Serological Reactivity and Identification of IgE-Binding Polypeptides of Ganoderma applanatum Crude Spore Cytoplasmic Extract in Puerto Rican Subjects

Abstract

Background

The allergenic potential of Ganoderma applanatum basidiospores has been demonstrated previously in Puerto Rico. However, basidiomycete allergens are not available for inclusion in allergy diagnostic panels. Therefore, we sought to confirm allergic sensitization towards G. applanatum crude spore cytoplasmic extract (CSCE) through reactivity in serological assays and detection of IgE-binding polypeptides.

Methods

With an indirect ELISA, serological reactivity was compared between groups of individuals with different allergic profiles. Group 1 (n = 51) consisted of individuals with sIgE to allergens included in diagnostic panels; group 2 (n = 14) were individuals with no sIgE to the allergens tested; and group 3 (n = 22) were individuals with no allergic history. To visualize IgE-binding polypeptides, group 1 sera were examined with Western blot (WB). Polypeptide bands with the highest reactivity were analyzed by mass spectrometry (MS) for putative identification.

Results

Serological reactivity of group 1 was significantly higher than that of group 3 in indirect ELISA (p = 0.03). Sixty five percent of group 1 individuals showed reactivity to polypeptide bands in WB. Bands of 81 and 56 kDa had the highest reactivity proportions among the reactive sera, followed by a 45 kDa band. MS analysis of these three polypeptides suggests they are basidiomycete-derived enzymes with aconitate hydratase, catalase, and enolase functions.

Conclusions

G. applanatum spores have allergenic components recognized by Puerto Rican individuals, which could eventually be considered as markers in cases of fungal allergy and be included in diagnostic allergen panels in Puerto Rico and tropical regions.

INTRODUCTION

Spores from basidiomycetes, such as Ganoderma applanatum, are prevalent in the atmosphere in many areas of the world and tropics, including in Puerto Rico, where basidiospore allergy has been reported [1–10]. Contrary to mitosporic fungi, which produce spores asexually and are mostly composed of asexual states of ascomycetes (e.g. Aspergillus spp., Penicillium spp.), the allergenic potential of basidiomycetes has been underestimated and only few allergens have been characterized, such as Cop c 1 to 7 (Coprinus comatus), Psi c 1 and 2 (Psilocybe cubensis), and Sch c 1 (Schizophyllum commune) [2, 11–13]. In addition, because of the difficulties encountered in collecting them on the field, and obtaining the necessary amount of spore material, extracts are not easily obtained [5–7]. As a result, basidiomyecetes are less studied and there is a lack of basidiospore extracts for allergy panels [5]. Considering that basidiospores constitute approximately two thirds of the total fungal spores in outdoor air samples, these panels may not be representative of the composition of the Puerto Rican airborne fungal spores with allergenic potential [4].

Skin test reactivity to crude extracts of G. applanatum spores was previously demonstrated by our group among Puerto Rican allergic subjects [7]. G. applanatum had a higher reactivity (30% vs 6%) than all the commercial extracts of mitogenic fungi tested (Penicillium spp., Aspergillus spp., Fusarium spp., Alternaria spp., and Chaetomium spp.). Furthermore, the level of sensitivity among asthmatic patients to G. applanatum and mites was 44% for both allergen extracts; the latter is an allergen source frequently associated with respiratory allergic diseases [14, 15]. However, to confirm antibody reactivity against G. applanatum spore allergens and identify immunoreactive components, serological tests that detect these interactions are needed.

The current pilot study investigates serological reactivity of Puerto Rican subjects to G. applanatum crude spore cytoplasmic extract (CSCE) using in vitro tests. An indirect enzyme-linked immunosorbent assay (ELISA) was designed to detect IgE antibodies and compare the serological reactivity among individuals with different allergic profiles. Furthermore, Western blot (WB) analysis was performed to identify IgE-binding polypeptides using the sera from allergic individuals previously tested in the indirect ELISA. The polypeptide bands with highest proportion of reactivity were analyzed and putatively identified by mass spectrometry (MS). Based on our previous findings and the high prevalence of respiratory diseases present among Puerto Ricans, we expected to find a high serological reactivity rate to G. applanatum CSCE among the serum samples tested [7, 16, 17].

MATERIALS AND METHODS

Spore Print Collection

Fruiting bodies of G. applanatum were searched in forested and green areas of Puerto Rico. The spore prints were collected in the field with spore traps, which were replaced every two to four days. The spore traps consisted of sterile Petri dishes placed under the sporocarp of the fruiting body, secured with brite aluminum screening (Phifer, Tuscaloosa, AL) and tacks. A cover slip was included in each spore trap to verify the purity of the spore print with microscopy. Once collected, the dishes with the spore print deposits were placed in a desiccator for at least 24 h or until dry. The dry material was stored at −80°C until used for extract preparation. If the spore print material showed any sign of contamination, it was discarded.

