Characterization of Recombinant and Brain Neuropsin, a
Plasticity-related Serine Protease*
Chigusa
Shimizu
,
Shigetaka
Yoshida
,
Masao
Shibata§,
Keiko
Kato
,
Yoshiharu
Momota
,
Kazumasa
Matsumoto
,
Takahiko
Shiosaka¶,
Ryosuke
Midorikawa
,
Tomohiro
Kamachi
,
Akiko
Kawabe§, and
Sadao
Shiosaka
From the
Division of Structural Cell Biology, Nara
Institute of Science and Technology, Nara Institute of Science and
Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, the
§ Medical and Biological Laboratories Company, Limited,
1063-103 Ohara, Ina, Nagano 396, and the ¶ Department of
Medical Laboratory Technology, Ehime College of Health Science,
543 Takaoda, Iyo, Ehime 791-21, Japan
 |
ABSTRACT |
Activity-dependent changes in
neuropsin gene expression in the hippocampus implies an involvement of
neuropsin in neural plasticity. Since the deduced amino acid sequence
of the gene contained the complete triplet (His-Asp-Ser) of the serine
protease domain, the protein was postulated to have proteolytic
activity. Recombinant full-length neuropsin produced in the
baculovirus/insect cell system was enzymatically inactive but was
readily converted to active enzyme by endoprotease processing. The
activational processing of prototype neuropsin involved the specific
cleavage of the Lys32-Ile33 bond near its
N terminus. Native neuropsin that was purified with a purity of
1,100-fold from mouse brain had enzymatic characteristics identical to
those of active-type recombinant neuropsin. Both brain and recombinant
neuropsin had amidolytic activities cleaving Arg-X and
Lys-X bonds in the synthetic chromogenic substrates, and
the highest specific activity was found against
Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. The active-type
recombinant neuropsin effectively cleaved fibronectin, an extracellular
matrix protein. Taken together, these results indicate that this
protease, which is enzymatically novel, has significant limbic effects
by changing the extracellular matrix environment.
 |
INTRODUCTION |
Some proteases have been suggested to be related to neural cell
dynamics in such processes as cell death, migration, cell-to-cell adhesion and de-adhesion, process elongation, pathfinding, and axonal
rearrangement (1-5). These phenomena have been investigated by
supplying known proteases involved in blood coagulation, fibrinolysis, or digestion to neural cell cultures. However, the observations that
the proteases are mainly localized in and released from non-neural cells do not support all of such neural effects (5-7). Thus, we
postulated that neurons themselves may produce and release their own
proteases to participate in the neural cell dynamics described
above.
Neuropsin (NP)1 was cloned
from the mouse brain and was shown to be localized in mouse
hippocampal pyramidal neurons (8). These results and the observation
that its mRNA showed marked activity-dependent changes
caused by plasticity-inducible stimuli are suggestive of some neural
effects in limbic plasticity (8, 9). However, it is still not known
whether NP protein has enzyme activity as suggested by the deduced
amino acid sequence (8). We postulated that the enzyme activity might
be a molecular basis for the physiological responses induced by various
stimuli. Therefore, in the present study, we examined whether
recombinant NP (r-NP) and brain NP had proteolytic activity against
synthetic and natural substrates.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Mono S, Sepharose 2B, CNBr-activated Sepharose 4B
and CL-6B, Superdex-75HR, Superose 12, Resource S, and Protein
G-Sepharose were from Amersham Pharmacia Biotech. Silver staining kits
were from Bio-Rad. Diisopropyl fluorophosphate (DFP), benzamidine, bestatin, soybean trypsin inhibitor, human plasma thrombin (EC 3.4.4.13), and TNM-FH insect cell medium were purchased from Sigma.
[1,3-3H]DFP was from NEN Life Science Products.
D-Pro-Phe-Arg-pNA, D-Val-Leu-Arg-pNA, and
D-Val-Leu-Lys-pNA were from Daiichi Pure Chemicals (Tokyo, Japan). N-Bz-DL-Lys-pNA, dimethyl sulfoxide,
aprotinin, protease 1 (Achromobacter lyticus, EC 3.4.21.50),
trypsin (EC 3.4.21.4), and polyethylene glycol 20,000 were from Wako
Pure Chemical Inc. (Osaka, Japan). Bz-Tyr-pNA, L-Leu-pNA,
Bz-L-Arg-pNA, Boc-Val-Pro-Arg-MCA, Z-Pro-Phe-Arg-MCA,
Boc-Glu-Lys-Lys-MCA, Pyroglutamyl-Gly-Arg-MCA, glutaryl-Gly-Arg-MCA,
Boc-Phe-Ser-Arg-MCA, succinyl-Leu-Leu-Val-Tyr-MCA, Boc-Asp-Pro-Arg-MCA, Boc-Glu-Gly-Arg-MCA, Boc-Leu-Arg-Arg-MCA, Boc-Leu-Thr-Arg-MCA, Boc-Gly-Arg-Arg-MCA,
Boc-Ala-Gly-Pro-Arg-MCA, N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl-agmatin
(E-64), pepstatin A, leupeptin, chymostatin, and antipain were from
Peptide Institute Inc. (Osaka, Japan). Bicinchoninic acid protein assay kit was from Pierce. pVL1392 transfer vector was from Invitrogen. BaculoGold transfection kit was from PharMingen (San Diego, CA). Serum-free medium for insect cells, SF-900II, and human plasma fibronectin were from Life Technologies, Inc. Fetal bovine serum and
(4-amidinophenyl)methanesulfonyl 1-fluoride were from Boehringer Mannheim (Germany). Horseradish peroxidase-labeled goat anti-rat IgG
was purchased from Cappel. All other reagents used were of analytical
grade.
