Rosa M. de Lederkremer

Direct sialic acid transfer from a protein donor to glycolipids of trypomastigote forms of Trypanosoma cruzi.

Labeled sialoglycolipids were purified from tissue culture-derived trypomastigotes incubated with [3H]fetuin. Thin layer chromatography of [3H]sialoglycolipids showed three components with the same migration as gangliosides extracted from parasites incubated with [3H]palmitic acid. Neuraminidase treatment or mild acid hydrolysis confirmed the presence of [3H]sialyl residues in sialoglycolipids synthesized after [3H]fetuin incubation. Labeling was not observed when parasites were incubated with free [3H]sialic acid (C7 derivative), suggesting that sialyl residues are directly transferred in vivo to gangliosides, by an enzymatic reaction possibly catalysed by a sialyl transferase (transglycosylase). Sonicated extracts of trypomastigotes incubated with [3H]fetuin catalysed the labeling of endogenous glycoconjugates as well as of bovine brain gangliosides. The transglycosylase activity was found associated with the particulate fraction and could be solubilized with Triton X-100. The specific activity of the sialic acid transglycosylase in epimastigotes is 17% of that found in trypomastigotes. Addition of an excess free sialic acid did not inhibit the reaction, suggesting that transfer does not occur via a pool of free sialic acid.

Deoxy sugars: occurrence and synthesis.

Publisher Summary This chapter describes the occurrence and synthesis of deoxy sugars. Several deoxy sugars, notably 2-deoxy- D -erythro-pentose (2-deoxy- D -ribose)—the sugar component of DNA, 6-deoxy- L -mannose ( L -rhamnose), 6-deoxy- L -galactose ( L -fucose), 6-deoxy- D -glucose (quinovose), and their derivatives, occur very widely in natural product. The iodo and bromo derivatives of monosaccharides can be reduced by a variety of reducing agents to afford the corresponding deoxy sugar. The more stable chloro derivatives can be reduced with Raney nickel. Selective reduction of a secondary chloride with respect to a primary chloride may be achieved if the reduction is performed in the presence of triethylamine. Mesylates or tosylates may be reduced directly or through the intermediate halides or epoxides. Epoxides are involved in the reduction of tosylates by lithium aluminum hydride or lithium triethyl borohydride. A general method for the synthesis of 2-deoxyaldoses utilizes a reaction sequence involving the formation and subsequent reduction of ketene dithioacetal intermediates.

Trans-sialidase and mucins of Trypanosoma cruzi: an important interplay for the parasite.

A dense glycocalix covers the surface of Trypanosoma cruzi, the agent of Chagas disease. Sialic acid in the surface of the parasite plays an important role in the infectious process, however, T. cruzi is unable to synthesize sialic acid or the usual donor CMP-sialic acid. Instead, T. cruzi expresses a unique enzyme, the trans-sialidase (TcTS) involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. The mucins are the major glycoproteins in the insect stage epimastigotes and in the infective trypomastigotes. Both, the mucins and the TcTS are anchored to the plasma membrane by a glycosylphosphatidylinositol anchor. Thus, TcTS may be shed into the bloodstream of the mammal host by the action of a parasite phosphatidylinositol-phospholipase C, affecting the immune system. The composition and structure of the sugars in the parasite mucins is characteristic of each differentiation stage, also, interstrain variations were described for epimastigote mucins. This review focus on the characteristics of the interplay between the trans-sialidase and the mucins of T. cruzi and summarizes the known carbohydrate structures of the mucins.

