From the
Heme in aqueous solutions actively promotes free radical
reactions leading to degradation of biological molecules. The
blood-sucking insect Rhodnius prolixus has a heme-binding
protein (RHBP) in its hemolymph (Oliveira, P. L., Kawooya, J. K,
Ribeiro, J. M. C., Meyer, T., Poorman, R., Alves, E. W., Walker, F.,
Padovan, G. J., and Masuda, H. (1994) J. Biol. Chem. 270,
10897-10901. Here we show that this protein inhibits
heme-dependent peroxidation of both linolenic acid liposomes and
lipophorin, the main lipoprotein of insect hemolymph. The oxidized
lipophorin is functionally impaired, being defective both in its
capacity to be loaded with phospholipids from the fat body as well as
in its ability to deliver phospholipids to the growing oocytes. RHBP
prevents the heme-induced oxidative damage to lipophorin. It is
proposed that in vivo RHBP binds the heme derived from
digestion of blood hemoglobin, suppressing the generation of activated
oxygen species and protecting the insect against oxidative stress
throughout the feeding cycle.
Free radical reactions injure cells and tissues by causing
oxidative damage to several classes of biomolecules
(1, 2) . Iron and iron-containing organic molecules such
as heme (Fe-protoporphyrin IX) are well-known catalysts of the
formation of activated oxygen species
(2, 3, 4, 5, 6) . Several
defensive mechanisms have arisen during the course of evolution to
protect cells from iron-induced oxidative injury
(2) . These
include proteins such as ferritin, transferrin, haptoglobin, and
hemopexin, which are capable of binding iron or iron-containing
molecules to form complexes that do not promote formation of free
radicals
(7) .
Hematophagous insects usually ingest in a
single meal large amounts of vertebrate blood
(8) , which has
about 10 mM heme bound to hemoglobin. After the meal, water is
rapidly excreted by the Malpighian tubules
(9) , leading to even
higher heme concentrations in the lumen of the digestive apparatus.
These insects therefore face an oxidative challenge due to heme release
(and probably also release of adventitious iron) following digestion of
hemoglobin by midgut proteinases. In Rhodniusiron
accumulates in pericardial cells and midgut
(10) , and in ticks,
host blood heme is utilized for synthesis of their own hemeproteins
(11, 12) . Very little is known, however, about intra-
and extracellular antioxidant defenses of blood-sucking insects.
The
accompanying article describes a heme-binding protein that is found in
the hemolymph of Rhodnius prolixus(13) . Here
we show that this protein, RHBP,
Hemin is known to increase in vitro peroxidation of
polyunsaturated fatty acids leading to formation of several breakdown
products, including malondialdehyde
(4) . The presence of RHBP
blocked the heme-induced fatty acid peroxidation (,
experiment 1).
When the insect lipoprotein lipophorin was the target
for heme-promoted radical reactions, the lipids bound to the protein
were also susceptible to peroxidation (, experiment 2). As
before, the reaction was blocked by RHBP. also shows that
RHBP is as effective as butylated hydroxytoluene, a general
radical-scavenging antioxidant. In these experiments deferoxamine, an
Fe
An experiment showing the time
course of linolenic acid peroxidation by hemin is depicted in
Fig. 1A, where O
During oogenesis in
R. prolixus, the ovary is the principal acceptor of the
phospholipids carried by the lipophorin particles
(24) . When
[
The foregoing results suggest a
physiological role for RHBP, provided that the hemin that is released
during digestion of a blood meal actually crosses the digestive system
wall and reaches the hemolymph. To test this possibility insects were
fed with rabbit plasma enriched with 0.1-1 mM hemin, and
the level of heme-RHBP appearing in the hemolymph was monitored by
recording its absorption spectrum (Fig. 2). An increase in the
RHBP Soret band at 412 nm occurred in parallel with the concentration
of hemin added to the plasma, indicating that hemin from the gut in
fact reaches the hemolymph.
Iron in the form of redox-active chelates such as heme causes
a number of free-radical reactions
(2) . Reactions of this sort
have been shown to occur in several pathological conditions where there
is uncontrolled availability of iron such as in iron overload or
chemically induced oxidative stress
(1) . Keeping the
concentrations of these compounds as low as possible is therefore a
major task of most organisms
(31) . This is achieved in part by
the action of proteins that are capable of binding these compounds,
thus creating ``safe,'' redox-inactive complexes
(7) .
In the accompanying article we have described a heme-binding
protein, RHBP, from the hemolymph of the blood-sucking insect, R.
prolixus. Here we present evidence that RHBP acts as an
antioxidant capable of blocking hemin-induced lipid peroxidation. Two
independent methods were used to show that purified RHBP effectively
protects linolenic acid micelles from oxidation when present in molar
excess to hemin (Fig. 1 A and ). Lipophorin,
a lipoprotein from the insect's own hemolymph, also is protected
by RHBP from the oxidative challenge (Fig. 1 B and
).
