From the Department of Biochemistry, University of Melbourne, Parkville 3052, Victoria, Australia
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glycosylphosphatidylinositol (GPI) glycolipids are major cell surface constituents in the Leishmania parasites. Distinct classes of GPI are present as membrane anchors for several surface glycoproteins and an abundant lipophosphoglycan as well as being the major glycolipids (GIPLs) in the plasma membrane. In this study we have identified putative precursors for the protein and lipophosphoglycan anchors and delineated the complete pathway for GIPL biosynthesis in Leishmania mexicana promastigotes. Based on the structural analyses of these GPI intermediates and their kinetics of labeling in vivo and in cell-free systems, we provide evidence that the GIPLs are the products of an independent biosynthetic pathway rather than being excess precursors of the anchor pathways. First, we show that the similar glycan head groups of the GIPL and protein/lipophosphoglycan anchor precursors are assembled on two distinct pools of PI corresponding to 1-O-(C18:0)alkyl-2-stearoyl-PI and 1-O-(C24:0/C26:0)-2-stearoyl-PI, respectively. These PI species account for 20 and 1% of the total PI pool, respectively, indicating a remarkable specificity in their selection. Second, analysis of the flux of intermediates through these pathways in vivo and in a cell-free system suggests that the GIPL and anchor pathways are independently regulated. We also show that GIPL biosynthesis requires fatty acid remodeling, in which the sn-2 stearoyl chains are replaced with myristoyl or lauroyl chains. Fatty acid remodeling is dependent on CoA and ATP and occurs on pre-existing but not on de novo synthesized GIPLs. We suggest that the compartmentalization of different GPI pathways may be important in regulating the species and stage-specific expression of different GPI structures in these parasites.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GPI1 glycolipids are important membrane constituents in all eukaryotic cells. They anchor a variety of proteins to the outer leaflet of the plasma membrane and may also have a role in the intracellular trafficking, transmembrane signaling, and intercellular transport of these proteins (reviewed in Refs. 1-3). Non-protein-linked GPI glycolipids may also be abundant in the plasma membrane, although the function and biosynthetic relationship between these glycolipids and the anchor GPIs in higher eukaryotes is unclear (4-6).
GPI glycolipids are particularly abundant in lower eukaryotes such as yeast and the parasitic protozoa where they are commonly utilized as membrane anchors for the major surface proteins (2). In the trypanosomatid parasites, non-protein-linked GPIs can also be the major cellular glycolipids (2). This is the case for the sandfly-transmitted Leishmania parasites which cause a range of important diseases in humans. Leishmania parasites alternate between a flagellated promastigote stage that resides within the digestive tract of the sandfly vector and a non-motile amastigote stage that proliferates within the phagolysosome of macrophages in the mammalian host. The major macromolecule on the promastigote surface is a complex lipophosphoglycan (LPG), which contains a polydisperse phosphoglycan chain and a novel GPI anchor with the structure Gal3(Glc-1-PO4)Man2GlcN-lyso-PI (Fig. 1) (2, 7, 8). This anchor is distinct from the protein anchors of Leishmania and other eukaryotes, which have the core structure: ethanolamine-PO4-Man3GlcN-PI (Fig. 1) (2, 9). Interestingly, despite these differences in glycan structure, both the protein and LPG anchors contain a PI lipid moiety which is highly enriched for 1-O-alkylglycerols with very long (C24:0 or C26:0) alkyl chains (7-9). The LPGs are essential virulence factors which have been shown to mediate the attachment of promastigotes to the insect midgut and mammalian macrophages, protect the parasite surface in these hostile environments, and modulate signal transduction pathways involved in activating various microbicidal processes in the host macrophage (2, 7). Intriguingly, the surface expression of both the LPG- and GPI-anchored proteins is massively down-regulated in the intracellular amastigote stages of several species (10-12), suggesting that the surface expression of these macromolecules is either not required or not compatible for life in the macrophage phagolysosome.
|
Leishmania spp. also synthesize glycoinositol phospholipids (GIPLs), a family of low molecular weight GPI glycolipids which are not linked to either protein or polysaccharide. Unlike the LPG- and GPI-anchored proteins, the GIPLs are major surface constituents (107 copies/cell) on both the promastigote and amastigote stages (11, 13-15). Depending on the species and developmental stage, the glycan moieties of the GIPLs may be homologous to the protein anchors (type I GIPLs), the LPG anchors (type 2 GIPLs), or contain elements of both anchors (hybrid type) (2). Despite having similar glycan moieties to the protein or LPG anchors, the GIPLs characteristically have distinct lipid compositions which are often enriched with alkylacyl-PI containing shorter C18:0-alkyl chains (11, 13). Less is known about the function of the GIPLs, but as the major cell surface constituents of the amastigote stage they may have specific role(s) in protecting the parasite surface and/or modulating signal transduction pathways in the macrophage (14, 16, 17). However, structurally related GIPLs are also abundant in the plasma membranes of other trypanosomatid organisms which have quite different hosts (ranging from humans to insects and plants) (2, 18), suggesting that these glycolipids may have a more general function in regulating the physicochemical properties of cellular membranes.
The biosynthesis of protein anchor precursors has been extensively
studied in a number of protozoan parasites such as Trypanosoma brucei (19-21), Trypanosoma cruzi (22),
Plasmodium falciparum (23), and Toxoplasma gondii
(24). However, little is known about the biosynthesis of the
non-protein-linked GPI glycolipids in these organisims. In particular,
nothing is known about the relationship between the different GPI
anchor pathways in Leishmania and whether the GIPLs
represent excess intermediates from these pathways or whether they are
the products of a separate pathway. Two recent studies in
Leishmania major suggest that the early steps in GIPL/LPG
anchor biosynthesis are the same as those involved in protein anchor
biosynthesis, involving the transfer of GlcNAc to a PI precursor to
form GlcNAc-PI, followed by the de-N-acetylation of GlcNAc
and transfer of a mannose residue to form Man1-4GlcN-PI (25, 26).
Other intermediates, which are likely to be precursors of the LPG
anchor and type-2 GIPLs have also been partially characterized. However, because the mature products of these pathways have identical core structures, it has been difficult to determine whether the LPG
anchors and the GIPLs of this species are synthesized by the same or
two different pathways.
