Hemoglobin Endocytosis in Leishmania Is Mediated
through a 46-kDa Protein Located in the Flagellar Pocket*
Shantanu
Sengupta
,
Jalaj
Tripathi
,
Ruchi
Tandon
,
Manoj
Raje§,
Rajendra P.
Roy
,
Sandip K.
Basu
¶, and
Amitabha
Mukhopadhyay
From the
National Institute of Immunology, Aruna Asaf
Ali Marg, New Delhi 110067 and the § Institute of Microbial
Technology, Sector 39A, Chandigarh 160014, India
 |
ABSTRACT |
Four lines of evidence indicate that a specific
high affinity binding site on the surface of Leishmania
donovani promastigotes mediates rapid internalization and
degradation of hemoglobin. 1) Binding and uptake of
125I-hemoglobin by Leishmania followed
saturation kinetics and were competed by unlabeled hemoglobin but not
by globin or hemin or other heme- or iron-containing proteins. 2)
Immunogold labeling studies revealed that, at 4 °C, hemoglobin
binding was localized in the flagellar pocket of the promastigotes.
Indirect immunofluorescence assays showed that, at 37 °C, the bound
hemoglobin in such cells entered an endocytic compartment within 2 min
and dispersed throughout the cell body by 15 min. 3) After incubation
with hemoglobin-gold conjugates at 25 °C or 37 °C, the particles
accumulated in discrete intracellular vesicles. 4) A single
biotinylated protein of 46 kDa was revealed when solubilized membranes
from surface biotinylated intact Leishmania adsorbed by
hemoglobin-agarose beads were subjected to SDS-polyacrylamide gel
electrophoresis and Western blotting with avidin-horseradish
peroxidase. Considered together, these data indicate that this 46-kDa
protein on the cell surface of L. donovani promastigotes
mediates the binding of hemoglobin and its rapid internalization
through a vesicular pathway characteristic of receptor-mediated endocytosis.
 |
INTRODUCTION |
In both eukaryotic and prokaryotic cells, endocytosis is essential
for uptake of nutrients, down-regulation of cell surface receptors, and
maintenance of cell homeostasis (1-3). Among the protozoans,
endocytosis has been widely studied in trypanosomatids like
Trypanosoma and Leishmania (4-6). These
hemoflagellates are found predominantly in the blood stream and tissues
of mammalian host, where they rapidly multiply. Receptor systems have
been identified on trypanosomatid parasites, which mediate uptake of low density lipoprotein and transferrin presumably for efficient supply
of nutrients required for rapid growth such as cholesterol and metal
ions, which usually occur as tightly bound complexes with carrier
proteins (7, 8).
Leishmania sp. are protozoan parasites
responsible for several diseases varying from a single cutaneous lesion
to fatal visceral leishmaniasis, affecting millions of people worldwide
(9). Leishmania donovani is the etiologic agent of
kala-azar, a chronic and often fatal form of human visceral
leishmaniasis (10).
In common with most trypanosomatids, Leishmania lack a
complete heme biosynthetic pathway and must therefore acquire heme from
external sources (11-13). In the animal host, heme is present mainly
as hemopexin and hemoglobin of which hemoglobin is the most important
reservoir (14). This potential source may be available when
erythrocytes are lysed by hemolysins or natural degradation of
hemoglobin in macrophages or by hitherto unknown mechanisms. Galbraith
et al. (15) have reported the presence of specific
heme-binding sites on Leishmania mexicana amazonensis promastigotes. Since Leishmania promastigotes can also be
grown in vitro in blood agar medium without addition of heme
(16), it is possible that Leishmania might have evolved
mechanisms to internalize intact hemoglobin and generate heme
intracellularly. Hemoglobin-binding proteins have been identified in
some pathogens, suggesting that selective recognition of hemoglobin
occurred at the cell surface (17-21). However, no specific
hemoglobin-binding site has so far been reported to be present on
Leishmania.
