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1.
Carbohydr Res ; 333(2): 123-8, 2001 Jul 03.
Article in English | MEDLINE | ID: mdl-11448672

ABSTRACT

The development of beta-D-galactofuranosidase inhibitors provides a good chemotherapeutic target for treatment of major human diseases, because beta-D-galactofuranose is a constituent of important pathogen microorganisms but is absent in mammals. With this purpose we have prepared beta-D-galactofuranosyl nucleoside analogues, derived by the addition of nucleophiles to perbenzoylated beta-D-galactofuranosyl isothiocyanate, a compound previously prepared in this laboratory. N-beta-D-Galactofuranosyl-O-ethylthiourethane, N-beta-D-galactofuranosyl-4-oxoimidazolidine-2-thione, N-beta-D-galactofuranosyl-4-imidazoline-2-thione, and N-beta-D-galactofuranosyl-4-methoxyimidazolidine-2-thione, were prepared. The biological assays showed that imidazoline and imidazolidine-2-thione derivatives act as a new type of exo beta-D-galactofuranosidase inhibitor.


Subject(s)
Galactosides/chemical synthesis , Nucleosides/chemical synthesis , beta-Galactosidase/antagonists & inhibitors , Galactosides/pharmacology , Humans , Molecular Structure , Nucleosides/pharmacology , Penicillium/chemistry , Penicillium/enzymology , Thioglycosides/chemistry , beta-Galactosidase/metabolism
2.
J Biol Chem ; 275(36): 27671-80, 2000 Sep 08.
Article in English | MEDLINE | ID: mdl-10843987

ABSTRACT

The protozoan parasite Trypanosoma cruzi, the agent of Chagas disease, has a large number of mucin molecules on its surface, whose expression is regulated during the life cycle. These mucins are the main acceptors of sialic acid, a monosaccharide that is required by the parasite to infect and survive in the mammalian host. A large mucin-like gene family named TcMUC containing about 500 members has been identified previously in T. cruzi. TcMUC can be divided into two subfamilies according to the presence or absence of tandem repeats in the central region of the genes. In this work, T. cruzi parasites were transfected with one tagged member of each subfamily. Only the product from the gene with repeats was highly O-glycosylated in vivo. The O-linked oligosaccharides consisted mainly of beta-d-Galp(1-->4)GlcNAc and beta-d-Galp(1-->4)[beta-d-Galp(1-->6)]-d-GlcNAc. The same glycosyl moieties were found in endogenous mucins. The mature product was anchored by glycosylphosphatidylinositol to the plasma membrane and exposed to the medium. Sera from infected mice recognized the recombinant product of one repeats-containing gene thus showing that they are expressed during the infection. TcMUC genes encode a hypervariable region at the N terminus. We now show that the hypervariable region is indeed present in the exposed mature N termini of the mucins because sera from infected hosts recognized peptides having sequences from this region. The results are discussed in comparison with the mucins from the insect stages of the parasite (Di Noia, J. M., D'Orso, I., Sánchez, D. O., and Frasch, A. C. C. (2000) J. Biol. Chem. 275, 10218-10227) which do not have variable regions.


Subject(s)
Mucins/chemistry , Mucins/genetics , Trypanosoma cruzi/physiology , Amino Acid Sequence , Animals , Antigens, Protozoan/immunology , Base Sequence , Carbohydrate Sequence , Chagas Disease/immunology , Epitopes/chemistry , Genetic Variation , Glycosylation , Glycosylphosphatidylinositols/metabolism , Mice , Molecular Sequence Data , Mucins/immunology , Oligosaccharides/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Transfection , Trypanosoma cruzi/genetics
3.
Carbohydr Res ; 323(1-4): 7-13, 2000 Jan 12.
Article in English | MEDLINE | ID: mdl-10782280

