Molecular and biochemical characterization of a serine proteinase predominantly expressed in the medulla oblongata and cerebellar white matter of mouse brain.

A full-length cDNA clone of a serine proteinase, mouse brain serine proteinase (mBSP), was isolated from a mouse brain cDNA library. mBSP, which has been recently reported to be expressed in the hair follicles of nude mice, is most similar (88% identical) in sequence to rat myelencephalon-specific protease. The mBSP mRNA was steadily expressed in the brain of adult mice with a transient expression in the early fetal stage during development. The genomic structure of the mouse gene for mBSP was determined. The gene, which is mapped to chromosome 7B4-B5, is about 7.4 kilobases in size and contains 7 exons. Interestingly, the 5'-untranslated region of the mBSP gene was interrupted by two introns. In situ hybridization analyses revealed that mBSP is expressed in the white matter of the cerebellum, medulla oblongata, and capsula interna and capsula interna pars retrolenticularis of mouse brain. Further, mBSP was immunolocalized to the neuroglial cells in the white matter of the cerebellum. Recombinant mBSP was produced in the bacterial expression system and activated by lysyl endopeptidase digestion, and the activated enzyme was purified for characterization. The enzyme showed amidolytic activities preferentially cleaving Arg-X bonds when 4-methylcoumaryl-7-amide-containing peptide substrates were used. Typical serine proteinase inhibitors, such as diisopropyl fluorophosphates, phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, leupeptin, antipain, and benzamidine, strongly inhibited the enzyme activity. The recombinant mBSP effectively hydrolyzed fibronectin and gelatin, but not laminin, collagens I and IV, or elastin. These results suggest that mBSP plays an important role in association with the function of the adult mouse brain.

The ability of tumor cells to invade into the extracellular matrix has been linked to enzymes that are released by tumor cells or associated with the plasma membrane of tumor cells.
Proteinases have been implicated in tumor cell invasion and metastasis by numerous laboratories (1)(2)(3). One such enzyme is tissue-type plasminogen activator (tPA), 1 a serine proteinase widely distributed in tissues and organs (4). It has been established that tPA can be purified from the culture medium of human melanoma cell lines (Bowe) in two molecular forms of a single polypeptide chain (M r ϭ 64,000) and two polypeptide chains (M r ϭ 32,000 and 30,000), and that a proteinase(s) is involved in the proteolytic conversion of a single-chain form to the two-chain enzyme. Ichinose et al. (5) suggested that tissue kallikrein or a tissue kallikrein-like proteinase is responsible for the conversion. We recently demonstrated that, like human melanoma cells, mouse Lewis lung carcinoma and B16 melanoma cells secrete a common serine proteinase capable of rapidly converting a single-chain tPA to the two-chain enzyme (6).
In an attempt to search a mouse cancer cell cDNA library for a cDNA clone encoding the tPA-converting enzyme by means of polymerase chain reaction (PCR) using a series of degenerated oligonucleotide primers corresponding to the consensus sequences of the serine proteinase active sites, we happened to find a PCR product representing a unique proteinase that is expressed almost exclusively in the mouse brain. In the present study, we undertook molecular and biochemical analyses of this enzyme, designated mouse brain serine proteinase (mBSP), including the genomic structure and chromosomal localization, tissue distribution of the mBSP mRNA and protein product in the brain, and characterization of the recombinant enzyme. The results clearly show that mBSP is the homologue of rat myelencephalon-specific protease first reported by Scarisbrick et al. (7), and is the same enzyme very recently reported as brain and skin serine protease (BSSP) by Meier et al. (8). The current results also show that the recombinant mBSP is capable of degrading casein, fibronectin, and gelatin, suggesting a role of this enzyme in extracellular matrix protein degradation in the brain.
Animals-Mice (C57BL/6NCrj strain, female) were killed by cervical dislocation, and the brains were rapidly removed, frozen in liquid N 2 , and stored at Ϫ80°C until use.
RNA Isolation-Total RNAs were prepared from frozen tissues or confluent monolayers of the melanoma and carcinoma cells using the guanidine isothiocyanate-cesium chloride method (9). Poly(A) ϩ RNAs were selected by oligo(dT)-cellulose column chromatography.
