Isolation and characterization of allergens from curvularia lunata 4

CHAPTER 4: CROSS-COMPARISON OF VARIOUS HOMOLOGOUS ALLERGEN GROUPS 130 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 131 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 132 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 133 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 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. 134 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 135 be harvested from the rabbits after the initial immunization and to 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 Bcells 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 seriallydiluted 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 136 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 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 137 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 138 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, samples from bedroom carpets, 13 samples from living room floors, 17 samples from living room carpets and 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. 139 Chapter Figure 4.21: 3D models of various Cyps α helices are shown in red, β sheets in yellow and random coils in green. Figure 4.22: Hydropathy plots for carious Cyps Three possibly cross-reactive epitopic regions are showed in black circles. Gaps show deletion in the particular protein at that region. 173 Chapter ClHsp showed sequence similarity with other Hsps from fungi. Hence, these homologs were then amplified, cloned, and expressed. For the present study, Hsps from A. fumigatus (AfHsp: P40292) S. cerevisiae (ScHsp: AAC04952), A. alternata (AaHsp: U87808) and H. sapiens (HsHsp: MHS1010-58242) were used along with ClHsp. AfHSp and AaHsp are known allergens Asp f 12 and Alt a from Aspergillus fumigatus and Alternaria alternata, respectively. These allergen homologs were tested on four different populations as shown earlier. Hsps were found to bind with very low IgE binding frequencies in the Italian and Indian populations (data not shown). In the Colombian and Singaporean populations, Hsp showed variable IgE binding patterns. But this might be due to the fact that some of the Hsps (AaHsp, ClHsp) were truncated. Nevertheless, IgE binding frequencies of Hsps was high, the intensities of reactions were lower as compared to the other allergen groups previously tested (Figure 4.23). To test the possible cross-reactivity amongst the Hsps, inhibition studies were done using a pool of Hsp reactive sera. ClHsp was inhibited by AfHsp and AaHsp at even higher level compared to self inhibition. ScHsp and HsHsp also inhibited the ClHsp by around 60% (Figure 4.24). This data suggested that AfHsp and AaHsp strongly inhibit ClHsp. Moreover, it appeared that AfHsp and AaHsp might be the primary sensitizers for the Hsp atopic population. Levels of Hsp allergen were also detected in the Singapore indoor as well as outdoor environments. Out of the 45 outdoor samples taken, only samples had detectable levels of Hsp, averaging around 1.5 ng/m3 air/week. Also, the data did not coincide with the Curvularia spore count (data not shown). 174 Chapter Figure 4.23: IgE binding study of Hsps over fungal atopic Singaporean and Colombian 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, Sc: Saccharomyces cerevisiae, Af: Aspergillus fumigatus, Aa: Alternaria alternata, Hs: Homo sapiens Singaporean Sera (N=76) Colombian Sera (N=118) B) IgE binding frequencies (%) of the allergens tested over various populations Allergen ClHsp AfHsp ScHsp HsHsp Populations tested Singaporean Colombian 28.9 44.1 61.8 54.2 27.6 42.4 56.6 50.8 175 Chapter Figure 4.24: Self inhibition of ClHsp and cross-inhibition by AfHsp, AaHsp, ScHsp and HsCyp compared with the ClHsp self inhibition The pool of sera from the Hsp reactive Colombian population was used. BSA was used as a heterologous protein showing no binding to human IgEs Cross inhibition of ClHsp by the above mentioned inhibitors. White colored columns show inhibitors tested (2.5µg) while the black colored columns show self inhibition of ClHsp. 70 60 40 30 %Inhibition 50 20 10 0.00025 0.0025 0.025 0.25 2.5 Amount of inhibitor (ug) 90 80 70 % Inhibition 60 50 40 30 20 10 AfHsp AaHsp ScHsp Inhibitors 176 HsHsp Chapter On the contrary, the indoor levels of Hsp allergens were quantifiable in all the niches sampled except the bedroom carpets. An average of 0.5ug of Hsp antigens were detected per g of dust in various indoor niches. Highest levels of Hsp allergens were detected in the living room dust screens (living room floor and living room carpets) where the allergens levels were as high as 0.9 ug/g of dust (Figure 4.25). Also, taking into consideration the extensive cross-reactivity amongst various Hsps, the levels of Hsps detected in the indoor might be a cumulative total of various Hsps present in the dust. The reason for Hsps being present in smaller amounts and at irregular intervals might be due to possible degradation of Hsps. Three dimensional structure comparisons were not done for Hsps as the model for ClHsp was not been able to be generated by the freeware. Also, primary sequence analysis was redundant as ClHsp and Aahsp were having truncated sequences and hence the analysis would not have been correct. 