Cloning, Recombinant Expression and Activity Studies of a Major Allergen “Enolase” from the Fungus Curvularia Lunata

Abstract

Recombinant allergens are required to study allergy at the molecular level and are helpful tools for the improvement of diagnosis and therapy. In the present study, enolase was expressed from Curvularia lunata and analyzed for its immunological reactivity as an allergen. cDNA library was synthesized in lambda zap vector and screened with sera obtained from C. lunata allergic patients. A cDNA clone with an ORF of 1.3 kb showed homology to enolases from different fungal sources. It was expressed in E. coli, purified from inclusion bodies yielding 0.5 mg/L and showed enzyme activity of 48 units/mg. It resolved as 48-kDa band on SDS-PAGE and was recognized by all the individual Curvularia positive patient sera in immunoblot and ELISA. r Cur l 2 stimulated patients' PBMCs and supernatant of these cells showed elevated levels of Th 2 cytokines. Ten B cell epitopes were predicted using computational software and one showed 90% homology to an important IgE epitope of Cla h 6. The various parameters predicted by computational approach can be validated later as a future study to draw conclusive evidence about putative antigenic epitopes. This can further help in generating knowledge about residues important for IgE binding and developing therapeutic modalities.

Isolation and characterization of α-enolase, a novel fibronectin-binding protein from Streptococcus suis

“…Enolase has also been identified as a novel plasminogen receptor on the surface of many eukaryotic cells (Miles et al, 1991; Nakajima et al, 1994). This unexpected feature has also been described in several bacteria (such as Staphylococcus aureus and many streptococci), fungi and nematodes (Bergmann et al, 2001;Jolodar et al, 2003;Jong et al, 2003;Molkanen et al, 2002;Pancholi & Fischetti, 1998;Sharma et al, 2006). While the role of plasminogen as an important component in streptoccoci adhesion is well established (Pancholi et al, 2003), the role of enolase in bacterial adherence and host cell invasion has not, to our knowledge, been investigated previously.…”

Molecular and immunological characterization of subtilisin like serine protease, a major allergen of Curvularia lunata

“…Three patients (nos.−3, 12, 15) showing low IgE binding with 54 kDa protein may be hypersensitive to other C. lunata protein(s). Previously, purified allergens of C. lunata (Cur l 1, Cur l 2 and Cur l 3) demonstrated IgE binding in the range of 67-80% of Curvularia allergic patients' sera (Gupta et al, 2004;Sharma et al, 2006Sharma et al, , 2008.…”

“…Subsequently, enolase and cytochrome c allergens were identified and produced as recombinant proteins that offer scope for component resolved diagnosis and therapy. (Gupta et al, 2004;Sharma et al, 2006Sharma et al, , 2008.…”

Production of aChaetomium globosumEnolase Monoclonal Antibody

“…(15) Enolase has been localized microscopically in the fungal cell wall and the cytosol, and is secreted during hyphal growth. Enolase has been sequenced, cloned, and identified as an immunodominant antigen for several fungal species including S. cerevisiae, (26)(27)(28) Candida albicans, (25,27,(29)(30)(31)(32) Cladosporium herbarum, (15) Curvularia lunata, (33) Alternaria alternata, (15) Penicillium citrinum,…”