ABSTRACT

Syntheses of 4-nitrophenyl beta-D-fucofuranoside (6) and beta-D-fucofuranosyl-(1-->3)-D-mannopyranose (10) are reported. These compounds, as analogues of galactofuranosides, were used for studying the influence of the hydroxyl group at C-6 in the interaction of the substrate with beta-D-galactofuranosidase. For the synthesis of the fucofuranosides, 2,3,5-tri-O-benzoyl-6-bromo-6-deoxy-D-galactono-1,4-lactone (1) was the key intermediate, which upon reduction of the lactone group with diisoamylborane, acetylation of the anomeric hydroxyl group, and catalytic hydrogenolysis of the bromine at C-6, led to 1-O-acetyl-2,3,5-tri-O-benzoyl-alpha,beta-D-fucofuranose (4), a convenient derivative for the preparation of fucofuranosides. Compound 4 was glycosylated in the presence of SnCl4, either with 4-nitrophenol for the preparation of 6, or with 2,5,6-tri-O-benzoyl-D-mannono-1,4-lactone (7), for the synthesis of disaccharide 10, via the glycosyl-aldonolactone approach. The synthetic route developed for the beta-D-fucofuranosides is simple and efficient. Compound 6 was not hydrolyzed by incubation with the exo beta-D-galactofuranosidase from Penicillium fellutanum, showing that HO-6 is essential for interaction of the substrate with the enzyme.


Subject(s)
Disaccharides/chemical synthesis , Fucose/analogs & derivatives , Glycoside Hydrolases , Nitrobenzenes/chemical synthesis , beta-Galactosidase/metabolism , Alcohols/chemistry , Catalysis , Fucose/chemical synthesis , Fucose/chemistry , Magnetic Resonance Spectroscopy , Models, Chemical , Penicillium/enzymology , Substrate Specificity , Temperature , beta-Galactosidase/chemistry
4.
Anal Biochem ; 279(1): 79-84, 2000 Mar 01.
Article in English | MEDLINE | ID: mdl-10683233

ABSTRACT

The O-linked N-acetylglucosamine oligosaccharides in the mucins of Trypanosoma cruzi may contain galactofuranose or galactopyranose, depending on the strain, one of the components being the disaccharide. Since galactofuranose is a site for antibody recognition, it is desirable to have a sensitive method for the detection of the galactofuranosyl structures. In this paper, we present procedures for the separation of Galfbeta1-->XGlcNAc and Galpbeta1-->XGlcNAc (X = 3, 4, and 6) as the corresponding alditols by high-pH anion-exchange chromatography with pulse amperometric detection. All the isomeric disaccharides could be resolved on a CarboPac PA-10 column, the galactofuranose-containing disaccharides being more retained in the column. GlcNAcol and Galfbeta1-->4(Galpbeta1-->6)GlcNAcol could be analyzed in the same run. The compounds could also be separated by thin-layer chromatography on silica gel 60, a convenient method for analysis of the radiolabeled alditols obtained by reductive beta-elimination in the presence of NaB(3)H(4). Both methods were applied for the analysis of the O-linked sugars in the mucins of T. cruzi CL 14 and revealed that they contained only N-acetylglucosamine and the disaccharide Galpbeta1-->4GlcNAc.


Subject(s)
Chromatography, Ion Exchange/methods , Chromatography, Thin Layer/methods , Disaccharides/isolation & purification , Mucins/chemistry , Trypanosoma cruzi/chemistry , Animals , Carbohydrate Sequence , Disaccharides/chemistry , Evaluation Studies as Topic , Hydrogen-Ion Concentration , Mucins/isolation & purification , Sugar Alcohols/chemistry , Sugar Alcohols/isolation & purification
5.
Biochem J ; 345 Pt 1: 77-84, 2000 Jan 01.
Article in English | MEDLINE | ID: mdl-10600641

ABSTRACT

The lipid moiety in the glycosylphosphatidylinositol anchors of glycoproteins of Trypanosoma cruzi consists of an alkylacylglycerol, a lysoalkylglycerol or a ceramide. Previously, we showed that the inositolphosphoceramides (IPCs) are the major components in the precursor inositolphospholipids of epimastigote and trypomastigote forms. Using (3)H-labelled subfractions of IPC, phosphatidylinositol (PI) and glycoinositolphospholipids (GIPLs) as substrates with a cell-free system, we now demonstrate the association of at least five enzyme activities with the trypanosomal membranous particulate material. These include: phospholipase A(1) and phospholipase A(2), enzymes that release free fatty acid from the PI and GIPLs; an acyltransferase responsible for the acylation of the generated monoacyl or monoalkylglycerolipids with endogenous unlabelled fatty acid; two activities of phospholipase C, one releasing ceramide from IPC and the other alkylacylglycerol, alkylglycerol or diacylglycerol from PI. The neutral lipids were also generated on incubation of the GIPLs. The phospholipase C activities were inhibited by p-chloromercuriphenylsulphonic acid, as reported for other PI phospholipases C. An IPC-fatty-acid hydrolase, releasing fatty acid from the labelled IPC, was also observed. The enzyme activities reported in the present study may be acting in remodelling reactions leading to the anchor of the mature glycoproteins of T. cruzi.


