresponding state of the
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target molecule was generated using the ‘magic fit’ option followed by the ‘aa making
clash’ option and then ‘fix selected side chain’ option to remove the clash. The project
file was then saved and sent to SWISSPLOT website for further adjustment. The
obtained results were then saved and used for comparison with other allergen
structures using the PyMol v0.99 (DeLano Scientific LLC) freeware.
4.2.6 Detection of allergen levels in the indoor and outdoor environments
4.2.6.1 Detection of allergen levels in the indoor environment
To detect the levels of the specific allergen/homologous allergen group in the indoor
environment, dust samples were collected from the two major types of households in
Singapore, namely flats and landed properties. Other than the Housing Development
Board (HDB) flats, high-rise apartments were also considered under the category of
flats. Landed households comprised detached, semi-detached and terraced houses. The
niches from which dust was collected in each household were the living room floor,
kitchen floor and a bedroom floor. If a carpet was present in either the living room or
the bedroom, it was vacuumed for dust as well. A total of 12 samples from bedroom
floors, 3 samples from bedroom carpets, 13 samples from living room floors, 17
samples from living room carpets and 9 samples from kitchen floors were used in the
following study. A modified Kirby Classic III vacuum cleaner (with a chamber to
collect dust onto a filter paper) was used for sampling. At the beginning of the period
of sample collection, dust samples were obtained by vacuuming an area of 1m
2

twice.
Cross contamination of samples was avoided by using a different filter paper at each
niche within each household. Dust samples were stored in zip-lock bags at 4°C during
transportation back to the laboratory and at -20°C until use.
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Protein extraction from the dust samples was carried out by adding 1ml PBS to 50mg
of dust weighed and incubating at 4°C overnight before centrifuging at 5,000 rpm for
20 min at 4°C. Precaution was taken to avoid hair and non-dust particles. The
supernatant was divided into aliquots and stored at -20°C until further analysis.
Only the dust samples with sufficient volume for all planned assays were analysed for
specific allergens using ELISA. A 10-point serial dilution of recombinant allergen
starting from 20µg/ml was prepared as a standard curve for every plate of the dust-
screening ELISA. This standard curve was used to determine the concentration (in
µg/ml) of the specific allergen found in each dust sample. Dust samples were coated at
a volume of 50µl per well. The remaining steps of the ELISA were performed as per
the antibody titering protocol. The concentrations of the individual allergens were
expressed as µg/g of dust after extrapolating from the standard curve by the ELISA
plate reader.
4.2.6.2 Detection of allergen levels in the outdoor environment
To detect the levels of the specific allergen/homologous allergen group in the outdoor
environment, air samples were collected using the Cyclone Sampler (Burkard
Manufacturing). It is a continuous non-filter based volumetric sampler with wind
orientation using a single reverse miniature Cyclone. The flow rate of the cyclone is
set to16.5 litres/min. The particulate matter in the air is collected into an eppendorf
tube and can be transferred or analysed by microscopy or immunoassay (In the present
case ELISA was used).
Samples were collected every week over a period of one year (From Jan 2004 to Jan
2005). A total of 45 samples were collected (around 7 samples were lost due to service
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breakdowns as well as other unpropitious reasons). The tubes collected from the
cyclone sampler were stored at -20°C until use.
Protein extraction from the air samples was carried out by adding 500µl PBS to the
collected tube and incubating at 4°C overnight before centrifuging at 5,000 rpm for
20mins at 4°C. The supernatant was stored at -20°C until further analysis.
The air samples were analysed for specific allergens using ELISA. A 10-point serial
dilution of recombinant allergen starting from 20µg/ml was prepared as a standard
curve for every plate of the air-screening ELISA. This standard curve was used to
determine the concentration (in µg/ml) of the specific allergen found in each air
sample. Dust samples were coated at a volume of 50µl per well. The remaining steps
of the ELISA were performed as per the antibody titering protocol. The concentrations
of the individual allergens were expressed as ng/m
3
/week of dust after extrapolating
from the standard curve by the ELISA plate reader. Also, the levels of the allergens in
the air were correlated with the levels of C. lunata spores in the air.

