Characterization and immunolocalization of an NTP diphosphohydrolase of Trypanosoma cruzi q Juliana L.R. Fietto, a Ricardo DeMarco, b Ivan P. Nascimento, b Ieso M. Castro, a T ecia M.U. Carvalho, c Wanderley de Souza, c Maria T. Bahia, a Maria J.M. Alves, b and Sergio Verjovski-Almeida b, * a N ucleo de Pesquisas em Ci^ encias Biol ogicas, Universidade Federal de Ouro Preto, 35400-000 Ouro Preto, MG, Brazil b Departamento de Bioqu  ımica, Instituto de Qu  ımica, Universidade de S~ ao Paulo, Caixa Postal 26077, 05599-970 S~ ao Paulo, SP, Brazil c IBCCF-Universidade Federal do Rio de Janeiro, 21246-900 Rio de Janeiro, RJ, Brazil Received 6 February 2004 Abstract An ecto-NTP diphosphohydrolase (NTPDase) activity, insensitive to inhibitors of ATPases and phosphatases, was characterized on the surface of live Trypanosoma cruzi intact parasites. The enzyme exhibits broad substrate specificity, typical of NTPDases, and a high hydrolysis rate for GTP. A 2282 bp message encoding a full-length NTPDase was cloned by RT-PCR using epimastigote mRNA. A single protein was immunoprecipitated from [ 35 S]methionine-labeled parasites using antibodies against Toxoplasma gondii NTPase I. This antibody localized an NTPDase on the external surface of all forms of T. cruzi, as seen by confocal immuno- fluorescence microscopy. The NTPDase could be part of the parasite’s purine salvage pathway. Additionally, trypomastigotes (infective form) presented a 2:1 ATP/ADP hydrolysis ratio, while epimastigotes (non-infective form) presented a 1:1 ratio, sug- gesting a possible role for the NTPDase in the parasite’s virulence mechanisms. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Trypanosoma cruzi; Ecto-NTPDase; Apyrase; Immuno-localization; Plasma-membrane; Purine salvage pathway Trypanosoma cruzi is the etiological agent of Chagas’ disease, an endemic illness that affects people in Latin America and remains a problem of public health. In vertebrates, T. cruzi is an obligate intracellular parasite that causes chronic infection in humans and a large number of other mammalian species [1]. Three mor- phologically distinct forms can be ascribed to T. cruzi: amastigotes, a dividing form found intracellularly in mammalian hosts, epimastigotes, a non-infecting form found in the insect vector’s digestive tract, and try- pomastigotes that occur in the lumen of the rectum of the insect and in the mammalian host. A number of different proteins have been identified on the surface of the different forms of Trypanosoma, which are involved in metabolite exchange with the host [2]. In particular, it has been characterized that T. cruzi is unable to syn- thesize the purine ring [3] and probably makes the protozoan dependent on the salvage pathway [2]. It is interesting to note that Toxoplasma gondii, an intracel- lular parasite that exhibits a purine salvage pathway, has a well-characterized NTPDase activity [4] which is se- creted into the parasitophorous vacuolar space and may influence the exit of parasites from cells [5]. The GDA1/CD39 (NTPDase) or apyrase family (EC 3.6.1.5) [6,7] is composed of enzymes that hydrolyze a variety of nucleoside tri- and diphosphates with the re- lease of NDP, NMP, and orthophosphate, the activity being dependent on the presence of divalent cations such as calcium and magnesium. It has been suggested that they might play a role in endoparasites by de-activating defense responses of the host which involve ATP or ADP on the surface of the cells, such as platelet acti- vation cytotoxicity and cytolytic T-lymphocyte reactiv- ity [8–11]. In addition, CD39 (an NTPDase) was shown to play a modulatory role in inflammation and immune q Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2004.02.071. * Corresponding author: Fax: +55-11-3091-2186. E-mail address: verjo@iq.usp.br (S. Verjovski-Almeida). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.071 Biochemical and Biophysical Research Communications 316 (2004) 454–460 BBRC www.elsevier.com/locate/ybbrc