Subject(s)
Glycosphingolipids/metabolism , Phosphatidylinositols/metabolism , Phospholipases/metabolism , Trypanosoma cruzi/enzymology , Animals , Cell-Free System , Enzyme Inhibitors/pharmacology , In Vitro Techniques , Phosphatidylinositol Diacylglycerol-Lyase , Phospholipases/antagonists & inhibitors , Phospholipases A/antagonists & inhibitors , Phospholipases A/metabolism , Substrate Specificity , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolism
6.
Carbohydr Res ; 320(3-4): 176-82, 1999 Aug 15.
Article in English | MEDLINE | ID: mdl-10573856

ABSTRACT

An alternative and fast method for the purification of an exo-beta-D-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-beta-D-galactofuranoside affinity chromatography system and specific elution with 10 mM D-galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE-Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM D-galactono-1,4-lactone in a 100-500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-beta-D-galactofuranosidase was ascertained through binding to Concanavalin A-Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and Km and Vmax values of 0.311 mM and 17 mumol h-1 microgram-1 respectively, when 4-nitrophenyl beta-D-galactofuranoside was employed as the substrate.


Subject(s)
Chromatography, Affinity/methods , Glycoside Hydrolases , beta-Galactosidase/isolation & purification , Galactosides , Hydrogen-Ion Concentration , Kinetics , Ligands , Penicillium/enzymology
7.
Org Lett ; 1(2): 245-7, 1999 Jul 29.
Article in English | MEDLINE | ID: mdl-10905870

ABSTRACT

In a continuation of our studies on the characterization of the glycoproteins of T. cruzi new galactofuranosyl disaccharides were synthesized. Beta-D-Galf-(1-3)-D-GlcNAc was prepared by employing the trichloroacetamidate procedure for the glycosylation step. The mild conditions of this reaction are appropriate for condensation of 2,3,5,6-tetra-O-benzoyl-beta-D-galactofuranosyl trichloroacetamidate with acid-labile benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D-glucopyranoside. On the other hand, tin(IV) chloride promoted condensation of benzyl 2-acetamido-3-O-benzoyl-2-deoxy-alpha-D-glucopyranoside with penta-O-benzoyl-alpha-beta-D-galactofuranose gave the derivative of beta-D-Galf-(1-6)-D-GlcNAc in 78% yield.


Subject(s)
Disaccharides/chemical synthesis , Tin Compounds , Animals , Catalysis , Disaccharides/chemistry , Glycosylation , Mucins/chemistry , Trypanosoma cruzi/chemistry
8.
J Comp Physiol B ; 168(7): 549-54, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9810719

ABSTRACT

Modifications in content and lipid composition induced by fasting were examined in fat bodies from adults of Triatominae, Dipetalogaster maximus, Triatoma infestans and Panstrongylus megistus. With fasting, total lipid stores dropped approximately 50% for T. infestans and more than 70% for P. megistus. Total lipids analyzed by thin layer chromatography and fractionated by column chromatography on Unisil showed triacylglycerols as the main component in the three species, although P. megistus showed high levels of diacylglycerols (31-46%). Cholesterol amounted to 8-15%. In diacylglycerol fractions, C16:0, C18:1 and C18:0 fatty acids were detected; their ratio varied with species but it was not dependent on nutritional status. In triacylglycerol fractions C18:1 fatty acid was the major component at different times (48-68%); the ratio of monounsaturated to saturated in this fraction was 1.3, 2.6 and 1.2 for D. maximus, T. infestans and P. megistus respectively. The remarkable drop in lipid stores without noticeable changes in their relative composition would suggest that all types of lipid are used at similar rates. The higher content of diacylglycerols in P. megistus may be associated with the better flight performance of this species.


Subject(s)
Fat Body/metabolism , Lipid Metabolism , Triatominae/metabolism , Animals , Cholesterol/analysis , Diglycerides/analysis , Fasting/metabolism , Fatty Acids/analysis , Insect Vectors/metabolism , Lipids/analysis , Panstrongylus/metabolism , Triatoma/metabolism
9.
Carbohydr Res ; 311(4): 183-9, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9825521