Crude Spore Cytoplasmic Extract (CSCE) Preparation

The G. applanatum CSCE was prepared following the method described by Rivera-Mariani et al. [7] with few minor adjustments. Briefly, spore cytoplasmic proteins were extracted from a suspension consisting of 1 g of spore print material and 10 ml of ammonium bicarbonate buffer (0.125 M, pH 8.2; Fisher Scientific, Fair Lawn, NJ) prepared with sterile pyrogen-free water (Baxter Healthcare, Deerfield, IL). No protease inhibitors were added to the suspension; however, the preparation and handling of the extracts were carried out under conditions that minimize proteolysis. The homogenate was centrifuged at 208.5 × g for 20 min at 4°C with a Sorvall RT6000B Refrigerated Centrifuge (Dupont, Wilmington, DE). The resulting supernatant was centrifuged at 216179.7 × g for 1 h at 4°C in an Optima L-100 XP Centrifuge (Beckman Coulter, Brea, CA). The supernatant was filter-sterilized with a Nalgene 115 ml Filter Unit (0.45 µm; Nalge Nunc International, Rochester, NY) and lyophilized for 24 h with an Edwards Modulyo Freeze-Dryer (Edwards High Vacuum International, Crawley, England). The resulting lyophilized solid was re-suspended in 20% of the original volume with sterile pyrogen-free water, and the re-suspended fractions pooled and stored at −80°C until used. This was the sample of CSCE used for the serological assays.

A small amount of CSCE was also prepared for skin prick test (SPT) administration. For this preparation, a portion of the lyophilized material was re-suspended in 20% of the original volume with a solution containing 50% glycerol (VWR International, West Chester, PA), 85 mM sodium chloride (Sigma-Aldrich, St. Louis, MO), and 30 mM sodium bicarbonate (AMRESCO, Solon, OH), with subsequent pooling of the fractions. The saline part of the solution was prepared using sterile pyrogen-free water. Sterility of the CSCE pool for SPT administration was confirmed by inoculating a loopful of the extract on blood agar (Columbia blood agar base [Becton, Dickinson and Company, Sparks, MD], 5% sheep RBC [Biolab, Bayamón, PR]). The dish was left incubating at 25°C for seven days, with no evidence of growth. This extract preparation was stored at 2–8°C until used.

Determination of Basidiospore Extract Protein Concentration

The protein concentration of the CSCEs was determined with the DC Protein Assay Kit II (Bio-Rad, Hercules, CA) according to manufacturer’s instructions. The protein concentrations of the CSCE pools used in the serological assays ranged from 1.0 to 4.0 mg/ml. The protein concentration of the CSCE prepared for SPT administration was 1.3 mg/ml. This protein concentration is in accordance with the recommendation from Greer Laboratories (Greer Laboratories, Lenoir, NC) for allergenic fungal extracts diluted in 50% glycerol to be used in SPTs.

Human Sera

With the approval of the University of Puerto Rico, Medical Sciences Campus (UPR-MSC) Institutional Review Board (IRB: 9830113), human sera remnants were donated from Toledo Clinical Laboratory (Arecibo, PR) and other sera collected from Puerto Rican volunteers from the UPR-MSC community without history of allergic disease. The sera remnants donated from the clinical laboratory came from individuals that had been tested for sIgE with RAST against various commercial allergens included in either one of two panels that consisted of respiratory allergens alone or respiratory and food allergens combined (Table 1); their clinical history were not available and the serum samples were not tested to the same battery of commercial allergens. Volunteers were adults from whom 20 cc of blood were obtained, and the serum was extracted in the laboratory. They had no symptoms of allergy, never received treatment for allergy or its manifestations, and had no family history of allergic disease. The clinical history of the volunteers was not available.

Table 1.

RAST commercial allergen panels previously tested on donated sera remnants by the reference clinical laboratory (Toledo Clinical Laboratory, Arecibo, PR)

Allergen Panel	Allergens
Respiratory*	Alternaria tenuis, American cockroach, Aspergillus fumigatus, Cat dander/epithelium, Chicken feathers, Cladosporium herbarum, Dermatophagoides farinae, Dog epithelia, Horse dander, Mouse epithelium, Penicillium notatum, Rat epithelium
Respiratory/Food*	Acacia, Alternaria tenuis, Aspergillus fumigatus, Australian pine, Bermuda grass, Cat dander/epithelium, Cladosporium herbarum, Cockroach, Corn, Cow milk, Dermatophagoides farinae, Dog epithelia, Egg, English plantain, Fusarium moniliforme, Lamb’s quarter, Mango, Mucor racemosus, Penicillium notatum, Phoma betae, Queen palm, Rough pigweed, Western ragweed, Wheat

^*Each serum sample was tested to one of the panels, not both, and not all serum samples were tested to the same battery of allergens.

