From the Department of Molecular Biology and the § Department of Cell Biology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
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ABSTRACT |
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Neuropsin is a novel serine protease, the
expression of which is highly localized in the limbic areas of the
mouse brain and which is suggested to be involved in kindling
epileptogenesis and hippocampal plasticity. The 2.1-Å resolution
crystal structure of neuropsin provides the first three-dimensional
view of one of the serine proteases highly expressed in the nervous
system, and reveals a serine protease fold that exhibits chimeric
features between trypsin and nerve growth factor- Proteases have been shown to play essential roles in the nervous
system, including those of neurite outgrowth (1), neural degeneration
(2), and synaptic plasticity (3). These actions are thought to be
mediated by the proteolytic cleavage of zymogen precursors, the
activation of specific cell surface receptors, or the degradation of
extracellular matrix proteins (4). Neuropsin was cloned from a mouse
hippocampal cDNA library using sequences for key regions of the
serine protease domain of nerve growth factor
(NGF)- Protein Preparation and Crystallization--
Neuropsin was
over-expressed in baculovirus-infected High Five insect cells,
purified, and crystallized as described previously (13). The resulting
sample and the crystallized protein were verified with N-terminal
analysis using an Applied Biosystem automatic analyzer 476A. Neuropsin
has a putative glycosylation site at Asn95 of the
kallikrein loop. Time-of-flight mass spectroscopy with PerSeptive
JMS-ELITE matrix-associated laser desorption/ionization time-of-flight
indicated its heterogeneous glycosylation. Two-dimensional high
performance liquid chromatography mapping (14) revealed that the
N-glycans contained 89% paucimannosidic structures with and
without attached fucose residue(s) at the innermost GlcNAc residues but
the glycosylation pattern exhibited high heterogeneity as found on many
glycoproteins (15). The detailed procedures and obtained structures
will be described elsewhere. The crystals belong to space group
P1 (a = 38.15 Å, b = 54.95 Å, c = 64.29 Å, Structure Determination--
Initial phases were calculated by
molecular-replacement with the program AMoRe (17) using a search model
based on the structure of bovine pancreatic Current Structure--
Two regions were poorly defined in the
map. The first is at the loop residues, Arg74 and
Asp75, and the second is at the three C-terminal residues.
These have uninterpretable densities implying complex disorder. The
current structure contains 194 water molecules. The R-factor is 18.6% (an Rfree of 22.7%) for all reflections to
2.1-Å resolution. The root-mean-square (r.m.s.) deviations from target
values are 0.008 Å for bond lengths, 1.535° for bond angles, and
1.139° for the peptide torsion angles. The averaged B-factor is 30.8 Å2. There is no residue in disallowed regions as defined
in PROCHECK (22), but 89.4% residues in the most favorable regions and
10.6% residues in the additional allowed regions.
Overall Structure--
Neuropsin consists of fourteen
One of the characteristic features of neuropsin is the
N-glycosylated loop D that corresponds to the so-called
"kallikrein loop." This loop, having an Asn-X-Ser
sequence, is typical for members of the kallikrein family that contains
NGF S1 Site--
Enzyme assay using several 4-methylcoumaryl-7-amide
(MCA) derivatives of oligopeptides (25) has shown that neuropsin
cleaves peptide bonds C-terminal to Arg or Lys. This primary
specificity is well interpreted by the S1 pocket, a deep cylindrical
pocket that is formed by two loops, G and H, and punctuated at its base by the side chain of Asp189. However, neuropsin has large
conformational changes of loop G with maximum displacements of 4.8-5.8
Å compared with those of NGF Kallikrein Loop--
The kallikrein loop of neuropsin differs
radically from those of NGF S2 Site--
The kallikrein loop of neuropsin overhangs toward the
active site cleft with a prominent Pro95D residue, which
suggests its role in substrate binding. Interestingly, superposition of
neuropsin on porcine pancreatic trypsin complexed with soybean trypsin
inhibitor (STI) (27) revealed steric clashes between the kallikrein
loop of neuropsin and the two STI loops facing toward neuropsin. This
was borne out by biochemical experiments in which high molecular weight
inhibitors, such as STI or S3/S4 Site--
Most striking is the structure of loop F that is
similar to that of trypsin rather than NGF
It is remarkable that the above differences in the loop F may also be
correlated with the conformational differences in the kallikrein loops.
