©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Heme-binding Protein from Hemolymph and Oocytes of the Blood-sucking Insect, Rhodnius prolixus
ISOLATION AND CHARACTERIZATION (*)

Pedro L. Oliveira (1)(§), John K. Kawooya (3), José M. C. Ribeiro (4), Terrance Meyer (5), Roger Poorman (3), Elias W. Alves (1)(¶), F. Ann Walker (6), Ednildo A. Machado (1), Roberto H. Nussenzveig (1), Gilberto J. Padovan (2), Hatisaburo Masuda (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 and the (2) Departamento de Clnica Médica e Centro Interdepartamental de Qumica de Protenas, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, 14049-900, Brasil, (3) Cephalon, Inc., West Chester, Pennsylvania 19380, and the (4) Departments of Entomology, (5) Biochemistry, and (6) Chemistry, University of Arizona, Tucson, Arizona 85721

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A heme-binding protein has been isolated and characterized from both the hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus. The protein from both sources is identical in most aspects studied. The Rhodnius heme-binding protein (RHBP) is composed of a single 15-kDa polypeptide chain coiled in a highly -helical structure which binds non-covalently one heme/polypeptide chain. This RHBP is not produced by limited degradation of hemoglobin from the vertebrate host, since specific polyclonal antibodies against it do not cross-react with rabbit hemoglobin, and since it differs from hemoglobin in having a distinct amino-acid composition and NH-terminal sequence. The spectrum of the dithionite-reduced protein has peaks at 426, 530, and 559 nm and resembles that of a b-type cytochrome.

RHBP from hemolymph is not saturated with heme and promptly binds heme added to the solution. The oocyte protein, on the other hand, is fully saturated and is not capable of binding additional heme.


INTRODUCTION

Hematophagy has evolved independently in several insect orders and a great diversity of ways to digest and use blood have arisen during the course of evolution (1) . In general, blood feeding is characterized by ingestion of enormous amounts of blood in a single meal, usually comprising several times the animal's own weight (2) .

A special problem generated by having vertebrate blood as the sole food source is the large amount of free hemin that is produced upon digestion of hemoglobin. Hemin is known to stimulate lipid peroxidation (3) as a consequence of increasing formation of oxygen radicals. In mammalian extracellular fluids, a heme-binding protein called hemopexin has been shown to diminish the effectiveness of heme as a pro-oxidant (4, 5) . Hemopexin also is involved in heme transport in vertebrate plasma (6) . The question of how blood-feeding insects deal with the large amounts of heme in their diet has received very little attention.

Here we describe the isolation and characterization of a heme-binding protein from the blood-sucking insect, Rhodnius prolixus.


EXPERIMENTAL PROCEDURES

Insects Insects were taken from a colony of R. prolixusmaintained at 28 °C and 70% relative humidity. Normal mated females were fed on rabbit blood at 2-week intervals. Hemolymph and Oocytes Four to 6 days after a meal, hemolymph was collected in the presence of phenylthiourea (30-130 µg/ml), 5 mM EDTA, and a mixture of protease inhibitors prepared in 0.15 M NaCl, with final concentrations of 0.05 mg/ml of soybean trypsin inhibitor, leupeptin, lima bean trypsin inhibitor and antipain, and 1 mM benzamidine. On the same day, chorionated oocytes were dissected and washed with ice-cold 0.15 M NaCl in order to remove ovarian debris prior to homogenization. Oocytes were homogenized in a Potter-Elvehjem homogenizer in the presence of the same mixture of protease inhibitors, buffered with 20 mM Tris-HCl, pH 7.0, (approximately 500 oocytes to 1 ml), and centrifuged at room temperature in a microcentrifuge at 11,000 g for 5 min. The floating lipids and the pellet were discarded, and the clear supernatant was used as the crude oocyte extract for protein purification. Purification of RHBP()

From Oocytes

Solid ammonium sulfate was added to bring the oocyte extract to 45% saturation, and the suspension was gently stirred for 20 min at 4 °C. After centrifugation at 11,000 g for 10 min, the precipitate was discarded, and the supernatant was brought to 60% saturation. This new precipitate was then washed twice with a 60% saturated ammonium sulfate solution and then was back-extracted by resuspending in a 45% saturated solution and centrifuging. The pellet was discarded and the supernatant was dialyzed against 0.15 M NaCl, 10 mM Tris-HCl, pH 7.0, and applied to a column of Sephadex G-200 (2.5 55 cm) equilibrated with the same solution. Protein content of fractions was measured by the absorbance at 280 nm. The colored fractions containing RHBP were pooled, dialyzed against deionized water, and lyophilized.