Reverse Transcriptase-PCR-The first strand cDNA was synthesized from poly(A) ϩ RNAs of mouse B16 melanoma and Lewis lung carcinoma using a SuperScript Preamplification System (Life Technologies, Inc.) according to the manufacturer's protocol. Two degenerated oligonucleotide primers for PCR were synthesized based on the cDNA sequence for key regions of histidine and serine residues in some mouse serine proteinases (sense: 5Ј-GT(G/T)(C/G)T(G/T)(A/T)C(A/T)GCTGC(A/T/C)CA-CTG-3Ј corresponding to the amino acid sequence Val-Leu-Thr-Ala-Ala-His-Cys, and antisense: 5Ј-AG(A/C/G)GG(A/G/T)CCICC(A/T/C)GA(A/G)-TC(A/G)CC-3Ј corresponding to the amino acid sequence Gly-Asp-Ser-Gly-Gly-Pro-Leu). Conditions for PCR were 94°C for 3 min, followed by 40 cycles of 94°C for 1 min, 52°C for 2 min, 72°C for 3 min, and then a final extension at 72°C for 7 min. Fragments between 0.4 and 0.5 kb in size were recovered from PCR products by agarose gel electrophoresis. The fragments were subcloned into pBluescript (II) KSϩ (Stratagene, La Jolla, CA) cut with EcoRV. Transformation of the recombinant plasmids into Escherichia coli JM109 cells resulted in acquisition of 80 clones each from PCR products with both cells. Among these, one clone from poly(A) ϩ RNA of Lewis lung carcinoma represented a unique serine proteinase. This clone was 434 bp long, and corresponded to the nucleotide sequence numbers 382-815 of the full-length cDNA deposited in the DDBJ/EMBL/GenBank TM Data Bank under accession number AB015206.
Northern Blotting Analysis-Mouse multiple tissue Northern blots were purchased from CLONTECH (Palo Alto, CA). For preparation of the blot loading total RNAs from mouse brains of various postnatal developmental stages, 30 g of the total RNA was electrophoresed on a formaldehyde/1.2% agarose gel and transferred to a Nytran-Plus membrane (Schleicher & Schuell, Dassel, Germany). The blots were hybridized with a 32 P-labeled probe at 42°C in a buffer containing 50% formamide (Roche Molecular Biochemicals, Mannheim, Germany), 5ϫ SSPE, 1% SDS, 5ϫ Denhardt's solution, and 100 g/ml denatured salmon sperm DNA, and washed with increasing stringency, with a final wash of 0.1ϫ SSC, 0.1% SDS at 50°C. The probe used was the 434-bp cDNA fragment described above.
Cloning of mBSP cDNA-A mouse brain cDNA library was constructed in gt10 with 5 g of mouse brain poly(A) ϩ RNA and packaged using Gigapack III packaging extract (Stratagene). Approximately 7ϫ10 5 plaques from the library were transferred to nylon membranes (Schleicher & Schuell) and hybridized at 65°C in a buffer containing 5ϫ SSPE, 0.5% SDS, 5ϫ Denhardt's solution (Wako, Osaka, Japan), and 100 g/ml denatured salmon sperm DNA with 1ϫ10 6 cpm/ml of the 32 P-labeled, 434-bp cDNA fragment. Filters were washed with increasing stringency, with a final wash of 0.1ϫ SSC, 0.1% SDS at 50°C. One positive clone was obtained. The phage DNA was subcloned into pBluescript (II) KSϩ for sequencing. The nucleotide sequence was determined using ABI automatic sequencer models 373 and 377 (Perkin Elmer-Applied Biosystems, Foster City, CA).
Cloning of the mBSP Gene-About 1ϫ10 6 clones of the mouse genomic library (C57Black/6, liver, female, 1 year, Stratagene) were screened with the 32 P-labeled mBSP cDNA. Two positive plaques (411 and 91) were isolated and digested with several restriction enzymes, and examination by Southern blotting with the 32 P-labeled mBSP cDNA revealed that 411 included additional exons. We then determined the restriction enzyme maps and sequenced the full length of 411 using an ABI automatic sequencer model 377.
Primer Extension-1 g of mouse brain poly(A) ϩ RNA or E. coli transfer RNA was hybridized at 65°C for 90 min in a total of 20 l of 10 mM Tris-HCl (pH 8.3), 1 mM EDTA, and 0.25 M KCl with 5 pmol of the oligonucleotide probe, 5Ј-CTGTAAGTCCCTCTGTGGGT-3Ј (1778 -1797, genomic sequence), which had been endo-labeled with T4 polynucleotide kinase (Takara, Tokyo, Japan) and [␥-32 P]ATP (Amersham Pharmacia Biotech, Tokyo, Japan) at 37°C for 30 min. The reactions were then allowed to cool for 90 min at room temperature. After adding 46 l of 65 mM Tris-HCl (pH 8.3), 4.3 mM MgCl 2 , 15 mM dithiothreitol, and 0.72 mM dNTP mixture, 200 units of Superscript II reverse transcriptase (Life Technologies, Inc.) was introduced into the reaction mixture, which was incubated at 42°C for 60 min. The reactions were phenol-extracted, ethanol-precipitated, and electrophoresed in a denaturing 6% polyacrylamide sequencing gel along with a sequence ladder generated with the unlabeled primers using [␣-32 P]dCTP (Amersham Pharmacia Biotech) and Sequenase version 2.0 (United States Biochemical Corp.). The dried gel was visualized by autoradiography.
