From the Laboratory of Malaria and Vector Research,
NIAID, National Institutes of Health and the ¶ Molecular
Interactions Resource, Division of Bioengineering and Physical Science,
Office of Research Services, Office of the Director, National
Institutes of Health, Bethesda, Maryland 20892
Received for publication, November 8, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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The saliva of the blood-feeding insect
Rhodnius prolixus contains numerous pharmacologically
active substances. Included among these are a number of lipocalin
proteins that bind various ligands important in hemostasis and
inflammation. One such protein is a biogenic amine-binding protein
(ABP) that binds serotonin, epinephrine, and norepinephrine. Based on
amino acid alignments, it is most similar to the nitrophorin group of
lipocalins found in the same insect species. Physiologically, this
protein appears to act as both a vasodilator and platelet aggregation
inhibitor. This protein inhibits smooth muscle contraction of the rat
uterus in response to serotonin and of the rabbit aorta in response to
norepinephrine. Platelet aggregation induced by a combination of low
concentrations of ADP and either serotonin or epinephrine is inhibited
because of the binding of serotonin and epinephrine. Potentiation of
aggregation induced by low concentrations of collagen along with
serotonin or epinephrine is also inhibited. Dissociation constants for
biogenic amines were measured using isothermal titration calorimetry
and the Hummel-Dreyer method of equilibrium gel filtration. In this manner, Kd values of 102, 24, and 345 nM were found for serotonin, norepinephrine, and
epinephrine, respectively. Molecular modeling of ABP suggests that
ligand binding is mediated by interaction with the side chains of
aromatic amino acids and charged residues that line the binding pocket.
A major function of the salivary secretion of blood-feeding
arthropods is to inhibit hemostasis and inflammation in the host tissues and blood (1). In the case of Rhodnius prolixus, the saliva contains potent inhibitors of platelet aggregation,
anticoagulants, vasodilators, antihistamines, and antiserotonins
(2-5). Many of the active components of the saliva belong to the
lipocalin protein family (6, 7). The structure of lipocalins is
characterized by an 8-stranded Serotonin and catecholamines are important mediators of platelet
aggregation, vasoconstriction, and inflammatory processes. Serotonin is
contained in the dense granules of platelets and is released upon
stimulation by various agonists. It acts to increase vascular
permeability and is itself also a weak platelet agonist, leading to the
activation of phospholipase C. The catecholamines norepinephrine and
epinephrine, released by local nerves, cause vasoconstriction in
response to bleeding. Epinephrine also potentiates platelet aggregation
by a Gi-dependent signaling mechanism, leading to a decrease in adenylyl cyclase activity (12).
The inhibition of serotonin-induced smooth muscle contraction by
R. prolixus salivary extracts has been shown. However, its mechanism remains to be determined. In this study, a lipocalin-encoding sequence that was encountered frequently during the random sequencing of a R. prolixus salivary gland library has been expressed
in Escherichia coli. The protein was refolded and shown to
be a biogenic amine-binding protein. It was found to bind serotonin as
well as epinephrine and norepinephrine. It inhibits the
serotonin-mediated contraction of the rat uterine horn, and the
norepinephrine-mediated contraction of the rabbit aorta. During
feeding, this protein also appears to function as a
platelet-aggregation inhibitor by sequestering the agonists serotonin
and epinephrine secreted by activated platelets and neural sources. It
also may act to inhibit inflammatory responses at the feeding site by
binding these proinflammatory molecules.
Materials--
Serotonin, epinephrine, and norepinephrine were
obtained from Sigma. The MicroFast Track mRNA preparation kit was
from Invitrogen (San Diego, CA), and the SMART library construction kit
from Clontech (Palo Alto, CA).
Cloning of ABP1
cDNA--
A salivary gland cDNA library was prepared using the
SMART cDNA library construction system (13, 14). Messenger RNA was prepared from 4 pairs of R. prolixus salivary gland pairs
(5, 7, 10, and 14 days after blood feeding) using the MicroFast Track system. The cDNA for the ABP was cloned as part of an expressed tag sequencing project in which ~500 clones from the R. prolixus salivary gland library were partially sequenced. The ABP
sequence was recognized as encoding a previously uncharacterized
lipocalin and was well represented in the library.
Protein Expression and Purification--
Using the ABP
N-terminal sequence as a guide, the cDNA was modified to exclude
the 5'-untranslated region and the region encoding the signal peptide.