Cloning of Full-length NP cDNA into Baculovirus--
The
866-base pair NP3 cDNA was subcloned into the NotI site
of the pVL1392 transfer vector to create plasmid pVL1392/NP3 (Fig. 1)
(8). Plasmid DNA was transferred into the AcNPV genome by homologous
recombination so that Sf21 cells were transfected with transfer
vector and AcNPV DNA. The presence of the NP3 cDNA was confirmed by dot
blot hybridization. Positive virus clones were harvested from the
culture medium, and the DNA was characterized by Southern blotting.
Production of r-NP in Insect Cells--
Sf21 insect cells
were grown at 27 °C in TNM-FH medium containing 10%
heat-inactivated fetal calf serum to a density of 7 × 106/75-cm2 tissue culture flask. For infection,
cells were centrifuged for 5 min at 500 × g at room
temperature and resuspended in serum-free SF900II medium. Cells at a
density of 1 × 106 cells/ml were infected with
recombinant baculovirus at a multiplicity of infection of 1. Three to
five days postinfection, the incubation medium was harvested by
removing the cells by centrifugation at 500 × g for 10 min at 4 °C. The supernatant was clarified by centrifugation at
4 °C, 25,000 × g, for 1 h and dialyzed against
10 mM HEPES buffer (pH 7.4) for 2 days. The supernatant was
then concentrated using Spectrapore Membranes
(Mr 3500) against polyethylene glycol 20,000 at
4 °C and dialyzed against 10 mM HEPES buffer (pH 7.4). This solution was subjected to anion exchange chromatography using a
6-ml column of Resource S. Bound protein was eluted with a linear gradient of 0 to 1 M NaCl in 50 min at a flow rate of 2 ml/min; NP was eluted at 0.44-0.58 M NaCl. For further
purification, the protein was concentrated by Centricon 10 (Amicon),
dialyzed against 50 mM HEPES, 0.15 M NaCl (pH
7.4), and gel-filtrated with a 10 × 300-mm column of Superdex 75 HR. The recombinant protein samples were then applied to SDS-PAGE,
followed by silver staining under both reducing and non-reducing
conditions as described below. The expressed recombinant protein had no
or only low amidolytic activity, and this was assumed to be pro-NP
(r-pro-NP) (Table I).
Production of Monoclonal Antibodies against r-pro-NP--
Wistar
rats were immunized by intraperitoneal injection of 50 µg of purified
r-pro-NP emulsified with Freund's adjuvant several times. After
titration of rat serum by Western blotting, the rats were
intraperitoneally boosted with 50 µg of the r-pro-NP on 2 days before
cell fusion. Isolated spleen cells from the immunized rats were fused
with Y3Ag.1.2.3 rat myeloma cells (10). After selection in
hypoxanthine/aminopterin/thymidine medium, the supernatants of
hybridomas were screened for production of specific antibodies to
r-pro-NP by Western blotting and enzyme-linked immunosorbent assay
using microplates coated with r-pro-NP. Positive clones were used for
the production of ascites in pristane-primed nude rats (F344/N Jcl). We
screened for the clone producing the most specific monoclonal antibody
with the highest titer. The monoclonal antibodies (mAbF12 and mAbB5)
were purified from ascites by affinity chromatography on protein
G-Sepharose. Neither antibody showed cross-reactivity against serine
proteases localized in the brain such as
-thrombin, trypsin,
kallikrein, tissue, or urokinase plasminogen activator (11).
Electrophoresis and Western Blotting--
SDS-PAGE was carried
out using 12.5% gels (12), followed by silver staining (13). The
electrophoresed protein was transferred from the gels onto
nitrocellulose membranes which were then incubated overnight with
anti-NP monoclonal antibody diluted with 0.1 M Tris-HCl
buffer (pH 7.5) containing 1% Tween 20 and 0.15 M NaCl at
4 °C. After washing with the same buffer, the blotted membranes were
incubated with horseradish peroxidase-labeled goat anti-rat IgG diluted
with 0.1 M Tris-HCl buffer (pH 7.5) containing 1% Tween
20, 0.15 M NaCl, and 5% bovine serum albumin at room
temperature for 1 h. Immunoreactivity was visualized with 0.04%
3,3'-diaminobenzidine and 1.2% NH3NiSO4 in 50 mM Tris-HCl buffer (pH 7.5) containing 0.04%
H2O2.
