Zentrum fu¨r Hygiene und Medizinische Mikrobiologie, Philipps-Universita¨t Marburg, Marburg, Germany Mannose analogues 2-deoxy-D-glucose, 2-deoxy-2-fluoro-D-glucose and 2-amino-2-deoxy-D
Trang 1Inhibition of glycosyl-phosphatidylinositol biosynthesis in Plasmodium
Cristiana Santos de Macedo, Peter Gerold, Nicole Jung, Nahid Azzouz, Ju¨rgen Kimmel and Ralph T Schwarz
Med Zentrum fu¨r Hygiene und Medizinische Mikrobiologie, Philipps-Universita¨t Marburg, Marburg, Germany
Mannose analogues (2-deoxy-D-glucose,
2-deoxy-2-fluoro-D-glucose and 2-amino-2-deoxy-D-mannose) have been
used to study glycosylphosphatidylinositol (GPtdIns)
biosynthesis and GPtdIns protein anchoring in protozoal
and mammalian systems The effects of these analogues on
GPtdIns biosynthesis and GPtdIns-protein anchoring of the
human malaria parasite Plasmodium falciparum were
evaluated in this study At lower concentrations of
2-deoxy-D-glucose and 2-deoxy-2-fluoro-D glucose (0.2
and 0.1 mM, respectively), GPtdIns biosynthesis is inhibited
without significant effects on total protein biosynthesis At
higher concentrations of 2-deoxy-D-glucose and
2-deoxy-2-fluoro-D-glucose (1.5 and 0.8 mM, respectively), the
incorporation of [3H]glucosamine into glycolipids was
inhibited by 90%, and the attachment of GPtdIns anchor to
merozoite surface protein-1 (MSP-1) was prevented
However, at these concentrations, both sugar analogues
inhibit MSP-1 synthesis and total protein biosynthesis In
contrast to 2-deoxy-2-fluoro-D-glucose and
2-amino-2-deoxy-D-mannose (mannosamine), the formation of new
glycolipids was observed only in the presence of tritiated or nonradiolabelled 2-deoxy-D-glucose Mannosamine inhibits GPtdIns biosynthesis at a concentration of 5 mM, but neither
an accumulation of aberrant intermediates nor significant inhibition of total protein biosynthesis was observed in the presence of this analogue Furthermore, the [3 H]manno-samine-labelled glycolipid spectrum resembled the one described for [3H]glucosamine labelling Total hydrolysis of mannosamine labelled glycolipids showed that half of the tritiated mannosamine incorporated into glycolipids was converted to glucosamine This high rate of conversion led
us to suggest that no actual inhibition from GPtdIns biosynthesis is achieved with the treatment with manno-samine, which is different to what has been observed for mammalian cells and other parasitic protozoa
Keywords: glycosylphosphatidylinositol; Plasmodium falciparum;D-mannosamine; 2-deoxy-D-glucose; 2-deoxy-2-fluoro-D-glucose
Glycosylphosphatidylinositols (GPtdIns) represent a class
of glycolipids responsible for the anchoring of proteins
on the outer leaflet of the plasma membrane (reviewed in
[1 – 6]) GPtdIns from parasitic protozoa have been related to
the pathology of many parasitic diseases [5] The human
malaria parasite, Plasmodium falciparum, has been shown
to synthesize a spectrum of GPtdIns molecules [7], which
represent a class of malarial toxins [6] These toxins are
involved in activation of host cell macrophages, induction of
NO release and up-regulation of endothelial cell markers
[8 – 10] Major surface proteins of P falciparum merozoites
[for example, merozoite surface protein (MSP)-1 and -2] are GPtdIns-anchored [11] Biosynthesis of GPtdIns in Plas-modium has been established by characterizing the structures of putative biosynthesis intermediates synthesized
by parasite cultures [7,12] More detailed understanding of the biosynthesis pathway and function of GPtdIns came from the use of specific inhibitors of the GPtdIns biosynthesis A recently established fungi metabolite (YW3548) was shown to inhibit GPtdIns-biosynthesis in yeast and mammalian cells but not in parasitic protozoa [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 but not from HeLa cells [14,15] Therefore, C-2 substituted mannose analogues are the only inhibitors known to affect GPtdIns-synthesis and GPtdIns-anchoring of surface molecules in mammalian cells and protozoa (reviewed in [16]) 2-Amino-2-deoxy-D-mannose (mannosamine) has been used to study GPtdIns biosynthesis in a variety of cell-types and organisms In all systems investigated so far, mannosamine was able to inhibit GPtdIns biosynthesis In
T brucei, mannosamine inhibits the incorporation of ethanolamine into GPtdIns protein by being incorporated into GPtdIns-biosynthesis intermediates [17] This leads to the accumulation of a ManN-Man-GlcN-PtdIns inter-mediate, which could not be mannosylated at the C-2 position [18] In mammalian cells there are discrepant data
Correspondence to R T Schwarz, Med Zentrum fu¨r Hygiene und
Medizinische Mikrobiologie, Philipps-Universita¨t Marburg,
Robert-Koch-Strasse 17, 35037 Marburg, Germany.