The modifications were performed using PCR with the primer
CGCACCATATGGCATCTGGTTGTTCTACTGTGGATACTG. The resulting PCR
product had a methionine (ATG) codon directly upstream of the first
codon of the mature polypeptide. The PCR product was cloned into the
vector PCR 2.1 and sequenced. An NdeI restriction site at
the 5' end of the cDNA, added as part of the PCR mutagenesis, enabled its insertion into the expression vector pET 17b. The expression construct was then moved into the E. coli strain
BL21(DE3) pLys-E.
For the production of protein, 4 liters of LB broth were inoculated
with the expression strain and shaken at 250 rpm and 37 °C until the
absorbance at 600 nm was about 0.7. Isopropyl-1-thio-
The cell pellet was suspended in 100 ml of 20 mM Tris-HCl,
pH 8.0, 0.2 mM phenylmethylsulfonyl fluoride, and the cells
were lysed using a probe sonicator. The lysate was centrifuged at
10,000 × g and 10 °C for 20 min. Analysis by
SDS-PAGE followed by staining with Coomassie Blue showed the protein to
be present as inclusion bodies. The insoluble pellet was extracted with
20 mM Tris-HCl, pH 8.0, 1% Triton X-100, followed by
centrifugation at 10,000 × g. The extracted pellet was
washed three times with 20 mM Tris-HCl, pH 8.0, with each
wash followed by centrifugation.
The protein was solubilized in 20 ml of 20 mM Tris-HCl, pH
8.0, 6 M guanidinium hydrochloride, 10 mM
dithiothreitol. This material was diluted to 250 ml in 20 mM Tris-HCl, pH 8.0, 0.4 M arginine. After
centrifugation, the supernatant was dialyzed against 2 changes of 20 mM Tris-HCl, pH 8.0. The dialyzed sample was concentrated
by ultrafiltration, centrifuged at 100,000 × g, and
purified by gel filtration chromatography on Sephacryl S-100 (16/60
column, Amersham Biosciences) using 20 mM Tris-HCl, pH 8.0, 0.2 M NaCl for elution.
Smooth Muscle Preparations--
Longitudinal contractions of
isolated pieces of rat uterus horns (animals were primed 24 h
before dissection with 50 µg of stilbestrol) were recorded
isotonically with transducers from Harvard Apparatus Inc. (Holliston,
MA). Muscles were suspended in a 0.5-ml bath kept at 30 °C, using
the solution recommended by Gaddum et al. (15). Rabbit
aortic ring contractions were evaluated isometrically using transducers
from the same company. The bath was kept at 37 °C, and Tyrode's
solution was used (16). Additions to the bath were never greater than
5% of the volume of the bath.
Preparation of Human Washed Platelets and Platelet Aggregation
Assays--
Platelet-rich plasma was obtained by platelet pheresis
from medication-free platelet donors at the DTM/NIH blood bank under the direction of Dr. S. Leitmann, as described (17). Briefly, after
addition of 0.2 units/ml of apyrase, platelet-rich plasma was
centrifuged at 1100 × g for 15 min and washed twice by
centrifugation in Tyrode's buffer (137 mM NaCl, 27 mM KCl, 12 mM NaHCO3, 0.42 mM NaH2PO4, 1 mM
MgCl2, 5.55 mM, 0.25% bovine serum albumin, pH 7.4). Platelets were resuspended in apyrase-free Tyrode's buffer, containing 0.5 mg/ml fibrinogen and adjusted to a concentration of 200,000-400,000 platelets/µl. Washed human platelets (300 µl) were placed in a Chrono-Log Lumi-aggregometer (Chrono-Log Corp., Havertown, PA) and stirred at 1,200 rpm at 37 °C for 1 min followed by addition of reagents as indicated in figure legends. In some experiments, platelet-rich plasma was incubated with 50 µM indomethacin for 30 min at room temperature, followed
by a washing procedure as described above.
