 |
INTRODUCTION |
Serine proteases are involved in many processes during development
and tissue homeostasis. Among other functions, they degrade components
of the extracellular matrix to allow outgrowth of neuronal processes
(1) or cell migration (2); they promote cell death (3) and act as
mitogenic or survival factors (4). The activity of the proteases is
regulated by their cognate inhibitors, which must act in an accurately
balanced fashion to ensure a normal development and homeostasis.
Disturbances of this balance in the nervous system have been proposed
to be involved in pathological disorders such as Alzheimer's disease
(5).
To date, several serine proteases have been detected in the central
nervous system, including tissue-type plasminogen activator (t-PA),1 chymotrypsin,
neuropsin, elastase, and thrombin (6). Many serine protease inhibitors
regulating the activity of these proteases are found in the brain.
However, protease nexin-1 (7) is the only known endogenous thrombin
inhibitor present in the central nervous system. In vitro,
the interplay of thrombin and PN-1 has been shown to modulate neurite
outgrowth of neuronal cells (8, 9) and the stellation of astrocytes
(10). Furthermore, PN-1 is highly expressed in response to injury of
the nervous system (11). Despite these observations mice lacking PN-1
show only a subtle phenotype in the nervous system (12), a result that can be explained by the existence of other unknown serine protease inhibitors that compensate for the lack of PN-1 function in these animals. To address this question, we searched for thrombin inhibitory activities in the brain of
PN-1(
/
) mice.
In this report, we present the characterization and purification of a
novel serine protease inhibitor that could be identified as the mouse
phosphatidylethanolamine-binding protein, PEBP. This 187-amino
acid-containing protein belongs to a family of phospholipid-binding proteins found in a wide range of species from flowering plants to
mammals (13). In most cases the physiological role of these proteins
in vivo is not yet understood. The demonstration of
inhibitory activity against serine proteases indicates a new biological
function for mouse PEBP that it might have in common with other members of the PEBP family.
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EXPERIMENTAL PROCEDURES |
Preparation of Brain Homogenates--
The brains were prepared
from C57bl/6 mice (RCC Ltd. Switzerland) of different ages. The deeply
anesthetized animals were pericardially perfused with PBS without
Ca2+ or Mg2+ for 5-10 min to obtain blood free
brains. These were homogenized either in parts (cerebellum, cortex,
remaining parts of the brain) or as complete brains for 40 s using
a Polytron homogenizer (Kinematica GmbH) in 10 mM Hepes, pH
7.5, 0.2% Tween 20, 320 mM sucrose, 1 mM EDTA.
The homogenates were cleared by centrifugation (12,000 × g, 30 min, 4 °C) and the supernatants filtrated through a
Millex-HA 0.45-µm filter unit (Amicon).
Protease Inhibition Assays--
The human
-thrombin was
previously prepared and characterized (14); all other proteases were
commercially available products (Sigma, Fluka). The proteases were
diluted in enzyme buffer (67 mM Tris, pH 8.0, 133 mM NaCl, 0.13% polyethylene glycol 6000; in case of
chymotrypsin: 50 mM Tris, pH 8.0, 380 mM NaCl,
3 mM CaCl2) and used in the assays with the
following final amounts: thrombin, trypsin, and chymotrypsin, 0.005 pmol; t-PA and neuropsin, 0.5 pmol; pancreatic elastase, 4 pmol. 80 µl of the samples were mixed with 10 µl of protease in a 96-well
plate and incubated for 30 min at 37 °C. After addition of 10 µl
of chromogenic substrate (H-D-Ile-Pro-Arg-para-nitroanilide,
Chromogenix, 1.25 mg/ml in H2O; for chymotrypsin inhibition
assay:
N-succinyl-Ala-Ala-Pro-Phe-para-nitroanilide, Sigma, 2.3 mg/ml in H2O), the remaining amidolytic activity
was determined by measuring the rate of the hydrolysis of the
chromogenic substrate at 405 nm over 30 min using a Thermomax
microplate reader (Molecular Devices).
