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Molecular and Biochemical Characterization of a Serine Proteinase Predominantly Expressed in the Medulla Oblongata and Cerebellar White Matter of Mouse Brain

2000; Elsevier BV; Volume: 275; Issue: 15 Linguagem: Inglês

10.1074/jbc.275.15.11050

1083-351X

Hitoshi Matsui, Atsushi P. Kimura, Naoko Yamashiki, Akihiko Moriyama, Motohiro Kaya, Ikuya Yoshida, Nobuo Takagi, Takayuki Takahashi,

Hedgehog Signaling Pathway Studies

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 situhybridization 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. 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 situhybridization 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. tissue-type plasminogen activator polymerase chain reaction mouse brain serine proteinase 4-methylcoumaryl-7-amide t-butyloxycarbonyl succinyl digoxigenin soybean trypsin inhibitor base pair(s) kilobase pair(s) phosphate-buffered saline polyacrylamide gel electrophoresis brain and skin serine protease glial fibrillary acidic protein 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.Ossowski L. Reich E. Cell. 1983; 35: 611-619Abstract Full Text PDF PubMed Scopus (569) Google Scholar, 2.$$$$$$ ref data missingGoogle Scholar, 3.Khokha R. Denhardt D.T. Invasion Metastasis. 1989; 9: 391-405PubMed Google Scholar). One such enzyme is tissue-type plasminogen activator (tPA),1 a serine proteinase widely distributed in tissues and organs (4.Rickles R.J. Strickland S. FEBS Lett. 1988; 229: 100-106Crossref PubMed Scopus (37) Google Scholar). 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 = 64,000) and two polypeptide chains (M r = 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.Ichinose A. Kisiel W. Fujikawa K. FEBS Lett. 1984; 175: 412-418Crossref PubMed Scopus (64) Google Scholar) 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.Matsui H. Ohnishi J. Takahashi T. Zool. Sci. 1998; 15: 499-505PubMed Google Scholar). 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.Scarisbrick I.A. Towner M.D. Isackson P.J. J. Neurosci. 1997; 17: 8156-8168Crossref PubMed Google Scholar), and is the same enzyme very recently reported as brain and skin serine protease (BSSP) by Meier et al. (8.Meier N. Dear T.N. Boehm T. Biochem. Biophys. Res. Commun. 1999; 258: 374-378Crossref PubMed Scopus (31) Google Scholar). 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. Mouse B16 melanoma (RCB 0557) and Lewis lung carcinoma (RCB 0558) were obtained from the RIKEN Cell Bank (Tsukuba, Japan). Both cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 0.3 mg/ml l-glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin (Life Technologies, Inc.). Mice (C57BL/6NCrj strain, female) were killed by cervical dislocation, and the brains were rapidly removed, frozen in liquid N2, and stored at −80 °C until use. Total RNAs were prepared from frozen tissues or confluent monolayers of the melanoma and carcinoma cells using the guanidine isothiocyanate-cesium chloride method (9.Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (17730) Google Scholar). Poly(A)+ RNAs were selected by oligo(dT)-cellulose column chromatography. The first strand cDNA was synthesized from poly(A)+ 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: 5′-GT(G/T)(C/G)T(G/T)(A/T)C(A/T)GCTGC(A/T/C)CACTG-3′ corresponding to the amino acid sequence Val-Leu-Thr-Ala-Ala-His-Cys, and antisense: 5′-AG(A/C/G)GG(A/G/T)CCICC(A/T/C)GA(A/G)TC(A/G)CC-3′ 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) KS+ (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)+ 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/GenBankTMData Bank under accession number AB015206. 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 μg 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 32P-labeled probe at 42 °C in a buffer containing 50% formamide (Roche Molecular Biochemicals, Mannheim, Germany), 5× SSPE, 1% SDS, 5× Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA, and washed with increasing stringency, with a final wash of 0.1× SSC, 0.1% SDS at 50 °C. The probe used was the 434-bp cDNA fragment described above. A mouse brain cDNA library was constructed in λgt10 with 5 μg of mouse brain poly(A)+RNA and packaged using Gigapack III packaging extract (Stratagene). Approximately 7×105 plaques from the library were transferred to nylon membranes (Schleicher & Schuell) and hybridized at 65 °C in a buffer containing 5× SSPE, 0.5% SDS, 5× Denhardt's solution (Wako, Osaka, Japan), and 100 μg/ml denatured salmon sperm DNA with 1×106 cpm/ml of the 32P-labeled, 434-bp cDNA fragment. Filters were washed with increasing stringency, with a final wash of 0.1× SSC, 0.1% SDS at 50 °C. One positive clone was obtained. The phage DNA was subcloned into pBluescript (II) KS+ for sequencing. The nucleotide sequence was determined using ABI automatic sequencer models 373 and 377 (Perkin Elmer-Applied Biosystems, Foster City, CA). About 1×106 clones of the mouse genomic library (C57Black/6, liver, female, 1 year, Stratagene) were screened with the 32P-labeled mBSP cDNA. Two positive plaques (λ411 and λ91) were isolated and digested with several restriction enzymes, and examination by Southern blotting with the 32P-labeled mBSP cDNA revealed that λ411 included additional exons. We then determined the restriction enzyme maps and sequenced the full length of λ411 using an ABI automatic sequencer model 377. 