©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification, Partial Characterization, and Cloning of Nitric Oxide-carrying Heme Proteins (Nitrophorins) from Salivary Glands of the Blood-sucking Insect Rhodnius prolixus(*)

Donald E. Champagne (§) , Roberto H. Nussenzveig , José M. C. Ribeiro

From the (1) Department of Entomology and Center for Insect Science, University of Arizona, Tucson, Arizona 85721

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Four nitric oxide (NO)-carrying proteins have been isolated from salivary glands of the blood-sucking insect Rhodnius prolixus. These have been given the collective name ``nitrophorins,'' and individual proteins are designated NP1-NP4 in order of their relative abundance in the glands. All four reversibly bind NO; spectral shifts associated with NO binding indicate the interaction of NO with an Fe(III) heme. Physical properties, amino acid composition, and amino-terminal sequences of the nitrophorins are reported. The most abundant nitrophorin was cloned, and its sequence was determined.


INTRODUCTION

For hematophagous arthropods, feeding success is enhanced by a variety of salivary factors that counter the host's hemostatic responses (1) . Rhodnius prolixus, a principal vector of Chaga's disease, has a salivary vasodilator that has been shown to have properties similar to nitrovasodilators, pharmacological agents which release nitric oxide (NO)()(2) . On molecular sieving columns, the vasodilatory activity was associated with abundant heme proteins, which give Rhodnius salivary glands (SG) their characteristic cherry red color. Subsequently, it was shown that the heme protein fraction of the SG homogenate reversibly binds NO, and the proportion of protein with ligated NO correlates with the vasodilatory activity (3) . The reversible binding of NO was attributed to the interaction of NO with an Fe(III) heme. Further, NO was rapidly released at high pH and was retained at low pH, with a p Kof 6.5, suggesting an interaction of histidine with the heme (3) . In the present study, we have investigated further the nature of this unique NO storage and transport system.


MATERIALS AND METHODS

Purification of Rhodnius Salivary Nitrophorins

Salivary glands (240 pairs) were dissected in phosphate-buffered saline (150 m M NaCl, pH 7.5), transferred to 100 µl of 25 m M triethanolamine HCl pH 8.4, homogenized in a Potter homogenizer, and centrifuged; the supernatant (diluted to 500 µl) was then chromatofocused on a Mono-P fast protein liquid chromatography column (Pharmacia Biotech Inc.) developed from pH 8.4 to 7.0 with Polybuffer HCl at a flow rate of 0.5 ml/min. Elution was monitored at 280 nm for total proteins and 422 nm for heme proteins. The HPLC system consisted of a LDC constaMetric 4100 pump, a Milton Roy SM4000 detector, and a Milton Roy CI-10B integrator interfaced with an IBM PC. Fractions were collected at 1-min intervals. The pH gradient was also monitored by determining the pH of every fifth fraction with a Beckman pH meter.

Heme protein fractions were further purified by HPLC on a Macrosphere SCX 300-micron-strong cation exchange column (Alltech Associates Inc., Deerfield, IL), using a 60-min gradient of 0-1 M NaCl, 25 m M sodium acetate, pH 5.0, at 0.5 ml/min.

Proteins were assayed for purity using reversed-phase HPLC. Aliquots were chromatographed on a Macrosphere 300 C18 5-micron column (Alltech), using a gradient of 20-60% acetonitrile, 0.1% trifluoroacetic acid developed over 50 min at a flow rate of 1 ml/min. The HPLC was as above, except for a Milton Roy CM4000 pump. Elution was monitored at 220 nm.

The molecular weight of the purified proteins was determined by laser desorption mass spectroscopy.

Reversible NO Binding by Purified Nitrophorins

Purified heme proteins in 150 m M NaCl, 10 m M sodium phosphate pH 7.2 buffer were deoxygenated by blowing water-saturated argon across the surface of the solution in a gas-tight cuvette for 2 h. Residual oxygen was removed from the argon by passing the gas through a column of Rydox (Sigma). Nitric oxide-saturated water was produced by deoxygenating water (4 ml) with argon (2 h), then bubbling nitric oxide for 1 min, followed by 2 min of argon flushing to the head space to remove undissolved NO. Nitric acid and anhydrides were removed from the NO by passing the gas over KOH. Proteins were ligated with NO by adding NO-saturated HO in 1-µl increments until the Soret band had shifted to 422 nm. Spectra were recorded from 250 to 700 nm on a Perkin Elmer -19 spectrophotometer.