4.3 RESULTS
The results for cross-comparison and characterization of the individual homologous
allergen groups are discussed one by one based on the biochemical group they are
classified in. For the groups with unknown function, they are labeled based on the
homology to the known allergen.
4.3.1 Manganese Superoxide Dismutase (MnSOD)
Superoxide dismutases (EC 1.15.1.1) are known to be metalloenzymes catalyzing the
dismutation of the superoxide radicals to oxygen and hydrogen peroxide. Superoxide
radicals are toxic to the cell and hence these enzymes form the first line of defence
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(Bannister et al., 1987). The free radicals generation and disturbance of redox status

can modulate inflammatory molecule expression leading to exacerbation of
inflammation and tissue damage (Matés et al., 1987). There are 3 classes of SOD
based on the metal ion used: a) Fe/MnSODs utilizing iron/manganese, b) Cu/ZnSODs
utilizing copper or zinc and c) NiSOD utilizing Nickel (Stalling et al., 1984).
Curvularia MnSOD (ClSOD) is a 195 amino acid long protein with predicted
molecular weight of 21.8kDa and calculated pI of 7.59. It was named as Cur l 3 for
submission to the IUIS allergen database. ClSOD showed sequence similarity with
other MnSODs from fungi. Hence, these homologs were then amplified, cloned, and
expressed. For the present study, MnSOD from A. fumigatus (AfSOD: U53561), S.
cerevisiae (ScSOD: NP_011872) and H. sapiens (HsSOD: MHS1010-74217) were
used along with ClSOD. Of these, AfSOD is already a known allergen (Asp f 6) which
was identified by Crameri et.al, (1996).
These allergen homologs were tested on four different populations as shown earlier.
Various MnSODs were found to bind patient IgEs from all the populations suggesting
that all the MnSODs tested are allergenic. Singaporean fungal atopic population was
found not to react strongly to the SODs suggesting SODs are not the major allergens
for these populations. Conversely, the other 3 populations were found to show higher
IgE binding frequencies, suggesting SOD to be an important allergen in those
populations. Colombian asthmatic populations were found to be more reactive to the
tested MnSODs with IgE binding frequencies ranging from 40 – 90%. ClSOD was
found to react with more than 40% in Singaporean, Italian and Colombian populations,
suggesting it to be a major allergen for these populations. Surprisingly, AfSOD was
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found to react with lower IgE binding frequency as compared to other SODs. This data
was contradictory to the fact that AfSOD is a known allergen. ScSOD and HsSOD
which were not known to be allergenic were found to react with high IgE binding
frequency (Figure 4.1). This data suggested that the sera from various populations
were polysensitized to different SODs. The reason why ScSOD and HsSOD were seen
to react might be due to the fact that SODs allergens might be cross-reactive. On the

skin prick testing, the equimolar mixture of SODs was found to react to 1 (3.4%) out
of the tested 34 fungal atopic patients from Singapore, confirming low reactivity of
Singaporean sera to SODs.
Also correlation studies done using the Colombian population (since it was most
reactive) showed that the various reactions for the SODs are correlated. A strong
correlation was observed between ClSOD and ScSOD (r=0.713**), suggesting that
most of the sera that react to ClSOD also react to ScSOD. Similarly, it was observed
that there existed strong correlation between ScSOD and HsSOD (r=0.886**) and so
on for all the tested SOD allergens (Figure 4.2). This data suggested that there might
be possible cross-reactivity amongst various SODs.
Hence, to test this hypothesis, inhibition studies were carried out to test for possible
cross-reactivity amongst various SODs. For the competitive inhibition ELISA, the
pool of five SOD reactive patients’ sera from the Colombian population were first
incubated with the inhibitors (AfSOD, ScSOD, HsSOD and BSA) at concentration 5,
2.5, 0.25, 0.025, and 0.0025 and 0.00025 ug overnight at 4ºC before being added into
the respective wells having 1ug of ClSOD. ClSOD was found to be inhibited more

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Figure 4.1: IgE binding study of MnSODs over four different populations
A) Intensities of the reactions for various allergens tested
Reaction above the intensity of 20 units is considered as a positive reaction.
Cl: Curvularia lunata,
Af: Aspergillus fumigatus,
Sc: Saccharomyces cerevisiae
Hs: Homo sapiens



B) IgE binding frequencies (%) of the allergens tested over various populations

Populations tested
Allergen Singaporean Indian Italian Colombian
ClSOD 44.7 23.5 44.4 68.6
AfSOD 18.4 47.1 1.3 42.4
ScSOD 57.9 52.9 45.0 89.8
HsSOD 32.9 35.3 27.5 90.7

Singaporean Sera (N=76) Indian Sera (N=17)
Italian Sera (N=160) Colombian Sera (N=118)
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Figure 4.2: Correlation studies of SODs
A) Correlation biplots for ClSOD Vs ScSOD and ScSOD Vs HsSOD
Correlation coefficient, r was analyzed using Spearman’s Correlation Test. p = 0.01**.

