Serine Proteinase Inhibitor 3 and Murinoglobulin I Are Potent Inhibitors of Neuropsin in Adult Mouse Brain*

Keiko KatoDagger §, Tadaaki KishiDagger , Tomohiro KamachiDagger , Morito AkisadaDagger , Takuya OkaDagger , Ryosuke MidorikawaDagger , Koji Takio||, Naoshi Dohmae||, Phillip I. Bird**, Jiuru Sun**, Fiona Scott**, Yoshimasa MiyakeDagger Dagger , Kazuhiko Yamamoto§§, Atsunori MachidaDagger , Tatsuya Tanaka¶¶, Kazumasa MatsumotoDagger , Masao Shibata||||, and Sadao ShiosakaDagger

From the Dagger  Division of Structural Cell Biology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101 Japan, || Biomolecular Characterization Division, RIKEN (The Institute for Physical and Chemical Research), Wako, Saitama 351-0198, Japan, ** Department of Biochemistry and Molecular Biology, P.O. Box 13D, Monash University 3800, Australia, the Dagger Dagger  Faculty of Pharmaceutical Science, Kinki University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan, the §§ Department of Biochemistry, Kinki University School of Medicine, 377-2 Oonohigashi, Sayama, Osaka 589-8511, Japan, ¶¶ Center for Research and Education, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan, and the |||| Department of Pharmaceutical Development, Medial and Biological Laboratories Co., Ltd., 1063-103 Ohara Terasawaoka, Ina Nagano 396-0002, Japan

Received for publication, November 28, 2000, and in revised form, February 2, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Extracellular serine protease neuropsin (NP) is expressed in the forebrain limbic area of adult brain and is implicated in synaptic plasticity. We screened for endogenous NP inhibitors with recombinant NP (r-NP) from extracts of the hippocampus and the cerebral cortex in adult mouse brain. Two SDS-stable complexes were detected, and after their purification, peptide sequences were determined by amino acid sequencing and mass spectrometry, revealing that target molecules were serine proteinase inhibitor-3 (SPI3) and murinoglobulin I (MUG I). The addition of the recombinant SPI3 to r-NP resulted in an SDS-stable complex, and the complex formation followed bimolecular kinetics with an association rate constant of 3.4 ± 0.22 × 106 M-1 s-1, showing that SPI3 was a slow, tight binding inhibitor of NP. In situ hybridization histochemistry showed that SPI3 mRNA was expressed in pyramidal neurons in the hippocampal CA1-CA3 subfields, as was NP mRNA. Alternatively, the addition of purified plasma MUG I to r-NP resulted in an SDS-stable complex, and MUG I inhibited degradation of fibronectin by r-NP to 24% at a r-NP/MUG I molar ratio of 1:2. Immunofluorescence histochemistry showed that MUG I localized in the hippocampal neurons. These findings indicate that SPI3 and MUG I serve to inactivate NP and control the level of NP in adult brain, respectively.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Extracellular proteolysis exerted by secretory serine proteases has been implicated in neural development, plasticity, and degeneration and regeneration in the nervous system (1) and might be controlled by specific inhibitors (2). Neuropsin (NP),1 a serine protease with a chimeric structure similar to trypsin and nerve growth factor-gamma (3), was found to be expressed in the nervous system (4) and has been demonstrated to be engaged in activity-dependent plasticity changes in neurons. NP mRNA and protein levels increased in the hippocampus after kindled seizures and injection of antibody against NP led to retardation of epilepticus in mice (5, 6). Furthermore, application of recombinant NP induced an increase in the amplitude of the tetanic stimulation-induced early phase long term potentiation in the Schaffer collateral pathway (7). It has been proposed that the plasticity changes are regulated by the balance between the accumulation and degradation of the extracellular matrix (ECM) proteins. There is, indeed, some evidence that the formation of hippocampal LTP is attributable to cell-ECM interactions, involving cadherin (8), integrin (9, 10), N-syndecan (11), cell adhesion molecules, NCAM, and L1 (12-14). NP acted to degrade ECM including fibronectin (15), for which integrins were receptors (16, 17), and L1.2 Therefore, rearrangement of these ECM components by NP and a specific inhibitor might implicated in the formation of LTP.

As another characteristic of NP, it has been shown that NP mRNA was restricted to neurons in the limbic areas of adult brain involving the CA1-CA3 subfields of the hippocampus, the amygdaloid nucleus, the cingulate cortex, the anterior olfactory nuclei, the septal nuclei, and the diagonal bands (4, 18). Recent studies have shown that the neuronal degeneration response to several pathological stimuli, including age-associated memory impairment, Alzheimer's disease, brain ischemia, and epilepsy, occurs in highly vulnerable regions including the CA1 subfield and hilar area of the hippocampal formation, the cingulate area, and the basal forebrain (19-22), and most of the vulnerable regions coincide with the areas expressing NP. Thus, control of NP activity by a specific inhibitor may play a role in the selective vulnerability. However, the specific inhibitor for NP has still not been identified.

Serine proteases are controlled by specific inhibitors called serpins (2) and by the alpha 2-macroglobulin (alpha 2M) family that acts as a panprotease inhibitor through a unique trapping mechanism (23). The neurally expressed serpins protease nexin-1 and neuroserpin are synthesized and secreted in most brain regions (24-27) and are proposed to inactivate extracellularly thrombin and tissue plasminogen activator as specific inhibitors, respectively (28, 29). On the other hand, intracellular protease inhibitors belonging to the ovalbumin serpin family have been recently identified in the immune system (30), and induction of serine proteinase inhibitor-3 (SPI3) mRNA was reported in brain ischemia (31). However, the target protease is not to be identified in brain. Second, alpha 2M is cleaved in a peptide bond in the "bait region" within alpha 2M by proteases. This cleavage allows alpha 2M to undergo a drastic conformational change and to form a cage-like structure around the protease, leading to loss of protease hydrolytic activity against macromolecules (32, 33). Additionally, conformational change also reveals binding sites for the alpha 2M receptor/low density lipoprotein receptor-related protein (LRP) (34), which mediates the internalization and lysosomal degradation of alpha 2M and proteases (32).