Modified Hummel-Dreyer Method of Equilibrium Gel
Filtration--
To measure binding of ABP to serotonin, ABP was
injected in a Pharmacia Superdex Peptide HR/10/30 column (Amersham
Biosciences) perfused at 0.5 ml/min with 0.15 M NaCl and 20 mM Tris-Cl, pH 7.3, and the indicated concentrations of
serotonin. The column was equilibrated with at least 5 volumes of the
solution before injection of ABP. The eluent absorption was monitored
at 280 nm by an absorbance detector (model SM4100 from ThermoSeparation Products, Rivera Beach, FL), and the total area under the ABP peak
determined. The fluorescence emission of the eluent at 340 nm
(excitation at 280 nm) was also measured using a fluorescence detector
(model FL 3000 from the same company), and the total area of the
fluorescence under the ABP peak determined. Previously, we have
evaluated binding of nucleotides to proteins by the blue shift of the
absorbance when the nucleotide is bound to the protein. Accordingly,
measuring the absorption of the eluent in 2 wavelengths allowed
estimation of binding constants (7, 17). In this case, binding of
serotonin does not change the position of the spectral maximum from its
position at 290 nm. The fluorescence emission increases because of the
binding, however, thus the measurement of the ratio of the fluorescence
emission to the absorbance at 280 nm allows estimation of binding.
Binding of serotonin to the protein increases the absorbance of the
protein at 280 nm, but the fluorescence increase is greater than the
absorbance increase, and thus their ratio represents a relevant
measurement because the ratio of two hyperbolae is represented by
another hyperbola. Theoretically, the fluorescence result alone would
be sufficient to estimate an increase in binding, but using the ratio
normalizes for several sources of error such as pipetting volumes,
column band spreading, etc. Three independent column runs were made for each serotonin concentration tested.
The change in the fluorescence emission at 340 nm divided by the
absorbance at 280 nm following serotonin binding was modeled as
described earlier (7, 17) by a hyperbolic equation of the type,
Isothermal Titration Calorimetry--
ITC measurements were made
on a Microcal (Northhampton, MA) VP-ITC calorimeter. The protein was
dialyzed against 20 mM Tris-HCl, pH 7.5, and the dialysis
buffer was used to prepare solutions of ligands, to dilute the protein,
and to evaluate the baseline heats of dilution. All solutions were
degassed under vacuum before use. Protein at concentrations between 1 and 5 µM, depending on the experiment, was inserted in
the cell, and ligand at concentrations of 10-50 µM was
used in the syringe. Heats of binding were measured at a temperature of
25 °C. After subtraction of the measured heats of dilution, the net
enthalpy data were analyzed with a single-site binding model using the
Microcal origin software package (18).
Modeling of ABP--
A homology-based molecular model of ABP was
constructed based on the coordinates of the nitrophorin 2 crystal
structure (19), with all water and heme atoms removed (Protein Data
Bank accession number 1EUO). The model was constructed by an automated
procedure using the web-based SwissModel (20). Equivalent positions in nitrophorin 2 and ABP were determined by alignment. The resulting model
was minimized by 200 cycles of steepest descents followed by 300 cycles
of conjugate gradient minimization. Serotonin coordinates were added to
the model and positioned manually using the Insight II (MSI, San Diego,
CA) modeling software.
Detection and Cloning of ABP--
ABP was first detected in a
survey of expressed tag sequences from a R. prolixus
salivary gland cDNA library. It was part of a group of related
clones encoding putative lipocalins of unknown function (Fig.
1). Members of the group were encountered
frequently, suggesting that the mRNAs were present in high
abundance. Analysis using the Signal-P algorithm (21), as well as
alignments with known lipocalin sequences from R. prolixus
saliva, showed a signal sequence to be present indicating that the
protein is probably secreted into the saliva. A subsequent proteomic
analysis of R. prolixus salivary gland homogenate that
combined chromatographic separation of salivary components with
N-terminal Edman degradation confirmed that members of this group were
present in the saliva.2 The
signal peptide cleavage position was also confirmed by this analysis.
The ABP is most closely related to the nitrophorin group of lipocalins
that is also found in R. prolixus. Alignment of the amino
acid sequence with the published nitrophorin sequences shows ABP to
match at least one of the four nitrophorins at 84 of 196 amino acid
positions (Fig. 2); however, the degree
of identity with any single nitrophorin sequence does not exceed 23%.
This is consistent with the low identity levels commonly seen among members of the lipocalin group. The alignment of all four cysteine residues in ABP with their nitrophorin counterparts suggests that two
disulfide bonds are present that correspond in position to those seen
in crystal structures of nitrophorins 1, 2, and 4. A second group of
R. prolixus salivary lipocalins, typified by the ADP-binding
protein RPAI-1, is less similar to ABP, having six cysteines and three
apparent disulfide bonds. The ABP shares only 11% identity with a
serotonin-histamine-binding lipocalin recently isolated from the saliva
of the tick Dermacentor reticulatus, with a different
distribution of cysteine residues, suggesting that the functional
relationship between these two molecules is because of convergence
rather than common lineage (22).