Genomic Southern Blotting Analysis-Mouse genomic DNA was extracted as described previously (10). 5 g of the genomic DNA was completely digested with EcoRI, HindIII, BamHI, and XhoI. The DNA was fractionated on a 0.7% agarose gel and alkaline-transferred to a Nytran membrane (Schleicher & Schuell). The blot was hybridized at 42°C for 16 h in 6ϫ SSPE, 50% formamide, 5ϫ Denhardt's solution, 1% SDS, and 100 g/ml denatured herring sperm DNA with the 32 P-labeled full-length mBSP cDNA including exons 2-7. The membrane was washed at 50°C in 0.1ϫ SSC, 0.1% SDS and exposed to Kodak Biomax film (Eastman Kodak Co.).
Chromosome Localization of the mBSP Gene-Metaphase preparations were obtained from concanavalin-stimulated splenocytes of normal male mice after bromodeoxyuridine incorporation. The mixture of two mBSP genomic DNA (91 and 411) was labeled with biotin-16-dUTP (Roche Molecular Biochemicals) by nick-translation. After hybridization, slides were washed, blocked, and incubated with goat antibodies against biotin (Vectashield Vector Laboratory, Burlingame, CA). Slides were incubated with fluorescein isothiocyanate-conjugated rabbit antibodies against goat IgG (American Qualex, La Mirada, CA) and then with Alexa 488-conjugated rabbit antibodies against fluorescein (Molecular Probes, Eugene, OR).
In Situ Detection of the mBSP mRNA-Antisense and sense RNA probes were prepared by in vitro transcription of a reverse transcriptase-PCR-amplified fragment of mBSP cDNA with T3 or T7 RNA polymerase using a DIG RNA labeling kit (Roche Molecular Biochemicals). Mouse brain sections (10 m) were cut on cryostat and thawmounted onto slides coated with silan. Sections on slides were fixed in 4% paraformaldehyde (Wako) in phosphate-buffered saline (PBS) for 15 min. After washing with PBS, the sections were treated with 20 g/ml proteinase K (Wako) in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA for 10 min at 37°C, postfixed in the same fixative, permeabilized in 0.2 M HCl for 10 min, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (Merck), and dehydrated through an ascending alcohol series. The DIG-labeled RNA probe (antisense or sense) in hybridization buffer containing 50% formamide, 0.5 M NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10% dextran sulfate (Wako), 1ϫ Denhardt's solution, 0.25% SDS, and 0.2 mg/ml tRNA (Roche Molecular Biochemicals) were placed on the sections, and then incubated at 50°C for 18 h. The sections were washed at 50°C in 50% formamide, 2ϫ SSC for 30 min. Sections were then treated with 40 g/ml RNase A in RNase buffer (0.5 M NaCl, 10 mM Tris-HCl (pH 7.6), 1 mM EDTA) for 30 min at 37°C. Subsequently, sections were washed in RNase buffer for 10 min at 37°C, rinsed in 2ϫ SSC for 20 min at 50°C, and then in 0.2ϫ SSC for 20 min at 50°C twice.
Immunohistochemical Detection of mBSP-Whole brains dissected from mouse (8 weeks old) were fixed in 4% paraformaldehyde/PBS (pH 7.0). After dehydration in serial concentrations of ethanol and xylene, they were embedded in paraffin and sectioned at a 10 m thickness. The sections were mounted on a coverslip, and dried overnight at 45°C. Following deparaffinization and hydration, the sections were immersed in PBS-BT buffer (0.1 g of bovine serum albumin, 50 l of Tween 20, 0.1 g of NaN 3 in 100 ml of PBS) and incubated in normal goat serum (Sigma) for 30 min at room temperature. Anti-mBSP antiserum/ PBS-BT (1:100 dilution) or rabbit polyclonal antibody against glial fibrillary acidic protein (GFAP; Sanbio, Netherlands)/PBS-BT (1:300 dilution) was applied on the coverslip for 2 h at room temperature. After washing three times with PBS-BT, the specimens were treated with fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin (Cappel Products, West Chester, PA)/PBS-BT (1:100 dilution) containing propidium iodide (Sigma) for 1 h at room temperature. The coverslip was mounted on a slide with 70% glycerin containing 5% n-propyl gallate. In control studies for mBSP-specific immunofluorescence, the specimens were incubated with normal rabbit serum. The stained sections were examined with a confocal laser scanning microscope (Fluoview, Olympus, Tokyo, Japan).
