Detoxification of the glycopeptide bleomycin is mediated by bleomycin hydrolase, a cysteine aminopeptidase identified in a variety of organisms. The opportunistic fungal pathogen Candida albicans is known to exhibit increased resistance to bleomycin when compared to other yeast. Presented here is the cloning of the C. albicans Lap3p aminopeptidase, predicted by sequence identity to be the Candida form of bleomycin hydrolase. C. albicans Lap3p is functionally capable to replace the Saccharomyces cerevisiae Lap3p in vivo. Furthermore, the Candida enzyme was found to function as a cysteine aminopeptidase in vivo. It is shown here that upon introduction into a lap3 deletion strain of S. cerevisiae, the C. albicans Lap3p aminopeptidase does not significantly alter the response of Saccharomyces to bleomycin. These results suggest that C. albicans Lap3p does not function as the sole factor involved in bleomycin detoxification, and may require an accessory protein or co-factor in order to efficiently mediate this process in Candida. This study provides the first evidence of a functional description of the C. albicans Lap3p cysteine aminopeptidase, and provides the foundation for further mechanistic studies of the role of this protein in the cellular processes of Candida.
Key words: Candida albicans, bleomycin detoxification, Lap3p aminopeptidase, cysteine protease.
BCA, Bicinchoninic acid; E-64, trans-Epoxysuccinyl-L-â€‹leucyl-amido(4-â€‹guanidino)â€‹butane; H-Arg-AMC, H-arginine-7-amino-4-methylcoumarin.2HCl; H-Cit-AMC, H-citrulline-7-amino-4-methylcoumarin.2HBr; YEP, yeast extract/peptone; YEPD, yeast extract/peptone/glucose; YEP-Gal, yeast extract/peptone/galactose; Ura (-), synthetic culture medium lacking uracil; PMSF, phenylmethanesulfonylfluoride; EDTA, disodium ethylenediaminetetraacetic acid; DTT, dithiothreitol; WCE, whole cell extract.
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