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Crystal Structure of Neuropsin, a Hippocampal Protease
Involved in Kindling Epileptogenesis*
(Received for publication, September 11, 1998)
Tadaaki Kishi‡, Masato Kato‡, Toshiyuki Shimizu, Keiko Kato§, Kazumasa Matsumoto§,
Shigetaka Yoshida§, Sadao Shiosaka§, and Toshio Hakoshima
¶
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
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 pro-
vides the first three-dimensional view of one of the ser-
ine proteases highly expressed in the nervous system,
and reveals a serine protease fold that exhibits chimeric
features between trypsin and nerve growth factor-
g
(NGF
g
), 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
g
. 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.
Proteases have been shown to play essential roles in the
nervous system, including those of neurite outgrowth (1), neu-
ral degeneration (2), and synaptic plasticity (3). These actions
are thought to be mediated by the proteolytic cleavage of zy-
mogen precursors, the activation of specific cell surface recep-
tors, 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)-
g
1
(5). In the brain, no NGF
g
has
been identified so far, though NGF
b
is present. Neuropsin is
one of the serine proteases highly expressed in the nervous
system (6–8). The expression of neuropsin is localized at high-
est concentration in the hippocampus and the amygdala, which
are important for acquisition of memory and emotional mem-
ory, 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-de-
pendent changes in expression of neuropsin have been ob-
served 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 injec-
tion 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 pro-
tease and also is important to the design of inhibitors that
might be useful in treatment of pathological conditions such as
epilepsy.
EXPERIMENTAL PROCEDURES
Protein Preparation and Crystallization—Neuropsin was over-ex-
pressed 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 Asn
95
of the kallikrein loop. Time-of-flight mass
spectroscopy with PerSeptive JMS-ELITE matrix-associated laser des-
orption/ionization time-of-flight indicated its heterogeneous glycosyla-
tion. Two-dimensional high performance liquid chromatography map-
ping (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 heteroge-
neity as found on many glycoproteins (15). The detailed procedures and
obtained structures will be described elsewhere. The crystals belong to
space group P1(a 5 38.15 Å, b 5 54.95 Å, c 5 64.29 Å,
a
5 95.72°,
b
5
90.03°, and
g
5 110.29°). X-ray diffraction data were collected with
Rigaku imaging plate area-detectors, R-Axis IV and R-Axis IIc, using
Cu-K
a
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 reso-
lution bin), an R
merge
of 6.0% (19.8%), a mean ratio of intensity, and
s
of 8.5 (3.1).
Structure Determination—Initial phases were calculated by molecu-
lar-replacement with the program AMoRe (17) using a search model
based on the structure of bovine pancreatic
b
-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
* This work was supported by Grants-in-Aid for Scientific Research
(09308025, 10359003), on Priority Areas (10179104, 09277102), Bio-
metalics (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. This 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@bs.aist-nara.ac.jp.
1
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, myelenceph-
alon-specific protease; r.m.s., root mean square.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 7, Issue of February 12, pp. 4220 –4224, 1999
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org4220
by guest on December 27, 2015http://www.jbc.org/Downloaded from
matching with the program DM (20). Six regions of insertions and
deletions were inspected on the resulting 2F
o
2 F
c
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, respec-
tively. The kallikrein loop had large but mostly poor density connects to
the side chain of Asn
95
. After several cycles of refinements incorporat-
ing solvent water molecules located at the regions other than the
kallikrein loop, we defined one residue of N-acetylglucosamine (Glc-
NAc) residue bonded to Asn
95
, 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.
Current Structure—Two regions were poorly defined in the map. The
first is at the loop residues, Arg
74
and Asp
75
, and the second is at the
three C-terminal residues. These have uninterpretable densities imply-
ing complex disorder. The current structure contains 194 water mole-
cules. The R-factor is 18.6% (an R
free
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 Å
2
. 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 addi-
tional allowed regions.
RESULTS
Overall Structure—Neuropsin consists of fourteen
b
-strands
(designated as
b
1–
b
14) that are extensively twisted, two
a
-hel-
ices (designated as
a
1 and
a
2), and one short 3
10
-helix (Fig. 1a).
Each seven-
b
-strand forms an antiparallel
b
-sheet folded in a
b
-sandwich with a cleft where the catalytic triad (Asp
57
, His
102
,
and Ser
195
) 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 struc-
tures of surface loops that surround the substrate-binding site.
Because the loops of neuropsin, which contains eight promi-
nent 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.
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
g
, which
exhibits relatively high (46%) sequence identity to neuropsin
(Fig. 2). Neuropsin, however, forms six disulfide bonds corre-
sponding to those of trypsin with an additional disulfide bond
(SS3 between Cys
128
and Cys
232
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 neu-
ropsin superimpose on the corresponding atoms of bovine pan-
creatic trypsin (18), mouse submaxillary gland NGF
g
in7S
NGF (23), an
a
2
b
2
g
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 sim-
ilar 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).