0
20
40
60
80
100
120
140
160
0 20 40 60 80 100 120 140 160
ClSOD
ScSOD

0

20
40
60
80
100
120
140
160
180
0 20 40 60 80 100 120 140 160 180
ScSOD
HsSOD

B) Correlations between various SODs tested
Correlation coefficient, r was analyzed using Spearman’s Correlation Test. p = 0.01**.


ClSOD

ScSOD HsSOD

AfSOD
0.449 0.886 0.348
ClSOD
0.713 0.582
ScSOD
0.886
HsSOD

r = 0.
713
**

r = 0.
886
**

146

than 80% by ScSOD and HsSOD. ClSOD self inhibition was found to be around 85%
which was same as that of ScSOD and HsSOD. AfSOD did inhibit ClSOD but the
inhibition was around 60% suggesting that ClSOD has some unique epitopes other
than AfSOD. On the other hand ClSOD, ScSOD and HsSOD shared the epitopes. The
non-specific protein (BSA) was not able to inhibit the IgE binding of ClSOD
suggesting that the sera were specific to SODs (Figure 4.3).
In order to map the possible epitopes involved in the cross-reactivity as well as the
ones which are unique to the individual SODs, structural comparison was also carried
out.
First of all, the primary structures of the various SODs were compared by protein
alignments. Multiple sequence alignments showed that the fungal SODs had 45%
sequence identity while the HsSOD was found to be less than 40% identical with other
fungal SODs (Figure 4.4). Moreover, the N and the C-termini of various SODs were
found to be less conserved suggesting that the cross-reactive epitopes might be present
somewhere in the middle of the sequence. Hence, sequence alignments were not able
to provide with sufficient information about the location of the epitopes. To overcome
this, three dimensional structural comparisons of various SODs were carried out.
AfSOD (1kkc) and HsSOD (1luv) 3D structures were downloaded from the PDB
server. Further, AfSOD (1kkc) structure was used as a template to model the 3D
structures of ClSOD and ScSOD.

MnSODs were seen to be tetrameric in nature. Overall, all the four SOD structures
showed high structural similarity. Some differences in the top and bottom of the
147

Figure 4.3: Self inhibition of ClSOD and cross-inhibition by AfSOD, ScSOD,
HsSOD
The pool of sera used for the experiment was taken from the SOD reactive Colombian
population
BSA was used as a heterologous protein showing no binding to human IgEs


0
10
20
30
40
50
60
70
80
90
2.50.250.0250.00250.00025
Amount of inhibitor (ug)
%Inhibition
BSA Af Cl Sc Hs

148

Figure 4.4: Sequence comparison of various SODs
A) Multiple sequence alignments for various SODs

Alignments and tree generated using DNAMAN ver 4.15

12ClSOD
37AfSOD
60ScSOD
58HsSOD
MELHHSKHHQTY
MSQQYTLPPLPYPYDALQPYISQQIMELHHKKHHQTY
MFAKTAAANLTKKGGLSLLSTTARRTKVTLPDLKWDFGALEPYISGQINELHYTKHHQTY
MLSRAVCGTSRQLAPALGYLGSRQKHSLPDLPYDYGALEPHINAQIMQLHHSKHHAAY
A
A
T
T
L
L
R
R
Q
Q
K
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T
T
L
L
L
P
P
P

D
D
L
L
L
P
P
Y
W
Y
D
D
Y
F
Y
D
G
G
A
A
A
L
L
L
Q
E
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Y
Y
I
I
I
S
S
Q
Q
Q
I
I
I
M
M
M
E
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Q
L
L
L
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H
H
H
H
H
H

