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The Axonally Secreted Serine Proteinase Inhibitor, Neuroserpin,
Inhibits Plasminogen Activators and Plasmin but Not Thrombin*
(Received for publication, June 4, 1997, and in revised form, September 29, 1997)
Thomas Osterwalder‡, Paolo Cinelli, Antonio Baici§, Amedea Pennella, Stefan Robert Krueger,
Sabine Petra Schrimpf ¶, Marita Meins储, and Peter Sonderegger**
From the Institute of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, the §University
Hospital, Department of Rheumatology, CH-8091 Zurich, and the 储Friedrich Miescher Institute, Postfach 2543,
CH-4002 Basel, Switzerland
Neuroserpin is an axonally secreted serine proteinase
inhibitor that is expressed in neurons during embryo-
genesis and in the adult nervous system. To identify
target proteinases, we used a eucaryotic expression sys-
tem based on the mouse myeloma cell line J558L and
vectors including a promoter from an Ig-
-variable re-
gion, an Ig-
enhancer, and the exon encoding the Ig-
constant region (C
) and produced recombinant neuro-
serpin as a wild-type protein or as a fusion protein with
C
. We investigated the capability of recombinant neu-
roserpin to form SDS-stable complexes with, and to re-
duce the amidolytic activity of, a variety of serine pro-
teinases in vitro. Consistent with its primary structure
at the reactive site, neuroserpin exhibited inhibitory
activity against trypsin-like proteinases. Although neu-
roserpin bound and inactivated plasminogen activators
and plasmin, no interaction was observed with throm-
bin. A reactive site mutant of neuroserpin neither
formed complexes with nor inhibited the amidolytic ac-
tivity of any of the tested proteinases. Kinetic analysis of
the inhibitory activity revealed neuroserpin to be a slow
binding inhibitor of plasminogen activators and plas-
min. Thus, we postulate that neuroserpin could repre-
sent a regulatory element of extracellular proteolytic
events in the nervous system mediated by plasminogen
activators or plasmin.
Extracellular proteolysis exerted by serine proteinases has
been implicated in a variety of processes in the nervous system
during development and in adulthood. Among the serine pro-
teinases recently reported to play a role in neural development
and function, there are several well known proteins that had
previously been found and characterized in nonneuronal func-
tions, in particular blood coagulation and fibrinolysis. For ex-
ample, tissue-type plasminogen activator (tPA)
1
and urokinase
plasminogen activator (uPA) were found to be expressed in the
nervous system (1, 2), and they have been demonstrated to be
engaged in developmental processes such as cerebellar granule
cell migration (3, 4), Schwann cell migration, and wrapping of
axons (5), or neuromuscular synapse elimination (6). In the
period of neurite outgrowth, plasminogen activators (PAs) have
been found to be secreted at the growth cones of cultured
neurons or neuronal cell lines (7, 8), and they were demon-
strated to modify the molecular composition of the neurites’
substrata in vitro (9). In the adult nervous system, tPA is
induced in the hippocampus after seizure, kindling, and long
term potentiation (LTP) (10) and in the cerebellum after motor
learning tasks (11), and mice lacking the gene for tPA (12) show
a different form of hippocampal LTP (13, 14). Furthermore, tPA
has been demonstrated to be involved in excitotoxin-induced
neuronal cell death in the murine hippocampus by converting
locally secreted plasminogen to active plasmin (15, 16). Throm-
bin, which has been extensively characterized due to its impor-
tant function in the blood clotting system, has been reported to
be expressed in the nervous system (17). It has been found to
negatively affect neurite outgrowth in vitro (18) by inducing
growth cone collapse via proteolytic activation of the thrombin
receptor (19). Recently, four novel extracellular proteinases
have been reported in the nervous system. A serine proteinase
termed “erase” was proposed to be associated with membranes
of neurons from the peripheral but not from the central nervous
system (20), and the cDNAs of three serine proteinases called
“neuropsin,” “neurosin,” and “neurotrypsin,” respectively,
which are preferentially expressed in the nervous system, have
been cloned and characterized (21–23).
In analogy to processes of tissue remodeling, blood coagula-
tion, and fibrinolysis, one would expect specific inhibitors of
serine proteinases belonging to the structural class of the ser-
pins (serine proteinase inhibitors; for a review, see Ref. 24) to
act as regulators of proteolytic activity in the nervous system.
Guenther et al. (25) and Gloor et al. (26) have found a glial cell
line-derived activity, which promotes neurite outgrowth in
vitro and which is identical to protease nexin-1 (PN-1), a mem-
ber of the serpin family. PN-1 is directed against thrombin; it is
widely distributed in the nervous system (27) and might serve
as a physiological regulator of thrombin (28). Recently, a novel
member of the serpin family has been purified from bovine
brain (29). It has been shown to be expressed in neurons and in
* This work was supported by the Olga Mayenfisch Stiftung, the
Helmut Horten Stiftung, the Ciba-Geigy-Jubila¨umsstiftung, the Hart-
mann Mu¨ller-Stiftung, the EMDO-Stiftung, the Wolfermann-Na¨geli-
Stiftung, and the Union Bank of Switzerland on behalf of a client. The
costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “adver-
tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
‡ Current address: Dept. of Biology, Yale University, New Haven, CT
06511.
¶Current address: Dept. of Human Anatomy, The Biomedical Cen-
tre, S-75123 Uppsala, Sweden.
** To whom correspondence should be addressed. Tel.: 41-1-635 5541;
Fax: 41-1-635 6805; E-mail: pson@bioc.unizh.ch.
1
The abbreviations used are: tPA, tissue-type plasminogen activator;
BSA, bovine serum albumin; C
, constant region of Ig-
; cNS, recom-
binant chicken neuroserpin; cNS-C
, chicken neuroserpin fusion pro-
tein with C
; cNS
EP
-C
, chicken neuroserpin reactive site mutant fu-
sion protein with C
; DRG, dorsal root ganglion; FCS, fetal calf serum;
hNS-H
6
, recombinant human neuroserpin fusion protein tagged with 6
C-terminal histidines; LTP, long term potentiation; PA, plasminogen
activator; PAGE, polyacrylamide gel electrophoresis; PAI-1, plasmino-
gen activator inhibitor-1; PAI-2, plasminogen activator inhibitor-2;
PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PN-1,
protease nexin-1; SI, stoichiometric index; uPA, urokinase plasminogen
activator; VF, vitreous fluid.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 4, Issue of January 23, pp. 2312–2321, 1998
© 1998 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.org2312
at UZH Hauptbibliothek / Zentralbibliothek Zuerich on March 11, 2014http://www.jbc.org/Downloaded from
glial cells and to interact in vitro with several serine protein-
ases (e.g. thrombin) (30, 31). In contrast to thrombin, the PAs in
the brain have not been associated with a regulatory serpin.
The plasminogen activator inhibitors of nonneuronal tissues
(PAI-1 and PAI-2) were found in the nervous system (e.g. Refs.
32 and 33), but they are not coexpressed with PAs in a pattern
suggestive for a role of their physiological regulators in the
brain.
