From the Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, New York 10010
Received for publication, July 18, 2002, and in revised form, October 5, 2002
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ABSTRACT |
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African trypanosomes are lipid auxotrophs that
live in the bloodstream of their human and animal hosts. Trypanosomes
require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol, and
phospholipids to trypanosomes. We show evidence for the existence of a
trypanosome lipoprotein scavenger receptor, which facilitates the
endocytosis of both native and modified lipoproteins, including HDL and
LDL. This lipoprotein scavenger receptor also exhibits selective lipid
uptake, whereby the uptake of the lipid components of the lipoprotein
exceeds that of the protein components. Trypanosome lytic factor
(TLF1), an unusual HDL found in human serum that protects from
infection by lysing Trypanosoma brucei brucei, is also
bound and endocytosed by this lipoprotein scavenger receptor. HDL and
LDL compete for the binding and uptake of TLF1 and thereby attenuate
the trypanosome lysis mediated by TLF1. We also show that a mammalian
scavenger receptor facilitates lipid uptake from TLF1 in a manner
similar to the trypanosome scavenger receptor. Based on these results
we propose that HDL, LDL, and TLF1 are all bound and taken up by
a lipoprotein scavenger receptor, which may constitute the parasite's
major pathway mediating the uptake of essential lipids.
Exogenous lipids play indispensable roles in trypanosome cell
structure and metabolism. African bloodstream-form trypanosomes are
single-celled parasites that appear not to synthesize fatty acids
de novo (1-3), with the exception of myristate
([14C]fatty acid). Trypanosomes have an atypical type II
fatty acid synthase that utilizes exogenously supplied butyrate to
generate myristate, which is used exclusively for
glycosylphosphatidylinositol anchor biosynthesis (4, 5). Despite having
a variety of enzymes that catalyze metabolic lipid-modifying pathways
(6-9), trypanosomes are lipid auxotrophs. They require lipoproteins in addition to other serum components in order to multiply under axenic
culture conditions (10, 11). Delipidation of the lipoproteins abrogates
their capacity to support trypanosome growth. Both major classes of
serum lipoproteins, LDL1 and
HDL, are primary sources of lipids, delivering cholesterol esters,
cholesterol and phospholipids to trypanosomes (12, 13).
Trypanosomes endocytose HDL and LDL through their flagellar pocket (10,
13). All endocytosis and exocytosis in trypanosomes occurs at this
site. It is a specialized invagination in the cell membrane, which is
not lined with the microtubule network that encases the rest of the
cell that precludes any vesicular fusion or fission. At physiological
concentrations (~1 mg/ml), specific binding and uptake of the protein
component of both LDL and HDL has been demonstrated (12-14). In
contrast, at subphysiological concentrations (1-50 µg/ml) there was
no detectable uptake of the apolipoproteins themselves, whereas the
lipid components of HDL and LDL were taken up at rates that exceeded
fluid phase endocytosis by 1000-fold, suggesting that specific binding
sites were probably involved (15). A putative LDL receptor protein has
been purified but not yet cloned (16, 17), while there has been no
molecular identification of an HDL receptor in bloodstream-form trypanosomes.
Trypanosome lytic factors are HDL-related particles found in human
plasma. TLF1 contains lipid, apolipoprotein A-I (apoA-I), paraoxonase, and haptoglobin-related protein (Hpr) (18), while TLF2 is
a lipid-poor molecule that contains apoA-I, Hpr, and IgM (19). Both
high and low affinity binding sites for TLF1 on trypanosomes have been
reported in experiments using purified preparations of TLF1 (20). The
low affinity binding site can be competed by HDL whereas the high
affinity binding site is partially competed by reconstituted nonlytic
HDL containing Hpr (21), which led to the proposal that Hpr can mediate
TLF1 binding to trypanosomes through a haptoglobin-like receptor.
Many lipoprotein receptors have been characterized in eukaryotes, to
date only cubilin (22) and members of the CD36 superfamily of scavenger
receptors (23-25) bind native HDL (without requiring ApoE as a
component). The CD36 superfamily of scavenger receptors bind and take
up both native HDL and LDL as well as other polyanionic ligands,
including oxidized and acetylated LDL (26). Some of these scavenger
receptors mediate bi-directional lipid flux and exhibit a process
called selective lipid uptake. In polarized cells selective lipid
uptake is characterized by receptor-mediated uptake of the lipoprotein,
distribution of the lipid within the cell, and recycling of the
apolipoprotein to the cell surface (27). In non-polarized cells there
does not appear to be any uptake of the holoparticle, rather binding to
the surface of the cell via lipoprotein scavenger receptors facilitates
the transfer of lipid from the lipoprotein into cell membranes and
intracellular vesicles (28). After lipid transfer, the lipid-depleted
particle is released intact from the cell. One of the members of this
family, SR-BI (scavenger receptor class BI), mediates the highest level of selective lipid uptake analyzed to date (29, 30).