In the present investigation, we have shown that a limited number of
sites localized in the flagellar pocket of L. donovani promastigotes mediate uptake and degradation of hemoglobin with saturation kinetics. Furthermore, we also report the presence of a
46-kDa protein on the cell surface of L. donovani
promastigotes that specifically binds hemoglobin.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Hemoglobin, hemin, globin, transferrin,
hemocyanin, myoglobin, colloidal gold particles, rabbit anti-hemoglobin
antibodies, and hemoglobin-agarose affinity matrix were purchased from
Sigma. Goat anti-rabbit IgG conjugated with 20-nm colloidal gold was purchased from Jackson ImmunoResearch Laboratory, West Grove, PA.
N-Hydroxysuccinimido-biotin, avidin-horseradish peroxidase (avidin-HRP),1 and
bicinchoninic acid reagents were purchased from Pierce. Anti-hemoglobin antibody in mice was raised by standard technique (22). FITC-labeled horse anti-mouse IgG was purchased from Vector Laboratories,
Burlingame, CA. Other reagents used were of analytical grade.
Leishmania--
Promastigotes of L. donovani (UR 6)
were obtained from the Indian Institute of Chemical Biology, Calcutta,
India. The cells were routinely maintained on solid blood agar slants
containing glucose, peptone, sodium chloride, beef heart extract, and
rabbit blood with gentamycin (23, 24). For experimental purposes, cells
were harvested from 3-day-old blood agar slants by scraping into
phosphate-buffered (10 mM, pH 7.2) saline (0.15 M).
Radioiodination of Hemoglobin--
Hemoglobin was labeled with
Na125I by the iodine monochloride-catalyzed reaction (2).
More than 98% of the radioactivity was acid precipitable, and the
specific activity varied between 100 and 200 cpm/ng of hemoglobin from
batch to batch.
Binding of 125I-Hemoglobin to L. donovani
Promastigotes at 4 °C--
Promastigotes were suspended in RPMI
medium and washed three times with the same media. Cells (5 × 107) were resuspended in RPMI containing 1 mg/ml bovine
serum albumin (BSA) and incubated at 4 °C with varying
concentrations of 125I-hemoglobin. After 4 h, the
cells were washed three times with PBS containing 1 mg/ml BSA and then
three times with PBS to remove unbound radioactivity. The cell pellet
was finally dissolved in 0.1 N NaOH. The cell-associated
radioactivity was then measured by placing an aliquot in a
counter.
Cellular protein content was estimated by bicinchoninic acid reagent
(25). The results were expressed as femtomoles of hemoglobin bound/µg
of cell protein. To determine the specificity, binding of
125I-hemoglobin (6 µg/ml) with promastigotes was carried
out in the presence of different competitors as indicated.
Uptake and Degradation of 125I-Hemoglobin by
Leishmania at 37 °C--
Cells (5 × 107) were
incubated with increasing concentrations of 125I-hemoglobin
in 1 ml of RPMI 1640 medium containing 1 mg/ml BSA at 37 °C. After
3 h the cells were pelleted by centrifugation at 500 × g for 10 min. Aliquots of the supernatant were processed for
the determination of trichloroacetic acid-soluble non-iodide radioactivity after extraction with chloroform (2). Thin layer chromatographic analysis showed that about 80% of the trichloroacetic acid-soluble radioactivity released into the medium consisted of
monoiodotyrosines. The cells were washed as indicated in the binding
experiment, and an aliquot was measured to determine the cell-associated radioactivity.
Electron Microscopy--
Leishmania cells (5 × 105) were incubated with 6 µg/ml hemoglobin for 2 h
at 4 °C in RPMI 1640 medium containing 1 mg/ml BSA. The cells were
washed five times with ice-cold PBS to remove unbound hemoglobin and
fixed in 1% glutaraldehyde and 1% paraformaldehyde in PBS, pH 7.2, for 20 min at 4 °C. Subsequently, the cells were washed, dehydrated
in ethanol, and embedded in LR White resin. Ultrathin sections of the
LR White-embedded cells were blocked overnight with 2.5% casein in
0.001% Tween 20 in PBS. Sections were washed five times with PBS-Tween
20 and incubated with rabbit anti-hemoglobin antibody (1:100) for
2 h at room temperature. Sections were washed five times in
similar manner (5 min each time), and they were incubated with goat
anti-rabbit IgG conjugated with 20-nm colloidal gold (1:10) for 1 h at 37 °C to allow the detection of primary antibody binding.