1 μg of mouse brain poly(A)+RNA or E. coli transfer RNA was hybridized at 65 °C for 90 min in a total of 20 μl of 10 mm Tris-HCl (pH 8.3), 1 mm EDTA, and 0.25 m KCl with 5 pmol of the oligonucleotide probe, 5′-CTGTAAGTCCCTCTGTGGGT-3′ (1778–1797, genomic sequence), which had been endo-labeled with T4 polynucleotide kinase (Takara, Tokyo, Japan) and [γ-32P]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 μl of 65 mm Tris-HCl (pH 8.3), 4.3 mmMgCl2, 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 [α-32P]dCTP (Amersham Pharmacia Biotech) and Sequenase version 2.0 (United States Biochemical Corp.). The dried gel was visualized by autoradiography. Mouse genomic DNA was extracted as described previously (10.Kimura A. Yoshida I. Takagi N. Takahashi T. J. Biol. Chem. 1999; 274: 24047-24053Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). 5 μg 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 6× SSPE, 50% formamide, 5× Denhardt's solution, 1% SDS, and 100 μg/ml denatured herring sperm DNA with the32P-labeled full-length mBSP cDNA including exons 2–7. The membrane was washed at 50 °C in 0.1× SSC, 0.1% SDS and exposed to Kodak Biomax film (Eastman Kodak Co.). Metaphase preparations were obtained from concanavalin-stimulated splenocytes of normal male mice after bromodeoxyuridine incorporation. The mixture of two mBSP genomic DNA (λ91 and λ411) 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). 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 μm) were cut on cryostat and thaw-mounted 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 μg/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 mmTris-HCl (pH 8.0), 1 mm EDTA, 10% dextran sulfate (Wako), 1× 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, 2× SSC for 30 min. Sections were then treated with 40 μg/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 2× SSC for 20 min at 50 °C, and then in 0.2× SSC for 20 min at 50 °C twice. The hybridized probes were detected using a DIG nucleic acid detection kit (Roche Molecular Biochemicals). Sections were treated with 1% blocking reagent in DIG buffer 1 (100 mm Tris-HCl (pH 7.5), 150 mm NaCl) for 60 min, and incubated in 500× diluted anti-DIG antibody in DIG buffer 1 for 30 min. Sections were washed with DIG buffer 1, equilibrated with DIG buffer 3 (100 mmTris-HCl (pH 9.5), 100 mm NaCl, 50 mmMgCl2), and then incubated with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in DIG buffer 3 for 12 h. 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 μm 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 μl of Tween 20, 0.1 g of NaN3 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). The antigen used was the recombinant mBSP eluted from an Ni2+ column as described in the following section. Specific antiserum was raised by injecting female rabbits (2 months old) with 200 μg 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 μg of the antigen emulsified with Freund's incomplete adjuvant. The antisera were obtained from the blood collected after three booster injections. 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 intoEcoRI site of pET30a (Novagen, Madison, WI). PCR was performed with oligonucleotide primers pro-mBSP (5′-GGAATTCGCCTGGTCGGAGGAACAGGA-3′) and mBSP-AS (5′-GGAATTCTCACAGCCACTTGTTTCTGA-3′) to create EcoRI sites at the 5′ and 3′ ends. The reaction product was sequentially digested withEcoRI, gel purified, and ligated in-frame in theEcoRI 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. colistrain BL21 (DE3) pLysS (Novagen), and the cells were grown at 37 °C in 500 ml of 2% LB medium containing 30 μg/ml kanamycin and 34 μg/ml chloramphenicol until the optical density at 600 nm reached 0.5. Isopropyl-1-thio-β-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 × 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 Ni2+-chelate column (5 ml volume, Novagen) previously equilibrated with 50 mm Tris-HCl (pH 7.6) containing 0.5m 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 mmTris-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 0.1m Tris-HCl buffer (pH 9). NH2-terminal amino acid sequence analysis of the activated mBSP was performed using an ABI sequenator model 477A. 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. mBSP activity was determined with MCA substrate according to the method of Barrett (11.Barrett A.J. Biochem. J. 1980; 187: 909-912Crossref PubMed Scopus (394) Google Scholar) 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.Matsui H. Ohnishi J. Takahashi T. Zool. Sci. 1998; 15: 499-505PubMed Google Scholar). Casein and gelatin zymography were performed according to the methods previously described (12.Heussen C. Dowdle E.B. Anal. Biochem. 1980; 102: 196-202Crossref PubMed Scopus (1861) Google Scholar). 