Table 1.
Clinical characteristics of the mold allergy patients selected for the present study.
Fig 1.
Purification of native nRhi o 1.
(A) I. Chromatogram of anion exchange chromatography (Q-HP) of the fraction obtained from 60% ammonium sulfate cut of the total protein extracts. Third fraction (Fr:3) contains the 44 kD Rhi o 1. II. Chromatogram of the first round gel filtration chromatography (SuperdexTM 75) with Fr:3. Second fraction (Fr:3B) contains the desired allergen mixed with low molecular weight proteins. III. Chromatogram of the second round of gel filtration chromatography with Fr:3B. Peak (Fr:3BII) represents native Rhi o 1 with desired purity. (B) SDS-PAGE of all the Rhi o 1 containing fractions obtained during three round of column chromatography. Lane: M is molecular wt. marker and Lane: T is fraction obtained from 60% ammonium sulfate cut of the total protein. (C) Screening of these three fractions by IgE-immunoblot with RO positive patient sera to check for the presence of the allergen. Final fraction Fr:3BII showed the presence of a single IgE reactive band at 44 kDa implying the homogeneity of the purified allergen. (D) Specific IgE reactivity of the purified nRhi o1 by ELISA using individual sera from fourteen RO positive patients. ‘Y’ axis represents the P/N ratio, which was observed to be > 4.0for nRhi o 1 (blue bars). This was compared with P/N of the crude allergen extract of RO as positive control (red bars).
Fig 2.
(A) Purified nRhi o 1 was resolved in 2D gel and the observed pI was same with the theoretical pI of the allergen. Single spot on 2D gel suggests the absence of any isoform of Rhi o 1. (B) Results of peptide mass fingerprint of trypsin digested Rhi o 1. MS spectra showed homology with Endopeptidase of RO (gi|384498765) in NCBI database. (C) Mass spectra of Rhi o 1 generated in MALDI-TOF. The box represents the results of MALDI-TOF/TOF of eight unique matched peptides and their corresponding ion scores. MS/MS of all the peptides displayed significant match with the same protein gi|384498765, where the significance level was p > 0.05.
Fig 3.
Full length cDNA and amino acid sequence of Rhi o 1.
Amino acids, which are bold and underlined, represent those identified by N-terminal sequencing of nRhi o 1 suggesting the presence of an N terminal 20 amino acid long signal peptide. Amino acids shown as bold and within boxes, are those identified by MS/MS.
Fig 4.
Recombinant rRhi o 1 and its allergenecity.
(A) Recombinant expression of Rhi o 1 in E. coliinduced by IPTG. Lane: M is the molecular wt marker; Lane: T is the total protein after 0.5 mM IPTG induction of E. coli harboring pRSETA-Rhi o 1 cDNA construct and Lane: P is the Ni-NTA purified rRhi o 1. (B) IgE-western blots of rRhi o 1 with individual serum of ten RO positive patients (lane 1–10), which shows the presence of IgE reactivity in rRhi o 1. Lane-c represents the negative control blot with pool of sera from six healthy subjects in which no IgE-reactive bands are visible. (C) Mediator (histamine) release efficiency of rRhi o 1 from effector cells through passive sensitization with sera from ten allergy patients (1–10). HS1 and HS2 represent sensitization with two non-atopic healthy sera as negative control showing minimum percentage of histamine release. Challenge with BSA instead of Rhi o 1 also served as negative control with no histamine release.
Fig 5.
Enzyme assay for characterization of Rhi o 1 as aspartic protease.
(A) In-gel: 0.02% Gelatin zymography of purified nRhi o 1 (lane 1) and rRhi o 1 (lane 2) in 7% polyacrylamide gel. Due to protease activity colorless zones appeared at region corresponding to Rhi o 1 position on gel and remaining gel stained blue with CBB-R250 due to presence of gelatin. (B) In-sol: Spectrophotometric assay of aspartic protease with BSA as substrate. 100 nM Rhi o 1 catalyzed degradation of 2% BSA in presence of 50 mM sodium citrate (pH 3.2). After terminating reaction with perchloric acid, precipitated proteins were removed and absorbance of the clear supernatant containing degraded products of BSA was taken at 280 nm. Purified nRhi o 1 (blue line) and rRhi o 1 (red line) displayed time dependent increase in A280 of the supernatant.
Fig 6.
CD spectra of nRhi o 1 (solid line) and rRhi o 1 (discontinuous line) within a range from 200 to 260 nm at constant temperature.
Almost similar folding pattern was observed for both the proteins with high content of beat-sheets.
Fig 7.
Bioinformatic analysis of aspartic protease allergens.