We have recently purified a neuronal serpin, neuroserpin,
from ocular vitreous fluid (VF) of chicken embryos and have
cloned the cDNA (34). The amino acid sequence of neuroserpin
is highly conserved between chick, rodents, and man (35),
2
especially in the region of the reactive site loop (between P17
and P5⬘; following the standard nomenclature introduced by
Schechter and Berger (36)) (Fig. 1). A considerable amount of
work over the past decade revealed the unusually flexible re-
active site loop to be essential for the activity and the specificity
of serpins (e.g. Refs. 37– 40). Based on its amino acid sequence
within the reactive site loop, neuroserpin belongs to the inhib-
itory Arg serpins and might therefore be an inhibitor of trypsin-
like serine proteinases, and the high similarity of neuroserpin
sequences of chicken, mice, and men within this region (Fig. 1)
suggests a high conservation of target specificity. However, the
speculations about inhibitory activity and possible target pro-
teinases were solely based on amino acid sequence compari-
sons, and no antiproteolytic activity of neuroserpin has been
demonstrated so far. The purpose of the presented study was to
determine experimentally whether neuroserpin is an inhibi-
tory serpin and whether it is targeted against serine protein-
ases expressed in the nervous system. We used complex forma-
tion assays and inhibition assays to investigate the interaction
between recombinant neuroserpin and the neural serine pro-
teinases tPA, uPA, plasmin, and thrombin. We found that the
inhibitory activity of neuroserpin is directed specifically
against PAs and plasmin, whereas no activity versus thrombin
was observed.
EXPERIMENTAL PROCEDURES
Construction of the Expression Vectors pcNS, pcNS-C
, and pcNS
EP
-
C
—The cDNA fragments termed cNS-wt and cNS-fus were amplified
with polymerase chain reaction (PCR), using the full-length cDNA clone
Sc3a4 as a template and the oligonucleotide primer pairs cNS-for/cNS-
wt-back and cNS-for/cNS-fus-back, respectively. For site-directed mu-
tagenesis of the reactive site, the mutagenic backward primer cNS-mt-
back was combined with cNS-int-for to amplify the XbaI-ScaI fragment
of neuroserpin with a mutated reactive site (Fig. 2A). (cNS-for, 5⬘-GTC
TTA AGA GCT CAC AAC ATG TAT TTC C-3⬘; cNS-int-for, 5⬘-GTA TCT
ACC AAG TTC TAG AAA TAC C-3⬘; cNS-wt-back, 5⬘-GGG AAG CTT
ACT TAC CTA AAG CTC TTC AAA GTC ATG GCC-3⬘; cNS-fus-back,
5⬘-GGG AAG CTT ACT TAC CTA GCT CTT CAA AGT CAT GGC C-3⬘;
cNS-mt-back, 5⬘-GGA TAC AGT ACT GCA GGT TCG CTA ATG GC-3⬘.)
PCR amplification was carried out in a reaction mixture containing
0.025 units/ml AmpliTaq DNA Polymerase (Perkin-Elmer), 50
Meach
of dATP, dCTP, dGTP, and dTTP, 1.3 mMMg
2⫹
(3 mMfor cNS-mt), 200
nMamounts of each primer, and 0.25
g of template. Sc3a4 was linear-
ized by KpnI digestion, and cNS-wt or cNS-fus, respectively, were
amplified in a 50
l volume in a 16-cycle amplification (hot start with
denaturation for 5 min at 95 °C; cycles 1–5, annealing 1 min at 60 °C;
elongation 1.5 min at 72 °C; Denaturation 1 min at 95 °C; Cycles 6–16,
annealing and elongation 2 min at 72 °C; denaturation 1 min at 95 °C;
7 min completing at 72 °C). For the amplification of cNS-mt, KpnI-
linearized pBluescript containing cNS-fus was used as template, and
the PCR conditions were as above, except the annealing temperature
(59 °C for cycles 1–5 and 60 °C for cycles 6–16) and an elongation
time of 1 min in all cycles. cNS-wt and cNS-fus were digested with
SacI and HindIII (all restriction endonucleases from New England
Biolabs, Beverly, MA; recognition sites underlined in the primers)
and ligated into the identically digested vector pCD4-FvCD3-C
(kind-
ly provided by Dr. K. Karjalainen), resulting in the expression vectors
pcNS for wild-type and pcNS-C
for the fusion protein, respectively
(Fig. 2B). For mutant neuroserpin, the SacI-HindIII fragment of
pcNS-C
was excised and subcloned in pBluescript SK(⫹) (Stratagene,
La Jolla, CA), and the wild-type XbaI-ScaI fragment covering the reac-
tive site loop was replaced by the mutated fragment cNS-mt amplified
by PCR. Replacing the SacI-HindIII fragment of pcNS-C
with the
mutated neuroserpin yielded the expression vector pcNS
EP
-C
for mu-
tant neuroserpin as a fusion protein with C
. The integrity of the
resulting constructs was confirmed by double-strand sequencing using
the dideoxy chain termination method (41) with Sequenase 2.0 (U. S.
Biochemical Corp.) or with the SequiTherm Long Read Cycle Sequencing
Kit-LC (Epicentre Technologies, Madison, WI).
Protoplast Fusion, Selection, and Screening for Positive Clones—
Stable transfectants were obtained by transfecting pcNS, pcNS-C
,or
pcNS
EP
-C
, respectively, into the mouse myeloma cell line J558L by
protoplast fusion (42) as described by Oi and colleagues (43). To achieve
independent transfectants, the cells were diluted in Dulbecco’s modified
Eagle’s medium containing 10% fetal calf serum (FCS; both from Life
Technologies, Basel, Switzerland) and cultivated in 96-well microtiter
plates at 37 °C in 10% CO
2
. After 2 days, selection of transfectants was
started by adding 5 mML-histidinol (Sigma). Two weeks later, histidi-
nol-resistant colonies were expanded and cultivated for 3– 4 days in
24-well plates containing 600
l of selection medium (5 mML-histidinol
in Dulbecco’s modified Eagle’s medium, 10% FCS) per well. To screen
for recombinant wild-type neuroserpin (cNS) and for neuroserpin fusion
protein with C
(cNS-C
), 50
Ci/ml [
35
S]methionine (1000 Ci/mmol,
NEN Life Science Products) was then added, and the expanding colo-
nies were metabolically labeled for 20 h. Afterward, the relative expres-
sion levels of cNS and cNS-C
of different clones were compared by
immunoprecipitation of the recombinant protein from the supernatants
using the polyclonal antiserum R35 as described below, followed by
SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography.
For mutant neuroserpin fusion protein (cNS
EP
-C
), supernatants of the
surviving colonies were incubated with a sheep antibody against mouse
C
(The Binding Site, Birmingham, UK) dotted on nitrocellulose, and
the expression levels of the tested cell lines were compared by incubat-
ing the dot blot with a peroxidase-conjugated sheep antibody against
mouse IgG (H ⫹L; from Kirkegaard & Perry Laboratories, Gaithers-
burg, MD) and by developing with 0.5 mg/ml 4-chlor-1-naphthol (Merck,
Dietikon, Switzerland) in Tris-buffered saline (50 mMTris-HCl, pH 7.4,
200 mMNaCl). The clones with the highest expression were subcloned
and adapted to low serum conditions (Dulbecco’s modified Eagle’s me-
dium with 2% FCS).