Finally, the sections were stained with uranyl acetate and viewed at 80 kV in a transmission electron microscope (Joel 1200 EX 11). Cells
without hemoglobin bound was used as control.
To determine the intracellular location of hemoglobin inside the
Leishmania, 5 × 105 cells were incubated
with gold particles (20 nm) conjugated with hemoglobin (1 µg of
hemoglobin/ml) as described by De Mey (26) for 1 h at 25 °C or
at 37 °C in RPMI 1640 medium containing 1 mg/ml BSA. The cells were
washed five times with cold PBS to remove unbound gold and fixed in
2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH
7.3, washed, and treated with 1% OsO4 in 0.1 M
sodium cacodylate buffer pH 7.3. The cells were rinsed and dehydrated
in ethanol and embedded in araldite (27). Thin sections were
double-stained with uranyl acetate and lead citrate and examined at 60 kV with an electron microscope. BSA-gold was used as control. Finally,
quantitation was carried out by counting the number of gold particles
internalized by Leishmania for each set of experimental conditions.
Immunofluorescence Microscopy--
L.
donovani promastigotes (5 × 107) were
washed three times with PBS, incubated with 5 µg/ml hemoglobin for
2 h at 4 °C, washed, and incubated for different periods at
37 °C to allow internalization of the hemoglobin bound at 4 °C.
Finally, the cells were washed and processed as described for the
binding experiment.
Cells were resuspended in PBS, and thin smears of cells were prepared
on glass slides. Subsequently, the cells were fixed in precooled
(-20 °C) methanol for 20 min (28). After fixation, cells were
washed three times in PBS containing 0.2% Triton X-100 with 5 min of
incubation at room temperature each time. Cells were then incubated
with mouse anti-hemoglobin primary antibody in PBS for 1 h at
37 °C. After washing the cells three times with PBS (5 min/each),
they were incubated with horse anti-mouse FITC in PBS for 1 h at
37 °C to allow the detection of primary antibody binding. Cells were
then washed once with PBS containing 0.2% Triton X-100 and three times
with PBS (5 min/each). The slides were then mounted in glycerol
containing para-phenylenediamine and viewed in a
fluorescence microscope (Olympus).
Biotinylation of Leishmania Surface Proteins--
Cell surface
proteins of intact L. donovani promastigotes were
biotinylated using standard procedure (29). Briefly, cells were washed
and incubated with 500 µg/ml N-hydroxysuccinimido-biotin for 1 h at 4 °C. Subsequently, the cells were washed three
times with PBS containing 50 mM NH4Cl to remove
excess biotin, followed by one wash with PBS.
Preparation of Membrane Fraction--
Biotinylated L. donovani promastigotes were washed three times in PBS and
then kept for 1 h at 4 °C in hypotonic Tris-HCl buffer (5 mM, pH 7.2). Then, the cells were broken by sonication (3 × 20 s) and the unbroken cells and nucleus were separated
by low speed centrifugation (200 × g for 10 min.). The
resultant postnuclear supernatant was again centrifuged at 100,000 × g for 1 h. The membrane pellet was treated with PBS
containing 2%
-octyl glucoside and kept at 4 °C overnight.
Finally, extracted membrane proteins were separated from the debris by
centrifugation at 100,000 × g for 1 h at 4 °C.
The supernatant thus obtained contained biotinylated membrane proteins.