Fax: 1 49 6421 2868 976, Tel.: 1 49 6421 2865149,
E-mail: schwarz@mailer.uni-marburg.de
(Received 22 June 2001, revised 26 September 2001, accepted
3 October 2001)
Abbreviations: GPtdIns, glycosylphosphatidylinositol; PtdIns,
phosphatidylinositol; Man, mannose; ManN, mannosamine; GlcN,
glucosamine; EtN, ethanolamine, 2dGlc, 2-deoxy- D -glucose,
Dol-P-Man, dolichol-phosphate-mannose; HPAEC, high pH anion exchange
chromatography; GPtdIns-PLC,
phospholipase C; GPtdIns-PLD,
glycosylphosphatidylinositol-phospholipase D; MSP-1, merozoite surface protein-1; MDCK,
Madin– Darby canine kidney; TLC, thin-layer chromatography.
Trang 2on the incorporation of mannosamine into GPtdIns
precursors [19,20] Using Madin – Darby canine kidney
(MDCK) cells, mannosamine was shown to be incorporated
into GPtdIns biosynthesis intermediates [19] whereas no
incorporation of mannosamine into GPtdIns was observed
using HeLa or lymphoma cells [20], despite of the inhibition
of GPtdIns biosynthesis with the accumulation of
Man2-GPtdIns These data suggested a direct inhibition
of the enzyme a1,2-mannosyltransferase by mannosamine
Nonetheless, mannosamine inhibits the labelling of
GPtdIns-anchored proteins by tritiated ethanolamine and
mannose, and the polarized distribution of
GPtdIns-anchored proteins in polarized epithelial cells [17] In
L mexicana, synthesis of glycosylated inositol
phospholi-pids was inhibited by incorporation of mannosamine,
whereas the formation of lipophosphoglycans was inhibited
without mannosamine incorporation [21]
Mannose analogues such as 2-deoxy-2-fluoro-D-glucose
and 2-deoxy-D-glucose have been shown to inhibit GPtdIns
biosynthesis (reviewed in [16,22,23]), as both of them
inhibit the formation of dolichol-phosphate-mannose
[24,25], the donor for the mannose residues in GPtdIns
biosynthesis [26] Incubation of mammalian cells with
2-deoxy-2-fluoro-D-glucose led to an inhibition of
GPtdIns-anchoring of alkaline phosphatase and accumulation of a
precursor protein having an uncleaved GPtdIns-attachment
peptide [27] 2-Deoxy-D-glucose has not been described to
inhibit GPtdIns biosynthesis until now
Inhibition of malaria parasite P falciparum
multipli-cation in culture has been described for mannose analogues
[28 – 31] However, it remains unclear if these effects were
due to an inhibition of GPtdIns synthesis and/or protein
biosynthesis, the inhibition of glucosamine uptake or other
effects of mannose analogues on the parasite It is known
that light microscopy is a very sensitive method to obtain
information about the viability of malaria parasites, and is
used routinely to check multiplication and development of
P falciparum cultures
For a more detailed understanding and to establish
mannose analogues as potential specific inhibitors of the
biosynthesis of GPtdIns, we tested the in vivo effects of the
C-2 substituted mannose analogues mannosamine,
2-deoxy-2-fluoro-D-glucose and 2-deoxy-D-glucose on the
biosyn-thesis of free and protein-bound GPtdIns in Plasmodium
falciparum in comparison to total protein biosynthesis
M A T E R I A L S A N D M E T H O D S
Materials
D-[2-3H]mannose, 2-deoxy-D-[1-3H]glucose,
GDP-[2-3H]mannose and [35S]methionine were purchased from
Amersham (Germany) D-[6-3H]Glucosamine
hydrochlo-ride was obtained from Hartmann (Germany)
D-[6 –3H]Mannosamine was from ARC-Biotrend
(Germany) Mannosamine was obtained from Sigma
(Germany) 2-Deoxy-D-glucose was from Serva (Germany)
and 2-deoxy-2-fluoro-D-glucose was from Calbiochem All
solvents used were of analytical or high-performance liquid
chromatography grade and were obtained from
Riedel-de-Haen (Germany) Thin-layer chromatography (TLC) plates
were from Merck (Germany)
Parasites
P falciparum strain FCBR was obtained from B Enders, Behring Co (Marburg, Germany) It was maintained as previously described [32] Development and multiplication