Reports of lipoprotein oxidative damage in
vertebrates describe lipid peroxidation, protein aggregation, and loss
of biological function
(32) . Oxidized lipophorin lacks the
capacity to either load or unload phospholipids normally (Tables II and
III), and RHBP protects lipophorin by blocking the oxidative injury. As
a hydrophobic molecule, heme is expected to partition into the
lipophorin particle as a consequence of its high lipid content. Thus,
in the absence of RHBP, lipophorin would be a major target for radical
reactions occurring in the hemolymph due to hemin absorbed from the gut
after a blood meal (Fig. 2).
The effect of lipophorin
oxidation on its performance as a reusable lipid shuttle is a clear
example of the deleterious consequences of hemin-induced radical
reactions. However, hemin may affect other biological functions through
oxidative damage to proteins and DNA
(5, 33) . There are
also several reports of heme binding to phospholipid bilayers
(34) and partition of heme into membranes has been implicated in
toxicity to malaria parasites
(35) . Additional experiments will
be needed in order to determine whether in Rhodniusother biological functions beside lipid transport are affected by
heme-stimulated radical reactions.
In order to fulfill the role of
an antioxidant, apoRHBP should occur in significant amounts in the
hemolymph. Fig. 3shows that this was the case, suggesting that
the insect is always protected against hemin-induced free radicals.
Hemopexin, a 60-kDa heme-binding protein present in the plasma of
vertebrates
(36) has also been ascribed an antioxidant role
(4, 5) . In the preceding article we describe several
characteristics of RHBP that indicate absence of homology with
hemopexin
(13) . Besides serving as an antioxidant, hemopexin is
involved in heme transport
(37) . A similar function may also
apply to RHBP, as postulated by Wigglesworth
(10) . We have
preliminary evidence suggesting that, when heme-RHBP is injected into
adult insects, heme is taken up into several organs without
accumulation of the RHBP polypeptide chain.
To our knowledge, this is the first report of a
physiological antioxidant in a hematophagous insect. This protein may
have evolved as an adaptation to allow Rhodniusto feed
on blood. From the data available on antioxidant defenses in mammals,
it seems that rather than a single mechanism, the rule is to find
several lines of defense in the same organism, such as antioxidant
enzymes, radical scavengers, iron chelators, or chain reaction-breaking
agents. The relative importance of RHBP in comparison with other
physiologically relevant protectors against oxidative stress in
Rhodniusremains to be evaluated.
Fatty acid micelles or lipophorin were incubated at 37
°C in 0.1 M sodium phosphate, pH 7.2, and 0.1 mM deferoxamine. The reactions were stopped after 90 min by addition
of 0.1 mM butylated hydroxytoluene (BHT) and assayed for
malondialdehyde production. RHBP-free heme-binding sites concentrations
are indicated.
[
We express our gratitude to Dr. Martha M. Sorenson for
a critical reading of the manuscript and to Rosane O. M. M. Costa,
José S. Lima, Jr., and José F. Sourza Neto for excellent
technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
can inhibit
heme-induced lipid peroxidation and counteract the deleterious effects
of heme on an essential biological function: interorgan lipid transport
by lipophorin particles, the major lipoprotein in insect hemolymph.
This effect of RHBP is consistent with a protective role against
heme-induced oxidative stress.
Carrier
free P
Purification
P
purchased from Comissão Nacional
de Energia Nuclear (São Paulo, Brazil) was purified by means of
a Dowex 1
-10 column
(14) .
Insects
Insects were taken from a colony of
R. prolixus maintained at 28 °C and 70-80%
relative humidity. The experimental animals were adult, mated females
fed on rabbit blood at 3-week intervals.
Protein Purification
Hemolymph was collected as
described in the accompanying article
(13) , centrifuged at room
temperature for 5 min at 13,000 g, and the pellet
containing cells was discarded. The supernatant was stored under liquid
N
until protein purification. For the preparation of
lipophorin, the cell-free hemolymph was diluted to 5 ml with PBS (10
mM sodium phosphate, pH 7.4, 0.15 M NaCl) plus 5
mM EDTA and 1.25 g of KBr was added, followed by
centrifugation at 159,000
g at 4 °C for 20 h
(15) . Purified lipophorin was collected from the top of the
gradient, dialyzed against PBS and 5 mM EDTA, and stored under
liquid N
until use. The same protocol was used to purify
radioactive lipophorin from animals which had been fed with blood
enriched with
P
, using a special feeder
described by Garcia et al.(16) . Lipophorin obtained
in this way is labeled only in the phospholipid moiety
(15) .
