Mouse Homologue of Skin-specific Retroviral-like Aspartic Protease Involved in Wrinkle Formation
2006; Elsevier BV; Volume: 281; Issue: 37 Linguagem: Inglês
10.1074/jbc.m603559200
ISSN1083-351X
AutoresTakeshi Matsui, Yoko Kinoshita-Ida, Fumie Hayashi-Kisumi, Masaki Hata, Kaho Matsubara, Megumi Chiba, Sayaka Katahira-Tayama, Kazumasa Morita, Yoshiki Miyachi, Shöichiro Tsukita,
Tópico(s)Ocular Surface and Contact Lens
ResumoRetroviral proteases are encoded in the retroviral genome and are responsible for maturation and assembly of infectious virus particles. A number of retroviral protease sequences with retroviral elements are integrated in every eukaryotic genome as endogenous retroviruses. Recently, retroviral-like aspartic proteases that were not embedded within endogenous retroviral elements were identified throughout the eukaryotic and prokaryotic genomes. However, the physiological role of this novel protease family, especially in mammals, is not known. During the high throughput in situ hybridization screening of mouse epidermis, as a granular layer-expressing clone, we identified a mouse homologue of SASPase (Skin ASpartic Protease), a recently identified retroviral-like aspartic protease. We detected and purified the endogenous 32-kDa (mSASP32) and 15-kDa (mSASP15) forms of mSASP from mouse stratum corneum extracts and determined their amino acid sequences. Next, we bacterially produced recombinant mSASP15 via autoprocessing of GST-mSASP32. Purified recombinant mSASP15 cleaved a quenched fluorogenic peptide substrate, designed from the autoprocessing site for mSASP32 maximally at pH 5.77, which is close to the pH of the epidermal surface. Finally, we generated mSASP-deficient mice that at 5 weeks of age showed fine wrinkles that ran parallel on the lateral trunk without apparent epidermal differentiation defects. These results indicate that the retroviral-like aspartic protease, SASPase, is involved in prevention of fine wrinkle formation via activation in a weakly acidic stratum corneum environment. This study provides the first evidence that retroviral-like aspartic protease is functionally important in mammalian tissue organization. Retroviral proteases are encoded in the retroviral genome and are responsible for maturation and assembly of infectious virus particles. A number of retroviral protease sequences with retroviral elements are integrated in every eukaryotic genome as endogenous retroviruses. Recently, retroviral-like aspartic proteases that were not embedded within endogenous retroviral elements were identified throughout the eukaryotic and prokaryotic genomes. However, the physiological role of this novel protease family, especially in mammals, is not known. During the high throughput in situ hybridization screening of mouse epidermis, as a granular layer-expressing clone, we identified a mouse homologue of SASPase (Skin ASpartic Protease), a recently identified retroviral-like aspartic protease. We detected and purified the endogenous 32-kDa (mSASP32) and 15-kDa (mSASP15) forms of mSASP from mouse stratum corneum extracts and determined their amino acid sequences. Next, we bacterially produced recombinant mSASP15 via autoprocessing of GST-mSASP32. Purified recombinant mSASP15 cleaved a quenched fluorogenic peptide substrate, designed from the autoprocessing site for mSASP32 maximally at pH 5.77, which is close to the pH of the epidermal surface. Finally, we generated mSASP-deficient mice that at 5 weeks of age showed fine wrinkles that ran parallel on the lateral trunk without apparent epidermal differentiation defects. These results indicate that the retroviral-like aspartic protease, SASPase, is involved in prevention of fine wrinkle formation via activation in a