Purification of cNS, cNS-C
, and cNS
EP
-C
—Recombinant cNS was
partially purified from supernatants of transfected myeloma cells by a
two-step chromatographic procedure. The supernatants were first
passed through a column containing Blue Sepharose (CL6B, Pharma-
cia, Du¨bendorf, Switzerland). The flow-through was dialyzed against
ion exchange chromatography loading buffer (50 mMTris-HCl, pH 8.0,
200 mMNaCl), and proteins were fractionated using an anion exchange
column (MonoQ HR5/5, Pharmacia, Du¨bendorf, Switzerland) and a
linear gradient from 200 to 500 mMNaCl. The fractions from 350 to 400
mMNaCl were highly enriched with cNS; they were dialyzed against
phosphate-buffered saline (PBS; 10 mMphosphate buffer, pH 7.2, 140
mMNaCl, 4 mMKCl) and stored frozen until further use. For the
isolation of cNS-C
and cNS
EP
-C
, respectively, supernatants of the
transfected myeloma cells were passed through an immunoaffinity
column with the immobilized monoclonal antibody 187.1 (Rat anti-
mouse-C
IgG, ATCC). Bound antigen was eluted with 50 mMdiethyl-
amine, pH 11.5. The eluate was immediately neutralized by adding 230
mMTris-HCl, pH 6.5, and then dialyzed against PBS. All procedures,
except ion exchange chromatography, were carried out at 4 °C.
Procaryotic Expression and Purification of Recombinant Human
Neuroserpin (hNS-H
6
)—Human neuroserpin (PI12) was cytoplasmi-
cally expressed in Escherichia coli with a stretch of six histidines fused
to the C terminus of the protein. Briefly, a fragment of the human
neuroserpin cDNA encoding amino acids 1–394 of human neuroserpin
(according to the numbering of Schrimpf et al. (35)) was amplified in a
PCR using the oligodeoxynucleotide primers 5⬘-AAT TTC TAG AGA
AAG GAG ATA CAT ATG ACA GGG GCC ACT TTC CCT-3⬘and
5⬘-GGG AAG CTT CTA GTG GTG ATG GTG GTG GTG AAG TTC TTC
GAA ATC ATG GTC C-3⬘. The cDNA fragment was cloned into the
vector pAK400 (44) via the XbaI and HindIII sites of the vector, allow-
ing expression of the cDNA from the lac operator/promoter located
immediately upstream. For expression, a colony of E. coli strain
BL21DE3 harboring the expression plasmid was precultured overnight
2
Krueger, S. R., Ghisu, G. P., Cinelli, P., Gschwend, T. P., Oster-
walder, T., Wolfer, D. P., and Sonderegger, P. (1997) J. Neurosci. 17,
8984 – 8996.
Target Proteinases of Neuroserpin 2313
at UZH Hauptbibliothek / Zentralbibliothek Zuerich on March 11, 2014http://www.jbc.org/Downloaded from
at 37 °C in 100 ml of LB medium containing 30
g/ml chloramphenicol.
After inoculation of the same medium with the preculture, bacteria
were grown at 25 °C and induced with 1 mMisopropyl-1-thio-

-D-galac-
tosidase at an A
600
of 0.5. The bacteria were harvested by centrifugation
6 h after induction, resuspended in Ni-NTA-binding buffer (1 MNaCl,
50 mMTris-Cl, pH 8.0), and disrupted in a French press. The soluble
protein extract was incubated overnight at 4 °C with 0.4 ml of Ni-NTA
resin (Qiagen, Chatsworth, CA). Following extensive washing with
Ni-NTA binding buffer, bound proteins were eluted with Ni-NTA bind-
ing buffer containing 200 mMimidazole. The eluted protein was dia-
lyzed against PBS and immediately frozen at ⫺80 °C.
Generation of Polyclonal Antisera Against Neuroserpin—Generation
and specificity of the rabbit antiserum R35 against neuroserpin purified
from VF has been described earlier (34). The rabbit antiserum R61
against recombinant neuroserpin was generated by intramuscular in-
jection of 10 –20
g of cNS-C
in complete Freund’s adjuvant followed
by two booster injections of cNS-C
in incomplete Freund’s adjuvant 2
and 4 months later. As shown in Fig. 2D, the antiserum R61 against
cNS-C
obtained 1 week after the second booster injection was specific
for neuroserpin of both conditioned medium of cultured dorsal root
ganglion (DRG) neurons and VF.
Cell Culture, Metabolic Labeling, Immunoprecipitation, and Ocular
Vitreous Fluid—VF of 14-day-old chicken embryos was prepared as
described earlier (45). DRG neurons were dissected from 10-day-old
chicken embryos and cultured essentially as described by Sonderegger
and co-workers (46). Selective labeling of newly synthesized proteins
was carried out essentially as described by Stoeckli and co-workers (47).
The labeling medium consisted of growth medium containing 50
Ci/ml
[
35
S]methionine (1000 Ci/mmol, NEN Life Science Products), and the
incubation time was 24 – 48 h. Immunoprecipitation of neuroserpin
using the polyclonal antiserum R61 was carried out as described
previously (45).
Enzymes, Inhibitors, and Substrates—Human tPA (Activase
®
, re-
combinant Alteplase, 580,000 IU/mg; kindly provided by Genentech,
South San Francisco, CA) was dissolved in water to a final concentra-
tion of 1 mg/ml and stored in aliquots at ⫺80 °C; human uPA (100,000 –
300,000 Plough units/mg, Sigma U-8627) was delivered in a concentra-
tion of 1 mg/ml and stored at 4 °C; porcine plasmin (3–5 units/mg,
Sigma, P-8644) and human thrombin (50 –100 NIH units/mg, Sigma,
T-4648) were dissolved in water to final concentrations of 2 and 1
mg/ml, respectively. The enzyme substrate S-2288 (H-D-Ile-Pro-Arg-
para-nitroanilide) was purchased from Chromogenix (Mo¨lndal, Swe-
den), dissolved in water to a concentration of 25 mg/ml, and stored
frozen until use. Active enzyme concentrations were determined by
measuring the amidolytic activity of the proteinases in the presence of
1m
MS-2288, using values for substrate turnover of ⌬A
405
⫽0.275
min
⫺1
cm
⫺1
, 0.031 min
⫺1
cm
⫺1
, 0.030 min
⫺1
cm
⫺1
, and 0.042 min
⫺1
cm
⫺1
,for4nMthrombin, uPA, single chain tPA, and plasmin, respec-
tively, as indicated by the substrates’ supplier. Recombinant neuroser-
pin was prepared as detailed above, stored frozen, and thawed imme-
diately before use if not indicated otherwise. The concentrations of
cNS-C
and cNS
EP
-C
were determined by amino acid analysis on an
Aminoquant II equipped with the fluorescence detector 1046A
(Hewlett-Packard, Palo Alto, CA) using standard procedures. The con-
centration of cNS was estimated by SDS-PAGE and silver staining. The
concentration of hNS-H
6
was estimated using the Bradford protein
assay (Bio-Rad, Glattbrugg, Switzerland) in combination with densito-
metric analysis of SDS-polyacrylamide gels stained with Coomassie
Brilliant Blue. Recombinant PN-1 (active concentration 1.2 mg/ml) was
kindly provided by Dr. D. Monard and was stored frozen until use.
Reaction Buffers—Chemicals for reaction buffers were purchased
from Sigma, unless indicated otherwise. For complex formation assays,
a complexation buffer containing 67 mMTris-HCl, pH 8.0, 133 mM
NaCl, and 0.13% PEG 8000 was used. Inhibition buffer contained 10
mMphosphate buffer, pH 7.2, 140 mMNaCl, 4 mMKCl, 0.1% PEG 8000,
and 0.2 mg/ml bovine serum albumin (BSA; from Serva, Heidelberg,
Germany). Coating solution contained 1% BSA, 0.5% w/v PEG 8000,
and 0.01% v/v Triton X-100.