The bottom fractions of the KBr gradient were pooled and used for RHBP
isolation
(13) . The degree of purification was monitored by
SDS-polyacrylamide gel electrophoresis
(17) , and the protein
concentrations were estimated according to Lowry et al.(18) , using bovine serum albumin as standard.
Determination of RHBP-free Binding Sites
The
concentration of free heme-binding sites in the hemolymph or in each
batch of purified RHBP was monitored by measuring the absorbance of the
Soret band (412 nm) while progressively adding a solution of 1
mM hemin in 0.1 M NaOH
(13, 19) . The
amount of hemin needed to fully saturate the RHBP was determined from
the break in the plot of absorbance at 412 nm against the amount of
added hemin. The difference in absorbance at 412 nm before addition of
hemin and after saturation is reached is proportional to the
concentration of free heme-binding sites, and the amount of hemin
required to saturate the protein provides a measure of total RHBP
levels.
Thiobarbituric Acid Assay
Linolenic acid micelles
were prepared by vortexing in 0.1 M sodium phosphate buffer,
pH 7.2
(20) . Micelles (0.5 mg) or lipophorin solutions (0.23 mg
of protein) were incubated at 37 °C in 0.5 ml of the same buffer
with 0.1 mM deferoxamine added. Hemin and RHBP were added as
indicated for each experiment. Reactions were stopped after 90 min by
adding 0.1 mM butylated hydroxytoluene. Malondialdehyde was
measured by adding 0.2 ml of thiobarbituric acid (1% w/v), incubating
samples at 4 °C for 1 h, followed by 98 °C for 15 min, and
extracting with 0.5 ml of n-butanol before measuring the
absorbance of the organic phase at 532 nm
(21) .
O
O Uptake
consumption
during lipid peroxidation of linolenic acid micelles or lipophorin was
assayed using a Clark-type electrode (YSI, model 5775, Yellow Springs,
OH), calibrated to 100% with air-saturated buffer at room temperature
(4) . Reactions were carried out in 0.1 M sodium
phosphate buffer, pH 7.2, with 0.05 mM deferoxamine and were
started by the addition of 6 mM
-mercaptoethanol. Hemin
and RHBP were included as indicated in the figure legends.
Oxidized Lipophorin
Oxidized lipophorin for the
lipid transport experiments was obtained by preincubation at 37 °C
for 90 min in 0.1 M sodium phosphate buffer, pH 7.2, 20
µM hemin, and 0.1 mM deferoxamine. After this
preincubation, the protein was centrifuged in a ``spin
column'' of Sephadex G-50
(22) previously equilibrated
with culture medium or with PBS, depending on the experiment. The spin
column retains any hemin that is free in solution.
Phospholipid Transfer from
Fat bodies were prelabeled by feeding
adult females with blood enriched with P-Fat Bodies to
Lipophorin
(23
Pi. Two days later,
the insects were dissected, and the radioactive fat bodies were left
adhered to the abdominal cuticle and washed with Rhodnius saline
(9) . To each organ was added 20 µl of culture
medium (Sigma, 199) containing non-radioactive purified lipophorin (4
mg protein/ml). After incubation with the
P-fat body at 28
°C for 15 min, 10 µl of the incubation medium was diluted to
100 µl with PBS, centrifuged in a spin column equilibrated with
PBS, in order to separate lipophorin from small phosphorylated
molecules, and the
P measured in a liquid scintillation
counter. Controls for
P release into the incubation medium
were done by incubating
P-fat bodies in culture medium
without lipophorin. Phospholipid Transfer from [
P]Lipophorin to
Ovaries-Five µl of [
P]lipophorin
(18,000 counts/min) was injected into adult females fed 2 days
beforehand. After 4 h at 28 °C, the ovaries were dissected, washed,
and homogenized for counting as described
(24) . Controls were
injected with [
P]lipophorin and kept for 4 h at
4 °C in order to inhibit metabolism-dependent phospholipid uptake.
Passage of Ingested Heme from Digestive System to
Hemolymph
Adult females were fed with rabbit plasma enriched
with different concentrations of hemin. After 4 h the hemolymph was
collected, centrifuged, and diluted 1:50 in 0.1 M sodium
phosphate buffer, pH 7.2. The heme transferred from the digestive
system to hemolymph was monitored by the increase in the RHBP Soret
band compared to the controls feeding on plasma alone.
chelator, was present to ensure that the
malondialdehyde formation was due to the hemin added and not to free
iron released during incubation.
consumption was measured
with an oxygen electrode. A rapid fall in O
concentration
was observed when both hemin and
-mercaptoethanol (needed to
recycle the reduced heme) were present ( curve 1). Addition of
RHBP to provide an excess of heme-binding sites compared to the hemin
concentration blocked fatty acid oxidation ( curve 2), and
further additions of hemin increased the rate of oxidation toward the
value observed in the absence of RHBP. An experiment with lipophorin is
shown in Fig. 1 B. The rapid decrease in O
concentration in the presence of hemin was blocked by the
addition of RHBP ( curve 2).