(A) Dendrogram showing evolutionary relationship among twelve aspartic protease allergens (data retrieved from AllFam database) including Rhi o 1 and one unrelated protein (Human Glyceraldehyde phosphate dehydrogenase) as negative control. (B) Multiple sequence alignment of the amino acid sequences of four officially recognized aspartic protease allergens including Rhi o 1 (data retrieved from IUIS allergen database) showing substantial homology. The conserved aspartate residues of the catalytic sites are highlighted in Red box.
Fig 8.
Cross reactivity between Rhi o 1 and Bla g 2.
(A) IgE ELISA: rBla g r was tested for its IgE binding to ten Rhi o 1 allergic sera and eight of them were IgE reactive to rBla g 2 (except patient no. 5 and 8) (B) IgE ELISA inhibition: These eight Rhi o 1 positive sera with high anti-Bla g 2 IgE titer were pooled and pre-incubated with increasing concentrations of rBla g 2 (fluid phase inhibitor). rRhi o 1 and BSA instead of rBla g 2 were used as positive (auto inhibition) and negative control (no inhibition) respectively. IgE Binding to plate bound 1 μg of rRhi o 1 was inhibited by rBla g 2 in a dose dependent manner. IC50 for rBla g 2 is 20 ng and maximum 58% inhibition was observed with 100 ng rBla g 2. (C) A reciprocal IgE-ELISA inhibition where plate bound 1 μg of rBla g 2 in solid phase was incubated with sera mixed with rRhi o 1 or rBla g 2. In case of rRhi o 1 the IC50 is 9 ng and maximum ~93% was achieved at 1000 ng. In case of auto inhibition with rBla g 2 full inhibition was observed. (D) Cross-stimulation experiment: PBMC’s sensitized with eight anti-Rhi o 1 IgE antibody were stimulated with rBla g 2 and histamine release was observed within a range 29% to 36%. No histamine release observed for healthy sera (HS1 and HS2) sensitization and BSA challenge as negative controls.
Fig 9.
IgE binding to denatured Rhi o 1: (A) IgE blot inhibition: 5 μg rRhi o 1 on PVDF membrane was probed with patient sera containing increasing concentrations (0.1 to 10 μg) of Bla g 2. No visible inhibition of IgE binding to Rhi o 1 was observed in this case, suggesting the role of conformational epitope in cross reactivity. Whatever IgE reactive bands of Rhi o 1 appeared was due to non-cross reactive linear IgE epitopes of Rhi o 1. For auto inhibition positive control, 5μg of self protein (i.e. rRhi o 1) almost fully inhibited IgE binding to membrane bound Rhi o 1. (B) rRhi o 1 was heat denatured and then immediately dotted on PVDF membrane for IgE-blotting with three patient sera.
In all the cases heat denatured Rhi o 1 was found to bind IgE as efficiently as the non denatured Rhi o 1. BSA dotted on the same membrane served as negative control. Healthy serum and no serum buffer control are shown in the two lower most panels.
Fig 10.
Identification of cross reactive conformational epitope on Rhi o 1 using Bla g 2 specific mAb’s.
(A) Sandwich ELISA: Increasing concentration of rRhi o 1 was added to four solid phase plate bound anti-Bla g 2 mAb’s 4C3, 2F1, 1F3 and 7C11. Anti-aspartyl protease pAb in rabbit was used as detection Ab and AP tagged anti-rabbit IgG was used as secondary Ab. Only mAb 4C3 displayed dose dependent binding to Rhi o 1 whereas 7C11, 2F1 and 1F3 did not bind to Rhi o 1. (B) Effect of heat denaturation on mAb4C3 binding: Binding of mAb4C3 to rRhi o 1 treated at higher temperatures (>50°C) was almost abolished, suggesting the relevance of a conformational epitope in mAb4C3 binding. (C) Inhibition of IgE binding to Rhi o 1 by mAb 4C3: ELISA plates were coated with fixed concentration of rRhi o 1 followed by addition of increasing concentration of 4C3 and cross reactive patient sera. 4C3 showed dose dependent inhibition of IgE binding to Rhi o 1. Maximum 56% inhibition was observed at 10 μg/ml 4C3 concentrations. Anti-aspartyl protease pAb (maximum inhibition) and anti-6x His pAb (no inhibition) instead of 4C3 were used as positive and negative control respectively.
Fig 11.
Mapping of cross reactive 4C3 recognizable conformational epitope on Rhi o 1.
Superimposition of Rhi o 1 model on Bla g 2 crystal structure allowed identification of common structural elements in the conformational epitope. (A) Alignment of ribbon diagram of Bla g 2 crystal structure (pink) and Rhi o 1 model (yellow) identifies a region on Rhi o 1 (within box) structurally very similar to 4C3 binding region of Bla g 2. (B) Molecular docking of 4C3 crystal structure on Rhi o 1 model: H-bond interaction between the Rhio1 allergen (Brown) docked on mAb 4C3 (Light chain in Green and Heavy chain in Violet colour). The respective residues are labeled and colored accordingly. A Tyrosine274 was found to be critical for antibody binding.