From the
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.
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)
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.
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
Evidence from EPR spectroscopy and the
p K
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,
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.
(
)(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 K
of 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.
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.
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 H
O, 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
10
recombinants. A total of 15
10
recombinants
(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.
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.
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) .
of 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.
(
)
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
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.