Selection of Controls

A positive control was randomly selected from the donated sera remnants who had sIgE to one or more fungal allergens. This individual had sIgE to the following allergens: Alternaria tenuis, Aspergillus fumigatus, Cladosporium herbarum, Penicillium notatum, Dermatophagoides farinae, American cockroach, and rat epithelium. The sera remnant was further analyzed at a reference clinical laboratory for total IgE. The total IgE reported was 171 kU/L, which is higher than normal IgE level for adults (as per http://www.questdiagnostics.com/). The random negative control (selected from the volunteer’s sera) had no history of allergic disease and its total IgE was 4.20 kU/L, which is normal total IgE level (as per http://www.questdiagnostics.com/). In addition, SPTs were performed on the negative control volunteer by a board-certified allergist from the UPR-MSC Allergy Clinics with a commercial allergen panel (Greer Laboratories) and the G. applanatum CSCE prepared in the laboratory for SPT administration. The commercial allergen extracts consisted of mitosporic fungi such as Penicillium spp., Aureobasidium spp., Alternaria spp., Cladosporium spp., Fusarium spp., Aspergillus spp., and Acremonium spp.; ascomycetes such as Chaetomium spp.; pollen of trees, Bermuda, Johnson, and Bahia; pet allergens such as dog and cat dander; cockroaches; the mite allergens Der p and Der f; positive (histamine 15 mg/ml) and negative (glycerol-phosphate buffered saline [PBS]) controls. No positive results were recorded for the allergenic extracts tested, including CSCE.

Indirect Enzyme Linked Immunosorbent Assay (ELISA)

Indirect ELISA against CSCE was performed on 87 collected human sera following the protocol described by Figueroa-Santiago et al. [18] with minor adjustments. Reagents in the assay were optimized by checkerboard titration. Human sera were grouped as shown in Table 2. High-binding polystyrene microplates (Costar, Corning, NY) were coated with 5 µg/ml of CSCE. Human sera and conjugated antibody (polyclonal goat anti-human IgE labeled with horseradish peroxidase [HRP], 1.0 mg/ml; KPL, Gaithersburg, MD) were used at dilutions 1:100 and 1:2,500, respectively. Finally, substrate solution (10 mg o-phenylenediamine dihydrochloride [Sigma-Aldrich] + 10 ul 30% H₂O₂ [Sigma-Aldrich] in 0.1 M citrate-phosphate buffer, pH 5.0) was added to each well and the reaction was stopped with 10% HCl (Sigma-Aldrich). The absorbance at 490 nm (A₄₉₀) was immediately measured using a microplate reader (Model 680; Bio-Rad). Each determination was performed in duplicate, and the results expressed as the mean A₄₉₀ value for each determination. Positive and negative controls were included on each microplate.

Table 2.

G. applanatum crude spore cytoplasmic extract (CSCE) indirect ELISA sample groups

Serum Samples with Different Allergic Profiles	Origin	Number
Group 1†: Individuals* with sIgE reactive to one or more allergens of the RAST allergens tested	Donated sera remnants from Toledo Clinical Laboratory, Arecibo, PR	51
Group 2†: Individuals* with no reactive sIgE to the RAST allergens tested	Donated sera remnants from Toledo Clinical Laboratory, Arecibo, PR	14
Group 3††: Individuals* with no previous history of allergic disease based on self-reported symptomatology	Puerto Rican adult volunteers from the UPR-MSC community	22
Total		87

^†Individuals in these groups were suspected of having allergic symptomatology due to the medical referral for sIgE testing. The serum samples were not tested to the same battery of commercial allergens.

^††This group is not considered a negative control group because no skin or serological tests were performed to confirm absence of allergic sensitization.