Heterologous Expression of ABP--
ABP was expressed at high
levels in E. coli as insoluble inclusion bodies. After
solubilization in guanidine and reduction with dithiothreitol, the
protein was folded by dilution in arginine-containing buffer, followed
by dialysis. Gel filtration chromatography produced purified ABP as a
soluble monomer. Because the protein is related to the heme-containing
nitrophorins, heme was added to a portion of the refolded ABP followed
by gel filtration chromatography. Monitoring of the eluent at both 280 and 405 nm indicated that no heme was associated with the monomeric
protein (data not shown).
Inhibitory Effects of ABP on the Rat Uterus--
Serotonin is a
known mediator of mammalian uterine contraction via interaction with
the 5-HT2A receptor. R. prolixus salivary homogenates and a partially purified salivary protein have been shown
to inhibit this activity. In this study, both the R. prolixus salivary gland homogenate (results not shown) and the
purified, reconstituted recombinant ABP completely abrogated the
response of the rat uterus to serotonin when supplied in sufficient
quantity (Fig. 3). When 25 µg of ABP
was added to the uterine preparation, no response to incremental
additions of serotonin were seen until the cumulative concentration of
agonist in the bath just exceeded the concentration of ABP (Fig. 3).
The approximate 1:1 protein-agonist stoichiometry required for minimal
inhibition suggests that each molecule of ABP binds a single molecule
of serotonin (Fig. 3). Replacement of the saline solution containing
ABP with fresh solution caused the preparation to regain its normal
response to serotonin, indicating that the inhibition is reversible
(Fig. 3).
Inhibitory Effects of ABP on the Rabbit Aortic
Ring--
Norepinephrine is important in the maintenance of vascular
tone by inducing the contraction of smooth muscle via interaction with
Inhibition of Platelet Aggregation by ABP--
Serotonin and
epinephrine are secreted by platelets upon activation as constituents
of the dense granules. Serotonin is an agonist of platelet aggregation
acting through activation of the 5-HT2A receptor (12).
Binding to this receptor activates phospholipase C, resulting in
platelet shape change, but not aggregation. Serotonin alone has the
effect of potentiating aggregation induced by subthreshold levels of
other agonists such as collagen and ADP. Given in combination with
epinephrine, an agonist of
We evaluated the effects of ABP on platelet aggregation in the presence
of serotonin and epinephrine. When platelets are treated with the
cyclooxygenase inhibitor indomethacin, the effects of added serotonin
and epinephrine on platelet activation can be evaluated in the absence
of thromboxane A2 secretion. This limits the contribution
of secreted agonists, such as ADP to platelet activation. ABP was found
to completely eliminate, in a concentration-dependent manner, the shape change response of washed, indomethacin-treated human
platelets to serotonin, consistent with the observed serotonin-binding activity of the protein (Fig.
5A). Conversely, ABP had no
effect on platelet shape change induced by low concentrations of ADP or
on the aggregation response induced by higher agonist concentrations, indicating that ADP does not bind with ABP (Fig. 5B).
Serotonin and epinephrine are important as potentiators of platelet
agonists such as ADP and collagen. When ADP is administered to
indomethacin-treated platelets at concentrations below the threshold
for platelet aggregation, only shape change is seen (Fig.
5C). At higher concentrations of ADP, an aggregation
response is observed (Fig. 5C). However, when low
concentrations of ADP are added along with serotonin or epinephrine, a
full aggregation response is seen, indicating that serotonin and
epinephrine potentiate the response to ADP. In the presence of ABP, no
potentiation of aggregation by serotonin or epinephrine is observed
(Fig. 5, C and D). This suggests binding of these
two compounds by ABP eliminates any potentiation of the ADP response. A
similar effect is observed on the potentiation by serotonin or
epinephrine of collagen-mediated aggregation of nonindomethacin-treated
platelets (Fig. 6, A and B). The results indicate that in the natural physiological
system, where thromboxane A2 synthesis is not suppressed,
ABP increases the threshold concentration of collagen necessary for an
aggregation response.
Determination of ABP Ligand Binding Affinities--
The binding
affinity of ABP for serotonin was determined using both the
Hummel-Dreyer method of equilibrium gel filtration and ITC. Fig.
7 shows a modified Hummel-Dreyer analysis
of serotonin binding when the ligand concentration changed from 10 nM to 3 µM. The changes in optical properties
were saturable with increasing ligand concentration, and allowed
determination of a dissociation constant of 201 ± 71 nM.