Complex Formation Assays—In an Eppendorf reaction tube, protein-
ases and inhibitors were mixed in 30
l of complexation buffer and
incubated for 30 min at 37 °C if not indicated otherwise. For experi-
ments with hNS-H
6
, incubation was 15 min at 4 °C. The final amounts
of enzymes and inhibitors are indicated in Figs. 3–5. The reaction was
stopped by adding an equal volume of 2-fold concentrated sample buffer
for SDS-PAGE (containing 6% SDS, 10%

-mercaptoethanol, 30% glyc-
erol, 31.3 mMTris-HCl, pH 6.8) and by immediately boiling the sample
for 5 min.
SDS-PAGE and Immunoblotting—SDS-PAGE was carried out ac-
cording to Laemmli (48), and 2-dimensional SDS-PAGE was according
to O’Farrell (49) as modified by Sonderegger et al. (46). For autoradiog-
raphy, the PhosphorImager (Molecular Dynamics, Sunnyvale, CA) was
used. Silver staining was performed according to the procedure of
Switzer et al. (50) as modified by Oakley et al. (51). Carbonic anhydrase
(29 kDa), ovalbumin (45 kDa), BSA (66 kDa), phosphorylase (97 kDa),

-galactosidase (116 kDa), and myosin (205 kDa, all from Sigma) were
used as molecular mass markers throughout the study. Electrotransfer
of resolved proteins onto nitrocellulose (Schleicher & Schuell, Dassel,
Germany) was carried out according to Towbin et al. (52) at 30 V for 16 h
or at 100 V for 1–2 h at 4 °C. Molecular mass markers were visualized
by 3 min incubation of the membranes in 0.1% Ponceau S (Sigma
P-3504) in 1% acetic acid followed by destaining with distilled water.
Immunodetection of neuroserpin was performed using the polyclonal
antisera R35 or R61 and the BM Chemiluminescence Western blotting
kit (Boehringer, Mannheim, Germany) according to the supplier’s rec-
ommendations, or goat anti-rabbit IgG conjugated to peroxidase (Bio-
Science products, Emmenbru¨cke, Switzerland) at a dilution of 1/1,000.
Amidolytic Assays—Enzyme inhibition was determined by mixing
enzyme and inhibitor in a 96-well plate in 98
l of inhibition buffer. The
final concentrations of enzymes were as follows: uPA, 19.1 nM(9.1 nM
for experiments with hNS-H
6
shown in Fig. 5); tPA, 7.9 nM; thrombin,
18.8 nM; plasmin, 12.7 nM. Concentrations of inhibitor were 5 and 25 nM
(cNS-C
) or 30 and 150 nM(hNS-H
6
). cNS-C
was preincubated for 20
min at 37 °C, while hNS-H
6
was 10 min at room temperature. After
preincubation, the amidolytic reactions were started simultaneously by
adding 2
l of substrate solution (25 mg/ml S-2288) to each well.
Residual amidolytic activity was determined by measuring the hydrol-
ysis over time (velocity) using an enzyme-linked immunosorbent assay
reader (Dynatech, Denkendorf, Germany).
Determination of Kinetic Parameters—The kinetics of the interaction
between cNS-C
and tPA, uPA, or plasmin, was determined by the
progress curve method (53). Polystyrene cuvettes were coated for 1– 4 h
at room temperature with coating solution. Reactions were started by
adding a constant, catalytic amount of enzyme (tPA, 1.6 nM; uPA, 3.6
nM; plasmin, 1.4 nM) to inhibition buffer containing a fixed substrate
concentration (1.08 mMS-2288) and variable inhibitor concentrations
(ranging from 4.6 to 46.8 nM), preincubated at 37 ⫾1 °C. Tight-binding
conditions were avoided by using sufficiently high substrate and inhib-
itor concentrations. Since the interaction between serpins and serine
proteinases is assumed to follow slow binding kinetics, product forma-
tion was described with Equation 1.
关P兴⫺
st⫹共
z⫺
s兲共1⫺e⫺k⬘t兲/k⬘⫹d(Eq. 1)
s
and
z
represent the velocities at steady state and at zero time,
respectively; k⬘represents the apparent first-order rate constant for
approach to the steady state, and dis a displacement factor compen-
sating for small uncertainties in absorbance at the start of the reaction.
For each of several inhibitor concentrations,
s
,
z
,k⬘, and dwere
determined by fitting Equation 1 to the data sampled from progress
curves. The association and dissociation constants were determined
from the relationship (53) shown in Equation 2.
k⬘⫽kd⫹ka
1⫹关S兴/Km
关I兴(Eq. 2)
K
m
of S-2288 was 3 ⫻10
⫺6
M,2⫻10
⫺4
M,1⫻10
⫺3
M, and 9 ⫻10
⫺3
M
for thrombin, uPA, single chain tPA, and plasmin, respectively, as
indicated by the supplier. An absorption coefficient
⑀
405
⫽10,500 M
⫺1
cm
⫺1
for the released para-nitroaniline was used to determine the
product concentrations.
RESULTS
Heterologous Expression of cNS, cNS-C
, and cNS
EP
-C
—
Only small amounts of denatured neuroserpin could be purified
from chicken embryonic VF by the three-step purification strat-
egy detailed earlier (34). Therefore, we decided to recombi-
nantly express neuroserpin in a heterologous system. Since
neuroserpin contains two potential sites for N-glycosylation, of
which at least one is used, we have chosen a eucaryotic expres-
sion system based on myeloma cells (54, 55) that are able to
produce large amounts of glycosylated, neuronally secreted
proteins (56). We amplified three different forms of the chicken
neuroserpin cDNA by PCR, which all cover the entire open
reading frame (Fig. 2A); cNS-wt was amplified using the back-
Target Proteinases of Neuroserpin2314
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ward primer cNS-wt-back with the naturally occurring stop
codon TAA mutated to TAG to generate the splice consensus
donor site (AGGTAAGT) immediately downstream of the cod-
ing region; cNS-fus was amplified with the backward primer
cNS-fus-back designed to replace the stop codon TAA with a G
which, after splicing, generates a continuous open reading
frame with the sequence of the constant region of the
light
chain (C
). Either of these fragments were cloned via SacI and
HindIII into the eucaryotic expression vector pCD4-FvCD3-C
(54, 55), replacing the region coding for CD4 and FvCD3, and
giving rise to the neuroserpin expression vectors pcNS and
pcNS-C
, respectively (Fig. 2B). The mutant form of neuroser-
pin was generated using the mutagenic primer cNS-mt-back, in
which the putative reactive site positions P1 and P1⬘(see Fig.
1) were mutated (P1[R362E] and P1⬘[M363P]) to generate an
inactive form of neuroserpin. The XbaI-ScaI fragment carrying
this mutation was introduced into pcNS-C
replacing the wild-
type reactive site loop and thereby yielding pcNS
EP
-C
. The
myeloma cell line J558L was transfected with either of the
vectors by protoplast fusion (42), and the expression levels of
histidinol-resistant clones were tested by analysis of superna-
tants by immunoprecipitation of metabolically labeled, recom-
binant protein with the antibody R35 against purified chicken
neuroserpin (34) or with a sandwich dot-blot test using two
different antibodies against mouse IgG. The clones DG3 for
cNS, 3B6 for cNS-C
, and F2 for cNS
EP
-C
, respectively, were
subcloned, adapted to low serum conditions, and expanded. The
fusion proteins were purified from supernatants by affinity
chromatography using a monoclonal antibody against C
,
whereas cNS was only enriched after removing most of the
albumin by passing through a Blue Sepharose column and by
fractionation over an anion exchange column. As shown in Fig.