^*Clinical history of individuals is unknown.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of CSCE, Silver Staining, and Protein Transfer

The protein components in CSCE were separated by molecular weight (MW) under denaturing conditions using one-dimensional SDS-PAGE following the method described by Laemmli [19]. The process was carried out on a 15% gel with 30 µg of CSCE per lane under cold conditions at 200 V for 1 h and 45 min. The resolved polypeptides were silver stained with the Silver Stain Plus Kit (Bio-Rad) according to manufacturer’s instructions, or transferred to a nitrocellulose membrane (NCM) (0.45 µm; Bio-Rad). For polypeptides being transferred, a stained (Rainbow) full range MW marker (2.0 mg/ml; GE Healthcare Bio-Sciences, Piscataway, NJ) was used for every CSCE sample. The transfer of resolved polypeptides was carried out under cold conditions in a Mini-PROTEAN Tetra Cell (Bio-Rad) in Tris-glycine buffer (pH 8.0) containing 20% methanol, at 200 V for 2 h and 10 min.

Western Blot (WB)

Optimization of the assay was based on a compromise between band intensity in the positive control and less reactivity in the negative control, which was assisted by image-analysis performed for each sample and its corresponding marker using the open-source software ImageJ® (National Institutes of Health). Density plots were produced, in which the x-axis represents the distance along the membrane and the y-axis the averaged pixel intensity, represented as a peak. Each peak in the plot corresponds to a band in the membrane. Once the assay was optimized, WBs were performed in duplicate for each serum in group 1. After transference, the NCM was cut into strips and blocked with 5% bovine serum albumin (BSA) (AMRESCO) in PBS (0.01 M, pH 7.4) containing 0.05% Tween 20 (PBST-20) for 1 h at room temperature (RT). Each strip was incubated overnight (O/N) at 4°C with a serum sample diluted 1:100 in 3% BSA prepared in PBST-20. After several washes with PBST-20, incubation with biotinylated polyclonal goat anti-human IgE antibody (0.5 mg/ml; KPL), diluted 1:5,000 in blocking solution, followed for 4 h at RT. The strips were washed before incubating with avidin-HRP (1.0 mg/ml; Sigma-Aldrich), diluted 1:10,000 in blocking solution, for 20 min at RT. After washing, substrate solution (0.025 g of 3,3′-diaminobenzidine [Sigma-Aldrich] + 50 µl of 30% H₂O₂ in 50 ml PBS 1X 0.01 M, pH 7.4) was added and left incubating for 30 min at RT. The solution was discarded and the reaction stopped with ultrapure water. Image-analysis was performed with the software ImageJ® as previously described to assist in the identification of IgE-binding polypeptides. Once the reactive bands were identified, the marker was used to construct a reference standard curve from which the approximate MW of the reactive CSCE polypeptide bands was calculated based on their relative mobility in the membrane.

Ultrafiltration of CSCE with subsequent SDS-PAGE and Silver Staining

Ultrafiltration of CSCE was performed to concentrate and separate the extract proteins with MW above 30 kDa, which were the most reactive in the WBs. It was performed in an Amicon Filter Unit (Millipore, Beverly, MA) using a regenerated cellulose membrane with a 30 kDa cut-off (Millipore). The retentate, labeled as CSCE > 30, was collected and the protein concentration determined (3.2 mg/ml) as previously described for the CSCE. SDS-PAGE and silver staining for CSCE > 30 were performed as described for the CSCE with a slight modification: the electrophoresis run time was increased to 2 h and 30 min to allow more separation between the polypeptide bands.

Mass Spectrometry (MS)

Bands corresponding to three IgE-binding polypeptides identified to have the highest proportion of reactivity in the WBs were excised from a silver stained gel of a CSCE > 30 SDS-PAGE and sent O/N to the Proteomics and Mass Spectrometry Facility at Cornell University Institute of Biotechnology in Ithaca, NY for MS analysis and database search. The process consisted of an in-gel digestion with trypsin and extraction followed by nano liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis in an Orbitrap Elite (Thermo Fisher Scientific, Waltham, MA) instrument. Since there is no current G. applanatum database available, the MS data was compared against the National Center for Biotechnology Information (NCBI) database using other Fungi taxonomy (http://www.ncbi.nlm.nih.gov/protein/). The search was done using Mascot database search engine using the following search setting: one missed cleavage site by trypsin allowed with fixed carboxamidomethyl modification of cysteine and variable modifications of methionine oxidation and deamidation of asparagine and glutamine residues. Mascot reports for each band were then interpreted for a putative protein identification.