Serine proteases and their inhibitors form SDS-stable complexes (2, 32). In the present study, we investigated whether there were specific inhibitors that form a complex with NP in the hippocampus and cerebral cortex of the adult mouse brain in vivo. The search revealed two distinct complexes and SPI3 and MUG I as NP-specific. We propose that SPI3 and MUG I regulate NP activity in adult brain.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NP and Antibody-- Recombinant neuropsin (r-NP) was produced as precursor in cultured medium using a baculovirus expression system and purified as described previously (15). The r-proNP was activated by treatment with lysyl endopeptidase (EC 3.4.21.50) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) conjugated with Sepharose 4B (Amersham Pharmacia Biotech) at 37 °C for 50 min (r-actNP). The amidolytic activity of r-NP was determined using a synthetic chromogenic substrate, t-butyloxycarbonyl-Val-Pro-Arg-4-methylcoumaryl-7-amide (Peptide Institute, Inc., Osaka, Japan) (15). NP cDNA and the mutant cDNAs (DS211VA, D206V, C7S, and C7S/C108S the numerals shown in clone names indicate the amino acid number counted from start codon, Met, DDBJ accession number D30785) in the pED1 vector containing the cytomegalovirus enhancer and chicken beta -actin promoter (35, 36) were constructed3 and transfected into neuro2a cells (Institute for Fermentation, Osaka, Japan). The amount of r-NP and r-NP-mutant proteins (N2a) in the cultured supernatant was determined based on band density stained using colloidal properties of Coomassie G-250 (GelCode Blue, Pierce) followed by SDS-PAGE. Purified r-NP (Baculo) was used as a control of the amount. Anti-NP monoclonal antibodies (F12mAb and B5mAb) (6) and rat IgG conjugated with Affi-Gel Hz beads (Bio-Rad) or rabbit anti-NP polyclonal antibody (11pAb)3 were used for immunoprecipitation and affinity purification of NP-specific inhibitors or for Western blot analysis, respectively.

Preparation of Crude Extracts-- The hippocampus and cerebral cortex were dissected from adult mice (8 weeks old, Slc:ddY, Japan SLC, Shizuoka, Japan), and the tissues were mixed and homogenized using a Dounce homogenizer with 1 ml of ice-cold 20 mM Tris-HCl, pH 7.4, and 0.15 M NaCl for the tissues recovered from one mouse. After incubation on ice for 30 min and centrifugation at 10,000 × g for 10 min, the soluble fraction was recovered (0.15 M NaCl-soluble fraction). The sediment was resuspended with an equal volume of ice-cold 20 mM Tris-HCl, pH 7.4, and 2% Triton X-100, homogenized, and stood on ice for 30 min. After centrifugation at 102,000 × g for 10 min, the Triton-insoluble residue was pelleted; resuspended with an equal volume of ice-cold 20 mM Tris-HCl, pH 7.4, and 1% Triton X-100; homogenized; and stood on ice for 30 min. After further centrifugation at 208,000 × g for 10 min, the second Triton-insoluble fraction was homogenized in an equal volume of ice-cold 20 mM Tris-HCl, pH 7.4, and 0.5 M NaCl; incubated on ice for 30 min; and then centrifuged at 383,000 × g for 10 min. Finally, the supernatant was recovered as a cytoskeleton-rich fraction (15, 37). The protein contents were determined with BCA assay reagent (Pierce) using an albumin standard (Pierce).

Detection of NP Protease Inhibitors-- Crude extracts (3 mg) were incubated with r-NP (2 µg) on ice for 1 h and rotated in the presence of Affi-Gel Hz beads (Bio-Rad) conjugated with rat IgG (more than 20 µg of IgG/2 µg of r-NP) at 4 °C for 30 min. After a brief centrifugation, the supernatant was mixed with Affi-Gel Hz beads conjugated with F12mAb (more than 20 µg of IgG) in 20 mM Tris-HCl, pH 7.5, and 0.15 M NaCl and rotated at 4 °C for 15 h. The beads were washed, mixed with an equal volume of SDS-containing buffer w/o 100 mM dithiothreitol (DTT), boiled for 5 min, and then subjected to SDS-PAGE using a 5-15% gradient of acrylamide, according to Laemmli (38). The proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad). After blocking with 5% skim milk in 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.1% Tween 20 (TTBS) for 30 min at room temperature, the membranes were reacted with rabbit anti-NP polyclonal antibody (11pAb) and then anti-rabbit IgG conjugated with alkaline phosphatase (Bio-Rad) in 5% skim milk-TTBS. The secondary antibody was detected by enhanced chemiluminescence (Immun-Star Substrate; Bio-Rad). After development, the band intensities were analyzed using one-dimensional gel image analysis software (Quantity One software, PDI, Toyobo Co., Ltd., Osaka, Japan).

Isolation of 65-kDa Complex-- After incubation of r-actNP and Triton-soluble fractions (430 mg) on ice for 30 min, the Affi-Gel Hz beads conjugated with F12mAb were mixed, rotated, and washed sequentially based on the above immunoprecipitation. The r-actNP/Triton-soluble fractions/F12mAb ratio was 1:7600:10 (w/w/w). The F12mAb beads were packed in a Sepacol column (Seikagaku Corp., Tokyo, Japan). The column was washed with five bed volumes of 20 mM Tris-HCl, pH 7.5, and 0.15 M NaCl and of 20 mM Tris-HCl, pH 7.5, and 0.5 M NaCl. The 65-kDa complex was eluted with 0.2 M glycine-HCl, pH 2.5. After dialysis with a 2000-fold volume of 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5 mM DTT, and 1 mM phenylmethylsulfonyl fluoride at 4 °C for 2.5 h, the sample was freeze-dried. The concentrated sample was then resuspended with distilled water and subjected to 10% SDS-PAGE.