H
S
S
K
K
K
K
H
H
H
H
H
H
H
H
Q
Q
Q
T
T
T
Y
Y
Y
Y
65ClSOD
90AfSOD
119ScSOD
81HsSOD
ITNLNALLKTQAEAVSTLDIP SQVAVQQGIKFNAGGHINHSLFWQNLAPASSN

VNGLNAALEAQKKAAEATDVP KLVSVQQAIKFNGGGHINHSLFWKNLAPEKSG
VNGFNTAVDQFQELSDLLAKEPSPANARKMIAIQQNIKFHGGGFTNHCLFWENLAPE.SQ
VNNLNVTEEKYQEALAKGRFQAE
I
V
V
V
N
N
N
G
G
L
L
F
L
N
N
N
N
A
AA
A
L
L
V
E
D
E
Q

Q
Q
Q
E
E
E
A
A
A
L
L
D
D
P
P K
K
V
V
I
A
A
V
V
I
Q
Q
Q
Q
Q
Q

I
I
I
K
K
K
F
F
F
N
N
H
A
G
G
G
G
G
G
G
G
H
H
I
I
N
N
N
H
H

H
S
S
L
L
L
F
F
F
W
W
W
N
N
N
L
L
L
A
A
A
P
P
P
E
E
S
S
S
125ClSOD

148AfSOD
178ScSOD
115HsSOD
EAKSSAAPELIKRIKAIWGDEDKFREAFNAALLGIQGSGWGWLVKIEVGKEQRLSIVTTK
GGKIDQAPVLKAAIEQRWGSFDKFKDAFNTTLLGIQGSGWGWLVTDGP.KGK.LDITTTH
GGGEPPTGALAKAIDEQFGSLDELIKLTNTKLAGVQGSGWAFIVKNLSNGGK.LDVVQTY
RREAVPGRGD PREPGPIRTGLSVEENSLRICTGS
E
G
G
A
G
G
K
K
A
A
P
P
L
L
L
K
K
A
A
I
I
I
E

D
E
A
A
W
W
F
G
G
G
G
S
S
D
D
D
D
K
K
F
F
L
A
A
F
F
N
N
N
T

T
L
L
L
L
L
G
G
G
I
I
V
Q
Q
Q
G
G
G
S
S
S
G
G
G
E
W
W
W
G
G

A
G
W
W
F
L
L
I
I
V
V
V
K
K L
L
S
S
K
K
E
G
G
E
K
K
L
L
L
L
D

D
I
I
V
I
V
V
T
T
T
T
T
T
185ClSOD
206AfSOD
233ScSOD
140HsSOD
DQDPVVGKGETPIFGIDMWEHAYYLQYQNGKAAYVQNIWNVINWKTAEERYLGSRTDAFS
DQDPVTG AAPVFGVDMWEHAYYLQYLNDKASYAKGIWNVINWAEAENRYIAGDKGGHP
NQDTVTG.PLVPLVAIDAWEHAYYLQYQNKKADYFKAIWNVVNWKEASRRFDAGKI
EFSRHDS LSFK HMVYLIVEG VPRWV
D
D
N
E
Q
Q
Q
D
D

D
P
P
V
V
V
T
T
G
G
G
S
P
P
P
F
F
L
G
G
A
S
I
V
I
F
D
D
D
M

M
W
W
W
E
E
E
H
H
H
H
A
A
A
Y
Y
Y
Y
Y
Y
Y
L
L
L
L
Q
Q
Q
Y
Y

Y
Q
Q
E
N
N
N
K
K
K
A
A
A
Y
Y
Y
K
K
I
I
I
V
W
W
W
N
N
N
V
V

V
I
I
V
V
N
N
N
W
W
W
K
K
E
E
A
A
A
E
E
R
R
R
Y
Y
F
G
A
A
S

G
G
191ClSOD
210AfSOD
233ScSOD
140HsSOD
ILKASI
FMKL


K
K


B) Homology tree for various SODs

Similarity = 45%
149

structure where the β sheets were more compactly placed together in the case of
human SOD as compared to all the fungal SODs where they were placed a bit farther
apart (Figure 4.5). Some obvious differences were observed in between ClSOD and
AfSOD. ClSOD lacks β sheet on the corner of the structure which is present in
AfSOD. Some differences in the loop region were also present (Figure 4.6).
As per Sabine et al., 2000, the solvent accessible residues found could probably be the
critical residues. These important residues were found to be somewhat similar between
various fungal SODs. These critical residues might be imparting the observed cross-
reactivity to the SODs. The smaller differences within fungal as well as those between
fungal and human SODs might explain the differential reactions (Figure 4.7). These
residues could be possible candidates for future mutagenesis in order to prove the

