The use of Pseudomonas acyl-CoA synthetase to form acyl-CoAs from dicarboxylic fatty acids.

Pseudomonas acyl-CoA synthetase is shown to act on saturated dicarboxylic acids with a chain length of C10 or greater to produce conjugates containing a single CoA unit. The synthetase can, therefore, be used to generate novel acyl-CoA analogues for studies on proteins that utilise, bind to, or are modulated by acyl-CoAs.

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.

Binding site differences revealed by crystal structures of Plasmodium falciparum and bovine acyl-CoA binding protein.

Acyl-CoA binding protein (ACBP) maintains a pool of fatty acyl-CoA molecules in the cell and plays a role in fatty acid metabolism. The biochemical properties of Plasmodium falciparum ACBP are described together with the 2.0 A resolution crystal structures of a P. falciparum ACBP-acyl-CoA complex and of bovine ACBP in two crystal forms. Overall, the bovine ACBP crystal structures are similar to the NMR structures published previously; however, the bovine and parasite ACBP structures are less similar. The parasite ACBP is shown to have a different ligand-binding pocket, leading to an acyl-CoA binding specificity different from that of bovine ACBP. Several non-conservative differences in residues that interact with the ligand were identified between the mammalian and parasite ACBPs. These, together with measured binding-specificity differences, suggest that there is a potential for the design of molecules that might selectively block the acyl-CoA binding site.

Transformation of monomorphic Trypanosoma brucei bloodstream form trypomastigotes into procyclic forms at 37 degrees C by removing glucose from the culture medium.

African trypanosomes have been shown previously to undergo efficient transformation from bloodstream forms to procyclic (insect dwelling) forms in vitro by adding citrate and/or cis-aconitate to the culture medium and lowering incubation temperature to 27 degrees C. In this paper, it is shown that strain 427 monomorphic bloodstream forms of Trypanosoma brucei grown in axenic culture at 37 degrees C can be transformed to procyclic forms by simply replacing the glucose carbon source in the culture medium with glycerol. The removal of glucose from the medium results in the loss of the variant surface glycoprotein, the acquisition of cell surface procyclic acidic repetitive protein, the synthesis of procyclic-specific glycosylphosphatidylinositol precursors and the acquisition of substantial resistance to salicyl hydroxamic acid and glycerol within 72 h. A procyclic-specific cytoskeletal protein, known to be a marker of the late stage of transformation, is fully expressed by 96 h but full trans-sialidase activity appears only after 18-30 days. The transformation process described here is slower and less efficient than that previously described for monomorphic trypanosomes, using citrate and/or cis-aconitate and temperature shift as triggers. However, the separation of the transformation process from these stimuli is significant and the effects of glucose deprivation described here may reflect some of the events that occur in vivo in the tsetse fly midgut, where glucose levels are known to be very low.

Cloning, expression, and characterization of the acyl-CoA-binding protein in African trypanosomes.

African trypanosomes are shielded from their hosts' defenses by a coat of variant surface glycoprotein molecules, each of which is attached to the plasma membrane by a glycosylphosphatidylinositol anchor. During the later stages of glycosylphosphatidylinositol biosynthesis, myristic acid is incorporated into the anchor from the donor myristoyl-CoA by a series of unique fatty acid remodeling and exchange reactions. We have cloned and expressed a recombinant trypanosome acyl-CoA-binding protein that has a preference for binding relatively short chain acyl-CoAs and that has a high affinity for binding myristoyl-CoA (K(d) = 3.5 x 10(-10) M). This protein enhances fatty acid remodeling of glycosylphosphatidylinositol precursors in the trypanosome cell-free system. We speculate that the trypanosome acyl-CoA-binding protein plays an active role in supplying myristoyl-CoA to the fatty acid remodeling machinery in the parasite.

Inhibition of the GlcNAc transferase of the glycosylphosphatidylinositol anchor biosynthesis in African trypanosomes.