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 0.1m 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. 4 μg of human plasma fibronectin (Chemicon, Temecula, CA), 4 μg of mouse laminin (Biomedical Technologies Inc., Stoughton, MA), 20 μg of acid-soluble type I collagen from calf skin (Sigma), and 20 μg 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 μl. 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.Apple W. Bergmeyer H.U. Methods of Enzymatic Analysis. 2. Academic Press, New York1974: 1041-1045Crossref Google Scholar). Samples were subjected to SDS-PAGE (14.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (211971) Google Scholar) under reducing and nonreducing conditions, and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) by the method of Towbin et al. (15.Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (46066) Google Scholar). 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. 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.Scarisbrick I.A. Towner M.D. Isackson P.J. J. Neurosci. 1997; 17: 8156-8168Crossref PubMed Google Scholar). 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.1 A, 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/GenBankTM Data Bank under accession number AB015206). This clone was 1,115 bp long, including the 5′-noncoding region (213 bp), the coding nucleotide sequence (738 bp), and the 3′-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.Kozak M. Cell. 1986; 44: 283-292Abstract Full Text PDF PubMed Scopus (3774) Google Scholar), 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.Meier N. Dear T.N. Boehm T. Biochem. Biophys. Res. Commun. 1999; 258: 374-378Crossref PubMed Scopus (31) Google Scholar) 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.Scarisbrick I.A. Towner M.D. Isackson P.J. J. Neurosci. 1997; 17: 8156-8168Crossref PubMed Google Scholar). mBSP also exhibited 67% identity to human zyme (17.Little S.P. Dixon E.P. Norris F. Buckley W. Becker G.W. Johnson M. Dobbins J.R. Wyrick T. Miller J.R. MacKellar W. Hepburn D. Corvalan J. McClure D. Liu X. Stephenson D. Clemens J. Johnstone E.M. J. Biol. Chem. 1997; 272: 25135-25142Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), neurosin (18.Yamashiro K. Tsuruoka N. Kodama S. Tsujimoto M. Yamamura Y. Tanaka T. Nakazato H. Yamaguchi N. Biochim. Biophys. Acta. 1997; 1350: 11-14Crossref PubMed Scopus (165) Google Scholar), or protease M (19.Anisowicz A. Sotiropoulou G. Stenman G. Mok S.C. Sager R. Mol. Med. 1996; 2: 624-636Crossref PubMed Google Scholar); 45% identity to mouse neuropsin (20.Chen Z.L. Yoshida S. Kato K. Momota Y. Suzuki J. Tanaka T. Ito J. Nishino H. Aimoto S. Kiyama H. Shiosaka S. J. Neurosci. 1995; 15: 5088-5097Crossref PubMed Google Scholar); and 41% identity to mouse preprotrypsin (21.Stevenson B.J. Hagenbuchle O. Wellauer P.K. Nucleic Acids Res. 1986; 14: 8307-8330Crossref PubMed Scopus (52) Google Scholar). In adult mice, the mBSP mRNA was exclusively detected in the brain among the tissues examined (Fig.1 A), confirming the result of Meier et al. (8.Meier N. Dear T.N. Boehm T. Biochem. Biophys. Res. Commun. 1999; 258: 374-378Crossref PubMed Scopus (31) Google Scholar). 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)+ RNA of the 7-day fetus, but virtually disappeared thereafter (Fig. 1 B). Expression of the mRNA was resumed with the mouse brain of postnatal day 28 (Fig. 1 C). The screening described under “Experimental Procedures” resulted in isolation of two genomic clones (λ91 and λ411) 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/GenBankTM 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) interrupted the 5′-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.Shapiro M.B. Senapathy P. Nucleic Acids Res. 1987; 15: 7155-7174Crossref PubMed Scopus (2040) Google Scholar). The active site residues, His62, Asp106, and Ser197 (numbered from the putative translation initiation Met), of the catalytic triad are encoded by exons 4, 5, and 7, respectively.Table IExon-intron organizationExon no.Exon sizeSequence at exon/intron junctionPhaseIntron size5′ splice donor3′ splice acceptor1174GAC TTA CAGgtaggtgagttcatgcagGTC GGA TCA9172156CAC CAG CAGgtgagtgaacctctcaagATG CCC ATG391361AAA TCA GgtgatttaactccctcagCC TGG TCG1681Lys Ser A1a Trp Ser4157AAA AAA CCgtgagtttctcttcccagG AAT CTG21466Lys Lys Phe Asn Leu5248GAA AAT GgtcagtgagacctcctagGT GAC TTC1464Glu Asn Gly Asp Phe6137TCC TGT CAGgtaagaccgtccacacagGGT GAT TCT02214Ser Cys GlnGly Asp Ser7296TAG TGA CACagagcacag Open table in a new tab The nucleotide sequence of the 5′-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. We performed Southern blot hybridization using the cDNA encompassing exons 2–7 as a probe. When mouse genomic DNA was digested with EcoRI and BamHI, two hybridization-positive bands (5.0 and 6.5 kb for EcoRI; 1.2 and 6.8 kb for BamHI) were detected. On the other hand, digestion with HindIII andXbaI produced three bands (1.8, 2.8, and 10.0 kb forHindIII; 2.0, 7.3, and 8.5 kb for XbaI) (data not shown). The patterns were consistent with the numbers of the respective restriction enzyme sites present in the g