Statistical Analysis

The statistical analysis was performed in R version 3.2.3 (R Foundation for Statistical Computing, Vienna, Austria). Before performing a one-way ANOVA and Bonferroni’s pairwise comparison test to determine mean differences in indirect ELISA A₄₉₀ for individuals in groups 1, 2, and 3, the normal distribution of A₄₉₀ values and equal variances for each group was confirmed with a Shapiro-Wilk test and a Bartlett’s test, respectively. A Fisher’s exact test, with 95% confidence intervals (CI), was performed to compare the proportion of reactive sera for each polypeptide band detected in the WBs among group 1 sera reactive to CSCE. To evaluate if there is an association between indirect ELISA A₄₉₀ and WB reactivity, group 1 sera were grouped as reactive (if reactive to one or more polypeptide bands in WB) vs. non-reactive (not reactive to any polypeptide band in WB). Equal variances of the indirect ELISA A₄₉₀ values between groups was confirmed with an F-test, as well as normality in each group with a Shapiro-Wilk test prior to performing a Student’s two-tailed t-test (assuming equal variance) to compare differences in mean indirect ELISA A₄₉₀.

RESULTS

Indirect ELISA reactivity against the whole CSCE differed between Group 1 and 3

To detect IgE antibodies and compare serological reactivity to CSCE, an indirect ELISA was performed on sera from individuals with three different allergic profiles (Figure 1). The mean A₄₉₀ was highest among individuals from group 1 (0.5157 ± 0.1637); group 2 and group 3 mean A₄₉₀ were 0.4963 (± 0.1565) and 0.4162 (± 0.1100), respectively. The ANOVA test showed statistical significant difference (p = 0.04) in the mean A₄₉₀ among the three groups. After performing a Bonferroni’s pairwise comparison, the mean A₄₉₀ was statistically significantly higher only when group 1 was compared to group 3 (p = 0.03). However, no statistically significant mean differences were found between group 1 and group 2 (p = 1.00), nor between group 2 and group 3 (p = 0.37). Positive and negative control sera mean A₄₉₀ values were 0.6240 and 0.2885, respectively. These results suggest that the group with allergic history, based on sIgE to commercial extracts, was more reactive to the CSCE than the group with no reported history of allergies (based on self-reported symptomatology).

Fig. 1.

Serological reactivity of human sera against G. applanatum crude spore cytoplasmic extract (CSCE) when tested with indirect ELISA (A₄₉₀). The x-axis represents the three groups (group 1, n = 51; group 2, n = 14; group 3, n =22) of different allergic profiles and the y-axis the mean A₄₉₀ for each individual. The red square within each group represents the group mean. ** Mean difference was statistically significant (p < 0.05) compared to group 1.

Three out of five serum samples from group 1 were reactive to CSCE in the Western blot

In order to visualize IgE-binding polypeptides present in CSCE, Western blots (WBs) were performed with individual serum samples from group 1. We used sera from group 1 because they demonstrated the highest reactivity to CSCE in indirect ELISA compared to the other two groups, as well as having allergic sensitization to one or more allergens as demonstrated by RAST. Sixty five percent (33/51) of individuals were reactive to one or more polypeptide bands (Table 3). The reactivity against the IgE-binding polypeptides was confirmed through image-analysis by the presence of respective corresponding peaks in the membrane plots generated with the software ImageJ®. Seven IgE-binding polypeptides were detected in a variety of MWs: 19, 24, 33, 45, 56, 75, and 81 kDa (Figure 2). The positive control serum was reactive to the polypeptide band of approximately 45 kDa, whereas the negative control showed no reactivity. These results further support the reactivity of the CSCE as seen earlier with the indirect ELISA.

Table 3.

Western blot reactivity among serum samples from group 1 (n = 51)

Western Blot Reactivity	Number of Reactive Polypeptide Bands	Number of Reactive Serum Samples	Total Number of Sera
Reactive	1	3	33
	2	19
	3	6
	4	5
Non-Reactive	0	18	18

Fig. 2.

Plots of the serum samples in which the seven IgE-binding polypeptides, and their corresponding molecular weights, were immunodetected with the Western blot. Each band is represented by arrows of different colors and their corresponding molecular weight. Plot A represents a negative serum sample. Plot B represents a serum sample immunoreactive to the 81 (blue arrow), 56 (green arrow), 45 (red arrow), and 19 kDa (grey arrow) IgE-binding polypeptides; plot C a serum sample immunoreactive to the 33 (purple arrow) and 24 kDa (yellow arrow) IgE-binding polypeptides; plot D a serum sample immunoreactive to the 75 kDa (orange arrow) IgE-binding polypeptide. M = markers; 1 = lane-1.

The 56 and 81 kDa IgE-binding polypeptides had the highest proportion of reactivity

To determine the IgE-binding polypeptides with the highest reactivity among the 33 reactive serum samples from group 1, a Fisher’s exact test was performed to compare the proportion of reactive serum samples against each IgE-binding polypeptide. As shown in Table 4, proportion of reactivity were highest for the 56 (87.9%, CI 71.8 – 96.6%) and 81 (97.0%, CI 84.2 – 99.9%) kDa IgE-binding polypeptides.