Activation of r-Pro-NP by Endoproteases--
Protease 1 (EC
3.4.21.50) and trypsin (EC 3.4.21.4) were used for activational
processing of r-pro-NP, the enzymatically inactive form. Protease 1 more effectively processed r-pro-NP than trypsin, converting it to the
active form, and was thus used in the subsequent experiments. Protease
1 was immobilized on CNBr-activated Sepharose 4B prior to use and was
used to convert r-pro-NP to r-NP. Briefly, Sepharose 4B was washed
several times with coupling buffer (0.5 M NaCl in 0.1 M NaHCO3 (pH 8.3)). Ten mg of protease 1 dissolved in the coupling buffer was added to 1 g of the gel. After incubation in 0.2 M glycine (pH 8.0) to block free
CNBr residues, the Sepharose 4B-immobilized protease 1 was washed
several times with either 0.5 M NaCl in 0.1 M
Tris-HCl (pH 8.0) or 0.5 M NaCl in 0.1 M
acetate buffer (pH 4.0). The immobilized protease 1 was stored in 0.1 M Tris-HCl buffer (pH. 8.0) containing 0.5 M
NaCl. In special cases, protease 1 which was not immobilized on the gel
was also used (cf. Fig. 2).
N-terminal Sequencing of r-NP--
N-terminal amino acid
sequencing of r-NP was performed with an ABI 477 pulsed liquid
sequencer using standard sequencing chemistry. Purified samples were
spotted onto Immobilon membranes (Millipore) 5 mm in diameter and were
put on top of a UF membrane. The Immobilon membranes with bound protein
were then washed with 50% methanol and sequenced by Edman
degradation.
Time-of-flight Mass Spectrometry--
Matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)
was performed using PerSeptive Biosystems (Framingham, MA) Voyager
Elite. The accelerating voltage of the ion source was 25 kV. Data were
acquired with a 20 MHz digitizer. The matrix used for r-NP and r-pro-NP
was sinapinic acid. The matrix material was dissolved in aqueous
CH3CN (33%, v/v) to give a saturated solution. Ten pmol of
r-NP or r-pro-NP was mixed with the matrix prior to loading on the
plate. Bovine serum albumin and 2-thioredoxin were used as molecular
mass standards.
Platelet Aggregation Assay--
Platelet-rich plasma was
obtained from human blood from a volunteer donor containing 3.5%
sodium citrate. Platelets were isolated by gel filtration with a
Sepharose 2B column in HEPES-Tyrode's buffer. Aggregation was
performed at 37 °C with 200 µl of 2 × 108
platelets/ml containing 150 µg of fibrinogen in an aggregometer (Aggretec model TE-500 ERMER). Varying concentrations of thrombin (0.1 and 0.5 units) or r-NP (0.1, 1, and 2 units) were added to the
aggregometer tube.
Cleavage of Fibronectin by r-NP--
Aliquots of 4 µg of human
plasma fibronectin were incubated with 0.4 µg of r-pro-NP or r-NP in
20 µl of 0.2 M Tris-HCl buffer (pH 8.0). Reaction was
performed at 37 °C for 1, 2, 4, 8, and 16 h and was then
terminated by boiling for 3 min with loading buffer (5 µl of 60 mM Tris-HCl (pH. 6.8) containing 2% SDS, 7% glycerol, and
0.05% bromphenol blue). Samples were electrophoresed on 6.25%
polyacrylamide gels, followed by Coomassie Blue staining.
To analyze cleavage sites of fibronectin by r-NP, the digestion
products were electrophoresed and transferred onto Immobilon membranes.
The transferred bands on membranes corresponding to degraded
fibronectin were cut out and subjected to N-terminal sequencing as
described above.
Immunoaffinity Purification of Mouse Brain NP--
Affinity gel
was prepared using the monoclonal antibody produced as described above.
Six mg of mAbB5 was dialyzed against 0.1 M carbonate buffer
(pH 9.0) overnight. CNBr-activated Sepharose CL-6B was activated and
coupled to mAbB5.
Adult BALB/c mouse brains (10 g) were homogenized with 10 mM HEPES-NaOH (pH 7.4), 1% Triton X-100 and centrifuged at
10,000 × g for 1 h. The supernatant was applied
to a mAbB5-coupled affinity column equilibrated with 10 mM
sodium phosphate buffer (pH 7.4) containing 0.3 M NaCl.
Elution was performed with 0.17 M glycine HCl (pH 2.3) into
tubes containing 1 M Tris-HCl (pH 9.0), for neutralization.
Fractions were analyzed by 12.5% SDS-PAGE-silver staining by Western
blotting and by assay for amidolytic activity (described below).
Measurement of Amidolytic Activity of r-NP and Brain
NP--
Amidolytic activities of r-NP and brain NP were monitored by
release of MCA or pNA from the synthetic substrates listed above. Assay
mixtures containing chromogenic substrates at 100 µM and 45 mM Tris-HCl buffer (pH 8.0) and 10 µl of r-NP or brain
NP samples were incubated at 37 °C for 30 min. Released MCA and pNA
were measured with a Hitachi fluorescent colorimeter equipped with 370-nm excitation and 460-nm absorption filters, and with a Bio-Rad microplate reader with a 405-nm absorbance filter, respectively.
Kinetic analysis of r-NP and brain NP was performed by incubation of
100 ng of enzyme and increasing concentrations of amidolytic substrates
at 37 °C for 30 min. The kinetic parameters of amidolysis were
determined using a double-reciprocal Lineweaver-Burk plot for the rate
of release of MCA versus substrate.
Protein concentration was assayed by bicinchoninic acid protein assay
using bovine serum albumin as a standard (14).
