Initial Characterization of a cDNA Encoding a Heat Shock Protein Homolog From Stachybotrys chartarum

Abstract

RATIONALE: Exposure to Stachybotrys chartarum has been implicated in a number of adverse health effects. The purpose of this study was to identify and clone IgE binding proteins from S. chartarum for potential use as biomarkers of exposure.METHODS: Immunoblots with an extract of S. chartarum mycelia were probed with sera from fungal sensitized patients. IgE binding proteins were subjected to N-terminal sequence analysis. Degenerative primers were designed for RACE-PCR experiments using cDNA transcribed from S. chartarum mycelia total RNA. Based on the obtained sequence for one particular clone of interest, we devised a primer cDNA walking strategy and ultimately amplified the S. chartarum cDNA. The full-length cDNA was then amplified using gene specific primers.RESULTS: Western blot analysis revealed several proteins in the S. chartarum mycelia extract that specifically bound to IgE from fungal-sensitized patients. The N-terminus for a number of these proteins was blocked. Using the sequence obtained from a 15.5kDa allergen and RACE-PCR, several clones were produced and their sequence analyzed. Sequence analysis of the full-length cDNA of approximately 850bp revealed a high degree of homology to the Heat Shock Protein 40 family and exhibited a conserved DnaJ_C binding domain.CONCLUSIONS: Heat shock proteins (HSPs) have been identified in a number of pathogenic fungi and shown to be allergens. Furthermore, elevated levels of fungal HSPs have been documented upon human infection. Future studies will investigate the role of the S. chartarum HSP homolog by evaluating IgE-reactivity towards the recombinant protein. RATIONALE: Exposure to Stachybotrys chartarum has been implicated in a number of adverse health effects. The purpose of this study was to identify and clone IgE binding proteins from S. chartarum for potential use as biomarkers of exposure. METHODS: Immunoblots with an extract of S. chartarum mycelia were probed with sera from fungal sensitized patients. IgE binding proteins were subjected to N-terminal sequence analysis. Degenerative primers were designed for RACE-PCR experiments using cDNA transcribed from S. chartarum mycelia total RNA. Based on the obtained sequence for one particular clone of interest, we devised a primer cDNA walking strategy and ultimately amplified the S. chartarum cDNA. The full-length cDNA was then amplified using gene specific primers. RESULTS: Western blot analysis revealed several proteins in the S. chartarum mycelia extract that specifically bound to IgE from fungal-sensitized patients. The N-terminus for a number of these proteins was blocked. Using the sequence obtained from a 15.5kDa allergen and RACE-PCR, several clones were produced and their sequence analyzed. Sequence analysis of the full-length cDNA of approximately 850bp revealed a high degree of homology to the Heat Shock Protein 40 family and exhibited a conserved DnaJ_C binding domain. CONCLUSIONS: Heat shock proteins (HSPs) have been identified in a number of pathogenic fungi and shown to be allergens. Furthermore, elevated levels of fungal HSPs have been documented upon human infection. Future studies will investigate the role of the S. chartarum HSP homolog by evaluating IgE-reactivity towards the recombinant protein.