Characterization of a heavy metal ATPase from the apicomplexan Cryptosporidium parvum.
P1-ATPases are transporters which pump heavy metals across membranes, either to provide enzymes with essential cofactors or to remove excess, toxic metal cations from the cytosol. The first protist P1-ATPase (CpATPase2) has been isolated from the apicomplexan Cryptosporidium parvum, an opportunistic pathogen of AIDS patients. This single copy gene encodes 1260 amino acids (aa), predicting a protein of 144.7 kDa. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis confirmed CpATPase2 expression. Immunofluorescence microscopy of C. parvum sporozoites using rabbit antiserum raised against a glutathione-S-transferase (GST) fusion protein suggests that CpATPase2 is associated with the plasma- and cytoplasmic membranes. The protein shares greatest overall sequence similarity to previously characterized copper P1-ATPases. Expression and subsequent biochemical analyses of the N-terminal heavy metal binding domain (HMBD, GMxCxxC) of CpATPase2 as a maltose-binding protein (MBP) in Escherichia coli reveals that the protein specifically binds reduced copper, Cu(I), in vitro and in vivo, and that the cysteine residues of HMBD are responsible for heavy metal coordination. Overall, these data show that the apicomplexan C. parvum possesses a heavy metal P-ATPase transporter with a specificity for reduced copper. Since this discovery represents the first time a heavy metal P-ATPase has been identified and characterized from a protist, further molecular and biochemical studies are needed to understand the roles heavy metal P-ATPases play in heavy metal metabolism and potential virulence for this and other apicomplexa.
LaGier
M J
MJ
Wadsworth Center, New York State Department of Health, P.O. Box 22002, Albany, New York 12201-2002, USA.
Zhu
G
G
Keithly
J S
JS
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002412:Cations / Q000378:metabolism; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D016785:Cryptosporidium parvum / Q000201:enzymology / Q000235:genetics*; D003545:Cysteine / Q000378:metabolism; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019216:Metals, Heavy / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002412:Cations; D016054:DNA, Protozoan; D019216:Metals, Heavy; D011993:Recombinant Fusion Proteins; D003300:Copper; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases; D003545:Cysteine