Purification, cloning, and characterization of an acidic ectoprotein phosphatase differentially expressed in the infectious bloodstream form of Trypanosoma brucei.

We purified an ecto-phosphatase of 115 kDa (TryAcP115) specifically expressed by bloodstream forms of Trypanosoma brucei. The corresponding gene coded for a 45-kDa protein potentially including a signal peptide, a membrane-spanning domain and an N-terminal domain containing 8 N-glycosylation sites. There was no significant sequence homology with other phosphatases. Antiserum to the Escherichia coli recombinant N-terminal domain, Petase7, recognized a protein of 55 kDa in Western blots after deglycosylation of the TryAcP115 protein by N-glycosidase F. Immunofluorescence and trypsin treatment of living parasites showed that TryAcP115 was localized to the surface of the parasite and that its N-terminal domain was oriented extracellularly. The recombinant N-terminal domains, expressed in E. coli and Leishmania amazonensis, harbored phosphatase activity against Tyr(P)-Raytide, Ser(P)-neurogranin, and ATP. The enzymatic properties of native TryAcP115 and the recombinant proteins for the substrate Tyr(P)-Raytide were virtually identical and included: (i) K(m) and V(max) values of 15 nM and 200 pmol/min/mg, (ii) no requirement for divalent cations, and (iii) sensitivity to vanadate, sodium fluoride, and tartrate, but insensitivity to okadaic acid and tetramisole. Although the function of TryAcP115 remains unknown, a differentially expressed, unique ecto-phosphatase could regulate growth or influence parasite-host interactions and might provide a useful target for chemotherapy.