Isolation of 230-kDa Complex-- Crude extracts (150 mg) in the 0.15 M NaCl-soluble fraction were dialyzed with a dialysis membrane (Seamless Cellulose Tube, molecular weight cut-off 12,000-14,000; Wako) in a 100-fold volume of 20 mM Tris-HCl, pH 7.5, and 0.25 M NaCl at 4 °C for 4 h. After centrifugation and filtration with a 0.22-µm filter, the extract was applied to a Resource Q column (Amersham Pharmacia Biotech, Tokyo, Japan) equilibrated with 20 mM Tris-HCl, pH 7.5, and eluted with a linear gradient of 0.05-0.6 M NaCl (FPLC LCC-500 PLUS system; Amersham Pharmacia Biotech) for 144 min at a flow rate of 1 ml/min. The fractions with the greatest binding activity for r-actNP were eluted at 0.2-0.3 M, pooled, and subjected to affinity chromatography with r-actNP and Affi-Gel Hz beads conjugated with F12mAb according to the above procedures for purification of 65-kDa complex. The r-actNP/pooled Resource Q eluant/F12mAb ratio was 1:800:7 (w/w/w). Eluant with an A280 of more than 0.01 was pooled, and the pH was adjusted to 7.5 with 1 M Tris-HCl, pH 9.0. Then the mixture was diluted 5-fold with 12.5% glycerol and 0.125 M NaCl. Samples were applied to a column of Mono Q PC connected with a precolumn under the Smart system (Amersham Pharmacia Biotech). Anion exchange resin was equilibrated with 20 mM Tris-HCl, pH 7.5, and 10% glycerol. Bound proteins were eluted with NaCl in a linear gradient from 0.05 to 0.6 M for 67 min at a flow rate of 0.1 ml/min. Purity and molecular weight were assessed by SDS-PAGE using a 5-15% gradient of acrylamide, silver staining, and Western blot. The band densities visualized by Western blot and Coomassie Blue R-250 were determined using one-dimensional gel image analysis software (Quantity One software).

Peptide Sequence Determination-- The 65- and 230-kDa complexes were subjected to SDS-PAGE in a gel containing, respectively, 10% and a 5-15% gradient of acrylamide and stained with Coomassie Blue R-250. Each band was excised and digested with 3 µg/ml Achromobacter lyticus protease I (39, 40) in 50 mM Tris-HCl, pH 9.0, 1 mM EDTA, and 0.1% SDS at 37 °C overnight. The digests were separated using a model 1100 liquid chromatography system (Hewlett Packard Company, California) on a DEAE-5PW column (1 × 20 mm; Tosoh, Tokyo, Japan) and a CAPCELL PAK RP18 (1 × 100 mm; Shiseido, Tokyo, Japan), which were linked tandemly, with a linear gradient of 0-48% acetonitrile in 0.1% trifluoroacetic acid for 96 min for the 65-kDa complex and with linear gradients of 0.8% (0 min) to 10% (10 min) to 40% (130 min) to 80% (135 min) acetonitrile in 0.1% trifluoroacetic acid for the 230-kDa complex at a flow rate of 30 µl/min. Fractionated peptides were subjected to protein sequencing and mass spectrometry as described previously (41).

SPI3 and NP Kinetics-- Recombinant SPI3 (r-SPI3) was prepared with the Pichia pastoris expression system (42). To obtain an SDS-stable complex of r-SPI3 and r-actNP (Baculo), 200 ng of r-SPI3 was added to 100 ng of r-actNP and incubated at 37 °C for 20 min. Samples containing either r-SPI3 or r-actNP alone were incubated in parallel. Three identical sample sets were separated by reducing 12.5% SDS-PAGE. One set was silver-stained, and the other two sets were transferred to nitrocellulose and immunoblotted with either rabbit anti-NP (11pAb) or rabbit anti-SPI3 antibody.

Next, the stoichiometry was measured by incubating increasing amounts of r-SPI3 with r-actNP (40 nM) in a total volume of 100 µl of 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.01% bovine serum albumin in individual wells of a microtitration plate previously blocked with buffer containing 0.1% bovine serum albumin. The reactants were incubated for 30 min at 37 °C, and residual amidolytic activity was measured by 100 µl of 100 µM t-butoxycarbonyl-Val-Pro-Arg-methylcoumarin in the same buffer for a final concentration of 50 µM and monitoring the fluorescence change using a Biolumine read. The data were used to plot the enzymatic rate of substrate hydrolysis as a function of the amount of SPI-3 added to the reaction well. Linear regression to the x axis was used to calculate the precise amount of SPI-3 required to inhibit rNP completely.

Furthermore, the association rate constant between r-SPI3 and r-actNP was determined. A constant amount of r-actNP (0.2 nM) was mixed with different concentrations of r-SPI3 and excess substrate (40 µM; t-butoxycarbonyl-Val-Pro-Arg-4-methylcoumaryl-7-amide) in a final volume of 200 µl in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% polyethylene glycol 8,000. The reactions were monitored at 37 °C for 2 h with a PerkinElmer Life Sciences LS50B spectrofluorometer using an excitation wavelength of 370 nm and an emission wavelength of 450 nm, with residual activity determined periodically. The interactions of recombinant SPI3 with r-actNP were determined with the progress curve method followed by slow binding inhibition kinetics (43),
Y=v<SUB>s</SUB>t+(v<SUB>0</SUB>−v<SUB>s</SUB>) ∗ (1−e<SUP>−k′t</SUP>)/k′ (Eq. 1)
where Y represents the amount of product at time t, k' is the apparent first order rate constant, and v0 and vs are the initial and steady state velocities, respectively. The association (Ka) was calculated using the equation,
K<SUB>a</SUB>=K<SUB><UP>obs</UP></SUB>(1+[<UP>S</UP>]/K<SUB>m</SUB>) (Eq. 2)
where Kobs = k', [S] represents the concentration of substrate, and Km is the Michaelis constant. Ki is essentially obtained by the Guy Salvesen and Hideaki Nagase method (44),
K<SUB>i</SUB>=K<SUB>i</SUB>(<UP>app</UP>)/1+[<UP>S</UP>]/K<SUB>m</SUB> (Eq. 3)
where V0/Vi = 1 + [I]/Ki(app) and V0 represents the enzyme-catalyzed hydrolysis rate in the absence of an inhibitor. Vi is the inhibited rate. Values were determined from Dixon plots. In the present study, Km, kcat, and kcat/Km of r-NP were 123.6 µM, 4.2 s-1, and 33,900 M-1 s-1, respectively.

