Cellular Consequences of Diminished Protein O-Mannosyltransferase Activity in Baker's Yeast.

O-Mannosylation is a type of protein glycosylation initiated in the endoplasmic reticulum (ER) by the protein O-mannosyltransferase (PMT) family. Despite the vital role of O-mannosylation, its molecular functions and regulation are not fully characterized. To further explore the cellular impact of protein O-mannosylation, we performed a genome-wide screen to identify Saccharomyces cerevisiae mutants with increased sensitivity towards the PMT-specific inhibitor compound R3A-5a. We identified the cell wall and the ER as the cell compartments affected most upon PMT inhibition. Especially mutants with defects in N-glycosylation, biosynthesis of glycosylphosphatidylinositol-anchored proteins and cell wall ß-1,6-glucan showed impaired growth when O-mannosylation became limiting. Signaling pathways that counteract cell wall defects and unbalanced ER homeostasis, namely the cell wall integrity pathway and the unfolded protein response, were highly crucial for the cell growth. Moreover, among the most affected mutants, we identified Ost3, one of two homologous subunits of the oligosaccharyltransferase complexes involved in N-glycosylation, suggesting a functional link between the two pathways. Indeed, we identified Pmt2 as a substrate for Ost3 suggesting that the reduced function of Pmt2 in the absence of N-glycosylation promoted sensitivity to the drug. Interestingly, even though S. cerevisiae Pmt1 and Pmt2 proteins are highly similar on the sequence, as well as the structural level and act as a complex, we identified only Pmt2, but not Pmt1, as an Ost3-specific substrate protein.

Discovery of genetic biomarkers contributing to variation in drug response of cytidine analogues using human lymphoblastoid cell lines.

BACKGROUND: Two cytidine analogues, gemcitabine and cytosine arabinoside (AraC), are widely used in the treatment of a variety of cancers with a large individual variation in response. To identify potential genetic biomarkers associated with response to these two drugs, we used a human lymphoblastoid cell line (LCL) model system with extensive genomic data, including 1.3 million SNPs and 54,000 basal expression probesets to perform genome-wide association studies (GWAS) with gemcitabine and AraC IC50 values. RESULTS: We identified 11 and 27 SNP loci significantly associated with gemcitabine and AraC IC50 values, respectively. Eleven candidate genes were functionally validated using siRNA knockdown approach in multiple cancer cell lines. We also characterized the potential mechanisms of genes by determining their influence on the activity of 10 cancer-related signaling pathways using reporter gene assays. Most SNPs regulated gene expression in a trans manner, except 7 SNPs in the PIGB gene that were significantly associated with both the expression of PIGB and gemcitabine cytotoxicity. CONCLUSION: These results suggest that genetic variation might contribute to drug response via either cis- or trans- regulation of gene expression. GWAS analysis followed by functional pharmacogenomics studies might help identify novel biomarkers contributing to variation in response to these two drugs and enhance our understanding of underlying mechanisms of drug action.

New insights into the early steps of phosphatidylinositol mannoside biosynthesis in mycobacteria: PimB' is an essential enzyme of Mycobacterium smegmatis.

Phosphatidyl-myo-inositol mannosides (PIMs) are key glycolipids of the mycobacterial cell envelope. They are considered not only essential structural components of the cell but also important molecules implicated in host-pathogen interactions. Although their chemical structures are well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still incomplete. Here we show for the first time that although both mannosyltransferases PimA and PimB' (MSMEG_4253) recognize phosphatidyl-myo-inositol (PI) as a lipid acceptor, PimA specifically catalyzes the transfer of a Manp residue to the 2-position of the myo-inositol ring of PI, whereas PimB' exclusively transfers to the 6-position. Moreover, whereas PimB' can catalyze the transfer of a Manp residue onto the PI-monomannoside (PIM1) product of PimA, PimA is unable in vitro to transfer Manp onto the PIM1 product of PimB'. Further assays using membranes from Mycobacterium smegmatis and purified PimA and PimB' indicated that the acylation of the Manp residue transferred by PimA preferentially occurs after the second Manp residue has been added by PimB'. Importantly, genetic evidence is provided that pimB' is an essential gene of M. smegmatis. Altogether, our results support a model wherein Ac1PIM2, a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA, followed by PimB', and finally the acyltransferase MSMEG_2934. The essentiality of these three enzymes emphasizes the interest of novel anti-tuberculosis drugs targeting the initial steps of PIM biosynthesis.