Complex Formation Assay--
8 and 24 µl of brain homogenate
(3.2 µg of protein/µl) of a perfused
PN-1(
/
) mouse were incubated
with 40 ng of human
-thrombin in a total volume of 40 µl in enzyme
buffer for 30 min at 37 °C. After preincubation, 5 µl of
nonreducing sample buffer were added, and the proteins resolved by
SDS-PAGE without previous boiling. The resolved proteins were
electroblotted on Protran nitrocellulose transfer membrane (Schleicher
& Schuell) in a Transblot SD semidry transfer cell (Bio-Rad) at 3 mA/cm2 for 40 min. The immunodetection of thrombin was
performed using a polyclonal rabbit anti-human thrombin antibody
(American Diagnostics, no. 4702) as primary and a horseradish
peroxidase-coupled donkey anti-rabbit antibody (Amersham Pharmacia
Biotech) as secondary antibody. The Western blot was visualized using
the ECL detection kit (Amersham Pharmacia Biotech).
Coimmunoprecipitation--
Two wild type mouse brains were
homogenized. One half of the sample was incubated with 60 µl of
thrombin (1 nM), the other half with 60 µl of enzyme
buffer for 30 min at 37 °C. Monoclonal antibodies against thrombin,
EST-6 (American Diagnostics), were chemically cross-linked to protein
A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) as described
elsewhere (15). EST-6 is a monoclonal antibody that recognizes free
thrombin and also thrombin complexed with inhibitors (e.g.
antithrombin III) (16). 50 µl of the EST-6 coated beads were added to
the samples and the mixtures incubated at 4 °C overnight with gentle
shaking. The beads were then washed three times with 1 ml of enzyme
buffer each, and then resuspended in 50 µl of sample buffer,
denatured at 95 °C for 5 min, and the supernatants loaded on a
12.5% SDS-PAGE. The gel was then silver-stained following the
manufacturer's protocol (Bio-Rad Silver Stain).
Gel Filtration--
A Superdex 200 16/60 gel filtration column
(Amersham Pharmacia Biotech) was equilibrated with 50 mM
Tris, pH 8.0, 100 mM NaCl at 4 °C. The brain homogenate
was applied to the column at a flow rate of 0.25 ml/min with the
equilibration buffer. Fractions of 0.25 ml were collected and assayed
for thrombin inhibitory activity.
Purification by Ion Exchange Chromatography--
Brain
homogenates for ion exchange chromatography were prepared as above
using 20 mM ethanolamine, pH 9.0, 100 mM NaCl,
0.2% Tween 20, 320 mM sucrose, 1 mM EDTA as
homogenization buffer. The cleared and filtered homogenates were
applied on a 1-ml HiTrap Q-Sepharose column directly connected to a
1-ml HiTrap heparin-Sepharose column (Amersham Pharmacia Biotech) with
20 mM ethanolamine, pH 9.0, 100 mM NaCl at a
flow rate of 2 ml/min. Buffer exchange of the flow-through (2 ml) was
achieved by dilution with 10 ml of 50 mM acetate, pH 5.0, 70 mM NaCl, followed by concentration to 3 ml using a
Centricon YM-10 (5,000 × g, 1 h, 4 °C).
Subsequently, the sample was loaded on a 1-ml HiTrap SP-Sepharose
column (Amersham Pharmacia Biotech) with the same buffer at a flow rate
of 2 ml/min. The flow-through was concentrated to 300 µl as above.
Protein Sequencing--
The protein separated by SDS-PAGE was
excised from the gel, reduced with dithiothreitol, alkylated with
iodoacetamide, and cleaved with trypsin (sequencing grade, Promega) as
described (17). The extracted tryptic peptides were desalted with 5%
formic acid, 5% methanol in H2O on a 1-µl Poros P20
column and concentrated to 1 µl with 5% formic acid, 50% methanol
in H2O directly into the nanoelectrospray ionization
(NanoESI) needle. NanoESI mass spectrometry was performed according to
the published method of Wilm et al. (18). The mass spectra
were acquired on an API 300 mass spectrometer (PE Sciex) equipped with
a NanoESI source (Protana).
Generation of an Expression Construct for Recombinant Mouse PEBP
(PEBP-H6)--
The cDNA coding for the mouse PEBP was
amplified with Pwo DNA polymerase (Roche) from IMAGE clone
1921274 (Sugano mouse, kidney) using the following oligonucleotides:
5'-CTC TAA GCT TCC ATG GCC GCC GAC ATC-3' and 5'-TCA AAG CGG CCG CTA
CTT CCC TGA ACA GCT GCT CGT TAC AGC CTT GGG CAC ATA GTC ATC CCA CTC-3'.
