Tracing heme in a living cell: hemoglobin degradation and heme traffic in digest cells of the cattle tick Boophilus microplus
1 Departamento de Bioquímica Médica, ICB, Universidade Federal
do Rio de Janeiro, Brazil
2 Departamento de Virologia, IMPPG, Universidade Federal do Rio de Janeiro,
Brazil
3 Departamento de Patologia Veterinária, Universidade Estadual
Paulista UNESP, Campus Jaboticabal, São Paulo, Brazil
* Author for correspondence (e-mail: pedro{at}bioqmed.ufrj.br)
Accepted 13 June 2005
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Summary |
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We followed the fate of hemoglobin and albumin in primary cultures of digest cells by incubation with hemoglobin and albumin labeled with rhodamine. Uptake of hemoglobin by digest cells was inhibited by unlabeled globin, suggesting the presence of receptor-mediated endocytosis. After endocytosis, hemoglobin was observed inside large digestive vesicles. Albumin was exclusively associated with a population of small acidic vesicles, and an excess of unlabeled albumin did not inhibit its uptake. The intracellular pathway of the heme moiety of hemoglobin was specifically monitored using Palladiummesoporphyrin IX (Pd-mP) as a fluorescent heme analog. When pulse and chase experiments were performed using digest cells incubated with Pd-mP bound to globin (Pd-mP-globin), strong yellow fluorescence was found in large digestive vesicles 4 h after the pulse. By 8 h, the emission of Pd-mP was red-shifted and more evident in the cytoplasm, and at 12 h most of the fluorescence was concentrated inside the hemosomes and had turned green. After 48 h, the Pd-mP signal was exclusively found in hemosomes. In methanol, Pd-mP showed maximal emission at 550 nm, exhibiting a red-shift to 665 nm when bound to proteins in vitro.
The red emission in the cytosol and at the boundary of hemosomes suggests the presence of heme-binding proteins, probably involved in transport of heme to the hemosome. The existence of an intracellular heme shuttle from the digestive vesicle to the hemosome acting as a detoxification mechanism should be regarded as a major adaptation of ticks to a blood-feeding way of life. To our knowledge, this is the first direct observation of intracellular transport of heme in a living eukaryotic cell. A similar approach, using Pd-mP fluorescence, could be applied to study heme intracellular metabolism in other cell types.
Key words: palladium mesoporphyrin, endocytosis, hematophagy, hemosome, cattle tick, Boophilus microplus
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Introduction |
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The final step of heme biosynthesis, the insertion of the metal in the
porphyrin ring, catalyzed by ferrochelatase, occurs inside the mitochondria;
assembly of hemeproteins occurs not only in the mitochondria, but also in the
cytosol and inside the endoplasmic reticulum
(Asagami et al., 1994;
Ponka, 1999
). In addition,
heme degradation by the microsomal heme oxygenase plays a critical role in
cellular metabolism (Maines,
2003
), being induced in response to stress in several organisms
(Shibahara et al., 1987
). Heme
oxygenase acts not only as a protection against the potential toxicity of
heme, but also as a source of CO and biliverdin, which are important in cell
signaling and as free radical scavengers
(Dore et al., 1999
;
Stocker and Peterhans, 1989
).
However, very little information is available on the precise pathways involved
in heme traffic inside eukaryotic cells.
Hematophagous arthropods face a very special situation in relation to heme
metabolism since they ingest several times their own mass in vertebrate blood,
which contains approximately 10 mmol l1 heme bound to
hemoglobin. Insects such as Triatominae bugs and mosquitoes digest their blood
meals extracellularly, and most of the heme is retained in the gut lumen by
means of extracellular protective matrixes secreted by gut epithelial cells.
In the gut lumen of the kissing bug Rhodnius prolixus, a vector of
Chagas disease, perimicrovillar phospholipid membranes convert heme into a
special aggregate, hemozoin, also found in the malaria parasite (Oliveira et
al., 1999,
2000
). In the mosquito
Aedes aegypti, heme is retained in the peritrophic matrix, a layer of
protein and polysaccharides that covers the midgut and separates gut cells
from the blood bolus (Páscoa et
al., 2002
). In contrast, the luminal region of tick midgut is
crowded with digest cells, which degrade blood components intracellularly.