Preparation of Antiserum-The antigen used was the recombinant mBSP eluted from an Ni 2ϩ column as described in the following section. Specific antiserum was raised by injecting female rabbits (2 months old) with 200 g each of the above protein emulsified with Freund's complete adjuvant. Boosting was started 2 weeks later by injecting each at 2-week intervals with 200 g of the antigen emulsified with Freund's incomplete adjuvant. The antisera were obtained from the blood collected after three booster injections.
Preparation of Recombinant mBSP-The expression vector for mBSP was constructed by inserting its cDNA (amino acids 14 -246) including the complete pro-enzyme region of the mBSP gene into EcoRI site of pET30a (Novagen, Madison, WI). PCR was performed with oligonucleotide primers pro-mBSP (5Ј-GGAATTCGCCTGGTCGGAGGAACAGGA-3Ј) and mBSP-AS (5Ј-GGAATTCTCACAGCCACTTGTTTCTGA-3Ј) to create EcoRI sites at the 5Ј and 3Ј ends. The reaction product was sequentially digested with EcoRI, gel purified, and ligated in-frame in the EcoRI site of expression vector pET30a. The orientation and sequence of the BSP cDNA in the pET plasmid were confirmed by DNA sequencing. The ligated vector was transformed to E. coli strain BL21 (DE3) pLysS (Novagen), and the cells were grown at 37°C in 500 ml of 2% LB medium containing 30 g/ml kanamycin and 34 g/ml chloramphenicol until the optical density at 600 nm reached 0.5. Isopropyl-1thio-␤-D-galactoside was added to a final concentration of 1 mM, followed by incubation at 37°C for an additional 3 h. The cells were harvested by centrifuging at 6,000 ϫ g for 10 min, and treated by freeze-thawing to lyse the cells. After washing with 0.5% Triton X-100 twice, the sample was solubilized by dissolving and incubation in 50 ml of 50 mM Tris-HCl (pH 7.6) containing 6 M urea and 0.5 M NaCl for 12 h at room temperature. Solubilized materials were directly applied to an Ni 2ϩ -chelate column (5 ml volume, Novagen) previously equilibrated with 50 mM Tris-HCl (pH 7.6) containing 0.5 M NaCl and 6 M urea. The retained materials were eluted with 50 mM histidine in the same buffer containing 0.5 M NaCl and 6 M urea. The eluted protein (30 ml) was dialyzed against 2 liters of 20 mM Tris-HCl (pH 8.5) twice. The sample was then incubated in 20 mM Tris-HCl (pH 8.5) with immobilized lysyl endopeptidase on Sepharose 4B at 37°C for 6 h. The immobilized protease was removed by filtration, and the resulting filtrate was directly applied to a column of soybean trypsin inhibitor (SBTI)-Sepharose 4B (1 ml bed volume) previously equilibrated with 50 mM Tris-HCl (pH 8.0) containing 0.2 M NaCl. The retained materials were eluted with 0.1 M glycine-HCl buffer (pH 3). Fractions of 1 ml were collected in a tube that had contained 0.2 ml each of 0.1 M Tris-HCl buffer (pH 9). NH 2 -terminal amino acid sequence analysis of the activated mBSP was performed using an ABI sequenator model 477A.
Preparation of Affinity Gel-Immobilization of lysly endopeptidase (Wako) and SBTI on cyanogen bromide-activated Sepharose 4B gel (Amersham Pharmacia Biotech) was conducted according to the manufacturer's protocol.
Enzyme Activity Assay-mBSP activity was determined with MCA substrate according to the method of Barrett (11) with slight modification. The assay was carried out at 37°C for 30 min in a 0.5-ml reaction mixture comprising 0.1 M Tris-HCl buffer (pH 8.0) and 0.1 mM substrate. The reaction was terminated by the addition of 2.5 ml of 0.1 M sodium acetate buffer (pH 4.3) containing 0.1 M monochloroacetic acid. The amount of 7-amino-4-methylcoumarin released was spectrofluorophotometrically measured (6).
Zymography-Casein and gelatin zymography were performed according to the methods previously described (12). Briefly, mBSP was electrophoresed on 12% SDS-PAGE gels containing 0.1% casein (Wako) or 1 mg/ml type A porcine skin gelatin (Sigma) under nonreducing conditions. After electrophoresis, gels were washed twice in 2.5% Triton X-100 for 30 min, and then incubated with shaking in 0.1 M glycine-NaOH (pH 8.3) for 18 h at 37°C. The gels were stained with 0.25% Coomassie Brilliant Blue to visualize zones of lysis.
Degradation of Extracellular Matrix Components by mBSP-4 g of human plasma fibronectin (Chemicon, Temecula, CA), 4 g of mouse laminin (Biomedical Technologies Inc., Stoughton, MA), 20 g of acidsoluble type I collagen from calf skin (Sigma), and 20 g of acid-soluble type IV collagen from human placenta (Sigma) were each incubated at 37°C for 18 h in 50 mM Tris-HCl buffer (pH 8.0), with mBSP (500 ng) in a final volume of 20 l. Reactions were stopped with SDS sample buffer, and the reaction mixtures were boiled and subjected to SDS-PAGE using a 6% gel. After electrophoresis, gels were stained with 0.25% Coomassie Brilliant Blue.