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 Asp
189
. However,
neuropsin has large conformational changes of loop G with
maximum displacements of 4.8–5.8 Å compared with those of
NGF
g
, kallikrein, and trypsin (Fig. 3). The conformational
changes in neuropsin seem to be caused by the one-residue
(Gly
186B
) deletion in loop G. In addition, loop H of neuropsin
also displays large displacements from these proteases because
FIG.1.Overall structure of neuropsin. a, ribbon representation of neuropsin. Seven-stranded
b
-sheets (the top and bottom halves) are
sandwiched with the catalytic triad at the cleft of the
b
-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 Asn
95
with one visible GlcNAc residue. The side chains of the catalytic triad, Asp
189
at the S1-specific pocket, Lys
175
at the S3/4 site, Glu
149
and Asp
218
at the rim of the S1 pocket, and Leu
40
and Ile
41
at the S19 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 S19 sites
and characteristic surface residues are labeled.
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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, Pro
219
, that is conserved in neuropsin, NGF
g
,
and kallikrein. This deletion induces a larger displacement of
neuropsin loop H from trypsin (4.3 Å) than from NGF
g
(3.4 Å)
or kallikrein (3.2 Å). Interestingly, the P1 specificity of neurop-
sin for arginine is comparable with that for lysine. This is in
sharp contrast to NGF
g
, kallikrein, and trypsin, in which a
significant preference for arginine exists. Among key residues
of the S1 pocket, Gly
226
of neuropsin has relatively large dis-
placements from NGF
g
(0.7 Å) and kallikrein (1.2 Å). Alterna-
tively, Ser
217
of neuropsin has a relatively large displacement
from trypsin (1.3 Å). Compared with NGF
g
, the changes of the
neuropsin loop structures result in positional displacements
(0.4–0.8 Å) of Asp
189
, Thr
190
, and His
217
, which have been
reported to form hydrogen bonds to the P1 arginine of NGF
b
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 Arg
15
and
Ile
16
, neuropsin tucks the newly formed amino group of Ile
16
into the pocket to form multiple hydrogen bonds with the main
chains of loop E and to form an ion pair with Asp
194
of loop G.
Kallikrein Loop—The kallikrein loop of neuropsin differs
radically from those of NGF
g
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 neurop-
sin. It seems unlikely that the N-glycan bound to Asn
95
partic-
ipates 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.
S2 Site—The kallikrein loop of neuropsin overhangs toward
the active site cleft with a prominent Pro
95D
residue, which
suggests its role in substrate binding. Interestingly, superpo-
sition 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
a
-antitrypsin, were found to have little effect on the
neuropsin activity, whereas low molecular weight inhibitors,
such as leupeptin, markedly inhibited the activity. The over-
hanging kallikrein loop forms a narrow pocket (the S2 site) in
which Asp
102
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 struc-
ture of thrombin complexed with
D-Phe-Pro-Arg that loop B of
thrombin compresses the S2 site with the inserted residues,
Tyr
60A
and Trp
60D
, to deduce the P2 specificity of the enzyme
for proline (28). Superposition of neuropsin on thrombin shows
Pro
95D
of neuropsin located nearby Tyr
60A
and Trp
60D
of throm-
bin, but no contact between Pro
95D
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
g
.
S3/S4 Site—Most striking is the structure of loop F that is
similar to that of trypsin rather than NGF
g
and kallikrein,
where significant conformational changes of loop F from neu-
ropsin occur with large displacements, 5.2 and 6 Å, respec-
tively, accompanied by movements of helix
a
1 (Fig. 3). Loop F
is one of the main elements forming the S3/S4 site. It is notable
that this loop has Tyr
172
, which forms hydrogen bonds with the
main chains of loop H and which is conserved in trypsin but is
replaced by histidine in NGF
g
and kallikrein. This residue has
been elucidated to be one of the distal determinant residues for
the substrate specificity (29). Moreover, Gly
174
, which is con-
served in neuropsin and trypsin, is one of the key residues for
the loop F structure because this residue is an essential com-
ponent 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 confor-
mational resemblance of loop F with that of trypsin through
contacts with strand
b
13 that associated with the C-terminal
b
-strand of loop F,
b
11.
FIG.2. Secondary structural elements and sequence align-
ment of neuropsin and the related proteases, mouse submaxil-
lary gland NGF
g
, porcine pancreatic kallikrein A, and bovine
pancreatic
b
-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
b
-strands (
b
1–
b
14), cylinders for
a
-helices (
a
1,
a
2), and 3
10
-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. Asp
189
in the S1-specific pocket is marked by a
blue star. Gly
216
and Gly
226
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, Pro
147
and Pro
219
, and Tyr
172
-Pro
173
-Gly
174
of loop F are
boxed.