From Hemolymph

Hemolymph (approximately 3 ml) was diluted to 5 ml with phosphate-buffered saline (0.15 M NaCl, 0.1 M sodium phosphate, pH 7.0) and 1.25 g of KBr was added. The solution was centrifuged at 80,000 g for 20 h at 4 °C. The fractions at the bottom of the tube were collected and dialyzed against deionized water until an abundant precipitate had formed. The solution was then centrifuged at 11,000 g for 10 min at 4 °C. The supernatant was brought to 10 mM with Tris base and applied to a column (1.5 18 cm) of DEAE-Toyopearl, equilibrated with 10 mM Tris-HCl, pH 8.4. The column was first washed with 20 ml of the same buffer and then eluted with an NaCl gradient (0-100 mM). The fractions containing RHBP were pooled and applied to a Sephadex G-75 column (1.5 80 cm) equilibrated with 0.15 M NaCl, 10 mM Tris-HCl, pH 7.0. Fractions containing the RHBP were pooled, dialyzed against deionized water, and lyophilized. Polyacrylamide Gel Electrophoresis Polyacrylamide gradient (6-22.5%) gels (10 10 cm 1 mm) were run in the presence of SDS (7) at a constant current of 22 mA. Gels were stained with Coomassie Brilliant Blue R and destained with 7% acetic acid in 40% methanol.

Molecular weights of polypeptides were measured by SDS-PAGE using the following protein standards: myosin (205 kDa), -galactosidase (116 kDa), phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), soybean trypsin inhibitor (20 kDa), and -lactalbumin (14 kDa). Antibodies and Western Blot RHBP (1 mg) purified from oocytes was emulsified with an equal volume of Freund's adjuvant and injected subcutaneously into the back of a rabbit. Boosters of 0.5-1.0 mg of protein were injected at 2-month intervals, and blood was collected from an ear vein 3 months after the first interval.

Purified RHBP (0.5 mg) was adsorbed to a piece of nitrocellulose filter that, after saturation with a 5% solution of powdered milk, was used as an affinity matrix to isolate anti-RHBP IgG. The procedure has been described in detail elsewhere (8) . This IgG was used to perform Western blots according to Towbin et al.(9) . Amino Acid Analysis RHBP isolated from oocytes (4 nmol) was hydrolyzed for 22 h at 110 °C in 0.5 ml constant boiling 6 N HCl containing 0.01% phenol in an evacuated sealed tube. Amino acid analysis was carried by the method of Spackman et al.(10) using an automatic instrument (11) . No corrections were made for losses during acid hydrolysis. Hydrolysis with lithium hydroxide (12) was used for determination of tryptophan. NH-terminal Sequence Purified RHBP (100 pmol) was submitted twice to automatic Edman degradation using a liquid phase sequencer (Porton PI 2020/2090). Phenylthiohydanthoine amino acids were identified using a Hewlett Packard HPLC with a reverse phase AminoQuant analytical column (2.1 250 mm). The initial and repetitive yields obtained were 52 and 96%, respectively. Sequence comparisons were performed according to Devereux et al.(13) . Visible Absorption Spectra Visible spectroscopy experiments were carried out in 60 mM NaCl, 20 mM Tris-HCl, pH 8.0, on a Cary 100 spectrophotometer. The reduced form of RHBP was obtained by addition of small amounts of sodium dithionite. Reoxidation was achieved by progressive addition of potassium ferricyanide until dithionite consumption and reappearance of the ferric spectrum. Heme Extraction The heme group of RHBP was extracted by a modification of the acetone-HCl method (14) , using a final concentration of HCl of 25 mM and washing the protein pellet until total extraction of heme. Circular Dichroism The circular dichroism spectrum between 190 and 250 nm was obtained with the protein from oocytes, in an Aviv 60 DS circular dichroism spectropolarimeter (Aviv Associates, Inc., Lakewood, NJ) and a 0.05-mm quartz cell. Data were analyzed according to Chang et al.(15) . Five spectra were averaged and smoothed. Errors in the spectra were insignificant in comparison with errors in protein concentration and pipetteting. Gel Filtration The apparent molecular weight of RHBP from both the oocytes and the hemolymph was determined with the Sephadex G-75 column used during isolation from the hemolymph. The column was calibrated with proteins of known molecular weight.

