Characterization of a heavy metal ATPase from the apicomplexan Cryptosporidium parvum

P1-ATPases are transporters which pump heavy metals across membranes, tithe r to provide enzymes with essential cofactors or to remove excess, toxic me tal cations from the cytosol. The first protist P1-ATPase (CpATPase2) has b een 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-polymeras ch ain reaction (RT-PCR) and western blot analysis confirmed CpATPase2 express ion. Immunofluorescence microscopy of C. parvum sporozoites using rabbit an tiserum raised against a glutathione-S-transferase (GST) fusion protein sug gests that CpATPase2 is associated with the plasma- and cytoplasmic membran es. The protein shares greatest overall sequence similarity to previously c haracterized copper P1-ATPases. Expression and subsequent biochemical analy ses of the N-terminal heavy metal binding domain (HMBD, GMxCxxC) of CpATPas e2 as a maltose-binding protein (MBP) in Escherichia coli reveals that the protein specifically binds reduced copper, Cull), in vitro and in vivo. and that the cysteine residues of HMBD are responsible for heavy metal coordin ation. Overall. these data show that the apicomplexan C. parvum possesses a heavy metal P-ATPase transporter with a specificity for reduced copper, Si nce this discovery represents the first time a heavy metal P-ATPase has bee n identified and characterized from a protist, further molecular and bioche mical studies an needed to understand the roles heavy metal P-ATPases play in heavy metal metabolism and potential virulence for this and other apicom plexa. (C) 2001 Elsevier Science B.V. All rights reserved.