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

Dostál, Jiřı́; Brynda, J.; Hrušková−Heidingsfeldová, Olga; Sieglová, Irena; Pichová, Iva; Řezáčová, P.

doi:10.1016/j.jsb.2009.04.004

Cited by 19 publications

(14 citation statements)

References 44 publications

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“…In this study, we focused on the C. parapsilosis enzyme Sapp1p, which is induced in a similar manner as Sap2 of C. albicans but displays certain enzymological and structural differences. 23,26 To study the passage of Sapp1p through the yeast cell wall during secretion into the extracellular The band containing Sapp1p was cut from a gel and digested with trypsin or chymotrypsin (see Materials and Methods for details). B, number of peptide fragments containing bitonylated residue; S, total number of relevant peptide fragments; n.d., peptide fragment was not detected.…”

Section: Discussionmentioning

confidence: 99%

“…Labeling of cell surface proteins with a biotinylation reagent has been successfully used for identification of yeast cell wall proteins. 21,24 The recently solved Sapp1p X-ray structure 26 showed that the active molecule has an open structure and that lysine residues are present in both N-and C-terminal proteinase domains. Moreover, all 15 lysines are surface-exposed, which makes it possible to efficiently label Sapp1p with biotin.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2011

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Pathogenic yeasts of the genus Candida produce secreted aspartic proteinases, which are known to enhance virulence. We focused on Sapp1p proteinase secreted by Candida parapsilosis and studied the final stage of its passage through the cell wall and release into the extracellular environment. We found that Sapp1p displays enzyme activity prior to secretion, and therefore, it is probably fully folded within the upper layer of the cell wall. The positioning of cell surface-associated Sapp1p was detected by cell wall protein labeling using biotinylation agents, extraction of cell wall proteins by b-mercaptoethanol, immunochemical detection, and mass spectrometry analysis. All lysine residues present in the structure of soluble, purified Sapp1p were labeled with biotin. In contrast, the accessibility of individual lysines in cell wall-associated Sapp1p varied with the exception of four lysine residues that were biotinylated in all experiments performed, suggesting that Sapp1p has a preferred orientation in the cell wall. As the molecular weight of this partially labeled Sapp1p did not differ among the experiments, we can assume that the retaining of Sapp1p in the cell wall is not a totally random process and that pathogenic yeasts might use this cell-associated proteinase activity to enhance degradation of appropriate substrates.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

et al. 2011

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“…The refined Sapp1p-RTV structure shows no significant conformational changes in the protein chain compared to the structure of the same protein in complex with pepstatin A (PDB code 3FV3 17 ). The RMSD for superposition of 339 Cα atoms of different protein chains was 0.445-0.502 Å.…”

Section: Crystal Structurementioning

confidence: 99%

“…The efficiency of purification steps was analyzed using SDS-PAGE, Western blotting, and activity assays. Protein analyses and proteolytic activity assays were previously described 17 .…”

Section: Protein Preparationmentioning

confidence: 99%