ITC was used to evaluate the binding of serotonin, norepinephrine, and
epinephrine (Fig. 8). The results show
that the primary amine norepinephrine binds with the highest affinity
(Kd = 24 ± 6 nM) whereas the
secondary amine epinephrine binds with ~10-fold lower affinity
(Kd = 345 ± 67 nM). The affinity for serotonin is intermediate with a Kd value of
102 ± 38 nM, agreeing reasonably well with the result
of the Hummel-Dreyer experiment. Analysis of the ITC data using a
single-site binding model gave binding stoichiometries (N) of 0.85 ± 0.07, 0.80 ± 0.01, and 1.28 ± 0.03 for serotonin,
norepinephrine, and epinephrine, respectively; this further
strengthened the evidence for a single binding site for each ligand. To
determine whether serotonin and the catecholamines bind to separate
sites, the heats generated on titration of ABP with norepinephrine were
measured in the presence of saturating (1 mM)
concentrations of serotonin. After correcting for heats of dilution, no
significant binding of norepinephrine could be detected, suggesting
that identical, or considerably overlapping, binding sites are used for
both serotonin and the catecholamines.
Modeling of ABP--
The ABP sequence was aligned with that of
nitrophorin 2 and modeled using the SwissModel automated program with
the coordinates for the nitrophorin 2 crystal structure (Fig.
9). The resulting model shows the seventh
strand of the The Lipocalin Structure and Ligand Binding Function--
The
lipocalin fold is employed in many salivary functions that involve
ligand binding and protein-protein interactions (24, 25). Three major
R. prolixus salivary lipocalin groups have been
characterized to date. The nitrophorins are a group of heme-binding nitric oxide transporters that also bind histamine with high affinity (3). RPAI-1 is a high affinity ADP-binding protein that also binds a
variety of other adenosine derivatives (7). Finally, ABP binds the
biogenic amines serotonin, epinephrine, and norepinephrine.
On a structural level, the propensity of lipocalins to bind so many
ligand types derives from the flexibility of the
The high affinity binding of two structurally different amine types,
having either hydroxyindole or catechol nuclei in the same binding
site, suggests flexibility in the ABP-binding pocket. In the crystal
structure of a histamine-binding lipocalin from saliva of the tick
Rhiphicephalus appendiculatus, pi-pi stacking interactions
are important for the binding of aromatic ligands. In each of the two
histamine-binding sites, the planar imidazole ring of histamine is
stabilized by aromatic amino acid side chains (11). The aliphatic amino
group is also stabilized by electrostatic interactions with a number of
polar and acidic side chains, as well as ordered water molecules. Like
the tick proteins, ABP contains numerous aromatic side chains that
would be expected, from the results of alignments and modeling studies,
to pack the ligand binding pocket. These side chains could potentially
stabilize any appropriately sized aromatic group, and allow for the
relatively broad specificity for various aromatic ligands displayed by
ABP.
When ABP is modeled based on the nitrophorin 2 crystal structure, the
putative ligand binding pocket is seen as a cleft lined with the side
chains of Phe45, Phe59, Tyr111,
Tyr133, and Tyr135. When serotonin is manually
placed in the cleft, its indole nucleus is surrounded by three aromatic
side chains (Fig. 9) that form both stacking and end-on interactions
between the protein and ligand. The acidic side chains of
Glu57 and Asp113 as well as the hydroxyl group
of Tyr88 are appropriately positioned to stabilize the
aliphatic amino group of the ligand. The model is, of course,
hypothetical, but these types of binding interactions may explain the
combination of submicromolar binding affinity with the relative lack of
specificity indicated by the ability to accommodate both hydroxyindole
and catechol-type amines in the same binding site.
Antihemostatic Functions of ABP--
Serotonin and catecholamines
play important roles in numerous biologic processes. Inhibition of many
of these would be of obvious benefit to a blood-feeding insect. The
release of norepinephrine from the sympathetic nervous system occurs on
wounding and results in the constriction of blood vessels to prevent
blood loss. The highly abundant nitrophorins shuttle the vasodilator
nitric oxide from the R. prolixus salivary gland to the host
skin, and are apparently the primary mechanism for increasing blood
flow around the feeding site. However, the binding of norepinephrine by
ABP may complement the effect of nitric oxide by preventing an
antagonistic constriction response. A functionally similar role is
played by the salivary catechol oxidase of the mosquito Anopheles
albimanus. This enzyme destroys norepinephrine oxidatively and
apparently serves as a vasodilator (26).