Hemoglobin is thought to be hydrolyzed in the digestive vacuoles of these
cells (Agbede et al., 1985; Walker and
Fletcher, 1987
) by the action of acid proteases (Mendiola et al.,
1997), therefore releasing huge amounts of heme inside these cells. We have
recently shown that most of the heme produced by this process is ultimately
accumulated inside a specialized organelle called a hemosome, forming a unique
type of heme aggregate (Lara et al.,
2003
). Using globin reconstituted with Pd-mP, a fluorescent
metalloporphyrin employed here as a heme analog, we studied hemoglobin
degradation in living digest cells of the cattle tick Boophilus
microplus. We present evidence characterizing an intracellular pathway
that starts with hemoglobin endocytosis, followed by hydrolysis of the globin
polypeptide chain in a specific population of vesicles, then transport
mediated by cytosolic proteins, and finally concentration of heme inside
hemosomes.
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Materials and methods |
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The partially engorged females were mechanically detached from Babesia spp.-free cattle infested by Boophilus microplus, in the Centro de Pesquisas em Sanidade Animal (CPPAR), UNESP-Jaboticabal, São Paulo, Brazil.
Protein labeling
Globin and albumin were labeled for 1 h with rhodamine isothiocyanate
(Molecular Probes, Eugene, OR, USA) in 0.1 mol l1 sodium
bicarbonate buffer, pH 9.0. The reaction was stopped by adding 1.5 mol
l1 hydroxylamine, pH 8.5
(www.probes.com/media/pis/mp00143.pdf).
Unbound probe was removed by sequential dialysis against 0.15 mol
l1 NaCl, 10 mmol l1 sodium phosphate, pH
7.2 (PBS).
Pd-mP-globin
The globin solution was prepared as follows: bovine hemoglobin was
denatured and heme removed in acetoneHCl 0.02 mol l1
kept in an ethanoldry ice bath, as described
(Ascoli et al., 1981).
Precipitated protein was washed once and renaturated by sequential dialysis
against water. An equimolar amount of Pd-mP (Frontier Scientific, Logan, UT,
USA) was added to the refolded protein at a final concentration of 100 µmol
l1 and a new dialysis against water was performed, in order
to remove unbound Pd-mP. The Pd-mP-globin solution was centrifuged at 12 000
g and the supernatant was filtered using a sterile 0.22 µm
pore size membrane and stored at 4°C.
Artificial feeding of Boophilus microplus
Partially engorged females were fed by means of a 100 µl glass capillary
filled with 100 µmol l1 Pd-mP-globin prepared as
described above. Ticks were fed for 12 h at 38°C and 20% relative
humidity, as described by de la Vega et al.
(2000). Subsequently, the
females were dissected in PBS after different intervals, and anterior midguts
were fixed and processed as described below.
Digest cells culture
Fully engorged females from the second day after a blood meal (ABM) were
rinsed in 70% ethanol for 1 min, and dissected in sterile PBS containing 200 U
ml1 streptomycin and penicillin. To obtain primary cultures,
midguts were isolated in sterile Petri dishes and digest cells were detached
from the gut wall with sterile tweezers. Cells were carefully collected using
a 1 ml pipette tip, washed three times in the same buffer and then placed in a
12-well culture plate with L-15 Leibowitz's medium supplemented with 150 mmol
l1 NaCl plus 100 U ml1 each streptomycin
and penicillin. Bovine fetal serum proved to be cytotoxic and was omitted from
all incubations. Cells were kept at 28°C until use and were viable for
several weeks under these conditions. In endocytosis experiments, cells were
incubated with culture medium containing rhodaminealbumin,
rhodaminehemoglobin (20 µmol l1) or Pd-mP-globin
(100 µmol l1) for 2 or 4 h, washed twice and observed
immediately after the incubations. Alternatively, in pulse-chase experiments,
cells were washed and incubated in medium without Pd-mP-globin at different
time intervals as described in the figure legends.
Histological preparation and fluorescence microscopy
Engorged females were dissected in modified Karnovsky's fixative (2.5%
glutaraldehyde, 4% paraformaldehyde, 0.1 mol l1
CaCl2 and 0.1 mol l1 sodium cacodylate buffer, pH
7.3). Tissues were transferred to fresh fixative and kept at 4°C for 12 h.
Segments of the anterior portion of the midgut were then washed in 0.1 mol
l1 sodium cacodylate buffer pH 7.3. The tissues were
dehydrated in ethanol and embedded in Historesin (Leica). Semi-thin (5 µm)
sections were observed by differential interference contrast (DIC) using an
Axioplan 2 microscope (Zeiss). The fluorescence images were obtained using a
filtered 100 W mercury lamp as the excitation light source with a filter set
Zeiss-09 (BP450490 nm/FT 510 nm/LP 515 nm).