While studying trypanosome lytic factors, which are by definition
lipoproteins, we decided to revisit lipoprotein receptors. We found
evidence that Trypanosoma brucei brucei has a lipoprotein scavenger receptor that mediates the selective uptake of lipid over the
protein component of both HDL and LDL. The same receptor can also
mediate the uptake of oxidized lipoproteins. TLF1 is also bound and
endocytosed by this lipoprotein scavenger receptor. We show that HDL
and LDL compete for the binding and uptake of TLF1 and therefore
attenuate the trypanosome lysis mediated by TLF1.
Materials
The fluorescent probes: Alexa Fluor-488,
22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3 Methods
Purification of Lipoproteins--
Normal human serum, plasma or
bovine serum was adjusted to a density of 1.25 g/ml with KBr and
ultracentrifuged in a near vertical NVTi 65 rotor (Beckman) for 16 h at 49,000, 10 °C (31). The top 2 ml ( Purification of Trypanosome Lytic Factors--
Isolation of
human HDL and purification of TLF1 from normal human serum (Hp 1-1) was
performed as described by Raper et al. (19).
Fluorescent Labeling of Lipoproteins--
Purified lipoproteins
were incubated with Alexa Fluor-488 protein labeling kit (Ex 494, Em
519) according to manufacturers instructions. In order to label
lipoprotein lipids, 20 ml of normal human serum were incubated with 50 µl of 1 mg/ml NBD-cholesterol (Abs 469, Em 537) in DMF, NBD
C12 PtdCho (Abs 465, Em 534) in Me2SO, or NBD
PtdEth (Abs 463, Em 536) in MeOH for 16 h at 37 °C.
Lipoproteins were purified according to the above protocol. The
specific activity (absorbance/mg of protein) of each fluorophore incorporated into HDL was determined in a 96-well fluorometer (Labsystems Fluoroskan II).
Labeling of human HDL with DiI was carried out according to Calvo
et al. (24). Briefly, lipoproteins were incubated with the
DiI probe in lipoprotein-deficient serum for 12 h at 37 °C, using the following relative amounts: 300 µg DiI, 3 mg of
lipoprotein, and 2 ml of lipoprotein-deficient serum. The labeled
lipoproteins were subsequently re-isolated by ultracentrifugation at
100,000 × g for 2.3 h at 10 °C in a Beckman table
top ultracentrifuge (TLA 1.3 rotor). DiI-labeled HDL was then sized on
a Superdex HR 200 column (Amersham Biosciences). DiI-labeled TLF1 was
obtained by affinity purification of DiI-labeled HDL using a mouse
anti-human haptoglobin monoclonal (H-6395,Sigma) coupled to a HiTrap
column (Amersham Biosciences). The fractions containing Hpr were pooled and concentrated.
Radiolabeling Lipoproteins--
Purified TLF1 was radiolabeled
by the [125I]ICl technique with 125I to a
specific activity of 300-600 dpm/ng. Bound 125I was
separated from unbound by gel filtration on a PD10 column. Trichloroacetic acid-precipitable counts were >95%. The radiolabeled proteins were used on the day they were labeled.
Trypanosome Isolation--
Swiss Webster mice were inoculated
intraperitoneally with serum-sensitive T. b. brucei ETat
1.9s, and the trypanosomes were harvested 2 days later from infected
mouse blood as described previously (32). Parasites were resuspended at
2 × 107/ml in Dulbecco's modified medium (DMEM)
supplemented with 0.2-2% BSA.
Uptake Analyses--
Increasing concentrations of labeled
lipoprotein were incubated at 37 °C for various times with 2 × 107 trypanosomes in DMEM containing 0.2-2% BSA
supplemented with the following protease inhibitors: 0.3 mg/ml
antipain-HCl, 0.05 mg/ml bestatin, 0.1 mg/ml chymostatin, 0.3 mg/ml
E-64, 0.05 mg/ml leupeptin, 0.05 mg/ml pepstatin, 0.3 mg/ml
phosphoramidon, 2.0 mg/ml Pefabloc SC, 1.0 mg/ml EDTA, and 0.05 mg/ml
aprotinin (miniTab, Roche Molecular Biochemicals). Cells were washed
twice with bicine-buffered saline with glucose (BBSG), transferred to a
black 96-well plate, and lysed in 0.5% SDS. Lysates were read in a
Fluoroskan II at the wavelengths for the specific fluorophore.