ABSTRACT

A convenient synthesis of free beta-D-Galf-(1-->3)-D-Manp (8a) is reported. The disaccharide is present as external unit in the lipopeptidophosphoglycan (LPPG) of Trypanosoma cruzi and internally in the lipophosphoglycan (LPG) of Leishmania. Condensation of 2,5,6-tri-O-benzoyl-D-mannono-1,4-lactone (1) with 1,2,3,5,6-penta-O-benzoyl-D-galactofuranose, promoted by SnCl4, led to the beta-glycosyl-lactone, a key intermediate for disaccharide 8a, readily obtained by successive reduction of the lactone with diisoamylborane and debenzoylation. As in the LPG of Leishmania the HO-3 group of the galactofuranose is glycosylated by alpha-D-Galp, we also synthesized 3-deoxy-beta-D-xylo-hexofuranosyl-(1-->3)-D-Manp (8b) and p-nitrophenyl 3-deoxy-beta-D-xylo-hexofuranoside for studying the influence of HO-3 in the interaction with specific glycosidases. The disaccharide 8a, and its corresponding alditol, were good substrates for the beta-D-galactofuranosidase from Penicillium fellutanum, whereas the 3-deoxyglycosides were not hydrolyzed by the enzyme.


Subject(s)
Disaccharides/chemical synthesis , Glycoside Hydrolases , beta-Galactosidase/metabolism , Carbohydrate Sequence , Glycosphingolipids/metabolism , Magnetic Resonance Spectroscopy , Models, Chemical , Molecular Sequence Data , Penicillium/enzymology , Substrate Specificity
10.
Glycobiology ; 8(9): 901-4, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9675223

ABSTRACT

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D-galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4-aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.


Subject(s)
Galactose/analogs & derivatives , Thioglycosides/chemical synthesis , Thioglycosides/pharmacology , beta-Galactosidase/antagonists & inhibitors , Chromogenic Compounds/metabolism , Galactosides/metabolism , Penicillium/enzymology
11.
Steroids ; 63(2): 70-5, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9516715

ABSTRACT

In the present paper, we report that an inositolphosphoglycan (IPG), derived from a Trypanosoma cruzi glycoinositolphosphoceramide (LPPG), is able to inhibit ACTH-mediated accumulation of a glucocorticoid, cortisol, in calf adrenocortical cells. This IPG is also able to inhibit the stimulation by ACTH of the production of the main glucocorticoid, corticosterone and the main mineralocorticoid, aldosterone, in rat adrenocortical cells. Nitrous acid deamination confirmed that IPG is responsible for this inhibition. In order to study the involvement of glycosylphosphatidylinositol (GPI) in ACTH response in rat adrenal cortex, the activation of a phospholipase that hydrolyzes GPI (GPI-PLC) was evaluated. It was found that the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium is increased in rat adrenocortical cells by ACTH treatment. In addition, ACTH stimulates the release of ceramide from the glycoinositolphosphoceramide purified from T. cruzi. These data suggest that ACTH activates a GPI-PLC in rat adrenal cortex, which is in agreement with our previous data in calf adrenocortical cells; thus, the hydrolysis of GPI provoked by ACTH takes place in different mammals and the IPG released could inhibit ACTH-mediated synthesis of aldosterone, corticosterone and cortisol.


Subject(s)
Adrenal Cortex/drug effects , Adrenocorticotropic Hormone/pharmacology , Glucocorticoids/biosynthesis , Mineralocorticoids/biosynthesis , Oligosaccharides/pharmacology , Type C Phospholipases/metabolism , Adrenal Cortex/metabolism , Aldosterone/biosynthesis , Animals , Cattle , Corticosterone/biosynthesis , Enzyme Activation/drug effects , Glycolipids/chemistry , Glycolipids/metabolism , Glycosylphosphatidylinositol Diacylglycerol-Lyase , Hydrocortisone/biosynthesis , Inositol Phosphates/chemistry , Inositol Phosphates/metabolism , Male , Phosphatidylinositol Diacylglycerol-Lyase , Polysaccharides , Rats , Rats, Sprague-Dawley , Trypanosoma cruzi/chemistry
12.
Mol Biochem Parasitol ; 97(1-2): 123-31, 1998 Nov 30.
Article in English | MEDLINE | ID: mdl-9879892

