Terry K. Smith

The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness.

African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents.

Cloning and Characterization of a Novel Human Dual Flavin Reductase

Flavoprotein reductases play a key role in electron transfer in many physiological processes. We have isolated a cDNA with strong sequence similarities to cytochrome P-450 reductase and nitric-oxide synthase. The cDNA encodes a protein of 597 amino acid residues with a predicted molecular mass of 67 kDa. Northern blot analysis identified a predicted transcript of 3.0 kilobase pairs as well as a larger transcript at 6.0 kilobase pairs, and the gene was mapped to chromosome 9q34.3 by fluorescencein situ hybridization analysis. The amino acid sequence of the protein contained distinct FMN-, FAD-, and NADPH-binding domains, and in order to establish whether the protein contained these cofactors, the coding sequence was expressed in insect cells and purified. Recombinant protein bound FMN, FAD, and NADPH cofactors and exhibited a UV-visible spectrum with absorbance maxima at 380, 460, and 626 nm. The purified enzyme reduced cytochromec, with apparent K m andk cat values of 21 μm and 1.3 s−1, respectively, and metabolized the one-electron acceptors doxorubicin, menadione, and potassium ferricyanide. Immunoblot analysis of fractionated MCF7 cells with antibodies to recombinant NR1 showed that the enzyme is cytoplasmic and highly expressed in a panel of human cancer cell lines, thus indicating that this novel reductase may play a role in the metabolic activation of bioreductive anticancer drugs and other chemicals activated by one-electron reduction.

Parasite and mammalian GPI biosynthetic pathways can be distinguished using synthetic substrate analogues

Glycosylphosphatidylinositol (GPI) structures are attached to many cell surface glycoproteins in lower and higher eukaryotes. GPI structures are particularly abundant in trypanosomatid parasites where they can be found attached to complex phosphosaccharides, as well as to glycoproteins, and as mature surface glycolipids. The high density of GPI structures at all life‐cycle stages of African trypanosomes and Leishmania suggests that the GPI biosynthetic pathway might be a reasonable target for the development of anti‐parasite drugs. In this paper we show that synthetic analogues of early GPI intermediates having the 2‐hydroxyl group of the D‐myo‐inositol residue methylated are recognized and mannosylated by the GPI biosynthetic pathways of Trypanosoma brucei and Leishmania major but not by that of human (HeLa) cells. These findings suggest that the discovery and development of specific inhibitors of parasite GPI biosynthesis are attainable goals. Moreover, they demonstrate that inositol acylation is required for mannosylation in the HeLa cell GPI biosynthetic pathway, whereas it is required for ethanolamine phosphate addition in the T.brucei GPI biosynthetic pathway.

Chemical validation of GPI biosynthesis as a drug target against African sleeping sickness

It has been suggested that compounds affecting glycosylphosphatidylinositol (GPI) biosynthesis in bloodstream form Trypanosoma brucei should be trypanocidal. We describe cell‐permeable analogues of a GPI intermediate that are toxic to this parasite but not to human cells. These analogues are metabolized by the T. brucei GPI pathway, but not by the human pathway. Closely related nonmetabolizable analogues have no trypanocidal activity. This represents the first direct chemical validation of the GPI biosynthetic pathway as a drug target against African human sleeping sickness. The results should stimulate further inhibitor design and synthesis and encourage the search for inhibitors in natural product and synthetic compound libraries.

Cloning of Trypanosoma brucei and Leishmania major Genes Encoding the GlcNAc-Phosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis That Is Essential to the African Sleeping Sickness Parasite

The second step of glycosylphosphatidylinositol anchor biosynthesis in all eukaryotes is the conversion of D-GlcNAcα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol (GlcNAc-PI) tod-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol by GlcNAc-PI de-N-acetylase. The genes encoding this activity are PIG-L and GPI12 in mammals and yeast, respectively. Fragments of putative GlcNAc-PI de-N-acetylase genes from Trypanosoma bruceiand Leishmania major were identified in the respective genome project data bases. The full-length genes TbGPI12and LmGPI12 were subsequently cloned, sequenced, and shown to complement a PIG-L-deficient Chinese hamster ovary cell line and restore surface expression of GPI-anchored proteins. A tetracycline-inducible bloodstream form T. brucei TbGPI12conditional null mutant cell line was created and analyzed under nonpermissive conditions. TbGPI12 mRNA levels were reduced to undetectable levels within 8 h of tetracycline removal, and the cells died after 3–4 days. This demonstrates thatTbGPI12 is an essential gene for the tsetse-transmitted parasite that causes Nagana in cattle and African sleeping sickness in humans. It also validates GlcNAc-PI de-N-acetylase as a potential drug target against these diseases. Washed parasite membranes were prepared from the conditional null mutant parasites after 48 h without tetracycline. These membranes were shown to be greatly reduced in GlcNAc-PI de-N-acetylase activity, but they retained their ability to make GlcNAc-PI and to processd-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol to later glycosylphosphatidylinositol intermediates. These results suggest that the stabilities of other glycosylphosphatidylinositol pathway enzymes are not dependent on GlcNAc-PI de-N-acetylase levels.