Figure 1:
Inhibition by RHBP of hemin-induced
O consumption. Lipid peroxidation of linolenic acid
micelles ( A) or lipophorin ( B) was monitored by
measuring O
consumption with a Clark-type oxygen electrode
at room temperature. Cuvettes contained 3.1 ml of 0.1 M sodium
phosphate, pH 7.2, 0.05 mM deferoxamine, and either 3.1 mg of
linolenic acid micelles plus 12 nmol hemin ( A, curve
1) plus 22 nmol of free RHBP heme-binding sites ( A,
curve 2) or 18.6 mg of lipophorin ( B, curve
2) plus 20 nmol of hemin ( B, curve 1). Reactions
were started (
) by addition of 18.6 µmol of
-mercaptoethanol; subsequent additions of hemin ( H) or
RHBP ( R) are shown in the figure.
In vivo, lipophorin
transports lipids from the site of production (principally the fat
body) to other organs, including the ovary
(23, 24, 25, 26, 27, 28) .
In this role, it acts as a reusable shuttle; that is, after delivering
its load, it can be recharged 29-30). In the next experiments we
examined the effects of heme-induced oxidation and RHBP on the
performance of lipophorin as a reusable phopholipid transporter. When
P-labeled fat bodies are incubated in culture medium, the
release of [
P]phospholipids is increased by the
addition of the lipophorin (). This increase reflects the
loading of the lipoprotein particle with phospholipids
(23) .
Lipophorin preincubated with hemin before the incubation with the fat
body had a reduced [
P]phospholipid loading
capacity when compared with the control preincubated in the absence of
heme. When RHBP was present during the preincubation with hemin, normal
lipophorin loading was observed ().
P]lipophorin is injected into adult
vitellogenic females, the ovaries accumulate
[
P]phospholipids (I). Pretreatment
of [
P]lipophorin with hemin before the injection
reduced phospholipid transfer to a level only slightly higher than the
low temperature control. The presence of RHBP during the pretreatment
with hemin completely blocked the heme-induced reduction in
phospholipid transfer to the ovary (I). These results
(Tables II and III) show that the capacity of lipophorin to accept and
transfer phospholipid is impaired by heme-induced oxidation and that
RHBP can prevent this effect.
Figure 2:
Passage of ingested heme from digestive
system to hemolymph. Adult females were fed with rabbit plasma enriched
with different concentrations of hemin. Four h after the meal,
hemolymph was collected, and absorption spectra measured against PBS
were recorded. Numbers inside the figure are final
concentrations of hemin in rabbit plasma in
mM.
When the levels of heme-RHBP and total
RHBP were measured in the hemolymph during the days following a blood
meal (Fig. 3), the maximal capacity of RHBP for binding heme
( upper curve) was always in excess of the heme-RHBP actually
formed in vivo ( lower curve). This observation
indicates that apoRHBP is present throughout the feeding cycle.
Figure 3:
RHBP levels in the hemolymph of
Rhodnius after a blood meal. After a blood meal, hemolymph (20
µl pooled from four females) was collected at the times shown on
the abscissa and diluted in 1 ml of 0.1 M sodium
phosphate buffer, pH 7.2. After heme-RHBP levels were measured as the
absorbance at 412 nm (), the samples were titrated with hemin and
the level of total RHBP was obtained from the saturation point as
described under ``Materials and Methods'' (
). Symbols
show mean ± S.E. of four independent
determinations.
(
)
Table:
Inhibition by RHBP of hemin-induced lipid
peroxidation
Table:
Phospholipid transfer from
fat body to lipophorin
P-Fat bodies were incubated for
15 min at 28 °C in culture medium containing lipophorin (80 µg
of protein) that had been pretreated by incubation for 90 min at 37
°C in 20 µl of 0.1 M sodium phosphate buffer, pH 7.2,
0.1 mM deferoxamine with the additions indicated in the table.
An aliquot of each mixture was diluted with PBS, and
[
P]phospholipid transferred from the organ to
the lipophorin particle was measured as described under
``Materials and Methods.'' As a control for
[
P]phospholipid release not dependent on
lipophorin, fat bodies were also incubated in culture medium alone.
Table:
Phospholipid
transfer from P to the ovary
P]Lipophorin (5 µl) preincubated with or
without hemin and RHBP was injected into vitellogenic females, and the
[
P]phospholipid transferred from lipophorin to
the ovaries at 4 or 28 °C was measured 4 h later as described under
``Materials and Methods.'' Pretreatments were carried out for
90 min at 37 °C in 0.1 M sodium phosphate buffer, pH 7.2,
containing 0.1 mM deferoxamine and the additions indicated in
the table.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.