Fungal glycopeptide processing is a series of complex events that result in the modification of cell wall, membrane, and extracellular structures. The major goal of this subproject is to explore the hypothesis that a family of fungal acid phosphomonoesterase derived from Penicillium fellutanum are involved in the processing of glycopeptides from the membrane, cell wall, and extracellular fractions. In order to test this hypothesis, we propose to (1) isolate the phosphomonoesterase using established protein isolation (chromatographic) methods; (2) characterize the protein structure; (3) establish kinetic parameters for the enzyme; and (4) establish natural substrates for the enzyme by testing its activity on Penicillium cell wall, plasma membrane, and extracellular glycopeptides. Early reports by Gander and coworkers established the existence of at least three distinct acid phosphatase proteins and that some Penicillium acid phosphatase activities are inducible under conditions of phosphate limitation. To test the hypothesis that there are distinct inducible and constitutive acid phosphatases, we propose to (1) isolate and characterize gene sequences from Penicillium fellutanum DNA with homology to eukaryotic acid phosphatases; (2) create a cDNA library and identify cDNAs with homology to acid phosphatases; (3) clone the gene(s) for the acid phosphatases and express them in a suitable vector; (4) compare the effects of heat shock, phosphate deprivation, and pH on the amount of mRNA and DNA encoding for the acid phosphatase genes. We also propose to localize the acid phosphatases by fluorescence microscopy of cells, membranes, and cell walls. Discovery of the role of acid phosphatases in fungal metabolism and physiology is expected to provide information that can be applied to combat disseminated mycoses, such as those caused by Penicillium marneffei, in HIV- infected and other immunocompromised individuals.