weakly acidic stratum corneum environment. This study provides the first evidence that retroviral-like aspartic protease is functionally important in mammalian tissue organization. Proteases play an important role in many physiological processes by regulating the activation, synthesis, and turnover of proteins (1Barret A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, San Diego1998Google Scholar). Proteases are classified into five distinct classes as follows: aspartic, metallo-, cysteine, serine, and threonine proteases. Of these, aspartic proteases, also designated as acidic proteases, are a widely distributed family of proteolytic enzymes known to exist in vertebrates, invertebrates, fungi, plants, and retroviruses (2Rao J.K.M. Erickson J.W. Wlodawer A. Biochemistry. 1991; 30: 4663-4671Crossref PubMed Scopus (92) Google Scholar, 3Tang J. Wong R.N.S. J. Cell Biochem. 1987; 33: 53-63Crossref PubMed Scopus (251) Google Scholar). Eukaryotic aspartic proteases are ∼330-residue monomeric enzymes that consist of two homologous domains. Each domain contains an active site centered on a catalytically essential aspartic residue. These enzymes are synthesized as zymogens that are subsequently proteolytically processed. On the other hand, retroviruses are known to encode aspartic proteases in the viral genome (4Ratner L. Haseltine W. Patarca R. Livak K.J. Starcich B. Josephs S.F. Doran E.R. Rafalski J.A. Whitehorn E.A. Baumeister K. Ivanoff L. Petteway Jr., S.R. Pearson M.L. Lautenberger J.A. Papas T.S. Ghrayeb J. Chang N.T. Gallo R.C. Wong-Staal F. Nature. 1985; 313: 277-284Crossref PubMed Scopus (1728) Google Scholar). This retroviral aspartic protease is expressed as part of a large polyprotein precursor, with the mature protease released via autoprocessing activity. The primary structure of the enzyme corresponds to a single domain that undergoes autoprocessing in viral particles to produce an active one that exists as a homodimer. Recent analysis of human, mouse, and rat genomes have revealed that 553, 628, and 626 genes, respectively, are encoded as proteases or protease homologues (5Puente X.S. Sánchez L.M. Overall C.M. López-Otín C. Nature Rev. Genet. 2003; 4: 544-558Crossref PubMed Scopus (745) Google Scholar, 6Puente X.S. López-Otín C. Genome Res. 2004; 14: 609-622Crossref PubMed Scopus (159) Google Scholar). In the human genome, 21 genes are eukaryotic aspartic proteases, and more than 150 sequences are related to aspartic proteases that are embedded in endogenous retroviral elements as human endogenous retroviruses (5Puente X.S. Sánchez L.M. Overall C.M. López-Otín C. Nature Rev. Genet. 2003; 4: 544-558Crossref PubMed Scopus (745) Google Scholar). Most of these human endogenous retroviruses are believed to have been acquired 10-100 million years ago (7Lower R. Lower J. Kurth R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5177-5184Crossref PubMed Scopus (626) Google Scholar). Previously, Krylov and Koonin (8Krylov D.M. Koonin E.V. Curr. Biol. 2001; 11: R584-R587Abstract Full Text Full Text PDF PubMed Google Scholar) described new subfamilies of predicted retroviral-like aspartic proteases that include several human paralogues. These genes encode enzymes with some similarity to retroviral aspartic proteases, although they are not embedded within endogenous retroviral elements. They included the DNA damage-inducible UbL-UbA proteins Ddi1p and Ddi2p, neuron-specific nuclear receptors NIX1, NRIP2, and NRIP3, and other Ddi1p-related aspartic proteases (5Puente X.S. Sánchez L.M. Overall C.M. López-Otín C. Nature Rev. Genet. 