Purification and characterization of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase and comparison with the human enzyme

Keough, Dianne T.; Ng, A.; Winzor, Donald J.; Emmerson, B. T.; Jersey, John de

doi:10.1016/s0166-6851(98)00139-x

Cited by 86 publications

(136 citation statements)

References 26 publications

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“…The sequence for HGXPRT from P. yoelii is not present in the database. Recombinant P. berghei or P. yoelii HGXPRT was not available, but Plasmodium falciparum HGXPRT (pfHGXPRT) was available (20) and could stimulate protective band-specific T cells (Fig. 6A).…”

Section: Resultsmentioning

confidence: 99%

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Makobongo

Riding

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4 ؉ T cells, have never been defined. We generated CD4 ؉ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-␥, and tumor necrosis factor-␣, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. Nterminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.A lthough it is known that immunity to malaria can be mediated by different immune mechanisms (1-4), to date only target antigens for antibodies have been defined, and these targets are either variant or demonstrate allelic polymorphism (5). CD4 ϩ T cells can adoptively transfer resistance to malaria (3, 6, 7) in rodent models, but target antigens have not been defined. Of interest, CD4 ϩ T cells displaying a Th1 cytokine profile (IL-2-and IFN-␥-secreting) and specific for the major merozoite surface protein 1 (MSP1 19 ) of Plasmodium yoelii (a leading vaccine candidate homologue) are unable to transfer resistance, and immunization of mice with defined T cell epitopes from MSP1 19 did not render the mice resistant (8). Identification of a target antigen(s) would provide an additional vaccine strategy for vaccine design. The goal of this study was to define such target antigens in the rodent model, P. yoelii. Materials and MethodsMice. Four-to 6-week-old normal BALB͞c, athymic BALB͞c nude, and BALB͞c severe combined immunodeficient (SCID) mice were used when 6-8 weeks old.Parasites and Parasitemia. P. yoelii 17XNL was maintained by passaging between infected and uninfected mice via the i.p. route. A thin blood smear was air-dried and stained using Diff-Quick (Lab Aids, Narrabeen, Australia).Preparation of Parasite Antigens. Preparation of whole parasite antigen (pRBC). Parasitized red blood cells (pRBC) in PBS were lysed by incubation in erythrocyte lysis buffer (0.17 M Tris-hydroxymethyl aminomethane͞0.16 M ammonium chloride; pH 7.2) at 37°C for 10 min. The parasites were then freeze-thawed at least three times, sonicated at 4°C to break up the parasites, aliquoted, and stored at Ϫ70°C to be used for in vitro cell cultures. Preparation of soluble parasite antigen (sAg).RBCs were lysed by incubation of the blood in 0.01% saponin͞PBS at 37°C for 20 min in the presence of protease inhibitors (Sigma). The blood was then given another wash in saponin͞PBS buffer before the pRBCs were sonicated in cold PBS at 4°C.After sonication, the lysate was ...

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Section: Resultsmentioning

confidence: 99%

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Makobongo

Riding

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…Previous studies on P. falciparum-infected erythrocytes have shown that following uptake, adenosine is deaminated to inosine by adenosine deaminase (Gero et al, 1988), which has a K m for adenosine of 29 µM (Ting et al, 2005). Inosine is then converted to hypoxanthine by PNP, which has a K m for inosine of 4.7 µM (Shi et al, 2004), and hypoxanthine is then phosphoribosylated to IMP via the parasite's hypoxanthine-guanine-xanthine phosphoribosyl transferase, which has an apparent K m for hypoxanthine of ∼0.9 µM (at pH 8.5; Keough et al, 1999). It is likely to be this high-affinity phosphorylation step that results in the 'metabolic trapping' of the radiolabelled purines within the parasite, and hence the accumulation to distribution ratios >5 (Fig.…”