MUG I-NP Interaction Assays-- MUG I (sequential concentration, 0.28-4.48 µg) purified from mouse serum (45) was incubated with r-actNP (Baculo) (40 ng) at 25 °C for 15 min, and the mixture was subjected to nonreducing SDS-PAGE to determine whether it formed an SDS-stable complex. To determine the effect of MUG I on the proteolytic activity of NP for fibronectin, a mixture of r-actNP (Baculo) (20 ng) and plasma MUG I (sequential concentration, 0.14-2.28 µg) was incubated with fibronectin (400 ng; human plasma; Life Technologies, Inc.) at 37 °C for 16 h as described previously (15). Sample was subjected to nonreducing SDS-PAGE and electrotransferred to polyvinylidene difluoride membrane. The membranes were reacted with mouse anti-fibronectin monoclonal antibody (clone10; Transduction Laboratories, Lexington, KY) and then anti-mouse IgG conjugated with alkaline phosphatase (Bio-Rad). After detection of the secondary antibody by enhanced chemiluminescence, the band density of 170-kDa fragments of fibronectin was determined with one-dimensional gel image analysis software.

In Situ Hybridization-- For preparation of riboprobes, the SPI3 target sequence was amplified with a single cDNA synthesized from the hippocampal total RNA by one round of polymerase chain reaction using primers 5'-CCAAAGTTTAAGCTGGAGGAGAA-3'/5'-CTGACAGGAATACCCTTTGCTCA-3' (338 bp; bp 887-1224), and the polymerase chain reaction fragment was subcloned into pGEM-T easy vector. alpha -35S-Labeled riboprobes were prepared according to the manufacturer's instructions (Roche Molecular Biochemicals): antisense, NcoI and SP6 RNA polymerase; sense, SalI and T7 RNA polymerase. In situ hybridization histochemistry was performed as previously described (4). To determine areas labeled with SPI3 and NP (B41) (4) riboprobes, 14-µm-thick coronal sections were cut on a cryostat and thaw-mounted onto slides coated with 0.1% 3-aminopropyltriethoxy silane (Sigma-Aldrich) in acetone. No signals were detected on the adjacent sections incubated in control hybridization mixture containing sense probe (see Fig. 6C).

Immunofluorescence-- Coronal sections (14 µm thick) of adult mouse (8 weeks old, Slc:ddY) were cut on a cryostat. Sections on slides were fixed in 4% paraformaldehyde in Dulbecco's phosphate-buffered saline containing 0.7 mM CaCl2 and 0.5 mM MgCl2, for 1 h at 4 °C. Sections were treated with 20 mM glycine and phosphate-buffered saline for 10 min; washed with 0.15 M NaCl, 20 mM boric acid, and 5 mM sodium tetraborate decahydrate, pH 8.0 (BBS); and incubated with rabbit serum anti-MUG I antibody (diluted to 1:500) or the preimmune rabbit serum (diluted to 1:500) (46) in 3% bovine serum albumin and 3% goat serum in BBS overnight at 4 °C. After a wash with BBS, goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (diluted 1:600; BIOSOURCE International, Camarillo, CA) in 5% bovine serum albumin and BBS was applied overnight at 4 °C. After being washed, coverslips were mounted in glycerol containing Mowiol 4-88 (Calbiochem) and 1,4-diazobicyclo-(2,2,2)-octane (Sigma-Aldrich) (47) and were observed with an Axioplan2 microscope (CarlZeiss, Tokyo, Japan). No signals were detected on the adjacent sections incubated with preimmune serum before the rabbit was immunized with MUG I (see Fig. 7, G-I).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Detection of r-NP-binding Proteins in Mouse Hippocampal and Cerebral Cortical Extracts-- We screened for NP-specific inhibitors present in adult mouse brain in vivo. We monitored whether r-NP (Baculo), originating from the baculovirus expression system, formed SDS-stable complexes with molecules within extracts prepared from adult mouse hippocampi and cerebral cortices, in which NP mRNA is expressed most abundantly (4). The tissues were homogenized and fractionated into 0.15 M NaCl-soluble, Triton-soluble, and cytoskeleton-rich (0.5 M NaCl-soluble) fractions as described under "Experimental Procedures." As shown in Fig. 1, the 65-kDa complex was detected in all fractions with both r-actNP and r-proNP, while the 230-kDa complex was detected in the 0.15 M NaCl fraction with r-actNP but not with r-proNP (Fig. 1A, left panel). Both complexes were still detected when mixtures were treated with DTT prior to electrophoresis (Fig. 1A, right panel, lane 4). No 65- or 230-kDa complex was detected without the addition of r-actNP in the 0.15 M NaCl-soluble fraction (Fig. 1, A (right panel, lanes 1 and 3) and B (lane 2)). Since the 65-kDa complex detected with r-actNP was expressed with strong intensity in all fractions (Fig. 1A, left panel, lanes 6-9), a large amount of each fraction was immunoprecipitated with anti-NP antibody and subjected to SDS-PAGE and Western blotting (Fig. 1B). The result, that endogenous brain NP and the 65-kDa complex were detected in the 0.15 M NaCl- and Triton-soluble fraction, respectively, showed that the 65-kDa complex was obtained from endogenous NP and the target molecule.


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Fig. 1.   Identification of NP-specific inhibitors. A, detection of SDS-stable complexes with r-NP (Baculo), prepared from a baculovirus expression system, in adult mouse hippocampal and cortical extracts. The hippocampal and cortical tissues were dissected and fractionated to a 0.15 M NaCl-soluble fraction, first Triton-soluble fraction, second Triton-soluble fraction, and cytoskeleton-rich fraction as described under "Experimental Procedures." Immunoprecipitants of each fraction (3 mg) and purified r-NP (Baculo; 2 µg) obtained using Affi-Gel Hz beads conjugated with F12mAb (20 µg IgG) were run on a 5-15% polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane and then immunostained with anti-NP polyclonal antibody (11pAb). B, detection of 65-kDa complex with endogenous NP (star). Each fraction (33 mg) was immunoprecipitated with Affi-Gel Hz beads conjugated with B5mAb (450 µg IgG), and 1/4 volume of each precipitant was run on a 10% polyacrylamide gel and immunoblotted. Film was overexposed relative to A.