The activity of mBSP on elastin was assessed using elastin-orcein (Elastin Products Co., Inc., Owensville, MO) as a substrate according to the method of Apple (13).
Western Blotting Analysis-Samples were subjected to SDS-PAGE (14) under reducing and nonreducing conditions, and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) by the method of Towbin et al. (15). The blotted membrane was incubated with rabbit anti-mBSP antiserum at 1:3000 dilution and subsequently with goat anti-rabbit IgG antibody (Amersham Pharmacia Biotech). Immunoreactive signals were detected using an ECL Western blot detection kit (Amersham Pharmacia Biotech) according to the protocol provided by the manufacturer.

Molecular
Cloning of mBSP-mRNAs prepared from mouse Lewis lung carcinoma cells and B16 melanoma cells were used for reverse transcriptase-PCR with two degenerated oligonucleotide primers as described under "Experimental Procedures." Among the 160 PCR products analyzed, one with a length of 434 bp was found to have a nucleotide sequence highly homologous to that of the serine protease, rat myelencephalon-specific protease, which has been reported by Scarisbrick et al. (7). This 434-bp cDNA fragment was then used as a probe for Northern blot analysis conducted with mRNAs isolated from several mouse tissues. As shown in Fig. 1A, an intense band of 1.3 kb was detected only in the brain. Based on this observation, we screened a mouse brain cDNA library using the same cDNA fragment as a probe, and isolated a full-length cDNA clone (the nucleotide sequence has been deposited in the DDBJ/EMBL/GenBank TM Data Bank under accession number AB015206). This clone was 1,115 bp long, including the 5Ј-noncoding region (213 bp), the coding nucleotide sequence (738 bp), and the 3Ј-noncoding region (164 bp). There are 5 ATG codons between nucleotide positions 193 and 222. Since the codon at positions 214 -216 best meets the criteria for the initiation site of the translation (16), we tentatively assume that translation starts at this ATG codon. As a result, the open reading frame codes for a protein of 246 amino acids with a molecular weight of 26,664.
During the course of this study, Meier et al. (8) reported a cDNA clone encoding a novel serine protease designated BSSP. The authors demonstrated a predominant expression of this gene in the hair follicles of nude mice. mBSP is undoubtedly the same molecule as BSSP, and is highly homologous to rat myelencephalon-specific protease (88% identical in amino acids, Ref. 7). mBSP also exhibited 67% identity to human zyme (17), neurosin (18), or protease M (19); 45% identity to mouse neuropsin (20); and 41% identity to mouse preprotrypsin (21).
Expression of mBSP-In adult mice, the mBSP mRNA was exclusively detected in the brain among the tissues examined (Fig. 1A), confirming the result of Meier et al. (8). To determine the changes of mBSP gene expression during development, a time-course experiment was conducted by Northern blot analysis using RNAs obtained from various stages of developing mice. In the prenatal periods, the mRNA was transiently detected with poly(A) ϩ RNA of the 7-day fetus, but virtually disappeared thereafter (Fig. 1B). Expression of the mRNA was resumed with the mouse brain of postnatal day 28 (Fig. 1C).
The mBSP Gene Structure and Its Transcription Initiation Site-The screening described under "Experimental Procedures" resulted in isolation of two genomic clones (91 and 411) that hybridized to the mBSP cDNA. The complete nucleotide sequence of the mBSP gene, including introns, was determined (the sequence data are available from the DDBJ/ EMBL/GenBank TM Data Bank under accession number AB032402). Sequence comparison with the cDNA revealed that the mBSP gene encompassed a 7.4-kb region and consisted of 7 exons (Fig. 2). The first two introns (introns 1 and 2) inter-rupted the 5Ј-untranslated region and four introns (introns 3, 4, 5, and 6) interrupted the coding sequence. The coding regions of mBSP are contained in exons 3-7. As shown in Table I, all intron-exon splice junctions follow the GT-AG rule of Shapiro et al. (22). The active site residues, His 62 , Asp 106 , and Ser 197 (numbered from the putative translation initiation Met), of the catalytic triad are encoded by exons 4, 5, and 7, respectively.
The nucleotide sequence of the 5Ј-flanking region of the mBSP gene is shown in Fig. 3. To determine the transcription initiation site, we performed a primer extension experiment. The labeled antisense primer corresponding to the region from nucleotide residue 1778 to residue 1797 was hybridized to mouse brain RNA and subjected to reverse transcription. This analysis demonstrated a single band having the size of 174 nucleotides (data not shown), indicating that transcription initiates at the adenine residue 1624. A CAAT box, but not a TATA box, was detected in the vicinity of the transcription initiation site. A computer search for consensus binding sites of transcription factors indicated the presence of motifs for Sp1, AP1, and C/EBP.
