The Axonally Secreted Serine Proteinase Inhibitor, Neuroserpin,
Inhibits Plasminogen Activators and Plasmin but Not Thrombin*
Thomas
Osterwalder
,
Paolo
Cinelli,
Antonio
Baici§,
Amedea
Pennella,
Stefan Robert
Krueger,
Sabine Petra
Schrimpf¶,
Marita
Meins
, 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
Friedrich Miescher Institute, Postfach 2543,
CH-4002 Basel, Switzerland
 |
ABSTRACT |
Neuroserpin is an axonally secreted serine
proteinase inhibitor that is expressed in neurons during embryogenesis
and in the adult nervous system. To identify target proteinases, we
used a eucaryotic expression system based on the mouse myeloma cell line J558L and vectors including a promoter from an Ig-
-variable region, an Ig-
enhancer, and the exon encoding the Ig-
constant region (C
) and produced recombinant neuroserpin as a wild-type protein or as a fusion protein with C
. We investigated the
capability of recombinant neuroserpin to form SDS-stable complexes
with, and to reduce the amidolytic activity of, a variety of serine proteinases in vitro. Consistent with its primary structure
at the reactive site, neuroserpin exhibited inhibitory activity against trypsin-like proteinases. Although neuroserpin bound and inactivated plasminogen activators and plasmin, no interaction was observed with
thrombin. A reactive site mutant of neuroserpin neither formed complexes with nor inhibited the amidolytic activity of any of the
tested proteinases. Kinetic analysis of the inhibitory activity revealed neuroserpin to be a slow binding inhibitor of plasminogen activators and plasmin. Thus, we postulate that neuroserpin could represent a regulatory element of extracellular proteolytic events in
the nervous system mediated by plasminogen activators or plasmin.
 |
INTRODUCTION |
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 proteinases 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 functions, in particular blood coagulation
and fibrinolysis. For example, 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 demonstrated 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). Thrombin, which has been extensively
characterized due to its important 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 coagulation, and
fibrinolysis, one would expect specific inhibitors of serine
proteinases belonging to the structural class of the serpins (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 member 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 glial cells and to interact in vitro with several
serine proteinases (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 reactive 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 inhibitory 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
proteinases were solely based on amino acid sequence comparisons, 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 inhibitory serpin and whether it is targeted against serine proteinases expressed in the nervous system. We used
complex formation assays and inhibition assays to investigate the
interaction between recombinant neuroserpin and the neural serine
proteinases 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
pcNSEP-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 mutagenesis 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 µM each of dATP, dCTP, dGTP, and dTTP, 1.3 mM
Mg2+ (3 mM for cNS-mt), 200 nM
amounts of each primer, and 0.25 µg of template. Sc3a4 was linearized
by KpnI digestion, and cNS-wt or cNS-fus, respectively, were
amplified in a 50 µl 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
(kindly 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
reactive 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 pcNSEP-C
for mutant 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 pcNSEP-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%
CO2. After 2 days, selection of transfectants was started by adding 5 mM L-histidinol (Sigma). Two weeks
later, histidinol-resistant colonies were expanded and cultivated for
3-4 days in 24-well plates containing 600 µl of selection medium (5 mM L-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 µCi/ml [35S]methionine (1000 Ci/mmol, NEN Life Science Products) was then added, and the expanding
colonies were metabolically labeled for 20 h. Afterward, the
relative expression 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
(cNSEP-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 incubating the dot blot with a
peroxidase-conjugated sheep antibody against mouse IgG (H + L; from
Kirkegaard & Perry Laboratories, Gaithersburg, MD) and by developing
with 0.5 mg/ml 4-chlor-1-naphthol (Merck, Dietikon, Switzerland) in
Tris-buffered saline (50 mM Tris-HCl, pH 7.4, 200 mM NaCl). The clones with the highest expression were subcloned and adapted to low serum conditions (Dulbecco's modified Eagle's medium with 2% FCS).