Competition Analyses--
The ligand under study was mixed with
competitors at 30-100-fold excess in DMEM/0.2-2% BSA. Then prewarmed
T. b. brucei (2 × 107) were added and
incubated at 37 °C for 30 min. For radioactive ligands, cells were
washed three times with DMEM/BSA and transferred to a clean tube prior
to quantitating radioactivity in a gamma counter. Fluorescent-labeled
cells were washed twice with BBSG, transferred to black 96-well plates,
and lysed and read as above.
Fluorescence Microscopy--
Live trypanosomes were incubated in
DMEM/0.2% BSA with either 300 µg/ml of NBD cholesterol/cholesterol
ester-labeled plasma HDL or Alexa-labeled plasma HDL, 150 µg/ml of
rhodamine-concanavalin A, or 300 µg/ml Alexa-HDL combined with 150 µg/ml rhodamine-concanavalin A at 37 °C for 30 min. Cells were
washed three times in PBSG and resuspended in 3%
paraformaldehyde/PBSG. Cells were washed with PBSG and dried onto
12-well slides (Erie Scientific Co.). DAPI-containing mounting medium
was used to adhere coverslips to slides. Slides were viewed on a Nokia
fluorescent microscope.
Trypanolytic Assays--
To measure lytic activity, 2 × 106 trypanosomes were incubated in the presence of TLF1
(1-1.5 LU (5-40 µg/ml), or TLF1 combined with human LDL (0.75-1
mg/ml) or bovine HDL (1-1.6 mg/ml). LU is the lytic unit wherein 50%
of 2 × 106 trypanosomes are lysed in 150 min at
37 0C in a final volume of 200 µl. Following incubation
for 150 min at 37 °C, parasite lysis was determined using a
previously described calcein fluorescence-based assay (33).
Cell Culture and Maintenance--
LdlA[mSR-BI] and ldlA7 cells
were grown in Ham's F-12 medium supplemented with 5% (v/v) fetal calf
serum, 2 mM glutamine with or without 500 µg/ml G-418,
respectively. All cells were maintained in a 37 °C humidified 95%
air, 5% CO2 incubator.
Flow Cytometry--
LdlA[mSR-BI] and parental ldlA7 cells were
plated overnight in 6- or 12-well plates washed and incubated for 2-4
h in Ham's medium supplemented with 0.2% BSA. The cells were then
incubated at 37 °C for 2 h with 2.5 µg/ml DiI-HDL or DiI-TLF1
in Ham's F12 supplemented with 0.2% BSA. Cells were washed twice with
PBS, detached from the plate with cell stripper solution and
resuspended in PBS, then fixed with an equal volume of 4% paraformaldeyde.
For immunofluorescence, LdlA[mSR-BI] and ldlA7 cells were resuspended
in 100 µl of Ham's F12, 5% FCS to 106/ml. Anti-SR-BI
antibody (100 µl) to a final dilution 1:1000 was added and incubated
for 30 min on ice. Cells were then washed three times, resuspended to
100 µl in PBS, 0.1% BSA, and 100 µl of anti-rabbit
IgG-FITC-labeled antibody was added to a final dilution of 1:50. After
a 30-min incubation on ice, the cells were washed three times with PBS,
then resuspended to 100 µl in PBS and fixed with 100 µl of 4%
paraformaldehyde. Samples were subjected to flow cytometric analysis
using a Becton Dickinson FACScan flow cytometer. The following
excitation/emission were used: 488/525 nm for FITC-conjugated antibody
and 488/575 nm for DiI-labeled HDL and TLF1. Mean relative fluorescence
of cells was recorded and results are expressed as a percentage of control.
HDL and LDL Compete for HDL Uptake by Trypanosomes--
We labeled
HDL and LDL with Alexa, a fluorophore that conjugates to the free amino
groups in the protein components of these lipoproteins. We found that
trypanosomes accumulated HDL protein (2.25 pmol, calculated based on a
molecular mass of 350,000 Da by size exclusion chromatography, 50% of
which is protein) and LDL protein (2 pmol, calculated based on the
molecular mass of apoB, 550,000 Da) (Fig.