Activation of platelets by strong agonists such as thrombin or collagen
induces the release of dense granules that contain the weak agonists
ADP, serotonin, and epinephrine. Aggregation induced by weak agonists
requires activation of phospholipase C and inhibition of adenylyl
cyclase via a Gi-coupled receptor pathway. At high
concentrations, ADP stimulates both pathways sufficiently to induce
aggregation. At low concentrations, only shape change is seen because
of stimulation of phospholipase C via the P2Y1 receptor. Serotonin
activates the phospholipase C pathway via the 5-HT2A
receptor but not the Gi-dependent pathway, and
consequently induces only shape change when administered alone (12).
Epinephrine binds with Gi-coupled adrenergic receptors, inhibiting adenylyl cyclase, and eliciting no detectable aggregation response by itself (12). Administration of serotonin or epinephrine, along with low concentrations of ADP, induces a complete aggregation response, apparently by increasing the level of stimulation to the
necessary signal transduction pathways (Fig. 5). Notably, addition of
serotonin or epinephrine along with subthreshold levels of collagen
produces a full aggregation response, indicating that these molecules
are active in reducing the concentration threshold for
collagen-mediated aggregation (Fig. 6). The presence of ABP in the
circulation would therefore cause a localized increase in the agonist
threshold concentration for platelet aggregation. This protein would
act in concert with RPAI-1, the ADP-binding protein, and salivary
apyrase to reduce the concentration of weak agonists in the vicinity of
the feeding site, thereby attenuating the overall stimulus for platelet aggregation.
Hemostasis and inflammation are complex and redundant host reactions
(1, 27) against which Rhodnius has evolved a complex salivary mixture of antagonists. ABP is the latest such component to be
described. Many of these antagonists act on the same physiologic processes, such as platelet aggregation, but are aimed at different biochemical pathways. Based on the properties of individual components of R. prolixus salivary secretion, an increasingly complex
model is emerging to describe the interactions between the insect and the host hemostatic system. Nitrophorins released into the blood and
skin provide a burst of nitric oxide that dilates blood vessels and
inhibits platelet aggregation (3, 10). Nitrophorin 2 acts as a specific
inhibitor of coagulation by interacting with the proteolytic factor
Xase complex (28). ABP binds norepinephrine and inhibits the host
vasoconstriction response to blood loss. When platelets are activated
and the contents of dense granules are released, ABP binds the agonists
serotonin and epinephrine, whereas salivary apyrase degrades the
agonist ADP (29). Trace amounts of ADP are removed by binding with
lipocalin RPAI-1 (7). Additionally, any inflammation that may induce a
defensive response from the host is inhibited via histamine binding by
the four nitrophorins (4).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel having a generally
hydrophobic ligand-binding pocket in its interior (8). The entry to the
binding pocket is surrounded by a number of flexible loops that can
modulate binding through ligand-induced conformational changes (9, 10). A common mechanism of action for salivary lipocalins is the removal of
small molecule receptor agonists from the feeding area (7, 11). High
affinity binding of the pharmacologically active compounds histamine
and ADP by the lipocalins nitrophorin and RPAI-1, respectively, has
been demonstrated in R. prolixus (4, 7).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside was added to
a concentration of 1 mM and the flasks were shaken under
the same conditions for another 3 h. At the end of this period,
the cells were harvested by centrifugation and washed with 20 mM Tris-HCl, pH 8.0.
where r = observed fluorescence to absorbance
ratio; Ro = ratio in the absence of a ligand;
Rm = ratio when protein is saturated with ligand;
K0.5 = ligand concentration determining
1/2 of sites occupied in the protein; and [L] = molar concentration of the ligand. A bootstrap method was used to
evaluate the affinity of ABP to serotonin (17).
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
DNA sequence and deduced amino acid sequence
of ABP (GenBankTM accession number AY186251).
The signal sequence is underlined.
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Fig. 2.
Amino acid alignment of ABP with the four
nitrophorin (np 1-4) sequences. Alignment was
performed using the Clustal algorithm in the DNAStar package. Residues
conserved among all five sequences are shaded. Conserved
cysteines are shown as white within a black
box.
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Fig. 3.
