The secreted aspartic proteinases of Candida albicans : a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Institute of Molecular BioSciences at Massey University, New Zealand
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Date
1999
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Massey University
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Abstract
The common human fungal pathogen, Candida albicans, possesses at least nine genes encoding secreted aspartic proteinases (SAPs). Saps are widely regarded as virulence factors, despite historical controversy surrounding their actual roles in the onset and development of candidosis. While Sap1, Sap2 and Sap3 had been previously studied at the biochemical level, Sap4 and Sap5 had not been detected, purified or characterised. To facilitate analysis of the proteins, SAP4 and SAP5 were amplified by PCR and cloned. The nucleotide changes in SAP4 were silent, and SAP5 contained one conserved amino acid substitution, compared with the published sequences. The methylotrophic yeast Pichia pastoris was used as a host for heterologous expression of SAP4 and SAP5, and the respective proteins were purified to homogeneity. Purification of Sap4 involved Mono Q anion exchange chromatography at pH 7.0, while Sap5 was purified by cation exchange chromatography on Resource S at pH 7.0. C. albicans SAP1 was also over-expressed in P. pastoris as a control. Biochemical analysis of the recombinant proteins revealed that Sap4 and Sap5 were optimally active at pH 4.5, 1-2 pH units higher than the optima of Saps1, 2 and 3. At optimum pH, the specific activities of Sap4 and Sap5 were 239 and 33 µg tyrosine equivalents/min, respectively. These isoenzymes also retained significant activity at pH 7.0, which suggested roles in the disease process at host sites of neutral pH. Sap4 and Sap5 showed decreased specific activity towards denatured globin, and increased specific activity towards a fluorocasein substrate, when compared with Saps1-3. Substrate specificity analyses (performed using a peptide substrate, glucagon, and MALDI-TOF of the purified peptide fragments), showed that Sap4 and Sap5 hydrolysed the glucagon at the same sites, but the analysis did not reveal a consensus cleavage sequence. The deduced masses of Sap1, Sap4 and Sap5 were 36,179, 36,995 and 37,256, respectively. However, ES-MS indicated the masses of the recombinant Sap1 and Sap4 were larger than expected, by 2232 and 2041 respectively. Glycopeptide fragment ion analysis suggested the additional mass was due to attached sugar residues. Carbohydrate chromatography confirmed the presence of mannose and N-acetyl glucosamine. The presence of N-acetyl glucosamine species, and the lack of consensus N-linked glycosylation sites in the Sap1, Sap4 and Sap5 proteins suggests a novel pattern of O-linked glycosylation in P. pastoris. The purified enzymes were subjected to crystallisation trials and some promising crystals were produced. Previous studies showed that SAP4, 5 and 6 are expressed during serum-induced germ-tube formation, but this was not confirmed in this study.
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Keywords
Aspartic proteinases, Candida albicans, Genetics