ABSTRACT

Both, culture-derived and metacyclic trypomastigotes of Trypanosoma cruzi shed a glycoprotein, the shed acute phase antigen, that is responsible for the trans-sialidase activity. In the present work the structure of the glycosylphosphatidylinositol membrane anchor of the trans-sialidase isolated from metacyclic forms was determined. Parasites were metabolically labelled with [9, 10(n)3H]-palmitic acid and the glycoprotein was purified by immunoprecipitation with a monoclonal antibody directed against the repetitive aminoacid sequence. Treatment of the glycoprotein with phosphatidylinositol phospholipase C (PI-PLC) from Bacillus thuringiensis rendered a lipid that comigrated in TLC with a standard of ceramide. No alkylglycerol was detected in contrast with the results previously obtained for the trans-sialidase isolated from culture-derived trypomastigotes where both lipids were found. Chemical and chromatographic analysis showed that the lipid moiety is palmitoyldihydrosphingosine with a minor amount of stearoyldihydrosphingosine. The glycan constituent of the glycosylphosphatidylinositol-anchor was analysed by nitrous acid deamination of the aqueous phase of the PI-PLC treatment, followed by reduction with NaBH4 and hydrolysis of the phosphodiester with aqueous hydrofluoric acid. A major oligosaccharide was obtained and enzymatic treatment with exoglycosidases and further chromatography in a high pH anion exchange system showed that the trimannosyl core backbone is substituted by an alpha-galactose. A comparison between the lipid constituent of the glycosylphosphatidylinositol anchor of several proteins and their spontaneous shedding by the action of an endogenous PI-PLC was made.


Subject(s)
Antigens, Protozoan/chemistry , Glycoproteins/chemistry , Glycosylphosphatidylinositols/chemistry , Neuraminidase/chemistry , Trypanosoma cruzi/chemistry , Animals , Antigens, Protozoan/drug effects , Antigens, Protozoan/isolation & purification , Ceramides/analysis , Chromatography, Ion Exchange/methods , Chromatography, Thin Layer/methods , Glycoproteins/drug effects , Glycoproteins/isolation & purification , Glycosylphosphatidylinositols/isolation & purification , Hydrogen-Ion Concentration , Life Cycle Stages/physiology , Neuraminidase/drug effects , Neuraminidase/isolation & purification , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Trypanosoma cruzi/growth & development , Type C Phospholipases/pharmacology
13.
Glycobiology ; 7(6): 731-5, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9376675

ABSTRACT

The trans-sialidase from the trypomastigote stage of Trypanosoma cruzi was metabolically labeled with [3H]-palmitic acid and purified by immunoprecipitation with a monoclonal antibody. The action of PI-PLC on the immunoprecipitate released a lipid that was analyzed by TLC. Lyso-1-O-hexadecylglycerol and N-palmitoyl-sphinganine were obtained in a 1:3 ratio. A comparison with the GPI anchors present in the different stages of T. cruzi was made.


Subject(s)
Glycosylphosphatidylinositols/metabolism , Membrane Lipids/metabolism , Neuraminidase/metabolism , Trypanosoma cruzi/enzymology , Animals , Chromatography, Thin Layer , Mice , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Substrate Specificity , Type C Phospholipases/metabolism
14.
Exp Parasitol ; 87(1): 8-19, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9287953

ABSTRACT

In trypanosomatids, little is known about the biosynthetic pathways involved in the metabolism of ethanolamine. In an attempt to clarify this point, an exhaustive analysis of the chloroform:methanol extract of T. cruzi trypomastigotes metabolically labeled with [14C]ethanolamine, in comparison with the lipids from [3H]palmitic acid-incorporated parasites, was performed. In both cases, phosphatidylethanolamine and lysophosphatidylethanolamine were detected, while phosphatidylcholine and lysophosphatidylcholine were only labeled with the fatty acid precursor. However, dimethylphosphatidylethanolamine was isolated from parasites labeled with the base precursor, indicating the ability of trypanosomes to methylate phosphatidylethanolamine to dimethylphosphatidylethanolamine. Fatty acids of the labeled phospholipids were analyzed by reverse-phase thin-layer chromatography and fluorography. Interestingly, phospholipids from the trypomastigote stage show palmitic acid (C16:0) and stearic acid (C18:0) as the only labeled components. The same saturated fatty acids were found free and as components of the radioactive triglycerides. No unsaturated fatty acids were detected, in accordance with the results obtained with inositolphospholipids. Conversely, when the fatty acids of phospholipids purified from nonlabeled parasites were analyzed by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry, C18:1 was also detected. A striking finding was the presence of a considerable amount of free lignoceric acid (C24:0). Also, the C24:0 fatty acid was identified in the triglyceride fraction and as a component of phosphatidylcholine. The limited capacity of trypomastigote forms to elongate fatty acids was determined. In contrast with the results reported for other noninfective forms of the parasite, the absence of unsaturated fatty acids due to a low activity of desaturases was observed.