Purification of cNS, cNS-C
, and
cNSEP-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, Pharmacia, Dübendorf,
Switzerland). The flow-through was dialyzed against ion exchange
chromatography loading buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl), and proteins were fractionated using an anion
exchange column (MonoQ HR5/5, Pharmacia, Dübendorf, Switzerland)
and a linear gradient from 200 to 500 mM NaCl. The
fractions from 350 to 400 mM NaCl were highly enriched with
cNS; they were dialyzed against phosphate-buffered saline (PBS; 10 mM phosphate buffer, pH 7.2, 140 mM NaCl, 4 mM KCl) and stored frozen until further use. For the
isolation of cNS-C
and cNSEP-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 mM diethylamine, pH 11.5. The eluate was immediately
neutralized by adding 230 mM Tris-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-H6)--
Human neuroserpin (PI12) was
cytoplasmically 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, allowing 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 at 37 °C in 100 ml of LB medium
containing 30 µg/ml chloramphenicol. After inoculation of the same
medium with the preculture, bacteria were grown at 25 °C and induced
with 1 mM isopropyl-1-thio-
-D-galactosidase at an A600 of 0.5. The bacteria were harvested
by centrifugation 6 h after induction, resuspended in
Ni-NTA-binding buffer (1 M NaCl, 50 mM Tris-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 binding buffer
containing 200 mM imidazole. The eluted protein was
dialyzed 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 injection of 10-20 µg 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 µCi/ml [35S]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®, recombinant Alteplase, 580,000 IU/mg; kindly
provided by Genentech, South San Francisco, CA) was dissolved in water
to a final concentration 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 concentration 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 (Mölndal, Sweden), 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 1 mM S-2288, using values for substrate turnover of
A405 = 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, for 4 nM thrombin, uPA, single chain
tPA, and plasmin, respectively, as indicated by the substrates'
supplier. Recombinant neuroserpin was prepared as detailed above,
stored frozen, and thawed immediately before use if not indicated
otherwise. The concentrations of cNS-C
and cNSEP-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 concentration of cNS was estimated by
SDS-PAGE and silver staining. The concentration of hNS-H6
was estimated using the Bradford protein assay (Bio-Rad, Glattbrugg,
Switzerland) in combination with densitometric 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 mM Tris-HCl, pH
8.0, 133 mM NaCl, and 0.13% PEG 8000 was used. Inhibition
buffer contained 10 mM phosphate buffer, pH 7.2, 140 mM NaCl, 4 mM KCl, 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,
proteinases and inhibitors were mixed in 30 µl of complexation buffer
and incubated for 30 min at 37 °C if not indicated otherwise. For
experiments with hNS-H6, 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% glycerol, 31.3 mM Tris-HCl, pH
6.8) and by immediately boiling the sample for 5 min.
SDS-PAGE and Immunoblotting--
SDS-PAGE was carried out
according to Laemmli (48), and 2-dimensional SDS-PAGE was according to
O'Farrell (49) as modified by Sonderegger et al. (46). For
autoradiography, 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
recommendations, or goat anti-rabbit IgG conjugated to peroxidase
(Bio-Science products, Emmenbrücke, 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 µl of inhibition
buffer. The final concentrations of enzymes were as follows: uPA, 19.1 nM (9.1 nM for experiments with
hNS-H6 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-H6). cNS-C
was preincubated for 20 min at 37 °C, while hNS-H6 was 10 min at room
temperature. After preincubation, the amidolytic reactions were started
simultaneously by adding 2 µl of substrate solution (25 mg/ml S-2288)
to each well. Residual amidolytic activity was determined by measuring the hydrolysis 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 mM S-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 inhibitor concentrations. Since the interaction between
serpins and serine proteinases is assumed to follow slow binding
kinetics, product formation was described with Equation 1.
|
(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 d is a displacement factor compensating for small
uncertainties in absorbance at the start of the reaction. For each of
several inhibitor concentrations,
s,
z,
k
, and d were 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.
|
(Eq. 2)
|
Km 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
cNSEP-C
--
Only small amounts of denatured
neuroserpin could be purified from chicken embryonic VF by the
three-step purification strategy detailed earlier (34). Therefore, we
decided to recombinantly 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 expression 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
backward 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 coding 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
neuroserpin 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 pcNSEP-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 supernatants by immunoprecipitation of
metabolically labeled, recombinant 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 cNSEP-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 antiserum R35 (Blot). Interestingly,
cNS-C
always appeared as a double band. This effect was observed
earlier with recombinant C
fusion
proteins3 but could not be
explained so far. Although the immunoaffinity purification yielded
electrophoretically pure cNS-C
and cNSEP-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 recognized 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 identical N terminus as found in chicken neuroserpin purified from VF, indicating correct signal peptide cleavage (data not shown).

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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 neuroserpin from four different species.
Dark shading indicates amino acids identical to chicken
neuroserpin, and light shading indicates conservative
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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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 pcNSEP-C , which gave rise to the mutant fusion
protein cNSEP-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 ,
cNSEP-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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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 presence of reducing
agents (57). Since neuroserpin is predominantly 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 nervous 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 approximately 80, 86, and 112 kDa with uPA and tPA,
respectively, that matched the expected sums of the N-terminal part of
native neuroserpin (Phe17-Arg362, approximately 49 kDa) plus the catalytic subunits of the proteinases (uPA, approximately
30 kDa; two-chain tPA, approximately 35 kDa, single-chain tPA,
approximately 65 kDa). cNS behaved more substrate-like with plasmin, as
this enzyme cleaved cNS into one major fragment with the expected size
of native neuroserpin minus the C-terminal part
(Met363-Leu410). Only a small amount of cNS appeared
to form SDS-stable complexes of the expected size (approximately 75 kDa) with plasmin. No complex 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.