2003; 4: 544-558Crossref PubMed Scopus (745) Google Scholar, 6Puente X.S. López-Otín C. Genome Res. 2004; 14: 609-622Crossref PubMed Scopus (159) Google Scholar, 9Clarke D.J. Mondesert G. Segal M. Bertolaet B.L. Jensen S. Wolff M. Henze M. Reed S.I. Mol. Cell. Biol. 2001; 21: 1997-2007Crossref PubMed Scopus (76) Google Scholar, 10Gardner R.D. Burke D.J. Trends Cell Biol. 2000; 10: 154-158Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 11Shirayama M. Toth A. Galova M. Nasmyth K. Nature. 1999; 402: 203-207Crossref PubMed Scopus (294) Google Scholar, 12Greiner E.F. Kirfel J. Greschik H. Huang D. Becker P. Kapfhammer J.P. Schule R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7160-7165Crossref PubMed Scopus (43) Google Scholar). They were encoded not only in eukaryotes but also prokaryotes. Recently, Bernard et al. (13Bernard D. Méhul B. Thomas-Collignon A. Delattre C. Donovan M. Schmidt R. J. Investig. Dermatol. 2005; 125: 278-287Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) have separated total protein extracts of human reconstructed epidermis and identified a novel retroviral-like aspartic protease, SASPase (Skin ASpartic Protease). Human SASPase (hSASP) 3The abbreviations used are: hSASP, human SASPase; mSASP, mouse SASP; Nma, 2-(N-methylamino)benzoyl; Dnp, 2,4-dinitrophenyl; RT, reverse transcription; PBS, phosphate-buffered saline; EST, expressed sequence tag; GST, glutathione S-transferase; pAb, polyclonal antibody; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; nano-ESI MS/MS, nano-electrospray ionization tandem mass spectrometry; MS, mass spectrometry; HIV, human immunodeficiency virus; SC, stratum corneum; SCE, stratum corneum extracts; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; rmSASP, recombinant mSASP; ES, embryonic stem. was expressed in the granular layer of human epidermis, and immunoblotting of human epidermal extract revealed the expression of two forms of the enzyme, the 28- and 14-kDa forms. Similar to other retroviral proteases, recombinant SASPase undergoes auto-activation processing in vitro, and subsequently a 14-kDa protein is generated. The amino acid sequences of this recombinant 14-kDa product (hSASP14) were determined and shown to correspond with the protease domain. However, it was not clarified whether hSASP14 possessed protease activity, and its optimum pH was not determined. More importantly, the physiological role of SASPase is uncertain. We have previously performed high throughput in situ hybridization screening against sections of mouse foot sole epidermis using an equalized mouse back skin cDNA library (14Matsui T. Hayashi-Kisumi F. Kinoshita Y. Katahira S. Morita K. Miyachi Y. Ono Y. Imai T. Tanigawa Y. Komiya T. Tsukita S. Genomics. 2004; 84: 384-397Crossref PubMed Scopus (58) Google Scholar). Among them, we identified a mouse homologue of SASPase as a clone specifically expressed in the granular layer of epidermis. Here we report the purification of processed forms of endogenous mouse SASPase (mSASP) from mouse stratum corneum extracts (SCE) and the determination of their amino acid sequences. From these sequences, we succeeded in producing and purifying active recombinant protein in bacteria and performed biochemical analysis. Furthermore, to study the physiological role of SASPase, we generated SASPase-deficient mice. They unexpectedly formed fine skin wrinkles. Our data are the first evidence that genome-integrated retroviral-like aspartic protease is functionally important in mammalian tissue architecture. Materials—Oligonucleotide primers were purchased from Proligo Japan (Kyoto, Japan). N-terminal amino acid sequence analysis and mass spectrometric analysis were performed by Aproscience Co. Ltd. (Tokushima, Japan). Peptide (Nma-LFANSMG-K(Dnp)rrr-NH2) was synthesized and purified by high pressure liquid chromatography by Peptide Inc. (Osaka, Japan) and dissolved in Me2SO. FRETS-25-STD1 and FRETS-25-STD2 were also purchased from Peptide Inc. Generation of knock-out mice was performed by Kurabo, Inc. (Osaka, Japan). All animal studies have been approved by the Institutional Review Board of the KAN Research Institute, Inc. In Situ Hybridization—In situ hybridization was performed as described previously (14Matsui T. Hayashi-Kisumi F. Kinoshita Y. Katahira S. Morita K. Miyachi Y. Ono Y. Imai T. Tanigawa Y. Komiya T. Tsukita S. Genomics. 