Segregation of Glycosylphosphatidylinositol Biosynthetic Reactions in a Subcompartment of the Endoplasmic Reticulum

Glycosylphosphatidylinositols (GPIs) are synthesized in the endoplasmic reticulum (ER) via the sequential addition of monosaccharides, fatty acid, and phosphoethanolamine(s) to phosphatidylinositol (PI). While attempting to establish a mammalian cell-free system for GPI biosynthesis, we found that the assembly of mannosylated GPI species was impaired when purified ER preparations were substituted for unfractionated cell lysates as the enzyme source. To explore this problem we analyzed the distribution of the various GPI biosynthetic reactions in subcellular fractions prepared from homogenates of mammalian cells. The results indicate the following: (i) the initial reaction of GPI assembly, i.e. the transfer of GlcNAc to PI to form GlcNAc-PI, is uniformly distributed in the ER; (ii) the second step of the pathway, i.e.de-N-acetylation of GlcNAc-PI to yield GlcN-PI, is largely confined to a subcompartment of the ER that appears to be associated with mitochondria; (iii) the mitochondria-associated ER subcompartment is enriched in enzymatic activities involved in the conversion of GlcN-PI to H5 (a singly mannosylated GPI structure containing one phosphoethanolamine side chain; and (iv) the mitochondria-associated ER subcompartment, unlike bulk ER, is capable of the de novosynthesis of H5 from UDP-GlcNAc and PI. The confinement of these GPI biosynthetic reactions to a domain of the ER provides another example of the compositional and functional heterogeneity of the ER. The implications of these findings for GPI assembly are discussed.

Mitochondrial fatty acid synthesis is required for normal mitochondrial morphology and function in Trypanosoma brucei.

Trypanosoma brucei use microsomal elongases for de novo synthesis of most of its fatty acids. In addition, this parasite utilizes an essential mitochondrial type II synthase for production of octanoate (a lipoic acid precursor) as well as longer fatty acids such as palmitate. Evidence from other organisms suggests that mitochondrially synthesized fatty acids are required for efficient respiration but the exact relationship remains unclear. In procyclic form trypanosomes, we also found that RNAi depletion of the mitochondrial acyl carrier protein, an important component of the fatty acid synthesis machinery, significantly reduces cytochrome‐mediated respiration. This reduction was explained by RNAi‐mediated inhibition of respiratory complexes II, III and IV, but not complex I. Other effects of RNAi, such as changes in mitochondrial morphology and alterations in membrane potential, raised the possibility of a change in mitochondrial membrane composition. Using mass spectrometry, we observed a decrease in total and mitochondrial phosphatidylinositol and mitochondrial phosphatidylethanolamine. Thus, we conclude that the mitochondrial synthase produces fatty acids needed for maintaining local phospholipid levels that are required for activity of respiratory complexes and preservation of mitochondrial morphology and function.