DFP Affinity Labeling of r-NP and Brain NP--
r-NP and brain
NP in 10 mM HEPES buffer (pH 7.4) were mixed with
[3H]DFP (222 Bq/mmol) and incubated at 37 °C for
10 h. Nonspecific binding of [3H]DFP was removed by
addition of 2 µl of cold 5.4 M DFP and 10 µl of bovine
serum albumin (1 mg/ml) and washing with ice-cold acetone (100%). The
samples were precipitated at 12,000 × g for 15 min and
subjected to SDS-PAGE. The polyacrylamide gel was fixed with distilled
water:isopropyl alcohol:acetate (65:25:10) and exposed to Fuji x-ray
film for 3 days.
Subcellular Fractionation of Mouse Hippocampus
Homogenate--
All fractionation steps in Fig. 7A were
carried out at 4 °C. The tissue extraction procedure followed that
of Yen et al. (15). The hippocampi and the cerebrums from 12 ddY mice were homogenized with 12 ml of ice-cold Tris-buffered saline
(20 mM Tris-HCl (pH 7.4), 0.15 M NaCl),
incubated for 15 min, and then centrifuged at 10,000 × g for 10 min. The supernatant was referred to as 0.15 M NaCl-soluble fraction (A). The pellet was rehomogenized
with an equal volume of 20 mM Tris-HCl (pH 7.4), 2% Triton
X-100, incubated for 10 min, and then ultracentrifuged at 102,000 × g for 10 min. The supernatant was referred to as Triton
X-100 soluble fraction (B). The pellet was further rehomogenized with
an equal volume of 20 mM Tris-HCl (pH 7.4), 1% Triton
X-100, incubated for 10 min, and ultracentrifuged at 208,000 × g for 10 min. The supernatant was referred to as Triton
X-100 soluble fraction (C). The pellet was further rehomogenized with
an equal volume of 20 mM Tris-HCl (pH 7.4), 0.5 M NaCl, incubated for 10 min, and then ultracentrifuged at
383,000 × g for 10 min. The supernatant was referred
to as cytoskeleton-rich fraction (D). Each fraction was then
immunoprecipitated with anti-NP antibody (mAbB5) and immunoblotted with
anti-NP polyclonal rabbit antiserum. SDS-PAGE was performed as
described above.
 |
RESULTS |
Production, Purification, and Activation of r-pro-NP--
The full
murine NP sequence was subcloned into the NotI site of the
pVL1392 transfer vector (Fig.
1A) (8). By SDS-PAGE and
silver staining, a major 32-kDa product was detected in the medium
derived from infected cells (Fig. 1, B and C) but
not from uninfected cells (data not shown). The 32-kDa protein was
immunoreactive with anti-NP monoclonal antibody (mAbB5) on Western
blotting, and this band was therefore presumed to correspond to
recombinant NP protein. However, conditioned media from infected and
non-infected insect cells both lacked amidolytic activity for various
synthetic substrates (Table I).
Therefore, we speculated that the 32-kDa protein is a non-active
prototype NP protein (r-pro-NP). Conditioned medium from infected
insect cells showed high amidolytic activity following the endoprotease
processing (fluorescence intensity was 5130 per 1 µl of medium; see
details below; see also Figs. 1B and 2 and Table I), whereas
the medium from non-infected cells still showed very low amidolytic
activity even after endoprotease treatment (60.3 fluorescence intensity
per 1 µl of medium).

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Fig. 1.
Construction of the pVL1392/NP3 plasmid and
purification of r-pro-NP. A, an 866-base pair
NotI restriction fragment of NP3 cDNA containing the
entire open reading frame of mouse pro-NP was inserted into the unique
NotI cloning site under the control of the strong polyhedrin
promoter in the vector pVL1392. The sequence at the insertion sites is
highlighted to indicate the detailed positioning of the coding region
relative to the polyhedrin promoter and the multicloning site of
pVL1392. B, amidolytic activity of the eluate from Superdex
75HR gel filtration for purification of r-pro-NP. Fractions (0.5 ml)
were collected at a flow rate of 0.5 ml/min. Enzyme activities were
measured using aliquots of each fraction (11-26) after activation of
r-NP (see "Experimental Procedures"). , fluorescence intensity
of MCA released from Boc-Val-Pro-Arg-MCA ( em 370 nm and
ex 460 nm); , protein content. C, SDS-PAGE
and silver staining showed a single band of r-pro-NP in fraction
19 (left). The fractions were characterized by
immunodetection using anti-NP antibody (right). Purified
r-pro-NP migrated at a position corresponding to approximately 32 kDa.
Molecular masses of standards (kDa) are indicated in the
left and right margins.
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Table I
Enzyme activities of r-proNP, r-NP, and brain NP toward various
synthetic substrates
Enzyme activities were determined as described under "Experimental
Procedures" and are expressed as specific activities. Percentage of
activity toward Boc-Val-Pro-Arg-MCA is shown in parentheses in r-NP and
brain NP.
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The culture medium derived from infected insect cells was subjected to
anion exchange chromatography in one step to purify r-pro-NP. One major
peak of protein eluted by a linear NaCl gradient was collected. As the
fractions contained minor cell products, they were pooled and
concentrated before being applied to gel chromatography in the second
step to remove the minor components. The fractions eluted from gel
chromatography were used for assay of amidolytic activity after
activation by gel-immobilized protease 1 (Fig. 1B,
cf. Fig. 2). A single peak of
cleavage activity of Boc-Val-Pro-Arg-MCA was observed in
fractions 17-20 (Fig. 1B). To check the purity,
SDS-PAGE of the fractions was performed, and proteins were transferred
onto nitrocellulose membranes. Fraction 19 showed a single band by
SDS-PAGE followed by silver staining and Western blotting with mAbB5
and was thus used as purified r-pro-NP (Fig. 1C).