Modular approach to triazole-linked 1,6-α-D-oligomannosides to the discovery of inhibitors of Mycobacterium tuberculosis cell wall synthetase.

Aiming at developing inhibitors of mannosyltransferases, the enzymes that participate in the biosynthesis of the cell envelope of Mycobacterium tuberculosis, the synthesis of a range of designed triazole-linked 1,6-oligomannosides up to a hexadecamer has been accomplished by a modular approach centered on the Cu(I)-catalyzed azide-alkyne cycloaddition as key process. The efficiency and fidelity of the cycloaddition are substantiated by high yields (76-96%) and exclusive formation of the expected 1,4-disubstituted triazole ring in all oligomer assembling reactions. Key features of oligomers thus prepared are the anomeric carbon-carbon bond of all mannoside residues and the 6-deoxymannoside capping residue. Suitable bioassays with dimer, tetramer, hexamer, octamer, decamer, and hexadecamer showed variable inhibitor activity against mycobacterial α-(1,6)-mannosyltransferases, the highest activity (IC(50) = 0.14-0.22 mM) being registered with the hexamannoside and octamannoside.

Epimeric and amino disaccharide analogs as probes of an alpha-(1-->6)-mannosyltransferase involved in mycobacterial lipoarabinomannan biosynthesis.

Mycobacterial lipoarabinomannan (LAM) is an important, immunologically active glycan found in the cell wall of mycobacteria, including the human pathogen Mycobacterium tuberculosis. At the core of LAM is a mannan domain comprised of alpha-(1-->6)-linked-mannopyranose (Manp) residues. Previously, we and others have demonstrated that alpha-Manp-(1-->6)-alpha-Manp disaccharides (e.g., Manp-(1-->6)-alpha-ManpOctyl, ) are the minimum acceptor substrates for enzymes involved in the assembly of the LAM mannan core. We report here the synthesis five epimeric and three amino analogs of , and their subsequent biochemical evaluation against an alpha-(1-->6)-ManT activity present in a membrane preparation from M. smegmatis. Changing the manno- configuration of either residue of to talo- or gluco- led to a reduction or loss of activity, thus confirming earlier work showing that the C-2 and C-4 hydroxyl groups of each monosaccharide were important for enzymatic recognition. Characterization of the products formed from these analogs was done using a combination of mass spectrometry and glycosidase digestion, and full substrate kinetics were also performed. The analogs in which the acceptor hydroxyl group had been replaced with an amino group were, as expected, not substrates for the enzyme, but were weak inhibitors.

Rationally designed squaryldiamides - a novel class of sugar-nucleotide mimics?

Sugar-nucleotides such as GDP-mannose, GDP-fucose and UDP-glucose are important biomolecules with a central role in carbohydrate and glycoconjugate biosynthesis, metabolism and cell signalling. Analogues and mimics of naturally occurring sugar-nucleotides are sought after as chemical tools and inhibitor candidates for sugar-nucleotide-dependent enzymes including glycosyltransferases. Many sugar-nucleotides bind to their target glycosyltransferases via coordination of the diphosphate group to a divalent metal cofactor in the active site. The identification of uncharged, chemically stable surrogates for the diphosphate group, with the ability to coordinate to a divalent metal, is therefore an important design criteria for the development of sugar-nucleotide mimics. Here, we describe the rational design and synthesis of a novel class of sugar-nucleotide mimics based on a squaryldiamide scaffold, an uncharged phosphate isostere. We demonstrate by comprehensive NMR titration experiments that the new sugar-nucleotide mimics coordinate efficiently to Mg(2+), and provide results from biological studies with a therapeutically relevant mannosyltransferase from Trypanosoma brucei. Our findings suggest that squaryldiamides are a promising template for the development of sugar-nucleotide mimics, and illustrate the considerable potential of the squarylamide group as a fragment for inhibitor design.