2C, all recombinant proteins had the expected size (65 and 54
kDa, respectively) and were recognized by the polyclonal anti-
serum R35 (Blot). Interestingly, cNS-C
always appeared as a
double band. This effect was observed earlier with recombinant
C
fusion proteins
3
but could not be explained so far. Although
the immunoaffinity purification yielded electrophoretically
pure cNS-C
and cNS
EP
-C
, fractions of cNS still contained
30 –50% serum proteins, which were not recognized by R35
(Fig. 2C,lanes 1 and 2). With cNS-C
as immunogen, the
polyclonal antiserum R61 was raised in rabbit; it precipitated
neuroserpin under native conditions from medium conditioned
by primary DRG neurons (DRG CM), and specifically recog-
nized neuroserpin among the proteins in embryonic chicken VF
on Western blots (Fig. 2D). Different isoelectric variants of the
recombinant proteins, most likely due to glycosylation, were
observed. N-terminal sequencing of cNS-C
revealed an iden-
tical N terminus as found in chicken neuroserpin purified from
VF, indicating correct signal peptide cleavage (data not shown).
Complex Formation of cNS with Neural Serine Proteinases—
Upon binding of their target proteinases, serpins form highly
stable complexes that resist dissociation by SDS in the pres-
ence of reducing agents (57). Since neuroserpin is predomi-
nantly expressed in the nervous system, we tested the serine
proteinases tPA, uPA, plasmin, and thrombin, which exhibit
trypsin-like substrate specificity and are expressed in the nerv-
ous system (1, 2, 16, 17), for their ability to form SDS-stable
complexes with neuroserpin. cNS was incubated with different
concentrations of the respective proteinases, and the samples
were analyzed by SDS-PAGE and Western blotting (Fig. 3).
The estimated concentrations of inhibitors and proteinases are
indicated. cNS formed high molecular mass complexes of ap-
proximately 80, 86, and 112 kDa with uPA and tPA, respec-
tively, that matched the expected sums of the N-terminal part
of native neuroserpin (Phe
17
–Arg
362
, approximately 49 kDa)
plus the catalytic subunits of the proteinases (uPA, approxi-
mately 30 kDa; two-chain tPA, approximately 35 kDa, single-
chain tPA, approximately 65 kDa). cNS behaved more sub-
strate-like with plasmin, as this enzyme cleaved cNS into one
major fragment with the expected size of native neuroserpin
minus the C-terminal part (Met
363
–Leu
410
). Only a small
amount of cNS appeared to form SDS-stable complexes of the
expected size (approximately 75 kDa) with plasmin. No com-
plex formation and only very marginal proteolytic cleavage of
cNS were observed with thrombin. PN-1, which was used as a
control, readily formed complexes of approximately 65 and 54
kDa, with thrombin.
Complex Formation and Inhibitory Activity of cNS-C
and
cNS
EP
-C
—The question, whether the observed complex for-
mation was accompanied by an inhibition of the target protein-
ases, was studied using the fusion protein cNS-C
. Although
partially purified cNS still contained 30 –50% serum proteins
(see Fig. 2C,lane 2), the fusion proteins could be purified to
apparent homogeneity by a single step affinity chromatography
(see Fig. 2C, lanes 4 and 6). The ability of cNS-C
to form
complexes was tested in the same assay as used previously for
cNS. We found that cNS-C
formed complexes of the same
apparent molecular masses as the recombinant wild-type neu-
roserpin (Fig. 4A). In particular, cNS-C
readily formed com-
plexes with tPA and uPA, but a substrate-like reaction with
only a small portion of stable complexes was observed with
plasmin, and no complex formation and marginal proteolytic
cleavage was seen with thrombin. This suggested that the
presence of the Ig domain C
at the C terminus did not inter-
fere with the ability of recombinant neuroserpin to form SDS-
stable complexes with its target proteinases. To test whether
the complex formation reflected an antiproteolytic activity of
neuroserpin, we measured the residual amidolytic activities of
the proteinases after complex formation using an appropriate
chromogenic enzyme substrate (Fig. 4C). In accordance with
the results of the complex formation assays, we found that tPA
and uPA were inhibited by cNS-C
in a dose-dependent man-
ner, but the proteolytic activity of thrombin was not affected.
Interestingly, cNS-C
also inhibited plasmin, although the re-
3
C. Rader, personal communication.
FIG.1.Alignment of the reactive site loops of different serpins.
Reactive site loops of serpins directed against plasminogen activators,
plasmin, or thrombin are aligned to the corresponding region of neuro-
serpin from four different species. Dark shading indicates amino acids
identical to chicken neuroserpin, and light shading indicates conserv-
ative changes. A frame is drawn around the putative P1 and P1⬘amino
acids at the reactive site. Rat, human, and murine neuroserpin show an
identity of 91, 95, and 100%, respectively, to the chicken sequence in the
region from P17 to P5⬘but diverge strongly from plasminogen activator
inhibitors 1 and 2 (PAI-1, PAI-2), glia-derived nexin/protease nexin-1
(GDN/PN-1), antithrombin III (AT III), and antiplasmin.
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sults of the complex formation assays with neuroserpin and
plasmin pointed toward a more substrate-like interaction (see
Fig. 3 and Fig. 4A). Together, these results suggested that
neuroserpin inhibits the PAs via the formation of a tight, stoi-
chiometric complex, whereas it interacts with plasmin in a
serpin-like mechanism with a higher partition ratio.
It had been shown previously that the inhibitory activity and
specificity of serpins critically depend on their amino acid com-
position at the reactive site (37, 38). In particular, PAI-1, which
is a close relative of neuroserpin according to its amino acid
sequence, had been studied in detail by site-directed mutagen-
esis. Although PAI-1 must carry an arginine or a lysine at the
P1 position, P1⬘is more promiscuous, allowing every residue
except proline (37). To test whether a mutation found to be
“lethal” in PAI-1 abolishes the inhibitory activity of neuroser-
pin, we produced a fusion protein carrying a mutation of both
P1 and P1⬘(cNS
EP
-C
, R362E, M363P). As expected, the ability
to form stable complexes with any of the tested proteinases was
completely lost in the mutant neuroserpin (Fig. 4B). In line
with this observation, cNS
EP
-C
was unable to reduce the
FIG.3.Complex formation between neuroserpin and different
serine proteinases. Approximately 2 pmol of recombinant wild-type
neuroserpin (cNS) or 2.8 pmol of protease nexin-1 (PN-1) were incu-
bated either alone (⫺), or with uPA, tPA, thrombin, or plasmin and
analyzed by SDS-PAGE and Western blot. Numbers on the top indicate
amounts of proteinases in picomoles, and numbers on the left indicate
the molecular masses of marker proteins in kDa. Arrowhead on the
right indicates the molecular masses of the free inhibitors.
