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Background/objective: Epitope identification assists in developing molecules for clinical applications and is useful in defining molecular features of allergens for understanding structure/function relationship. The present study was aimed to identify the B cell epitopes of alcohol dehydrogenase (ADH) allergen from Curvularia lunata using in-silico methods and immunoassay., Method: B cell epitopes of ADH were predicted by sequence and structure based methods and protein-protein interaction tools while T cell epitopes by inhibitory concentration and binding score methods. The epitopes were superimposed on a three dimensional model of ADH generated by homology modeling and analyzed for antigenic characteristics. Peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by ELISA using individual and pooled patients' sera., Result: The homology model showed GroES like catalytic domain joined to Rossmann superfamily domain by an alpha helix. Stereochemical quality was confirmed by Procheck which showed 90% residues in most favorable region of Ramachandran plot while Errat gave a quality score of 92.733%. Six B cell (P1-P6) and four T cell (P7-P10) epitopes were predicted by a combination of methods. Peptide P2 (epitope P2) showed E(X)(2)GGP(X)(3)KKI conserved pattern among allergens of pathogenesis related family. It was predicted as high affinity binder based on electronegativity and low hydrophobicity. The computational methods employed were validated using Bet v 1 and Der p 2 allergens where 67% and 60% of the epitope residues were predicted correctly. Among B cell epitopes, Peptide P2 showed maximum IgE binding with individual and pooled patients' sera (mean OD 0.604±0.059 and 0.506±0.0035, respectively) followed by P1, P4 and P3 epitopes. All T cell epitopes showed lower IgE binding., Conclusion: Four B cell epitopes of C. lunata ADH were identified. Peptide P2 can serve as a potential candidate for diagnosis of allergic diseases.

Exposure to Epicoccum purpurascens is implicated in respiratory allergies and asthma. Several allergens of clinical importance were identified in Epicoccum extract (EE), but only one allergen has been isolated and characterized. In the present study, a 12-kDa allergen was isolated from an Epicoccum spore-mycelial extract by concanavalin-A sepharose, reverse-phase hydrophobic and gel filtration chromatography. The purified protein was recognized as a single 12-kDa allergen on immunoblot with a serum pool of Epicoccum-sensitive patients. Of the 94 respiratory allergy patients tested intradermally, 17 showed marked positive skin reactions to EE and 12 of them reacted with the 12-kDa protein, indicating a diagnostic sensitivity of 70%. More than 80% patients' sera showed immunoglobulin E (IgE) reactivity to the purified protein in enzyme-linked immunosorbent assay and immunoblot, identifying it as a major allergen. Preincubation of pooled serum with the protein led to inhibition of IgE binding to solid-phase-bound EE (effective concentration 50%=180 ng). Twelve of the 17 serum samples showed significant basophil histamine release upon stimulation with purified protein. The protein induced significant proliferation of peripheral blood mononuclear cells of 13 patients. A high level of interleukin-4 in the culture supernatant of these cells indicated induction of a T-helper type 2 response. The purified 12-kDa protein is a clinically relevant allergen and has potential for the diagnosis and therapy of Epicoccum allergies.

Rationale: Studies with mite allergens demonstrated that proteolytic activity augments allergic airway inflammation. This knowledge is limited to few enzyme allergens., Objective: The objective of this study is to investigate the effect of serine protease Cur l 1 from Curvularia lunata in airway inflammation/hyper-responsiveness., Methods: Cur l 1 was purified and inactivated using a serine protease inhibitor. Balb/c mice were sensitized with enzymatically active Cur l 1 or C. lunata extract. Sensitized mice were given booster dose on day 14 with active or inactivated Cur l 1. Intranasal challenge was given on day 28, 29, and 30. Airway hyper-responsiveness was measured by plethysmography. Blood, bronchoalveolar lavage fluid (BALF), spleen, and lungs from mice were analyzed for cellular infiltration, immunoglobulins, and cytokine levels., Results: Mice challenged with enzymatically active Cur l 1 demonstrated significantly higher airway inflammation than inactive Cur l 1 group mice (p < 0.01). There was a significant difference in serum IgE and IgG1 levels among mice immunized with active Cur l 1 and inactive Cur l 1 (p < 0.01). IL-4 and IL-5 were higher in BALF and splenocyte culture supernatant of active Cur l 1 than inactive Cur l 1 mice. Lung histology revealed increased eosinophil infiltration, goblet cell hyperplasia and mucus secretion in active group., Conclusion: Proteolytic activity of Cur l 1 plays an important role in airway inflammation and the inactivated Cur l 1 has potential to be explored for immunotherapy.

