Methods: Spore mycelia 2D proteome was confronted with IgE of asthma patient sera and the immunoreactive proteins were identified via MALDI-TOF/TOF.The native allergen was purified via column chromatography and N-terminal sequencing was done for reconfirmation. Recombinant form of this antigen was prepared by cloning and expressing the corresponding 1206 bp cDNA ORF in pRSETA vector using E. coli (BL21) system and purified by Ni-column. Both the native and recombinant allergens were further tested for immunoreactivity. Structural modeling predicted a refined 3D conformation of this antigen with 3 potential B-cell epitopes. The critical residue(s) in one such epitope was converted by inducing base pair substitution mutation using single point site-directed mutagenesis kit and the mutant forms were tested for sero-reactivity. The non-mutant form was studied for cross reactivity with Asp f 10 and Bla g 2.
Results: The purified native and recombinant antigens were identified as a 44 kD aspartyl endopeptidase and found to have retained IgE-immunoreactivity upon western blotting. Multiple sequence alignment and 3D structure of this allergen resembles with Bla g 2 and Asp f 10. Three mutant forms of the antigen was not able to bind with IgE and IgG.
Conclusions: This is the first potent immune-dominant major allergen reported from this Mucormycosis agent (RO). This allergen is cross reactive with Bla g 2 and Asp f 10. The 3D conformation of the antigen has a potential immundominant surface epitope, which upon mutation, has lost its ability to act as an immunodeterminant. This wild and hypoantigenic forms, can be further studied in great detail for its potential use as infection biomarker and vaccination agent respectively.