In NGF A tripeptide substrate preferred for thrombin, Val-Pro-Arg-MCA,
has been found to exhibit the highest sensitivity for neuropsin to
date. However, poor structural homology between neuropsin and thrombin
is evident from the large r.m.s. deviation of 2.7 Å for 78 identical
residues. Moreover, thrombin cleaves Val-Pro-Arg-MCA much faster
(33-fold) than Phe-Ser-Arg-MCA, which is one of the preferred
substrates of trypsin, whereas the activities of neuropsin for these
substrates are comparable. In addition, thrombin also exhibits a
significant preference for arginine at the P1 position, but no such
preference was observed for neuropsin, as described above. These
results, together with structural differences of several loops, verify
the unique substrate specificity of neuropsin, even if the P2
preference for proline is analogous to thrombin.
Compared with the subsite preferences on the N-terminal side of the
scissile bond, little is known about subsite preferences on the
C-terminal side at present. However, a shallow bowl formed by
Cys42, Ile41, and Leu40 of strand
It is an interesting question whether neuropsin could process NGF In addition to neuropsin, three other serine proteases have been
reported to be more highly expressed in the central nervous system than
in most peripheral tissues. These include myelencephalon (MSP)-specific
protease (7), neurosin (8), and neurotrypsin (6). MSP and neurosin
exhibit sequence identities to neuropsin of 48 and 46%, respectively.
Neurotrypsin, which is a multidomain serine protease whose expression
is most prominent in the cerebral cortex, hippocampus, and amygdala,
has a protease domain exhibiting 33% sequence identity to neuropsin.
Sequence alignments with neuropsin indicate that these proteases would
have different structures of surface loops surrounding the
substrate-binding site. Remarkably, loop D of either of these proteases
has no N-glycosylation site and no inserted residues. This
lack of a kallikrein loop would result in their P2 specificities
differing from that of neuropsin. Moreover, loop G of MSP and neurosin
has no one-residue deletion, which causes significant structural
changes of loops G and H forming the S1 pocket. Alternatively, compared
with neuropsin, neurotrypsin has a three-residue insertion in loop G
and a one-residue deletion in loop H. These differences would endow
these other proteases with substrate specificities different from that
of neuropsin.
In conclusion, many aspects of neuropsin structure and function reveal
that this hippocampal serine protease displays chimeric structural
features of trypsin and NGF (NGF
), a member
of the kallikrein family. Neuropsin possesses an
N-glycosylated "kallikrein loop" but forms six
disulfide bonds corresponding to those of trypsin. The ordered
kallikrein loop projects proline toward the active site to restrict
smaller residues or proline at the P2 position of substrates. Loop F,
which participates in forming the S3/S4 sites, is similar to trypsin
rather than NGF
. The unique conformations of loops G and H form an
S1 pocket specific for both arginine and lysine. These characteristic
loop structures forming the substrate-binding site suggest the novel
substrate specificity of neuropsin and give a clue to the design of its specific inhibitors.
INTRODUCTION
Top
Abstract
Introduction
References
1 (5). In the brain,
no NGF
has been identified so far, though NGF
is present.
Neuropsin is one of the serine proteases highly expressed in the
nervous system (6-8). The expression of neuropsin is localized at
highest concentration in the hippocampus and the amygdala, which are
important for acquisition of memory and emotional memory, respectively.
This localization is in contrast to that of tissue plasminogen
activator (tPA), which is well documented to play a crucial role in the
nervous system by mediating plasticity but is distributed more
uniformly across the other brain regions and throughout other organs
(9). Activity-dependent changes in expression of neuropsin
have been observed upon direct hippocampal stimulation and induction of
kindling, which is a model for epilepsy and neuronal plasticity
characterized by the progressive development of electrographic and
behavioral seizures (5, 10). A single intraventricular injection of
monoclonal antibodies specific to neuropsin reduces or eliminates the
epileptic pattern (11). Moreover, oxidative stress is shown to effect
the expression of neuropsin in the limbic areas, which might be related
to the disturbance in shock-avoidance learning of mice (12). These
activity-dependent changes and the specific localization of
neuropsin indicate the involvement of this protease in hippocampal
plasticity and its pathogenesis. Knowledge of the three-dimensional
structure of neuropsin provides clues to the biological activity of
this protease and also is important to the design of inhibitors that
might be useful in treatment of pathological conditions such as epilepsy.