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Fig. 2.
Activational processing of r-pro-NP by
endoprotease. The horizontal axis indicates the
incubation time of r-pro-NP with various amounts of protease I. Enzyme
activities were measured with Pro-Phe-Arg-MCA as a substrate, because
no amidolytic activity was identified on this substrate by protease 1 itself. The reaction of r-NP was started by addition of the substrate.
The error bars indicate S.D. One unit of activity was
defined as that required to hydrolyze 1 µmol/min chromogen.
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Endoprotease treatment of r-pro-NP induced rapid induction of
amidolytic activity by conversion to r-NP (Fig. 2). The enzyme activity
in this experiment was observed using Z-Pro-Phe-Arg-MCA, whereas it was
cleaved less effectively than Boc-Val-Pro-Arg-MCA because it was not
cleaved by protease 1 itself (Table I). To examine the time course of
the induction of activity, various concentrations of protease 1 were
incubated with purified r-pro-NP. The r-pro-NP was processed in a
dose-dependent manner after 5 min of incubation with
protease 1 (Fig. 2). Longer incubation resulted in the enzyme activity
reaching a plateau with almost the same activity at 2, 4, and 16 ng of
protease 1 (Fig. 2). No induction of the enzyme activity was observed
when protease 1 was omitted (Fig. 2, open squares). Enzyme
activity of r-NP was characterized using this activated recombinant
protein produced by endoprotease processing of r-pro-NP. The effects of
pH on r-NP amidolytic activity were examined using four different
buffers as follows: acetate (pH 3.5-5.5), phosphate (pH 5.5-7.5),
Tris-HCl (pH 7.0-9.0), and carbonate buffer (pH 9.0-10.0). As shown
in Fig. 3, the pH optimum for the enzyme
activity was around pH 8.0.

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Fig. 3.
Effects of pH on activity of r-NP.
Enzyme activity of r-NP was measured by addition of Boc-Val-Pro-Arg-MCA
as a substrate. The buffers used were acetate buffer, pH 3.5-5.5
( ); phosphate buffer, pH 5.5-7.5 ( ); Tris-HCl buffer (pH
7.0-9.0) ( ); and carbonate buffer, pH 9.0-10.0 ( ) at a final
concentration of 0.1 M.
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To confirm that r-pro-NP is a zymogen of r-NP, we performed N-terminal
amino acid sequencing of both r-pro-NP and r-NP. The sequenced
N-terminal peptide of r-NP was comprised of 7 amino acids and started
at Ile33, 33 amino acids downstream of the deduced amino
acid sequence of the NP3 cDNA (data not shown). On the other hand, the
N terminus of r-pro-NP was blocked and not sequenced. To identify the
N-terminal amino acid of r-pro-NP, the difference of molecular weight
between r-NP and r-pro-NP was analyzed by matrix-assisted
laser/desorption ionization time-of-flight mass spectrometry
(MALDI-TOFMS). MALDI-TOFMS revealed that the molecular masses of
r-pro-NP and r-NP were 26.613 and 26.229 kDa, respectively, and thus
r-NP was 384 daltons less than r-pro-NP. This difference was suggested
to be caused by an N-terminal peptide of r-pro-NP composed of 4 amino
acid residues, Gln29-Lys32 (calculated
Mr = 400). Some unknown modification(s),
probably cyclization, of the N-terminal Gln of r-pro-NP might have been responsible for the inability to determine its N-terminal sequence. Hydrophobicity plot analysis of the deduced amino acid sequence of the
NP3 cDNA showed that the peptide from
Met1-Ala28 is highly hydrophobic (16), and this
was postulated to be the signal sequence. Therefore, we concluded that
secreted r-pro-NP from infected insect cells undergoes activational
processing at the Lys32-Ile33 bond as shown in
the model (Fig. 4).

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Fig. 4.
Model of activational processing of
r-NP. Starts of r-pro-NP (Gln29) and r-NP
(Ile33) were revealed by MALDI-TOFMS and N-terminal
sequencing analyses, respectively. N-terminal signal sequence
(Met1-Ala28) of recombinant protein that was
translated from the entire open reading frame of the NP3 cDNA (8) was
removed (r-pro-NP) and secreted from insect cells as an enzymatically
inactive zymogen (open arrow). The r-pro-NP readily
underwent activational processing by protease 1, a lysine-specific
endopeptidase, to generate the enzymatically active form (r-NP) by
removal of Gln29-Lys32 (filled
arrow).
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As described above, the molecular mass of r-pro-NP was determined to be
26.6 kDa by MALDI-TOFMS analysis. This is in good agreement with the
calculated molecular mass (25.5 kDa) from the amino acid content
deduced from the cDNA (8). The molecular mass was estimated by
SDS-PAGE under reducing and non-reducing conditions as 32 (Fig.
1C) and 26 kDa (Fig. 7B), respectively, and by
Superose 12 size exclusion chromatography at 29 kDa, suggesting that
the enzyme exists as a monomer.