of plasmodial cultures was followed by microscopic evaluation of Giemsa-stained smears Parasite cultures were routinely checked for Mycoplasma contamination Inhibition of parasite multiplication was assessed as described [29]
Metabolic labelling of parasites
To test for the effects of mannose analogues on the incorporation of radioactive precursors, parasite cultures were preincubated with inhibitors for 2 h prior to the start of labelling Metabolic labelling of parasite cultures using tritiated glucosamine, mannose, mannosamine,
2-deoxy-D-glucose or [35S]methionine was performed as described previously [7,11] Incubations were performed for 3 h (glycolipids) or 8 h (glycoproteins) at 37 8C
Viability of parasites After 10 h of incubation, the viability of parasites was assessed by light microscopy of Giemsa-stained smears and measured by [35S]methionine incorporation into total proteins by liquid scintillation counting, after trichloroacetic acid precipitation of proteins on filter membranes [29]
Extraction and purification of lipids Glycolipids were extracted with chloroform/methanol/water (10 : 10 : 3, v/v/v) as described [32] The chloroform/ methanol/water-extracted glycolipids were dried in a Speed-vac concentrator (Savant Inc.), subjected to repeated ‘Folch’ washings, and finally, partitioned between water and water-saturated n-butanol Washed glycolipid extracts were analysed on silica gel 60 TLC plates using chloroform/ methanol/water (4 : 4 : 1, v/v/v) as the solvent system After chromatography, the plates were dried and scanned for radioactivity using a Berthold LB 2842 automatic TLC scanner or analysed by a BAS-1000 Bio-Imaging Analyser (Fuji Film)
Total hydrolysis of glycolipid extracts Glycolipid extracts labelled with tritiated glucosamine or mannosamine were hydrolysed with 4M HCl for 4 h at
100 8C After treatment samples were washed with methanol, resuspended in water, and filtered through a 0.2-mm filter Monosaccharides were analysed by high pH anion exchange chromatography (HPAEC) on a Dionex Basic Chromatography System (Dionex Corp.) using a CarboPac PA-1 column (4 mm 250 cm, Bio-LC, Dionex Co., Sunnyvale, CA, USA), and isocratic conditions (10 mM NaOH) Fractions of 0.3 mL were collected and subjected to liquid scintillation Elution positions of nonradioactive coinjected mannosamine and glucosamine standards were detected using a pulsed amperometric detector
Trang 3Analysis of parasite proteins
Incorporation of radioactivity into total parasite
proteins was estimated by liquid scintillation counting
after trichloroacetic acid precipitation of proteins on
filter membranes [29] Incorporation of radioactivity
into the MSP-1 was investigated after immunopurification
of the protein using the monoclonal antibody 111.4,
specifically raised against MSP-1 (kindly provided by
A A Holder, Division of Parasitology, National
Institute for Medical Research, Mill Hill, London, UK)
[11,33]
Preparation of GDP-[3H]Man Standards
Washed parasites (30 – 40 h post-invasion) were harvested
by saponin lysis [7] Parasite lysates were prepared
essentially as previously described [34] Briefly, about
5 109 parasites were hypotonically lysed and
homogen-ized by 20 strokes of a Dounce homogenizer An equal
volume of double isotonic strength buffer was added This
preparation was designated parasite lysate All experiments
involving parasite lysates were performed with freshly
prepared lysates For cell-free labelling about 5 108
parasite-equivalents, processed as parasite lysates or
membrane preparations, were supplemented with 5 mM
MnCl2, 1 mM Coenzyme A (CoA), 1 mM ATP and 2 mCi
GDP-[2-3H]mannose Incubations were performed for
45 – 90 min at 37 8C Glycolipids were processed as
described above
R E S U L T S
Mannose analogues effect GPtdIns biosynthesis in
P falciparum
To test the effects of mannose analogues on the GPtdIns