EXPERIMENTAL PROCEDURES
= 95.72°,
= 90.03°, and
= 110.29°). X-ray diffraction data were collected with Rigaku
imaging plate area-detectors, R-Axis IV and R-Axis IIc, using
Cu-K
radiation and also with a Weissenberg camera at the
BL-18B beamline station of the Photon Factory, Tsukuba using 1-Å
radiation. Intensities were evaluated with the program DENZO/SCALEPAK
(16), which yielded 25,778 independent combined reflections
corresponding to 90.7% completeness at 2.1-Å resolution (74.5% in
the highest resolution bin), an Rmerge of 6.0%
(19.8%), a mean ratio of intensity, and
of 8.5 (3.1).
-trypsin (PDB code 4PTP)
(18). Rigid body refinements of the searched model were performed with
the program X-PLOR (19), followed by density averaging/histogram matching with the program DM (20). Six regions of insertions and
deletions were inspected on the resulting
2Fo
Fc map, which was
generated with the program O (21). The structure was built and refined
through alternating cycles using the programs O and X-PLOR,
respectively. The kallikrein loop had large but mostly poor density
connects to the side chain of Asn95. After several cycles
of refinements incorporating solvent water molecules located at the
regions other than the kallikrein loop, we defined one residue of
N-acetylglucosamine (GlcNAc) residue bonded to
Asn95, as well as weaker density for additional sugar
residues that we have been unable to identify definitively. The
residual weak density is extending toward a large solvent channel in
the crystal.
RESULTS
-strands
(designated as
1-
14) that are extensively twisted, two
-helices (designated as
1 and
2), and one short
310-helix (Fig.
1a). Each seven-
-strand forms an antiparallel
-sheet folded in a
-sandwich with a cleft where the catalytic triad (Asp57, His102, and
Ser195) is located (Fig. 1, a and b).
This overall structure is homologous to those of the chymotrypsin-type
serine proteases, which share an identical catalytic mechanism, but
among which the substrate specificity varies. Many known structures of
these proteases delineate a clear framework, demonstrating that this
variety is a function of evolved diversity in the structures of surface
loops that surround the substrate-binding site. Because the loops of
neuropsin, which contains eight prominent loops
(A-H in Fig. 1a), are conserved in
their relative positions with respect to the active site, general themes for their individual functions can be derived.
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Fig. 1.
Overall structure of neuropsin. a,
ribbon representation of neuropsin. Seven-stranded -sheets (the top
and bottom halves) are sandwiched with the catalytic triad at the cleft
of the
-sandwich. The surface loops (A-H) forming the
substrate-binding site are colored with labels. Six
disulfide bonds are shown by bridges in white, and the
disulfide bond (SS3), which is conserved in trypsin but not
in kallikrein, was labeled. Loop D is the kallikrein loop
that has an N-glycosylated Asn95 with one
visible GlcNAc residue. The side chains of the catalytic triad,
Asp189 at the S1-specific pocket, Lys175 at the
S3/4 site, Glu149 and Asp218 at the rim of the
S1 pocket, and Leu40 and Ile41 at the S1' site,
are also shown with stick representations with one-letter
amino acid labels. b, molecular surfaces of neuropsin viewed
from nearly the same direction of panel a. Surface
electrostatic potentials calculated and rendered using GRASP (negative
potentials are in red and positive in blue). The
S1-S4 and S1' sites and characteristic surface residues are
labeled.