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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 incubated 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.
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Complex Formation and Inhibitory Activity of cNS-C
and
cNSEP-C
--
The question, whether the observed complex
formation was accompanied by an inhibition of the target proteinases,
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 neuroserpin
(Fig. 4A). In particular,
cNS-C
readily formed complexes 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 interfere 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 manner, but the proteolytic activity of
thrombin was not affected. Interestingly, cNS-C
also inhibited
plasmin, although the results 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, stoichiometric complex, whereas it interacts with plasmin
in a serpin-like mechanism with a higher partition ratio.

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Fig. 4.
Complex formation and inhibitory activity of
neuroserpin fusion proteins. A and B, complex
formation between 0.9 pmol of cNS-C or 0.9 pmol of
cNSEP-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. C and 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
cNSEP-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
cNSEP-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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It had been shown previously that the inhibitory activity and
specificity of serpins critically depend on their amino acid composition 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 mutagenesis.
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 neuroserpin, we produced a fusion protein
carrying a mutation of both P1 and P1
(cNSEP-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, cNSEP-C
was unable to reduce the proteolytic activity of
either of the tested proteinases more than 15% (Fig.
4D).
Complex Formation and Inhibitory Activity of
hNS-H6--
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 capacity of human neuroserpin to form complexes with, and its inhibitory activity toward, uPA, tPA, thrombin, and plasmin. We found that hNS-H6 formed SDS-stable complexes with uPA, tPA, and
plasmin, but no reaction with thrombin could be observed (Fig.
5A). Moreover, a significant
amount of neuroserpin was proteolytically cleaved by the target
proteinases, but the two shorter forms of neuroserpin (Fig. 5A,
open arrowheads) appeared to be inactive and are most probably
produced by alternative usage of translation start signals or
N-terminal proteolytic degradation of hNS-H6 by the
bacterial host strain. Amidolytic assays revealed a
dose-dependent inhibitory activity of hNS-H6
against uPA, tPA, and plasmin, whereas thrombin was not affected (Fig.
5B). These results are qualitatively in accordance with the
results obtained for chicken neuroserpin.

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Fig. 5.
Complex formation and inhibitory activity of
human neuroserpin. A, approximately 2 pmol of
procaryotically expressed hNS-H6 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 molecular 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 inhibitory
activity of hNS-H6 is shown by plotting the residual
amidolytic activity of the indicated proteinases after 10 min
preincubation with two different concentrations of hNS-H6
(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).
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Stability of Complexes and Latency of Neuroserpin--
Complexes
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 incubation 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 included 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 absence 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 for 5 h on ice prior to
mixing with the proteinases (lanes 2). A slightly reduced
reactivity after preincubation for 5 h at 37 °C was observed
for both cNS and cNS-C
, as indicated by the higher intensity of the
bands representing free cNS (Iw) or cNS-C
(If) at 54 or 65 kDa, respectively (lane
3). Once formed, a large proportion of the complexes remained stable for at least 5 h on ice (lane 5), whereas
incubation at 37 °C lead to a slightly increased decay (lane
4), as indicated by the higher intensity of the band
Icl representing the proteolytically cleaved
form of neuroserpin at 49 kDa.

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Fig. 6.
Latency of neuroserpin and decay of complexes
with tPA or uPA. An antiserum against neuroserpin recognizes
cleaved neuroserpin (Icl, 49 kDa) and free cNS
(Iw, 54 kDa) and cNS-C (If, 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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Kinetics of the Interaction between cNS-C
and Trypsin-like
Proteinases--
Second-order rate constants, ka,
for the interaction 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 published
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
departure 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. These properties justified the
calculation of the association and dissociation constants according to
Equation 2 (53). Fig. 7 exemplifies 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 ka can be considered
sufficiently precise, kd 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
kd for uPA and tPA can be calculated from the
Ki 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
thrombin was detected, even at an 80-fold cNS-C
concentration over
enzyme (data not shown).

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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. Inhibition 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 nM tPA, 1.08 mM substrate, and cNS-C as
indicated by numbers near the curves. The
first-order rate constants (k ) were calculated for each
inhibitor concentration 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
Km using Equation 2.