Formation and Remodeling of Inositolphosphoceramide during Differentiation of Trypanosoma cruzi from Trypomastigote to Amastigote

ABSTRACT Differentiation of Trypanosoma cruzi trypomastigotes to amastigotes inside myoblasts or in vitro, at low extracellular pH, in the presence of [3H]palmitic acid or [3H]inositol revealed differential labeling of inositolphosphoceramide and phosphatidylinositol, suggesting that a remodeling process takes place in both lipids. Using 3H-labeled inositolphosphoceramide and phosphatidylinositol as substrates, we demonstrated the association of at least five enzymatic activities with the membranes of amastigotes and trypomastigotes. These included phospholipase A1, phospholipase A2, inositolphosphoceramide-fatty acid hydrolase, acyltransferase, and a phospholipase C releasing either ceramide or a glycerolipid from the inositolphospholipids. These enzymes may be acting in remodeling reactions leading to the anchor of mature glycoproteins or glycoinositolphospholipids and helping in the transformation of the plasma membrane, a necessary step in the differentiation of slender trypomastigotes to round amastigotes. Synthesis of inositolphosphoceramide and particularly of glycoinositolphospholipids was inhibited by aureobasidin A, a known inhibitor of fungal inositolphosphoceramide synthases. The antibiotic impaired the differentiation of trypomastigotes at acidic pH, as indicated by an increased appearance of intermediate forms and a decreased expression of the Ssp4 glycoprotein, a characteristic marker of amastigote forms. Aureobasidin A was also toxic to differentiating trypomastigotes at acidic pH but not to trypomastigotes maintained at neutral pH. Our data suggest that inositolphosphoceramide is implicated in T. cruzi differentiation and that its metabolism could provide important targets for the development of antiparasitic therapies.

CHARACTERIZATION OF INOSITOLPHOSPHOLIPIDS IN TRYPANOSOMA CRUZI TRYPOMASTIGOTE FORMS

In vivo labeling experiments with [3H]palmitic acid, [3H]inositol, and [3H]glucose allowed the identification of two main classes of inositolphospholipids (IPLs) from the trypomastigote stage of Trypanosoma cruzi. Purification of these compounds was achieved by ion-exchange chromatography, high performance liquid chromatography and thin layer chromatography. Specific phosphatidyl-inositol phospholipase C digestion, dephosphorylation and acid methanolysis showed a ceramide structure for the lower migrating IPL1. Palmitoyldihydrosphingosine and palmitoylsphingosine were detected by reverse-phase thin-layer chromatography. On the other hand, IPL2 showed to be a mixture of diacylglycero- and alkylacylglycero-phospholipids in a 1:1 ratio. After PI-PLC digestion, the lipids were separated by preparative TLC and individually analysed. The diacylglycerol contained mainly C18:0 fatty acid together with a low amount of C16:0. Hexadecylglycerol esterified with the C18:0 fatty acid was the only alkylacylglycerol detected. The C18:2 and C18:1 fatty acids, preponderant in the PI molecules of epimastigote forms, were not detected in trypomastigote forms. This is the first report on inositol phospholipids, putative precursors of lipid anchors in the infective stage of T. cruzi.

Ceramide and inositol content of the lipopeptidophosphoglycan from Trypanosoma cruzi.

Abstract Inositol (2.5%), 17-methyl-sphinganine (4.8%) and sphinganine (1.5%) have been identified as constituents of the lipopeptidophospholycan isolated from whole cells of epimastigote forms of Trypanosoma cruzi . The branched chain base was characterized by combined gas chromatography — mass spectrometry.

Developmentally regulated expression of ceramide in Trypanosoma cruzi

Amastigote forms of T. cruzi express the specific Ssp-4 surface antigen which is progressively shed, by the action of an endogenous phosphatidylinositol-phospholipase C, during their development into epimastigotes (Andrews et al., J. Exp. Med., 167 (1988) 300-314). We show now that the lipid moiety of the anchor of Ssp-4 is a ceramide which was metabolically labelled with [3H]palmitic acid. The lipid could be cleaved by PI-PLC digestion in vitro, and was identified by methanolysis and reverse phase thin layer chromatography of the products, as palmitoyldihydrosphingosine. Also, the free biosynthesized lipids were investigated in parasites obtained after 0, 24, 48 and 72 h differentiation of trypomastigotes and further incubated with [3H]palmitic acid for 2 h. A maximum of free ceramide was found in the 24 h point, in accordance with the maximum of amastigote forms. In contrast only traces of free ceramide were found in trypomastigotes. The major ceramide (more than 90%) is palmitoyldihydrosphingosine, which is the same as found in the anchor of Ssp-4. The ceramide could play an important role in the cell biology of the parasite as previously found for mammalian cells.