1A) with similar kinetics,
approximating steady state within 30 min. Trypanosomes also accumulated
NBD-cholesterol/cholesterol ester (NBD-C/CE) from HDL (lipid uptake
equivalent to 350 pmol of protein) and LDL (lipid uptake equivalent to
425 pmol of protein; Fig. 1B), approximating steady state
within 30 min. TLC analysis indicated that the NBD cholesterol labeled
both free cholesterol and cholesterol esters in the lipoproteins (data
not shown). Therefore, results shown in Fig. 1B represent
total cholesterol uptake. As HDL and LDL have similar kinetics of
protein and lipid uptake (Fig. 1, A and B), they
were used in competition assays. The competition was assessed at 30 min, such that the uptake was close to steady state (Fig. 1,
A and B). Both unlabeled HDL and LDL were
effective competitors of protein (Fig. 1D) and lipid (Fig.
1C) uptake from labeled HDL. Fig. 1, panel D
shows that 4 times more HDL than LDL was needed to give a 50%
reduction in the uptake of HDL protein. This suggests that the putative
lipoprotein receptor has higher affinity for LDL than HDL. Although
Fig. 1, panel C displays a similar trend in LDL
versus HDL competition, we found that some of the
NBD-cholesterol was transferred to the non-labeled lipoprotein, which
may contribute to the enhanced competition of lipoprotein lipid uptake.
This desorption diffusion process may also occur to some extent when
NBD-cholesterol HDL is incubated with trypanosomes.
HDL Lipids Are Selectively Taken Up Over HDL Protein--
To date
the only characterized eukaryotic lipoprotein receptors that bind both
native HDL and LDL have the unique characteristic of selective lipid
uptake over protein. In light of the above findings we examined if
there was a differential uptake of other lipid components over the
protein components of HDL. We extended this study to include HDL
labeled with fluorescent phospholipids NBD-phosphatidylcholine
(NBD-PtdCho), NBD-phosphatidylethanolamine (NBD-PtdEth) as well as
NBD-cholesterol/cholesterol ester (NBD-C/CE). The specific activity
(fluorescence/µg of protein) of each fluorescent-labeled HDL was
taken into consideration, such that the uptake of each fluorescent
lipid was calculated from a standard curve of µg of protein
versus fluorescent units. The results are expressed as microgram of protein equivalents taken up rather than fluorescent units. We found that all classes of lipid molecules in HDL were taken
up 50-100-fold more than the protein component (Fig.
2). It is apparent that the uptake of
free cholesterol and cholesterol ester exceeds that of phospholipids.
In addition NBD-PtdEth was taken up almost 2-fold more than NBD-PtdCho.
The uptake of individual lipid species does not correlate with their
concentrations found in a typical human HDL, which are 55, 7, and 27 weight% for phosphatidylcholine, cholesterol, and cholesterol esters
respectively (34).
Localization of Lipid Versus Protein--
Since all classes of
lipid molecules were taken up more avidly than protein, we examined the
distribution of the fluorescent lipid and protein molecules by
fluorescent microscopy. NBD-cholesterol and cholesterol esters
(NBD-C/CE) were rapidly distributed throughout the parasite, and all of
the parasites were labeled (Fig.
3A). Confocal analysis
revealed diffuse staining throughout the cytosol of the parasites (data
not shown). The labeled phospholipids, NBD-PtdCho and NBD-PtdEth also
demonstrated a staining pattern similar to that of NBD-C/CE (data not
shown). In contrast, the apolipoprotein (Alexa-HDL) uptake by
trypanosomes was visualized in the flagellar pocket and intracellular
vesicles (Fig. 3B) with a distribution similar to
endocytosed concanavalin A (ConA) (Fig. 3C). All of the
parasites accumulated detectable Alexa-HDL. Concanavalin A has been
shown to distribute within endocytic vesicles of trypanosomes when
endocytosed by live trypanosomes (35, 36); in contrast ConA labels the
VSG coat when used on fixed trypanosomes presumably due to the exposure
of carbohydrate epitopes upon fixation. Coincubation with
rhodamine-ConA and Alexa-HDL revealed colocalization in some endocytic
vesicles (yellow) near the flagellar pocket but not all
endocytic vesicles (red) (Fig. 3D).
HDL Competes for the Binding of TLF1 to Trypanosomes--
All of
the data thus far point to the presence of a lipoprotein receptor in
trypanosomes that can facilitate the uptake of both native HDL and LDL.