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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
g
and kallikrein, the highly mobile
kallikrein loops heavily interact with loop F although the kal-
likrein loop of neuropsin has no direct interaction with loop F,
as it does with loop D of trypsin. One of the interesting conse-
quences 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
g
. The aromatic rings of Trp
215
, Tyr
172
, and
His
99
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 hav-
ing hydrophobic residues in the P3 position. Moreover, in neu-
ropsin, Lys
175
of loop F is projected toward the S3/S4 site (Fig.
1b), whereas Lys
175
of NGF
g
is projected away from the S3/S4
site. It is interesting that Lys
175
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 pro-
tein 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 Lys
175
.
DISCUSSION
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. devi-
ation 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 prefer-
ence was observed for neuropsin, as described above. These
results, together with structural differences of several loops,
FIG.3.Comparison of the surface loops forming the substrate-binding site between neuropsin (colored) and the related proteases
(gray): NGF
g
-NGF
b
in 7 S NGF (a) and trypsin-leupeptin complexes (b). The C
a
-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
b
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
g
and trypsin are also shown with white labels. These are Trp
215
, His
172
, Lys
175
, Asp
189
, and the disulfide bond SS6
of NGF
g
in panel a, and Trp
215
, Tyr
172
, Asp
189
, and the disulfide bond SS6 of trypsin. The hydrogen bonds between Tyr
172
(His
172
of NGF
g
) and
loop H are indicated by white (black for His
172
) broken lines.
FIG.4. Comparison of the surface loops forming the S2 site
between neuropsin (colored) and
a
-thrombin (gray). The C
a
-car-
bon atom tracings of loops of neuropsin, colored as in Fig. 1a, are
superimposed on
a
-thrombin complexed with D-Phe-Pro-Arg-chloro-
methylketone (green). The van der Waals surfaces of the inhibitor
peptide and P95D are shown with dot-surface representations. Labels
are as in Fig. 3.
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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 prefer-
ences on the C-terminal side at present. However, a shallow
bowl formed by Cys
42
, Ile
41
, and Leu
40
of strand
b
3 seems to
provide a hydrophobic S19 site (Fig. 1b). The shape of the
substrate-binding surface and the surface electrostatic distri-
bution 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, Asp
218
and
Glu
149
, expose the side chains to the solvent region. Glu
97
of the
kallikrein loop also is projected from the surface. These char-
acteristics may be related to the specificity that are distinct
from those of other proteases. Neuropsin exhibits weak proteo-
lytic activities against gelatin and collagen but effectively
cleaves fibronectin, as already mentioned. By changing the
extracellular environment, neuropsin may exert its limbic
effects.
It is an interesting question whether neuropsin could process
NGF
b
precursor and form 7 S NGF instead of NGF
g
.In7S
NGF, the active site of NGF
g
was occupied by the C-terminal
Arg
118
of the mature NGF
b
, as a cleaved product, with exten-
sive interactions of the large nicked kallikrein loop with the
C-terminal regions and
b
-strand of NGF
b
. Docking studies
suggest that neuropsin would lose most of these interactions
although the smaller residues, Thr
117
and Ala
116
,ofNGF
b
could fit to the S2 and S3 sites of neuropsin. In addition, the
small cavity of NGF
g
for Ala
116
of NGF
b
is missing in neurop-
sin. Moreover, Lys
192
, which is located at the rim of the active
site of NGF
g
and forms hydrogen bonds to main chain carbon-
yls of Thr
117
and Lys
74
of NGF
b
, is replaced by Gln
192
in
neuropsin. In 7 S NGF, a zinc ion was located at the interface
between NGF
g
and NGF
a
to stabilize the complex. This coor-
dination is also lost when NGF
g
is replaced by neuropsin
because zinc-coordinated His
217
and Glu
222
of NGF
g
are re-
placed 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
b
precursor. However,
this will require further investigation.
In addition to neuropsin, three other serine proteases have
been reported to be more highly expressed in the central nerv-
ous 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. More-
over, 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 neurop-
sin, 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 func-
tion reveal that this hippocampal serine protease displays chi-
meric structural features of trypsin and NGF
g
with novel sub-
strate 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.
Acknowledgments—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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Crystal Structure of Brain Neuropsin4224
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Toshio Hakoshima
Shigetaka Yoshida, Sadao Shiosaka and
Shimizu, Keiko Kato, Kazumasa Matsumoto,
Tadaaki Kishi, Masato Kato, Toshiyuki
Kindling Epileptogenesis
Hippocampal Protease Involved in
Crystal Structure of Neuropsin, a
STRUCTURE:
PROTEIN CHEMISTRY AND
doi: 10.1074/jbc.274.7.4220
1999, 274:4220-4224.J. Biol. Chem.
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