Chromosome Localization of the mBSP Gene-We performed Southern blot hybridization using the cDNA encompassing exons 2-7 as a probe. When mouse genomic DNA was digested  We next attempted to determine the chromosomal localization of the mBSP gene by in situ hybridization with the mBSP clone on R-banded metaphase chromosomes that had been prepared from concanavalin-stimulated normal male mouse splenocytes. As shown in Fig. 4, fluorescence signals were detected on chromosome 7B4-B5 and this result was reproducible. These results indicate that mBSP is encoded by a single, unique gene that resides on chromosome 7B4-B5.
Histological Studies-Sections of adult mouse brains were analyzed for in situ detection of the mBSP mRNA using DIGlabeled RNA probes. The antisense probe detected specific signals in the white matter of the cerebellum (Fig. 5, C and D). The molecular layer, Purkinje cell layer, and granular layer did not show any positive signals. Clear signals were also observed in the medulla oblongata (Fig. 5, E and F), and capsula interna (CAI) and capsula interna pars retrolenticularis (CAIR) (Fig. 5,  G and H). No significant expression of mBSP mRNA was detected in any other part of the brain.
To obtain information on the identity of mBSP-producing cells, immunohistochemical analyses were conducted with the cerebellum. Two adjacent saggital sections were stained with specific antibody against mBSP or antibody against GFAP, a well known marker protein for astrocytes. As shown in Fig. 6A, the mBSP antibody produced strong fluorescent signals almost exclusively in the cerebellar white matter. This is in good agreement with the result obtained by in situ hybridization analysis as described above. mBSP is apparently distributed in areas surrounding the nuclei of mBSP-producing cells. As expected, GFAP-immunoreactive astrocytes were found only in the white matter of the cerebellum (Fig. 6B). The distribution of GFAP-immunoreactive astrocytes corresponds in part to that of the mBSP-immunoreactive cells, suggesting that astrocytes are at least one kind of cells producing mBSP in vivo. However, the current immunohistochemical data also indicate that other type(s) of neuroglial cells may produce mBSP in the mouse cerebellum.
Characterization of the Recombinant mBSP-Since the peptide bond Ser 13 -Ala 14 is presumed to be the putative cleavage site by a signal peptidase (23), the nucleotide sequence, which starts with the Ala 14 codon and ends with the stop codon, was subcloned into the EcoRI site of the E. coli expression vector pET30. Transformation of this construct into E. coli resulted in production of a 38-kDa protein (data not shown). Considering that the recombinant protein should contain 51 extra amino acid residues, which originate from the plasmid sequence, at the NH 2 terminus, in addition to 233 residues of its own pro-mBSP, the size was somewhat greater than expected. This fusion protein, which had been obtained by fractionating on an Ni 2ϩ -chelate column, was digested with an immobilized lysyl endopeptidase, and the activated enzyme was purified using a SBTI-Sepharose 4B column (Fig. 7). The direct Edman degradation of the purified sample gave a single NH 2 -terminal amino acid sequence of Val-Val-His-Gly-Gly-Pro-X-Leu-(the 7th residue was not clearly identified). However, two polypeptides with a close molecular weight were separated at around 22,000 in the SDS-PAGE (Fig. 7). These polypeptides were reactive with the rabbit anti-mBSP antibody as examined by Western blot analysis (data not shown). Since the polypeptides were both retained on and recovered from the affinity column of SBTI-Sepharose, they are presumed to be enzymatically active. Based on these results, we concluded that activation with lysyl endopeptidase occurred by cleaving at the peptide bond of Lys 21 -Val 22 . A plausible explanation for the presence of two active enzymic forms in the recombinant enzyme preparation is that part of the activated mBSP might have undergone an additional hydrolysis by lysyl endopeptidase at a peptide bond near the COOH terminus.
When the purified, active mBSP was kept at 4°C or frozen at Ϫ20°C in 50 mM Tris-HCl buffer (pH 8.5) for 2 weeks, the enzyme activity was reduced by 67% and 45%, respectively. Electrophoretic analysis of the samples stored as above revealed that this loss of enzyme activity was accompanied by degradation of the polypeptide due to autolysis.
Enzyme activity of the recombinant mBSP was measured at various pH levels using Boc-Pro-Phe-Arg-MCA as substrate. The activity was detected at pH range 6 -10 in a typical bell shape, and the optimum pH was 8.5.
