Cloning and expression of the adenine phosphoribosyltransferase gene from Leishmania donovani

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Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme in the purine salvage pathway of many protozoan parasites. The predicted amino acid sequences of certain HGPRT proteins from parasites of the Trypanosomatidae family reveal a COOH-terminal tripeptide signal that is consistent with the degenerate topogenic signal targeting proteins to the glycosome, a fuel-metabolizing microbody unique to these parasites. To determine definitively the intracellular milieu of HG-PRT in these pathogens, polyclonal antiserum to the purified recombinant HGPRT from Leishmania donovani was generated in rabbits, and confocal and immunoelectron microscopy were employed to establish that the L. donovani HGPRT is localized exclusively to the glycosome. No HGPRT protein was detected in ⌬hgprt null mutants in which both alleles of the HGPRT locus had been replaced by a drug-resistance cassette. Transfectants of the ⌬hgprt knockout strain in which a wildtype HGPRT was amplified on an expression plasmid contained augmented amounts of HGPRT, all of which was localized to the glycosome. ⌬hgprt transfectants containing amplified copies of a mutated HGPRT construct in which the Ser-Lys-Val COOH-terminal targeting signal had been deleted expressed HGPRT throughout the parasite, including subcellular organelles such as the nucleus and flagellum. These data demonstrate that the L. donovani HGPRT is compartmentalized exclusively within the glycosome and that the COOH-terminal tripeptide of the protein is necessary to achieve targeting to this organelle.

Section: Discussionmentioning

confidence: 99%

Localization and Targeting of the Leishmania donovaniHypoxanthine-Guanine Phosphoribosyltransferase to the Glycosome

Shih¹,

Hwang²,

Carter³

et al. 1998

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“…AAH Activity Measurements in Intact Parasites-The ability of wild type and ⌬aah promastigotes and axenic amastigotes to deaminate [8-14 C]adenine (50 mCi/mmol) into [8][9][10][11][12][13][14] C]hypoxanthine was compared using TLC. 7.0 ϫ 10 7 parasites were washed and resuspended in 30 l of PBS, and [8-14 C]adenine (50 mCi/mmol) was added to a final concentration of 67 M. At various time points over a 1-h time course, 5 l of the parasite mixture were added to 2 l of glacial acetic acid to lyse the cells and terminate the reaction (25), spotted onto a PEI-cellulose TLC plate (Scientific Adsorbents Inc., Atlanta, GA), and run in n-butanol/water/glacial acetic acid at a ratio of 20:6:4 (v/v) (25).…”

Section: Materials Chemicals and Reagents-[8-mentioning

confidence: 99%

“…5-l samples were spotted at the 0-and 2-h time points, and the TLC plate was run, exposed, and developed as described above. [8][9][10][11][12][13][14] C]Adenine (50 mCi/mmol) was used as a control to verify that the AAH protein employed in the assay was enzymatically active.…”

Section: Materials Chemicals and Reagents-[8-mentioning

confidence: 99%

Adenine Aminohydrolase from Leishmania donovani

Boitz

Strasser

Hartman

et al. 2012

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Background: Purine salvage in Leishmania is an indispensable nutritional process. Results: Adenine aminohydrolase, a key purine enzyme in Leishmania, has been characterized biochemically and genetically. Conclusion: Adenine aminohydrolase is a unique enzyme in purine salvage that converts 6-aminopurines into 6-oxypurines. Significance: Functional characterization of key enzymes is crucial for understanding purine salvage and ultimately for targeting the pathway with drugs.

“…APRT Utilizes Hypoxanthine as a Substrate-The L. donovani APRT has been previously reported to exclusively recognize adenine as a substrate (23,34). However, the correlation between the capacity of ⌬hgprt/⌬xprt[Ino/Hyp] to incorporate and grow in hypoxanthine (Figs.…”

Section: Resultsmentioning

confidence: 99%

“…Immunoblotting and DNA Manipulations-Monospecific polyclonal antibodies raised against purified recombinant L. donovani APRT, HGPRT, and XPRT proteins in rabbits have been described previously (23)(24)(25), and Western blotting protocols were performed as detailed (26). Monoclonal anti-␣-tubulin antibody (DM1A) produced in mice was obtained from EMD Chemicals (Gibbstown, NJ).…”

Section: Methodsmentioning

confidence: 99%

Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases

Boitz

Ullman

2010

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Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal ⌬hgprt/⌬xprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084 -16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the ⌬hgprt/⌬xprt knockout cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of ⌬hgprt/⌬xprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which ⌬hgprt/⌬xprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured ⌬hgprt/⌬xprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of ⌬hgprt/⌬xprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which ⌬hgprt/⌬xprt parasites displayed persistence and increased virulence in mice.Leishmania donovani is a protozoan parasite that is the causative agent of visceral leishmaniasis, a debilitating and often fatal disease in humans. Leishmania spp. are digenetic protozoan parasites that exist as flagellated, motile promastigotes within the alimentary tract and salivary glands of their insect vector, members of the Phlebotomine sandfly family and as nonflagellated, amotile amastigotes within macrophages and other reticuloendothelial cells of the mammalian host. No effective vaccines are available for visceral leishmaniasis-or for that matter any disease caused by protozoan parasites, and therefore chemotherapy offers the only means of defense for the treatment and prevention of leishmaniasis and other diseases of parasitic origin. Unfortunately, the current armamentarium of drugs employed against visceral and other forms of leishmaniasis is far from ideal and is adversely affected by toxicity, protracted and invasive routes of administration, and therapeutic unresponsiveness. As a result, there is an acute need for better and more efficacious drugs to combat the disease.The establishment of an efficacious, parasite-specific ...