The PCR product was cloned via the NotI and
HindIII sites into the expression vector pCEP-Pu,
i.e. pCEP4 (Invitrogen) with a puromycin instead of a
hygromycin resistance gene. A cDNA coding for PEBP with a stretch
of six histidine residues fused to the carboxyl terminus was amplified
from this construct using the oligonucleotides: 5'-CTC TAA GCT TCC ATG
GCC GCC GAC ATC-3' and 5'-TCA AAG CGG CCG CTT AAT TAA CGT GAT GGT GAT
GGT GAT GCT TCC CTG ACA GCT GCT CG-3'. The correct structure of both
constructs was confirmed by DNA sequencing. The sequencing reactions
were performed using Dye Terminators (BigDye, PE Biosystems) with a
GeneAmp PCR system 9700 or 2400 thermocycler (PerkinElmer Life
Sciences) and analyzed on an ABI Prism 377 DNA sequencer.
Expression and Purification of Recombinant
PEBP-H6--
Rat-1 cells (4 × 105) were
plated in a 10-cm cell culture dish in 10 ml of normal growth medium
(Dulbecco's modified Eagle's medium with 10% fetal calf serum).
After 24 h, the cells were transfected with 4 µg of
pCEP-Pu-PEBP-H6 using the FuGENE 6 transfection reagent
(Roche Molecular Biochemicals) according to supplier's instructions.
24 h after transfection, the growth medium was exchanged with
serum-free Dulbecco's modified Eagle's medium supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, 16 µg/ml putrescine, and 10 ng/ml progesterone. 72 h after
transfection, the conditioned medium of four dishes was collected and
incubated with 0.5 ml of Ni2+-NTA-agarose (Qiagen) for
2 h at 4 °C. Ni2+-NTA-agarose was collected by
centrifugation and extensively washed three times with 5 ml of enzyme
buffer complemented with 20 mM imidazole. The recombinant
protein was eluted three times with 2 ml of 20 mM
sodium phosphate, pH 6.8, 320 mM sucrose, 0.2% Tween 20, 1 mM EDTA, 250 mM imidazole.
Characterization of Inhibitory Properties--
The inhibitory
activity of purified PEBP-H6 was tested against several
serine proteases. To determine the inhibition constants, the rates for
the hydrolysis of the chromogenic substrates were measured as described
above at fixed enzyme concentrations and substrate concentrations
ranging from 3.5 to 216 µM and PEBP-H6 concentrations between 0 and 2.9 µM. The obtained values
were fitted with the software GraFit 4.0 (Erithacus Software) to the equation for a competitive inhibitor.
|
(Eq. 1)
|
[S] is the substrate concentration,
[I] the total inhibitor concentration,
Km the Michaelis-Menten constant, and Ki the inhibition constant.
Immunocytochemistry--
To raise a polyclonal antiserum against
PEBP, two COOH-terminal of the mouse PEBP (amino acids 144-159 and
174-187) were cross-linked to ovalbumin and injected into rabbits
following standard protocols. The specificity of the immune serum was
assessed by the detection of a single 21-kDa band on a immunoblot of
mouse brain homogenate. Rat-1 fibroblasts were washed three times with
PBS, fixed for 20 min with 4% paraformaldehyde in PBS at room
temperature, and again washed with PBS. For permeabilization, the cells
were fixed in 4% paraformaldehyde with 15% picric acid in PBS (20 min, room temperature), and washed with PBS with 0.2% Triton X-100.
After blocking for 30 min with 3% BSA in PBS, the cells were incubated for 1 h with antiserum (1:1500 in blocking solution) and then washed with PBS. Immunofluorescence detection was performed using the
Alexa 488 goat anti-rabbit IgG conjugate (Molecular Probes) as a
secondary antibody.
 |
RESULTS |
Detection of Thrombin Inhibitory Activity in
PN-1(
/
) Brains--
Perfused brains of
PN-1(
/
) and wild type mice were
divided into cerebellum, cortex, and the remaining parts of the brain.