Substrate specificity of mBSP was examined using various MCA-containing peptide substrates, and the results are shown in Table II. The recombinant pro-mBSP showed little or no enzyme activity toward any substrate, but was dramatically activated by lysyl endopeptidase treatment. Among substrates tested, the best was Boc-Val-Pro-Arg-MCA. Boc-Phe-Ser-Arg-MCA was the second best substrate, and Boc-Gln-Ala-Arg-MCA was also a good substrate. Benzoyl-Arg-MCA and Lyscontaining substrate Boc-Val-Leu-Lys-MCA were hydrolyzed very little by the enzyme. The chymotrypsin substrates, Suc-Ala-Ala-Pro-Phe-MCA and Suc-Leu-Leu-Val-Tyr-MCA, were resistant to the enzyme. Table III shows the effects of proteinase inhibitors on the enzyme activity. Diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, SBTI, aprotinin, leupeptin, antipain, and benzamidine strongly inhibited the activity. The results are consistent with the idea that mBSP is a serine proteinase.
Examination of Enzyme Action on Some Extracellular Matrix Proteins-To gain some insight into the biological role of mBSP, its action on protein substrates, including several extracellular matrix proteins, was examined. Zymographic analysis revealed that heat-denatured casein and gelatin were degraded by mBSP (Fig. 8A). Five extracellular matrix proteins were tested for the enzyme. Incubation of the enzyme with fibronectin apparently produced several degraded polypeptides (Fig. 8B), whereas laminin, collagen I, collagen IV, and elastin were resistant to the enzyme action (data not shown). Finally, we examined the effect of mBSP on human tPA. The recombinant enzyme was unable to convert single-chain tPA to its two-chain form at a significant rate. These results suggest that fibronectin and gelatin could be candidate protein substrates of mBSP in vivo.

DISCUSSION
Our attempt to search for the serine proteinase responsible for proteolytic conversion of single-chain tPA to two-chain tPA using mouse cancer cells eventually led to identification of mBSP. The mRNA of this proteinase was expressed exclusively in the mouse brain under normal physiological conditions. Very recently, however, Meier et al. (8) isolated from nude mouse skin a cDNA clone encoding BSSP and having the same amino acid sequence as mBSP. Further comparison of the mBSP protein sequence with those of other related proteins reported to date indicated that rat myelencephalon-specific protease (7) shows the highest homology (88% identity) to the current proteinase. Human zyme (17), which is also known as neurosin (18) and protease M (19), is in the next place in homology with 67% identity. This value is significantly low in comparison with that of rat myelencephalon-specific protease. We believe that mBSP is a mouse homologue of rat myelencephalon-specific protease. This idea is supported by following the similarities in the regiospecificity of gene expression of these two serine proteases. 1) Both genes are specifically expressed in the central nervous system. 2) Within the central nervous system, rat myelencephalon-specific protease is expressed at the highest level in the medulla oblongata and spinal cord. Similarly, the region exhibiting predominant expression of the mBSP gene in  the mouse brain is the medulla oblongata. 3) Like rat myelencephalon-specific protease, mBSP is expressed in the cortex of the cerebellum.
Although the mBSP mRNA is transiently detected with poly(A) ϩ RNA isolated from developing mouse embryos, the proteinase is a brain-specific protein. The expression of mBSP in the brain was found to initiate at 28 days after birth. This observation suggests the presence of factors and mechanisms that strictly control mBSP gene expression. To establish a basis for further investigation into the regulatory mechanism of the gene expression, the structure, including its 5Ј-flanking region, of the mBSP gene was determined in this study. The gene spans 7.4 kb in total, and contains 7 exons. The exon/intron organization in the coding region was basically the same as those of trypsin-family genes such as trypsin (AB017032), cathepsin G (24), and granzymes (25). Interestingly, the 5Ј-untranslated region of this gene was interrupted by two introns. Obviously this is a unique feature of the mBSP gene, inasmuch as no intron has been reported at this position for trypsin-type serine proteinase genes, with the exception of the neuropsin gene, which contains an intron in the 5Ј-untranslated region (26). Comparison of our genomic sequence (DDBJ/EMBL/Gen-Bank TM AB032402) against data base entries revealed that, like the mBSP gene, the human zyme (GenBank TM AF149289) exhibits a similar exon/intron organization, having two intervening sequences in its 5Ј-untranslated region. These findings may indicate that the mBSP gene is derived from an ancestor gene common to the trypsin family proteinases, and that the current gene, perhaps together with the human zyme gene, subsequently evolved in a manner different from that of trypsin (5 exons) or neuropsin (6 exons). In this context, we should note the further similarity between the mBSP and human zyme genes with respect to their chromosome localizations. In the present study, the mBSP gene was localized to mouse chromosome 7B4-B5, while Little et al. (17) mapped the human zyme gene to chromosome 19q13.3. Interestingly, this region of human chromosome 19 is known to share a region of synteny with mouse chromosome 7. Despite such common features between these two genes, we tentatively assume that they are not homologous genes because of the noticeable sequence differences described above. In order to validate this assumption, however, it will be necessary to identify the human homologue of mBSP.   