synthesis in Plasmodium, parasite cultures containing late
trophozoites (34 – 42 h post-infection) were pretreated with
various concentrations of the mannose analogues
manno-samine, 2-deoxy-2-fluoro-D-glucose and 2-deoxy-D-glucose
followed by metabolic labelling using tritiated glucosamine
in the presence of sugar analogues Glycolipids were
extracted by organic solvents and the radioactivity
present was determined by scintillation counting The
stage of the parasites developmental cycle used for
these experiments incorporates almost exclusively tritiated
glucosamine into GPtdIns [7,32] Therefore, radioactivity
found in glycolipid extracts of glucosamine labelled
parasites is indicative of GPtdIns synthesis The addition
of increasing amounts of 2-deoxy-D-glucose led to a
decrease in the incorporation of tritiated glucosamine
into GPtdIns (Fig 1A) The presence of 0.2 mM of
2-deoxy-D-glucose was sufficient to reduce the
radioactivity found in GPtdIns by 71% whereas 94%
inhibition was achieved by 1.5 mM of 2-deoxy-D-glucose
(Table 1) Having present 2-deoxy-2-fluoro-D-glucose or
mannosamine gave similar results (Table 1) The
incorpor-ation of glucosamine into GPtdIns was inhibited by 61%
using about 0.1 mM 2-deoxy-2-fluoro-D-glucose (Fig 1B
and Table 1) or mannosamine (Fig 1C and Table 1)
Complete inhibition ( 90%) of GPtdIns biosynthesis
using these two inhibitors was achieved using 0.8 m and
5 mM, respectively (Table 1) These data suggest an effective inhibition of malarial GPtdIns synthesis using concentrations of the inhibitors that have been described in other systems [16]
The incorporation of tritiated glucosamine into manno-sylated and nonmannomanno-sylated GPtdIns was inhibited using the three mannose analogues (Fig 1A – C) The presence of 2-deoxy-D-glucose led to the formation of new glycolipids, despite the presence of 2-deoxy-2-fluoro-D-glucose or mannosamine However, in the presence of 2-deoxy-2-fluoro-D-glucose, it was observed that the formation of late mannosylated intermediates (Man4-GlcN-PtdIns and Man3-GlcN-PtdIns) was specifically blocked In the case of mannosamine, neither aberrant GPtdIns biosynthetic inter-mediates nor inhibition of the synthesis of any specific intermediate were observed, in contrast to the findings of Naik et al [31] We found a dose-dependent inhibition of all species of GPtdIns, and neither mannosylated nor nonmannosylated intermediates accumulated in the pre-sence of mannosamine The abpre-sence of new mannosamine-containing GPtdIns and the inhibition of incorporation of tritiated glucosamine into GPtdIns may point to an inhibition of GPtdIns biosynthesis by a mechanism which is different from the ones observed in other systems, that is, the incorporation of mannosamine into the GPtdIns trimannosyl-core glycan and further accumu-lation of aberrant intermediates, or inhibition of mannosyltransferases
Fig 1 TLC analyses of P falciparum glycolipids synthesized in the presence of mannnose analogues Parasites were treated in vivo with different concentrations (mM) of 2-deoxy- D -glucose (A), 2-deoxy-2-fluoro- D -glucose (B) and mannosamine (C), then labelled with tritiated glucosamine and extracted with chloroform/methanol/water (10 : 10 : 3, v/v/v) The chloroform/methanol/water extracts were subjected to repeated ‘Folch’ partitions, dried and partitioned between water and water-saturated n-butanol Glycolipids recovered in the butanol phase were analysed on silica TLC plates using a chloroform/ methanol/water solvent system (4 : 4 : 1, v/v/v) Plates were then exposed on an imaging plate, which was analysed by a BAS-1000 Bio-Imaging Analyser (Fuji Film Co.) O, origin; F, front The structure of previously characterized glycolipids are indicated in (A) Uncharacter-ized glycolipids are indicated with (*) E, ethanolamine; M, mannose;
G, glucosamine; aPtdIns, acyl-phosphatidylinositol.