Inhibition of serotonin-induced uterine
contraction by ABP. Time and contraction distance scales are
indicated. Arrows above the plots indicate additions of
serotonin hydrochloride; amount of serotonin added (ng/ml) is shown
above each arrow. Arrows below the
plots indicate addition of 25 µg of ABP. A,
left-hand trace shows the response of the uterine
preparation to 60 and 300 ng/ml addition of serotonin. Center
trace shows the responses to addition of 60 ng/ml serotonin
followed by two additions of 300 ng/ml, administered after addition of
25 µg of ABP. Right-hand trace shows the response of the
same preparation to 60 and 300 ng/ml additions of serotonin after
removal of ABP and addition of fresh saline. B,
left-hand trace shows response of the uterine preparation to
100 and 300 ng/ml additions of serotonin. Center trace shows
the response to five successive 100 ng/ml additions of serotonin
administered after addition of 25 µg (~2.3 µM) of
ABP. Note that the inhibition is largely overcome at 500 ng/ml (2.4 µM) serotonin. Right-hand trace shows the
response to 100 ng/ml serotonin after removal of ABP and addition of
fresh saline.
-adrenergic receptors. The release of norepinephrine at sympathetic
presynaptic membranes in response after wounding leads to rapid
vasoconstriction (23). Scavenging of norepinephrine would benefit a
feeding insect by inhibiting vasoconstriction, thereby allowing
unimpeded blood flow to the feeding site. The effect of ABP on
norepinephrine-induced vasoconstriction was evaluated using a rabbit
aortic ring preparation. Addition of ABP (to a concentration of 2 µM) to a preparation stimulated to contract with 1 µM norepinephrine caused relaxation that began
immediately after addition of protein (Fig.
4). When the protein was removed from the
bath, the preparation could again be induced to contract by addition of
agonist. This reversible antagonism of norepinephrine is consistent
with binding of this compound by ABP.
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Fig. 4.
Inhibition of norepinephrine-induced
contraction of the rabbit aorta by ABP. Rabbit aortic rings in
Tyrode's solution were contracted by addition of norepinephrine to a
concentration of 1 µM. Norepinephrine binding by ABP was
measured as a relaxation after addition of recombinant protein.
Control indicates addition of buffer only, whereas ABP
indicates addition of 25 µg (~2.3 µM) of recombinant
protein. Arrow indicates point of addition.
2-adrenergic receptors,
serotonin induces an aggregation response. Epinephrine alone does not
produce a detectable effect on platelet aggregation, but like serotonin it potentiates the effect of other agonists (12).
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Fig. 5.
ABP inhibits platelet aggregation in
indomethacin-treated platelets. A, ABP inhibits
serotonin-induced platelet shape change. Platelets were incubated with
phosphate-buffered saline (PBS) or ABP (0.2-0.8
µM) followed by addition of serotonin (5-HT,
0.5 µM) as indicated. B, ABP does not affect
ADP-induced platelet aggregation. Platelets were incubated with
phosphate-buffered saline or ABP (0.8 µM) followed by
addition of ADP as indicated. C, ABP inhibits
serotonin-mediated potentiation of ADP-induced platelet aggregation.
Platelets were incubated with phosphate-buffered saline
(vehicle) or ABP (0.8 µM) followed by addition
of ADP (1.3 µM) plus serotonin (0.4 µM) as
indicated. D, ABP inhibits epinephrine
(EPI)-mediated potentiation of ADP-induced platelet
aggregation. Platelets were incubated with phosphate-buffered saline
(vehicle) or ABP (0.8 µM) followed by addition of ADP
(1.3 µM) plus epinephrine (0.5 µM) as
indicated.
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Fig. 6.
ABP inhibits platelet aggregation in the
absence of indomethacin treatment. A, ABP inhibits
epinephrine-mediated potentiation of collagen (COLL)-induced
platelet aggregation. Platelets were incubated with phosphate-buffered
saline (PBS) (vehicle) or ABP (0.8 µM)
followed by addition of epinephrine (EPI, 0.5 µM) plus collagen (0.75 µg/ml) as indicated. From
left to right traces show shape change with
collagen alone, no detectable response with epinephrine alone,
potentiation of the collagen response by co-addition of epinephrine
resulting in a full aggregation response, and loss of potentiation by
epinephrine in the presence of ABP. B, ABP inhibits
serotonin-mediated potentiation of collagen-induced platelet
aggregation. Platelets were incubated with phosphate-buffered saline
(vehicle) or ABP (0.8 µM) followed by addition of
serotonin (5-HT, 0.5 µM) plus collagen (0.75 µg/ml) as indicated. From left to right traces
show shape change induced by collagen alone, shape change induced by
serotonin alone, potentiation of the collagen response by co-addition
of serotonin resulting in a full aggregation response, and loss of
potentiation by serotonin in the presence of ABP.