Homogenates of these samples were checked for the presence of thrombin
inhibitory activity. Stepwise dilutions of aliquots of the homogenates
containing equal amounts of proteins revealed the presence of a
thrombin inhibitory activity in all investigated brain compartments
(Fig. 1). The level of this inhibitory
activity was equivalent in the different parts of the brain tested. As
expected, the thrombin inhibition was significantly higher in the wild
type brains than in the PN-1(
/
)
brains. However, the fact that thrombin inhibition was also detected in
the PN-1(
/
) brains indicated
indeed the presence of an additional thrombin inhibitor present in the
mouse brain.

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Fig. 1.
Thrombin inhibitory activity in brains of
wild type and
PN-1( / )
mice. Different parts of wild type ( , cerebellum; , cortex;
, remaining parts) and
PN-1( / ) ( , cerebellum; ,
cortex; , remaining parts) brains were homogenized and their protein
content measured. Aliquots with equal protein contents were stepwise
diluted and assayed for thrombin inhibition.
|
|
Complex Formation Assay--
An electrophoretic mobility shift
assay was performed to address the question whether a complex is formed
between thrombin and one or more components of
PN-1(
/
) brains. Aliquots of
brain homogenates from perfused
PN-1(
/
) mice were preincubated
with human
-thrombin and applied to an SDS-PAGE under nonreducing,
semi-native (0.1% SDS) conditions. The resolved proteins were analyzed
for high molecular weight complexes by subsequent immunoblot analysis
with a polyclonal anti-thrombin antibody (Fig.
2A). Preincubation of the
brain homogenates with thrombin resulted in the formation of complexes
of ~60 kDa. This size suggested a complex between thrombin (37 kDa)
and a second protein of ~23 kDa.

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Fig. 2.
Complex formation and coimmunoprecipitation
of brain homogenates with human
-thrombin. Panel A, the indicated
protein amounts of cleared homogenates from
PN-1( / ) brains were
preincubated with 40 ng of thrombin for 30 min at 37 °C and then
applied to an SDS-PAGE gel under nondenaturing conditions.
Immunoblotting was performed with a polyclonal anti-human thrombin
antibody. Panel B, brain homogenates were preincubated with
buffer or with thrombin and then coimmunoprecipitated with protein
A-Sepharose beads coated with a monoclonal antibody against thrombin.
The proteins were resolved under denaturing conditions on an SDS-PAGE
gel and visualized by silver staining. A protein of approximately 20 kDa (arrowhead) coimmunoprecipitated after preincubation
with thrombin.
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|
Coimmunoprecipitation--
A coimmunoprecipitation was performed
to confirm the formation of such a complex between thrombin and an
inhibitory protein. Brain homogenates were incubated with human
-thrombin for 30 min at 37 °C. After preincubation an
immunoprecipitation was performed with protein A-Sepharose beads coated
with the anti-thrombin monoclonal antibody EST-6. Brain homogenate
preincubated with buffer only was used as a negative control. The
coimmunoprecipitated proteins were applied to an SDS-PAGE and
visualized by silver staining (Fig. 2B). As the binding
properties between thrombin and the putative inhibitor were not known,
the coimmunoprecipitation was performed at very low stringency
conditions, resulting in a high background. Nevertheless, a protein of
~20 kDa was coimmunoprecipitated after preincubation with human
-thrombin, again indicating the existence of an inhibitory thrombin
binding protein of 20-23 kDa.
Gel Filtration--
To partially purify the inhibitor, brain
homogenates from PN-1(
/
) mice
of different ages were fractionated according to their molecular weight
with a Superdex-200 gel filtration column. The fractions were assayed
for the presence of thrombin inhibitory activity. As shown in Fig.
3, thrombin inhibitory activity eluted
from the column in fractions 56-63, corresponding to an elution volume of 14-15.75 ml. Calibration of the gel filtration column with ribonuclease A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), and bovine serum albumin (67 kDa) under the same conditions indicated a molecular mass for the putative inhibitor of 21-24 kDa.
