The precursor of secreted aspartic proteinase Sapp1p from Candida parapsilosis can be activated both autocatalytically and by a membrane-bound processing proteinase

Abstract

Opportunistic pathogens of the genus Candida produce secreted aspartic proteinases (Saps) that play an important role in virulence. Saps are synthesized as zymogens, but cell-free culture supernatants of Candida spp. contain only mature Saps. To study the zymogen conversion, the gene encoding a precursor of C. parapsilosis proteinase Sapp1p was cloned, expressed in E. coli and the product was purified. When placed in acidic conditions, the precursor was autocatalytically processed, yielding an active proteinase. The self-activation proceeded through an intermediate product and the resulting enzyme was one amino acid shorter than the authentic enzyme. This truncation did not cause changes in proteinase activity or secondary structure compared to the authentic Sapp1p. Accurate cleavage of the pro-mature junction, however, required a processing proteinase. A crude membrane fraction prepared from C. parapsilosis cells contained an enzyme with Kex2-like activity, which processed the Sapp1p precursor at the expected site. The pro-segment appeared to be indispensable for Sapp1p to attain an appropriate structure. When expressed without the pro-segment, the Sapp1p mature domain was not active and had a lower content of alpha-helical conformation, as measured by circular dichroism. A similar effect was observed when a His(6)-tag was linked to the C-terminus of Sapp1p or its precursor.

Candida parapsilosis, an Emerging Fungal Pathogen

“…Three Saps have been identified in C. parapsilosis, two of which remain largely uncharacterized (175). The Sapp1p isoenzyme has been biochemically characterized (75,92,219). Although originally classified as a pseudogene, SAPP2P produces a functional proteinase, Sapp2p, which constitutes about 20% of the Saps isolated from a culture supernatant (93).…”

The crystal structure of the secreted aspartic protease 1 from Candida parapsilosis in complex with pepstatin A

“…Ser change to the P3 0 residue of PepA in our Sapp1p structure encouraged us to revisit the results of our previous work (Dostal et al, 2005), which described processing of a recombinant Sapp1p precursor. This precursor was expressed in Escherichia coli, contained Leu at the position 193, and was capable of autocatalytic activation, although the cleavage occurred one amino acid downstream from the expected promature junction.…”

“…While the SAPP3 gene product remains enigmatic, the Sapp1p and Sapp2p isoenzymes have already been subjected to enzymological studies (Dostal et al, 2005;Merkerova et al, 2006). Both of these enzymes have broad substrate specificity, but they differ in catalytic activities and expression pattern.…”

Evidence for the presence of proteolytically active secreted aspartic proteinase 1 of Candida parapsilosis in the cell wall

“…Only Sapp1p and Sapp2p proteinases have been biochemically characterized. [12][13][14] Production of Sapp1p is induced in the presence of an exogenous protein as a sole nitrogen source, as in the case of the Sap2 enzyme from C. albicans. Sapp1p, the main secreted isoenzyme of C. parapsilosis, has a broad substrate specificity and higher catalytic activity than Sapp2p, which has been shown to be secreted constitutively under all conditions tested but in a concentration at least one order of magnitude lower.…”

“…The signal peptide is removed in the rough endoplasmic reticulum, and the zymogen is activated after transport to the Golgi apparatus by Kex2 proteinases or alternative pathways. 13,16,17 The mature proteinases are transported via the secretory pathway to the cell surface and have to pass the protective cell wall, which consists of two main layers of different electron density. The inner layer contains a network of 1,3-b-glucan, 1,6-b-glucan, and chitin molecules, and the dense outer layer is enriched with mannoproteins covalently linked to the nonreducing ends of 1,6-b-glucans via glycosyl phosphatidylinositol (GPI) anchors.…”