Affinity Chromatography of the Biotinylated Membrane
Proteins--
The extract (100 µl) containing biotinylated membrane
proteins (0.6 mg) was added to hemoglobin-agarose beads (100 µl of
packed gel containing 1.6 mg of hemoglobin) equilibrated with 10 mM phosphate buffer containing 1 M NaCl in a
microcentrifuge tube and incubated overnight at 4 °C. The beads were
then washed five times with the same buffer to remove the proteins
bound nonspecifically with the affinity matrix. Subsequently, 30 µl
of sodium dodecyl sulfate-polyacrylamide gel elctrophoresis (SDS-PAGE)
sample buffer (0.0625 M Tris-HCl, pH 6.8, 2% SDS, 10%
glycerol, 0.002% bromphenol blue) was added to the washed beads and
kept in a boiling water bath at 100 °C for 5 min. The sample was
allowed to cool, and then the affinity beads were pelleted by
centrifugation at 10,000 × g for 5 min. The
supernatant was transferred to a clean microcentrifuge tube, and 20 µl of the sample was subsequently applied to a 10%
SDS-polyacrylamide gel for analysis by SDS-PAGE. Finally, proteins were
transferred onto nitrocellulose membranes and Western blot analysis was
carried out to detect the biotinylated surface proteins of
Leishmania using avidin-HRP and developed by
-chloronaphthol.
 |
RESULTS |
Binding of 125I-Hemoglobin at 4 °C--
The data
presented in Fig. 1a show the
amount of 125I-hemoglobin binding at 4 °C to the cells
as a function of the concentration of 125I-hemoglobin in
the incubation medium. Half-maximal binding of 125I-hemoglobin occurred at a concentration of about 0.09 µM hemoglobin (6 µg/ml). Scatchard plot (30) of the
data in Fig. 1a exhibited a straight line
(Kd = 0.25 µM). The number of
hemoglobin binding sites per promastigote was found to be 1.2 × 105. The binding of 125I-hemoglobin to
Leishmania was effectively inhibited by unlabeled hemoglobin
(Fig. 1b) with 50% inhibition achieved at about 0.09 µM (6 µg/ml) hemoglobin. When cells were incubated with
0.09 µM (6 µg/ml) of 125I-hemoglobin at
4 °C along with 10-fold excess of hemocyanin, transferrin, hemin,
globin, and myoglobin, the binding of 125I-hemoglobin was
not affected (Fig. 2), indicating the
specificity of these binding sites for hemoglobin.

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Fig. 1.
a, binding of
125I-hemoglobin to Leishmania donovani
promastigotes at 4 °C. Promastigotes (5 × 107/ml)
were incubated with 125I-hemoglobin at 4 °C for 4 h
and processed as described under "Experimental Procedures." Data,
expressed as femtomoles of labeled protein bound/µg of cellular
protein, represent an average of three determinations ± S.E.
Inset shows the Scatchard plot of the binding data.
Kd value as determined from the slope was found to
be 0.25 µM while the number of sites per promastigote was
derived by dividing the product of the abscissa and Avogadro's number
by the number of cells taken per milliliter. b, competition
by unlabeled hemoglobin for binding of
125I-hemoglobin by Leishmania.
Promastigotes (5 × 107/ml) were incubated
with 125I-hemoglobin (0.09 µM) and indicated
concentrations of unlabeled hemoglobin for 4 h at 4 °C. Data
represent an average of three determinations ± S.E.
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Fig. 2.
Effect of various compounds on the binding of
125I-hemoglobin at 4 °C. Promastigotes
(5 × 107/ml) were incubated with 0.09 µM labeled hemoglobin in the absence or presence of
10-fold excess of the indicated compounds at 4 °C for 4 h.
Data, expressed as percentage of hemoglobin bound by the cells in the
absence of any competitor (100% bound), represent an average of three
determinations ± S.E.
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Uptake and Degradation of 125I-Hemoglobin at
37 °C--
When promastigotes were incubated with different
concentrations of 125I-hemoglobin at 37 °C for 3 h,
cell-associated radioactivity increased in a saturable fashion while a
part of the added radioactivity was released into the medium as
trichloroacetic acid-soluble material (Fig.