Table 4.

Percentage of reactivity to IgE-binding polyleptides among the 33 reactive serum

IgE-Binding Polypeptide (kDa)	% of Reactive (n = 33)	95% CI
19	6.1	0.7 – 20.2
24	3.0	0.1 – 15.8
33	18.2	7.0 – 34.5
45	24.2	11.1 – 42.3
56	87.9	71.8 – 96.6
75	3.0	0.1 – 15.8
81	97.0	84.2 – 99.9

samples in the Western blot

There was no association between indirect ELISA A₄₉₀ and Western blot reactivity

To evaluate if there was an association between indirect ELISA A₄₉₀ and WB reactivity, indirect ELISA values were grouped by WB reactivity among group 1 serum samples: if reactive to one or more IgE-binding polypeptides or no reactivity at all (Figure 3). Although group 1 reactive serum samples had a higher mean A₄₉₀ (0.5285 ± 0.1658) than the non-reactive serum samples (0.4922 ± 0.1617), the mean difference was not statistically significant (p = 0.45) after performing a two-tailed Student’s t-test (assuming equal variance). Therefore, the reactivity of the serum samples in the indirect ELISA was not associated with their reactivity in the WBs.

Fig. 3.

Comparison of group 1 indirect ELISA A₄₉₀ between non-reactive (n=18) and reactive (n=33) individuals in Western blot. The x-axis represents Western blot reactivity and the y-axis the mean A₄₉₀ for each individual. The red square within each group represents the group mean.

Mass spectrometry analysis putatively identified three IgE-binding polypeptides as basidiomycete-derived enzymes

The three IgE-binding polypeptides with the highest reactivity in WB, of approximate MWs of 81 (P1), 56 (P2), and 45 (P3) kDa, were analyzed via MS for a putative identification. Identification for each polypeptide, along with information derived from the Mascot reports, is listed in Table 5. Reports were interpreted for the best hit based on the highest protein score which also matched the polypeptides’ approximate MW. This resulted in choosing the protein hit with the third highest protein score for P1 and the protein hits with the highest protein score for P2 and P3, all being basidiomycete-derived enzymes.

Table 5.

Putative identification of IgE-binding polypeptides by mass spectrometry from NCBI database using other Fungi taxonomy

Polypeptide Band	1 (81 kDa)	2 (56 kDa)	3 (45 kDa)
NCBI Accession Number	2::gi|170094674	2::gi|754378673	2::gi|630186999
Protein Name	Aconitate hydratase	Hypothetical protein PHLGIDRAFT_83711	Enolase
Species	Laccaria bicolor S238N-H82	Phlebiopsis gigantea 11061_1 CR5-6	Moniliophthora roreri MCA 2997
Biological Function	Reversible isomerization of citrate and isocitrate as part of the tricarboxylic acid (Krebs) cycle	Putative catalase function (Hori et al. 2014): decomposition of hydrogen peroxide to molecular oxygen and water; inorganic ion transport and metabolism	Reversible dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate as part of the glycolytic and gluconeogenesis pathways
Cellular Location	Mitochondria	Unknown	Cytoplasm
Protein Score	206	1141	1030
Molecular Weight (Da)	82,919	57,439	47,270
Isoelectric Point	6.02	6.39	5.65
Peptide Numbers Matching to the Protein	10	28	37
Significant Peptide Numbers Matching to the Protein	10	28	37
Unique Peptide Numbers to the Protein	3	2	8
Unique Peptide Numbers to the Protein with Significance	3	2	8
Protein Sequence Coverage	4.2	5.9	19.1
Exponentially Modified Protein Abundance Index (emPAI)*	0.26	0.18	2.03
Molar %**	14.61	2.679	9.107

^*emPAI = 10^^{Protein abundance index (PAI)}-1, where PAI is defined as the number of detected peptides divided by the number of observable peptides per protein and has a linear relationship with the logarithm of protein concentration in LC-MS/MS experiments, making emPAI proportional to protein content in a protein mixture (Ishihama et al. 2005).

^**Molar % = emPAI / Σ(emPAI) × 100.

Hori C, Ishida T, Igarashi K, Samejima M, Suzuki H, Master E, Ferreira P, Ruiz-Dueñas FJ, Held B, Canessa P, Larrondo LF, Schmoll M, Druzhinina IS, Kubicek CP, Gaskell JA, Kersten P, St John F, Glasner J, Sabat G, Splinter BonDurant S, Syed K, Yadav J, Mgbeahuruike AC, Kovalchuk A, Asiegbu FO, Lackner G, Hoffmeister D, Rencoret J, Gutiérrez A, Sun H, Lindquist E, Barry K, Riley R, Grigoriev IV, Henrissat B, Kües U, Berka RM, Martínez AT, Covert SF, Blanchette RA, Cullen D, Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood. PLoS Genetics 2014;10 :e1004759.doi:10.1371/journal.pgen.1004759.

Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, Mann M, Exponentially Modified Protein Abundance Index (emPAI) for Estimation of Absolute Protein Amount in Proteomics by the Number of Sequenced Peptides per Protein. Molecular & Cellular Proteomics 2005;4: 1265–72.

DISCUSSION

In this study, the serological reactivity of the Puerto Rican population to G. applanatum crude spore cytoplasmic extract (CSCE) was evaluated. Serum samples from allergic individuals with sIgE against commercial extracts (group 1), no sIgE to commercial extracts (group 2), or no self-reported history of allergies (group 3) were tested with indirect ELISA and Western blotting (WB). Indirect ELISA revealed that serological reactivity was higher among individuals from group 1, and further evaluation of this group with WB yielded a serological reactivity prevalence above 60%. Mass spectrometry (MS) revealed that the IgE-binding polypeptides with highest serological reactivity were enzymes with metabolic properties. This study provides further evidence of the allergenic potential of airborne basidiospores endemic in tropical settings, such as those from G. applanatum.

Seven G. applanatum CSCE IgE-binding polypeptides were detected in WB, with molecular weights (MW) ranging from 19 to 81 kDa. Two of them, with approximate MWs of 81 (P1) and 56 (P2) kDa, had the highest proportions of reactivity, 97.0% and 87.9%, respectively. These results are similar to the results obtained by Vijay et al. [20], in which they found eight G. applanatum spore allergens in a comparable MW range (18 to 82 kDa). To our knowledge, this is the only previous study identifying G. applanatum spore IgE-binding polypeptides. Even though no sensitization prevalence was reported by the authors (only three atopic patients were studied), strong reactivity of sIgE antibodies was observed at six different MWs. These reactivities included one at approximately 82 kDa, and the strongest at approximately 45 kDa. Interestingly, this last IgE-binding polypeptide has the same approximate MW (P3) as one reported in this study, and which also reported one of the highest reactivities. These allergenic components recognized by the majority of sensitized individuals could eventually be considered markers in cases of fungal allergy. Differences in the allergenic polypeptides detected between the studies may arise from the different extraction protocols, the population tested (possibly sensitized to different allergenic components), or from intrinsic allergen spore differences between places, as it has been demonstrated that spore extracts from different locations can vary [21–25].

MS analysis of P1, P2, and P3 putatively identified these three polypeptides as metabolic enzymes derived from fungal species belonging to the Basidiomycota division, the same as G. applanatum. Aconitate hydratase and enolase are involved in cellular metabolic pathways, while catalase is part of the oxidative stress response proteins (Table 5). It is not uncommon to see proteins involved in intrinsic cellular metabolic pathways acting simultaneously as allergens, and they have been described for fungi in both categories [26]. Each one of the enzymes have also been described as an allergen for different species. For instance, aconitate hydratase has been reported as an allergen of the Asian wasp’s venom, and proteins with catalase and enolase activity have been identified as fungal allergens in several fungal genera, including Aspergillus and Penicillium [27–30]. In fact, enolases are known to be a class of highly conserved fungal allergens and major allergens of prominent fungal species inducing type I allergy such as Cladosporium herbarum and Alternaria alternata [31, 32]. Identification of metabolic enzymes as prevalent allergens in G. applanatum might suggest these enzymes can be allergen sources in other basidiomycete species as well.

Western blotting of G. applanatum CSCE with individuals from group 1 resulted in more than 60% of the reactive serum samples having sIgE antibodies towards one or more spore polypeptides. A high prevalence of sensitization to these basidiospores could be the result of certain attributes which make G. applanatum a good allergen source. The fruiting bodies are present all year long in different areas of Puerto Rico, as evidenced by finding basidiocarps in different municipalities throughout the island in any season, in both urban and forested scenarios. In turn, each basidiocarp can potentially release trillions of spores daily, causing exposure of spore allergens to individuals in clinically relevant amounts, a prerequisite for the development of allergic sensitization to an allergen source [5, 7, 33]. Even if the basidiospore source is not close to populated areas, the spores can still reach urban places by wind dispersion, as demonstrated by the daily reports at the San Juan Station certified by the National Allergy Bureau of the American Academy of Allergy Asthma and Immunology (http://pollen.aaaai.org/nab/index.cfm?p=allergenreport&stationid=168). Continuous breathing of high numbers of spores results in IgE development towards spore antigens in susceptible individuals; therefore, it is not surprising to see a considerable proportion of reactivity to G. applanatum among the individuals evaluated in the study [5, 12, 24]. These results suggest that fungal allergens not being routinely tested in diagnostic panels, like those from G. applanatum spores, should be considered among the panels of allergens for skin and serological testing in tropical settings.

