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.
Bakalara
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Laboratoire de Parasitologie Moléculaire UMR CNRS 5016, Université Victor Segalen Bordeaux II, 33076 Bordeaux Cedex, France. bakalara@hippocrate.u-bordeaux2.fr
Santarelli
X
X
Davis
C
C
Baltz
T
T
D000143:Acids; D000818:Animals; D001769:Blood / Q000469:parasitology; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology*; D003001:Cloning, Molecular; D008562:Membrane Glycoproteins / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015800:Protozoan Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity
D000143:Acids; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D015800:Protozoan Proteins; D010749:Phosphoprotein Phosphatases; C406810:Try-AcP115 protein, Trypanosoma brucei