3a). The half-maximal value
for uptake and degradation was achieved at about 0.065 µM
hemoglobin. The data in Fig. 3b show, that when the
promastigotes were incubated with varying concentrations of
125I-hemoglobin at the optimal growth temperature of
25 °C, the content of cell-associated radioactivity
reached saturation with a half-maximal value of 0.06 µM
indicating expression of hemoglobin-binding sites of similar affinity
and number as at 37 °C. The amount of trichloroacetic acid-soluble
radioactivity released in the medium, however, was about one-fifth of
that at 37 °C. These results indicate that, even at 25 °C,
Leishmania takes up hemoglobin and then degrades it, albeit
to a lesser extent than at 37 °C. When Leishmania
promastigotes were incubated with 125I-hemoglobin (0.09 µM) at 37 °C for different time periods, cellular radioactivity reached a steady state plateau within 30 min while trichloroacetic acid-soluble radioactivity continued to increase at a
linear rate (Fig. 4), indicating the
simultaneous uptake and degradation of 125I-hemoglobin.

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Fig. 3.
Concentration dependence of uptake and
degradation of 125I-hemoglobin by L. donovani
promastigotes at 37 °C or 25 °C.
Promastigotes (5 × 107/ml) were incubated with
indicated concentrations of 125I-hemoglobin at 37 °C
(a) or 25 °C (b). After incubation for 3 h, the amounts of radioactivity accumulated in the cells and the
trichloroacetic acid-soluble (non-iodide) degradation products of the
labeled protein released in the medium were determined as described
under "Experimental Procedures." Data, expressed as femtomoles of
labeled protein taken up or degraded/µg of cellular protein,
represent an average of three determinations ± S.E.
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Fig. 4.
Time course of uptake and degradation of
125I-hemoglobin at 37 °C. Promastigotes
(5 × 107/ml) were incubated with 0.09 µM 125I-hemoglobin at 37 °C. After
indicated time intervals, cell-associated radioactivity and
trichloroacetic acid-soluble non-iodide products of
125I-hemoglobin degradation released in the medium were
determined. Data represent an average of three determinations ± S.E.
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Uptake of 125I-hemoglobin at 37 °C was inhibited by
unlabeled hemoglobin, whereas globin and hemin did not significantly
inhibit the uptake of hemoglobin, indicating that the binding sites
recognized hemoglobin as a whole (Fig.
5a). Unlabeled hemoglobin, but
not globin, competed for the degradation of 125I-hemoglobin
(Fig. 5b). Surprisingly, about 80% of the
125I-hemoglobin degradation was inhibited by 45 µM hemin (Fig. 5b). We also noted that hemin
(15 µM) inhibited the scavenger receptor-mediated degradation of maleylated bovine serum albumin in a macrophage cell
line (data not shown). In contrast, hemoglobin degradation by
Leishmania could not be inhibited by lysosomotropic agents chloroquine, monensin, or ammonium chloride (data not shown), which are
potent inhibitors of lysosomal degradative processes in mammalian
cells.

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Fig. 5.
Effect of hemin, globin, and unlabeled
hemoglobin on the uptake and degradation of 125I-hemoglobin
by L. donovani promastigotes. Promastigotes (5 × 107/ml) were incubated with 0.09 µM
125I-hemoglobin at 37 °C in presence of indicated
concentrations of unlabeled hemoglobin, hemin and globin. The
cell-associated radioactivity (a) and trichloroacetic
acid-soluble degradation products (b) were expressed as
femtomoles of protein taken up or degraded/µg of cellular protein.
Results are an average of three determinations ± S.E.
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Internalization of Hemoglobin by Leishmania--
When
Leishmania previously incubated with hemoglobin at 4 °C
were treated with a rabbit anti-hemoglobin antibody followed by an
anti-rabbit IgG conjugated with colloidal gold, accumulation of gold
particles could be seen in the flagellar pocket (Fig. 6, b and e). In
contrast, no gold particles were detected in the cells not incubated
with hemoglobin (Fig. 6, a and d) or
anti-hemoglobin antibody (Fig. 6, c and f).

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Fig. 6.