FIG. 8. Activities of purified recombinant mBSP on protein substrates. A, for casein and gelatin zymography, 0.5 g of purified recombinant mBSP was loaded on 12% SDS-PAGE gels containing 0.1% casein or 1 mg/ml gelatin. Molecular masses (kDa) of standard proteins are indicated at left. B, human fibronectin (4 g) was separately incubated with (ϩ) or without (Ϫ) mBSP (0.5 g) for 18 h at 37°C, and was subjected to SDS-PAGE. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250. Molecular masses (kDa) of standard proteins are indicated at left. Meier et al. (8) have isolated a full-length cDNA from a dE18 mouse embryonic skin library exhibiting a different 5Ј-end distinct from that of our cDNA. The present data on the genomic structure clearly demonstrate that such a difference is due to an alternative splicing event occurring at the 5Ј-untranslated region of the mBSP gene. The 5Ј-end guanine of the isolated skin cDNA corresponded to the guanine (2655) of the gene (AB032402), which was found in intron-1 of the mBSP primary transcript in mouse brain and 60 nucleotides upstream of the intron-1/exon-2 boundary. The exact role of the different 5Ј-noncoding sequences obtained by alternative RNA processing has not yet been elucidated. However, it is well accepted that alternative RNA splicing of a single gene is a ubiquitous mechanism for the generation of multiple protein isoforms or tissue-specific gene regulation. Similar alternative splicing has been found in aromatase (27), prolactin receptor (28), and human estrogen sulfotransferase (29) genes. The availability of the mBSP gene sequence offers an opportunity to study in detail the regulation and tissue-specific expression of this gene.
The recombinant mBSP shows trypsin-like enzyme activity toward MCA-containing peptide substrates. The inhibitor profiles of mBSP are compatible with this idea, except that 1-chloro-3-tosylamido-7-amino-2-heptanone is not a potent inhibitor of the enzyme. At present, it is unknown why the inhibitor does not show a significant inhibition for mBSP. The enzyme may have a unique structure distinct from other trypsin-like proteinases in its active site. mBSP preferentially hydrolyzes substrates containing Arg in the P1 position. In a broad sense, the substrate specificity of mBSP apparently resembles that of mouse neuropsin (30), although only 45% identity is found in the amino acid sequences of these proteins. More importantly, mBSP was demonstrated to exhibit a hydrolyzing activity toward protein substrates. Fibronectin, gelatin, and casein were found to be degraded effectively by the enzyme. The ability of mBSP to degrade the former two extracellular matrix proteins (fibronectin and gelatin) is of particular interest from a biological point of view, since the enzyme is thought to function in the extracellular space. If mBSP is spatially localized with these extracellular matrix proteins in the same tissues, the proteinase could hydrolyze them in vivo. Existing evidence indicates that fibronectin is a major extracellular matrix protein expressed in the nervous system (31). Alternatively, mBSP may be involved in the neural injury leading to neuronal and glial cell death, phagocytosis, glial cell proliferation, and migration. Extensive degenerative changes associated with this process definitely require the degradation of a variety of proteins by many extracellular proteinases. mBSP could be a proteinase for this process. Indeed, the involvement of rat myelencephalon-specific protease, here demonstrated to be a mBSP homologue, in the process of spinal cord injury was suggested by Scarisbrick et al. (7). In this connection, it would be interesting to examine whether mBSP mRNA would be induced after brain injury. It should be noted, however, that mBSP plays a fundamental role in normal brain homeostasis, since the mBSP gene is steadily expressed in adult mice.
We initially isolated a PCR fragment of the mBSP clone from mRNAs of mouse cancer cells, such as Lewis lung carcinoma and B16 melanoma cells. Since these cancer cells commonly secrete a serine proteinase capable of converting human singlechain tPA to its two-chain enzyme (6), we were interested in whether or not mBSP is the enzyme in question. However, no such converting activity was demonstrated for the current enzyme, indicating that the putative tPA-converting enzyme is distinct from mBSP. Nevertheless, mBSP may play a role in the invasive and metastatic process of cancer cells, given that it effectively degrades fibronectin and gelatin.
In conclusion, the present report provides new information on mBSP that has not been described in the two previous related papers (7,8). This information includes the mouse gene structure, chromosome localization, and histological localization of the mRNA and protein product in the brain. In addition, this paper describes for the first time the enzymic function of mBSP using a recombinant protein, and demonstrates its action on the extracellular matrix proteins, fibronectin and gelatin. The present data should help elucidate the biological role of the enzyme not only in the central nervous system but also in the skin.