Digest cells were observed in culture using a coverslip with a 10 µm spacer. Fluorescence images were obtained using the same filter set described above or, alternatively, using a filter set Zeiss-02 (G 365 nm/FT 395 nm/LP 420 nm). The confocal images were acquired in a Zeiss Meta 510 laser scanning microscope, with an excitation laser of 514 nm. Emission spectra from selected areas of the image (hemosome, cytosol and digestive vesicle) were obtained using the Zeiss Meta 510 software.
Fluorescence and absorption spectroscopy
Palladium meso-porphyrin IX stock solution (10 mmol l1)
was prepared in 0.1 mol l1 NaOH and diluted to 1 mmol
l1 in methanol. 10 µmol l1 Pd-mP
solutions were made in water, methanol or PBS with globin (10 µmol
l1) or albumin (10 µmol l1). Absorption
spectra were acquired in a GBC 920 (Camberra, Melbourne, Australia)
spectrophotometer. Fluorescence spectra and fluorescence decay time course
were recorded on a Varian Eclipse spectrofluorimeter (Palo Alto, CA, USA).
Liposomes of soybean phosphatidylcholine (Sigma, St Louis, MO, USA) were
prepared as described (Persechini et al.,
1992) and used at 0.015% (w/v) in 10 mmol l1
Hepes buffer, pH 7.2.
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Results |
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Pd-mP-globin uptake by digest cells in vivo
Partially engorged adult females were fed using bovine plasma enriched with
Pd-mP-globin for 12 h, using an artificial feeding system. Ticks were
dissected 2 and 6 h after feeding and midguts were fixed and embedded in
methacrylate. At 2 h (Fig. 5A),
Pd-mP was visible as a yellow-green fluorescence, associated mainly with the
large vesicles of digest cells (the vesicles implicated in hemoglobin uptake
in Fig. 1A), but some
fluorescence was also found in the cytosol, in contrast to hemosomes, which
were almost devoid of label at this time
(Fig. 5A). At 6 h
(Fig. 5B), the Pd-mP
fluorescence had reached the hemosomes, showing that the Pd-mP generated from
digestion of Pd-mP-globin in vivo followed the same path suggested
previously for heme accumulation in the digest cell
(Lara et al., 2003).
|
|
In order to compare the fluorescence profile of the images obtained in the microscope with the emission spectra observed in Fig. 3, the fate of Pd-mP-globin after endocytosis by digest cells was evaluated by spectral analysis using confocal microscopy with excitation using a 514 nm laser (Fig. 7A). Images were acquired at a succession of defined wavelengths over the 530700 nm range. Representative areas of the image were selected and analysis of their emission spectra (Fig. 7B) showed the predominance of a red emission in the cytosol, contrasting with the higher intensity of the green emission in the hemosome. Digestive vacuoles exhibited an intermediate profile, probably as a consequence of Pd-mP-globin degradation, as described in Fig. 4.
|
The time course of hemoglobin digestion was also followed using an excitation bandpass filter of 350400 nm (Fig. 8). This excitation wavelength resulted in a higher level of autofluorescence of digest cells. However, the higher emission intensity allowed a brief Pd-mP labeling pulse of only 2 h, which resulted in a more precise discrimination of events during traffic of heme in the cell. By 6 h Pd-mP was clearly detected in the cytosol as a red fluorescence (Fig. 8), suggesting association of the probe with proteins. At the same time, Pd-mP labeling reached the hemosomes, several showing fluorescence concentrated at the border of the organelle as a bright red emission, suggesting interaction with proteins (Fig. 8, inset). Interestingly, a few hemosomes already showed the characteristic green emission associated to the hemosome core, similar to what was observed in Fig. 6.
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Discussion |
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Literature on the biology of ticks has described digest cells as phagocytic
cells (Agbed and Kemp, 1985; Walker and
Fletcher, 1987). However, the difficulty in obtaining evidence for
phagocytosis of red blood cells led some authors to postulate that endocytosis
of blood proteins may occur by pinocytosis
(Koh et al., 1991
). Here we
show that albumin and hemoglobin, the two main proteins of vertebrate blood,
are not handled in the same way (Fig.