Constructing a human complex type N-linked glycosylation pathway in Kluyveromyces marxianus.

Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (ß-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.

Inactivation of Mycobacterium tuberculosis mannosyltransferase pimB reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death.

The Mycobacterium tuberculosis (M.tb) cell wall contains an important group of structurally related mannosylated lipoglycans called phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM), and mannose-capped lipoarabinomannan (ManLAM), where the terminal alpha-[1-->2] mannosyl structures on higher order PIMs and ManLAM have been shown to engage C-type lectins such as the macrophage mannose receptor directing M.tb phagosome maturation arrest. An important gene described in the biosynthesis of these molecules is the mannosyltransferase pimB (Rv0557). Here, we disrupted pimB in a virulent strain of M.tb. We demonstrate that the inactivation of pimB in M.tb does not abolish the production of any of its cell wall mannosylated lipoglycans; however, it results in a quantitative decrease in the ManLAM and LM content without affecting higher order PIMs. This finding indicates gene redundancy or the possibility of an alternative biosynthetic pathway that may compensate for the PimB deficiency. Furthermore, infection of human macrophages by the pimB mutant leads to an alteration in macrophage phenotype concomitant with a significant increase in the rate of macrophage death.

First small molecular inhibitors of T. brucei dolicholphosphate mannose synthase (DPMS), a validated drug target in African sleeping sickness.

Drug-like molecules with activity against Trypanosoma brucei are urgently required as potential therapeutics for the treatment of African sleeping sickness. Starting from known inhibitors of other glycosyltransferases, we have developed the first small molecular inhibitors of dolicholphosphate mannose synthase (DPMS), a mannosyltransferase critically involved in glycoconjugate biosynthesis in T. brucei. We show that these DPMS inhibitors prevent the biosynthesis of glycosylphosphatidylinositol (GPI) anchors, and possess trypanocidal activity against live trypanosomes.

Efficient antibody production upon suppression of O mannosylation in the yeast Ogataea minuta.

When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.

A new small-molecule KRE2 inhibitor against invasive Candida parapsilosis infection.

AIM: To investigate the antifungal activity of MOL3, a small molecule that was selected by virtual screening, against Candida spp. MATERIALS & METHODS: The antifungal activity of MOL3 was evaluated using standard strains and clinical isolates. Activity was evaluated in both in vitro tests and animal models. RESULTS: The minimum fungicidal concentration of MOL3 against Candida spp. ranged from 16 to 128 mg/l. MOL3 at the sub-minimum fungicidal concentration inhibited hyphal elongation. The remaining yeast cells presented morphological changes and were metabolically inactive. MOL3 was toxicologically inert both in vitro and in the animal model. MOL3 also reduced experimental systemic infection by C. parapsilosis in mice. CONCLUSION: The selection of MOL3 by virtual screening was successful, revealing a promising antifungal candidate.

Evaluation of monovalent and multivalent iminosugars to modulate Candida albicans ß-1,2-mannosyltransferase activities.

ß-1,2-Linked oligomannosides substitute the cell wall of numerous yeast species. Several of those including Candida albicans may cause severe infections associated with high rates of morbidity and mortality, especially in immunocompromised patients. ß-1,2-Mannosides are known to be involved in the pathogenic process and to elicit an immune response from the host. In C. albicans, the synthesis of ß-mannosides is under the control of a family of nine genes coding for putative ß-mannosyltransferases. Two of them, CaBmt1 and CaBmt3, have been shown to initiate and prime the elongation of the ß-mannosides on the cell-wall mannan core. In the present study, we have assessed the modulating activities of monovalent and multivalent iminosugar analogs on these enzymes in order to control the enzymatic bio-synthesis of ß-mannosides. We have identified a monovalent deoxynojirimycin (DNJ) derivative that inhibits the CaBmt1-catalyzed initiating activity, and mono-, tetra- and polyvalent deoxymannojirimycin (DMJ) that modulate the CaBmt1 activity toward the formation of a single major product. Analysis of the aggregating properties of the multivalent iminosugars showed their ability to elicit clusterization of both CaBmt1 and CaBmt3, without affecting their activity. These results suggest promising roles for multivalent iminosugars as controlling agents for the biosynthesis of ß-1,2 mannosides and for monovalent DNJ derivative as a first target for the design of future ß-mannosyltransferase inhibitors.