To elucidate whether the formation of the two SDS-stable complexes was dependent on the catalytic structure of NP, we prepared r-actNP proteins from neuro2a cells transfected with wild type NP and mutant plasmids with disruptions of the protease active pocket, DS211VA and D206V, and of cysteine residues with amidolytic activity, C7S and C108S (Fig. 2). The protease active pocket mutants of r-NP (N2a) did not form SDS-stable complexes, while wild type and other mutants of r-NP (N2a) with amidolytic activity interacted with the two target molecules of 65- (Fig. 2C) and 230-kDa (Fig. 2B) complexes. It was indicated that formation of the two SDS-stable complexes was dependent on the presence of the active pocket of NP.


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Fig. 2.   NP structures necessary for the formation of the SDS-stable complexes. A, amidolytic activities of r-actNP proteins prepared from neuro2a cells (N2a) transfected with wild type NP and the mutant plasmids (at 25 °C). WT, wild type r-NP; D206V, disruption of S1-specific pocket; DS211VA, disruption of catalytic triad; C7S and C108S, disruption of free cysteine residues. B, mixtures of the pooled Resource Q eluant (20 µg) and each r-actNP (0.6 µg) were immunoprecipitated and applied to a 5-15% linear gradient acrylamide gel under nonreducing conditions for SDS-PAGE before being subjected to Western blot. C, mixtures of the 0.15 M NaCl soluble fraction (1.5 mg) and each r-actNP (0.6 µg) were immunoprecipitated and subjected to reducing SDS-PAGE (100 mM DTT), followed by Western blot. Note that no SDS-stable binding of D206V- and DS211VA-r-NP, treated with lysyl endopeptidase conjugated with Sepharose 4B, to target molecules was observed. The 230-kDa complex with r-actNP (N2a) was sensitive to reduction, being different from the complex with r-actNP (Baculo).

NP contains an N-glycosylation site (48), and the band of r-NP (N2a) migrated more slowly than that of r-NP (Baculo) in SDS-PAGE (Fig. 2C). Treatment of these r-NPs with N-glycosidase resulted in similar band shifts,3 suggesting that the molecular weights of r-NP (Baculo) and r-NP (N2a) were different due to species and the length of the sugar chain. There was no effect of differential glycosylation on the formation of the 65-kDa complex, while glycosylation of r-NP in neuro2a cells led to the disappearance of the 230-kDa complex in the presence of DTT, suggesting that the sugar chain affected the formation of the 230-kDa complex.

Since interactions of two specific inhibitors with NP in vivo were proposed, 65- and 230-kDa complexes were next purified by the procedure described under "Experimental Procedures."

Purification and Identification of Inhibitors Specific for NP-- Concerning the 65-kDa complex, the Triton-soluble fraction containing r-actNP (Baculo) was applied to an F12mAb-Affi-Gel Hz affinity column, and the 65-kDa complex was eluted with a 1.6 × 103-fold purification and a yield of 34%. To determine the target molecule of 65-kDa complex by microsequencing, the eluant was freeze-dried and subjected to SDS-PAGE. A portion was, however, degraded after being freeze-dried to a final yield of 1.5%, and the 41-kDa band appeared (Fig. 3A). Concerning the 230-kDa complex, the 0.15 M NaCl soluble fraction was subjected to Resource Q chromatography to separate 230- from 65-kDa complex. The fractions of 0.2-0.3 M NaCl containing r-actNP (Baculo) were subjected to F12mAb-Affi-Gel Hz affinity and Mono Q chromatography to concentrate the immunoaffinity eluant (Fig. 3B). Coomassie Blue staining revealed the presence of a 54-kDa species after immunoaffinity chromatography (Fig. 3B), but Western blot analysis did not (data not shown), showing degradation of the target molecule in the 230-kDa complex by proteolytic activity of r-actNP (Baculo). Finally, the 230-kDa complex was purified 4.3 × 103-fold with a yield of 11%.


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Fig. 3.   Affinity purification of 65- and 260-kDa SDS-stable complex. A, purification of 65-kDa complex. Mixtures of Triton-soluble fractions and r-actNP were subjected to affinity purification with Affi-Gel Hz beads conjugated with F12mAb as described under "Experimental Procedures." After SDS-PAGE, gels were subjected to silver staining, Western blotting, and Coomassie Blue R-250 staining. Lanes 1 and 3, mixtures of Triton-soluble fractions and r-actNP (25 µg; fraction/r-actNP ratio of 7600:1 (w/w)); lanes 2 and 4, eluants for F12mAb immunoaffinity chromatography (0.3 µg); lane 5, sample (1.2 µg) dialyzed, freeze-dried, and resuspended with distilled water. Note that 65- and 41-kDa bands on the gel following Coomassie Blue staining were subjected to peptide sequencing. B, Mono Q chromatography following F12mAb-immunoaffinity chromatography for purification of 260-kDa complex. After application of F12mAb-immunoaffinity eluant to a Mono Q column for chromatography, a peak of 0.2 M NaCl eluant (star) containing 1.5 µg was subjected to SDS-PAGE followed by staining with Coomassie Blue R-250 (inset): 230 kDa, 1.1 µg; 54 kDa, 0.4 µg. Note that the 230- and 54-kDa bands on the gel following Coomassie Blue staining were subjected to peptide sequencing.

Purified 65- and 230-kDa complexes were subjected to SDS-PAGE, each band (65-, 41-, 230-, and 54-kDa) was treated with A. lyticus protease I, and peptide sequences were determined using a protein sequencer. Peptide sequencing identified the inhibitors to be SPI3 for 65-kDa complex and MUG I for 230-kDa complex, and peptide mass chromatography confirmed these identifications. Fifty-seven percent and 27.3% of SPI3 and MUG I were sequenced, respectively. In addition, we screened an adult mouse hippocampal cDNA library by polymerase chain reaction with primers based on the peptide sequence obtained from the degraded 65-kDa complex (41-kDa species), which resulted in amplification of the SPI3 cDNA. Thus, it was found that the addition of r-NP to adult mouse hippocampal and cortical extracts resulted in the appearance of SPI3-NP (65-kDa) and MUG I-NP (230-kDa) complexes. We next investigated whether the inhibitors formed complexes with r-NP (Baculo) or inhibited the proteolytic activities of r-NP (Baculo).