observed fact.
Lastly, the level of SOD allergens in the Singapore environment was tested. For this,
dust and air samples were collected from indoor and outdoors respectively. A total of
54 dust samples were collected from indoor environments in Singapore. Levels of
SOD allergen were tested using rabbit polyclonal antibodies generated against ClSOD.
Allergen levels were measured in ug/g of dust. An average of 5ug of SOD antigens
were detected per gm of dust in various indoor niches screened. The highest levels of
SOD allergens were detected in the living room dust screens (living room floor and
living room carpets) where the allergens levels were as high as 10-15 ug/gm of dust
(Figure 4.8). Furthermore, it was observed that anti-ClSOD rabbit polyclonal
antibodies which were used for screening were found to cross-react with other SODs
from human and fungi suggesting that the levels of SODs in the indoor
150

Figure 4.5: 3D models of various MnSODs
Monomeric and tetrameric (top view) subunit of the SOD is taken from Sabine et al., (2002). α helices are shown in red, β sheets in
yellow. Obvious difference between human and other fungal SODs is shown by an empty circle.
151

Figure 4.6: Overlap of AfSOD and ClSOD 3D models
AfSOD is shown in red while ClSOD is shown in green.
The structural differences between both of them are showed in black empty circles.






152

Figure 4.7: Critical residues for SOD cross-reactivity
The highlighted residues were shown to be involved in cross-reactivity amongst
various SODs by Sabine et al., (2000).

12ClSOD
37AfSOD
60ScSOD
58HsSOD
MELHHSKHHQTY
MSQQYTLPPLPYPYDALQPYISQQIMELHHKKHHQTY
MFAKTAAANLTKKGGLSLLSTTARRTKVTLPDLKWDFGALEPYISGQINELHYTKHHQTY
MLSRAVCGTSRQLAPALGYLGSRQKHSLPDLPYDYGALEPHINAQIMQLHHSKHHAAY
A
A
T
T
L
L
R
R
Q
Q
K
K
T
T
L
L
L
P

P
P
D
D
L
L
L
P
P
Y
W
Y
D
D
Y
F
Y
D
G
G
A
A
A
L
L
L
Q
E
E
P

P
P
Y
Y
I
I
I
S
S
Q
Q
Q
I
I
I
M
M
M
E
E
E
Q
L
L
L
L
H
H
H
H

H
H
H
S
S
K
K
K
K
H
H
H
H
H
H
H
H
Q
Q
Q
T
T
T
Y
Y
Y
Y
65ClSOD
90AfSOD
119ScSOD

81HsSOD
ITNLNALLKTQAEAVSTLDIP SQVAVQQGIKFNAGGHINHSLFWQNLAPASSN
VNGLNAALEAQKKAAEATDVP KLVSVQQAIKFNGGGHINHSLFWKNLAPEKSG
VNGFNTAVDQFQELSDLLAKEPSPANARKMIAIQQNIKFHGGGFTNHCLFWENLAPE.SQ
VNNLNVTEEKYQEALAKGRFQAE
I
V
V
V
N
N
N
G
G
L
L
F
L
N
N
N
N
A
AA
A
L
L
V
E
D

E
Q
Q
Q
Q
E
E
E
A
A
A
L
L
D
D
P
P K
K
V
V
I
A
A
V
V
I
Q
Q
Q
Q

Q
Q
I
I
I
K
K
K
F
F
F
N
N
H
A
G
G
G
G
G
G
G
G
H
H
I
I
N
N
N

H
H
H
S
S
L
L
L
F
F
F
W
W
W
N
N
N
L
L
L
A
A
A
P
P
P
E
E
S
S

S
125ClSOD
148AfSOD
178ScSOD
115HsSOD
EAKSSAAPELIKRIKAIWGDEDKFREAFNAALLGIQGSGWGWLVKIEVGKEQRLSIVTTK
GGKIDQAPVLKAAIEQRWGSFDKFKDAFNTTLLGIQGSGWGWLVTDGP.KGK.LDITTTH
GGGEPPTGALAKAIDEQFGSLDELIKLTNTKLAGVQGSGWAFIVKNLSNGGK.LDVVQTY
RREAVPGRGD PREPGPIRTGLSVEENSLRICTGS
E
G
G
A
G
G
K
K
A
A
P
P
L
L
L
K
K
A
A
I
I