Novel prenyl-linked benzophenone substrate analogues of mycobacterial mannosyltransferases.

PPM (polyprenol monophosphomannose) has been shown to act as a glycosyl donor in the biosynthesis of the Man (mannose)-rich mycobacterial lipoglycans LM (lipomannan) and LAM (lipoarabinomannan). The Mycobacterium tuberculosis PPM synthase (Mt-Ppm1) catalyses the transfer of Man from GDP-Man to polyprenyl phosphates. The resulting PPM then serves as a donor of Man residues leading to the formation of an alpha(1-->6)LM intermediate through a PPM-dependent alpha(1-->6)mannosyltransferase. In the present study, we prepared a series of ten novel prenyl-related photoactivatable probes based on benzophenone with lipophilic spacers replacing several internal isoprene units. These probes were excellent substrates for the recombinant PPM synthase Mt-Ppm1/D2 and, on photoactivation, several inhibited its activity in vitro. The protection of the PPM synthase activity by a 'natural' C(75) polyprenyl acceptor during phototreatment is consistent with probe-mediated photoinhibition occurring via specific covalent modification of the enzyme active site. In addition, the unique mannosylated derivatives of the photoreactive probes were all donors of Man residues, through a PPM-dependent mycobacterial alpha(1-->6)mannosyltransferase, to a synthetic Manp(1-->6)-Manp-O-C(10:1) disaccharide acceptor (where Manp stands for mannopyranose). Photoactivation of probe 7 led to striking-specific inhibition of the M. smegmatis alpha(1-->6)mannosyltransferase. The present study represents the first application of photoreactive probes to the study of mycobacterial glycosyltransferases involved in LM and LAM biosynthesis. These preliminary findings suggest that the probes will prove useful in investigating the polyprenyl-dependent steps of the complex biosynthetic pathways to the mycobacterial lipoglycans, aiding in the identification of novel glycosyltransferases.

Lead selection and characterization of antitubercular compounds using the Nested Chemical Library.

Discovering new drugs to treat tuberculosis more efficiently and to overcome multidrug resistance is a world health priority. To find novel antitubercular agents several approaches have been used in various institutions worldwide, including target-based approaches against several validated mycobacterial enzymes and phenotypic screens. We screened more than 17,000 compounds from Vichem's Nested Chemical Library™ using an integrated strategy involving whole cell-based assays with Corynebacterium glutamicum and Mycobacterium tuberculosis, and target-based assays with protein kinases PknA, PknB and PknG as well as other targets such as PimA and bacterial topoisomerases simultaneously. With the help of the target-based approach we have found very potent hits inhibiting the selected target enzymes, but good minimal inhibitory concentrations (MIC) against M. tuberculosis were not achieved. Focussing on the whole cell-based approach several potent hits were found which displayed minimal inhibitory concentrations (MIC) against M. tuberculosis below 10 µM and were non-mutagenic, non-cytotoxic and the targets of some of the hits were also identified. The most active hits represented various scaffolds. Medicinal chemistry-based lead optimization was performed applying various strategies and, as a consequence, a series of novel potent compounds were synthesized. These efforts resulted in some effective potential antitubercular lead compounds which were confirmed in phenotypic assays.

Specificity of GDP-Man:dolichyl-phosphate mannosyltransferase for the guanosine diphosphate esters of mannose analogues containing deoxy and deoxyfluoro substituents.