Localization of hemoglobin in the flagellar
pocket by immunogold labeling. Leishmania promastigotes
(5 × 105) were incubated with (b,
c, e, and f) or without (a
and d) 6 µg/ml hemoglobin for 2 h at 4 °C The
cells were washed to remove the unbound hemoglobin, fixed, and
processed for immunogold labeling as described under "Experimental
Procedures." Panels c and f show
cells incubated with hemoglobin but without primary anti-hemoglobin
antibody. Panels a, b, and
c represent the longitudinal section of the flagellar
pocket, and panels d, e, and
f represent the cross-section of the flagellar pocket.
Original magnification, ×50,000 (a-c), ×70,000
(d and f), and ×40,000 (e).
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To explore the fate of hemoglobin bound with high affinity to the sites
in the flagellar pocket, we incubated the Leishmania with
hemoglobin-gold for 1 h either at 37 °C (Fig.
7, a and b) or at
25 °C (Fig. 7, c and d) and localized the gold
particles by electron microscopy. The results presented in Fig. 7
(a and c) show that the gold particles are
localized in discrete intracellular vesicles, suggesting the
internalization of hemoglobin at both temperatures. This result also
indicated that transport of hemoglobin in Leishmania is
mediated through vesicle fusion. The uptake of hemoglobin-gold
particles was inhibited by incubating the cells with free hemoglobin in
similar condition at both temperatures (Fig. 7, b and
d), suggesting that uptake is mediated through specific
binding sites as evidenced in the biochemical assay. In order to
quantitate the uptake of hemoglobin-gold by Leishmania, we
counted the number of gold particles internalized by 50 cells. The data
in Table I show that
Leishmania internalized about 371 gold particles in discrete
vesicular structures when the cells were incubated at 37 °C with
hemoglobin-gold. In contrast, only 86 particles were detected when the
Leishmania were incubated with the same concentration of
hemoglobin-gold in presence of excess unlabeled hemoglobin. The uptake
of BSA-gold, presumably through nonspecific fluid phase endocytosis,
was about 10% of that of hemoglobin-gold. Similar results were also
obtained when the same experiments were carried out at 25 °C (Table
I).

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Fig. 7.
Electron micrograph showing the intracellular
localization of the hemoglobin-gold inside the Leishmania.
Promastigotes (5 × 105) were incubated with
hemoglobin-gold (1 µg/ml) for 1 h in the absence (a
and c) or presence (b and d) of excess
unlabeled hemoglobin at 37 °C (a and b) or
25 °C (c and d). The cells were washed and
processed for electron microscopy. Original magnification,
×25,000.
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To follow the kinetics of hemoglobin uptake, after binding hemoglobin
at 4 °C the washed cells were incubated for different periods of
time at 37 °C, treated with anti-hemoglobin antibodies, and
visualized by subsequent probing with secondary antibodies labeled with
FITC. Immediately after binding at 4 °C hemoglobin was visualized as
a bright fluorescent spot (Fig.
8a) in the apical region of
Leishmania. After warming to 37 °C, progressive dispersal of the staining pattern began by 2 min, and by 15 min the fluorescence dispersed throughout the cell body (Fig. 8d).

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Fig. 8.
Time course of internalization at 37 °C of
hemoglobin bound at 4 °C by Leishmania
promastigotes. Cells (5 × 107) were
incubated at 4 °C for 2 h with hemoglobin (5 µg/ml) to allow
binding with cell surface. Subsequently, cells were washed to remove
unbound hemoglobin and incubated at 37 °C for 0 min (a),
2 min (b), 5 min (c), and 15 min (d).
Hemoglobin molecules were visualized by anti-hemoglobin antibody probed
with respective anti-mouse FITC-labeled second antibody. There was no
staining in cells incubated in absence of hemoglobin or the cells
processed without anti-hemoglobin antibodies.
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Identification of Putative Hemoglobin-binding Proteins on
Leishmania--
In order to identify the putative hemoglobin-binding
sites, surface membrane fractions prepared from intact biotinylated
promastigotes were solubilized with detergent and subjected to affinity
chromatography on hemoglobin-agarose beads. The proteins from the
Leishmania surface membranes bound to hemoglobin-agarose
affinity matrix were separated by SDS-PAGE, and the biotinylated
proteins were detected by Western blot analysis using avidin-HRP, which
revealed a single band with a molecular mass of 46 kDa (Fig.