I
E
D
E
A
A
W
W
F
G
G
G
G
S
S
D
D
D
D
K
K
F
F
L
A
A
F
F
N
N

N
T
T
L
L
L
L
L
G
G
G
I
I
V
Q
Q
Q
G
G
G
S
S
S
G
G
G
E
W
W
W

G
G
A
G
W
W
F
L
L
I
I
V
V
V
K
K L
L
S
S
K
K
E
G
G
E
K
K
L
L
L

L
D
D
I
I
V
I
V
V
T
T
T
T
T
T
185ClSOD
206AfSOD
233ScSOD
140HsSOD
DQDPVVGKGETPIFGIDMWEHAYYLQYQNGKAAYVQNIWNVINWKTAEERYLGSRTDAFS
DQDPVTG AAPVFGVDMWEHAYYLQYLNDKASYAKGIWNVINWAEAENRYIAGDKGGHP
NQDTVTG.PLVPLVAIDAWEHAYYLQYQNKKADYFKAIWNVVNWKEASRRFDAGKI
EFSRHDS LSFK HMVYLIVEG VPRWV
D
D
N
E
Q
Q
Q

D
D
D
P
P
V
V
V
T
T
G
G
G
S
P
P
P
F
F
L
G
G
A
S
I
V
I
F
D
D

D
M
M
W
W
W
E
E
E
H
H
H
H
A
A
A
Y
Y
Y
Y
Y
Y
Y
L
L
L
L
Q
Q
Q

Y
Y
Y
Q
Q
E
N
N
N
K
K
K
A
A
A
Y
Y
Y
K
K
I
I
I
V
W
W
W
N
N
N

V
V
V
I
I
V
V
N
N
N
W
W
W
K
K
E
E
A
A
A
E
E
R
R
R
Y
Y
F
G
A

A
S
G
G
191ClSOD
210AfSOD
233ScSOD
140HsSOD
ILKASI
FMKL


K
K



153

Figure 4.8: Levels of SOD allergen in the indoor dust samples
Anti-ClSOD rabbit polyclonal antibodies were used to detect the SOD levels in the dust.
A total of 54 dust samples [12 samples from bedroom floor (BRF), 3 samples from bedroom carpet (BRC), 13 samples from living
room floor (LRF), 17 samples from living room carpet (LRC) and 9 samples from kitchen floor (KF)] were screened.

L R F L R C B R F B R C K F
0
5
1 0
1 5
Va r i o u s N i c h e s S am p l e d

C on c ent r at i on ( ug /gm )
Chapter 4


154

environment detected were cumulative levels of all the cross-reactive SODs that might
be present in the dust (Figure 4.9).
For the detection of the SODs in the outdoor environment, 45 air samples were
collected from the outdoors environments in Singapore. As in the case of dust
sampling, the levels of SOD allergen were tested using rabbit polyclonal antibodies
generated against ClSOD. Allergen levels were expressed in terms of ng/m
3
/week of
the sampled air. The levels of the allergens were 1000-fold lower in the air as
compared to the sampled dust which was so because of the low amount of particulate
matter in the air. Around 2-7 ng of SOD antigens were detected per cubic meter of air
in one week. A distinct increase in the SOD allergen levels in the environment was
observed between the months of January to Match and June to July. The SOD allergen
levels in air were also seen to be comparable with the average Curvularia spore counts
over the year. As observed in the case of the SOD allergens, a distinct peak in spore
numbers was observed between Jan-Mar and between Jun-Jul, suggesting that the
SOD allergens were coming from excessive sporulation occurring in the environment
(Figure 4.10). As in case of the dust screening, in this case too the antibodies used
were cross-reacting with various SOD allergens. Hence the level of the SOD allergens
detected in the environment was actually the cumulative levels of all the cross-reactive
SODs present.