TLF1 is a subclass of HDL and is composed of lipids, apolipoprotein
A-I, paraoxanase, and haptoglobin-related protein. Lipids and
apolipoprotein A-I are the common components of all HDLs, whereas Hpr
is unique to TLF particles. There are studies documenting the specific
and saturable binding of TLF1 and HDL to the flagellar pocket of
African trypanosomes (14, 20). We found that TLF1 (20 µg/ml (60 pmol,
calculated based on a molecular mass of 550,000 Da by size exclusion
chromatography, 60% of which is protein)) and HDL (200 µg/ml (1440 pmol) could compete for binding of 125I-TLF1 to T. b.
brucei (Fig. 4). We did not
investigate the effect of LDL on the binding of TLF1 to T. b.
brucei, because LDL takes 6 h to reach equilibrium binding to
trypanosomes whereas HDL and TLF take 30 min. Therefore we could not
have a fair competition for binding and evaluated uptake only.
HDL and LDL Can Attenuate TLF1-mediated Trypanosome
Lysis--
Given that we observed competition for binding of TLF1 to
trypanosomes by HDL, we evaluated the effect of non-lytic bovine HDL on
TLF1-mediated trypanolysis. We observed that non-lytic bovine HDL was
able to attenuate trypanosome lysis by purified TLF1 (Fig.
5). Non-lytic human LDL was also
effective in attenuating trypanolysis by TLF1.
TLF1 Binds to Mouse Scavenger Receptor Class B Type I and Donates
Lipids--
Our results support the presence of a lipoprotein
scavenger receptor in trypanosomes that can bind multiple lipoprotein
ligands such as TLF1, HDL, LDL, and oxidized LDL (not shown) and
exhibit a process called selective lipid uptake similar to eukaryotic lipoprotein scavenger receptors. To directly address whether TLF1 could
bind to a eukaryotic lipoprotein scavenger receptor with the same
ligand binding characteristics as the trypanosome lipoprotein receptor,
we examined the binding of TLF1 to a CHO cell line that overexpresses
mouse Scavenger Receptor-Class BI (mSR-BI). The parental CHO
ldlA[clone 7] cells do not express the LDL receptor and were stably
transfected with a vector expressing mouse scavenger receptor-class BI
to create ldlA[mSR-BI] cells (37). These cell lines were first
validated with antibodies raised against mSR-BI; the parental ldlA
cells showed little staining, while the transfected ldlA[mSR-BI]
cells stained readily with anti-mSR-BI (Fig.
6, inset). HDL labeled with
the fluorescent lipid DiI exhibited lipid uptake into cells expressing
mSR-BI that was 30-fold greater than the uptake by the parental ldlA
cells (Fig. 6). TLF1 labeled in the lipid component with DiI to the
same specific activity as DiI-HDL, was taken up to a 5-fold greater
extent by cells expressing mSR-BI than that shown by the parental ldlA
cells (Fig. 6).
Lipoprotein receptors that can bind more than one ligand are known
as lipoprotein scavenger receptors. Our results suggest that
bloodstream-form trypanosomes have a single lipoprotein scavenger receptor that can facilitate the uptake of all major lipoprotein classes including HDL, LDL, oxidized LDL (results not shown), and TLF1.
Moreover, like eukaryotic scavenger receptors, the trypanosome receptor
shows selective uptake of lipid over protein from the lipoprotein
particle. Trypanosomes are lipid auxotrophs, and host lipoproteins are
required for their survival. This putative lipoprotein scavenger
receptor may well constitute the primary pathway by which the parasite
acquires essential host lipids, and would therefore represent an
important therapeutic target. Very few bloodstream-form trypanosome
receptors have been previously characterized biochemically. These
include an LDL receptor (10) and a HDL receptor (13) both of which may
be identical to the scavenger receptor described here (see below), a
haptoglobin-like receptor, which may also be a TLF receptor (21), and a
receptor for transferrin, which has been molecularly cloned
(38-43).
The characterization of this trypanosome scavenger receptor serves to
unify a variety of disparate data regarding the utilization of
lipoproteins and TLF by the parasite. Vandeweerd and Black (15) showed
that the uptake at 37 °C of radiolabeled lipid components in either
HDL or LDL was inhibited (50-85%) by unlabeled HDL or LDL (15). It
was concluded that the uptake process did not discriminate between HDL
or LDL. In this study we have confirmed and extended these
observations. We also found that accumulation of HDL-labeled protein or
HDL-labeled lipid, was inhibited by increasing concentrations of HDL
and LDL (Fig. 1, C and D). Previous
investigations of lipoprotein binding to a different isolates of
trypanosomes indicated that HDL binding could be partially competed by
LDL (13) although LDL binding could not be competed by HDL (17).