4.3.5 Alcohol Dehydrogenase (Alc) Alcohol dehydrogenase (EC 3.4.11.4) is a group of dehydrogenase enzymes which helps in the inter-conversion of alcohols to aldehydes/ketones. This enzyme aids in detoxifying the alcohol present in the body whilst in the yeast they catalyze the reverse reaction fermenting the alcohol. Alcohol dehydrogenases have also been thought to activate cinnamaldehyde from its prohapten form to the active hapten form which can then cause allergic contact dermatitis in humans, suggesting that alcohol dehydrogenase has an important role in allergy (Smith et al., 2000). 177 Chapter Figure 4.25: Levels of Hsp allergen in the indoor dust samples Anti ClHsp rabbit polyclonal antibodies were used to detect the hsp levels in the dust. A total of 54 dust samples [12 samples from bedroom floor (BRF), samples from bedroom carpet (BRC), 13 samples from living room floor (LRF), 17 samples from living room carpet (LRC) and samples from kitchen floor (KF)] were screened. 178 Chapter Curvularia alc (ClAlc) is a 352 amino acid long protein with predicted molecular weight of 37.5kDa and calculated pI of 7.02. It was named as Cur l 10 for submission to the IUIS allergen database. ClAlc showed sequence similarity with other Alcs. Hence, these homologs were then amplified, cloned, and expressed. For the present study, H. sapiens (HsAlc: EHS1001_7517365) were used along with ClAlc. Alcohol dehydrogenase from Candida albicans (Can a 1) was known to be allergenic (P43067). Not much has been studied for this group of allergen. These allergen homologs were tested on four different populations as shown earlier. Alcs were found to bind patient IgEs from all the populations with high frequencies suggesting them to be allergenic and hence important. The Singaporean fungal atopic population was found to react strongly to Alcs with IgE binding frequencies of 60% and higher suggesting Alc to be a major allergenic protein. Also, ClAlc showed around 60% or more IgE binding frequency (except Italian population). Surprisingly, HsAlc also showed high IgE binding over the tested populations (ranging from 35 to 70%). The Singaporean and Indian sera were found to be more reactive to Alcs as compared to the Colombian and Italian populations respectively (Figure 4.26). Also, biplots of ClAlc Vs HsAlc showed strong correlation (r = 0.743**) hinting possible cross-reactivity amongst the tested Alcs (Figure 4.27). In order to confirm the cross-reactivity, inhibition studies were carried out where ClAlc was inhibited around 80% by HsAlc (Figure 4.28). A pool of fungal atopic Singaporean patients` sera was used fore this experiment. This data hence suggests that like cyclophilins, alcohol dehydrogenase also tend to cross-react. Structural comparisons were not possible due to absence of suitable template for 3-D modelling. 179 Chapter Figure 4.26: IgE binding study of Alcs 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, Hs: Homo sapiens Singaporean Sera (N=76) Indian Sera (N=17) Italian Sera (N=160) Colombian Sera (N=118) B) IgE binding frequencies (%) of the allergens tested over various populations Allergen ClAlc HsAlc Singaporean 60.5 68.4 Populations tested Indian Italian 70.6 25.6 41.2 35.0 180 Colombian 58.5 51.7 Chapter Figure 4.27: Correlation biplots for ClAlc Vs HsAlc Correlation coefficient, r was analyzed using Spearman’s Correlation Test. p = 0.01**. 120 r=0.743** 100 HsAlc 80 60 40 20 0 20 40 60 80 100 120 ClAlc Figure 4.28: Self inhibition of ClAlc and cross-inhibition by HsAlc The pool of sera used for the experiment was taken from the Alc reactive Singaporean population BSA was used as a heterologous protein showing no binding to human IgEs 100 90 80 60 50 40 30 20 10 0.09375 0.1875 0.375 0.75 Amount of inhibitor (ug) BSA Cl Hs 181 1.5 %Inhibition 70 Chapter Levels of Alc in the environments were detected both in indoor as well as outdoor samples. An average of 2.5ug of Alc antigen was detected per g of dust in various indoor niches. Alc were detected in all the niches suggesting a strong presence of these antigens in the Singapore environment. Highest levels of Alc allergens were detected in the kitchen where the allergens levels were as high as ug/g of dust (Figure 4.29). Also, taking into consideration the extensive cross-reactivity