, which exhibits relatively high (46%) sequence identity to
neuropsin (Fig. 2). Neuropsin, however,
forms six disulfide bonds corresponding to those of trypsin with an
additional disulfide bond (SS3 between Cys128 and
Cys232 in Fig. 1a) that is missing in members of
the kallikrein family. Large differences exist in the loop regions
surrounding the substrate-binding site, whereas the core region
contains only minor variations. Excluding the insertion and deletion
residues, the main-chain atoms of neuropsin superimpose on the
corresponding atoms of bovine pancreatic trypsin (18), mouse
submaxillary gland NGF
in 7 S NGF (23), an
2
2
2 complex of NGF, and
pancreatic porcine kallikrein (24) with r.m.s. deviations of 1.26, 1.43, and 1.84 Å, respectively. The geometry of the catalytic triad is
highly similar to those of the serine proteases with r.m.s. deviations in a range of 0.2-0.24 Å. Neuropsin has no prominent structural similarity to tPA, showing a high r.m.s. deviation of 2.74 Å for 79 identical residues (37% sequence identity).
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Fig. 2.
Secondary structural elements and sequence
alignment of neuropsin and the related proteases, mouse submaxillary
gland NGF , porcine pancreatic kallikrein A, and bovine pancreatic
-trypsin. The sequential numbering of neuropsin is shown at the
top, and the chymotrypsinogen based used throughout this
paper is at the bottom. The secondary structural elements of
neuropsin are shown at the top with arrows for
-strands (
1-
14), cylinders for
-helices (
1,
2), and
310-helix. The loops (A-H) forming the
substrate-binding site are marked with colored bars with
labels. Insertion or deletion sites are marked by heavy line
boxes. The disulfide-bond-forming cysteines are boxed
and marked by labels (SS1-SS6), which correspond to six
disulfide-bonds, respectively. The catalytic triad is marked by
red circles. Asp189 in the S1-specific pocket is
marked by a blue star. Gly216 and
Gly226 of the rim of the S1 pocket are marked by
black stars. The putative N-glycosylation
sequence of Asn-X-Ser, which is conserved in members of the
kallikrein family, two cis prolines of neuropsin,
Pro147 and Pro219, and
Tyr172-Pro173-Gly174 of loop F are
boxed.
, kallikrein, and trypsin (Fig.
3). The conformational changes in
neuropsin seem to be caused by the one-residue (Gly186B)
deletion in loop G. In addition, loop H of neuropsin also displays large displacements from these proteases because loop H is heavily interacted with loop G in all the proteases. It is noteworthy that loop
H of trypsin has a one-residue deletion of the cis proline, Pro219, that is conserved in neuropsin, NGF
, and
kallikrein. This deletion induces a larger displacement of neuropsin
loop H from trypsin (4.3 Å) than from NGF
(3.4 Å) or kallikrein
(3.2 Å). Interestingly, the P1 specificity of neuropsin for arginine
is comparable with that for lysine. This is in sharp contrast to
NGF
, kallikrein, and trypsin, in which a significant preference for
arginine exists. Among key residues of the S1 pocket,
Gly226 of neuropsin has relatively large displacements from
NGF
(0.7 Å) and kallikrein (1.2 Å). Alternatively,
Ser217 of neuropsin has a relatively large displacement
from trypsin (1.3 Å). Compared with NGF
, the changes of the
neuropsin loop structures result in positional displacements (0.4-0.8
Å) of Asp189, Thr190, and His217,
which have been reported to form hydrogen bonds to the P1 arginine of
NGF
in 7 S NGF (23). These local differences may be responsible for
the unique P1 specificity of neuropsin. Like other serine proteases,
which are activated by cleavage of the bond between Arg15
and Ile16, neuropsin tucks the newly formed amino group of
Ile16 into the pocket to form multiple hydrogen bonds with
the main chains of loop E and to form an ion pair with
Asp194 of loop G.
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Fig. 3.
Comparison of the surface loops forming the
substrate-binding site between neuropsin (colored) and the
related proteases (gray): NGF -NGF
in 7 S NGF
(a) and trypsin-leupeptin complexes (b).