2004; 84: 384-397Crossref PubMed Scopus (58) Google Scholar, 15Komiya T. Tanigawa Y. Hirohashi S. Anal. Biochem. 1997; 254: 23-30Crossref PubMed Scopus (24) Google Scholar). cDNA Cloning and Recombinant Protein Expression—Mouse back skin total RNA was isolated from 8-week-old female BALB/c mice according to the method described by Chomczynski and Sacchi (16Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63184) Google Scholar). First strand cDNA was prepared by Superscript II reverse transcriptase (Invitrogen) from mouse back skin total RNA. The DNA fragment encoding open reading frame of the mouse SASPase 32-kDa form (mSASP32) was amplified from a mouse back skin cDNA library by PCR using 5′-EcoRI-mSASP32 primer (5′-ATATGAATTCGCCACCATGGCCACCAGCGGAGTCAG-3′) and 3′-NotI-mSASP32 primer (5′-AATTGCGGCCGCTTAGTGGGAGCCCTCCGGTG), respectively. These primers were designed using the sequence from GenBank™ accession number BC057938. After digestion with EcoRI and NotI, the cDNA was subcloned into EcoRI-NotI sites of pGEX4T-1 (GE Healthcare) to yield pGEX-mSASP32. Protease-dead mutant, pGEX-mSASP32(D210A), was generated by using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) from pGEX-mSASP32 by mutating the active aspartic acid residue (amino acid 210) to alanine. To prepare antibodies against mSASP, the cDNA fragment encoding residues 182-300 of mSASP32(D210A) (mSASP-(182-300)(D210A)) was amplified from pGEX-mSASP32(D210A) by PCR using 5′-SalI-mSASP-PR primer (5′-ATATGTCGACGCCACCGAAGAGATTTTGTTTGCCAACAGC-3′) and 3′-NotI-mSASP-PR primer (5′-AATTGCGGCCGCGGTGCGGTGTTCGAAGTCCAGCAC-3′). To produce GST fusion protein of mSASP-(182-300)(D210A), the PCR product was digested with SalI and NotI and then subcloned into SalI-NotI sites of pGEX4T-3 to yield pGEX-mSASP-(182-300)(D210A). To produce His-tagged mSASP-(182-300)(D210A), the NotI site was introduced between XhoI and PstI sites of pRSET-A (Invitrogen) to yield pRSET-A(NotI), and the SalI/NotI-digested PCR-product was subcloned into XhoI-NotI sites of pRSET-A(NotI) to yield pRSET-mSASP-(182-300)(D210A). Recombinant protein, GST-mSASP32, GST-mSASP32-(D210A), GST-mSASP-(182-300)(D210A), and His6-mSASP-(182-300)(D210A) were produced and purified according to the manufacturer's instructions. Purified GST-mSASP-(182-300) (D210A) was cleaved with thrombin to remove the GST to yield the mSASP-(182-300)(D210A) protein by the thrombin cleavage capture kit (Novagen, Madison, WI). Northern Blotting and Quantitative Real Time RT-PCR—Northern blotting and quantitative real time RT-PCR were performed as described previously (14Matsui T. Hayashi-Kisumi F. Kinoshita Y. Katahira S. Morita K. Miyachi Y. Ono Y. Imai T. Tanigawa Y. Komiya T. Tsukita S. Genomics. 