One-pot synthesis of β-d-Gal>(1 → 4)[β-d-Galp(1 → 6)]-d-GlcNAc, a ‘core’ trisaccharide linked O-glycosidically in glycoproteins of Trypanosoma cruzi

Abstract Tin(IV) chloride-promoted condensation of benzyl 2- acetamido -3-O- benzoyl -2- deoxy -α- d -glucopyranoside ( 4 ) with penta -O- benzoyl -β- d -galactopyranose ( 6 ) gave the derivative of β- d -Gal p-(1 → 6)-α- d -GlcNAc 7 in 80% yield. This was glycosylated with penta -O- benzoyl -α,β- d -galactofuranose ( 5 ), employing the same catalyst, to afford the protected benzyl per -O- benzoyl -β- d -Gal >(1 → 4)[β- d -Gal p(1 → 6)] d -GlcNAc 10 in 41% yield. Alternatively, compound 10 was obtained directly in a one-pot reaction from 4 , by sequential addition of 6 and 5 (34% yield). β-Glycosidic linkages were diastereoselectively formed. De- O -benzoylation of 10 , followed by heterogeneous catalytic transfer hydrogenolysis of the benzyl group afforded the free trisaccharide β- d -Gal >(1 → 4)[β- d -Gal p(1 → 6)]- d -GlcNAc ( 14 ) in 98% yield from 10 . Sodium borohydride reduction of 14 gave the corresponding alditol, whose spectral data were identical to those reported for the alditol obtained from the 38–43 kDa cell-surface glycoprotein of Trypanosoma cruzi .

The structure of an α-D-glucan from Cyttaria harioti Fischer

Abstract A homogeneous glucan has been isolated from the fruiting bodies of Cyttaria harioti Fischer. Partial acid hydrolysis produced major amounts of isomaltose, whereas acetolysis gave maltose and maltotriose. Enzymic hydrolysis with amylo-glucosidase and pullulanase indicated a structure based on maltotriose residues connected by (1→6)-α- D linkages. This conclusion was supported by periodate-oxidation data which also showed that 3–7% of the glucose resisted oxidation. Methylation analysis confirmed the presence of (1→6) and (1→4) linkages in the ratio 1:2.4.

The reaction of 2-hydroxyglycal esters with alcohols in the presence of N-iodosuccinimide, stereoselective synthesis of α anomers of alkyl 3-deoxyhex-2-enopyranosides and 3,4-dideoxyhex-3-enopyranosid-2-uloses

Abstract Reaction of 2,3,4,6-tetra-O-acetyl-1,5-anhydro- d -arabino-hex-1-enitol (1; 2-hydroxyglucal tetraacetate) with 1.0–1.5 mol of primary secondary, or tertiary alcohols, in the presence of 0.1–1.0 mol of N-iodosuccinimide (NIS) in acetonitrile as solvent, afforded the α anomers of alkyl 3-deoxyhex-2-enopyranosides in very good yields. The reaction proceeded more slowly, and with poorer yields, in the case of the tetrabenzoate corresponding to 1. When 1 was treated with higher concentrations of 2-propanol, increasing proportions of a reaction by-product, 2-propyl 6-O-acetyl-3,4-dideoxy-α- d -glycero-hex-3-enopyranosid-2-ulose (10), were produced. Such sugar enones as 10 were more readily formed when the starting 2-hydroxyglycal acetate had the lyxo configuration. Thus, two cholesteryl α-hex-3-enopyranosid-2-ulose derivatives were each prepared in one step and with good yields. Remarkable stereoselectivity for the formation of the α anomers was observed in all of these reactions.