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De Macedo Et Al 2001
De Macedo Et Al 2001
De Macedo Et Al 2001
Mannose analogues (2-deoxy-D -glucose, 2-deoxy-2-fluoro- glycolipids was observed only in the presence of tritiated or
D -glucose and 2-amino-2-deoxy-D -mannose) have been nonradiolabelled 2-deoxy-D -glucose. Mannosamine inhibits
used to study glycosylphosphatidylinositol (GPtdIns) GPtdIns biosynthesis at a concentration of 5 mM , but neither
biosynthesis and GPtdIns protein anchoring in protozoal an accumulation of aberrant intermediates nor significant
and mammalian systems. The effects of these analogues on inhibition of total protein biosynthesis was observed in
GPtdIns biosynthesis and GPtdIns-protein anchoring of the the presence of this analogue. Furthermore, the [3H]manno-
human malaria parasite Plasmodium falciparum were samine-labelled glycolipid spectrum resembled the one
evaluated in this study. At lower concentrations of described for [3H]glucosamine labelling. Total hydrolysis of
2-deoxy-D -glucose and 2-deoxy-2-fluoro-D glucose (0.2 mannosamine labelled glycolipids showed that half of the
and 0.1 mM , respectively), GPtdIns biosynthesis is inhibited tritiated mannosamine incorporated into glycolipids was
without significant effects on total protein biosynthesis. At converted to glucosamine. This high rate of conversion led
higher concentrations of 2-deoxy-D -glucose and 2-deoxy- us to suggest that no actual inhibition from GPtdIns
2-fluoro-D -glucose (1.5 and 0.8 mM , respectively), the biosynthesis is achieved with the treatment with manno-
incorporation of [3H]glucosamine into glycolipids was samine, which is different to what has been observed for
inhibited by 90%, and the attachment of GPtdIns anchor to mammalian cells and other parasitic protozoa.
merozoite surface protein-1 (MSP-1) was prevented.
However, at these concentrations, both sugar analogues Keywords: glycosylphosphatidylinositol; Plasmodium
inhibit MSP-1 synthesis and total protein biosynthesis. In falciparum; D -mannosamine; 2-deoxy-D -glucose; 2-deoxy-
contrast to 2-deoxy-2-fluoro-D -glucose and 2-amino- 2-fluoro-D -glucose.
2-deoxy-D -mannose (mannosamine), the formation of new
Glycosylphosphatidylinositols (GPtdIns) represent a class [for example, merozoite surface protein (MSP)-1 and -2] are
of glycolipids responsible for the anchoring of proteins GPtdIns-anchored [11]. Biosynthesis of GPtdIns in Plas-
on the outer leaflet of the plasma membrane (reviewed in modium has been established by characterizing the
[1–6]). GPtdIns from parasitic protozoa have been related to structures of putative biosynthesis intermediates synthesized
the pathology of many parasitic diseases [5]. The human by parasite cultures [7,12]. More detailed understanding of
malaria parasite, Plasmodium falciparum, has been shown the biosynthesis pathway and function of GPtdIns came
to synthesize a spectrum of GPtdIns molecules [7], which from the use of specific inhibitors of the GPtdIns
represent a class of malarial toxins [6]. These toxins are biosynthesis. A recently established fungi metabolite
involved in activation of host cell macrophages, induction of (YW3548) was shown to inhibit GPtdIns-biosynthesis in
NO release and up-regulation of endothelial cell markers yeast and mammalian cells but not in parasitic protozoa
[8–10]. Major surface proteins of P. falciparum merozoites [13]. Structural analogues of GPtdIns having modified
hydroxylgroups at the inositol were shown to inhibit
selectively GPtdIns-biosynthesis in cell-free systems pre-
pared from Trypanosoma brucei and Leishmania mexicana
Correspondence to R. T. Schwarz, Med. Zentrum für Hygiene und
but not from HeLa cells [14,15]. Therefore, C-2 substituted
Medizinische Mikrobiologie, Philipps-Universität Marburg,
Robert-Koch-Strasse 17, 35037 Marburg, Germany.