Guanosine diphosphate (GDP) esters of 2-deoxy-D-glucose (2dGlc), 2-deoxy-2-fluoro-D-mannose (2FMan), 3-deoxy-D-mannose (3dMan), 4-deoxy-D-mannose (4dMan) and 6-deoxy-D-mannose (6dMan) have been synthesised and tested for their ability to act as inhibitors of dolichyl phosphate mannose synthesis (enzyme: GDP-mannose:dolichyl-phosphate mannosyltransferase, EC 2.4.1.83) in chick embryo cell microsomal membranes. The following order of efficiency was found with the apparent Ki in parentheses: GDP-6dMan (0.40 microM +/- 0.15) greater than GDP-3dMan (1.0 microM +/- 0.1) = GDP-2dGlc (1.3 microM +/- 0.2) greater than GDP-4dMan (3.1 microM +/- 0.1) GDP-2FMan (15 microM +/- 0). For comparison the Km for GDP-Man was 0.52 microM +/- 0.02 and the Ki for GDP was 56 microM +/- 2. These results indicate that the 6-hydroxyl group of mannose is not crucial for enzyme-substrate recognition, whereas the 2- and 3-hydroxyls may have some involvement. The 4-hydroxyl appears to be an important determinant for enzyme-substrate recognition in this mannosyltransferase.

Inhibition of glycosyltransferases by bis-(p-nitrophenyl)phosphate: general effect and relation to their membrane integration.

The effect of bis-(p-nitrophenyl)phosphate on various glycosyltransferases involved in protein glycosylation (sialyl-, fucosyl-, galactosyl-, mannosyl- and glucosyltransferases) have been studied using crude enzyme preparations solubilized from rat spleen lymphocytes. Bis-(p-nitrophenyl)phosphate appears as a common inhibitor for every glycosyltransferase reaction utilizing sugar nucleotides as direct donors. In most cases 10 mM inhibitor is sufficient to obtain a 90 per cent inhibition. Kinetic studies achieved with a purified galactosyltransferase preparation reveal that bis-(p-nitrophenyl)phosphate exerts a competitive inhibition towards UDP-galactose binding. Concerning membrane-bound enzymes, the interaction of bis-(p-nitrophenyl)phosphate depends on its accessibility to the enzyme active site. This is shown by the different effect obtained with two UDP-Glc utilizing membrane-bound enzymes : UDP-Glc : phospho-dolichyl glucosyltransferase and UDP-Glc : ceramide glucosyltransferase : the first one not being affected but the second one being markedly inhibited under the same condition, although both are inhibited when the membrane environment is disturbed by detergent. Bis-(p-nitrophenyl)phosphate appears to be a tool to study membrane topology of glycosyltransferases.

Regulation of Paramecium primaurelia glycosylphosphatidyl-inositol biosynthesis via dolichol phosphate mannose synthesis.

A set of glycosylinositol-phosphoceramides, belonging to a family of glycosylphosphatidyl-inositols (GPIs) synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia has been described. The final GPI precursor was identified and structurally characterized as: ethanolamine-phosphate-6Man alpha 1-2Man alpha 1-6(mannosylphosphate) Man alpha 1-4glucosamine-inositol-phospho-ceramide. During our investigations on the biosynthesis of the acid-labile modification, the additional mannosyl phosphate substitution, we observed that the use of the nucleotide triphosphate analogue GTP gamma S (guanosine 5-O-(thiotriphosphate)) blocks the biosynthesis of the mannosylated GPI glycolipids. We show that GTP gamma S inhibits the synthesis of dolichol-phosphate-mannose, which is the donor of the mannose residues for GPI biosynthesis. Therefore, we investigated the role of GTP binding regulatory 'G' proteins using cholera and pertussis toxins and an intracellular second messenger cAMP analogue, 8-bromo-cAMP. All the data obtained suggest the involvement of classical heterotrimeric G proteins in the regulation of GPI-anchor biosynthesis through dolichol-phosphate-mannose synthesis via the activation of adenylyl cyclase and protein phosphorylation. Furthermore, our data suggest that GTP gamma S interferes with synthesis of dolichol monophosphate, indicating that the dolichol kinase is regulated by the heterotrimeric G proteins.

Possible localisation of dolichol-dependent mannosyltransferase of Trypanosoma brucei to the rough endoplasmic reticulum.