Characterization of the Heme Group

The heme groups of all four proteins were characterized by the pyridine-hemochromogen method (4) . A dilute solution of the protein (70 µl) was combined with 30 µl of pyridine, 2 µl of 5 N KOH, and crystals of dithionite. After mixing, 60 µl were transferred to a 1-cm path length cuvette, and three spectra were taken from 700 to 540 nm at 1-min intervals. The peak absorbance at 556 nm minus the absorbance at 700 nm was used to determine the hemin concentration, assuming an extinction coefficient of 32 m Mcm(4) . The ratio of heme to protein was determined using protein concentrations determined by duplicate amino acid analyses. As this procedure underestimates protein concentrations due to hydrolysis of some residues, a correction factor of 1.302 was derived by determining the concentration of a sample of the heme protein myoglobin spectroscopically (4) and by amino acid analysis. This correction factor was applied to estimate actual nitrophorin concentrations.

Tryptic Digestion

An aliquot of the most abundant protein, NP1 (1.25 nmol = 25 µg), was dissolved in 50 µl of 8 M urea, 0.4 M NHacetate reduced with 5 µl of 45 m M dithiothreitol for 15 min at 50 °C, then carboxyamidomethylated with 5 µl of 100 m M iodoacetamide at room temperature for 15 min (5) . After dilution to 200 µl with HO, 1 µg of trypsin (Boehringer Mannheim) was added, and the solution was incubated at 37 °C overnight. Peptides were separated by reversed-phase HPLC as described above for the purified heme proteins, except that the gradient was developed over 70 min.

Amino Acid Analysis and Edman Sequencing

The amino acid composition of duplicate 40-pmol aliquots of the four proteins was determined on a Beckman 7300 amino acid analyzer using the post-column ninhydrin ion-exchange method. The amino-terminal sequences of all heme proteins (1 nmol each) were determined using an ABI 477A pulsed-liquid sequencer. Two peptides from the tryptic digest of NP1 were also sequenced. Amino acid analysis and Edman degradation were carried out by the University of Arizona Macromolecular Structure Facility.

PCR Amplification of a Partial NP1 Clone

A 96-fold degenerate oligonucleotide primer was designed based on amino acid residues 12-19 of the amino-terminal sequence of NP1. A second degenerate primer was based on one of the peptides isolated from the tryptic digest. mRNA was isolated from 40 SG pairs and reverse transcribed into cDNA using Superscript (Life Technologies, Inc.) and the primer 5`-GGGGAGGCTCGAGTTTTTTTTTTTTTTTT-3`. First-strand cDNA was amplified in a PCR experiment using the above primers, with annealing at 55 °C, extension at 72 °C, and denaturing at 94 °C, for 1 min each in a Coy model 60 temp cycler. The reaction mix included 2.5 m M MgCl, 50 m M KCl, 10 m M Tris, pH 8.3, 0.01% gelatin, 0.2 m M of each dNTP, and 1.25 units of Ampli- Taq polymerase (Perkin-Elmer Corp.). PCR products were gel purified (Sephaglas BandPrep kit, Pharmacia) and cloned into the pCR II vector (TA Cloning, Invitrogen) for dideoxy sequencing with Sequenase II (U. S. Biochemical Corp.).

Library Construction and Cloning of NP1

Messenger RNA was isolated (Micro Fast-Track kit, Promega) from Rhodnius salivary glands taken at 2, 4, and 7 days after a blood meal (50 pairs each), at 14 days (250 pairs), and at 17 days (100 pairs). Total yield was 16 µg of mRNA, of which 5 µg were used to construct a cDNA library in ZAP (Stratagene). The unamplified library had a complexity of 1.38 10recombinants. A total of 15 10recombinants (from the unamplified library) were screened with a P-labeled probe made (using the Prime-It II kit, Stratagene) from a 120-bp PCR-generated fragment of NP1. Positive plaques were purified with a secondary screening and rescued in pBluescript (Stratagene). Plasmids were isolated using the Magic Mini-Prep kit (Invitrogen) and sequenced manually using Sequenase II or cycle sequenced at the University of Georgia Molecular Instrumentation facility. Two clones were sequenced in both directions.