FIG.2.Recombinant expression of neuroserpin. A, two cDNA fragments were amplified by PCR from the neuroserpin cDNA Sc3a4 using
the two primer pairs cNS-for/cNS-wt-back and cNS-for/cNS-fus-back, respectively. The primers were designed to introduce a SacI restriction site
upstream of the translation start signal (ATG) at the 5⬘end (cNS-for) and a splice donor site followed by a HindIII restriction site at the 3⬘end
(cNS-wt-back and cNS-fus-back) of neuroserpin. In the primer cNS-fus-back, the wild-type stop signal (TAA) was deleted. Using the primer
cNS-int-for and the mutagenic primer cNS-mt-back, an XbaI-ScaI fragment carrying the mutated reactive site (R362E, M363P, indicated by a star)
was amplified using the chicken cDNA as a template. B, PCR fragments encoding neuroserpin (cNS-wt and cNS-fus, respectively) were ligated into
the parental expression vector pCD4-FvCD3-C
(55) substituting for CD4 and FvCD3 and are therefore put under the control of an Ig V
promoter
(P
) and an Ig
enhancer (E
) and forced to splice onto an Ig C
exon (C
). The novel expression vectors pcNS and pcNS-C
gave rise to the
wild-type neuroserpin cNS and the fusion protein cNS-C
(with the deleted neuroserpin translation stop signal), respectively. To generate mutant
neuroserpin fusion protein, the mutated fragment (cNS-mt) was introduced into pcNS-C
using the parental XbaI and ScaI restriction sites,
yielding pcNS
EP
-C
, which gave rise to the mutant fusion protein cNS
EP
-C
with P1 and P1⬘mutated (amp, ampicillin resistance gene for
procaryotic selection; his, histidinol resistance gene for eucaryotic selection; S,E,H,X, and S⬘indicate recognition sites for the restriction
endonucleases SacI, EcoRI, HindIII, XbaI, and ScaI, respectively). C, SDS-PAGE followed by silver staining (Silver) or Western blot (Blot) using
rabbit anti-neuroserpin, R35, of partially purified wild-type (cNS) and affinity purified neuroserpin fusion proteins (cNS-C
, cNS
EP
-C
). D,
two-dimensional SDS-PAGE of immunoprecipitated, metabolically labeled neuroserpin from conditioned medium of primary DRG neurons (DRG
CM) and Western blot of neuroserpin in the ocular vitreous fluid (VF) of chicken embryos, using the rabbit antiserum R61 raised against the
recombinant fusion protein cNS-C
.Numbers on the left indicate the molecular masses of marker proteins in kDa, and the direction of the
isoelectric focusing is indicated at the top.
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proteolytic activity of either of the tested proteinases more than
15% (Fig. 4D).
Complex Formation and Inhibitory Activity of hNS-H
6
—The
extremely high conservation of the primary structure within
the reactive site loop of neuroserpin from different species (Fig.
1) suggested a conserved target specificity from birds to men.
To test this hypothesis experimentally, we examined the ca-
pacity of human neuroserpin to form complexes with, and its
inhibitory activity toward, uPA, tPA, thrombin, and plasmin.
We found that hNS-H
6
formed SDS-stable complexes with uPA,
tPA, and plasmin, but no reaction with thrombin could be
observed (Fig. 5A). Moreover, a significant amount of neuroser-
pin was proteolytically cleaved by the target proteinases, but
the two shorter forms of neuroserpin (Fig. 5A, open arrow-
heads) appeared to be inactive and are most probably produced
by alternative usage of translation start signals or N-terminal
proteolytic degradation of hNS-H
6
by the bacterial host strain.
Amidolytic assays revealed a dose-dependent inhibitory activ-
ity of hNS-H
6
against uPA, tPA, and plasmin, whereas throm-
bin was not affected (Fig. 5B). These results are qualitatively in
accordance with the results obtained for chicken neuroserpin.
Stability of Complexes and Latency of Neuroserpin—Com-
plexes between serpins and serine proteinases exhibit various
degrees of stability, depending on the nature of the serpin and
the cognate proteinase as well as on the reaction conditions
(58). Moreover, the well characterized serpin PAI-1 had been
demonstrated to become inactive after a relatively short incu-
bation by assuming a so-called “latent form” (59). We have
therefore investigated the complexes between neuroserpin and
the PAs with regard to their stability, and we have also in-
cluded tests of the stability of free neuroserpin in the absence
of a proteinase. cNS and cNS-C
were incubated at various
temperatures and over different times in the presence or ab-
sence of uPA or tPA. As shown in Fig. 6, the results were
similar for uPA (upper panels) and tPA (lower panels), and no
obvious difference was found between cNS (left panels) and
cNS-C
(right panels). At low temperature, no evidence for a
transition into a latent form was observed. The reactivity of
neuroserpin remained the same whether it was thawed and
immediately used for the complexation test (lanes 1)or
whether it was preincubated for5honiceprior to mixing with
the proteinases (lanes 2). A slightly reduced reactivity after
preincubation for5hat37°Cwasobserved for both cNS and
cNS-C
, as indicated by the higher intensity of the bands
representing free cNS (I
w
) or cNS-C
(I
f
) at 54 or 65 kDa,
respectively (lane 3). Once formed, a large proportion of the
complexes remained stable for at least5honice(lane 5),
whereas incubation at 37 °C lead to a slightly increased decay
(lane 4), as indicated by the higher intensity of the band I
cl
representing the proteolytically cleaved form of neuroserpin at
49 kDa.
Kinetics of the Interaction between cNS-C
and Trypsin-like
Proteinases—Second-order rate constants, k
a
, for the interac-
tion of cNS-C
with uPA, tPA, and plasmin, were determined
under pseudo first-order conditions using the progress curve
method (53). This also permitted a direct comparison between
the constants obtained for neuroserpin and previously pub-
lished second-order rate constants for PN-1 (60). Plots of k⬘
values versus the corresponding inhibitor concentrations were
linear in all cases. We performed a statistical test for a depar-
ture from linearity. The slope of the k⬘versus [I] plot was
significantly different from 0, and there was no indication that
a hyperbola fit the data better than a straight line. Similarly,
the dependence of
z
upon [I], in all cases, revealed a slope not
significantly different from 0, indicating the independence of
z
from [I]. The profiles of
s
versus [I] were hyperbolic and fit well
to a classical, fully competitive inhibition mechanism, whereas
fitting to a tight binding model gave considerably worse results.
FIG.4. Complex formation and inhibitory activity of neuroserpin fusion proteins. Aand B, complex formation between 0.9 pmol of
cNS-C
or 0.9 pmol of cNS
EP
-C
, respectively, and the proteinases uPA, tPA, thrombin, or plasmin are monitored using SDS-PAGE followed by
Western blotting. Numbers on the top indicate amounts of proteinases in picomoles, and numbers on the left indicate the molecular masses of
marker proteins in kDa. Arrowheads on the right indicate the molecular masses of the free inhibitors. Cand D, inhibitory activity of the fusion
proteins is shown by plotting the residual amidolytic activity of several proteinases after preincubation with two different concentrations of cNS-C
or cNS
EP
-C
, respectively. The final concentrations of enzymes were as follows: uPA, 19.1 nM; tPA, 7.9 nM; thrombin, 18.8 nM; plasmin, 12.7 nM.
Numbers at the bottom indicate the final concentrations (in nM) of cNS-C
and cNS
EP
-C
, respectively, and numbers on the left indicate the residual
amidolytic activity of the proteinases in % of the samples without inhibitor (white column, 100% per definition).
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These properties justified the calculation of the association and
dissociation constants according to Equation 2 (53). Fig. 7 ex-
emplifies the case of tPA, and the results are summarized in
Table I (the primary data for uPA and plasmin are not shown).
The behavior was typical for a slow, tight binding mechanism.
