PMID:19451239

Citation	Kennedy, CC, Kottom, TJ and Limper, AH (2009) Characterization of a novel ADAM protease expressed by Pneumocystis carinii. Infect. Immun. 77:3328-36
Abstract	Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. Recent evidence has suggested that unidentified proteases are involved in Pneumocystis life cycle regulation. Proteolytically active ADAM (named for "a disintegrin and metalloprotease") family molecules have been identified in some fungal organisms, such as Aspergillus fumigatus and Schizosaccharomyces pombe, and some have been shown to participate in life cycle regulation. Accordingly, we sought to characterize ADAM-like molecules in the fungal opportunistic pathogen, Pneumocystis carinii (PcADAM). After an in silico search of the P. carinii genomic sequencing project identified a 329-bp partial sequence with homology to known ADAM proteins, the full-length PcADAM sequence was obtained by PCR extension cloning, yielding a final coding sequence of 1,650 bp. Sequence analysis detected the presence of a typical ADAM catalytic active site (HEXXHXXGXXHD). Expression of PcADAM over the Pneumocystis life cycle was analyzed by Northern blot. Southern and contour-clamped homogenous electronic field blot analysis demonstrated its presence in the P. carinii genome. Expression of PcADAM was observed to be increased in Pneumocystis cysts compared to trophic forms. The full-length gene was subsequently cloned and heterologously expressed in Saccharomyces cerevisiae. Purified PcADAMp protein was proteolytically active in casein zymography, requiring divalent zinc. Furthermore, native PcADAMp extracted directly from freshly isolated Pneumocystis organisms also exhibited protease activity. This is the first report of protease activity attributable to a specific, characterized protein in the clinically important opportunistic fungal pathogen Pneumocystis.
Links	PubMed PMC2715687 Online version:10.1128/IAI.01383-08
Keywords	ADAM Proteins/genetics; ADAM Proteins/metabolism; Amino Acid Sequence; Base Sequence; Blotting, Northern; Catalytic Domain; Cloning, Molecular; Coenzymes/pharmacology; DNA, Fungal/chemistry; DNA, Fungal/genetics; Fungal Proteins/genetics; Fungal Proteins/metabolism; Gene Expression; Gene Expression Profiling; Molecular Sequence Data; Peptide Hydrolases/genetics; Peptide Hydrolases/metabolism; Pneumocystis carinii/enzymology; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Saccharomyces cerevisiae/genetics; Sequence Alignment; Sequence Analysis, DNA; Zinc/pharmacology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
PNECA:A3QZA9		GO:0008237: metallopeptidase activity	ECO:0000314:		F		See Figure 7	complete CACAO 10372
PNECA:A3QZA9	enables	GO:0008237: metallopeptidase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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