In the present study, we have investigated the biosynthesis of both the anchor and free GPIs in L. mexicana promastigotes. We have previously shown that the hybrid type GIPLs of this species have glycan and lipid moieties that are distinct from both the protein and LPG anchors (Fig. 1) (13, 27) making it possible to distinguish between intermediates from different pathways. We show that the distinct alkyl chain compositions of the L. mexicana anchor GPIs and the GIPLs are acquired by the selection of different pools of PI precursor species. In contrast, the short sn-2 acyl chains are incorporated into the GIPLs and protein anchors via highly specific fatty acid remodeling of later intermediates. Taken together, these data provide evidence that the anchor GPIs probably share a set of common early intermediates, whereas the GIPLs are the products of a completely separate pathway. The implications of these results for understanding the stage-specific regulation of GPI biosynthesis in Leishmania are discussed.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
Materials were obtained as follows: ATP and
coenzyme A were from Sigma. Jack bean -mannosidase and
Bacillus cereus phosphatidylinositol-specific phospholipase
C were from Boehringer Mannheim. [2-3H]Mannose (17 Ci/mmol), [6-3H]glucosamine (2 Ci/mmol),
[6-3H]glucose (3.1 Ci/mmol),
[9,10-3H]myristic acid (11.2 Ci/mmol),
[9,10-3H]palmitic acid (35.9 Ci/mmol),
[1-14C]stearic acid (55 mCi/mmol), and
GDP-[2-3H]Man (7 Ci/mmol) were from NEN Life Science
Products Inc. [14C]Octadecanol (52 mCi/mmol) was
from American Radiolabeled Chemicals and [3H]ethanolamine
(28 Ci/mmol) from Amersham.
Metabolic Labeling of L. mexicana Promastigotes-- Promastigotes of L. mexicana (strain MNYC/BZ/62/M379) were routinely cultivated at 27 °C in RPMI medium supplemented with 10% fetal bovine serum. For labeling with [3H]inositol or [3H]ethanolamine, mid-log growth phase promastigotes were resuspended in RPMI-fetal bovine serum medium (5 × 106 cell/ml) containing 10 µCi of [3H]inositol/ml or 20 µCi of [3H]ethanolamine and cultivated for 12-20 h (steady state labeling) at 27 °C before harvesting. For pulse-chase labeling of promastigotes with [3H]Man or [3H]GlcN, parasites were harvested by centrifugation and preincubated in RPMI containing 1% bovine serum albumin and 1 µl/ml tunicamycin for 30 min at 27 °C. Cells were washed with glucose-free RPMI containing 1% bovine serum albumin and 1 µl/ml tunicamycin and preincubated in the same medium for 10 min at 27 °C before addition of either [3H]Man (50 µCi/ml) or [3H]GlcN (20 µCi/ml). After 5 min, cells were rapidly harvested by brief centrifugation in a microcentrifuge (5,000 × g, 30 s) and resuspended at 108 cells/ml in RPMI medium containing 10% fetal bovine serum and 1 µg/ml tunicamycin. Aliquots (5 × 107 cell equivalents) were harvested by centrifugation (5,000 × g, 30 s) at defined time intervals and the GPI glycolipids extracted from the cell pellets as described below. For labeling with the [3H]- and [14C]fatty acids and [14C]octadecanol, promastigotes were harvested and washed with RPMI medium containing 2 mg/ml defatted bovine serum albumin. Fatty acids were added after 20 min preincubation in this medium, as an equimolar complex with defatted bovine serum albumin and aliquots (5 × 107 cell equivalents) of cell suspension removed at defined time intervals. In the pulse-chase experiment described in Fig. 5, promastigotes were labeled with [3H]myristic acid for 2 min, then resuspended in fresh medium containing 10% fetal calf serum and defatted bovine serum albumin (2 mg/ml) loaded with unlabeled myristic acid (1:1 molar complex). Aliquots were removed immediately and at the designated time points for processing.
Cell pellets were extracted twice in 300 µl of chloroform, methanol, water (1:2:0.8, v/v), with intermittent sonication for 2 h at 25 °C and insoluble material removed by centrifugation (16,000 × g, 5 min). The combined supernatants were dried under a stream of N2, and GPIs recovered by being partitioned in a biphasic system of 1-butanol (200 µl) and water (100 µl). After centrifugation (16,000 × g, 30 s), the lower aqueous phase was washed once with water-saturated 1-butanol (200 µl) and the combined organic phases back-extracted with water (150 µl) before being dried in a Speed-Vac (Savant) concentrator. GPI glycolipids recovered in the 1-butanol phase were resuspended in 40% 1-propanol for HPTLC analyses. In the experiment shown in Fig. 5, LPG was extracted from the delipidated pellet of [3H]Man-labeled promastigotes in 9% aqueous 1-butanol (2 × 300 µl) and purified on a small column (1 ml) of octyl-Sepharose (28). The dephosphorylated LPG anchor was obtained by mild acid hydrolysis of this extract in 40 mM trifluoroacetic acid (12 min, 100 °C), digestion of the hydrolysate with alkaline phosphatase (20 units/50 µl) in 0.5 M ammonium bicarbonate, pH 8.5 (18 h, 37 °C), and separation of the anchor from labeled LPG repeat units on a mini octyl-Sepharose column as described above (28).Radiolabeling of GPI Glycolipids in L. mexicana Cell-free Systems-- Promastigotes were preincubated in RPMI medium containing 10% fetal bovine serum and 1 µg/ml tunicamycin for 30 min at 27 °C, then hypotonically permeabilized in water containing 2 mM EGTA, 0.1 mM TLCK, 0.1 µM leupeptin, 1 mM dithiothreitol (108 cells/ml) for 10 min at 0 °C. Permeabilized cells were pelleted by centrifugation (16,000 × g, 5 min, 4 °C) and resuspended (109 cell equivalents/ml) in 50 mM Hepes-NaOH buffer, pH 7.4, containing 100 mM KCl, 2 mM EGTA, 1 mM ATP, 0.2 mM TLCK, 0.1 µM leupeptin, 0.5 mM dithiothreitol (buffer A). Labelings were initiated by adding 10 µl of GDP-[3H]Man (0.5 µCi) and UDP-GlcNAc (10 mM) to 50 µl of permeabilized cells. After 10 min, the label was chased by adding an equal volume of buffer A containing 1 mM UDP-GlcNAc and 1 mM GDP-Man. Aliquots (108 cell equivalents) were removed at the beginning and at the indicated time points during the chase for extraction of GPIs. In some experiments, the assay buffer contained CoA (0.2 mM) and myristic acid (0.1 mM).
