©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Antioxidant Role of Rhodnius prolixus Heme-binding Protein
PROTECTION AGAINST HEME-INDUCED LIPID PEROXIDATION (*)

Marlvia Dansa-Petretski (1)(§), José M. C. Ribeiro (2), Geórgia C. Atella (1), Hatisaburo Masuda (1), Pedro L. Oliveira (1)(¶)

From the (1) Departamento de Bioqumica Médica, Instituto de Cincias Biomédicas, Universidade Federal do Rio de Janeiro, P. O. Box 68041, Rio de Janeiro, RJ, CEP 21941-590, Brasil and the (2) Department of Entomology, University of Arizona, Tucson, Arizona 85721

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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,() 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.


MATERIALS AND METHODS

P Purification

Carrier free 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 Uptake

O 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 P-Fat Bodies to Lipophorin (23

Fat bodies were prelabeled by feeding adult females with blood enriched with 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.


RESULTS

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 chelator, was present to ensure that the malondialdehyde formation was due to the hemin added and not to free iron released during incubation.

An experiment showing the time course of linolenic acid peroxidation by hemin is depicted in Fig. 1A, where O 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 ().

During oogenesis in R. prolixus, the ovary is the principal acceptor of the phospholipids carried by the lipophorin particles (24) . When [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.

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.


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.




DISCUSSION

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.

  
Table: Inhibition by RHBP of hemin-induced lipid peroxidation

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.


  
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.



FOOTNOTES

*
This work was supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnológico and Financiadora de Estudos e Projetos in Brazil and from The MacArthur Foundation and NAID Grant 18694 (to J. M. C. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Submitted in partial fulfillment of the requirements for the degree of Ph.D. Present address: Universidade Estadual do Norte Fluminense, Centro de Biocincias e Biotecnologia, Campos, RJ, 28015-620, Brasil.

To whom correspondence should be addressed. Fax: 55-21-2708647; E mail: Pedro@server.biomed.ufrj.br.

The abbreviation used is: RHBP, Rhodnius heme-binding protein.

P. L. Oliveira, unpublished observations.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.