The glycosylphosphatidylinositol membrane anchor of variant surface glycoprotein of the African trypanosome Trypanosoma brucei contains several mannosyl residues for which dolichol phosphoryl mannose is supposed to be the precursor; this itself is probably synthesised by a dolichol-dependent mannosyltransferase. We have characterised and localised a mannosyltransferase activity of T. brucei which transfers mannose from GDP-[14C]mannose to exogenously added dolichyl phosphate. The enzyme was saturable for both its substrates and had a Km of 7.8 microM and 3.3 microM, respectively, for dolichyl phosphate and GDP-mannose. Mannosyltransferase was labile at 37 degrees C in the presence of Triton X-100, but its activity remained constant for at least 60 min at temperatures between 10-15 degrees C. The enzyme was inhibited by amphomycin and this inhibition was potentiated by the presence of 10 mM CaCl2. After subcellular fractionation of cell homogenates by differential centrifugation, mannosyltransferase was recovered mainly in the microsomal fraction and its distribution was very similar to that of RNA, a marker for the rough endoplasmic reticulum. After isopycnic centrifugation in a linear sucrose gradient the distribution of mannosyltransferase also resembled that of RNA. Both constituents exhibited a shift towards lower densities after pre-treatment of microsomal membranes with inorganic pyrophosphate, while other membrane markers such as acid phosphatase and nucleoside diphosphatase did not. It is concluded that the formation of dolichol phosphoryl mannose from GDP-mannose and dolichyl phosphate in T. brucei occurs mainly in the rough endoplasmic reticulum.

Comparative studies on mannosylphosphoryl dolichol and glucosylphosphoryl dolichol synthases.

Factors affecting the synthesis of mannosylphosphoryl dolichol and glucosylphosphoryl dolichol hen oviduct microsomes were compared in order to gain insight into the properties of their respective synthases. A stabilized form of mannosylphosphoryl dolichol synthase, but not glucosylphosphoryl dolichol synthase, was released from microsomes by freezing the membranes after exposure to the detergent CHAPSO. The activation energy for mannosylphosphoryl dolichol synthesis in membranes was 9.4 glucosylphosphoryl dolichol synthesis in membranes had a similar activation energy, 8.1 kcal/mol, but below 18 degrees C the value was 16.7 kcal/mol. Tryptic digestion of sealed microsomes preferentially inactivated mannosylphosphoryl dolichol synthase; however, both synthases were equally inactivated in detergent-permeabilized microsomes. Periodate-oxidized UDP-Glc was used to probe the topological orientation of glucosylphosphoryl dolichol synthase in rat liver microsomes. Sealed microsomes treated with oxidized UDP-Glc were inactive in synthesis of glucosylphosphoryl dolichol. However, when these treated microsomes were permeabilized, glucosylphosphoryl dolichol synthase activity was readily detected. From these studies we conclude that although mannosyl- and glucosylphosphoryl dolichol synthases catalyze chemically similar reactions in the endoplasmic reticulum, they differ in several respects. These differences were interpreted in terms of a topological model in which the active sites of the two enzymes reside on opposite faces of the endoplasmic reticulum, with that of the glucosyl lipid synthase facing the lumen and that of the mannosyl lipid synthase facing the cytosol.

Synthesis of some second-generation substrate analogues of early intermediates in the biosynthetic pathway of glycosylphosphatidylinositol membrane anchors.

1-D-6-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-2-O-octyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (23) and the corresponding 2-O-hexadecyl-D-myo-inositol compound 24 have been prepared as substrate analogues of an early intermediate in the biosynthetic pathway of glycosylphosphatidylinositol (GPI) membrane anchors. 1-D-6-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-octyl-sn-glycerol 3-phosphate) has also been prepared as a substrate analogue. Biological evaluation of the analogues 23 and 24 revealed that they are neither substrates nor inhibitors of GPI biosynthetic enzymes in the human (HeLa) cell-free system but are potent inhibitors at different stages of GPI biosynthesis in the Trypanosoma brucei cell-free system.