Substrates and Inhibitors of r-NP--
Table I shows amidolytic
activities of r-pro-NP and r-NP on various synthetic substrates. The
highest amidolytic activity was observed toward Boc-Val-Pro-Arg-MCA, a
substrate of
-thrombin. Boc-Phe-Ser-Arg-MCA, which is a substrate of
trypsin, was also a good substrate for r-NP. The effects of various
protease inhibitors on amidolytic activity of r-NP are shown in Table
II. The enzyme activities measured were
strongly inhibited by low molecular weight protease inhibitors that
bind to His and Ser residues in the active centers of serine proteases.
DFP, leupeptin, and (4-amidinophenyl)methanesulfonyl 1-fluoride belong
to this group. Inhibition was also seen with benzamidine and antipain.
Both pepstatin A, an aspartic protease inhibitor, and E-64, a cysteine
protease inhibitor, had no or only slight effects on r-NP activity. In
addition, the divalent cations Ca2+ and Mg2+
and metal ion chelators had little effect on r-NP activity. Thus, NP
was categorized as a serine protease and not an aspartic, cysteine, or
metalloprotease.
Classified inhibitors were applied to define the r-NP activity.
Specific low molecular weight inhibitors of chymotrypsin and trypsin,
chymostatin and aprotinin markedly inhibited the amidolytic activity of
r-NP. However, high molecular weight inhibitors of serine proteases did
not significantly inhibit the enzyme activity (Table II). Hence, NP has
a similar catalytic center to trypsin and chymotrypsin, but the
substrate specificity due to the structure of the active site might be
very different from those of these enzymes (see "Discussion").
No Platelet Aggregation Activity of r-NP--
As the r-NP
favorably cleaved Boc-Val-Pro-Arg-MCA, a synthetic substrate of
-thrombin, thrombin-like blood coagulation activity was analyzed.
Although 0.1 and 0.5 units of thrombin, used as a positive control, had
strong platelet aggregation activity, no activity was found in r-NP at
any concentration tested (0.1, 1, and 2 units; data not shown). These
results suggested that r-NP does not process thrombin receptor protein
(present results and see Refs. 17 and 18).
Degradation of Fibronectin by r-NP, but Not by r-pro-NP--
No or
only weak proteolytic activities of r-NP against gelatin and collagen
types I, III, IV, and VI were detected by zymography (data not shown).
Strong proteolytic activity of r-NP was found against fibronectin, an
extracellular matrix protein, which is widely distributed in the
nervous system. Human plasma fibronectin is composed mainly of a
440-kDa dimer with lesser but variable amounts of lower molecular
weight forms, when analyzed under non-reducing conditions (lane
1 in Fig. 5, A and
B). Upon incubation with r-NP, the molecular mass of
fibronectin gradually decreased from the 440-kDa dimer to 220-, 200-, and 170-kDa monomers (Fig. 5A, lanes 2-6), although no
degradation of fibronectin was induced by r-pro-NP (Fig. 5B,
lanes 2-6). The 220-kDa fragment, which is the monomeric form of
fibronectin (19), appeared initially, followed by the 200- and 170-kDa
degradation products in a time-dependent manner. Furie and
Rifkin (20) demonstrated that the interchain disulfide bridges of the
dimerized fibronectin are present very close to the C terminus.
Together with this finding, our results indicate that r-NP-mediated
cleavage might occur initially near the C-terminal region.

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Fig. 5.
Cleavage of fibronectin by r-NP but not by
r-pro-NP. Time courses of fibronectin digestion by r-NP
(A), and fibronectin digestion by r-pro-NP (B).
Human plasma fibronectin (4 µg) was incubated in the absence
(lane 1) or presence (lanes 2-6) of r-NP or
r-pro-NP at 37 °C. Samples were electrophoresed on a 6.25%
polyacrylamide gel under non-reducing conditions and analyzed by
staining with Coomassie Blue. Molecular masses of standards (kDa) are
indicated in the left margin. Fibronectin alone (lane
1) or fibronectin which was incubated with 0.4 µg of r-NP or
r-pro-NP for 1 h (lane 2), 2 h (lane
3), 4 h (lane 4), 8 h (lane 5),
and 16 h (lane 6) were electrophoresed. Fibronectin was
gradually cleaved into small fragments in A (r-NP), whereas
no cleavage was observed in B (r-pro-NP). C, a
diagram indicating the cleavage sites of fibronectin by r-NP. The
cleavage site between fifth and sixth fibronectin type 1 repeats of N
terminus (Arg-Ala) were shown.
|
|
Next, we performed N-terminal amino acid sequencing of the 200- and
170-kDa degradation products and found that these degradation products
started at the same site, Ala291. Therefore, r-NP cleaves
specific sites of fibronectin as follows: 1) the C terminus, and 2) a
site between the fifth and sixth fibronectin type I repeats (FnI). The
five FnI repeats in the N terminus are lost following r-NP treatment
for 2 h or more (Fig. 5, A and C). No
further degradation was observed with incubation up to 6 h (Fig.
5A). Thus, the degradation step of fibronectin was very specific and might be important for physiology of cells because the
N-terminal five FnI repeats contain important functional sites (Fig.
5C, Ref. 21; see also "Discussion").