amongst Alc, the levels of Alc detected in the indoor might be cumulative total of various cross-reactive Alcs in the dust. For the outdoor environment, substantial levels of Alcs were detected. The highest level of alc in the environment (8ng) was detected during the month of Jun-July (Figure 4.30). Unlike SODs, the levels of Alcs did not coincide with the Curvularia spore counts. This can be because the Alcs might be resistant to degradation and hence are prevalent for longer periods of time in the environment (indoor as well as outdoor). This may account for the high level of IgE reactivity of Singaporean patients to Alcs. 4.3.6 Miscellaneous allergen groups Due to the limitations in the number of available homologs (in a group) to compare, the details on the rest of the homologous allergen groups would be briefly explained and summarized under this section. As no homologs were available for Cur l and Cur l 13, cross-comparison studies for these were not carried out. In order to compare the IgE binding frequencies, the homologous allergens were expressed and tested over the 182 Chapter Figure 4.29: Levels of Alc allergen in the indoor dust samples Anti ClAlc rabbit polyclonal antibodies were used to detect the hsp levels in the dust. A total of 54 dust samples [12 samples from bedroom floor (BRF), samples from bedroom carpet (BRC), 13 samples from living room floor (LRF), 17 samples from living room carpet (LRC) and samples from kitchen floor (KF)] were screened. Average concentrations for individual niches are showed by cross-bars. Figure 4.30: Levels of Alc allergen in the outdoor air samples A total of 45 weekly air samples (spanning over a year) were screened. Concentration of allergen (ng/m3 air/week) Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan 183 Chapter earlier mentioned four populations. As expected, these allergens showed lower IgE binding frequencies as compared to the earlier groups. Out of all the miscellaneous homologous allergen groups, Calcium binding proteins (CaBP) showed lowest IgE binding frequencies (ranging from – 32%) over all the four populations tested as compared to other groups. No IgE binding towards CaBP was found in the Indian population. Cur l showed higher IgE binding frequencies as compared to the corresponding human CaBP (except for the Singaporean population). For the Asp f allergen homologs group, Cur l showed higher IgE binding frequencies as compared to Asp f (except for the Indian population). In the case of the Asp f 15 homologs group, Asp f 15 showed higher IgE binding frequencies as compared to that of Cur l 11 (except for the Indian population). Likewise, in the case of Asp f homologs group, Asp f showed higher IgE binding frequencies as compared to that of Cur l 12 (except for the Indian population). Overall, the Colombian population showed higher IgE binding frequencies to these allergens as compared to other populations (Table 4.1). Inhibition studies were carried out on these groups in order to establish cross-reactivity (if any) between the homologs. In the case of the CaBP, human CaBP was seen to inhibit Cur l around 70% suggesting it to be cross-reactive. For the Asp f homologs group, Asp f was only partially able to inhibit Cur l suggesting that there are some epitopes unique to Asp f and Cur l 9. For the Asp f 15 homologs group, Asp f 15 was able to inhibit Cur l 11 by 65%, suggesting possible cross-reactivity amongst these two allergens (Table 4.2). 184 Chapter Table 4.1: IgE binding frequencies of the miscellaneous homologous allergen groups Accession numbers for the known allergen homologs as well as the Open Biosystems accession numbers for human homolog are given in the bracket. Allergen groups are named as per the biochemical function of the protein. For the proteins with unknown functions, name of the known allergen homologs are used. Allergen Groups Homologs IgE binding frequency (%) Singaporean Italian Calcium binding proteins Asp f homologs Asp f 15 homologs Asp f homologs Cur l H. sapiens (MHS1011-58592) Cur l Asp f (U56938) Cur l 11 Asp f 15 (AJ002026) Cur l 12 Asp f (AJ223315) 185 7.9 30.3 40.8 36.8 38.2 53.9 43.4 47.4 30.6 11.9 28.8 11.9 33.1 40.0 18.8 35.0 Colombian Indian 32.2 16.1 66.1 60.2 56.8 60.2 50.8 59.3 0.0 17.6 52.9 76.5 82.4 58.8 70.6 47.1 Chapter Table 4.2: Self and cross-inhibition studies for the miscellaneous homologous allergen groups The pools of sera used for the experiment were taken from the specific reactive patients` sera from the Colombian population. BSA was used as a heterologous protein showing no binding to human IgEs. Cross inhibition of the specific C. lunata allergens by the corresponding homolog while self inhibition by the respective C. lunata allergen was carried out. Sequence Identity is the percentage sequence identity