9, Control). Preincubation of
the membrane preparation with hemoglobin effectively blocked the
adsorption of the 46-kDa protein to hemoglobin-agarose beads, as can be
seen by the reduced intensity of the band in the middle
lane. In contrast, preincubation with hemin did not affect
the binding of the protein to hemoglobin-agarose (last lane). Therefore, the 46-kDa band is likely to be the
putative hemoglobin-binding protein on Leishmania
surface.

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Fig. 9.
Western blot analysis of proteins recovered
by hemoglobin-agarose affinity purification of detergent extracts of
L. donovani membranes. Promastigotes were
biotinylated, and the membrane fraction obtained thereafter was
solubilized and incubated with hemoglobin-agarose beads as described
under "Experimental Procedures." The proteins eluted from
hemoglobin-agarose beads were subjected to SDS-PAGE, transferred to
nitrocellulose membrane, and stained with avidin-HRP. Lane
1, control. Lanes 2 and 3,
membrane fractions pre-incubated with hemoglobin and hemin,
respectively.
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DISCUSSION |
Our study demonstrates absorptive endocytosis of hemoglobin by
L. donovani promastigotes in vitro. At 4 °C
125I-hemoglobin binds to Leishmania in a
saturable manner, indicating that it is recognized by a limited number
of binding sites on the cell surface (Fig. 1a). The binding
sites recognize hemoglobin as a whole, which was evident from the fact
that, although unlabeled hemoglobin effectively competed for the
binding of 125I-hemoglobin, free hemin and
globin (Figs. 1b and 2), as well as the heme-containing
and
chains of hemoglobin (data not shown) did not inhibit the
binding. The exquisite specificity of these binding sites for the
particular conformation of intact hemoglobin molecule is further
highlighted by the fact that hemoglobin binding to
Leishmania is not inhibited by myoglobin. Binding of hemoglobin was also not competed by hemocyanin (protein containing copper-tetrapyrrole) or transferrin (Fig. 2).
At higher temperatures (25 °C or 37 °C), hemoglobin is
internalized and degraded up to the monoiodotyrosine level (Fig. 3, a and b). Uptake of hemoglobin at 37 °C
reached a steady state within 60 min, while the degradation continued
to show a linear rise, indicating simultaneous uptake and degradation
of hemoglobin by the parasite (Fig. 4). Hemin and globin did not show
any significant competition for uptake (Fig. 5a). Globin
also did not compete for degradation of hemoglobin, but hemin caused
substantial inhibition of hemoglobin degradation (Fig. 5b).
It is possible that hemin-mediated impairment of hemoglobin degradation
in Leishmania might act as a feedback mechanism precluding
intracellular accumulation of excessive heme.
Three lines of evidence suggest that after initial binding with cell
surface receptor hemoglobin is internalized by Leishmania. 1) Electron microscopic studies showed that at 4 °C hemoglobin bound
to specific sites located in the flagellar pocket of
Leishmania (Fig. 6, b and e), whereas
at 37 °C the hemoglobin was found distributed throughout the cell.
After initial binding with the sites localized in the flagellar pocket
at 4 °C, hemoglobin was rapidly internalized by the cells as shown
by the fluorescence microscopic studies (Fig. 8). When the cells were
warmed for only 2 min, most of the hemoglobin was detected inside the
cell presumably in the early endosome-like compartments (Fig.
8b). After 5 min, hemoglobin was detected in discrete
intracellular vesicles (Fig. 8c) and was eventually
dispersed throughout the cell body by 15 min (Fig. 8d). 2)
When the cells were incubated with hemoglobin-gold at 37 °C or
25 °C for 1 h, gold particles were found to be localized in
discrete intracellular vesicles throughout the cell body (Fig. 7,
a and c). Competition of hemoglobin-gold uptake
by free hemoglobin indicated the specificity of hemoglobin endocytosis
(Fig. 7, b and d; Table I). 3) Biotinylated
hemoglobin bound by Leishmania at 4 °C could be detected
in endocytic vesicles isolated after short incubation of the cells at
37 °°C (data not shown). These results suggest that,
after high affinity binding with specific sites on the cell surface,
hemoglobin is internalized through a vesicular transport pathway.