The amount of this inhibitory activity was detected at equal levels in
brains of wild type and PN-1(
/
)
mice (data not shown). In addition the activity did not depend on the
age (from 14 days to 2 years). The peak in hydrolysis rate detected in
fractions 64-66 of brain homogenates obtained from older animals
corresponds to a proteinase, not further characterized, that seems to
be up-regulated with age.

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Fig. 3.
Thrombin inhibitory activity in gel
filtration fractions. Brains obtained from
PN-1( / ) mice of different ages
( , 14 days; , 6 months; , 1 year; , 2 years) were
homogenized and applied to a Superdex-200 gel filtration column
(dashed line, molecular weight calibration) and
the collected fractions assayed for thrombin inhibitory activity (×,
brain homogenation buffer only).
|
|
Taken together, the results of the thrombin assays, the complex
formation assay, the coimmunoprecipitation, and the gel filtration suggested an unidentified thrombin inhibitor in the mouse brain. The
finding that heat treatment of the brain homogenates (95 °C for 5 min) completely abolished the thrombin inhibition (data not shown)
supported the hypothesis that the inhibitory activity is indeed due to
a protein.
Purification and Identification of the Inhibitor--
The
inhibitor did not bind to Q- and heparin-Sepharose at pH 9.0 and
SP-Sepharose at pH 5.0 under the conditions we used. Nevertheless, we
succeeded to purify the inhibitor by a combined anion exchange and
heparin affinity chromatography followed by a cation exchange
chromatography. The activity was always recovered from the flow-through
of the columns. The heparin column was included to bind and thereby
remove PN-1 whose thrombin inhibitory activity is well established
(19). The cleared homogenate of two wild type brains was applied to the
combined Q and heparin columns; the flow-through containing the
inhibitor was recovered and concentrated. The sample was then diluted
with a buffer appropriate for the cation exchange chromatography and
loaded on an equilibrated SP-Sepharose column. The concentrated
flow-through of this purification step was applied to a 15% SDS-PAGE.
Silver staining of the gel revealed a single band of the expected size
of 20-25 kDa. The tryptic peptides derived from this SDS-PAGE
separated inhibitor were analyzed by tandem mass spectrometry. Sequence
tags from five peptides were obtained that all fitted to the amino acid
sequence of the mouse phosphatidylethanolamine-binding protein (Fig.
4, bold).

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Fig. 4.
Identification of the inhibitor by NanoESI
mass spectrometry. The tryptic peptides found to be identical to
mouse PEBP are shown in bold. The serine at position 116 originally
published (Swiss-Prot P70296) was found to be a glycine that is in fact
conserved in all known proteins belonging to the
phosphatidylethanolamine-binding protein family. This finding was
confirmed by DNA sequencing of IMAGE clone 1921274 that has as the
116th codon a GGT instead of the previously published AGT (GenBankTM
accession no. U43206).
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Recombinant Expression of Mouse PEBP-H6--
For the
cloning of the cDNA coding for mouse PEBP, we used IMAGE clone
1921274 (Sugano mouse, kidney) as a template. Sequencing of this clone
showed that the 3' end of this cDNA does not correspond to the
published sequences for the mouse PEBP mRNA. This would result in
an incorrect sequence for the last 10 amino acids of PEBP. As the
NanoESI mass spectrometry had shown that the COOH terminus of the
purified protein is identical with the published sequence for mouse
PEBP (GenBankTM accession no. P70296), we decided to use an antisense
primer in the PCR that codes for the correct last 10 amino acids. This
cDNA served as a template for the generation of a cDNA coding
for mouse PEBP fused to six histidine residues at the COOH terminus to
obtain highly purified protein. In initial experiments we found that
the recombinant PEBP-H6 is most easily purified from the
conditioned medium of transfected cells. The second elution fraction of
the purification using Ni2+-NTA-agarose resulted in a
single band after SDS-PAGE (Fig.
5A, lane
4). The identity of this protein with PEBP-H6
was confirmed by immunoblot analysis (data not shown). The inhibitory
properties of PEBP against thrombin, chymotrypsin, trypsin, t-PA,
elastase, and neuropsin were investigated using this highly purified
PEBP-H6 preparation. Thrombin (Fig. 5B) and
chymotrypsin (Fig. 5C) are competitively inhibited by
PEBP-H6 with apparent Ki values in the
nanomolar and low micromolar range, respectively (Table I). Neuropsin was inhibited as well (data
not shown), whereas the amidolytic activities of trypsin, tissue
plasminogen activator, and pancreatic elastase were not affected.