For labeling experiments with [3H]myristic and [3H]palmitic acids, permeabilized cells were suspended in buffer A containing CoA (0.2 mM), GDP-Man (1 mM), UDP-GlcNAc (1 mM) and added to a microcentrifuge tube in which 100 µCi of fatty acid (suspended in ethanol) had been dried. The cell suspension was vortex mixed gently and incubated at 20 °C for 30 min before extraction of lipids and processing as described above.High Performance Thin Layer Chromatography-- Samples were applied to Silica Gel 60 aluminum-backed HPTLC plates (Merck). Unless otherwise stated all samples containing GPI glycolipids were analyzed on plates developed for 10 cm in chloroform, methanol, 1 M ammonium acetate, 13 M NH4OH, water (180:140:9:9:23, v/v) (solvent A). For analysis of the phosphorylated LPG anchor, plates were developed in chloroform, methanol, 0.2% KCl (10:10:3, v/v) followed by 1-butanol, pyridine, 0.25% KCl (9:6:4, v/v) in the same dimension (28). For analysis of glycans released by nitrous acid deamination, the HPTLC sheets were developed in 1-propanol, acetone, water (9:6:5, v/v) (solvent B) (29). HPTLC plates were sprayed with EN3HANCETM (DuPont) and exposed to either XAR-5 or BioMax (Kodak) film. To quantitate incorporation of radiolabel into individual species, HPTLC plates were either scanned before fluorography on a Berthold LB2821 automatic TLC linear analyzer, or the intensity of bands on preflashed film determined by densitometry using a Molecular Dynamics 300A densitometer. In experiments described in Fig. 5, the Man2GlcN-PI and Man3GlcN-PI complexes from each time point were scraped from the HPTLC sheet after fluorography and the glycans obtained by nitrous acid deamination/reduction reanalyzed and quantitated by HPTLC and Dionex HPAEC, respectively, to determine incorporation of [3H]Man into the various comigrating species. For preparative purification of GPIs and inositol lipids, 2-mm bands of silica were scraped and extracted with chloroform, methanol, water (1:2:0.8, v/v) with intermittent sonication and each fraction reanalyzed by HPTLC.
Chemical and Enzyme Treatments--
Digestion with
B. cereus PI-specific phospholipase C was
performed in 50 mM triethanolamine-HCl buffer, pH 7.5, 10 mM EDTA, 0.16% (w/v) sodium deoxycholate for 15 h at
37 °C. Jack bean -mannosidase (50 units/ml) digestions were
performed in 0.1 M NaOAc, pH 5.0, containing 0.2%
taurodeoxycholic acid for 15 h at 37 °C. Mild base hydrolysis
was performed in 0.1 M methanolic-NaOH (50 µl) for 2 h at 37 °C and samples neutralized with 1 M acetic acid. In each case the lipidic products of these treatments were recovered by
1-butanol, water biphase partitioning as described above.
Analysis of Glycan Head Groups-- The HPTLC-purified glycolipids or crude lipid extracts were dried in a Speed-Vac concentrator and resuspended with sonication in freshly prepared 0.5 M NaNO2 in 0.5 M NaOAc buffer, pH 4.0 (20 µl), and incubated for 1 h at 50 °C. Two further additions of NaNO2 in NaOAc buffer, pH 4.0 (15 µl), were made at 1-h intervals, before the mixture was extracted with 100 µl of water-saturated 1-butanol to remove uncleaved GPIs (less than 5% of total). The pH of the aqueous phase was adjusted to pH 10 with 5 M NaOH and the released glycans reduced by addition of NaBH4 (20 mg/ml) in 0.1 M NH4OH and incubation for 2 h at 25 °C. Excess NaBH4 was destroyed by addition of 1 M acetic acid and the samples desalted by passage down a column of AG50 × 12 (H+) and repeated (× 5) drying of the eluent with methanol. The deaminated/reduced glycans were analyzed by HPTLC (see above), or Dionex HPAEC. HPAEC was performed using a CarboPac PA1 column on a Dionex HPLC, equipped with a pulsed amperometric detector. The column was eluted with 0.15 M NaOH, 12.5 mM sodium acetate for 1 min, then with a linear gradient of sodium acetate (12.5-55 mM) in 0.15 M NaOH over 60 min at a flow rate of 0.6 ml/min (28). The elution time of labeled species was determined relative to a mixture of coinjected dextran oligomers (detected by PAD) and expressed as glucose units.
MALDI-TOF-MS--
HPTLC-purified GPIs and inositol lipids were
analyzed on a Kratos MALDI-IV in negative ion mode. Lipid and
glycolipid samples in 60% 1-propanol (0.5 µl of 1 mM
solution) were analyzed in 0.5 µl of freshly prepared
-cyano-4-hydroxycinnamic acid (25 mg/ml) in 60% 1-propanol.
Calibration was carried out with previously defined GIPL standards.
Compositional Analysis-- HPTLC-purified samples were subjected to solvolysis in 0.5 M methanolic-HCl (50 µl) for 12 h at 80 °C. Samples were neutralized with 10 µl of pyridine, dried under vacuum, and derivatized with pyridine, hexamethyldisilazane, trimethylchlorosilane (9:3:1, v/v) for 10 min at 25 °C. The fatty acid methyl esters and the trimethylsilyl-derivatives of the released sugars and 1-O-alkylglycerols were detected by GC-MS, as described previously (15) using either a HP1 (Hewlett Packard) or a SE-54 (Alltech) capillary column.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Lipid Moieties of the Different GPI Classes Are Distinct from Each Other and from the Total Cellular Pool of Free Inositol Phospholipids
Previous analyses have shown that the GPI anchors and GIPLs of
L. mexicana promastigotes contain alkylacyl-PI (or
lyso-alkyl-PI) with C18:0 (in the GIPLs) or C24:0 and C26:0 (in the LPG
and protein anchors) alkyl chains. The sn-2 fatty acyl
composition of the GIPLs and protein anchors has not been examined in
detail. To determine to what extent these distinctive PI moieties are
represented of the total PI pool, we have analyzed the molecular
species composition of promastigote PI and also re-examined the acyl
chain composition of the GIPLs. After steady state labeling with
[3H]inositol, PI accounted for 58% of the inositol
lipids (Fig. 2A, first lane).
Mild base hydrolysis of this fraction showed that approximately 80% of
the PI contained a base-sensitive diacylglycerol lipid, while 20%
comigrated with lyso-PI, indicating the presence of an
alkylacylglycerol lipid (Fig. 2A, second lane). Analysis of
HPTLC-purified PI by MALDI-TOF-MS produced one major pseudomolecular ion at m/z 864 and minor ions at m/z 836 and 850 (Fig. 2B). These ions corresponded to the
[M-1] pseudomolecular ion of distearoyl-PI,
stearoyl-palmitoyl-PI, and
1-O-(C18:0)alkyl-2-O-stearoyl-PI, respectively,
as judged by GC-MS compositional analysis (data not shown). The
presence of the latter species was confirmed by analysis of the
base-treated fraction, which generated a single [M-1]
pseudomolecular ion at m/z 584, the expected mass of
1-O-(C18:0)alkyl-2-lyso-PI (Fig. 2C).