The C
-carbon atom tracings of loops B, D, and F-H of
neuropsin are colored as in Fig. 1a with both the
N- and C-terminal residue numbers in black and superimposed
on those of the proteases with the bound substrate or inhibitor
(green); the C-terminal Ala-Thr-Arg of NGF
in panel
a, or leupeptin in panel b. The side chains of the key
residues of the loops and the catalytic triad of neuropsin are shown
with yellow labels, but the others are not shown for the
clarity. Some of the side chains and the disulfide bond SS6 of NGF
and trypsin are also shown with white labels. These are
Trp215, His172, Lys175,
Asp189, and the disulfide bond SS6 of NGF
in panel
a, and Trp215, Tyr172, Asp189,
and the disulfide bond SS6 of trypsin. The hydrogen bonds between
Tyr172 (His172 of NGF
) and loop H are
indicated by white (black for His172)
broken lines.
and kallikrein. In these members of the
kallikrein family, the loop was cleaved into the highly mobile nicked
chains. In contrast, the loop of neuropsin is packed into an ordered
and relatively compact conformation without any nicked site: neuropsin has no arginine or lysine residue in the loop. Recently determined crystal structure (26) of mouse glandular kallikrein-13 shows an
ordered kallikrein loop, but no conformational similarity with the loop
of neuropsin. It seems unlikely that the N-glycan bound to
Asn95 participates directly in the substrate binding
because the GlcNAc residue and the residual density are oriented away
from the active site as in members of the kallikrein family.
-antitrypsin, were found to have little
effect on the neuropsin activity, whereas low molecular weight
inhibitors, such as leupeptin, markedly inhibited the activity. The
overhanging kallikrein loop forms a narrow pocket (the S2 site) in
which Asp102 is positioned at the base and would restrict
the size of the side chain in the P2 position of substrate peptides.
This is consistent with the results of a previous enzyme assay (25), in
which high activities of neuropsin were observed for peptide substrates
having smaller residues or proline in the P2 positions. It has been
well demonstrated by the crystal structure of thrombin complexed with D-Phe-Pro-Arg that loop B of thrombin compresses the S2
site with the inserted residues, Tyr60A and
Trp60D, to deduce the P2 specificity of the enzyme for
proline (28). Superposition of neuropsin on thrombin shows
Pro95D of neuropsin located nearby Tyr60A and
Trp60D of thrombin, but no contact between
Pro95D and the proline residue of D-Phe-Pro-Arg
bound to thrombin, which suggests that the P2 preference for proline
may be mediated by the kallikrein loop of neuropsin, instead of loop B
of thrombin, but rather weaker than that of thrombin (Fig.
4). Phenylalanine at the P2 position
remarkably reduces the neuropsin activity, which is one of the major
differences from kallikrein and NGF
.
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Fig. 4.
Comparison of the surface loops forming the
S2 site between neuropsin (colored) and -thrombin
(gray). The C
-carbon atom tracings of
loops of neuropsin, colored as in Fig. 1a, are superimposed
on
-thrombin complexed with
D-Phe-Pro-Arg-chloromethylketone (green).
The van der Waals surfaces of the inhibitor peptide and P95D are shown
with dot-surface representations. Labels are as in Fig.
3.
and kallikrein, where
significant conformational changes of loop F from neuropsin occur with
large displacements, 5.2 and 6 Å, respectively, accompanied by
movements of helix
1 (Fig. 3). Loop F is one of the main elements
forming the S3/S4 site. It is notable that this loop has
Tyr172, which forms hydrogen bonds with the main chains of
loop H and which is conserved in trypsin but is replaced by histidine
in NGF
and kallikrein. This residue has been elucidated to be one of
the distal determinant residues for the substrate specificity (29).
Moreover, Gly174, which is conserved in neuropsin and
trypsin, is one of the key residues for the loop F structure because
this residue is an essential component of the type-II reverse turn,
YPGK at 172-175. It should be pointed out that the disulfide bond SS3,
which is conserved in trypsin as already mentioned, may contribute to
the conformational resemblance of loop F with that of trypsin through
contacts with strand
13 that associated with the C-terminal
-strand of loop F,
11.