High performance liquid chromatography gel filtration was carried out using a TSK-125 column and pre-column equilibrated with 0.1 M sodium phosphate, pH 6.8, and eluted at 0.8 ml/min using a LDC series 4000 pump and detector (set at 412 nm) and a CI-10 integrator. Heme Binding Assay Binding of heme to RHBP was monitored by measuring the absorbance of the Soret band at 412 nm while progressively adding a solution of 1 mM hemin in 0.1 M KOH (16) . The absorbance was plotted against the molar ratio of added hemin to polypeptide ( M = 15,000), and the hemin necessary to fully form the holoprotein was determined from the break in the curve.


RESULTS

The pigment that gives Rhodniuseggs and hemolymph their characteristic pink color in both cases copurified throughout all protein isolation steps with a polypeptide of 15 kDa (Fig. 1, A and B).


Figure 1: Summary of RHBP purification from oocytes and hemolymph analyzed by SDS-PAGE. A, from oocytes: 1, crude oocyte extract; 2, after ammonium sulfate precipitation; 3, after Sephadex G-200 chromatography. Molecular weights at the left are vitellin apoproteins. B, from hemolymph: 1, crude hemolymph; 2, subnatant from KBr density centrifugation; 3, supernatant after precipitation against water; 4, after DEAE-Toyopearl column; 5, after Sephadex G-75 chromatography.



Both the hemolymph and oocyte proteins are monomeric, as indicated by an apparent molecular weight of 12,400 for the native protein as estimated by gel filtration chromatography (Fig. 2) and 15,000 by SDS-PAGE. The amino acid composition corresponding to this size is shown in .


Figure 2: Native molecular weight determination. The molecular weight of RHBP from both oocytes and hemolymph ( arrow) was determined using a Sephadex G-75 column. The following proteins were used as standards: albumin ( A), ovalbumin ( O), carbonic anhydrase ( C), cytochrome c ( Ci) , and aprotinin ( Ap). Ve;, elution volume; V, exclusion volume.



Analysis of the secondary structure by circular dichroism (Fig. 3) shows a mean residue ellipticity of -22,635 degcmdmol at 222 nm. The best estimate indicates a high proportion of -helix (75%).


Figure 3: Circular dichroism spectrum of RHBP. RHBP isolated from oocytes was used at a concentration of 0.1 mg/ml at 30 °C in 10 mM sodium phosphate buffer, pH 7.0.



In order to determine whether the oocyte and hemolymph proteins are related and whether they are derived from ingested proteins or synthesized by the insect itself, antibodies against the purified oocyte protein were raised in rabbits. The specific IgGs were purified as described under ``Experimental Procedures'' and used in a Western blot. Fig. 4shows that a 15 kDa band is specifically recognized in the hemolymph of females and oocytes. Interestingly, although hemolymph from fourth stage larvae is not as strongly colored as that from adult females, the same 15-kDa band is seen in the Western blot (Fig. 4, lane 2), indicating that the protein is not exclusively related to oogenesis.


Figure 4: Western blot of hemolymph and oocytes. Affinity-purified IgG against RHBP was used for a Western blot (see ``Experimental Procedures''). Samples were run on a 5-15% SDS-PAGE gel prior to electrophoretic transfer to a nitrocellulose membrane. Shown are: 1, crude oocyte extract; 2, fourth-stage larvae hemolymph; 3, adult female hemolymph; 4, rabbit hemoglobin; and 5, RHBP purified from oocytes.