Although our data for lipoprotein uptake at 37 °C suggest that
competition should have been reciprocal, it is possible that binding at
4 °C gives different results from uptake experiments performed under physiological conditions at 37 °C. The observation that the number of estimated HDL and LDL receptors in trypanosomes are roughly equivalent (30,000-52,000 LDL receptors/cell (10, 17, 44), 22,000-64,000 HDL receptors/cell (10, 13, 20, 21)) supports the notion
that a single scavenger receptor can mediate uptake of both HDL and
LDL. Interestingly, anionic phospholiposomes, which have been shown to
be effective inhibitors of trypanolysis mediated by TLF1 (45), are also
ligands for eukaryotic lipoprotein scavenger receptors (46, 47). Taken
together, these results suggest the presence of a lipoprotein scavenger
receptor in trypanosomes that can bind multiple ligands.
The trypanosome lipoprotein scavenger receptor shares characteristics
with certain subclasses of mammalian scavenger receptors. Members of
the CD36 superfamily can bind native HDL and LDL and exhibit selective
lipid uptake from both lipoproteins. Binding appears to be mediated by
a combination of apolipoprotein and lipid. These characteristics most
resemble what we have found for the putative trypanosome lipoprotein
scavenger receptor. We find that when we correct for the specific
activity of each labeled lipoprotein, the lipid components are
selectively accumulated more than the protein component (Fig. 2). It is
worth noting that although cholesterol/cholesterol ester is taken up
3-4-fold more than phospholipid, it only comprises 36% of native HDL
lipids relative to 55% for phosphatidyl choline. The selective uptake of lipoprotein cholesterol/cholesterol ester over phospholipid has also
been characterized in SR-BI scavenger receptors, which are a subclass
of the CD36 superfamily (28, 48).
Ligands other than native HDL and LDL have been identified for
eukaryotic lipoprotein scavenger receptors, such as oxidized lipoproteins (46). Oxidized LDL is a ligand for the trypanosome lipoprotein receptor, in that native HDL and LDL or oxidized
lipoproteins (not shown) were effective competitors for uptake. Native
HDL was a consistently better competitor than native LDL when measuring oxidized LDL uptake by trypanosomes. On the other hand, native LDL was
a better competitor than native HDL when measuring HDL uptake in
trypanosomes (Fig. 1, C and D). It has been shown
for CD36 that oxidized LDL competes more effectively than LDL for HDL
binding to CD36 (24).
The similar biochemical properties of the trypanosome lipoprotein
scavenger receptor and mammalian CD36 superfamily members compelled us
to analyze the interaction of TLF1 with the prototypical class B
eukaryotic lipoprotein scavenger receptor, SR-BI, which exhibits the
highest degree of selective lipid uptake (29, 30). We found that like
HDL, TLF1 is able to donate lipids via this eukaryotic lipoprotein
scavenger receptor (Fig. 6), indicating that TLF1 can bind to and
productively interact with SR-BI. Although there was a specific
association of TLF1 with CHO cells expressing SR-B1 we did not observe
any obvious toxicity at physiological concentrations of TLF1 (~20
µg/ml).
Both apolipoprotein and lipid are taken up by the parasite. The lipid
is selectively removed from the lipoprotein and distributed throughout
the cell (Fig. 3A). The apolipoprotein localizes to endocytic vesicles (Fig. 3D). The distribution of protein is
very different from that seen for lipid, suggesting that at some point after interaction with a receptor the lipid is selectively removed and
dispersed throughout the cell. The current hypotheses for HDL uptake in
eukaryotic cells involve either retro-endocytosis or the formation of a
non-polar channel, created by the binding of the apolipoprotein at the
cell surface (28), through which lipids are delivered. We have
demonstrated that HDL apolipoprotein is found inside the trypanosome
(Fig. 3D). Because we detect intracellular HDL and we do not
detect degradation (not shown), the majority of the endocytosed
apolipoprotein may be recycled back to the cell surface. Other
researchers have found that trypanosome endocytosis of HDL (13) and
more recently TLF1 (49) do not result in the degradation of the
apolipoproteins. In contrast, apolipoprotein B of LDL is rapidly
degraded during its transit through the endocytic machinery (50). The
reason for this difference in proteolytic processing is not known, it
may be due to differential routing of the ligands in the endocytic
pathway or differential sensitivity to endosomal and lysosomal proteases.
Physiological concentrations of HDL can compete for at least ~80% of
the binding of TLF1 to T. b. brucei (Fig. 4). It has been
proposed that the remaining ~20% of TLF1 is taken up by another trypanosome receptor that recognizes haptoglobin (21). Irrespective of
whether there are one or more receptors for TLF, if there is competition for binding of TLF, there should be competition for uptake.
