Molecular characterization and influence on fungal development of ALP2, a novel serine proteinase from Aspergillus fumigatus

A novel subtilisin-related serine proteinase (ALP2) [EC 3.4.21.48] with a b road range of activity between pH 4.5 and 11.0 was released from a cell wal l fraction of Aspergillus fumigatus by an alkaline pH shift. The enzyme whi ch was not detected in the culture supernatant was partially purified by ph enylbutylamine agarose chromatography. The N-terminal sequence revealed tha t ALP2 is the same protein identified as the major allergen of A. fumigatus in patients suffering from extrinsic bronchial asthma (Shen et al. 1999, I nt. Arch. Allergy Immunol. 119, 259-264). Based on this N-terminal sequence and on a conserved region of fungal subtilisins, a specific PCR probe was generated and the ALP2 genomic and cDNA were isolated from corresponding ph age libraries. ALP2 shares a 49 % identity with the vacuolar proteinase B ( PrB) of Saccharomyces cerevisiae. In addition there is a 78 % identity with PEPC, a serine proteinase which has been described in Aspergillus niger. T argeted disruption of the ALP2-encoding gene resulted in a slightly decreas ed speed of vegetative growth and in a more than 80% reduction of sporulati on in the alp2-negative mutants, correlated with an approximately 50 % redu ction of the median diameter of conidiophore vesicles. The requirement of A LP2 for regular sporulation, in addition to its role in allergic asthma, ra ises further interest in cellular proteinases in respect to morphogenesis a nd pathogenesis in A. fumigatus.