Enzymic characteristics of secreted aspartic proteases of Candida albicans

Candida yeasts are rarely infectious, but frequently cause life-threatening systemic infections in patients immunocompromised by AIDS or by immunosupp ressive therapeutics. The secreted aspartic proteases (Saps) are known viru lence factors of pernicious Candida species. The most virulent, Candida alb icans, possesses at least nine SAP genes, some of which are specifically ex pressed from cells with morphologies associated with virulence. Only one of these proteases, Sap2, has been previously purified from yeast in sufficie nt quantities for enzymic studies. The other enzymes are present in low amo unts in yeast culture and are difficult to purify. As a consequence, enzyme properties, including the substrate specificities, of all Saps are poorly studied. Therefore, four Saps that are known to be expressed in C. albicans , Sap1, Sap2, Sap3 and Sap6, were produced in Escherichia coli as recombina nt zymogens and purified in large quantities. These proenzymes were autoact ivated and purified as active proteases. The enzymic properties including t he substrate specificities at the P-1 and P-1' sites were determined using a competitive hydrolysis method employing synthetic substrate mixtures. All four Saps cleave peptide bonds between larger hydrophobic amino acids, but these somewhat broad specificities differ in detail among the four enzymes at both sites. At the P-1 site, Sap1, Sap2 and Sap6 prefer Phe while Sap3 prefers Leu. Positively charged amino acids are also accommodated, especial ly by Sap2 and Sap3. The specificities at P-1' are broader than at P-1 for all four enzymes. Sap6 prefers Ala, whereas other Saps prefer Tyr. Acidic s ide chains are also accommodated at this site. Analysis of substrates with a hydrophobic amino acid in P-1' reveals that all the Saps possess a unique preference for Ala at this site. The observed differences of residue prefe rences among Saps may be utilized for the design of specific substrates and inhibitors. (C) 2000 Elsevier Science B.V. All rights reserved.