Effects of SPI3 and MUG I on the Proteolytic Activities of NP-- We purified recombinant SPI3 (r-SPI3) with the P. pastoris expression system (42). The addition of r-SPI3 to r-actNP (Baculo) resulted in the formation of a 65-kDa complex, which was subjected to reduction, boiling, and SDS-PAGE (Fig. 4A). The complex was recognized by antibodies against both NP and SPI3. The stoichiometry of inhibition for r-SPI3 and r-NP was 1, indicating that only one molecule of SPI3 was required to inhibit one molecule of NP. The association rate constant Ka (Fig. 4, Kass), for interaction of r-actNP (Baculo) with r-SPI3, was determined under pseudo-first-order conditions using the progress curve method (43). The Ka and Ki for complex formation were calculated as 3.4 + 0.22 × 106 M-1 s-1 and 0.8 nM, respectively. This was well within the range for physiologically significant interactions (2, 49).


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Fig. 4.   Interaction of SPI3 and NP. A, detection of SDS-stable complex of r-NP and r-SPI3 with silver staining and Western blotting with anti-NP (11pAb) and rabbit anti-SPI3 antibodies, respectively. B, plot of the reciprocal residual NP activity (fluorescence units) against time in the presence of increasing concentrations of SPI3. C, observed inhibitory rate of different concentrations of SPI3, indicating a Ka of 3.4 ± 0.22 × 106 M-1 s-1 and Ki of 0.8 nM.

We purified plasma MUG I from adult mouse serum (45). The addition of plasma MUG I to r-actNP (Baculo) resulted in the formation of a 230-kDa complex, which was subjected to boiling and SDS-PAGE (Fig. 5A). The band density of the 230-kDa complex increased upon the addition of plasma MUG I at increasing molar ratios. Since fibronectin (15) and L12 among the components of the ECM have been proposed as NP substrates, we examined whether MUG I inhibited r-NP-mediated degradation of fibronectin. MUG I inhibited the degradation of fibronectin by r-actNP to 24% at a r-NP/MUG I molar ratio of 1:2. These results suggested that MUG I affected physiologically the biological effect of NP.


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Fig. 5.   Interaction of MUG I and NP. A, determination of SDS-stable complex between r-actNP (Baculo; 40 ng) and increasing molar ratios of plasma MUG I by Western blotting with 11pAb and silver staining. B, effect of plasma MUG I on the proteolytic activity of r-actNP (Baculo; 20 ng) for fibronectin as determined by Western blotting with mouse anti-fibronectin monoclonal antibody. Fibronectin was composed of a dimer (440 kDa) and monomer (240 kDa), and r-actNP hydrolyzed fibronectin to fragments of 220, 200, and 170 kDa. The 170-kDa fragment was the smallest product obtained by r-NP-mediated degradation as described previously (15). MUG I cross-reacted with secondary antibody against mouse IgG (star). A 230-kDa band also appeared (small arrowheads). The closed triangle shows co-aggregations of fibronectin and MUG I. C, plot of the residual NP activity in the presence of increasing concentrations of MUG I. Band densities of 170-kDa in B were determined using one-dimensional gel image analysis software. The error bars show the S.E. of three experiments.

Localization of SPI3 and MUG I in the Adult Mouse Brain-- We next determined whether native NP could co-localize with SPI3 and MUG I in vivo.

It has been suggested that SPI3 was not secreted and had an intracellular role in monocytes and granulocytes (50, 51). We, therefore, investigated whether areas of the brain expressing NP mRNA also expressed SPI3 mRNA by in situ hybridization histochemistry (Fig. 6). The SPI3 and NP mRNAs were expressed in mostly the same areas. In the hippocampal formation, SPI3 mRNAs were expressed in the CA3 pyramidal neurons very intensely, in the CA1 pyramidal neurons moderately intensely, and in the granule cells of the dentate gyrus weakly, while the CA1-CA3 subfields but not the dentate gyrus expressed NP mRNAs (4). In the frontal cortex, labeled neurons spread to layer V and, to a lesser extent, II and IV, a similar profile to that of NP (4). Neurons in the cingulate cortex were also labeled and the signal continued posteriorly into the retrosplenial cortex and presubiculum like the NP mRNA signals (4). The anterior olfactory nuclei, medial septal nucleus, diagonal bands, and amygdaloid complex were labeled as well as with NP probe (4). However, in the forebrain limbic area, the lateral septal nucleus was labeled with NP but not SPI3 riboprobe. In contrast, there were cells with intense SPI3 mRNA signals in the thalamus, hypothalamus, and choroid plexus, although no NP-positive signals were found in these areas.


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Fig. 6.   Distribution of SPI3 transcripts. In situ hybridization histochemistry in mouse adult frontal sections under dark-field illumination is shown. NP antisense (A), SPI3 antisense (B, D, and E), and SPI3 sense (C) riboprobes were used in 14-µm-thick coronal sections. Bar, 500 µm. I-V, layers of the frontal cortex; AL, amygdaloid complex; Ao, anterior olfactory nuclei; CA1-3, subfields CA1-3 of Ammon's horn; cc, corpus callosum; Cg, cingulate cortex, cp, choroid plexus in lateral ventricle; DG, dentate gyrus; Ent, entorhinal cortex; FR, frontal cerebral cortex; Hb, habenular nucleus; HDB, horizontal diagonal band; Hy, hypothalamus; LV, lateral ventricle; MS, medial septal nucleus; Th, thalamus; VDB, vertical diagonal band.

MUG I signals were detected as very small spots in areas of the hippocampus and cerebral cortex under low power magnification (data not shown) and in neuronal cell bodies and neurites in the hippocampus at high magnification (Fig. 7, A, B, E, and F). The intensity and frequency of the signals were also high in the cerebral cortex, thalamus, and hypothalamus as well as in the hippocampus (data not shown). In addition, MUG I also localized in the epithelium of the choroid plexus and the penetrating blood vessels (Fig. 7, C and D).