Determination of the NH-terminal amino acid sequence by automated Edman degradation revealed total identity of the oocyte and hemolymph proteins in the first 36 residues (Fig. 5).


Figure 5: NH-terminal amino acid sequence. The NH-terminal primary structure of RHBP from both oocytes and hemolymph was determined by automated Edman degradation and found to be identical.



Searching protein data bases (SWISS-PROT, GenBank, and EMBL) with this sequence did not indicate any significant similarity to already known polypeptides. This result clearly establishes that the protein is not derived from dietary hemoglobin, as suggested previously (17) , but is instead synthesized by the insect itself. The same conclusion is reinforced by the amino acid composition (), which is distinct from vertebrate hemoglobin (18, 19) and by the lack of immunological cross-reaction between rabbit hemoglobin and RHBP, as revealed by the Western blot (Fig. 4).

The absorption spectra of the proteins from oocyte and hemolymph are typical of a heme-containing protein (Fig. 6, A and B). The protein was isolated from both sources in the ferric state ( solid line), and spectra determined on fresh crude oocyte extracts and hemolymph also showed the pattern typical of the oxidized protein (data not shown). Reduction with sodium dithionite ( dashed line) changed both intensity and position of the major -peak, which shifted from 412 to 426 nm, and produced the characteristic - and -bands at 559 and 530 nm, respectively. This low spin state spectrum closely resembles that of b-type cytochrome, which are characterized by two histidine axial ligands (20, 21) . The absence of peaks at the 620 and 695 nm regions of the oxidized spectrum (high spin and methionine axial ligand indicators, respectively) also are similar to b-type cytochromes (data not shown). Nevertheless, more direct information on heme axial ligands ( e.g. magnetic CD spectra) is still lacking. The alkaline pyridine derivative obtained in 0.2 N NaOH and 20% pyridine gives, after reduction with dithionite, a typical protoheme band at 558 nm (data not shown).


Figure 6: Visible absorption spectra of oxidized and dithionite-reduced RHBP. Spectra from protein purified from oocytes ( A) or hemolymph ( B) were recorded in 60 mM NaCl, 20 mM Tris-HCl, pH 8.4. Shown are: -, native protein (no addition); - - - - , dithionite-reduced; , reoxidized by potassium ferricyanide.



In common with other proteins that exhibit a b-type cytochrome absorption spectrum, the heme group can be extracted by the acetone-HCl method (20) , and the resulting apoprotein can be titrated by adding back the hemin (Fig. 7). As hemin bound to the protein has a higher extinction coefficient at 412 nm than free hemin, the saturation of the apoprotein is indicated by a break in the straight line on the plot of absorbance at 412 nm against the amount of hemin added to the medium. The two lines intersect at a hemin-to-polypeptide ratio of approximately 1:1, demonstrating that only one heme group is bound by each polypeptide.


Figure 7: Heme-RHBP reconstitution. The heme group of RHBP from hemolymph was extracted by the acetone-HCl method. The apoprotein was diluted in 0.15 M NaCl, 20 mM HCl, pH 7.5, and titrated by addition of hemin and measurement of the absorbance at the Soret band (412 nm).



Although the native RHBP purified from hemolymph already has some heme bound to it, addition of hemin also produces a break in the plot (Fig. 8 A), revealing that the protein is not saturated and that about half of the protein molecules in the hemolymph are apoproteins. A different result is obtained with the oocyte protein, where addition of heme shows no available heme-binding sites, indicating that in the oocyte RHBP is fully saturated (Fig. 8 A). As a control, aliquots from the experiment shown in Fig. 8 A were applied to a gel filtration TSK-125 HPLC column while monitoring the absorbance of the eluant at 412 nm (Fig. 8 B) and observing the increase in the height of the peak. Saturation was obtained with the same amount of added hemin as in the previous experiment.