It has been reported that TLF1 exhibits both high affinity (0.75-3.6
µg/ml) and low affinity (80-175 µg/ml) binding to trypanosomes, and that only the low affinity sites are competed by HDL (20, 21). We
believe, as has been proposed by others for LDL binding to trypanosomes
(10, 51), that the low affinity sites for TLF1 may represent single
receptors along the flagellum and within the pocket, whereas high
affinity sites represent dimerized or clustered receptors within the
flagellar pocket. Complete inhibition of binding or uptake at the
receptor level, requires the competing ligand to be at least 100-fold
above its own Kd in order to saturate all of the
available receptors (52). Therefore complete inhibition of TLF1 binding
and uptake at the receptor level would require 2.7-8 mg/ml of HDL (13,
21) and 13-33 mg/ml LDL (44). These concentrations are above the
physiological levels found in plasma which are ~1 mg/ml. Therefore,
in vivo as in our assay, the lipoproteins would be able to
attenuate the killing by TLF1 but they would not inhibit the killing.
This is illustrated by the attenuation of TLF1-mediated lysis (Fig. 5)
in the presence of HDL (1-1.6 mg/ml) and LDL (0.75-1 mg/ml). Oxidized
lipoproteins were in themselves
trypanolytic,2 and we
therefore could not evaluate their capacity to attenuate TLF-mediated lysis.
Overall our results suggest that trypanosomes have a lipoprotein
scavenger receptor that can bind HDL, LDL, and TLF despite their
distinct apolipoprotein content. The characteristics of the lipoprotein
interactions with trypanosomes most closely resemble those of the class
B type lipoprotein scavenger receptors, which are the only receptors to
date that bind native HDL. Members of the CD36 superfamily of proteins
are found in eukaryotes ranging from mammals to fungi (53), and
generally have 30% amino acid sequence identity (26). However,
extensive searching of the trypanosome databases has not yet revealed
an identifiable homologue. This is not entirely surprising as
trypanosome proteins are often very divergent. Extensive searching of
the trypanosome databases with the eukaryotic transferrin receptor
reveals nothing, yet there is a non-homologous but well-characterized
transferrin receptor in trypanosomes (38-43). Other scavenger receptor
domains (SRCR, Pfam 00530) have been identified in Plasmodium
sp. (54). It is quite probable that scavenger receptors exist in
other lipid auxotrophic parasites, and these may become apparent as the
genomes of parasites are completed and fully annotated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ol
(NBD-cholesterol), NBD-PtdCho, NBD-PtdEth,
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholrate
(DiI), and Calcein-AM were purchased from Molecular Probes (Eugene,
OR). Tissue culture reagents: Ham's F12, DMEM, cell stripper solution,
and G418 were obtained from Cellgro. Rhodamine-concanavalin A and
Vectashield mounting medium with DAPI were obtained from Vector Labs.
Protease inhibitors were purchased from Roche Molecular Biochemicals.
Polyclonal rabbit anti-mouse SR-BI was obtained from Novus Biologicals.
[125I]Iodine was purchased from Amersham Biosciences.
Sephadex G-25 columns were bought from Isolab Inc. Mouse monoclonal
anti-human haptoglobin, anti-rabbit IgG-FITC (F-0511), and all other
chemicals and reagents were purchased from Sigma Chemical Co. T. b. brucei Etat 1.9s were kindly provided by Dr. Miki
Rifkin. CHO ldlA clone 7 and ldlA[mSR-BI] cells were generously
provided by Dr. Monty Krieger.
= 1.0-1.26 g/ml)
were collected and size-fractionated on a Superose 6 16/50 column
equilibrated with TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl). Proteins from fractions were separated on a
4-15% Tris-HCl gel and stained with Coomassie Blue. Fractions containing either ApoA-I (HDL) or Apo-B (LDL) were separately pooled
and concentrated.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
HDL and LDL compete for HDL uptake in
trypanosomes. Trypanosomes (300 µl of 2 × 108
parasites/ml) were incubated at 37 °C in the presence of 50 µg/ml
Alexa-labeled HDL (285 pmol; squares) (A) or LDL
(90 pmol; diamonds) or NBD-C/CE-labeled HDL or LDL
(B). Uptake was determined by reading fluorescence of cell
lysates at 0, 5, 15, 30, 45, and 60 min. Fluorescent units were
converted to mol of protein using a standard curve for each
fluorophore. Data are representative of two independent experiments.