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Fig. 7.
Binding of serotonin with ABP. A,
gel filtration chromatogram ABP with absorbance detection at 280 nm.
Solid line, ABP alone; dashed line, an equal
amount of ABP chromatographed with 1 µM serotonin added
to running buffer. Baseline is corrected for increased absorbance
because of free serotonin in the buffer. B, gel filtration
chromatogram ABP with fluorescence detection at 340 nm. Solid
line, ABP alone; dashed line, an equal amount of ABP
chromatographed with 1 µM serotonin added to running
buffer. Base line is corrected for increased fluorescence because of
free serotonin in the buffer. C, modified Hummel-Dreyer
method was applied at the indicated concentrations of serotonin.
Symbols and error bars indicate the mean ± S.E. of three experiments. The continuous line represents
the best fit obtained by 1,000 bootstraps of random nonlinear
regressions, giving an apparent kilodalton of 201 ± 71 nM.
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Fig. 8.
Binding of serotonin, epinephrine, and
norepinephrine by isothermal titration calorimetry. A,
enthalpies of binding from the titration of 50 µM
norepinephrine into 5 µM ABP (open squares).
The best fit with a single-site model (solid line) results
in a binding constant of 24 nM, H =
13.5 kcal/mol, and T
S =
3.1 kcal/mol,
with a stoichiometry of n = 0.81. For comparison, the
solid squares show the titration of norepinephrine to
protein in the presence of 1 mM serotonin under otherwise
identical conditions. B, titration of 10 µM
serotonin into 1 µM ABP (open triangles) and
best fit model (solid line) with
H =
14.2 kcal/mol, T
S =
4.6 kcal/mol,
n = 0.85. C, titration of 50 µM epinephrine into 5 µM ABP (open
circles) and best fit model (solid line) with
H =
8.5 kcal/mol, T
S = 0.3 kcal/mol, and n = 1.28.
-barrel moved toward the center of the protein
relative to the template, reducing the volume of the binding pocket. A
molecule of serotonin, manually inserted into the protein, is
surrounded by the side chains of the aromatic residues
Phe59, Tyr111, and Tyr133, which
stabilize the ligand via pi-pi interactions. Situated in this manner
the aliphatic amino group of the serotonin molecule would be further
stabilized by electrostatic interactions with the carboxylates of
Glu57 and Asp113 as well as the hydroxyl group
of Tyr88 (Fig. 9).
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Fig. 9.
Stereoview of a modeled ligand binding pocket
containing serotonin. Protein model constructed using the
SwissModel program and the coordinates of the nitrophorin 2 crystal
structure with heme removed. In the modeled pocket, the serotonin
molecule is surrounded by the aromatic side chains of
Phe59, Tyr111, and Tyr133.
Asp113 and Glu57 could potentially serve to
stabilize the aliphatic amino group by electrostatic interactions. The
figure was produced with Molscript (30).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel structure
and its ability to tolerate considerable amino acid side chain
variability while maintaining its fold (8). Sequence comparisons with
other R. prolixus lipocalins show that ABP is most closely
related to the nitrophorins (6). The positions of the cysteine
residues are equivalent in the two groups, suggesting that the
disulfide bonding pattern is the same in the two (Fig. 2). However, the
proximal histidine residue (His59 of nitrophorin 1), which
coordinates with the heme iron atom and is located on the third
-strand of the nitrophorins, is replaced with Asn61 (in
the fully processed protein) in ABP. Consequently, ABP shows no ability
to bind heme. ABP and the nitrophorins are apparently derived from a
common ancestor through gene duplication events. Divergence of the two
protein forms has resulted in highly different ligand-binding
specificities and, consequently, very different functional roles.
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ACKNOWLEDGEMENTS |
---|
We acknowledge Van My Pham for DNA sequencing, Roseanne Hearn for technical assistance, and Nancy Schulman for editorial work on the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1EUO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ To whom correspondence should be addressed: Rm. 126, Bldg. 4, Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, 4 Center Dr., Bethesda, MD 20892-0425. Tel.: 301-435-2967; Fax: 301-402-2201; E-mail: jandersen@niaid.nih.gov.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M211438200
2 J. M. C. Ribeiro, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: ABP, amine-binding protein; ITC, isothermal titration calorimetry.
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REFERENCES |
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