Figure 8: Binding of heme to the hemolymph protein. Association of heme with the protein was measured by the increase in absorbance at the Soret band (412 nm) as in the previous figure. Shown are: A, titration of RHBP purified from hemolymph () and oocytes (); B, gel permeation high performance liquid chromatography of native hemolymph RHBP after different additions of hemin. Numbers inside the figure indicate ratios of added hemin to polypeptide.




DISCUSSION

Our results show that the blood-sucking hemipteran R. prolixussynthesizes a unique heme-binding protein of 15 kDa, not previously described. To our knowledge, this is the first description of a heme-binding protein in a blood-feeding insect. In a classical report, Wigglesworth (17) showed that the pigment present in Rhodniushemolymph and eggs was associated with a protein that he believed was produced by partial digestion of hemoglobin from the vertebrate host. However, on the basis of the present work, this can be ruled out, since specific antibodies against RHBP do not cross-react with rabbit hemoglobin (Fig. 4). The conclusion that RHBP is a different protein, synthesized by the insect itself, is also supported by a clearly distinct amino acid composition ( and Ref. 18 and 19) and by its NH-terminal amino acid sequence (Fig. 5), which does not indicate significant homology to any known protein.

Titration of the apoprotein with hemin shows that there is only one binding site/polypeptide chain (Fig. 7). Addition of hemin to the native protein purified from the hemolymph, but not from the oocyte, is immediately followed by its binding to the protein (Fig. 8), indicating that the protein in the hemolymph is not saturated. The possibility that the heme-binding site in the purified protein is an artifact generated during the isolation procedure can be excluded, since titration of crude hemolymph with hemin gave similar results (data not shown).

Vertebrate plasma also has a heme-binding protein, hemopexin (22) . Hemopexin and RHBP share absorption spectra in the visible range typical of b-type cytochromes ( Fig. 6and Refs. 20, 21, and 23). RHBP (Fig. 2) is only one-fourth the size of hemopexin (60 kDa) (22) . Limited proteolysis of hemopexin produces a peptide that can still bind heme (24) . However, the CD spectrum of the insect heme-binding protein (Fig. 3) reveals a highly helical secondary structure instead of the 95% random-coil pattern displayed by hemopexin (25) . The amino acid sequence at the NH terminus of RHBP is also unique, reinforcing the same conclusion. These observations suggest an independent evolutionary origin for RHBP.

In the oocyte, RHBP is found inside the yolk platelets (data not shown), which are organelles specialized in storing materials for growth of the embryo (26) . Therefore, in the egg RHBP may be a source of heme or iron for insect embryogenesis, as the protein is accumulated in the oocyte in significant quantities, as indicated by the strong 15 kDa band in the crude oocyte extract (Fig. 1 A).

Hemin is a powerful generator of free radical reduction products of dioxygen that are capable of causing biological injury through peroxidation of lipids, proteins and DNA (3-5 and 27). The antioxidant role of RHBP is investigated in the following article (28) .

  
Table: Amino acid composition of heme RHBP from oocytes

Data are reported for a 22-h acid hydrolysis and amino acid analysis in duplicate. The integral molar values, residues/mol, are given in parenthesis. No corrections have been made for losses during acid hydrolysis. The minimal chemical molecular weight calculated for 132 residues/mol is 16,125.



FOOTNOTES

*
This work was supported by grants from Conselho Nacional de Desenvolvimento Cientfico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (Finep), Fundaão de Amparo Pesquisa do Estado do Rio de Janeiro (FAPERJ) in Brazil and from The MacArthur Foundation and NIAID 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.

§
To whom correspondence should be addressed. Fax: 55-21-2708647; E mail: PEORO@SERVER.BIOQMED.UFRJ.BR.

Present address: Universidade Estadual do Nore Fuminense, Centro de Biocincias e Biotecnologia, Campos, RJ, 28015-620, Brasil.

The abbreviations used are: RHBP, Rhodnius heme-binding protein; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We express our gratitude to Dr. Martha M. Sorenson and to Dr. John H. Law for a critical reading of the manuscript and to Rosane O. M. M. Costa, José S. Lima Jnior, José F. Souza Neto, and Sebastiana S. Santos for excellent technical assistance.


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