Trypanosomes (100 µl of 2 × 108/ml) were incubated
at 37 °C in the presence of 50 µg/ml (285 pmol) NBD-C/CE-labeled
HDL (C) or 50 µg/ml (285 pmol) Alexa-labeled HDL
(D) and increasing concentrations of unlabeled HDL
(squares) or LDL (diamonds). Panel C,
100% 1.9 µg (10 pmol); panel D, 100%
20 µg (114 pmol).
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[in a new window]
Fig. 2.
Trypanosomes exhibit selective lipid
uptake. Trypanosomes (2 × 107) were incubated in
a final volume of 150 µl in the presence of increasing concentrations
of Alexa-HDL (squares), NBD-PtdEth-HDL
(diamonds), NBD-PtdCho-HDL (triangles), or
NBD-C/CE-HDL (circles) for 30 min. at 37 °C. Cells were
washed and fluorescence was determined.
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[in a new window]
Fig. 3.
HDL lipid and protein are distributed
differently. Trypanosomes (3 × 106) were
incubated for 30 min at 37 °C in a final volume of 100 µl in the
presence of 300 µg/ml (1.7 nmol) of NBD-C/CE-labeled HDL
(A); 300 µg/ml (1.7 nmol) of Alexa-labeled plasma HDL
(B); 150 µg/ml (0.7 nmol) concanvalin A (C);
300 µg/ml of Alexa-labeled plasma HDL and 150 µg/ml ConA
(D). Cells were washed and nuclei and kinetoplast
(mitochondrial DNA) stained with DAPI and fixed and then viewed by
fluorescent microscopy.
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[in a new window]
Fig. 4.
HDL and TLF1 compete for TLF1 binding to
T. b. brucei. 200 ng/ml (0.6 pmol)
125I-TLF1 (*TLF1) was mixed with 20 µg/ml (60 pmol) TLF1
or 200-250 µg/ml (1.1-1.4 nmol) HDL. Trypanosomes (1 × 107) were added, and binding was allowed to reach
equilibrium at 4 °C. Each value for bound TLF is the mean of two
independent experiments done in triplicate.
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Fig. 5.
Lipoproteins attenuate trypanolysis by
TLF1. Trypanosomes (2 × 106) were incubated in a
final volume of 200 µl in the presence of TLF1 (1-1.5 LU, 5-40
µg/ml, 15-121 pmol) alone or TLF1 plus human LDL (0.75-1 mg/ml,
1.45-1.8 nmol) or bovine HDL (1-1.6 mg/ml, 5.7-9.1 nmol) for 150 min
at 37 °C. Cell lysis was determined microscopically and as described
under "Experimental Procedures." Results are expressed as % lysis
and represent the mean of five independent experiments.
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Fig. 6.
SR-BI mediates lipid uptake from TLF1.
Ldl 7[mSR-BI] and ldl A7cells were analyzed with fluorescent
anti-mSR-B1 to confirm the overexpression of mSR-B1. The cells lines
were then incubated in the presence of 2.5 µg/ml (14 pmol) DiI-HDL or
2.5 µg/ml (7.5 pmol) DiI-TLF1 for 2 h at 37 °C as described
under "Experimental Procedures." Red fluorescence (DiI)
or green fluorescence (anti-SR-BI FITC) was measured by flow
cytometry. Shown is mean relative fluorescence expressed as percent of
the control (control = 100%, see "Experimental
Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Isabelle Coppens, Paul Englund, Dan Eichinger, Mary Gwo-Shu Lee, and Photini Sinnis for critical reading of the article. J. R. thanks Fred Opperdoes, Joris Van Roy, and Pierre Courtoy for the introduction to lipoprotein receptor biology.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI01660 and AI41233 and a predoctoral fellowship (to H. G.) from the Ford Foundation.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.
To whom correspondence should be addressed. Tel.: 212-263-7632;
Fax: 212-263-8179; E-mail: jr57@nyu.edu.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M207215200
2 J. Raper, M. P. Molina Portela, and E. N. St. Jean, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: LDL, low density lipoprotein; apoA-I, apolipoprotein A-I; Hpr, haptoglobin-related protein; Hp 1-1, haptoglobin type 1-1; TLF, trypanosome lytic factor; HDL, high density lipoprotein; SR-BI, scavenger receptor class B type I; mSR-BI, murine SR-BI; ldlA7, ldlA (clone 7) LDL receptor-negative CHO cell mutant clone; CHO, Chinese hamster ovary; ConA, concanavalin A; C/CE, cholesterol/cholesterol ester; PtdEth, phosphatidylethanolamine; PtdCho, phosphatidylcholine; DMEM, Dulbecco's modified medium; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; DAPI, 4',6-diamidino-2-phenylindole.
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