2004; 84: 384-397Crossref PubMed Scopus (58) Google Scholar). The primer set was as follows: mouse mSASP (BC057938), forward primer (5′-AGAGGCTATTATTGGCACAGACGTC-3′) and reverse primer (5′-GGAGCAGGCGGAACTTCTTC-3′). Antibodies—The pAb specific for the C-terminal domain of mSASP32 was produced in rabbits against synthetic peptide (EFDLELIEEEEGSSAPEGSH) corresponding to the amino acids 320-339 of mSASP32 (mSASP-C pAb). The mSASP-C pAb was affinity-purified on this peptide covalently coupled to thiopropyl-Sepharose 6B (GE Healthcare). This pAb specifically recognized mSASP32 by immunoblotting. We raised two rabbit antisera specific for the protease domain of mSASP, mSASP-PR1 and mSASP-PR2 pAbs (where PR stands for protease domain). The mSASP-PR1 pAb was raised against the mSASP-(182-300)(D210A) protein and was affinity-purified on His6-mSASP-(182-300)(D210A) covalently coupled to a Hitrap NHS-activated HP 1-ml column (GE Healthcare). This pAb specifically recognized both mSASP32 and mSASP15 by immunoblotting. mSASP-PR2 pAb was raised against GST-mSASP32(D210A) and was affinity-purified on His6-mSASP-(182-300)(D210A) as described above. This pAb specifically recognized both mSASP32 and mSASP15 by immunoprecipitation. The flow-through fraction of antiserum during affinity purification of mSASP-PR2 pAb was used for immunoabsorption of mSASP32 (mSASP-N pAb antiserum). Mouse monoclonal antibody to keratin 1/10 (clone k8.60) was purchased from Sigma. Rabbit antibodies to keratin 14, keratin 1, involucrin, filaggrin, and loricrin were purchased from Covance (Denver, PA). A rabbit antibody against desmoglein 1 was purchased from Santa Cruz Biotechnology (Delaware, CA). Preparation of SCE from Nude Mice—Mice (8-week-old female BALB/c nude mice) were first anesthetized, and the surface of the skin was washed with 10 ml per mouse of a SCE buffer (50 mm sodium phosphate buffer, pH 7.0, 5 mm EDTA, 150 mm NaCl and 0.1% Tween 20 supplemented with protease inhibitor mixture (Nakalai Tesque, Tokyo, Japan)). Thereafter, the washed area was scraped with the edge of a microscope slide and continuously rinsed with SCE buffer. The buffer containing corneocytes was collected in a container placed below the mice. Corneocytes were removed by centrifugation at 3,000 × g for 30 min at 4 °C and then ultracentrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was passed over a Millex-GV (0.45 μm; Millipore, Billerica, MA) and then over a Millex-HV (0.22 μm; Millipore). The resulting SCE was ∼0.25 mg/ml. Immunoprecipitation of mSASP32/15 and N-terminal Amino Acid Sequence Analysis—First, 0.3 ml of protein G-Sepharose 4B fast flow (GE Healthcare) was incubated with 0.3 mg of mSASP-PR2 pAb for 1 h at 4 °C. After washing three times with SCE buffer, beads were incubated with 15 ml (3.75 mg of protein) of nude mouse SCE for 1 h at 4 °C. The beads were then extensively washed with PBS. The bound proteins were eluted by boiling the beads in an SDS sample buffer (62 mm Tris-Cl, pH 6.7, 3% SDS and 5% glycerol) for 5 min. The samples were separated by SDS-PAGE and stained by 2D Silver Stain II (DAIICHI Pure Chemicals, Tokyo, Japan) or Coomassie staining solution (Bio-Rad). The mSASP32 and mSASP15 bands were excised from the gel and subjected to N-terminal amino acid sequencing. Purification of mSASP15 from Mouse SCE and C-terminal Amino Acid Analysis—5 mg of mSASP-PR2 pAb was coupled to 0.8 ml of Hitrap NHS-activated HP column (GE Healthcare) according to the manufacturer's instruction and equilibrated with SCE buffer (mSASP-PR2 pAb column). 3 ml of mSASP-N pAb antiserum was incubated with 1 ml of protein G-Sepharose 4B fast flow for 4 h at 4 °C and equilibrated with SCE buffer. 30 ml of mouse SCE was incubated with these beads for 1 h at 4 °C to immunodeplete mSASP32. The absence of mSASP32 was confirmed by immunoblotting with mSASP-C pAb. After removal of mSASP32-bound beads, mSASP32-depleted SCE was then applied to the mSASP-PR2 pAb column and washed with 50 mm sodium phosphate, pH 7.0, containing 1 m NaCl, and bound mSASP15 was eluted with 0.1 m glycine, pH 2.0. Eluted mSASP15 was immediately neutralized with 1 m Tris-Cl, pH 9.5, and concentrated with an Ultrafree-0.5 centrifugal filter and tube (BioMax-5K NMWL; Millipore) and then dialyzed against 20 mm Tris-Cl, pH 