Partial Purification of Mouse Brain NP--
We focused on enzyme
characterization of native NP. For this purpose, brain NP from mouse
brain homogenate was partially purified (Table
III). One-step immunoaffinity
chromatography after detergent solubilization of brain lysate resulted
in purification with a 1,100-fold purity and a 72.7% recovery with a
major band on SDS-PAGE visualized by silver staining (Fig.
6A, asterisks in
fractions 3 and 4; Table III). The amidolytic activity measured with
Boc-Val-Pro-Arg-MCA as a substrate was eluted in fractions 3 and 4 (Fig. 6B). Western blotting analysis showed a dense band of
32 kDa in fractions 3 and 4 (Fig. 6C). A faint band of 30 kDa in fractions 3 was considered to be a degradation product of NP
because it showed immunoreactivity. SDS-PAGE of 3H-labeled
DFP-bound protein originating from fractions 3 and 4 followed by
autoradiography showed a single band of approximately 32 kDa (Fig.
6D). Therefore, the 32-kDa protein was considered to be
brain active-type NP. In addition, an approximately 50-kDa minor
component visualized by silver staining, but which was not immunoreactive on Western blotting and did not bind
[3H]DFP, was not eliminated by this experiment. This band
might correspond to an associated protein or an endogenous protease inhibitor of NP (Fig. 6, A-D).

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Fig. 6.
Partial purification of NP. The
mAbB5-coupled affinity gel (see "Experimental Procedures") was used
for purification of NP from brain. Fraction size was 1 ml each.
A, electrophoresis of aliquots (10 µl) of eluates was
performed in a 12.5% gel followed by silver staining and Western
blotting. Lane 1, eluate fraction; lane 2, pool
of wash; and lanes 3-5, eluted 1-ml fractions.
B, enzyme activity of each fraction measured by
Boc-Val-Pro-Arg-MCA as a substrate. Note that cleavage activity for the
chromogenic substrate was found in fractions 3 and 4. C,
Western blotting analysis of each fraction demonstrated that NP was
present in fractions 3 and 4. D, autoradiography of
[3H]DFP bound to partially purified NP and r-NP. A single
band of approximately 32 kDa was detected by [3H]DFP
autoradiography. Aliquots of 10 µl of partially purified brain NP
(NP, fraction 3) and 2 ng of r-NP were
electrophoresed.
|
|
Characterization of Brain NP--
The characteristics of enzyme
activity of brain NP were identical to those of r-NP (Table I). The
Km values were very similar between brain NP and
r-NP, suggesting that these molecules are enzymatically identical
(Tables I and IV). As shown in Table I,
brain NP had a lower specific activity than r-NP, which was thought to
be due to the presence of the NP binding protein that inhibits the
amidolytic activity of NP in the partially purified brain extracts. The
partially purified brain NP was itself enzymatically active (Table I).
The highest NP activity was found against Boc-Val-Pro-Arg-MCA (Table
I). Synthetic substrates were preferentially cleaved by the enzyme at
an Arg or Lys residue at the P1 position (Table I). Thus, enzyme
characterization studies of r-NP and NP clearly demonstrated that the
protease is a novel trypsin-type serine protease existing as a
naturally active enzyme (Tables I and IV).
Subcellular Localization of Brain NP in the Hippocampus--
To
define the distribution of endogenous brain NP in various subcellular
compartments, the mouse brain homogenate was fractionated, as
illustrated in Fig. 7A,
followed by immunoprecipitation and immunoblotting analysis. Major and
a minor weak bands corresponding to brain NP and its degradation
product, respectively, were detected in the soluble fraction
(A), whereas no band was detected in the Triton
X-100-soluble fraction (B and C). A faint band of
brain NP was also detectable in the cytoskeleton-rich fraction
(D).

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Fig. 7.
Subcellular fractionation of brain NP.
A, schematic illustration of tissue extraction used in the
present study. A detailed description and protocol is provided under
"Experimental Procedures." B, a major band and a weak
minor band of brain NP were detectable at approximately 28 and 25 kDa
in the buffered saline-soluble fraction (lane A). A faint
band of brain NP was also detectable in the cytoskeleton-rich fraction
(lane D). Molecular weight standards are indicated in the
left margin. The r-NP was electrophoresed at 26 kDa under
non-reducing conditions.
|
|
 |
DISCUSSION |
It has been suggested that neural plasticity is based on the
actions of a variety of proteases and their inhibitors (3, 22-25).
Various neurological stimuli induce tissue-type plasminogen activator
(tPA) mRNA in the brain, and it has been shown to be involved in
seizures, kindling, and neural degeneration (5, 23, 26). It was also
suggested that tPA might be related to repulsion of neural outgrowth,
because inhibitors of tPA increase neurite outgrowth in sympathetic
ganglion cells (27). Such induction of neurite retraction on the
cultured central and peripheral neurons was also found for thrombin (4,
28-31). However, these proteases have been shown to be involved only
in glial to neuronal interactions in the developing brain (1, 5, 24).
On the other hand, proteases relating to intersynaptic connections
among neurons to neurons in the matured brain have not been identified.
We hypothesized the existence of a novel protease localized in and
released from the pyramidal neurons of the hippocampus, and we cloned
the NP cDNA from these brain areas as described in our previous reports (8, 9, 32). The NP mRNA was expressed in pyramidal neurons of the
hippocampus with the highest density in the brain, and the neural
responses of this gene strongly suggested that this protease has
important physiological functions in the limbic brain.