Alkylacyl-PI molecular species with C24:0 and C26:0 alkyl chains
accounted for less than 5% of the plasmanyl-PI, as determined by
GC-MS, and were not detected by the less sensitive MALDI-TOF-MS
analyses.
|
In contrast, the [3H]inositol-labeled GIPL species, iM2,
iM3, and iM4, contained exclusively alkylacylglycerol lipids, as shown by their reduced HPTLC mobility after base treatment (Fig. 2A, second lane). GC-MS and MALDI-TOF-MS analyses confirmed the
presence of exclusively C18:0 alkyl chains in these glycolipids, but
also revealed the presence of a distinct sn-2 fatty acyl
composition. For example, the MALDI-TOF mass spectrum of iM2
(Man1-3Man
1-4GlcN-PI) contained one major [M-1]
pseudomolecular ion at m/z 1281 and a minor ion at 1252 (Fig. 3A). After base
treatment only a single pseudomolecular ion was detected at
m/z 1071 (Fig. 3B), which is the correct mass for Hex2HexN-1-O-(C18:0)alkyl-2-lyso-PI. These data
suggest that iM2 contains either
1-O-(C18:0)alkyl-2-myristoylglycerol (90%) or 1-O-(C18:0)alkyl-2-lauroylglycerol, which accounted for 95 and 5% of the molecular species, respectively, as judged by GC-MS compositional analysis. Identical lipid compositions were found in the
other two major GIPL species, iM3 and iM4 (Fig. 3, C and D). Thus the lipid moieties of the GIPLs differ from those
of the major alkylacyl-PI pool in having predominantly myristate instead of stearate. These data suggest that the GIPLs are either assembled on a very minor pool of alkylacyl-PI or that the predominant alkylacyl-PI molecular species are utilized but then subjected to fatty
acid remodeling.
|
Early Intermediates in GPI Biosynthesis Contain Unremodeled Alkylacyl-PI
We next identified early intermediates in these pathways to
determine at what stage the GIPLs and GPI anchors acquire their distinct alkyl/acyl chain compositions. All these glycolipids contain
the common core structure Man1-4GlcN
1-6-myoinositol and are
expected to be synthesized via the early intermediates, GlcNAc-PI,
GlcN-PI, and Man1GlcN-PI (1, 30, 31). Attempts to
metabolically label GlcNAc-PI and GlcN-PI with [3H]GlcN
were unsuccessful, as this monosaccharide is rapidly incorporated into
a variety of phospholipids, including PI and inositol phosphoceramide (IPC) which have a very similar HPTLC mobility to these intermediates (Fig. 4, lane 2, see also
labeling of PE and PC phospholipids). However, metabolic labeling with
[3H]Man specifically labeled a doublet
(1/M1VLAC and 2/M1) which had the expected HPTLC mobility
of Man1GlcN-PI (Fig. 4, lane 3; Fig.
5A). Nitrous acid deamination
and reduction of the upper and lower bands of this doublet released a
single glycan head group in each case which comigrated with
Man
1-4AHM on HPTLC (Fig. 6,
lanes 2 and 3). After base treatment these bands
had slower and distinct HPTLC mobilities (Fig.
7, lanes 1-4), suggesting that they contained alkylacyl-PI moieties and that the differences between the two M1 species resided in their alkyl chain composition. This was supported by GC-MS analysis of the HPTLC-purified bands, which
showed that the upper band in the doublet contained
1-O-alkylglycerols with C24:0 and C26:0 alkyl chains, while
the lower band contained predominantly 1-O-octadecylglycerol
(data not shown). When a fraction containing both bands was analyzed by
MALDI-TOF-MS, three major ions were evident at m/z 1174, 1258, and 1286 (Fig. 8A).
Reanalysis of this fraction after base treatment gave ions at
m/z 908, 992, and 1020, which are the expected
[M-1]
pseudomolecular ions for Hex·HexN·lyso-PI
with C18:0, C24:0, and C26:0 alkyl chains, respectively (Fig.
8B). These data show that early intermediates in the
pathways of GPI biosynthesis contain exclusively alkylacyl-PI molecular
species which are highly enriched for very long alkyl chains (VLAC).
They also show that the predominant sn-2-fatty acid in these
intermediates is stearoyl, indicating that later intermediates must
undergo fatty acid remodeling to achieve the sn-2-fatty acyl
composition of the mature GIPLs (see below).
|
|
|
|
|
Identification of Unremodeled GIPL Intermediates and Putative Anchor Precursors
Metabolic labeling of promastigotes with [3H]Man identified 13 PI-PLC-sensitive bands (Fig. 4, lanes 3 and 10), which corresponded either to the mature GIPL species (iM2, iM3, and iM4), or to unique intermediates in the pathways of GIPL, protein anchor, and LPG anchor biosynthesis. The [3H]Man-labeled species were HPTLC purified and further characterized to define the intermediates in these pathways and the steps involved in fatty acid remodeling.
Man2GlcN-PI Complex--
Bands 3/iM2VLAC
and 4/iM2' had a faster HPTLC mobility than mature iM2 and were
isolated as a mixture (Fig. 4, lane 3; Fig. 7, lane
5). HPTLC analysis of the deaminated/reduced glycan head groups
from this mixture indicated that both species had the same head group
as iM2 (i.e. Man1-3Man
1-4GlcN) (Fig. 6, lane
4), suggesting that they differed from mature iM2 in having more
hydrophobic lipid moieties. After mild base treatment, these bands
showed a similar shift to a slower HPTLC mobility, indicating that the differences in the lipid moieties resided primarily in their alkyl chain composition (Fig. 7, lanes 5 and 6).