A wide variety of eukaryotic membrane proteins are anchored to the cell surface by a covalent linkage to glycosylphosphatidylinositol. One of the best characterised examples is the variant surface glycoprotein of the protozoan parasite, Trypanosoma brucei. The pathway for the formation of the glycosylphosphatidylinositol precursor has been previously described, with the first step being the transfer of GlcNAc, from UDP-GlcNAc to endogenous phosphatidylinositol to form N-acetyl-glucosaminylphosphatidylinositol [Doering, T. L., Masterson, W. J., Hart, G. W. & Englund, P. T. (1989) J. Biol. Chem. 264, 11,168-11,173]. Here we report that low concentrations of sulphydryl alkylating reagents irreversibly inhibit this transferase in a trypanosome-derived cell-free system. The site of inactivation by N-ethylmaleimide appears to be at, or close to, the enzyme active site, since incubation of the enzyme preparation with the donor molecule UDP-GlcNAc substantially protects the enzyme from inactivation. The protection appears to be primarily dependent on the nucleotide portion of the molecule, since UMP and UDP can mimic the protection seen with UDP-GlcNAc.

A novel glycosylphosphatidylinositol in African trypanosomes. A possible catabolic intermediate.

The major glycosylphosphatidylinositols (GPIs) in African trypanosomes are glycolipid A, the precursor of the variant surface glycoprotein membrane anchor, and glycolipid C, a species identical to glycolipid A except that it contains an acylated inositol. Both glycolipids A and C contain dimyristoyl glycerol and are efficiently labeled with [3H]myristate in a cell-free system. We now report a novel GPI known as lipid X. This GPI is radiolabeled strongly with [3H]palmitate (and very poorly with [3H]myristate or [3H]stearate) in digitonin-permeabilized cells. The structure of lipid X is Man1GlcNAc-(2O-palmitoyl)-D-myo-inositol-1-HPO4-3(lyso-pa lmitoylglyce rol). Metabolically, lipid X exists as an intermediate, and can be detected only under conditions in which its formation is stimulated (e.g. by EDTA) or its breakdown is inhibited (e.g. by Co2+). Lipid X has not been observed previously because these conditions do not support GPI biosynthesis. We speculate that lipid X is an intermediate in the catabolism of conventional trypanosome GPIs, possibly deriving from breakdown of glycolipid C.

Myristate exchange on the Trypanosoma brucei variant surface glycoprotein.

The glycosyl-phosphatidylinositol (GPI) anchor of the Trypanosoma brucei variant surface glycoprotein (VSG) is unique in having exclusively myristate as its fatty acid component. We previously demonstrated that the myristate specificity is the result of two independent pathways. First, the newly synthesized free GPI, which is not myristoylated, undergoes fatty acid remodeling to replace both its fatty acids with myristate. Second, the myristoylated precursor, glycolipid A, undergoes a myristate exchange reaction, detected by the replacement of unlabeled myristate by [3H]myristate. Remodeling and exchange have different enzymatic properties and apparently occur in different subcellular compartments. We now demonstrate that the GPI anchor linked to VSG is the major substrate for myristate exchange. VSG can be efficiently labeled with [3H]myristate by exchange in the presence of cycloheximide, an inhibitor that prevents new VSG synthesis and thus anchor addition to protein. Not only is newly synthesized VSG subject to exchange, but mature VSG, possibly recycling from the cell surface, also undergoes myristate exchange.

The mechanism of inhibition of glycosylphosphatidylinositol anchor biosynthesis in Trypanosoma brucei by mannosamine.

The inhibition of glycosylphosphatidylinositol anchor biosynthesis by mannosamine has been described previously in the procyclic forms of Trypanosoma brucei and in mammalian cells (Lisanti, M. P., Field, M. C., Caras, I. W. J., Menon, A. K., and Rodriguez-Boulan, E. (1991) EMBO J. 10, 1969-1977). A recent report has suggested that mannosamine exerts these effects by becoming incorporated into glycosylphosphatidylinositol anchor intermediates (Pan, Y-T., Kamitani, T., Bhuvaneswaran, C., Hallaq, Y., Warren, C. D., Yeh, E. T. H., and Elbein, A. D. (1992) J. Biol. Chem. 267, 21250-21255). In this paper we have analyzed the effects of mannosamine on glycosylphosphatidylinositol anchor and variant surface glycoprotein biosynthesis in the blood-stream form of T. brucei. Trypanosomes were biosynthetically labeled with [3H]mannosamine, and [3H]glucosamine in the presence of mannosamine, and the structures of the labeled glycolipids which accumulated were determined. The main glycolipid metabolite of mannosamine was shown to be ManN-Man-GlcN-PI. A trypanosome cell-free system preloaded with this compound was significantly impaired in its ability to synthesize glycosylphosphatidylinositol anchor intermediates beyond Man alpha 1-6Man alpha 1-4GlcN alpha 1-6PI. This compound is therefore proposed to be an inhibitor of the Dol-P-Man:Man alpha 1-6Man alpha 1-4GlcNa alpha 1-6PI alpha 1-2-mannosyltransferase of the GPI biosynthetic pathway. In living trypanosomes, 4 mM mannosamine had no effect on protein synthesis but reduced the rate of formation of mature glycosylphosphatidylinositol anchor precursors by 80%. This reduction in anchor precursor synthesis was insufficient to prevent the attachment of glycosylphosphatidylinositol anchors to newly synthesized variant surface glycoprotein molecules. These data suggest that the rate of anchor precursor synthesis in the bloodstream form of T. brucei, in contrast to mammalian cells and the procyclic form of T. brucei, is in large excess of the cellular requirements for protein anchorage.