7.5. Mass Spectrometry—Molecular mass of purified mSASP15 was determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra in a Voyager-DE STR (Applied Biosystems), operated in the linear mode. For determination of C-terminal amino acid sequence of mSASP15, purified mSASP15 was subjected to proteolysis of lysyl endopeptidase at 35 °C for 20 h. This digest was desalted on a C18 Ziptip (Millipore), eluted in 2,5-dihydroxybenzoic acid, and subjected to MALDI-TOF MS analysis. The rest of the lysyl endopeptidase digest was also desalted on C18 Ziptip and eluted in 50% acetonitrile containing 1% formic acid and subjected to nano-electrospray ionization mass spectrometry (nano-ESI MS) in a Q-TOF2 mass spectrometer (Waters Micromass, Manchester, UK). The selected precursor ion was further analyzed by nano-electrospray ionization tandem mass spectrometry (nano-ESI MS/MS). In collision-induced dissociation experiments (collision gas argon, collision energy 30-40 eV), product ions were analyzed by the orthogonal TOF analyzer. Immunofluorescence—Mouse side skin was fixed in 2% paraformaldehyde/PBS for 1 h at room temperature. Next, samples were incubated with 10% sucrose in PBS for 3 h at 4 °C and then 20% sucrose in PBS overnight at 4 °C. Finally, samples were mounted in Tissue-Tek O.C.T. compound (Sakura Fine-technical, Tokyo, Japan) and frozen on dry ice. Frozen samples were cut into 5-μm thick sections (10-μm sections for confocal imaging) on a cryostat and put on silan-coated glass slides and air-dried. These samples were soaked in Block-Ace blocking solution (Dainippon Pharmacy, Osaka, Japan) for 1 h at room temperature and subsequently incubated in primary antibodies for 1 h at room temperature. Sections were washed three times with PBS and incubated with secondary antibodies for 30 min at room temperature. Alexa 488-labeled goat anti-rabbit IgG antibody (Molecular Probes, Eugene, OR) and SYTOX Green (Molecular Probes) were used as secondary antibodies. Samples were washed three times with PBS and mounted in PBS containing 50% glycerol. Photographs were recorded using a photomicroscope with an Olympus IX70 through a cooled CCD camera (model ORCA-ER; Hamamatsu Photonics K.K., Hamamatsu, Japan) controlled by Aquacosmos software (Hamamatsu Photonics). Confocal imaging was performed using LSM510 confocal laser scanning microscope (version 2.3; Carl Zeiss Inc., Jena, Germany). Purification of Recombinant mSASP15 (rmSASP15)—All procedures were performed at 4 °C, and chromatography was carried out on anÄKTAexplorer 10S chromatography system (GE Healthcare). We produced rmSASP15 via autoprocessing of GST-mSASP32 expressed in Escherichia coli. 500 ml of E. coli culture expressing GST-mSASP32 was collected by centrifugation, and cell pellets were dissolved in 9 ml of buffer H (50 mm sodium acetate, pH 5.5, 1 mm EDTA, 0.15 m NaCl, 0.1% Triton X-100) containing protease inhibitor mixture (Nakalai Tesque, Japan). After sonication, samples were centrifuged at 100,000 × g for 1 h, and the supernatant was then frozen at -80 °C. 2 ml of supernatant was thawed and dialyzed against buffer A (50 mm sodium phosphate buffer, pH 6.6, 1 mm EDTA) by using NAP-10 (GE Healthcare). The sample was passed over a HiTrap SP HP, 1 ml (GE Healthcare), equilibrated with buffer A at a flow rate of 1 ml/min. After washing with 3 column volumes of buffer A, bound rmSASP15 was eluted with a 6-ml linear gradient of NaCl (0-0.25 m) in buffer A, and fractions of 0.4 ml each were collected. Fractions containing rmSASP15 (fractions 1-16) were pooled and dialyzed against buffer B (50 mm sodium phosphate buffer, pH 7.0, 1 mm EDTA) and concentrated with Ultrafree-0.5 centrifugal filter and tube (BioMax-5K NMWL; Millipore). Samples were passed