Deacylated 4/iM2' comigrated with the lyso derivative of mature iM2
(Fig. 7, compare lanes 6 and 8), while deacylated
3/iM2VLAC had a significantly faster HPTLC mobility. It is
likely that 3/iM2VLAC corresponds to a iM2 species with
very long alkyl chains (presumably C24:0 and C26:0), while 4/iM2'
corresponds to a iM2 species with the same alkyl chain composition as
mature iM2 (i.e. C18:0) but with a more hydrophobic sn-2 fatty acid. Band 4/iM2' comigrated with a
[14C]stearate-labeled band (data not shown), suggesting
that the sn-2 fatty acyl chain may be stearate. These
results are consistent with 3/iM2VLAC being the first
committed precursor of the LPG anchor, and 4/iM2' being an unremodeled
iM2 species with a 1-octadecyl-2-stearoylglycerol lipid.
|
Man3GlcN-PI Complex--
Band 6/M3,iM3' had a slightly
faster HPTLC mobility than mature iM3 (Fig. 4 and Fig. 7, upper
band in lane 9) and contained Man3GlcN-PI
species from HPTLC analysis of the deaminated/reduced glycan head group
(Fig. 6, lane 6). Because linear and branched Man3AHM glycans are not resolved on HPTLC, the glycans
obtained from a more highly purified fraction of band 6/M3,iM3' were
further analyzed by Dionex HPAEC. Two glycans were resolved which
coeluted with the linear glycan, M3 (Man1-2Man
1-6Man
1-4AHM)
at 2.8 glucose units and the branched glycan, iM3
(Man
1-6[Man
1-3]Man
1-4AHM) at 3.2 glucose units (data not
shown), suggesting that band 6/M3,iM3' contained unbranched and
branched Man3GlcN-PI species in the ratio of 3:1. Both
Man3GlcN-PI species appeared to have the same lipid composition as they comigrated together with lyso-iM3 after base treatment (Fig. 7, lanes 9 and 10). Moreover,
only one GlcN-PI molecular species was generated by
-mannosidase
treatment of 6/M3,iM3' (Fig. 9, lane 7). The
-mannosidase-treated band comigrated with
GlcN-[1-O-(C18:0)-2-stearoyl]-PI derived from 2/M1 (Fig. 9, compare asterisked bands in lanes 3 and
7), suggesting that both the M3 and iM3 species in this band
have C18:0 alkyl chains and predominantly C18:0 fatty acids.
Man4GlcN-PI Complex-- Glycan head group analysis of a mixture containing bands 8/iM4' and 9/iM4 indicated that both these species corresponded to Man4GlcN-PI species. Band 8/iM4' migrated just above mature iM4, whereas 9/iM4 comigrated with iM4 on HPTLC (Fig. 4, lanes 1 and 4; Fig. 7, lane 11). However, both bands comigrated with lyso-iM4 after base treatment indicating that they probably contain a C18:0 alkyl chain (Fig. 7, lane 12). Band 8/iM4' comigrated with a band that was labeled with [14C]stearic acid (data not shown) and probably corresponds to an unremodeled iM4 species with 1-O-(18:0)alkyl-2-stearoylglycerol, while band 9/iM4 (which was prominantly labeled with [3H]inositol, [14C]octadecanol, and [3H]myristic acid (Fig. 4, lanes 1, 4, and 5)) corresponds to mature iM4 with 2-O-(C18:0)alkyl-2-myristoylglycerol. Glycan analysis of band 13/lyso-iM4 indicated the presence of a third Man4GlcN-PI species (Fig. 6, lane 9). This species comigrated with lyso-iM4, was base resistant (Fig. 7, lanes 15 and 16), and is likely to correspond to the deacylated intermediate in the fatty acid remodeling of iM4'.
Ethanolamine-containing GPIs--
Steady state labeling of
L. mexicana promastigotes with [3H]ethanoamine
identified three PI-PLC-sensitive bands (Fig. 4, lanes 7 and
14). Two of these [3H]ethanolamine-labeled
species comigrated with the ethanolamine-phosphate-modified forms of
iM3 (EPiM3, RF 0.1) and iM4 (EPiM4,
RF 0.06). Previous analyses have shown that these
species contain predominantly
1-O-(C18:0)alkyl-2-myristoylglycerol (13). The third GPI
species, band 10/EPM3VLAC (asterisked band Fig.
4, lane 7) was a minor band in the
[3H]ethanolamine profile (Fig. 4, lane 7,
RF 0.16), but was pulse-labeled with
[3H]Man more strongly than either EPiM3 or EPiM4 (Fig. 4,
lane 3), indicating that it has a faster turnover than the
ethanolamine-substituted GIPLs. Band 10/EPM3VLAC was
resistant to -mannosidase digestion (Fig. 9, lanes 8 and
9) and had a slower HPTLC mobility after mild base (data not
shown), indicating that the ethanolamine may cap the terminal mannose
residue and that this species has an alkylacyl-PI lipid. Although the
glycan and lipid head group of this species was not further
characterized, its faster HPTLC mobility relative to EPiM3 (both as
native and deacylated species) is consistent with it having the
structure; ethanolamine-PO4-Man3GlcN-PI with very long alkyl chains. This minor species is a putative precursor for
the protein anchors.
Pulse-Chase [3H]Man Labeling of GPI Intermediates
Precursor-product relationships between the various GPI
intermediates were further investigated with pulse-chase labeling experiments. Incorporation of [3H]Man into each species
was determined from the analysis of the labeled lipids as well as
glycan head group analysis of individual HPTLC bands that contained
more than one GPI species (see "Experimental Procedures").
[3H]Man was incorporated into the mature GIPL species,
5/iM2, 7/iM3, 9/iM4, and 12/EPiM3 (after 40 min lag) throughout the
chase, consistent with these species being metabolic end-products (Fig.
5, A and C). In contrast, all the other
intermediates were depleted after the 4-h chase. The rapid labeling and
chase kinetics of M1, M2, and M3 strongly suggest that these species
form the biosynthetic series M1 M2
M3 (Fig. 5B). The
cumulative label in these species was similar to that found in the
mature GIPLs at the end of the chase, consistent with these species
being unremodeled intermediates in the GIPL pathway. A
precursor-product relationship between M1, M2, and M3 and iM2', iM3',
and iM4', respectively, was suggested by the slower kinetics of
labeling of the latter relative to the former species (Fig.
5D). Labeling of lyso-iM3 and lyso-iM4 occurred with
essentially the same kinetics as the unremodeled acylated species,
although in some cases maximum labeling of the lyso species occurred
just before its putative precursor. This was probably due to the
presence of low levels of labeled
Man5GlcNAc2-P-P-Dol and
Man6GlcNAc2-P-P-Dol in the early time points
which comigrate with lyso-iM3 and
lyso-iM4.2 The presence of
these species reflects a low level of dolichol-oligosaccharide synthesis in the presence of tunicamycin.