Enzymes play an important role in inducing airway inflammation, but knowledge is limited to few proteins. This study was carried out to assess the role of Epi p 1, a serine protease of Epicoccum purpurascens, in inducing allergy and inflammation in a murine model. Balb/c mice were sensitized with Epi p 1 active protease (EAP) or Epicoccum extract. Subsequently, Epi p 1 sensitized mice were boosted on day 14 with EAP or inactivated protease (EIAP). Three intranasal challenges were given and mice were killed to obtain blood, bronchoalveolar lavage fluid (BALF), spleen and lung tissues. Cellular airways infiltration, immunoglobulin E (Ig)E titres and cytokine levels in BALF and splenocyte culture supernatant were compared. Mice immunized with EAP had higher Epi p 1-specific serum IgE and IgG1 than EIAP immunized mice (P < 0.01). There was a twofold difference in the number of eosinophils in BALF of EAP mice and EIAP mice (P < 0.01). A similar trend was recorded for eosinophil peroxidase activity (P < 0.05), indicating the role of proteolytic activity in inducing inflammation. Further, lung histology revealed increased leucocyte infiltration and airway narrowing, with higher inflammation scores in the EAP group than in the EIAP group. The lungs of EAP mice showed increased mucus and goblet cell metaplasia. Interleukin (IL)-4 and IL-5 levels were higher in BALF and splenocyte culture supernatant of EAP mice than in EIAP mice (P < 0.05), indicating a T helper 2 response. Proteolytic activity of Epi p 1 plays an important role in inducing allergic inflammation. The enzymatically inactive form may be investigated for immunotherapy.

Sensitization to allergens of Epicoccum purpurascens is reported from many places world over. Previously, the IgE binding proteins of Epicoccum were studied by one-dimensional immunoblotting (1-DE). However, the allergens recognized by 1-DE may be a mixture of proteins appearing at the same molecular weight. In the present study, IgE-binding components of E. purpurascens were detected using two-dimensional immunoblotting and mass spectrometry. Approximately 250 protein spots were identified by Coomassie staining in the range of 14-110kDa and pI 3.5-9.5. Of these, 147 showed specific IgE reactivity with pooled patients' sera whereas 16 protein components reacted with more than 50% of the individual patients' sera. The frequency of IgE reactivity was highest (87.5%) for a 25-kDa protein (pI-7.85). Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of 16 protein components led to identification of 6 important allergens as phospho-glycerate kinase (PGK), RNA-dependent RNA polymerase, pyrroline-5-carboxylate dehydrogenase, 40S ribosomal protein and two proteins of unknown functions. The major allergens identified may be assessed for cross reactivity with other fungi. In conclusion, 2-DE followed by immunoblotting allowed characterization of E purpurascens allergens. Six new allergens have been identified using this technique and mass spectrometry. The availability of such relevant allergens would help in component-based diagnosis and therapy of fungal allergy.

Objective: Attachment to host matrix is an important provisory step for the institution of any fungal infection. The present study investigates the role of glycoproteins of Epicoccum purpurascens in host-fungal adherence., Methods: Epicoccum spore-mycelial extract was fractionated on a concanavalin A-Sepharose column. Three glycoproteins of 12, 17 and 33 kDa (Epi p 1) were electroeluted and checked for hemagglutination and hemagglutination inhibition. The monosaccharide content of the highly potent protein Epi p 1 was determined by high-performance anion exchange chromatography and pulsed amperometric detection. The interaction of Epi p 1 with mannose-binding lectin (MBL) leading to the activation of the complement system was studied by immunoblot, ELISA and ELISA inhibition techniques. Immunoblot and immunoblot inhibition were carried out with culture filtrate to determine the nature of Epi p 1., Results: 33 (Epi p 1)-, 17- and 12-kDa proteins were 58, 46 and 38 times more potent than crude extract in hemagglutination activity (HA). The HA of Epi p 1 was inhibited by N-acetyl glucosamine, glucose and laminin. Epi p 1 had a high mannose content, showed MBL binding in ion-dependent manner and caused complement activation. The protein was detected in culture filtrate and thus seems to play a significant role in fungal invasion., Conclusion: Epi p 1, an allergenic glycoprotein of E. purpurascens, is involved in host-fungal interactions through MBL.