as obtained after protein alignments of the homologs using DNAMAN v4.15. Allergen Groups Calcium binding proteins Asp f homologs Asp f 15 homologs Asp f homologs Homologs Sequence Identity (%) Cur l H. sapiens (MHS1011-58592) Cur l Asp f (U56938) Cur l 11 Asp f 15 (AJ002026) Cur l 12 Asp f (AJ223315) 186 63% 30% 48% 23% IgE inhibition (%) 85% 68% 80% 30% 90% 65% N.D. N.D. Chapter Inhibition studies for the Asp f homologs group was not carried out since the IgE binding reaction on the bi-plots (data not shown) were not correlating strongly. Three dimensional structural comparisons were not possible for these groups as there was no template structure available to model these allergens. Sequence similarities suggested that CaBPs were highly conserved (63% identity) amongst themselves, thus explaining the observed cross-reactivity. Low sequence similarities were observed amongst the Asp f homologs group which also explains the low IgE inhibition of Cur l by Asp f 2. Asp f 15 homologs group was seen to have 50% identity amongst them. These identical residues might play a role in crossreactivity as observed amongst these homologs. No detectable levels of these miscellaneous allergen homologs groups were observed in both the indoor and the outdoor environments, suggesting that these allergens are present in very little amounts in the environments which in turn imply that there is a lesser chance of sensitization to these allergens. 4.4 DISCUSSION The exhaustive studies done on cross-comparison of various allergen homologs with the corresponding C. lunata allergens provides us with an impetus of understanding the C. lunata allergens and fungal allergens in general. First of all, the allergens from C. lunata as well as the corresponding protein homologs showed binding to the patient IgEs. The IgE binding frequencies are varied within and amongst the homologous allergen groups. As this study involves testing all the allergens of C. lunata as well as their homologs to be tested and comparing them over 187 Chapter the same sets of populations, it is easier to compare the reaction profiles of one group or a particular allergen with the other, thus giving a better and more correct picture of the IgE binding. Moreover, as more than one population of varied geography, demographics as well as atopy patterns were used, it aids in understanding the effect of a particular allergen or a group over a specific population or atopy. Also, this studies show that a particular allergen can have differential reactivities over various populations. Secondly, these studies show that majority of the Curvularia lunata allergens (fungal allergens in general) are cross-reactive. The reason behind this might be due to conserved phylogeny of these proteins, thus conferring them to bear shared epitopes generating cross-reactivity. Also, as observed for some of the groups, the member proteins shared sequence as well as the three dimensional structures. Finally, quantifiable levels of some of these allergens persist in the indoor as well as outdoor environments, making them responsible for possible sensitization amongst the atopic patients. 188 [...]... Italian 44 .7 23.5 44 .4 18 .4 47.1 1.3 57.9 52.9 45 .0 32.9 35.3 27.5 144 Colombian 68.6 42 .4 89.8 90.7 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** 160 r = 0.713** 140 120 ScSOD 100 80 60 40 20 0 0 20 40 60 80 100 120 140 160 ClSOD 180 r = 0.886** 160 140 HsSOD... with other SODs from human and fungi suggesting that the levels of SODs in the indoor 149 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 150 Figure 4. 6: Overlap of AfSOD and ClSOD 3D models... 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... 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... 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 1 54 Chapter 4 Figure 4. 9: Antigenic cross-reactivity... 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 158 Chapter 4 Figure 4. 11: IgE binding study of Trxs over four different populations A) Intensities of the reactions for various allergens tested... 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 4 C before being added into the respective wells having 1ug of ClSOD ClSOD was found to be inhibited more 143 Figure 4. 1: IgE binding study of MnSODs over four different... 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... (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. .. 140 ClSOD AfSOD ScSOD HsSOD ILKASI K FMKL K 191 210 233 140 152 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 C on c ent r at i on ( ug /gm ) 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 . (%) 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. 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. are mentioned 1 34 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