Trang 4Labelling of GPtdIns by tritiated mannose analogues
To check for the incorporation of mannose analogues into
malarial GPtdIns, parasite cultures were labelled with
equivalent amounts of tritiated mannosamine,
2-deoxy-D-glucose or mannose (as a control) Glycolipids were
extracted by organic solvents and analysed on TLC (Fig 2),
with GDP-[3H]Man labelled glycolipids as standards The
labelling efficiency differs between the radioactive
pre-cursors used Only 16% (^ 5%) and 34% (^ 7%) of the
radioactivity from tritiated 2-deoxy-D-glucose and
manno-samine (respectively) was incorporated into malarial
glycolipids, relative to the incorporation of tritiated
mannose Because of the lower labelling efficiency of
mannosamine and 2-deoxy-D-glucose, minor amounts
(about 1/10th of the applied quantity of 2-deoxy-D-glucose
and mannosamine) of mannose and glucosamine-labelled
glycolipids were used for comparison on the TLC analysis
Labelling with tritiated 2-deoxy-D-glucose showed that this
precursor is incorporated into three glycolipids (Fig 2)
These newly formed glycolipids were identified as GPtdIns
by their sensitivity towards GPtdIns-specific nitrous acid
deamination, GPtdIns-PLC and GPtdIns-PLD (not shown)
The spectrum of glycolipids labelled with tritiated
mannosamine resembles the one obtained for glucosamine
labelled parasites These glycolipids are GPtdIns as they
were sensitive towards GPtdIns-specific nitrous acid
deamination, GPtdIns-PLC and GPtdIns-PLD (data not
shown) Besides the mannosylated GPtdIns, tritiated
mannosamine also labelled the nonmannosylated GPtdIns
glucosamine-phosphatidylinositol (GlcN – PtdIns) and
glu-cosamine-acylphosphatidylinositol (GlcN-acyl – PtdIns)
(Fig 2) These data imply that mannosamine was
incorporated into GPtdIns instead of glucosamine and/or
that it was converted to glucosamine prior to incorporation
In order to determine the nature of the labelled sugar present
in GPtdIns, total glycolipids of parasites labelled with
tritiated mannosamine were subjected to total hydrolysis,
and monosaccharide composition was analysed by HPAEC
Radioactive profiles are shown in Fig 3 It was observed
that mannosamine-labelled glycolipids contained
approxi-mately half of the incorporated mannosamine converted to
Fig 2 TLC analyses of P falciparum glycolipids synthesized in the presence of tritiated mannose analogues Parasites were labelled in vivo for 3 h with the tritiated precursor indicated, in the presence or absence of mannose analogues Glycolipids were extracted and processed as described in Fig 1 The TLC plate was then exposed on
an imaging plate, which was analysed by a BAS-1000 Bio-Imaging Analyser (Fuji Film Co.) Control labellings with [3H]glucosamine and [3H]mannose were performed in parallel, and P falciparum GDP-[ 3 H]Man in vitro labelled glycolipids were run in the same plate The structure of previously characterized glycolipids are indicated O, origin, F, front 2dGlc, 2-deoxy- D -glucose; ManN, mannosamine; E, ethanolamine; M, mannose; G, glucosamine; aPtdIns, acyl-phospha-tidylinositol; Dol-P-Man, dolichol-phosphate-mannose Glycolipids synthesized in the presence of 2-deoxy- -glucose are indicated with (*).
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.
Inhibitor used m M
[ 3 H]GlcN-labelled protein-bound GPtdIns (%)
[ 35 S]Methionine-labelled total protein (%)
2-deoxy-2-fluoro- D -glucose 0 100 100
Trang 5glucosamine, whereas parallel controls with
glucosamine-labelled total glycolipids showed a single peak
correspond-ing to a nonradioactive coinjected glucosamine standard
The effect of mannose analogues on GPtdIns-anchor and
protein synthesis
Inhibition of GPtdIns-anchor precursor synthesis will affect
GPtdIns-attachment to parasite proteins To investigate
specific inhibition of GPtdIns-anchor synthesis by mannose
analogues, addition of GPtdIns to proteins and protein synthesis rates were investigated by labelling parasites with [3H]glucosamine or [35S]methionine in the presence
of different concentrations of inhibitors Proteins were
Fig 4 Effects of mannose analogues on GPtdIns anchored protein MSP-1 biosynthesis and anchoring Parasite proteins and GPtdIns anchors were labelled with [35S]methionine and [3H]glucosamine, respectively [11] Incorporation of radioactivity into MSP-1 and MSP-1 anchor were measured after immunoprecipitation of the protein with the monoclonal antibody 111.4 (A) 2-deoxy- D -glucose, (B) 2-deoxy-2-fluoro- D -glucose and (C) mannosamine treated parasites.