over a Hitrap Q HP, 1 ml equilibrated with buffer B, and then washed with 3 column volumes of buffer B. Bound rmSASP15 was eluted with a 6-ml linear gradient of NaCl (0-0.3 m) in buffer B. Fractions containing rmSASP15 (fractions 16-20) were pooled and dialyzed against buffer C (50 mm sodium phosphate buffer, pH 6.0, 1 mm EDTA) and concentrated as described above. Samples were then passed over a Hitrap SP HP, 1 ml equilibrated with buffer C, and washed with 3 column volumes of buffer C. Bound rmSASP15 was eluted with a 6-ml linear gradient of NaCl (0-0.25 m) in buffer C. Purified rmSASP15 (fractions 13-22) was pooled and concentrated as described above. For gel filtration, purified rmSASP15 was fractionated (0.5 ml/min) by Superdex 75 HR 10/30 gel filtration chromatography (GE Healthcare) and equilibrated in buffer C, and fractions of 0.5 ml each were collected. Peptide Cleavage Assay—For determination of specific activity, purified rmSASP15 (130pmol) was incubated with 0.1 mm peptide substrate (Nma-LFANSMG-K(D-np)rrr-NH2) in 100 μl of buffer A (50 mm sodium acetate, pH 5.29, 0.15 m NaCl), buffer B (50 mm sodium phosphate, pH 7.65, 0.15 m NaCl), or buffer C (50 mm sodium acetate, pH 5.38, 0.7 m NaCl) for indicated times at 37 °C. For determination of the optimum pH, purified rmSASP15 (25 pmol) was incubated with 0.1 mm peptide substrate in 100 μl of buffer D (50 mm sodium acetate, pH 4.32 or pH 4.96, 0.7 m NaCl) or buffer E (50 mm sodium acetate, pH 5.41-7.59, 0.7 m NaCl) for 60 min at 37 °C. All buffers contained 1 mm EDTA and protease inhibitor mixture (Nakalai Tesque) that consisted of 4-(2-aminoethyl)benzene-sulfonyl fluoride, aprotinin, E-64, and leupeptin to completely inhibit the activities of serine and cysteine protease. After incubation, fluorescence was measured using a VICTOR2 (PerkinElmer Life Sciences) with the excitation and emission wavelengths at 355 and 460 nm, respectively. Specific activities were calculated according to the fluorescence of FRETS-25-STD1 and FRETS-25-STD2 (Peptide Inc.). Generation of Knock-out Mice—The mSASP gene is encoded by a single exon. The 2.2-kb fragment upstream of mSASP was amplified by genomic PCR using the following primers: 5′-AATTCCGCGGATCCTGGGCTGTAGCGTGAGTTTCAG-3′ and 5′-ATATCCCGGGATCTTGAACTTCAGGGAGCGACGTCCT-3′. The 7.7-kb fragment downstream of mSASP was amplified by genomic PCR using the primers 5′-AATTGTCGACAGCCAGGAATGTCAATCTTGAGAGAGGACC-3′ and 5′-AATTCCGCGGTTTGCCCCCCTCATCTCAGTGTTCTAGCTC-3′ from genomic DNA of J1 ES cells. These primers were designed from publicly available genomic DNA sequences around the mSASP gene locus. The targeting vector was constructed by ligating a 2.2-kb SacII/SmaI fragment and a 7.7-kb blunted fragment, which were located upstream and downstream of the single exon of mSASP to the pgk-neo cassette, respectively. J1 ES cells were electroporated with the targeting vector and selected in the presence of G418. The G418-resistant colonies were removed and screened by Southern blotting with the 3′ external probe (Fig. 5A). Correctly targeted ES clones were identified by an additional 2.7-kb band together with the 6.8-kb band of the wild type allele when digested with EcoRI. The targeted ES cells obtained were injected into C57BL/6 blastocysts, which were in turn transferred into BALB/c foster mothers to obtain chimeric mice. Male chimeras were mated with C57BL/6 females, and agouti offspring were genotyped to confirm the germ line transmission of the targeted allele. The littermates were genotyped by Southern blotting. Heterozygous mice were then interbred to produce homozygous mice. Removal of Hair—The mice were anesthetized, and hair was removed by shaving, and hair remover (Kanebo, Tokyo, Japan) was rubbed on. After 