It is unknown whether ascorbate alone (vitamin C), its oxidized metabolite dehydroascorbic acid alone, or both species are transported into human cells. This problem was addressed using specific assays for each compound, freshly synthesized pure dehydroascorbic acid, the specially synthesized analog 6-chloroascorbate, and a new assay for 6-chloroascorbate. Ascorbate and dehydroascorbic acid were transported and accumulated distinctly; neither competed with the other. Ascorbate was accumulated as ascorbate by sodium-dependent carrier-mediated active transport. Dehydroascorbic acid transport and accumulation as ascorbate was at least 10-fold faster than ascorbate transport and was sodium-independent. Once transported, dehydroascorbic acid was immediately reduced intracellularly to ascorbate. The analog 6-chloroascorbate had no effect on dehydroascorbic acid transport but was a competitive inhibitor of ascorbate transport. The Ki for 6-chloroascorbate (2.9-4.4 microM) was similar to the Km for ascorbate transport (9.8-12.6 microM). 6-Chloroascorbate was itself transported and accumulated in fibroblasts by a sodium-dependent transporter. These data provide new information that ascorbate and dehydroascorbic acid are transported into human neutrophils and fibroblasts by two distinct mechanisms and that the compound available for intracellular utilization is ascorbate.

De-N-acetylation of N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) is the second step of glycosylphosphatidylinositol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent mannosylation of glucosaminyl-phosphatidylinositol (GlcN-PI) which leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. The substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells were studied with respect to the N-acyl groups (R) that could be removed from a series of GlcNR-PI substrates, where R = acetyl (Ac), propionyl (Pr), butyryl (Bu), isobutyryl (iBu), pentanoyl (Pen) or hexanoyl (Hex). The data show that the trypanosomal and HeLa enzymes had similar specificities and that the turnover of GlcNR-PIs by the trypanosomal enzyme was in the order GlcNAc-PI > GlcNPr-PI GlcNBu - PI ≈ GlcNiBu - PI ≈ GlcNPen - PI GlcNHex - PI. The trypanosome and HeLa de-N-acetylases were unable to de-N-acetylate mannosylated GlcNAc-PI intermediates, which explains why de-N-acetylation must precede mannosylation in the GPI biosynthetic pathway.

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.

The de-N-acetylation of N-acetyl-D-glucosaminylphosphatidylinositol (GlcNAc-PI) is the second step of mammalian and trypanosomal glycosylphosphatidylinositol biosynthesis. Glycosylphosphatidylinositol biosynthesis is essential for Trypanosoma brucei, the causative agent of African sleeping sickness, and GlcNAc-PI de-N-acetylase has previously been validated as a drug target. Inhibition of the trypanosome cell-free system and recombinant rat GlcNAc-PI de-N-acetylase by divalent metal cation chelators demonstrates that a tightly bound divalent metal cation is essential for activity. Reconstitution of metal-free GlcNAc-PI de-N-acetylase with divalent metal cations restores activity in the order Zn 2؉ > Cu 2؉ > Ni 2؉ > Co 2؉ > Mg 2؉ . Site-directed mutagenesis and homology modeling were used to identify active site residues and postulate a mechanism of action. The characterization of GlcNAc-PI de-N-acetylase as a zinc metalloenzyme will facilitate the rational design of antiprotozoan parasite drugs.

No abstract

A cell-free system based on washed Leishmania major membranes was labelled with GDP-[$H]Man in the presence of synthetic glucosaminyl-phosphatidylinositol (GlcN-PI) and N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI). In both cases, the major radiolabelled products were Manα1-4GlcNα1-6myo-inositol1-HPO % -(sn-1,2-dipalmitoylglycerol) and Manα1-4GlcNα1-6myo-inositol1-HPO % -(sn-1-palmitoyl-2-lysoglycerol), to which an additional -mannose residue was added when a chase with an excess of GDP-Man was performed. The L. major cell-free system can therefore be used to observe the actions of four enzymes, namely

De-N-acetylation of N-acetylglucosaminyl-phosphatidylino-sitol (GlcNAc-PI) is the second step of glycosylphosphatidylino-sitol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent processing of glucosaminyl-phosphatidylinositol (GlcN-PI) that leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. In this article, we used a direct de-N-acetylase assay, based on the release of [14C]acetate from synthetic GlcN[14C]Ac-PI and analogues thereof, and an indirect assay, based on the mannosylation of GlcNAc-PI analogues, to study the substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells. The HeLa enzyme was found to be more fastidious than the trypanosomal enzyme such that, unlike the trypanosomal enzyme, it was unable to act on a GlcNAc-PI analogue containing 2-O-octyl-d- myo -inositol or on the GlcNAc-PI diastereoisomer containing l- myo -inositol (GlcNAc-P(l)I). These results suggest thatselective inhibition of the trypanosomal de-N-acetylase may be possible and that this enzyme should be considered as a possible therapeutic target. The lack of strict stereospecificity of the trypanosomal de-N-acetylase for the d- myo -inositol component was also seen for the trypanosomal GPI alpha-manno-syltransferases when GlcNAc-P(l)I was added to the trypanosome cell-free system, but not when GlcN-P(l)I was used. In an attempt to rationalize these data, we modeled the structure and dynamics of d-GlcNAcalpha1-6d- myo -inositol-1-HPO4-( sn )-3-glycerol and its diastereoisomer d-GlcNAcalpha1-6l- myo -inositol-1-HPO4-( sn )-3-glycerol. These studies indicate that the latter compound visits two energy minima, one of which resembles the low-energy conformer of former compound. Thus, it is conceivable that the trypanosomal de-N-acetylase acts on GlcNAc-P(l)I when it occupies a GlcNAc-PI-likeconformation and that GlcN-P(l)I emerging from the de-N-acetylase may be channeled to the alpha-mannosyltransferases in this conformation.