Analyses of the deduced amino acid sequence of the NP3 cDNA
suggested that this protein has protease activity. Studies were begun
to produce a recombinant protein in the baculovirus-insect cell system
and to measure the protease activity of the recombinant protein.
However, in contrast to our initial assumption, the recombinant protein
did not show clear cleavage activity for any synthetic protease
substrates examined. The present study, however, clearly demonstrated
that recombinant prototype NP (r-pro-NP) secreted from insect cells was
processed by endoproteases and was converted to the enzymatically
active form (r-NP) as shown in Fig. 4. It is interesting that the
activational processing was brought about by the removal of only four
N-terminal amino acids.
Brain NP which was affinity purified from brain homogenate had
enzymatic properties identical to those of r-NP. The enzyme preferentially hydrolyzes Arg-X bonds and, to a lesser
extent, Lys-X bonds. In addition, the tripeptide MCA
substrate Z-Pro-Phe-Arg-MCA, but not the dipeptide MCA substrate
Z-Phe-Arg-MCA, was cleaved by the enzyme, and thus at least four
binding sites (S3, S2, S1, and
S1') seem to be a prerequisite for hydrolysis (33, 34). The
enzyme therefore appears to contain multiple amino acid side chain
binding sites in its active site. The S3 subsite appears to
favor hydrophobic (Val, Phe) side chains, judging from the higher
specific activities of NP for Boc-Val-Pro-Arg-MCA and
Boc-Phe-Ser-Arg-MCA than for Boc-Asp-Pro-Arg-MCA (see Table I). The
order of specific activities of the three highest activity substrates
was Boc-Val-Pro-Arg-MCA > Boc-Phe-Ser-Arg-MCA > D-Val-Leu-Arg-pNA (see Table I). Human thrombin cleaved
Boc-Val-Pro-Arg-MCA faster than Boc-Phe-Ser-Arg-MCA, but the activity
for the latter substrate was only 3% of that for the former
(cf. 48-78% in NP, Table I) (35). In addition, porcine
kallikrein and plasmin cleaved Boc-Val-Pro-Arg-MCA more slowly than
D-Val-Leu-Arg-pNA (35). Thus, the substrate specificity of
NP is very different from those of thrombin, tissue kallikrein, and
plasmin.
By the subcellular fractionation of mouse brain, NP co-fractionated
with the saline-soluble fraction which is composed of cytoplasm and
extracellular soluble components (Fig. 7 and Ref. 15). As the
hydrophobic signal sequence is encoded in the NP3 cDNA (8) and the
recombinant protein is released into medium from NP3 cDNA-infected
insect cells as shown in the present study and NP3 cDNA-transfected
neuroblastoma cells (Neuro
2a).2 Therefore, NP is
strongly suggested to be an extracellular protease. Recently, we
reported that intraventricular injection of monoclonal antibodies
specific to NP into mouse brain reduced the epileptic pattern of
electroencephalograms and epileptic behavior (11). As exogenously
applied antibodies can hardly pass across the plasma membrane into
living cells, the antibodies are thought to modify the activity of
extracellular NP. Taken together, these observations suggested that
brain NP secreted from hippocampal pyramidal neurons as a zymogen and
undergone an activational processing might be involved in neural
plasticity-related protease function (present study and Refs. 8, 9, and
11).
Since NP is considered to be a secretory serine protease as described
above, the extracellular matrix molecules are good candidates for the
physiological substrates of NP. r-NP effectively cleaved fibronectin
which is a major extracellular matrix protein expressed in the nervous
system (present study and Ref. 37). The specific cleavage by r-NP as
shown in the present study might directly affect fibronectin's
functions as a cell adhesion molecule, because the N-terminal 5 FnI
domains contain the main fibrin-binding site (21). Such cleavage
pattern of fibronectin by r-NP is analogous to that by plasmin but was
different from those by thrombin, plasminogen activator, and trypsin
(19, 38).
In conclusion, the present study clearly demonstrated characters of
r-NP and brain NP as a novel serine protease and also presented that
the protease might be involved in neural plasticity by potential
modification of the extracellular environments.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Fang-Sik Che of NAIST and
Takeshi Kato of Yokohama City University for their suggestions on
protein sequence analysis and activational processing of zymogen,
respectively. We also thank Junko Tsukamoto for performing TOFMS.
 |
FOOTNOTES |
*
This work was supported in part by a Grant-in-Aid for
Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan.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.
To whom correspondence should be addressed: Division of
Structural Cell Biology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Tel.: 81-74372-5410; Fax:
81-74372-5419; E-mail: sshiosak{at}bs.aist-nara.ac.jp
1
The abbreviations used are: NP, neuropsin; r,
recombinant; DFP, diisopropyl fluorophosphate; pNA,
p-nitroanilide; MCA, 4-methylcoumaryl-7-amide; Bz, benzyl;
Boc, t-butyloxycarbonyl; Z, benzyloxycarbonyl; E-64, N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl-agmatin;
PAGE, polyacrylamide gel electrophoresis; tPA, tissue-type plasminogen activator; MALDI-TOFMS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; FnI, fibronectin type I.
2
T. Oka, Y. Hashimoto, S. Shiosaka, and K. Kato,
unpublished observations.
 |
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