The labeling and chase kinetics of 1/M1VLAC were consistent with it being a precursor to 3/iM2VLAC and 10/EPM3VLAC which were maximally labeled after 40 min (Fig. 5E). Labeling of iM2VLAC preceeded that into the mature LPG anchor and was effectively chased over 2-3 h, while label in the LPG anchor increased over 2 h before leveling off (Fig. 5, Panel E), presumably due to the loss of newly synthesized LPG from the cell surface (25). A precursor-product relationship between 10/EPM3VLAC and the protein anchors was not determined from these analyses because of the difficulty of detecting significant and specific [3H]Man incorporation into the anchor moiety of newly synthesized proteins. Low levels of incorporation were not surprising given that GPI-anchored promastigote proteins are approximately 10-fold less abundant than the LPGs. Finally, the distinct differences in the turnover of the two Man1GlcN-PI bands, 1/M1VLAC and 2/M1, suggested that the relative flux of intermediates into the GIPLs is more than twice as fast as the flux of intermediates into combined anchor pathways (Fig. 5, B, E).
Fatty Acid Remodeling of Pre-existing Pools of GIPL and Anchor Intermediates in Vivo
The specificity of the fatty acid remodeling reactions was analyzed by continuous labeling of L. mexicana promastigotes with [3H]myristic, [3H]palmitic, or [14C]stearic acids. [3H]Myristic acid was rapidly incorporated into all the inositol lipids (PI, PIP, and IPC) as well as into the mature GIPLs (Fig. 10A). After 4 h labeling the majority of the label (>95%) was still present as myristate (data not shown). [3H]Myristic acid was also rapidly incorporated into 10/EPM3VLAC with similar kinetics to the GIPLs (Fig. 4, lanes 5 and 12; Fig. 10A). Importantly, the specific activity of the [3H]myristic acid-labeled GIPLs was comparable to that of the total PI pool at the earliest time point of 5 min (Fig. 10B), suggesting that the myristic acid was being incorporated directly into pre-existing GIPL pools rather than by the de novo synthesis of GIPLs from [3H]myristate-labeled PI. These kinetics were quite distinct from those found when promastigotes were metabolically labeled with [3H]inositol. Labeling of GIPLs with [3H]inositol was only detected after several hours, following strong labeling of the PI, PIP, and IPC pools (data not shown). Consequently, the GIPLs have a very low relative specific activity at early time points (Fig. 10B). The kinetics of incorporation of [3H]palmitic and [14C]stearic acids into the GIPLs were similar to those for [3H]inositol, suggesting that these fatty acids were also being incorporated via PI (Fig. 10B). Thus [3H]myristic acid is unusual in it being specifically incorporated into pre-existing GIPL pools, while fatty acids that are characteristic of unremodeled GIPL species are incorporated via 3H-labeled PI. Interestingly, the [3H]myristic acid-labeled GIPLs had a very slow turnover in pulse-chase experiments (Fig. 10C), suggesting that the myristoylated GIPLs are not extensively remodeled.
|
Fatty Acid Remodeling of GIPLs and Anchor Precursors in a Cell-free System
The fatty acid remodeling reactions were further investigated in a cell-free system. When permeabilized cells were incubated with CoA and either [3H]myristic or [3H]palmitic acids, only myristic acid was incorporated into the GIPLs (Fig. 11A). This labeling occurred in the absence of nucleotide sugar additives (GDP-Man or UDP-GlcNAc) suggesting that labeling was not dependent on continued GPI biosynthesis (Fig. 11A). Myristate labeling was dependent on the addition of ATP and was markedly stimulated by CoA (Fig. 11B), consistent with the major acyl donor being myristoyl-CoA. These data suggest that fatty acid remodeling of pre-existing pools of GIPL precursors can be reconstituted in vitro.
|
To investigate whether de novo synthesized (as distinct from pre-existing) GIPL pools were being remodeled in vitro, permeabilized cells were pulsed with GDP-[3H]Man and UDP-GlcNAc for 10 min, then chased in the presence of unlabeled GDP-Man, myristic acid, and CoA. As shown in Fig. 12, most of the GPI intermediates including the unremodeled and lyso-GIPL species were synthesized in the permeabilized cells. However, synthesis of fully remodeled GIPLs was very inefficient. As the remodeling enzymes are known to be active using these assay conditions (see above) it is possible that the de novo synthesized GIPLs must be translocated to a different compartment for myristoylation in a step that has not been reconstituted in vitro. Note that some of the label in the lyso species at early time points is due to the presence of variable amounts of comigrating dolichol-P-P-oligosaccharides (i.e. Man5-6GlcNAc2-P-P-dol).2
|
The putative anchor intermediates, 1/M1VLAC, 3/iM2VLAC, and 10/EPM3VLAC, were also labeled in these cell-free assays, although their turnover during the chase was very slow (Fig. 12, A and B). The slow turnover is most likely due to the depletion in suitable sugar donors (i.e. the galactofuranose donor for the next step in LPG anchor biosynthesis) or protein acceptors, which are required for further processing of these intermediates, respectively. In contrast, the labeling and turnover of the GIPL intermediates was comparable to that observed in vivo, suggesting that GIPL biosynthesis can be uncoupled from that of the protein and LPG anchor biosynthesis.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we have delineated the pathway for GIPL biosynthesis in L. mexicana promastigotes and identified several putative precursors for the protein and LPG anchors. We propose that the anchor pathways share a set of common early intermediates, whereas the GIPLs are the products of a separate pathway. We also show that intermediates in the GIPL, and possibly also the protein and LPG anchor pathways undergo highly specific fatty acid remodeling reactions that result in the incorporation of short chain fatty acids, primarily myristate into these glycolipids. These findings are incorporated into a new model for GPI biosynthesis in L. mexicana promastigotes which is summarized in Fig. 13, and described in more detail below.
|
The major GPI intermediates labeled in vivo and in the
cell-free system were intermediates in the pathway of GIPL biosynthesis based on the characterization of their glycan and lipid moieties and
their kinetics of labeling in the [3H]Man pulse-chase
experiments. The initial steps in this pathway appear to be the same as
for the assembly of the protein anchors except that all these
precursors contain a PI lipid moiety with C18:0 rather than C24:0 or
C26:0 alkyl chains which are characteristic of mature protein anchors
(Fig. 13). The earliest intermediates in this pathway, GlcNAc-PI and
GlcN-PI were not detected in the in vivo
[3H]GlcN labeling experiments because of conversion of
the label to comigrating PI and IPC lipids, but have been identified
after incubation of cell-free systems with
UDP-[3H]GlcNAc.2 These species are
mannosylated to form the unbranched GPI intermediates M1, M2, and M3
which are subsequently modified with a Man1-3 branch to form the
unremodeled GIPL species, iM2', iM3', and iM4' (Fig. 13). Although this
appears to be the main pathway for the assembly of the GIPL head
groups, it is possible that some of the Man
1-3 branched GIPLs may
be further extended on their
1-6 mannose arm (see ?