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Fig. 7.   Immunofluorescence micrographs obtained with anti-MUG I antibody in mouse adult hippocampus. A-F, anti-MUG I polyclonal antibody; G-I, preimmune rabbit serum. The CA3 subfield (A, B, and H), the choroid plexus (C, D, and G), the dentate gyrus (E), and the CA1 subfield (F and I) are illustrated. Bar, 20 µm. fi, fimbria; G, granule cell of the dentate gyrus (small arrow); P, pyramidal cell layer; s-o, stratum oriens. Note that signals immunoreactive with anti-MUG I antibody were observed in the cell body of neurons (arrowhead) and neurites (arrow). Images in G-I were overexposed relative to A-F.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we obtained the following evidence that SPI3 and MUG I are NP-specific inhibitors in adult brain: (i) purification of SDS-stable complexes containing endogenous inhibitors within brain extracts; (ii) detection of inhibitory activities for the proteolytic effect of r-NP; and (iii) co-localization of NP with the inhibitors in the hippocampus. Previous study has shown that NP modulated early phase long term potentiation dependent on the proteolytic activity (7) and that extracellular targets of NP include components of the ECM such as fibronectin and L1, but not laminin (15).2 There has, however, been no evidence of how the NP activity was regulated. The present identification of two inhibitors supports the hypothesis that SPI3 and MUG I regulate the proteolytic activity of NP for rearrangement of ECM proteins and, as a result, are implicated in synaptic modification.

Interaction of SPI3 with NP-- Previous reports have shown that SPI3 is the mouse homologue of proteinase inhibitor 6 (PI-6) (52), which shows inhibitory activity against trypsin, thrombin, and plasmin in vitro (42) and against cathepsin G (Ka = 6.8 × 106 M-1 s-1) in monocytes and granulocytes in vivo (51). The present study revealed that SPI3 was a potent inhibitor specific for NP (Ka = 3.4 × 106 M-1 s-1) in the nervous system. In the brain, neuroserpin and protease nexin-1 are proposed to be potent inhibitors of tissue-type plasminogen activator (1.5 × 105 M-1 s-1 (29)) and thrombin (6.0 × 105 M-1 s-1 (28)), respectively. The Ka of SPI3-NP was greater than the constant for protease nexin-1-thrombin and neuroserpin-tissue plasminogen activator, indicating that inhibition of NP by SPI3 occurs rapidly and extensively. It was therefore suggested that marked interaction occurred between SPI3 and NP in neurons in vivo.

The SPI3 P1 residue that interacts with NP remains to be identified but is predicted to be Arg353. Previous study has shown that PI-6 and SPI3 both utilize the P1 Arg for interactions with serine proteases possessing trypsin-like activity (52, 53), and NP is a trypsin-like serine protease showing amidolytic activity against the C terminus of Arg (15). Moreover, mutagenetic analysis of NP showed that SPI3 interacted specifically with the NP active site pocket. We therefore predict that SPI3 used P1 Arg for the interaction with NP.

Next, we discuss potential roles of SPI3 in the regulation of NP activity in the brain. The present study revealed co-localization of SPI3 and NP mRNAs in pyramidal neurons in the CA1-CA3 subfields of the hippocampus (Fig. 6, A and B), suggesting that SPI3 was also implicated in synaptic modification in the hippocampus. Previous studies have shown that SPI3 is an intracellular protease inhibitor (50), while NP functions extracellularly. In the present study, SPI3 was detected in a 65-kDa complex with r-NP in the 0.15 M NaCl-soluble, Triton-soluble, and cytoskeleton-rich fractions (Fig. 1A), suggesting that SPI3 both localized in the cytosol and associated with organelle membranes and the cytoskeleton. On the other hand, endogenous brain SPI3-NP complex was detected only in the Triton-soluble fraction (Fig. 1B), showing that the complex accumulated in this fraction. Additionally, r-proNP (Baculo) formed a 65-kDa complex with endogenous SPI3 like r-actNP (Fig. 1). Previous studies have suggested that NP is activated after it is secreted (6, 15). Therefore, the results from the present study indicate that SPI3 interacts with proNP before it is secreted or interacts with actNP after internalization. It remains to be determined directly when and where SPI3 regulates the proteolytic activity of NP.

It has recently been shown that PI-6 is a potent intracellular inhibitor specific for cathepsin G in monocytes and granulocytes (51). Cathepsin G is one of the azurophilic granule cytotoxins, which functions in the elimination of bacterial and fungal pathogens, and fibronectin is raised as one of the extracellular targets (54, 55). Additionally, recent findings show that cathepsin G activates a proapoptotic protease, caspase-7, suggesting that it has the potential to induce apoptosis intracellularly (56). Thus, the hypothesis has been proposed that PI-6 prevents cathepsin G-mediated damage in monocytes and granulocytes.

We propose that SPI3, the mouse homologue of PI-6, prevents NP-mediated damage in neurons. There is evidence that excessive stimulation of glutamate receptors induces a large increase in [Ca2+]i, leading to neuronal cell death (57-59). We recently found that glutamate stimulation induced secretion of NP in hippocampal primary cultures.3 Thus, overstimulation of the receptors might lead to secretion of excessive NP and failure to control NP by SPI3, resulting in neuronal cell death. Additionally, brain ischemia induced expression of SPI3 mRNA in pyramidal neurons of the hippocampus at 3 days after treatment (31), and oxidative stress in mouse brain induced a transient increase of NP mRNA in neurons of the hippocampus at 2 h after treatment (60). Under these pathological conditions, up-regulation of SPI3 expression might protect neurons from the excess of internalized actNP.

In situ hybridization histochemistry also showed that several areas of the brain expressed either SPI3 or NP mRNAs but not both (Fig. 6). The lateral septal nucleus was labeled with NP but not SPI3 riboprobe. Since we purified the 65-kDa complex from the hippocampal and cerebral cortical extracts but not from the basal forebrain, it remains unclear whether there are other specific inhibitors for NP in the lateral septal nucleus. SPI3 mRNA signals distributed in several areas not expressing NP mRNA, leading to the hypothesis that SPI3 could interact with other serine proteases in these areas.