Subject(s)
Phospholipids/isolation & purification , Trypanosoma cruzi/chemistry , Animals , Chromatography, Thin Layer , Fatty Acids/analysis , Gas Chromatography-Mass Spectrometry , Lipids/analysis , Lysophosphatidylcholines/analysis , Lysophosphatidylcholines/isolation & purification , Lysophospholipids/analysis , Lysophospholipids/isolation & purification , Phosphatidylcholines/analysis , Phosphatidylcholines/isolation & purification , Phosphatidylethanolamines/analysis , Phosphatidylethanolamines/isolation & purification , Phospholipids/analysis , Triglycerides/analysis
15.
Lipids ; 32(8): 907-11, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9270985

ABSTRACT

A simple method is presented to esterify 1-O-hexadecyl-rac-glycerol using lipases in different organic solvents. The following fatty acids were used: C14:0, C16:0, C18:0, C18:1, and C18:2. Monoesterification was achieved by using a limiting amount of fatty acid. Both the 1-O-hexadecyl-3-O-acylglycerol and the 2-O-acylglycerol were obtained in a total yield of 75% and a ratio 7:1 in dichloromethane after 3 d. Chromatographic data for the monoesters, useful for the identification of the natural products, are given (gas-liquid chromatography, thin-layer chromatography, reverse-phase thin-layer chromatography). The structure was confirmed by a chemical synthesis of 1-O-hexadecyl-2-O-hexadecanoylglycerol. The 3-O-glyceride was also formed by acyl migration, as the minor component. The monoesters were separated by column chromatography and characterized by 1H and 13C nuclear magnetic resonance spectra.


Subject(s)
Glycerides/chemical synthesis , Glyceryl Ethers/metabolism , Lipase/metabolism , Catalysis , Esterification , Fatty Acids/metabolism , Kinetics , Magnetic Resonance Spectroscopy , Molecular Structure , Pseudomonas/enzymology , Solvents , Temperature
17.
Carbohydr Res ; 305(2): 163-70, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9581272

ABSTRACT

Tin(IV) chloride-promoted condensation of benzyl 2-acetamido-3-O-benzoyl-2-deoxy-alpha-D-glucopyranoside (4) with penta-O-benzoyl-beta-D-galactopyranose (6) gave the derivative of beta-D-Galp-(1-->6)-alpha-D-GlcNAc 7 in 80% yield. This was glycosylated with penta-O-benzoyl-alpha, beta-D-galactofuranose (5), employing the same catalyst, to afford the protected benzyl per-O-benzoyl-beta-D-Galf(1-->4)[beta-D-Galp(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). beta-Glycosidic linkages were diastereoselectively formed. De-O-benzoylation of 10, followed by heterogeneous catalytic transfer hydrogenolysis of the benzyl group afforded the free trisaccharide beta-D-Galf(1-->4)[beta-D-Galp(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.


Subject(s)
Glycoproteins/chemistry , Protozoan Proteins/chemistry , Trisaccharides/chemical synthesis , Trypanosoma cruzi/chemistry , Animals , Carbohydrate Sequence , Molecular Sequence Data , Oxidation-Reduction , Sugar Alcohols/chemical synthesis
18.
Mol Biochem Parasitol ; 79(2): 143-51, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8855551

ABSTRACT

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.


Subject(s)
Ceramides/biosynthesis , Trypanosoma cruzi/metabolism , Variant Surface Glycoproteins, Trypanosoma/biosynthesis , Animals , Ceramides/isolation & purification , Chromatography, Thin Layer , Palmitic Acid/metabolism , Variant Surface Glycoproteins, Trypanosoma/isolation & purification
20.
Biochim Biophys Acta ; 1300(3): 233-9, 1996 May 20.
Article in English | MEDLINE | ID: mdl-8679689

ABSTRACT

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.


Subject(s)
Glycosylphosphatidylinositols/analysis , Phosphatidylinositols/analysis , Trypanosoma cruzi/chemistry , Animals , Ceramides/analysis , Ceramides/chemistry , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Diglycerides/analysis , Fatty Acids/analysis , Fatty Acids/chemistry , Glycosylphosphatidylinositols/chemistry , Inositol Phosphates/analysis , Molecular Structure , Palmitic Acid , Palmitic Acids/metabolism , Phosphatidylinositols/chemistry , Phosphatidylinositols/isolation & purification , Sphingosine/analysis
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