Selective inhibitors of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei

Synthetic analogues of D‐GlcNα1–6D‐myo‐inositol‐1‐HPO4‐3(sn‐1,2‐diacylglycerol) (GlcN‐PI), with the 2‐position of the inositol residue substituted with an O‐octyl ether [D‐GlcNα1–6D‐(2‐O‐octyl)myo‐inositol‐1‐HPO4‐3‐sn‐1,2‐dipalmitoylglycerol; GlcN‐(2‐O‐octyl) PI] or O‐hexadecyl ether [D‐GlcNα1–6D‐(2‐O‐hexadecyl)myo‐inositol‐1‐HPO4‐3‐sn‐1,2‐dipalmitoylglycerol; GlcN‐(2‐O‐hexadecyl)PI], were tested as substrates or inhibitors of glycosylphosphatidylinositol (GPI) biosynthetic pathways using cell‐free systems of the protozoan parasite Trypanosoma brucei (the causative agent of human African sleeping sickness) and human HeLa cells. Neither these compounds nor their N‐acetyl derivatives are substrates or inhibitors of GPI biosynthetic enzymes in the HeLa cell‐free system but are potent inhibitors of GPI biosynthesis in the T.brucei cell‐free system. GlcN‐(2‐O‐hexadecyl)PI was shown to inhibit the first α‐mannosyltransferase of the trypanosomal GPI pathway. The N‐acetylated derivative GlcNAc‐(2‐O‐octyl)PI is a substrate for the trypanosomal GlcNAc‐PI de‐N‐acetylase and this compound, like GlcN‐(2‐O‐octyl)PI, is processed predominantly to Man2GlcN‐(2‐O‐octyl)PI by the T.brucei cell‐free system. Both GlcN‐(2‐O‐octyl)PI and GlcNAc(2‐O‐octyl)PI also inhibit inositol acylation of Man1–3GlcN‐PI and, consequently, the addition of the ethanolamine phosphate bridge in the T.brucei cell‐free system. The data establish these substrate analogues as the first generation of in vitro parasite GPI pathway‐specific inhibitors.

The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol.

In bloodstream‐form Trypanosoma brucei (the causative agent of African sleeping sickness) the glycosylphosphatidylinositol (GPI) anchor biosynthetic pathway has been validated genetically and chemically as a drug target. The conundrum that GPI anchors could not be in vivo labelled with [3H]‐inositol led us to hypothesize that de novo synthesis was responsible for supplying myo‐inositol for phosphatidylinositol (PI) destined for GPI synthesis. The rate‐limiting step of the de novo synthesis is the isomerization of glucose 6‐phosphate to 1‐d‐myo‐inositol‐3‐phosphate, catalysed by a 1‐d‐myo‐inositol‐3‐phosphate synthase (INO1). When grown under non‐permissive conditions, a conditional double knockout demonstrated that INO1 is an essential gene in bloodstream‐form T.u2003brucei. It also showed that the de novo synthesized myo‐inositol is utilized to form PI, which is preferentially used in GPI biosynthesis. We also show for the first time that extracellular myo‐inositol can in fact be used in GPI formation although to a limited extent. Despite this, extracellular inositol cannot compensate for the deletion of INO1. Supporting these results, there was no change in PI levels in the conditional double knockout cells grown under non‐permissive conditions, showing that perturbation of growth is due to a specific lack of de novo synthesized myo‐inositol and not a general inositol‐less death. These results suggest that there is a distinction between de novo synthesized myo‐inositol and that from the extracellular environment.

Phosphatidylinositol synthesis is essential in bloodstream form Trypanosoma brucei

PI (phosphatidylinositol) is a ubiquitous eukaryotic phospholipid which serves as a precursor for messenger molecules and GPI (glycosylphosphatidylinositol) anchors. PI is synthesized either de novo or by head group exchange by a PIS (PI synthase). The synthesis of GPI anchors has previously been validated both genetically and chemically as a drug target in Trypanosoma brucei, the causative parasite of African sleeping sickness. However, nothing is known about the synthesis of PI in this organism. Database mining revealed a putative TbPIS gene in the T. brucei genome and by recombinant expression and characterization it was shown to encode a catalytically active PIS, with a high specificity for myo-inositol. Immunofluorescence revealed that in T. brucei, PIS is found in both the endoplasmic reticulum and Golgi. We created a conditional double knockout of TbPIS in the bloodstream form of T. brucei, which when grown under non-permissive conditions, clearly showed that TbPIS is an essential gene. In vivo labelling of these conditional double knockout cells confirmed this result, showing a decrease in the amount of PI formed by the cells when grown under non-permissive conditions. Furthermore, quantitative and qualitative analysis by GLC-MS and ESI-MS/MS (electrospray ionization MS/MS) respectively showed a significant decrease (70%) in cellular PI, which appears to affect all major PI species equally. A consequence of this fall in PI level is a knock-on reduction in GPI biosynthesis which is essential for the parasites survival. The results presented here show that PI synthesis is essential for bloodstream form T. brucei, and to our knowledge this is the first report of the dependence on PI synthesis of a protozoan parasite by genetic validation.