In contrast to mammalian cells, endocytosis occurs in the
trypanosomatid parasites through the flagellar pocket, although the
exact nature of these compartments is not known (4). It has been shown
that, after binding to specific receptors localized in the flagellar
pocket, rapid endocytosis of macromolecular nutrients such as low
density lipoprotein or transferrin occurs in trypanosomatid parasites.
We have demonstrated that the hemoglobin-binding sites in
Leishmania located in the flagellar pocket mediate rapid
endocytosis of bound hemoglobin. Transferrin receptor is known to be
recycled in mammalian cells (31). In contrast, it has been demonstrated in Trypanosoma that both transferrin receptor along with the
ligand are targeted to a lysosome-like compartment (32). Our data
showing degradation of 125I-hemoglobin to trichloroacetic
acid-soluble material primarily to the level of monoiodotyrosines are
also consistent with existence of such a degradative compartment in
Leishmania. The insensitivity of hemoglobin degradation by
Leishmania to potent inhibitors of lysosomal degradative
processes in mammalian cells chloroquine, monensin, or ammonium
chloride (33, 34) remains to be explained, but could presumably be
related to the relatively higher pH values (
6) of endolysosomal
compartments in protozoan parasites (35). It will be interesting to
determine the nature of the intracellular compartments to establish the
pathway of hemoglobin transport inside the cell. It would also be
interesting to determine whether the intracellular transport of
hemoglobin is regulated by vesicle fusion. Actually, in some of the
micrographs, we were able to detect dumbbell-shaped vesicles containing
gold particles, indicating the possible fusion of different
intracellular compartments (Fig. 7a). However, further work
would be needed to establish the nature of the compartments and the
mechanism of the fusion.
A single biotinylated protein of 46 kDa was revealed when solubilized
membranes from surface biotinylated intact Leishmania adsorbed by hemoglobin-agarose beads were subjected to SDS-PAGE and
Western blotting with avidin-HRP (Fig. 9). This further confirms that
the hemoglobin-binding protein is present on the Leishmania membrane, as only cell surface proteins would be biotinylated under the
conditions employed (29). Furthermore, preincubation of the membrane
fraction with hemoglobin could compete this band out, suggesting the
hemoglobin-binding specificity of the 46-kDa protein. Preincubation
with hemin did not result in any competition, indicating that the
hemoglobin-binding protein on the Leishmania membrane does
not interact with hemin.
Thus, in the present investigation, we have shown that hemoglobin binds
with high affinity through specific binding sites located in the
flagellar pocket of Leishmania. The binding of hemoglobin
with Leishmania surface membrane is mediated through a
putative receptor protein of apparent molecular mass of 46 kDa. After
high affinity binding with cell surface receptor, hemoglobin is rapidly
internalized into discrete intracellular compartments. To the best of
our knowledge, this is the first report demonstrating the existence of
a vesicular pathway for transport of hemoglobin in
Leishmania. It will be interesting to determine the
mechanism of hemoglobin trafficking inside the Leishmania
and the role of Rab-like GTPases in this transport. Further studies are
in progress to determine the role of signal transduction intermediates
in the mechanism of hemoglobin transport.
 |
ACKNOWLEDGEMENTS |
We thank Dr. S. N. Upadhyay for support
in carrying out electron microscopy studies and Dr. A. C. Banerjea, Dr. S. Rath, and Dr. C. M. Gupta for critically
reviewing the manuscript.
 |
FOOTNOTES |
*
This work was supported by grants from the Department of
Biotechnology to the National Institute of Immunology and Jawaharlal Nehru Center for Advanced Scientific Research (to S. K. B.).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.:
9111-617-5002; Fax: 9111-610-9433 or 9111-616-2125; E-mail:
sandip{at}nii.res.in.
The abbreviations used are:
HRP, horseradish
peroxidase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis.
 |
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