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Fig. 5.
Inhibitory properties of PEBP-H6.
Panel A, following expression in Rat-1 cells,
PEBP-H6 was purified using Ni2+-NTA-agarose.
Samples of the purification step containing 100 ng of protein were
subjected to electrophoresis on a 12.5% SDS-PAGE. The gel was
silver-stained. Lane 1, flow-through of
Ni2+-NTA-agarose; lane 2, first washing;
lane 3, first elution; lane 4, second elution.
Panel B, 0.1 nM thrombin was incubated with
increasing amounts of PEBP-H6 and the velocity of the
hydrolysis of the chromogenic substrate was determined as described
under "Experimental Procedures" (PEBP-H6
concentrations: , dashed and dotted line, 0 µM; , dotted line, 0.74 µM; , dashed line, 1.10 µM; , straight line, 1.47 µM). Panel C, 0.1 nM chymotrypsin
was incubated with increasing amounts of PEBP-H6
(PEBP-H6 concentrations: , dashed and dotted
line, 0 µM; , dotted
line, 1.48 µM; , dashed
line, 2.22 µM; , straight
line, 2.95 µM). The data represent the mean of
three separate determinations.
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An interference of the COOH-terminal histidine tag could be excluded by
the use of purified NH2-terminal His-tagged PEBP or partially purified PEBP with a NH2- or COOH-terminal
hemagglutinin tag, or, furthermore, by the use of the protein encoded
by IMAGE clone 1921274 with 10 different amino acids at the COOH
terminus. All these different proteins show similar inhibitory properties.
Extracellular Localization of PEBP--
As PEBP is thought to be a
cytoplasmic protein and lacks a secretion signal in its sequence, it
was somehow unexpected to detect it in the conditioned medium of
PEBP-expressing cells. To confirm this extracellular localization of
PEBP, Rat-1 fibroblast cells were immunostained with a polyclonal
antiserum directed against mouse PEBP. The detection of PEBP
immunoreactivity at the surface of nonpermeabilized Rat-1 cells (Fig.
6A) showed that an
extracellular localization of PEBP has to be considered as well.

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Fig. 6.
Cell surface localization of PEBP.
Nonpermeabilized (panel A) and permeabilized
(panel B) Rat-1 fibroblasts were immunostained with a
polyclonal antiserum against mouse PEBP. No signal was obtained with
the pre-immune serum on nonpermeabilized (panel C) and
permeabilized (panel D) cells.
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|
 |
DISCUSSION |
In this study, we report the detection of a novel serine protease
inhibitor in mouse brain. Purification and analysis by NanoESI mass
spectrometry identified this protein as PEBP. We demonstrate that PEBP
competitively inhibits the amidolytic activities of thrombin,
chymotrypsin, and neuropsin, whereas the activities of trypsin, t-PA,
and pancreatic elastase were not affected.
The phosphatidylethanolamine-binding protein was originally purified
from bovine brain and described as a soluble 23-kDa basic cytosolic
protein (20). Binding studies revealed its affinity for
phosphatidylethanolamine (21), nucleotides like GTP and GDP and small
GTP-binding proteins and other hydrophobic ligands (22). Independently,
PEBP was purified from human brain as neuropolypeptide h3 (23);
sequencing of this protein (24) showed 95% amino acid sequence
identity with the sequence for the bovine PEBP (25). The sequence of
PEBP shares no significant homology with other proteins so that it is
assumed to be the prototype member of the PEBP family. PEBP homologues
have been identified in other mammals including rat (26), mouse (27),
and monkey (28). Other members of the PEBP family include the putative
odorant-binding protein in Drosophila (29), the putative
PEBP of the malaria parasite Plasmodium falciparum (30), and
the Ov-16 antigen of Onchocera volvulus (31) and the
toxocara excretory-secretory antigen 26 of Toxocara canis
(32), two parasitic nematodes. A dosage-dependent suppressor of CDC25 mutations in Saccharomyces
cerevisiae, TFS1 (33), and several proteins in flowering plants
(Arabidopsis thaliana (Refs. 34-36) and
Antirrhinum (Ref. 37)) also belong to this family. Despite
this widespread expression and the resolution of the three-dimensional
structures of bovine and human PEBP by x-ray crystallography (38, 39),
only very little is known about the functions of this protein family.