arrows in Fig. 13). The sn-2 fatty acid (predominantly stearate) of these unremodeled intermediates is removed to form the
corresponding lyso derivative which is reacylated with myristate (or
laurate) to form the mature GIPL species (Fig. 13). The remodeling reactions appear to be specific for short chain fatty acids as neither
[3H]palmitic nor [14C]stearic acids are
incorporated into the GIPLs, either in vivo or in
vitro. The remodeling reactions require both CoA and ATP, essential factors in acyl-CoA synthesis, suggesting that the fatty acids are transferred from myristoyl- or lauroyl-CoA. Interestingly, while [3H]myristic acid was specifically incorporated
into pre-existing GIPL pools in the cell-free systems, remodeling of
de novo synthesized GIPLs was very inefficient even in the
presence of CoA and fatty acids. This raises the possibility that a
transport step is required before newly synthesized GIPLs can be
accessed by the remodeling enzymes. Finally, some of the iM3 and iM4
species are elaborated with an ethanolamine-phosphate residue which is
attached to the
1-6-linked mannose residue (13, 14). From the
kinetics of in vivo labeling of EPiM3 it is likely that this
ethanolamine-phosphate is only added to fatty acid remodeled GIPL
species. Only a minor pool of GIPLs are modified with
ethanolamine-phosphate in L. mexicana promastigotes, while
the converse is true in the amastigote stage (14). This modification
appears to be unique to the GIPLs and may be mediated by a second
ethanolamine-phosphotransferase or in a compartment from which
GPI-anchored proteins are excluded.
A number of GPI intermediates were identified which are likely to be precursors to the protein and LPG anchors. These included (i) the Man1GlcN-PI molecular species with very long alkyl chains (M1VLAC), which may be precursors for both anchor pathways, (ii) iM2VLAC which is likely to be the first committed intermediate in the LPG anchor pathway; and (iii) EPM3VLAC which may represent the final precursor in the protein anchor pathway. These analyses allow several conclusions to be made regarding the anchor pathways. First, they strongly suggest that the distinctive alkyl chain composition of both the protein and LPG anchors is acquired by the selection of specific alkylacyl-PI molecular species rather than by alkyl chain remodeling later in the pathway. This is consistent with the finding that neither LPG nor the GIPLs are substrates for the L. donovani glyceryl ether monooxygenase that is capable of cleaving 1-O-alkylglycerols (32). The exclusive use of PIVLAC in the protein and LPG anchors indicates the presence of a highly efficient selection mechanism as these species account for less than 1% of the total PI pool. However, it should be noted that there are still 2 × 106 molecules of PIVLAC per cell as judged by GC-MS mass analysis. This is comparable to the pool size of all GPI-anchored macromolecules (~6 × 106 molecules/cell), suggesting that PIVLAC will not be limiting for protein and LPG anchor biosynthesis. Why very long alkyl chains are selectively incorporated into the protein and LPG anchors is unknown. However, it is a highly conserved feature and may be required to stabilize the association of GPI-anchored macromolecules with the plasma membrane (25) or to modulate transmembrane signal transduction pathways in the mammalian host (Ref. 33 and references therein).
Second, it is likely that the protein anchor precursors are fatty acid remodeled in the same way as the GIPLs. This is suggested by the rapid labeling of 10/EPM3VLAC with [3H]myristic acid and the finding that the protein anchors of L. major are highly enriched for myristic acid (9). Whether the same fatty acid remodeling reactions also generate the unusual lyso-alkyl-PI lipid moiety of the LPG anchors is unclear. This type of lipid could be generated if the fatty acid remodeling reactions did not proceed beyond removal of the sn-2-fatty acid. Even though the final LPG anchor precursors are synthesized in the Golgi apparatus (34), it is possible that earlier intermediates (such as iM2VLAC) are partially remodeled and that these lyso intermediates are selectively utilized as LPG anchors. Indeed a significant proportion of the steady state pool of LPG anchor precursors and type-2 GIPLs contain a lyso-alkyl-PI anchor (15, 25). Alternatively, a second Golgi-located phospholipase A2 activity that only acts on LPG anchor intermediates (or type-2 GIPLs) may be responsible for modifying the LPG anchor.
Third, it is of interest that iM2VLAC was the only dedicated intermediate in the LPG anchor pathway to be detected in short term in vivo labeling experiments. This species is probably made in the endoplasmic reticulum,3 whereas the next step in the pathway, involving the addition of a galactofuranose residue, occurs in the Golgi apparatus (34). The absence of detectable levels of more polar LPG anchor intermediates suggests that the transport of iM2VLAC from the endoplasmic reticulum to the Golgi apparatus may be a rate-limiting step in this pathway. However, we have previously shown that the more polar intermediates are relatively abundant in L. mexicana promastigotes (13). It is possible that these GPIs represent excess LPG anchor intermediates which have escaped the site of LPG phosphoglycan synthesis and been transported to the cell surface. Depending on their rate of accumulation these species may not be labeled significantly in the short term labeling experiments used in this study.
Fourth, in contrast to the situation in mammalian cells (30, 31), yeast (35, 36), and some protozoa (19, 21-23), none of the GPI anchor intermediates in Leishmania are palmitoylated on the inositol ring. In mammalian cells, inositol palmitoylation precedes and may be required for the subsequent mannosylation of GlcN-PI (37). In the African trypanosomes, inositol palmitoylation occurs on later intermediates, is reversible, and is thought to be involved in translocation of intermediates across the endoplasmic reticulum membrane (21). These data suggest that the early mannosyltransferases of Leishmania and the African trypanosomes have distinct substrate requirements from those in mammalian cells, but also point to possible differences in the topology of some of the GPI biosynthetic reactions in African trypanosomes and L. mexicana.