Although k
a
can be considered sufficiently precise, k
d
values for
uPA and tPA were too small for experimental evaluation. The
estimated range in which their actual value may lie is shown
for qualitative purposes as the 95% confidence interval of the
intercept in the k⬘versus [I] plot. An estimate of k
d
for uPA and
tPA can be calculated from the K
i
values determined from the
dependence of
s
upon [I], namely 4.2 ⫻10
⫺5
s
⫺1
and 4.5 ⫻
10
⫺5
s
⫺1
for uPA and tPA, respectively. No inhibition of throm-
bin was detected, even at an 80-fold cNS-C
concentration over
enzyme (data not shown).
DISCUSSION
A detailed analysis of the amino acid composition within the
putative reactive site loop led us to predict that neuroserpin
could serve as an inhibitor of serine proteinases with trypsin-
like substrate specificity, and the high conservation of this
region further suggested a conservation of the target protein-
ases between species ranging from birds to men (Fig. 1). Among
the serine proteinases with trypsin-like substrate specificity,
we focused on tPA, uPA, thrombin, and plasmin, due to a
colocalization with neuroserpin in the nervous system (1, 2, 16,
17). Since the published purification procedure for chicken
neuroserpin from VF allowed the purification of only very small
amounts of SDS-denatured protein (34), we established a het-
erologous system for the eucaryotic expression of neuroserpin.
The recombinantly expressed proteins were of the expected
size, and the co- and posttranslational modifications, such as
signal peptide cleavage and glycosylation, were in agreement
with the characteristics found for purified neuroserpin. Fur-
thermore, the antiserum raised against recombinant neuroser-
pin was cross-reactive with chicken neuroserpin, and the anti-
serum against purified neuroserpin recognized cNS, cNS-C
,
and cNS
EP
-C
, both under native and denatured conditions.
Therefore, it seemed reasonable to base the functional study of
neuroserpin on recombinantly expressed protein.
Neuroserpin Is a Typical Serpin-like Inhibitor of Serine Pro-
teinases—The presented results characterized neuroserpin as a
mechanism-based (suicide substrate) inhibitor of tPA, uPA,
and plasmin. Neuroserpin formed SDS-stable complexes with
the PAs and plasmin, although no interaction with thrombin
was found. The two different complexes found with tPA most
probably represented complexes with the single chain form of
tPA and with the catalytic subunit in the proteolytically
cleaved two chain form. Both forms of tPA are proteolytically
active (61) and interact with PAI-1, their physiological inhibi-
tor in the blood (62). Since we used the recombinant single
chain form of tPA, the two chain form appears to be generated
by autoproteolytic cleavage. Whether this cleavage occurred
before or after the interaction with neuroserpin cannot be dis-
tinguished by our experiments. Interestingly, only a small pro-
portion of neuroserpin was found in an SDS-stable complex
FIG.5. Complex formation and inhibitory activity of human
neuroserpin. A, approximately 2 pmol of procaryotically expressed
hNS-H
6
were incubated either alone (⫺), or with uPA, tPA, thrombin, or
plasmin and analyzed by SDS-PAGE followed by Western blotting
using the polyclonal serum R35. Numbers on the top indicate amounts
of proteinases in picomoles, and numbers on the left indicate the mo-
lecular masses of marker proteins in kDa. Filled arrowhead on the right
indicates the molecular mass of the free inhibitor; open arrowheads
indicate N-terminally truncated forms of recombinant neuroserpin that
most probably arise from alternative usage of translation start signals
or from proteolytic cleavage by the bacterial host strain. B, the inhibi-
tory activity of hNS-H
6
is shown by plotting the residual amidolytic
activity of the indicated proteinases after 10 min preincubation with
two different concentrations of hNS-H
6
(indicated at the bottom in nM).
The final concentrations of protease were as follows: uPA, 9.1 nM; tPA,
7.9 nM, thrombin, 18.8 nM; plasmin, 12.7 mM.Numbers on the left
indicate the residual amidolytic activity of the proteinases in % of the
samples without inhibitor (white column, 100% per definition).
FIG.6. Latency of neuroserpin and decay of complexes with
tPA or uPA. An antiserum against neuroserpin recognizes cleaved
neuroserpin (I
cl
, 49 kDa) and free cNS (I
w
, 54 kDa) and cNS-C
(I
f
,65
kDa) or complexes with uPA (80 kDa) or tPA (86 kDa and 112 kDa) after
SDS-PAGE and Western blotting. Numbers in the middle indicate the
relative molecular masses of marker proteins in kDa. Samples were
treated as follows: n, no proteinase; 1, no preincubation of neuroserpin;
2, 5 h preincubation on ice; 3, 5 h preincubation at 37 °C before addition
of proteinases; 4, 5 h incubation at 37 °C; 5, 5 h incubation on ice after
addition of proteinases. Reaction time with proteinases was 15 min in
all cases. The reactions were stopped by addition of SDS-PAGE sample
buffer and boiling.
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with plasmin, where most of the inhibitor was found in a
modified form of approximately 49 kDa (43 kDa for hNS-H
6
),
most probably representing the thermodynamically stable ser-
pin core I* after cleavage between P1 and P1⬘(63, 64). This
suggested that neuroserpin interacts with plasmin by a serpin-
like mechanism with a higher partition ratio. Calculation of the
stoichiometric index (SI) from the residual activity of plasmin
after 30 min of preincubation with cNS-C
yielded an SI of
approximately 3 for plasmin, whereas the values for PAs are
close to 1, which is in agreement with the results of the complex
formation assays.
The size of the complexes with the proteinases as well as the
size of the cleaved form of neuroserpin were undistinguishable
for cNS or cNS-C
, and the patterns of complex formation were
identical for the wild-type and the fusion protein. Moreover, the
stability of both free neuroserpin and the complexes of neuro-
serpin with tPA and uPA were very similar for cNS and cNS-
C
. Due to the close resemblance with respect to selectivity and
stability, it seemed reasonable to base the determination of the
inhibitory activity of neuroserpin on the fusion protein cNS-C
.
Using cNS-C
was advantageous, because it could be obtained
as an apparently homogeneous protein without disturbing con-
taminations of serum proteins by a one-step immunoaffinity
purification.
The results of the inhibition assays confirmed the specificity
of neuroserpin determined by complex formation assays. Neu-
roserpin inhibited uPA, tPA, and plasmin in a dose-dependent
manner, although inhibition of plasmin had a higher SI. No
inhibition of thrombin was observed. The large, C-terminal Ig
domain did not interfere with the activity nor with the speci-
ficity of neuroserpin. The experiments with mutant neuroser-
pin provided strong evidence for the putative reactive site
P1-P1⬘(Arg
362
and Met
363
) being involved in the interaction
with all target proteinases, since the reactive site mutant
cNS
EP
-C
formed no complexes with either of the proteinases,
and the antiproteolytic activity was strongly reduced. The
weak inhibition of uPA by an excess of cNS
EP
-C
might repre-
sent a competitive effect that does not lead to the formation of
stable complexes. Altogether, the results characterized neuro-
serpin as a typical serpin-type inhibitor of tPA, uPA, and plas-
min, with the amino acids Arg
362
and Met
363
forming the
reactive site P1-P1⬘.
Chicken and Human Neuroserpin Exhibit the Same Target
Specificity—Complex formation tests and inhibition assays re-
vealed the same target proteinase preference for the human
and the chicken form of neuroserpin. Both hNS-H
6
and cNS-C
readily formed SDS-stable complexes with plasminogen activa-
tors and with plasmin but did not react with thrombin. Inhib-
itory assays with hNS-H
6
and with cNS-C
resulted in quali-
tatively the same target preference pattern. Both hNS-H
6
and
cNS-C
exhibit the strongest inhibitory activity against tPA,
although hNS-H
6
in general shows a lower specific activity.