In rat, PEBP was described to be the precursor of the hippocampal
neurostimulating peptide, an undecapeptide that is involved in the
differentiation of neurons in the medial septal nucleus, where it
enhances the synthesis of choline acetyltransferase (40). However,
being the precursor of hippocampal neurostimulating peptide cannot be
the only biological function of PEBP, as it is not only expressed in
the central nervous system but also in a wide variety of other tissues
including spleen, testis, ovary, muscle, and stomach (41, 42). The
members of the PEBP family are often highly expressed in growing or
elongated cells such as oligodendrocytes, spermatides, and the
inflorescence meristem of flowering plants (36). This expression
pattern and the binding to phospholipids located mainly on the inner
leaflet of the plasma membrane suggest a role of PEBP in the
organization of the plasma membrane during cell growth and development.
Serine proteases are known to inhibit or reverse morphological changes of neuronal cells in vitro; thrombin, for example, is
characterized as a neurite retraction signal (9). PEBP could therefore
be important in the outgrowth and maintenance of neuronal processes. PEBP was reported to be one of several cellular proteins present inside
the human immunodeficiency virus type 1 virions (43), again indicating
a possible role for PEBP in membrane organization.
On the other hand, the interaction between PEBP and small-GTP binding
proteins leads to speculation that PEBP could be involved in the
signaling machinery. Recently, the human PEBP was described as a Raf-1
kinase inhibitor protein that suppresses the mitogen-activated protein
kinase signaling (44). The authors of this study showed that PEBP
regulates the extracellular signal-regulated kinase pathway at the
Raf/mitogen-activated protein kinase/extracellular signal-regulated
kinase kinase interface by binding to Raf-1 and thus leading to a
competitive inhibition of mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase. They further
showed that the binding of PEBP/Raf kinase inhibitor protein to Raf-1
decreases during mitogenic stimulation and could characterize the
binding sites (45). Interestingly, thrombin is a mitogenic signal and
Raf-1 is phosphorylated upon binding of thrombin to its receptor, a G
protein-coupled receptor (46). Therefore, PEBP could modulate the
cellular response to stimulation of protease-activated receptors.
As PEBP shares no significant homology with other known classes of
serine protease inhibitors such as the serpins, the Kunitz, the Kazal,
or the Bowman-Birk family (47), the results of this study define it as
the prototype for a novel family of serine protease inhibitors. Of
special interest is the fact that PEBP inhibits a serine protease with
specificity for hydrophobic and aromatic amino acids (chymotrypsin) at
the P1 position as well as one that cleaves only after basic amino
acids (thrombin). The active site and the mechanism by which PEBP
inhibits the serine proteases remain to be determined.
Our finding that mouse PEBP acts as an inhibitor of trypsin-like serine
proteases is supported by a report that the yeast homologue TFS1 acts
as a high affinity inhibitor of the carboxypeptidase Y (48). TFS1 and
mouse PEBP share 31% identity at the amino acid level. The
carboxypeptidase Y contains a catalytic Ser, His, Asp triad and a
trypsin-like oxyanion hole. Its catalytic mechanism is therefore
believed to be similar to the serine proteases of the trypsin-type
(49). The fact that members of the PEBP family in yeast and mouse, two
evolutionarily very distant organisms, act as inhibitors of proteolytic
activity suggests that this might be a common feature of the PEBPs.
The amino acid sequence of PEBP contains no obvious secretion signal,
and previous immunohistochemical studies attributed a cytoplasmic
localization to this protein. The immunodetection of endogenous PEBP on
the cell surface and the presence of active PEBP-H6 in the
supernatant of transfected cells shown here demonstrate that, at least
under cell culture conditions, the localization of PEBP is not
restricted to the cytoplasm or the inner leaflet of the plasma
membrane. Whether this is due to shedding of membranous material as is
found for the nematodes' homologues of PEBP (32) or whether the
binding to phosphatidylethanolamine plays a role in a putative
transport across the plasma membrane remains to be investigated.