Section: Purine Uptake Into the Parasitementioning

confidence: 99%

Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1

Downie¹,

Saliba

Howitt³

et al. 2006

Molecular Microbiology

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SummaryLike all parasitic protozoa, the human malaria parasite Plasmodium falciparum lacks the enzymes required for de novo synthesis of purines and it is therefore reliant upon the salvage of these compounds from the external environment. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a nucleoside transporter that has been localized to the plasma membrane of the intraerythrocytic form of the parasite. In this study we have characterized the transport of purine and pyrimidine nucleosides across the plasma membrane of 'isolated' trophozoite-stage P. falciparum parasites and compared the transport characteristics of the parasite with those of PfENT1 expressed in Xenopus oocytes. The transport of nucleosides into the parasite: (i) was, in the case of adenosine, inosine and thymidine, very fast, equilibrating within a few seconds; (ii) was of low affinity [ K m (adenosine) = 1.45 ± 0.25 mM; K m (thymidine) = 1.11 ± 0.09 mM]; and (iii) showed 'crosscompetition' for adenosine, inosine and thymidine, but not cytidine. The kinetic characteristics of nucleoside transport in intact parasites matched very closely those of PfENT1 expressed in Xenopus oocytes [ K m (adenosine) = 1.86 ± 0.28 mM; K m (thymidine) = 1.33 ± 0.17 mM]. Furthermore, PfENT1 transported adenosine, inosine and thymidine, with a cross-competition profile the same as that seen for isolated parasites. The data are consistent with PfENT1 serving as a major route for the uptake of nucleosides across the parasite plasma membrane.

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“…A number of purine salvage enzymes have been described for Plasmodium spp. (2)(3)(4), and one, hypoxanthine-guanine-xanthine phosphoribosyltransferase, has been characterized in detail both biochemically (5) and structurally (6).…”

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confidence: 99%

Isolation and Functional Characterization of the PfNT1 Nucleoside Transporter Gene from Plasmodium falciparum

Carter¹,

Mamoun²,

Liu³

et al. 2000

Journal of Biological Chemistry

130

123

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Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is incapable of de novo purine synthesis, and thus, purine acquisition from the host is an indispensable nutritional requirement. This purine salvage process is initiated by the transport of preformed purines into the parasite. We have identified a gene encoding a nucleoside transporter from P. falciparum, PfNT1, and analyzed its function and expression during intraerythrocytic parasite development. PfNT1 predicts a polypeptide of 422 amino acids with 11 transmembrane domains that is homologous to other members of the equilibrative nucleoside transporter family. Southern analysis and BLAST searching of The Institute for Genomic Research (TIGR) malaria data base indicate that PfNT1 is a single copy gene located on chromosome 14. Northern analysis of RNA from intraerythrocytic stages of the parasite demonstrates that PfNT1 is expressed throughout the asexual life cycle but is significantly elevated during the early trophozoite stage. Functional expression of PfNT1 in Xenopus laevis oocytes significantly increases their ability to take up naturally occurring D-adenosine (K m ‫؍‬ 13.2 M) and D-inosine (K m ‫؍‬ 253 M). Significantly, PfNT1, unlike the mammalian nucleoside transporters, also has the capacity to transport the stereoisomer L-adenosine (K m > 500 M). Inhibition studies with a battery of purine and pyrimidine nucleosides and bases as well as their analogs indicate that PfNT1 exhibits a broad substrate specificity for purine and pyrimidine nucleosides. These data provide compelling evidence that PfNT1 encodes a functional purine/pyrimidine nucleoside transporter whose expression is strongly developmentally regulated in the asexual stages of the P. falciparum life cycle. Moreover, the unusual ability to transport L-adenosine and the vital contribution of purine transport to parasite survival makes PfNT1 an attractive target for therapeutic evaluation.

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Purification and characterization of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase and comparison with the human enzyme

Cited by 86 publications

References 26 publications

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1

Isolation and Functional Characterization of the PfNT1 Nucleoside Transporter Gene from Plasmodium falciparum

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