Interaction of MUG I with NP-- The alpha 2M family are known as panextracellular protease inhibitors; their interaction with proteases results in activation of the thiol ester, a change in conformation, and exposure of a latent alpha 2M receptor (LRP) binding site (32). In the present study, we showed that active forms of r-NP (Baculo) and r-NP (N2a) formed SDS-stable complexes with MUG I in adult mouse brain (Figs. 1A and 2B). Alternatively, it was likely that the binding was different between r-NP (Baculo) and r-NP (N2a). The MUG I-NP (Baculo) complex appeared on reducing SDS-PAGE (180-kDa species), while the complex with r-NP (N2a) disappeared under reducing conditions (Fig. 2C). Differential glycosylation between r-NP (Baculo) and r-NP (N2a) might lead to a change in the conformation of the MUG I-NP complex, and the sugar chain of NP might regulate the interaction of NP with MUG I in vivo. Additionally, the point mutation within the active site pocket of NP eliminated its capacity to bind MUG I, suggesting that the MUG I-NP complex was specific for the active site pocket of NP. On the other hand, no endogenous MUG I-NP complex was detected in the absence of r-NP (Fig. 1A). Previous study shows that endogenous brain NP is detected as proNP in vivo and the amidolytic activity is acquired after activation with lysyl endopeptidase (6, 15). We suggest that most of the endogenous NP in the 0.15 M NaCl-soluble fraction was proNP (Fig. 1B) and therefore unable to bind MUG I.

Most members of the alpha 2M family are tetramers (e.g. human alpha 2M, rat alpha 2M and alpha 1-macroglobulin, and mouse alpha 2M); however, dimeric (e.g. human PZP) and monomeric (e.g. mouse murinoglobulin (MUG) I, II, and IV and rat alpha 1I3) forms are also identified (32, 61). Monomeric MUG I is the most abundant of the alpha 2M in mouse plasma (46). Previous reports have shown that, in the mouse, the plasma content of MUG I was 4-fold that of alpha 2M (46) and that MUG I may act as a substitute for alpha 2M in alpha 2M-deficient mice (62). Thus, in humans, alpha 2M alone may perform the physiological functions that both MUG I and alpha 2M perform in mice.

In the present study, we observed that MUG I protein localized in neurons of the hippocampus (Fig. 7) and that plasma MUG I significantly inhibited cleavage of fibronectin by r-NP (Fig. 5C). A report has revealed that activated human alpha 2M, but not native alpha 2M, can inhibit LTP development in the CA1 subfield (63). NP has the potential to induce LTP in the CA1 of the mouse hippocampus in a "bell-shaped" dose-responsive manner. The addition of 2.5 nM r-actNP provides the greatest enhancement, while higher concentrations are less effective, and 170 nM completely inhibits LTP (7). As functional mechanisms, a critical small concentration of endogenous actNP might be necessary for modulation of LTP accompanied by degradation of components of the ECM. Accumulation of actNP might result in binding and inactivation by MUG I, resulting in a reduction of LTP. It was proposed that the alpha 2M family regulated degradation of ECM by target proteases (32). Moreover, an excess concentration of extracellular actNP may activate MUG I and cause it to inhibit LTP further.

While it had been suggested that there were no alpha 2M or MUG I mRNAs (64) in adult mouse brain, the present study showed the presence of MUG I protein in adult mouse brain. Previous reports have, indeed, shown the presence of alpha 2M in adult human brain (65, 66), confirming our immunofluorescence results. The immunofluorescence signals localized to neuronal cell bodies and neurites in the CA1-CA3 subfields of the hippocampus (Fig. 7, A, B, E, and F). It is suggested that endogenous NP interacts with MUG I extracellularly in neurons of the CA1 and CA3 subfields. However, the intensity and frequency of the signals of MUG I in other brain areas were similar to those in the hippocampus (data not shown). MUG I might interact with other proteases in neurons of other areas and function as a pan-protease inhibitor in vivo. MUG I also localized in the epithelium of the choroid plexus and penetrating blood vessels (Fig. 7, C and D). The results of the current study might suggest that MUG I is transported to neurons through the blood-brain barrier. On the other hand, the localization of MUG I was consistent with the localization of LRP described previously (67, 68). Additionally, since r-actNP only formed an SDS-stable complex with MUG I, endogenous actNP might interact with MUG I after degrading components of the ECM, and then the binding of MUG I to LRP might lead to endocytosis and degradation of NP (32). Furthermore, recent studies have provided evidence that alpha 2M and LRP are genetically associated with the pathogenesis of Alzheimer's disease (69, 70). It should be determined whether failure to control extracellular NP by alpha 2M is implicated in the pathogenesis of Alzheimer's disease.

Finally, the present study identified natural NP inhibitors, intracellular SPI3 and extracellular MUG I, in adult brain, suggesting that SPI3 and MUG I regulate the physiological role mediated by NP in adult brain.

    ACKNOWLEDGEMENT

We thank Dr. Mahito Nakanishi for providing pED1 vector.

    FOOTNOTES

* This work was supported in part by grants from the Ministry of Education, Science, Culture, and Sports in Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Division of Structural Cell Biology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101 Japan. Tel.: 81-74372-5415; Fax: 81-74372-5419; E-mail: kato@bs.aist-nara.ac.jp.

Supported by a research fellowship for young scientists from the Japan Society for the Promotion of Science.

Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M010725200

2 A. Ninomiya, and S. Shiosaka, manuscript in preparation.

3 T. Oka, S. Shiosaka, and K. Kato, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: NP, neuropsin; alpha 2M, alpha 2-macroglobulin; ECM, extracellular matrix; LRP, alpha 2M receptor/low density lipoprotein receptor-related protein; LTP, long term potentiation; MUG, murinoglobulin; r-NP (Baculo), recombinant NP prepared with a baculovirus expression system; r-NP (N2a), recombinant NP prepared from neuro2a cells transfected with NP cDNA; r-actNP, activated form; r-proNP, precursor form; PAGE, polyacrylamide gel electrophoresis; PI-6, proteinase inhibitor 6; SPI3, serine proteinase inhibitor 3; DTT, dithiothreitol.

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ABSTRACT
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RESULTS
DISCUSSION
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