Isolation and characterization of allergens from curvularia lunata 4

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

CHAPTER 4:

CROSS-COMPARISON OF VARIOUS HOMOLOGOUS

ALLERGEN GROUPS

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

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

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

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

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

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

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

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 http://www.rcsb.org/pdb/home/home.do)

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 (http://www.expasy.org/spdbv/) 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

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 1m2 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

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

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/m3/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

(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

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

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

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**

B) Correlations between various SODs tested

Correlation coefficient, r was analyzed using Spearman’s Correlation Test p = 0.01**

ClSOD ScSOD HsSOD

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

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

2.5 0.25

0.025 0.0025

Figure 4.4: Sequence comparison of various SODs

A) Multiple sequence alignments for various SODs

Alignments and tree generated using DNAMAN ver 4.15

12 ClSOD

37 AfSOD

60 ScSOD

58 HsSOD

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

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A A A

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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

65 ClSOD

90 AfSOD

119 ScSOD

81 HsSOD

L L V

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Q Q Q Q

E E E

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L L

D D

P

K

V V I

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125 ClSOD

148 AfSOD

178 ScSOD

115 HsSOD

P P

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A

A

W W F

G G G G

S S

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G G G E

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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

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V V

T T T

T T T

185 ClSOD

206 AfSOD

233 ScSOD

140 HsSOD

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

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Y Y Y

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N N N

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E E

R R R

Y Y F

G A A

S G G

191 ClSOD

210 AfSOD

233 ScSOD

140 HsSOD

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

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

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

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)

12 ClSOD

37 AfSOD

60 ScSOD

58 HsSOD

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

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Y F Y

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A A A

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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

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Y Y Y Y

65 ClSOD

90 AfSOD

119 ScSOD

81 HsSOD

L L V

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L L

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P

K

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H H H

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125 ClSOD

148 AfSOD

178 ScSOD

115 HsSOD

P P

L L L

K K

A A

I I I

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A

A

W W F

G G G G

S S

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A A

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N N N

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G G G

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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

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T T T

185 ClSOD

206 AfSOD

233 ScSOD

140 HsSOD

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

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R R R

Y Y F

G A A

S G G

191 ClSOD

210 AfSOD

233 ScSOD

140 HsSOD

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

0 5

Chapter 4

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/m3/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

Chapter 4

Figure 4.9: Antigenic cross-reactivity amongst various SODs

Anti ClSOD rabbit polyclonal antibodies were used

BSA was used as a heterologous protein showing no binding to human IgEs

0.500 0.250

0.125 0.063

0.031 0.016

Chapter 4

Figure 4.10: Levels of SOD allergen in the outdoor air samples

A total of 45 weekly air samples (spanning over a year) were screened

A) Levels of the detected SOD allergen in air over one year

B) Average Curvularia spore counts over one year

The coinciding peak periods of Curvularia sporulation and that of SOD in the air are

highlighted by empty black circles

Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May

Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May

Chapter 4

4.3.2 Thioredoxin (Trx)

Thioredoxins (EC 1.8.4.8) are small and multifunctional proteins They participate in various redox reactions through reversible oxidation of the active centre disulfide bond, playing a pivotal role in gene expression and proliferation Thioredoxins have also been reported to prevent nitrous oxide (NO) mediated cell injury in macrophage

cells (Cho et al., 2001)

Curvularia Trx (ClTrx) is a 112 amino acid long protein with predicted molecular weight of 12.3kDa and calculated pI of 4.84 It was named as Cur l 4 for submission to the IUIS allergen database ClTrx showed sequence similarity with other Trxs from

fungi Hence, these homologs were then amplified, cloned, and expressed For the present study, Trx from S cerevisiae (ScTrx: NP_011725) and H sapiens (HsTrx:

MHS1010-57478) were used along with ClTrx These allergen homologs were tested

on four different populations as shown earlier Various Trxs were found to bind patient IgEs from all the populations suggesting that all the Trxs tested were allergenic The Singaporean fungal atopic population was found to react strongly to the Trxs suggesting Trxs are the major allergens for these populations Conversely, the Indian and Italian populations were found to show lower IgE binding frequencies, suggesting Trx is not an important allergen in those populations Colombian asthmatic populations were found to be more reactive to the tested Trxs with IgE binding frequencies ranging from 40 – 70% ClTrx was found to react more than 40% in the Singaporean and Colombian populations, suggesting it to be a major allergen for these populations Surprisingly, ScTrx was found to react with higher IgE binding

Comparing the overall primary structures of various Trxs, it was evident that Trxs showed high sequence identity (around 58%) Moreover, all the Trxs showed more than 40% sequence identity to a known fungal thioredoxin allergen (Cop c 2) (Figure 4.14) Also, the Kyte and Dolittle plots generated using DNAMAN ver 4.15, showed that that the possible hydrophilic regions of the 3D structure were conserved across