Structural studies on a lipoarabinogalactan of Crithidia fasciculata.

The monosaccharide D-arabinopyranose has only been found in glycoconjugates of the trypanasomatid parasites Leishmania major, Endotrypanum schaudinni and Crithidia fasciculata. The donor molecule for the relevant arabinosyltransferases is known to be GDP-alpha-D-Arap in L. major and C. fasciculata, and the latter organism is being used to study the biosynthesis of GDP-alpha-D-Arap. In this study, we describe the structure of the terminal product of arabinose metabolism in C. fasciculata, namely lipoarabinogalactan. This molecule was purified by hydrophobic-interaction chromatography and studied by a variety of techniques, including gas chromatography-mass spectrometry, electrospray mass spectrometry and chemical and enzymic digestions. These data show that lipoarabinogalactan contains a previously described D-arabino-D-galactan polysaccharide component covalently attached to a glycosylphosphatidylinositol type of membrane anchor that is similar to, but not identical with, that found in the lipophosphoglycans of the Leishmania.

Partial purification and characterization of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol anchor biosynthesis in African trypanosomes.

N-Acetylglucosaminylphosphatidylinositol (GlcNAc-PI) de-N-acetylase was solubilized from the bloodstream form of African trypanosomes using Zwittergent 3-14. The solubilized GlcNAc-PI de-N-acetylase was assayed using radiolabeled GlcNAc-PI substrates. The enzyme was partially purified about 140-fold from washed trypanosome membranes using conventional liquid chromatography. The enzyme has a Km of 1.5 microM. Replacement of the di-O-substituted D-myo-inositol of the natural GlcNAc-PI substrate by the L-myo-inositol isomer did not significantly alter the ability of the compound to act as a substrate for the de-N-acetylase, suggesting that the C-2 to C-5 hydroxyl groups of the myoinositol ring do not play a critical role in substrate recognition. A substrate analogue lacking fatty acids was a relatively poor substrate for the enzyme, indicating that the lipid component plays an important role in substrate recognition and/or presentation of the substrate to the enzyme in detergent micelles. Substrate analogues lacking the glycerophosphate component were not recognized by the enzyme, suggesting that this component is important in the substrate recognition process.

Glycosyl-phosphatidylinositol molecules of the parasite and the host.

The glycosyl-phosphatidylinositol (GPI) protein-membrane anchors are ubiquitous among the eukaryotes. However, while mammalian cells typically express in the order of 100 thousand copies of GPI-anchor per cell, the parasitic protozoa, particularly the kinetoplastids, express up to 10-20 million copies of GPI-anchor and/or GPI-related glycolipids per cell. Thus GPI-family members dominate the cell surface molecular architecture of these organisms. In several cases, GPI-anchored proteins, such as the variant surface glycoprotein (VSG) of the African trypanosomes, or GPI-related glycolipids, such as the lipophosphoglycan (LPG) of the Leishmania, are known to be essential for parasite survival and infectivity. The highly elevated levels and specialised nature of GPI metabolism in the kinetoplastid parasites suggest that the GPI biosynthetic pathways might be good targets for the development of chemotherapeutic agents. This article introduces the range of GPI structures found in protozoan parasites, and their mammalian hosts, and discusses some aspects of GPI biosynthesis.