The higher stoichiometric indices observed for hNS-H
6
as com-
pared with cNS-C
could reflect a lower stability of the com-
plexes formed by human neuroserpin. Alternatively, this ob-
servation could be explained by the fact that the hNS-H
6
used
for these experiments was of procaryotic origin and, thus, less
stable due to a lack of glycosylation (65).
Neuroserpin Is a Slow Binding Inhibitor of tPA, uPA, and
Plasmin—A considerable amount of work has been done over
the last years to uncover the mechanism by which serpins
inhibit their target serine proteinases (64, 66, 67, for a recent
review, see Ref. 68). In accordance with a general mechanism of
serpins, we observed the generation of a modified inhibitor
species under particular experimental conditions, namely at
relatively high enzyme and inhibitor concentrations, with en-
zyme and inhibitor concentration of the same order of magni-
tude, and in the absence of substrate. Conversely, progress
curves of amidolytic activity were obtained with catalytic
amounts of enzyme, in the presence of a relatively high con-
centration of a synthetic substrate and at inhibitor concentra-
tions greatly exceeding those of the enzymes. Therefore, kinetic
parameters could be determined under fully competitive con-
ditions. Furthermore, the rate constants obtained under
pseudo first-order conditions showed a linear dependence upon
the inhibitor concentration, which allowed the usage of the
progress curve method (53) to calculate second-order rate con-
stants k
a
for the association of neuroserpin with uPA, tPA, and
plasmin. These data could be compared with analogous data
previously determined for PN-1 by Scott et al. (60), although
obtained under different experimental conditions. Neuroserpin
interacted relatively fast with tPA and plasmin and slightly
slower with uPA. However, the high stability of the complex
with tPA, in comparison to plasmin, as well as the pronounced
cleavage of neuroserpin by plasmin pointed toward tPA as the
most likely physiological target of neuroserpin. Interestingly,
the association of tPA with neuroserpin occurred about 2 orders
of magnitude faster than with PN-1, which is the closest rela-
tive of neuroserpin in the nervous system.
Is Neuroserpin the Physiological Inhibitor of tPA in the Nerv-
ous System?—There is growing evidence for tPA and thrombin
playing an important role in the nervous system. Secretion of
tPA by neurons in vitro was interpreted to reflect its role in
facilitating neurite growth (7, 8) and neuronal migration (4).
The strong expression of tPA in particular regions of the adult
FIG.7. Inhibition of tPA by cNS-C
under pseudo first-order
conditions. The interaction of tPA and cNS-C
was measured under
pseudo first-order conditions using the progress curve method. Inhibi-
tion of tPA at different concentrations of cNS-C
in inhibition buffer
was followed by measuring the product concentration every 300 s. A,
tPA progress curves were measured with 1.6 nMtPA, 1.08 mMsub-
strate, and cNS-C
as indicated by numbers near the curves. The
first-order rate constants (k⬘) were calculated for each inhibitor concen-
tration by a nonlinear regression fit using Equation 1. Best fit curves
are shown as solid lines.B, dependence of the first-order rate constant
(k⬘) on the concentration of inhibitor. A second-order rate constant
was obtained from the slope of this line and corrected with K
m
using
Equation 2.
Target Proteinases of Neuroserpin 2319
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brain (1) and the observation of tPA mRNA being up-regulated
after motor learning or experimental seizures, kindling, or LTP
(10, 11) led to the speculation that tPA could also be involved in
synaptic plasticity subserving learning and memory. In recent
studies of tPA
⫺/⫺
mice, indeed a retardation in cerebellar gran-
ule cell migration (74) as well as a different form of hippocam-
pal LTP (13, 14) were found. On the other hand, several lines of
evidence point toward a role of thrombin during neural devel-
opment and establishment of neuromuscular connectivity as
follows: prothrombin mRNA is expressed in the nervous system
and in muscles (17, 69); neurite retraction is induced by pro-
teolytic activation of the thrombin receptor in vitro (19); and
the proteolytic activity of thrombin is required for neuromus-
cular synapse elimination in vitro and in vivo (28, 69). The
inhibitory activity of neuroserpin is directed against tPA but
not against thrombin. This specificity is remarkable, since it is
complementary to the inhibitory activity of PN-1. PN-1 was
initially found to promote neurite outgrowth (25, 26). It now
seems clear that this activity is due to its fast and strong
inhibition of thrombin, which induces neurite retraction in
vitro (for a review, see Ref. 70). PN-1 only slowly interacts with
tPA, in particular when the latter is present in the single chain
form (71). Although tPA is converted into a two chain form by
several proteinases in vitro (61, 72, 73), the single chain form is
proteolytically active (62), and it is not certain in which form
tPA is present in the nervous system. Neuroserpin forms stable
complexes with both forms of tPA, and it interacts with single
chain tPA approximately 2 orders of magnitude faster than
PN-1. Together with the colocalization of tPA and neuroserpin
in the nervous system of the mouse,
2
these results make neu-
roserpin an interesting candidate for a physiological, local reg-
ulator of tPA in the nervous system. Based on results indicat-
ing a discrepancy between tPA expression and its proteolytic
activity in the hippocampus and in the cerebellum of mice,
Sappino et al. (1) recently proposed an inhibitor of tPA different
from PAI-1, PAI-2, or PN-1 to exist in the murine brain. It will
be of particular interest to clarify whether neuroserpin is re-
sponsible for the inhibition of tPA observed in their assay.
Despite indications for an interaction between neuroserpin
and tPA in the developing and in the adult nervous system,
there is good reason not to exclude other serine proteinases as
potential targets of neuroserpin. Since only one plasminogen
activator (namely uPA) is thought to exist in chicken, and so far
all attempts to find a chicken tPA failed, uPA might replace
tPA in its functions in the chicken nervous system. Therefore,
inhibition of uPA in vitro might reflect a regulatory function of
neuroserpin toward PA-mediated processes in birds. Moreover,
recently discovered serine proteinases such as neuropsin (21),
neurosin (23), or neurotrypsin (22) fulfill the prerequisites for a
target of neuroserpin (namely extracellular location, temporal
and spatial coexpression). They have not yet been available for
tests with recombinant neuroserpin. It will therefore be impor-
tant to test biochemically the interaction between neuroserpin
and new neuronal proteinases, to identify the physiological
pathways of proteolysis in the developing and the adult nerv-
ous system. In conclusion, the data presented here make tPA a
likely candidate for a physiological target of neuroserpin. The
striking differences in target specificity between the two neural
serpins, neuroserpin and PN-1, would allow the selective reg-
ulation of different proteolytic cascades in the extracellular
space of the developing and the adult nervous system.
Acknowledgments—We thank Dr. D. Monard for providing purified
PN-1 and antibodies against PN-1 and Dr. K. Karjalainen for providing
the eucaryotic expression vector pCD4-FvCD3-C
. Recombinant human
tPA was kindly provided by Genentech (San Francisco, CA). We further
acknowledge R. Sack for performing the amino acid analysis and T. P.
Gschwend for carefully reading the manuscript.
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Rate constants for endopeptidase inhibition by cNS-C
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Reaction conditions were as follows: PBS, 0.1% PEG 8000, 0.2 mg/ml BSA, pH ⫽7.2; temperature ⫽37 ⫾1 °C. k
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c
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