Fig 3 Dionex-HPAEC analysis of monosaccharides generated
from P falciparum glycolipids labelled with [ 3 H]mannosamine
and [3H]glucosamine Parasites were labelled in vivo for 3 h with
[3H]mannosamine and [3H]glucosamine Glycolipids were extracted
with chloroform/methanol/water (10 : 10 : 3, v/v/v) The chloroform/
methanol/water extracts were subjected to repeated ‘Folch’ partitions,
dried and partitioned between water and water-saturated n-butanol.
Glycolipids recovered in the butanol phase were submitted to total
hydrolysis (4 M HCl at 100 8C for 4 h), desalted with methanol,
resuspended in water and filtered through a 0.2-mm filter
Mono-saccharides were analysed by Dionex-HPAEC at 10 m M NaOH (A)
mixture of mannosamine and glucosamine radioactive standards; (B)
[3H]mannosamine labelled glycolipids; (C) [3H]glucosamine labelled
glycolipids The elution positions of coinjected nonradiolabelled
mannosamine and glucosamine standards are indicated on the top of A,
B and C.
Trang 6precipitated by trichloroacetic acid and washed with ethanol
prior to determine the incorporation of radioactive
precursors into proteins Addition of 0.2 mM
2-deoxy-D-glucose, 0.1 mM 2-deoxy-2-fluoro-D-glucose or 0.5 mM
mannosamine led to a decrease of the synthesis of
protein-bound GPtdIns anchors (determined by [3H]glucosamine
incorporation) by 71.2, 61.1 or 73.3%, respectively
(Table 1) Whole protein biosynthesis (determined by
[35S]methionine incorporation) was reduced by 7.3% using
2-deoxy-D-glucose whereas 2-deoxy-2-fluoro-D-glucose
and mannosamine did not affect protein synthesis at these
concentrations (Table 1) Higher concentrations of the
inhibitors led to more pronounced inhibitory effects on
GPtdIns-anchoring (Table 1) However, bulk protein
syn-thesis was significantly affected and reduced to less than
10% in the presence of 1.5 mM 2-deoxy-D-glucose or
0.8 mM 2-deoxy-2-fluoro-D-glucose (Table 1) Therefore,
higher concentrations of 2-deoxy-D-glucose and
2-deoxy-2-fluoro-D-glucose did not only affect the synthesis of
GPtdIns-anchors bound to proteins but also nonspecifically
decreased bulk parasite protein synthesis measured by
[35S]methionine incorporation In contrast, even the
presence of 5 mM mannosamine led only to a marginal
reduction in the incorporation of [35S]methionine into
whole parasite proteins by 4.6% whereas the synthesis of
GPtdIns-anchors bound to proteins was reduced by 94%
(Table 1)
To understand more specifically the effects of mannose analogues on the synthesis of GPtdIns-anchored parasite proteins, their effects on the synthesis of a major GPtdIns anchored parasite protein (MSP-1) have been investigated Parasite cultures were labelled with [3H]glucosamine or [35S]methionine in the presence or absence of different concentrations of mannose analogues In the presence of low concentrations of 2-deoxy-D-glucose (0.2 mM), 2-deoxy-2-fluoro-D-glucose (0.1 mM) or mannosamine (0.5 mM), the synthesis of GPtdIns-anchored MSP-1 determined by incorporation of [3H]glucosamine decreased by 80%, 82%
or 81%, respectively (Fig 4) Using higher concentrations
of 2-deoxy-D-glucose (1.5 mM), 2-deoxy-2-fluoro-D -glu-cose (0.8 mM) or mannosamine (5 mM) lead to an even more pronounced inhibition of glucosamine incorporation
by 97, 98 or 98%, respectively The synthesis of the MSP-1 determined by incorporation of [35S]methionine into immunoprecipitated protein was reduced by 57 and 88% using 2-deoxy-D-glucose, 72 and 92% using 2-deoxy-2-fluoro-D-glucose, and 27 and 32% using mannosamine These data indicate that concentrations of 2-deoxy-D -glu-cose and 2-deoxy-2-fluoro-D-glucose necessary to block the attachment of a GPtdIns-anchor onto parasite proteins effectively also inhibit protein synthesis In contrast, high concentrations of mannosamine leading to an almost complete block of the GPtdIns-anchor attachment onto the MSP-1 only partly inhibit protein synthesis
Viability of parasites after treatment with mannose analogues