1). Albumin seems to be taken up by non-specific, probably
fluid-phase, endocytosis, as there is no competition between labeled and
unlabeled protein. Once inside the cell, albumin is directed to a population
of small acidic vesicles (Figs
1B and
2). Hemoglobin, on the other
hand, seems to be recognized by specific cell-surface receptors, which target
it towards a population of large vesicles
(Fig. 1A) that are also capable
of acidification (Fig. 2). This
is consistent with the presence of proteases having acid pH optima in the
midgut of ticks (Mendiola et al.,
1996
), but the presence of two independent endocytotic pathways
points to the need for subcellular localization of each protease in order to
establish its precise role in blood digestion. In mammals, macrophages are the
main site of heme degradation, and an acute phase protein, CD163, has been
recently identified as the macrophage hemoglobin scavenger receptor
(Kristiansen et al., 2001
). In
this case, however, the receptor mediates endocytosis of the
haptoglobinhemoglobin complex, whereas tick cells are able to take up
hemoglobin alone. Whatever the molecular nature of the digest cell receptor
for hemoglobin, the existence of a distinct apparatus receptor, cell
compartments and possibly proteases and membrane transporters
dedicated to digestion of hemoglobin can be understood as a protection against
the potential toxicity of the heme group, and it illustrates the complexity of
adaptation to digestion of hemoglobin. In the case of mammalian macrophages,
several other protein ligands are taken up by endocytosis
(Cherukuri et al., 1998
), but
the possibility of a distinct path for hemoglobin hydrolysis has not been
investigated.
The Pd-mP fluorescence emission is sensitive to its molecular environment
and a marked red shift was produced when the probe is bound to globin/protein
(Fig. 3). During in
vitro proteolytic digestion of Pd-mP-globin, intermediate profiles were
obtained (Fig. 4) due to a
mixture of native, undigested globin, and the metalloporphyrin splitting out
from the heme pocket upon loss of tertiary structure that follows cleavage of
the globin polypeptide chain, similar to what happens during hemoglobin
digestion in the digestive vacuole of the malaria parasite
(Goldberg et al., 1990). In
living tick digest cells, similar intermediate emission spectra were also
observed during Pd-mP-globin digestion (Figs
6 and
7), resulting in the
characteristic yellow fluorescence of the digestive vesicles. Being a mixture
of green and red, a yellow emission might be due to association of Pd-mP with
a phospholipid bilayer (Fig.
5). However, as the large digestive vesicle is the site of
hemoglobin hydrolysis, a more plausible explanation may be that it contains
both undigested Pd-mP bound to globin together with substantial amounts of
free Pd-mP produced inside the large digestive vesicles. In contrast, when
Pd-mP was released from the digestive vesicles into the cytosol, its emission
spectrum shifted again to red, pointing to association with proteins (Figs
7 and
8), in accordance with the
general assumption that heme in the cytosol is always bound to proteins. An
even more intense red shift was observed when the fluorophore reached the edge
of the hemosome (Fig. 8),
suggesting that the initial events in the entrance of heme into the hemosome
may include an integral membrane protein transporter. ABC cassette
transporters have been implicated in translocation of heme across membranes
both in bacteria and mitochondria
(Köster, 2001
). However,
further Pd-mP accumulation into the hemosome resulted in replacement of the
red emission by the green fluorescence that predominates at later chase times
(Fig. 6CF), which is in
accordance with our previous result (Lara
et al., 2003
) showing that the hemosome core has a low protein
content and is made up of 90% heme. Heme segregation in the hemosome is
thought to be a protective mechanism, acting by reducing availability of heme
to participate in redox reactions that generate free radical species (not
shown), in a way analogous to the formation of hemozoin, the heme aggregate of
the malaria parasite. Hemozoin formation inhibited free radical production by
steric hindrance of substrate access to heme molecules in the interior of the
aggregate particle (Oliveira et al., 2002).
Ticks are obligate ectoparasites that have vertebrate blood as their only
source of nutrients. They are thought to have evolved a blood-feeding life
style in the Paleozoic era, before the advent of mammals
(Hoogstraal and Aeschlimann,
1982). The development of efficient heme detoxification
mechanisms, in particular hemosomes, should be regarded as an essential aspect
of their evolutionary track to hematophagy. Some of the protein components of
the intracellular machinery used to transport heme to the hemosome may be new,
tick-specific proteins; once identified, these may be the objects of future
research directed to the development of new methods of population control of
the parasite. On the other hand, it is plausible to speculate that conserved
proteins related to heme trafficking in other organisms may also have been
recruited in the tick to achieve safe, efficient heme accumulation in the
hemosome. Accordingly, the tick digest cell may prove useful as a general
model for studying the cell biology of heme.
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List of abbreviations |
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Acknowledgments |
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