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Table I
Rate constants for endopeptidase inhibition by cNS-C and PN-1
Reaction conditions were as follows: PBS, 0.1% PEG 8000, 0.2 mg/ml
BSA, pH = 7.2; temperature = 37 ± 1 °C.
ka and kd were calculated by the
progress curve method, and ka values for PN-1 were
taken from Ref. 60.
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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 proteinases 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 heterologous 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.
Furthermore, the antiserum raised against recombinant neuroserpin was
cross-reactive with chicken neuroserpin, and the antiserum against
purified neuroserpin recognized cNS, cNS-C
, and
cNSEP-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
Proteinases--
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 inhibitor 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
distinguished by our experiments. Interestingly, only a small proportion of neuroserpin was found in an SDS-stable complex with plasmin, where most of the inhibitor was found in a modified form of
approximately 49 kDa (43 kDa for hNS-H6), most probably
representing the thermodynamically stable serpin 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 neuroserpin 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
contaminations 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. Neuroserpin 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 specificity of neuroserpin.
The experiments with mutant neuroserpin provided strong evidence for the putative reactive site P1-P1
(Arg362 and
Met363) being involved in the interaction with all target
proteinases, since the reactive site mutant cNSEP-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 cNSEP-C
might represent a
competitive effect that does not lead to the formation of stable
complexes. Altogether, the results characterized neuroserpin as a
typical serpin-type inhibitor of tPA, uPA, and plasmin, with the amino
acids Arg362 and Met363 forming the reactive
site P1-P1
.
Chicken and Human Neuroserpin Exhibit the Same Target
Specificity--
Complex formation tests and inhibition assays
revealed the same target proteinase preference for the human and the
chicken form of neuroserpin. Both hNS-H6 and cNS-C
readily formed SDS-stable complexes with plasminogen activators and
with plasmin but did not react with thrombin. Inhibitory assays with
hNS-H6 and with cNS-C
resulted in qualitatively the same
target preference pattern. Both hNS-H6 and cNS-C
exhibit
the strongest inhibitory activity against tPA, although
hNS-H6 in general shows a lower specific activity. The
higher stoichiometric indices observed for hNS-H6 as
compared with cNS-C
could reflect a lower stability of the complexes
formed by human neuroserpin. Alternatively, this observation could be
explained by the fact that the hNS-H6 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 enzyme and inhibitor concentration of the same
order of magnitude, 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 concentration
of a synthetic substrate and at inhibitor concentrations greatly
exceeding those of the enzymes. Therefore, kinetic parameters could be
determined under fully competitive conditions. 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 constants
ka 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 relative of neuroserpin in the nervous
system.
Is Neuroserpin the Physiological Inhibitor of tPA in the Nervous
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 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 granule cell migration (74) as well as a
different form of hippocampal LTP (13, 14) were found. On the other
hand, several lines of evidence point toward a role of thrombin during
neural development 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 proteolytic
activation of the thrombin receptor in vitro (19); and the
proteolytic activity of thrombin is required for neuromuscular 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 neuroserpin an interesting candidate for a physiological,
local regulator of tPA in the nervous system. Based on results
indicating 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 responsible 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
important to test biochemically the interaction between neuroserpin and
new neuronal proteinases, to identify the physiological pathways of
proteolysis in the developing and the adult nervous 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 regulation of different proteolytic cascades in the
extracellular space of the developing and the adult nervous system.
 |
ACKNOWLEDGEMENTS |
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.
 |
FOOTNOTES |
*
This work was supported by the Olga Mayenfisch Stiftung, the
Helmut Horten Stiftung, the Ciba-Geigy-Jubiläumsstiftung, the Hartmann Müller-Stiftung, the EMDO-Stiftung, the
Wolfermann-Nägeli-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. The article
must therefore be hereby marked
"advertisement" 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
Centre, S-75123 Uppsala, Sweden.
**
To whom correspondence should be addressed. Tel.: 41-1-635 5541;
Fax: 41-1-635 6805; E-mail: pson{at}bioc.unizh.ch.
1
The abbreviations used are: tPA, tissue-type
plasminogen activator; BSA, bovine serum albumin; C
, constant region
of Ig-
; cNS, recombinant chicken neuroserpin; cNS-C
, chicken
neuroserpin fusion protein with C
; cNSEP-C
, chicken
neuroserpin reactive site mutant fusion protein with C
; DRG, dorsal
root ganglion; FCS, fetal calf serum; hNS-H6, recombinant
human neuroserpin fusion protein tagged with 6 C-terminal histidines;
LTP, long term potentiation; PA, plasminogen activator; PAGE,
polyacrylamide gel electrophoresis; PAI-1, plasminogen 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.
2
Krueger, S. R., Ghisu, G. P., Cinelli, P.,
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