and kallikrein, the highly mobile kallikrein loops heavily
interact with loop F although the kallikrein loop of neuropsin has no
direct interaction with loop F, as it does with loop D of trypsin. One
of the interesting consequences of these conformational characteristics
in loops D and F is that the S3/S4 site of neuropsin is similar to that
of trypsin rather than NGF
. The aromatic rings of
Trp215, Tyr172, and His99 provide a
shallow but wide hydrophobic depression for the S3/S4 site as in
trypsin and could explain the high activities of neuropsin observed for
synthetic tripeptide substrates having hydrophobic residues in the P3
position. Moreover, in neuropsin, Lys175 of loop F is
projected toward the S3/S4 site (Fig. 1b), whereas Lys175 of NGF
is projected away from the S3/S4 site. It
is interesting that Lys175 may play a role in the P3/P4
interaction with the substrates. Actually, one of the cleavage sites of
fibronectin (25), which is an extracellular matrix protein exhibiting
strong proteolytic sensitivity for neuropsin, had the N-terminal
sequence of Asp-Val-Arg, whose acidic residue at the P3 position may
interact with Lys175.
DISCUSSION
3 seems to provide a hydrophobic S1' site (Fig. 1b). The
shape of the substrate-binding surface and the surface electrostatic distribution of neuropsin display several differences in details from
other serine proteases. One of the pronounced characteristics is the
rim of the S1 pocket, where acidic residues, Asp218 and
Glu149, expose the side chains to the solvent region.
Glu97 of the kallikrein loop also is projected from the
surface. These characteristics may be related to the specificity that
are distinct from those of other proteases. Neuropsin exhibits weak
proteolytic activities against gelatin and collagen but effectively
cleaves fibronectin, as already mentioned. By changing the
extracellular environment, neuropsin may exert its limbic effects.
precursor and form 7 S NGF instead of NGF
. In 7 S NGF, the active
site of NGF
was occupied by the C-terminal Arg118 of the
mature NGF
, as a cleaved product, with extensive interactions of the
large nicked kallikrein loop with the C-terminal regions and
-strand
of NGF
. Docking studies suggest that neuropsin would lose most of
these interactions although the smaller residues, Thr117
and Ala116, of NGF
could fit to the S2 and S3 sites of
neuropsin. In addition, the small cavity of NGF
for
Ala116 of NGF
is missing in neuropsin. Moreover,
Lys192, which is located at the rim of the active site of
NGF
and forms hydrogen bonds to main chain carbonyls of
Thr117 and Lys74 of NGF
, is replaced by
Gln192 in neuropsin. In 7 S NGF, a zinc ion was located at
the interface between NGF
and NGF
to stabilize the complex. This
coordination is also lost when NGF
is replaced by neuropsin because
zinc-coordinated His217 and Glu222 of NGF
are replaced by serine and lysine in neuropsin. Taken together, these
results suggest that neuropsin is incapable of forming 7 S NGF, even if
neuropsin could process the NGF
precursor. However, this will
require further investigation.
with novel substrate specificity. These
findings could give a clue to the structure-based drug design useful in
treatment of pathological conditions such as epilepsy and also useful
in analyzing the processes of synaptic plasticity.
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ACKNOWLEDGEMENTS |
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We thank Drs. Ben Bax (Birkbeck College) and S. E. Won Suh (Seoul National University) for providing the coordinates of 7 S NGF and soybean trypsin inhibitor-trypsin complex, respectively; M. Suzuki for data collection at PF; and S. Takayama and J. Tsukamoto for help with the mass spectroscopy and N-terminal analysis.
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FOOTNOTES |
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* This work was supported by Grants-in-Aid for Scientific Research (09308025, 10359003), on Priority Areas (10179104, 09277102), Biometalics (09235220) (to T. H.) from the Ministry of Education, Science, Sports and Culture, 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.
The atomic coordinates and structure factors (code 1NPM) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
Supported by a research fellowship for young scientists from the
Japan Society for the Promotion of Science.
¶ A member of the TARA project of Tsukuba University. To whom correspondence should be addressed. Tel.: 0743-72-5570; Fax: 0743-72-5579; E-mail: hakosima{at}bs.aist-nara.ac.jp.
The abbreviations used are: NGF, nerve growth factor; tPA, tissue plasminogen activator; GlcNAc, N-acetylglucosamine; STI, soybean trypsin inhibitor; MCA, 4-methylcoumaryl-7-amide; MSP, myelencephalon-specific protease; r.m.s., root mean square.
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REFERENCES |
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