Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon y to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon y resulted in a significant reduction in the expression of iNOS mRNA and of NO synthesis, compared with cells preincubated with culture medium alone. The regulatory effect on the induction of iNOS by LPG is located in the LPG phosphoglycan disaccharide backbone. Synthetic fragments of this backbone had a similar regulatory effect on NO synthesis. Further, the production of NO by activated macrophages in the present system was correlated directly with the leishmanicidal capacity of the cells. These data therefore demonstrate that LPG glycoconjugates have a profound effect on the survival of Leishmania Leishmania major infection in the murine model is directly associated with the expression of cytokine-inducible NO synthase (iNOS) (4-7).Macrophages express iNOS following activation by a variety of immunological stimuli such as interferon y (IFN-'y), tumor necrosis factor a (TNF-a), and bacterial lipopolysaccharide (LPS) (for reviews, see refs. 8-10). iNOS catalyzes the synthesis of high concentrations of NO from L-arginine and molecular oxygen (for review, see ref. 11), and NO is involved in the killing of a range of microorganisms (for reviews, see refs. 12-14), of which L. major is an example (15-17). We report here that lipophosphoglycan (LPG), a predominant surface molecule of promastigotes, can regulate the expression of iNOS and influence the survival of the parasites.The basic LPG structure of all Leishmania species consists of four domains: (i) a 1-O-alkyl-2-lysophosphatidyl(myo)inositol anchor; (ii) a hexasaccharide core; (iii) a polymer of repeating phosphodisaccharides of galactose and mannose; and (iv) a neutral mannose cap (see Fig. 1), with some species specific differences in the carbohydrate side-chains of the helical phosphodisaccharide repeats (18,19 from Amersham. Phosphatidylinositol-specific phospholipase C (PI-PLC) was purchased from Oxford Glycosystems (Abingdon, U.K.). L-NG-monomethyl-arginine (L-NMMA), an inhibitor of NO synthase, and D-NG-monomethyl-arginine (D-NMMA), its inert enantiomer, were kindly provided by S. Moncada (The Cruciform Project, University College London). All other reagents were of analytical grade.Cell Culture. The murine macrophage cell line J774 was obtained from the American Type Culture Collection and was passaged in DMEM containing 2 mM L-glutamine, 100 units/ml penicillin, 100 ,tg/ml streptomycin, and 10% heat-inactivated fetal calf serum (FCS) 'To whom reprint requests should be addressed. 10984The publication costs of this article were defrayed in part by page charge payment. This article must the...

The biosynthesis of glycosylphosphatidylinositol (GPI) precursors in Trypanosoma brucei involves the D-mannosylation of D-GlcN␣1-6-D-myo-inositol-1-PO 4 -sn-1,2-diacylglycerol (GlcN-PI). An assay for the first mannosyltransferase of the pathway, Dol-P-Man:GlcN-PI ␣1-4-mannosyltransferase, is described. Analysis of the acceptor specificity revealed (a) that the enzyme requires the myo-inositol residue of the GlcN-PI substrate have the D configuration; (b) that the enzyme requires the presence of the NH 2 group of the D-GlcN residue; (c) that GlcNAc-PI is more efficiently presented to the enzyme than GlcN-PI, suggesting a degree of substrate channelling via the preceding GlcNAc-PI de-N-acetylase enzyme; (d) that the fatty acid and phosphoglycerol components of the phosphatidyl moiety are important for enhancing substrate presentation and substrate recognition, respectively; and (e) that D-GlcN␣1-6-D-myoinositol is the minimum structure that can support detectable acceptor activity. Analysis of the donor specificity revealed that short chain (C 5 and C 15 ) analogues of dolichol phosphate can act as substrates for the trypanosomal dolichol-phosphomannose synthetase, whereas the corresponding mannopyranosides cannot act as donors for the Dol-P-Man:GlcN-PI ␣1-4-mannosyltransferase.

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.

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.

The substrate speci®cities of Trypanosoma brucei and human (HeLa) GlcNAc-PI de-N-acetylases were determined using 24 substrate analogues. The results show the following. (i) The de-N-acetylases show little speci®city for the lipid moiety of GlcNAc-PI. (ii) The 3¢-OH group of the GlcNAc residue is essential for substrate recognition whereas the 6¢-OH group is dispensable and the 4¢-OH, while not required for recognition, cannot be epimerized or substituted. (iii) The parasite enzyme can act on analogues containing bGlcNAc or aromatic N-acyl groups, whereas the human enzyme cannot. (iv) Three GlcNR-PI analogues are de-N-acetylase inhibitors, one of which is a suicide inhibitor. (v) The suicide inhibitor most likely forms a carbamate or thiocarbamate ester to an active site hydroxy-amino acid or Cys or residue such that inhibition is reversed by certain nucleophiles. These and previous results were used to design two potent (IC 50 = 8 nM) parasite-speci®c suicide substrate inhibitors. These are potential lead compounds for the development of anti-protozoan parasite drugs.

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.

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