mannose analogues are the only inhibitors known to
Fax: 1 49 6421 2868 976, Tel.: 1 49 6421 2865149,
affect GPtdIns-synthesis and GPtdIns-anchoring of surface
E-mail: schwarz@mailer.uni-marburg.de
molecules in mammalian cells and protozoa (reviewed in
Abbreviations: GPtdIns, glycosylphosphatidylinositol; PtdIns, [16]). 2-Amino-2-deoxy-D -mannose (mannosamine) has
phosphatidylinositol; Man, mannose; ManN, mannosamine; GlcN, been used to study GPtdIns biosynthesis in a variety of
glucosamine; EtN, ethanolamine, 2dGlc, 2-deoxy-D -glucose, Dol- cell-types and organisms. In all systems investigated so far,
P-Man, dolichol-phosphate-mannose; HPAEC, high pH anion exchange mannosamine was able to inhibit GPtdIns biosynthesis. In
chromatography; GPtdIns-PLC, glycosylphosphatidylinositol- T. brucei, mannosamine inhibits the incorporation of
phospholipase C; GPtdIns-PLD, glycosylphosphatidylinositol- ethanolamine into GPtdIns protein by being incorporated
phospholipase D; MSP-1, merozoite surface protein-1; MDCK, into GPtdIns-biosynthesis intermediates [17]. This leads to
Madin–Darby canine kidney; TLC, thin-layer chromatography. the accumulation of a ManN-Man-GlcN-PtdIns inter-
(Received 22 June 2001, revised 26 September 2001, accepted mediate, which could not be mannosylated at the C-2
3 October 2001) position [18]. In mammalian cells there are discrepant data
14321033, 2001, 23, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1046/j.0014-2956.2001.02571.x by Univ of Sao Paulo - Brazil, Wiley Online Library on [26/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
6222 C. Santos de Macedo et al. (Eur. J. Biochem. 268) q FEBS 2001
M AT E R I A L S A N D M E T H O D S
Total hydrolysis of glycolipid extracts
Materials
Glycolipid extracts labelled with tritiated glucosamine or
3
D -[2- H]mannose, 2-deoxy- D -[1-3 H]glucose, GDP- mannosamine were hydrolysed with 4 M HCl for 4 h at
[2- H]mannose and [35S]methionine were purchased from
3
100 8C. After treatment samples were washed with
Amersham (Germany). D -[6-3H]Glucosamine hydrochlo- methanol, resuspended in water, and filtered through a
ride was obtained from Hartmann (Germany). 0.2-mm filter. Monosaccharides were analysed by high pH
3
D -[6 – H]Mannosamine was from ARC-Biotrend anion exchange chromatography (HPAEC) on a Dionex
(Germany). Mannosamine was obtained from Sigma Basic Chromatography System (Dionex Corp.) using a
(Germany). 2-Deoxy-D -glucose was from Serva (Germany) CarboPac PA-1 column (4 mm 250 cm, Bio-LC, Dionex
and 2-deoxy-2-fluoro-D -glucose was from Calbiochem. All Co., Sunnyvale, CA, USA), and isocratic conditions (10 mM
solvents used were of analytical or high-performance liquid NaOH). Fractions of 0.3 mL were collected and subjected to
chromatography grade and were obtained from Riedel-de- liquid scintillation. Elution positions of nonradioactive
Haen (Germany). Thin-layer chromatography (TLC) plates coinjected mannosamine and glucosamine standards were
were from Merck (Germany). detected using a pulsed amperometric detector.
14321033, 2001, 23, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1046/j.0014-2956.2001.02571.x by Univ of Sao Paulo - Brazil, Wiley Online Library on [26/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
q FEBS 2001 P. falciparum GPtdIns glycosylation inhibition (Eur. J. Biochem. 268) 6223
Table 1. Effects of mannose analogues on protein-bound GPtdIns and total protein biosynthesis. P. falciparum proteins and protein-bound
anchors were labelled in vivo in the presence of the inhibitors with [35S]methionine and [3H]glucosamine, respectively. Incorporation of radioactivity
into proteins and protein-bound anchors were measured by scintillation counting after trichloroacetic acid precipitation of proteins on filter
membranes. [35S]Methionine incorporation into total proteins was also used to assess parasite viability.
[3H]GlcN-labelled [35S]Methionine-labelled
Inhibitor used mM protein-bound GPtdIns (%) total protein (%)
17. Lisanti, M.P., Field, M.C., Caras, I.W., Menon, A.K. & Rodriguez--
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