In order to check the viability of parasites after treatment with mannose analogues, incorporation of [35S]methionine into total parasite proteins as well as light microscopy, which is a very sensitive method to evaluate the development of P falciparum cultures When parasites were pretreated for 2 h with 0.2 mM2-deoxy-D-glucose and 0.1 mM 2-deoxy-2-fluoro-D-glucose and labelled for 8 h with [35S]methionine, no decreasing on the incorporation of this precursor into total proteins (in comparison with controls) was observed (Table 1) This indicates that cells were still viable after 10 h [comprising both previous treatment (2 h) and labelling (8 h) periods] Light microscopy of Giemsa-stained smears of parasites treated with 0.2 mM 2-deoxy-D-glucose and 0.1 mM 2-deoxy-2-fluoro-D-glucose (Fig 5) showed no significant morpho-logical changes between treated and nontreated parasites after 10 h of exposition to the analogues At higher concentrations of 2-deoxy-D-glucose (1.5 mM) and 2-deoxy-2-fluoro-D-glucose (0.8 mM), parasites showed a very low incorporation (less than 10%) of [35S]methionine into total proteins (Table 1), which clearly indicates the nonviability of the parasites after treatment and consequent cell death, also observed by optical evaluation of parasites (Fig 5), confirming the data presented on Table 1
In the case of mannosamine, parasites showed no significant decrease on total protein biosynthesis after the treatment, either at lower (0.5 mM) or higher (5 mM) concentrations of the analogue (Table 1) In accordance with this data, no significant morphological differences were observed with light microscopy either, in comparison to control parasites (Fig 5)
Fig 5 Light microscopy of parasites after 10 h of treatment with
mannose analogues After 10 h of incubation in the presence of
mannose analogues, parasites were visualized by light microscopy in
Giemsa-stained thin smears The concentration of analogues is shown at
the top-right corner of each panel.
Trang 7D I S C U S S I O N
GPtdIns biosynthesis has shown to be essential for growth
and development of yeast and parasite cells whereas
mammalian cells survive even if their GPtdIns-synthesis is
deficient (reviewed in [1 – 6]) Differences in the
biosyn-thesis of GPtdIns in mammalian cells and some well-studied
parasitic protozoa are described in the literature [13 – 15]
Therefore, interfering with the GPtdIns-biosynthesis of
parasitic protozoa provides a potential drug target [13 – 15]
Potential inhibitors described to block dolichol-phosphate
dependent mannosylation are mannose analogues such as
mannosamine [17 – 21], 2-deoxy-D-glucose [24] and
2-deoxy-2-fluoro-D-glucose [25] (reviewed in [22,23])
The biosynthesis of GPtdIns in T brucei has been shown
to be sensitive towards mannose analogues such as
2-deoxy-D-glucose and mannosamine (reviewed in [16]), as the
formation of dolichol-phosphate linked intermediates of
these sugars lead to their incorporation into the growing
GPtdIns-core glycan As the C-2 hydroxylgroup of these
mannose analogues is modified, GPtdIns core glycan chain
elongation is blocked at this position For the human
malaria parasite P falciparum, 2-deoxy-2-fluoro-D-glucose
and 2-deoxy-D-glucose have been described to kill
parasites in the culture with the IC50 of 0.65 mM and
5.0 mM, respectively, ([28,29] and C Santos de Macedo and
P Goold, unpublished observations)
The labelling with 2-deoxy-D-glucose leads to the
formation of Dol-P-2dGlc [22], which inhibits the formation
of Dol-P-Man Probably 2-deoxy-D-glucose is incorporated
into the GPtdIns instead of mannose This leads to the
synthesis of three major 2-deoxy-D-glucose containing
GPtdIns These glycolipids are slightly more hydrophobic
than GPtdIns precursors EtN-Man4-GlcN-acyl-PtdIns,
EtN-Man3-GlcN-acyl-PI and Man2-GlcN-acyl-PI, respectively
The more hydrophobic character of 2-deoxy-D-glucose
would explain the hydrophobic TLC mobility of these
glycolipids When parasites are treated with
2-deoxy-D-glucose and labelled with glucosamine, it is observed that
increasing concentrations of 2-deoxy-D-glucose do not lead
to a further accumulation in these three 2-deoxy-D-glucose
containing GPtdIns but resulted in the down-regulation of
the synthesis of all GPtdIns (including the mannosylated and
the nonmannosylated ones) These data imply that higher
concentrations of 2-deoxy-D-glucose affect not only
GPtdIns mannosylation but also lead to more general
effects on parasite glycosylation Low concentrations of
2-deoxy-D-glucose were able to inhibit the synthesis of