The presence of distinct PI lipid moieties in the GIPL and GPI anchor
precursors as well as the marked differences in the flux of
intermediates through these pathways provides strong evidence that the
GIPLs are the products of a separate pathway rather than being excess
precursors of the anchor pathways. The selective incorporation of PI
with C18:0 and C24:0/C26:0 alkyl chains into the GIPL and anchor
GPIs, respectively, is striking and may reflect differences in the
substrate specificity of the early enzymes in each pathway or the
subcellular compartmentalization of the GIPL and anchor pathways into
different membranes with distinct alkylacyl-PI compositions. At
present, there is no evidence that early enzymes, such as the
N-acetylglucosaminyl transferase show a strong selectivity
for specific PI molecular species in either mammalian (Ref. 30 and
references therein) or parasite systems. In this regard, we have
recently shown that diacyl-PI can be incorporated into some leishmanial
GPI classes in
vivo,4 demonstrating a
lack of specificity by early GPI enzymes for alkylacyl-PI. The
mannosyltransferases involved in the assembly of the glycan head groups
also lack a pronounced specificity for GPI molecular species with
different lipid compositions as synthetic GlcN-PI acceptors containing
short (C8) and long (C16:0) acyl chains are utilized by early
mannosyltransferases in L. major (26), African trypanosome
(38), and mammalian (37) cell-free systems. Moreover, as shown in this
study, GPI precursors with different alkyl chain compositions
(i.e. M1 and M1VLAC) can be used by enzymes such
as the 1-3- and
1-6-mannosyltransferases (Fig. 13). In
contrast, several lines of evidence support the notion that there may
be some degree of subcellular compartmentalization of the GIPL and GPI
anchor biosynthetic pathways. First, it is clear from this study that a
1-3 mannose branch can be added to GPI precursors with the same
backbone sequence as the protein anchors. However, this branch is
completely absent from the protein anchors (and conversely the
1-6-linked mannose is absent from the LPG anchor) suggesting that
distinct enzyme complexes are involved in the biosynthesis of the three
classes of leishmanial GPIs. Second, the marked differences in the flux
of intermediates through each pathway, notably the 2-fold higher flux
through the GIPL pathway compared with the combined anchor pathways may
reflect the localization of these enzyme complexes to membranes with
different concentrations of substrates such as PI and Dol-P-Man. Third, from the kinetics of labeling of GPI intermediates in the cell-free assays, the pathway of GIPL biosynthesis appears to be uncoupled from
those of protein and LPG anchor biosynthesis (Fig. 12). The independent
regulation of these pathways is also supported by studies on the
in vivo expression of these molecules in different developmental stages of the parasite. In particular, the transformation of promastigotes to intracellular amastigotes is associated with a
dramatic down-regulation in the surface expression of GPI-anchored proteins and LPG, whereas the GIPLs are synthesized in high copy number
in both developmental stages (10-12, 14). Collectively, these
observations support the notion that there are three distinct pathways
of GPI biosynthesis in L. mexicana promastigotes and that
the regulation of these pathways requires a degree of
compartmentalization of overlapping enzymes. It is possible that a
similar degree of compartmentalization of GPI biosynthetic enzymes in
other eukaryotes may explain why many parasite and mammalian GPI
protein anchors contain alkylacylglycerols which are relatively minor
components of the total PI pool (30, 39).
The requirement for fatty acid remodeling in the synthesis of the GIPLs and possibly also the protein and LPG anchors was an unexpected finding. However, it may be important in other Leishmania spp. as the structurally distinct type-2 GIPLs of L. major also contain predominantly C14:0 and C12:0 fatty acids (15). These fatty acid remodeling reactions are similar to those that occur in the African trypanosomes, and which were previously thought to be unique to these parasites. As in Leishmania, fatty acid remodeling of the trypanosome GPIs occurs mainly after assembly of the glycan head group and involves the exchange of the sn-2 fatty acid (predominantly stearate) with myristoyl chains that are transferred from a myristoyl-CoA donor (19, 40). However, in African trypanosomes the sn-1 fatty acid is also replaced with myristate to generate a dimyristoylglycerol lipid in the mature protein anchor precursors (19). It is possible that L. mexicana contains the same enzymatic machinery but that the presence of an ether-linked aliphatic chain at the sn-1 position of the L. mexicana GIPLs prevents this chain from being remodeled. There are two other differences between the remodeling reactions of L. mexicana and T. brucei. First, the turnover of myristate in the leishmanial GIPLs is very slow (Fig. 10C), suggesting that remodeled species are not subjected to further rounds of remodeling. In contrast, myristoylated-GPI precursors and protein-linked GPIs are extensively remodeled in African trypanosomes in a series of "proof-reading" reactions that can be biochemically and topologically distinguished from the initial remodeling reactions (41). Second, shorter fatty acids than myristate (i.e. C12:0) are incorporated into the leishmanial but not the trypanosome GPIs, suggesting that there may be subtle differences in the substrate specificity of the acyltransferases of these parasites. The reason why both the trypanosomes and Leishmania incorporate myristate into their GPI glycolipids is unknown. However, it appears to be important as the trypanosomes will divert scarce myristate into GPI remodeling when exogenous supplies of this fatty acid are low (42) and myristate analogues have been found to have potent trypanocidal activity and cause disruption of the endomembrane system (43, 44). By analogy, it is possible that the incorporation of relatively short acyl chains into the abundant leishmanial GIPL species is crucial for maintaining the physical properties of the plasma membrane and that this process could be a target for anti-leishmanial drugs.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Gavin Reid (The Ludwig Institute, Melbourne Branch) for assistance with the MALDI-TOF analyses, Dr. Claire Masterson who was involved in early experiments, and Dr. Steven Ilgoutz for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Australian National Health and Medical Research Council and the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Wellcome Trust Senior Research Fellow. To whom correspondence
should be addressed: Dept. of Biochemistry and Molecular Biology, University of Melbourne, Parkville, 3052 Australia. Tel.:
613-9344-5681; Fax: 613-9347-7730; E-mail:
m.mcconville{at}biochemistry.unimelb.edu.au.
1
The abbreviations used are: GPI,
glycosylphosphatidylinositol; PI, phosphatidylinositol; IPC, inositol
phosphoceramide; GIPL, glycoinositol phospholipid; HPAEC, high pH anion
exchange chromatography; LPG, lipophosphoglycan; TLCK,
N-p-tosyl-L-lysine
chloromethyl ketone; HPTLC, high performance liquid chromatography;
MALD-TOF-MS, matrix-assisted laser desorption time of flight mass
spectrometry. The nomenclature used to describe the L. mexicana GIPLs and GPI precursors is as follows (see Fig. 13 for
structures): the letter M and numbers 1-4 refer to the number of
mannose residues in each structure. The prefix "i" indicates the
presence of an
1-3-linked mannose branch on the mannose proximal to
glucosamine (the iso series) (29). Structures without the i
prefix lack this branch and have part or all of the structure Man
1-2Man
1-6Man
1-4GlcN-PI. The prefix "EP" indicates
the presence of an ethanolamine-phosphate residue on the glycan
backbone (13), while the subscript "VLAC" refers to GPI species
which contain 1-O-alkylglycerols with very long alkyl chains
(C24:0, C26:0). HPTLC-derived fractions used in this study are denoted
by a band number and the major composite GPI species.
2 R. Ralton and M. J. McConville, unpublished data.
3 K. Mullin, S. Ilgoutz, J. Ralton, and M. J. McConville, unpublished data.
4 J. Zawadzski, C. Scholz, G. Currie, G. Coombs, and M. J. McConville, submitted for publication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|