RESULTS AND DISCUSSION

Purification of Rhodnius Nitrophorins

Chromatofocusing of 240 pairs of Rhodnius salivary glands yielded four major heme proteins, identified by their absorption at both 422 and 280 nm (Fig. 1). We propose the name ``nitrophorins'' for these proteins, to reflect their NO-transporting function, and designate the four proteins as NP1-NP4 as indicated in Fig. 1. Each of the four appeared to be double peaks, with the components separated by 0.03-0.06 pH units. Initially, it was thought that each component of the double peaks was a separate protein, but subsequent strong cation exchange chromatography (Fig. 2) and reversed-phase chromatography (not shown) indicated that the early- and late-eluting components of each peak were identical. The same result was obtained with polyacrylamide gel electrophoresis (data not shown), and pooled early and late peaks gave a single amino-terminal sequence. The peak splitting is attributed to the presence of a mixture of NO-bound and unligated protein. The unligated proteins had pI values of 7.97, 7.53, 6.94, and 7.75 for NP1 to NP4, respectively. Laser desorption mass spectrometry gave masses of 20,378 for NP1, 19,689 for NP2, 19,718 for NP3, and 20,914 for NP4 (Table I). Spectral characteristics of the four proteins are also reported in .


Figure 1: Isolation of heme proteins from R. prolixus salivary glands. 240 salivary gland pairs were chromatofocused over the pH range 8.4-7.0. Elution was monitored at 280 nm ( top panel) for total proteins and at 422 nm ( bottom panel) for heme proteins. The pH gradient was monitored by determining the pH of every fifth fraction. O.D., optical density.



The pyridine-hemochromogen spectra of NP1-NP3 show a peak at 556 nm, consistent with identification of the heme group as protoporphyrin IX (4) . The spectrum of NP4 has a peak at 556 nm and a shoulder at 580-595 nm, indicating the presence of other pigments in addition to hemin. The molar ratio of heme to protein is close to 1 for each of the proteins (). The total of the three proteins, 325 pmol/gland pair, is close to the estimated content of 282 pmol NO per SG pair (3) , which is also consistent with a single NO binding site per protein molecule.

NO Binding by Rhodnius Nitrophorins

At pH 7.2, all four nitrophorins bind NO, indicated by a shift in the Soret band. The Soret shifts from 404 to 422 nm for NP1, NP2, and NP3; ligated NP4 has a Soret maximum at 417.9 nm (Fig. 3). The direction of the shift indicates that all four nitrophorins bind NO with an iron III heme group, as was previously found for whole salivary gland homogenates (3) . Further, the and bands of the spectrum intensify on NO binding, indicating a transition from high to low spin (4) . All four nitrophorins release NO on dilution or with equilibriation with argon; the kinetics of NO binding and release will be reported elsewhere.

Amino Acid Analysis and Edman Sequencing

Amino acid analysis indicates that NP1 is most similar to NP4, and NP2 is most similar to NP3 (Table II). All four proteins contain an unusually high amount of tyrosine, 13 residues in the case of NP1. NP1 differs from NP4 only in a higher content of (Asn + Asp) and lower amounts of serine and glycine. NP2 appears to differ from NP3 in a higher content of (Gln + Glu), tyrosine, leucine, and lysine and a lower content of serine, glycine, and proline. The amino-terminal sequences also suggest the relationship of NP1 to NP4 and NP2 to NP3 (Fig. 4). NP1 and NP4 differ at only 2 of the first 15 residues, with substitutions of alanine for lysine in position 1 and isoleucine for leucine in position 7. NP2 and NP3 differ by only 1 of the first 20 residues. In contrast, NP1 and NP2 have only 8 of the first 20 residues in common. A blank cycle in position 2 of all four proteins was presumed to indicate cysteine, an assignment which is supported by the cDNA sequence of NP1 given below.

Cloning of NP1

Messenger RNA was isolated from 40 R. prolixus SG pairs, reverse transcribed into single-stranded cDNA, and used in a PCR experiment with degenerate oligonucleotide primers based on the partial amino acid sequences obtained from Edman analysis. A single band of 120 bp was produced. This product was purified by gel electrophoresis and cloned into the pCRII vector. Three transformed clones were sequenced in both directions; all three coded for NP1, based on amino acid sequence identity with residues 20-44 of the amino-terminal sequence. Two discrepancies with the Edman-derived sequence were readily resolved. One involved the misidentification of Tyr, tailing from the previous cycle, in a blank cycle produced by a cysteine in position 41. In position 39, the tailing Lys peak from the previous cycle was chosen over the Arg peak.

An aliquot of 15,000 recombinants was screened with the 120-bp NP1 PCR product; 132 positives were detected, and 15 were picked for a secondary screening. This indicates that about 0.9% of the mRNA in Rhodnius salivary glands codes for NP1. Ten plaque-purified clones were rescued in pBluescript. Restriction digestion of the resultant plasmids indicated that nine of these had inserts of about 900 bp, and one had a 700-bp insert. Two clones with the larger insert were completely sequenced in both directions. Both clones were identical and were consistent with the amino acid sequence derived from amino-terminal sequencing of NP1. The complete sequence of the NP1 clone is given in Fig. 5.