GPtdIns-anchors attached to proteins significantly without
affecting bulk protein synthesis, thus without affecting
parasite viability, as shown by light microscopy In contrast,
the formation of the GPtdIns-anchored MSP-1 was inhibited
significantly, probably because the inhibition of GPtdIns
synthesis would increase the number of non-GPtdIns
anchored MSP-1, which might not be stable and would be
readily degraded
Concerning the treatment with 2-deoxy-2-fluoro-D
-glu-cose, a similar set of results was found for the inhibition of
GPtdIns synthesis in P falciparum Although
2-deoxy-2-fluoro-D-glucose was not incorporated into GPtdIns, it
inhibited the formation of GPtdIns probably because of
the synthesis of GDP-2-deoxy-2-fluoro-D-glucose and
consequent reduction of endogenous Dol-P-Man levels
[22] The inhibition of GPtdIns-anchor synthesis by 0.1 mM 2-deoxy-2-fluoro-D-glucose without affecting bulk protein synthesis showed that the inhibitory effect is specific for GPtdIns biosynthesis This is in agreement with the finding that the synthesis of the GPtdIns-anchored MSP-1 is inhibited, as this inhibition is probably due to lack of GPtdIns-anchor attachment, resulting in a reduced stability
of this parasite protein In contrast, the presence of higher concentrations of this inhibitor during labelling led to a reduction of bulk protein and MSP-1 synthesis by more than 90%, and leading to parasite death, confirmed by light microscopy These data point to an unspecific inhibition of parasite metabolism in the presence of higher concentrations of 2-deoxy-2-fluoro-D-glucose Therefore, 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D-glucose showed
a specific effect on GPtdIns biosynthesis at low concen-trations At higher concentrations, these inhibitors were seen
to to strongly affect total protein biosynthesis, leading to cell death, as seen under light microscopy
Our results did not suggest that mannosamine would block GPtdIns biosynthesis by being incorporated into the GPtdIns trimmanosyl-core glycan and acting as chain terminator This is different from the findings in T brucei [18] and in mammalian cells [19], where the formation of ManN-Man-GlcN-PI was observed They are also different from the recent findings of Naik et al [31], where it was suggested that mannosamine inhibits P falciparum GPtdIns biosynthesis, preventing the attachment of the first mannose
to GlcN-PtdIns, leading to the accumulation of the latter Our data showed that the spectrum of mannosamine-labelled glycolipids resembled very much the spectrum of glucosamine-labelled glycolipids, without the accumulation
of any GPtdIns intermediate Total hydrolysis of manno-samine-labelled glycolipids showed that in P falciparum mannosamine is converted to glucosamine (as already described for T brucei and L mexicana [18,21]), which would explain the same spectrum of glycolipids Further-more, this finding explains the absence of detectable levels
of glucosamine-labelled glycolipids in the presence of high levels of nonradioactive mannosamine, as well as the lack of inhibition of protein biosynthesis and of parasite multipli-cation (C Santos de Macedo, unpublished observations) Light microscopy showed no morphological difference between mannosamine treated and nontreated parasites Therefore, in contrast to the findings in other systems, mannosamine seems to have no effect on P falciparum GPtdIns biosynthesis
These findings lead us to suggest that P falciparum synthesizes a large excess of GPtdIns It seems that this parasite possesses different mechanisms for GPtdIns biosynthesis than mammalian and other parasitic systems, which would indicate P falciparum GPtdIns biosynthetic pathway as a potential target for new therapies
A C K N O W L E D G E M E N T S
This work was supported by the Deutsche Forschungsgemeinschaft, Hessisches Ministerium fu¨r Kultur und Wissenschaft, Stiftung P.E Kempkes, the Human Frontier Science Program and Fonds der Chemischen Industrie C S de M receives a fellowship from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brası´lia, Brazil The authors thank Prof Dr Volker Kretschmer, the Blood Bank
of University of Marburg for providing human erythrocytes.
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