The higher reactivity in the indirect ELISA by individuals with sIgE to commercial extracts (group 1) compared to group 3, but not to group 2, was expected because individuals from both group 1 and group 2 had suspected allergic symptomatology (due to medical referrals for sIgE testing). The higher reactivity in individuals from group 1 compared to group 2, although not statistically significant, matches our expectations because many fungi-sensitized individuals have sIgE to other allergen sources as well, including inhalant allergens [33]. Sensitivity to simultaneous inhalant allergens have been reported in SPTs with G. applanatum antigen preparations, in which 96.7% of positive subjects were also positive to other common inhalant allergen sources [34]. The reactivity of individuals from group 2 in the indirect ELISA, which reported results between the reactivity of individuals with sIgE to commercial extracts and those with no self-reporter history of allergic symptoms, suggests that testing basidiomycetes’ allergens in Puerto Rico could be important in establishing an allergy diagnosis.

There was no relationship between the serological reactivity of individuals from group 1 in the indirect ELISA and the WB. Since the same extract pool was used for both assays, variability in the extract composition and/or proportion of allergens because of fungal strain or batch-to-batch differences was discarded [3]. A possible reason may be the nature of the antigen source, as discrepancies have been reported concerning the sensitivity and specificity of ELISA tests when crude extracts are used [35]. Spore crude extracts are known to contain a mixture of proteins and other spore non-protein components, like carbohydrates [22].Within the extract, these components can compete between them for attachment sites or form interactions that prevent binding of other molecules [35, 36]. These interferences of molecular nature, which are not present in Western blotting, might have an effect on ELISA’s sensitivity and specificity [36]. Furthermore, there might be a difference in the antigenicity of some native proteins compared to their denatured counterparts in the WB, as alteration in the structure of antigenic epitopes might alter serological and other immunological analysis of a protein [23, 37, 38].

The small number of serum samples, must be considered a limiting factor in the current study. Another limitation was not having all serum samples tested with RAST for the same battery of commercial allergen extracts, and lack of a more thorough clinical history of volunteers. This information may have allowed us to perform additional statistical analysis to examine the possibility of cross-reactivity or the relationship with other health factors. Because this was a pilot study, we did not expect to have a large sample size nor complete a comprehensive clinical history. As a follow up to this investigation, a clinical study recruiting patients from the UPR-MSC Allergy Clinics is under way, in which full clinical profiles for each individual will be available. The lack of protease inhibitors in the CSCE preparation may have influenced extract reactivity due to possible proteolytic degradation of allergens or alteration in their IgE-binding sites, but measures were taken to reduce this limitation (i.e. maintaining the extract at lower temperatures) [22, 23]. Protease inhibitors were added because some proteins require proteolytic activation to exhibit their allergenic potential [22, 39]. Reactivity could also have been influenced by other factors, such as diminished allergen recovery from the spores, the possibility of G. applanatum allergenic components having less sensitization capacity, or due to inter-individual variability in the immune responses [23, 33, 34]. Finally, the use of one-dimensional immunoblotting for the identification of CSCE IgE-binding polypeptides is a limitation for two reasons: first, reactive bands may contain more than one polypeptide with the same MW and, second, differences in antibody reactivity between protein isoforms, if any, are masked [40, 41]. Due to lack of resources, two-dimensional immunoblotting was not possible. Prospective studies will employ two-dimensional immunoblotting, complemented with MS analysis, to confirm the identity of the IgE-binding polypeptides putatively identified in this study.

In conclusion, this study provides evidence of the serological reactivity of Puerto Rican individuals to G. applanatum spore allergens. Also, the findings of this study support previous studies from our group about the allergenic potential of less-studied fungi, such as those from basidiomycetes [5–7]. The three most recognized allergens among CSCE sensitized individuals have been putatively identified; however, confirmation of each polypeptide’s identity along with their subsequent purification are needed for proper immunological/molecular characterization and assessment of clinical relevance. This is the first step towards the formulation of high quality G. applanatum allergen sources for use in allergy diagnostic tools, as well as in prevention strategies (i.e. vaccines) and allergen-specific immunotherapy in the tropics, including Puerto Rico.