MSK
Search
- Page Path
- HOME > Search
Research Support, Non-U.S. Gov't
- Saccharomyces cerevisiae Can Secrete Sapp1p Proteinase of Candida parapsilosis But Cannot Use It for Efficient Nitrogen Acquisition
- Zuzana Vinterová , Václava Bauerová , Ji&# , Hana Sychrová , Olga Hru&# , Iva Pichová
- J. Microbiol. 2013;51(3):336-344. Published online June 28, 2013
- DOI: https://doi.org/10.1007/s12275-013-2422-4
- 18 View
- 0 Download
- 2 Citations
-
Abstract
- Secreted aspartic proteinase Sapp1p of Candida parapsilosis represents one of the factors contributing to the pathogenicity of the fungus. The proteinase is synthesized as an inactive pre-pro-enzyme, but only processed Sapp1p is secreted into extracellular space. We constructed a plasmid containing the SAPP1 coding sequence under control of the ScGAL1 promoter and used it for proteinase expression in a Saccharomyces cerevisiae kex2Δ mutant. Because Sapp1p maturation depends on cleavage by Kex2p proteinase, the kex2Δ mutant secreted only the pro-form of Sapp1p. Characterization of this secreted proteinase form revealed that the Sapp1p signal peptide consists of 23 amino acids. Additionally, we prepared a plasmid with the SAPP1 coding sequence under control of its authentic CpSAPP1 promoter, which contains two GATAA motifs. While in C. parapsilosis SAPP1 expression is repressed by good low molecular weight nitrogen sources (e.g., ammonium ions), S. cerevisiae cells harboring this plasmid secreted a low concentration of active proteinase regardless of the type of nitrogen source used. Quantitative real-time PCR analysis of a set of genes related to nitrogen metabolism and uptake (GAT1, GLN3, STP2, GAP1, OPT1, and PTR2) obtained from S. cerevisiae cells transformed with either plasmid encoding SAPP1 under control of its own promoter or empty vector and cultivated in media containing various nitrogen sources also suggested that SAPP1 expression can be connected with the S. cerevisiae regulatory network. However, this regulation occurs in a different manner than in C. parapsilosis.
- Purification and characterization of extracellular aspartic proteinase of candida albicans
- Na, Byoung-Kuk , Lee, Seong Il , Kim, Sin Ok , Park, Young Kil , Bai, Gill Han , Kim, Sang Jae , Song, Chul Yong
- J. Microbiol. 1997;35(2):109-116.
- 14 View
- 0 Download
-
Abstract
- An extracellular proteinase of Candida albicans was purified by a combination of 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephacryl S-200 HR molecular sieve chromatography. Its mlecular weight was approximately 41 kDa on SDS-PAGE and isoelectric point was 4.4. The enzyme was inhibited by pepstain A. Optimum enzyme activity ranged from pH 2.0 to 3.5 with its maximum at pH 2.5 and a temperature of 45℃. The addition of divalent cations, Ca^2+, Zn^2+ and Mg^2+, resulted in no significant inhibition of enzymatic activity. However, some inhibitory effects were observed by Fe^2+, Ag^2+ and Cu^2+. With BSA as substrate, an apparent K_m was determined to be 7 × 10^7 M and K_I, using pepstatin A as an inhibitor, was 8.05 × 10^8 M. N-terminal amino acid sequence was QAVPVTLXNEQ. Degradation of BSA and fibronectin was shown but not collagen, hemoglobin, immunoglobulin G, or lysozyme. The enzyme preferred peptides with Glu and Leu at the P₁position, but the enzyme activity was highly reduced when the P₂position was Phe or Pro. This enzyme showed antigenicity against sera of patients with candidiasis.
First
Prev
TOP