As is typical for a secreted protein, there is a signal peptide sequence of 23 residues, 14 of which are hydrophobic. The mature protein (without the signal peptide) is 184 residues long and has a predicted mass of 20,482, within experimental error of the mass of NP1 (20, 378) determined by laser desorption mass spectroscopy. Although a single potential glycosylation site (6) is present at residue 147, mass spectrometry indicates NP1 is not glycosylated. The predicted pI of the clone (7.25) is lower than the observed pI of the native protein, but the pI would be expected to be altered by the presence of a heme group and secondary structure.

The predicted amino acid composition of the NP1 clone compares well with the composition of the native protein (); the only discrepancy was a greater than expected number of cysteines, but cysteines are typically underrepresented in amino acid analysis.

Data base searches found no similarity between the sequence of nitrophorin 1 and other proteins. However, the non-covalently bound protoporphyrin IX heme, the size of the protein, and the interaction of a histidine with the heme (indicated by the pH dependence of NO binding and the EPR spectrum of salivary homogenates) (3) suggests a possible relationship to hemoglobins or b cytochromes. Alignment of NP1 with hemoglobin sequences from the insect Chironomus (7) , the annelids Lumbricus and Tylorrhynchus (8, 9) , the mollusc Glycera (10) , the parasitic nematode Ascaris (11) , human chain hemoglobin, leghemoglobin, and vertebrate cytochromes (12) indicates an overall sequence similarity of 38-45%, but no pronounced regions of sequence identity were seen. However, the same result is produced by aligning sequences of various invertebrate and vertebrate hemoglobins, a consequence of the extreme sequence variability of this group of proteins (13, 14, 15) . Predictions of secondary structure, generated using the PeptideStructure program of the GCG sequence analysis software package (16) , also indicate little correspondence between NP1 and hemoglobins, but again the same result is obtained when, for example, Chironomus chain III hemoglobin is compared with human chain hemoglobin. If the Rhodnius salivary heme proteins are in fact derived from invertebrate hemoglobin, they conform to the pattern of insect hemoglobins in being single domain, single subunit molecules with a single heme binding site (14) .

Evidence from EPR spectroscopy and the p Kof NO release suggest that the imidazole of a histidine interacts with the heme (3) . The NP1 clones encode three histidines in positions 59, 120, and 124. Many hemoglobins from vertebrates, invertebrates, and plants contain a pair of histidines, separated by 2-6 amino acids, located in positions 118-125; usually, the histidine closer to the amino-terminal end is the proximal ligand that interacts with the heme (12) . In most hemoglobins and cytochromes, a third histidine, located 28-35 residues to the amino-terminal end from the proximal histidine, forms the distal ligand to the heme (12) ; in invertebrate hemoglobins, this histidine is often replaced with valine, leucine, or glutamine (14, 15) . The highly conserved CD1 phenylalanine, located 13-21 residues before the distal histidine, is required to anchor the heme in the heme pocket (12) . Alignment of NP1 with hemoglobin sequences suggests that the histidine at position 120 may form the proximal ligand; if so, the distal ligand may be replaced with a valine in position 89, and the conserved phenylalanine may be represented in position 68. The interaction of these residues with the heme will have to be confirmed by site-directed mutagenesis.

The similar amino-terminal sequence and amino acid composition of NP1 and NP4 suggest that these proteins are closely related and may even be allelic variants. NP2 and NP3 are also similar to one another, although they differ more in amino acid composition. The significance of multiple nitrophorins is unclear, but the four proteins differ from one another in their NO binding and release kinetics,() and, in combination, they may deliver NO to a greater length of the blood vessel. The presence of pigments other than hemin associated with NP4 suggests that this protein may help scavenge degraded hemin, which could result from reaction with NO. Given the unique nature of these NO storage and transport proteins and the varied roles of NO in biological systems (17) , their continued study is certain to prove of interest.

  
Table: Physical properties of nitrophorins from R. prolixus salivary glands


  
Table: Amino acid composition (as number of residues) of NP1-NP4

Values shown are based on between 2 and 6 analyses for each protein. The predicted amino acid composition of the NP1 clone is also shown.



FOOTNOTES

*
This work was supported by NIAID, National Institutes of Health Grants AI-18694 (to J. M. C. R.) and AI-35591 (to D. C.). 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 reprint requests should be addressed.

The abbreviations used are: NO, nitric oxide; bp, base pair(s); HPLC, high pressure liquid chromatography; PCR, polymerase chain reaction; SG, salivary glands.

D. E. Champagne, R. Nussenzveig, and J. M. C. Ribeiro, unpublished results.


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