10 min, the hair remover was rubbed off, and Acid Care Lotion (Kanebo) was applied to the surface of the skin. Preparation of Cornified Envelopes—For preparation of cornified envelopes, the tip of the ear was cut, placed in water containing 25 mm dithiothreitol and 2% SDS, heated to 100 °C for 15 min, and centrifuged. The pellet was resuspended in 10 mm Tris-Cl, pH 8.0, and 1 mm EDTA. Envelopes were examined in a hemocytometer under phase microscopy. Epidermal Protein Extraction and Immunoblot Analysis—The epidermis and dermis were separated by heating the skin for 5 min at 54 °C in 5 mm EDTA in PBS. Separated epidermis was extracted in a buffer containing 62.5 mm Tris-Cl, pH 6.8, 2% glycerol, 1% SDS, 5 mm EDTA, and a protease inhibitor mixture (Nakalai Tesque) followed by sonication on ice 5 times for 3 s and centrifuged at 15,000 × g for 20 min at room temperature. The supernatant was used as epidermal extract. Proteins were separated by SDS-PAGE on 15% acrylamide gels. After electrophoresis, proteins were electrophoretically transferred from gels onto nitrocellulose membranes that were then incubated with the first antibody. Bound antibodies were visualized with alkaline phosphatase-conjugated goat anti-rabbit IgG and the appropriate substrate as described by the manufacturer (GE Healthcare). To identify genes involved in epidermal differentiation, we previously performed "high throughput in situ hybridization screening" of ∼10,000 genes expressed in mouse skin and identified 116 unique clones that were expressed in a layer-specific manner in mouse foot pad epidermis (14Matsui T. Hayashi-Kisumi F. Kinoshita Y. Katahira S. Morita K. Miyachi Y. Ono Y. Imai T. Tanigawa Y. Komiya T. Tsukita S. Genomics. 2004; 84: 384-397Crossref PubMed Scopus (58) Google Scholar). Among them, we identified a novel cDNA fragment, SK082D11, of which the in situ hybridization signal was specifically detected in the granular layer (Fig. 1A). To obtain a full-length cDNA clone, the publicly available mouse EST data base was searched, and an EST was identified (BC057938) that contained the entire sequence of SK082D11 (Fig. 1B). Close comparison of BC057938 to the mouse genome data base revealed that 17 nucleotides of the 5′-end were not present. The deduced amino acid sequence of the corrected BC057938 revealed that it encoded 339 amino acids. Searching the publicly available data bases with these predicted amino acids revealed that it was a mouse homologue of human SASPase, a recently identified retroviral-like skin-specific aspartic protease (13Bernard D. Méhul B. Thomas-Collignon A. Delattre C. Donovan M. Schmidt R. J. Investig. Dermatol. 2005; 125: 278-287Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The PROSITE search also predicted a retroviral type aspartic protease signature with aspartic acid 210 as the putative active amino acid site. At the amino acid sequence level, mSASP and hSASP showed 72% identity (Fig. 1C). Next, the expression of mSASP mRNA in various mouse tissues was examined by Northern blotting (Fig. 2A). The SK082D11 probe detected a strong 1.5-kb band in the stomach and skin and a weaker band in the lung. Furthermore, we examined the expression of mSASP by quantitative RT-PCR (Fig. 2B). In good agreement with the Northern blot findings, mSASP mRNA was highly expressed in stratified epithelia such as the skin, tongue, esophagus, forestomach, and vagina. It was further expressed in the trachea and urinary bladder as well as in the thymus. It was undetectable in typical simple epithelia such as the liver and small intestine. In mouse embryonic development, mSASP mRNA expression was first detected at embryonic day 15.5, which coincided well with the emergence of epidermal stratification (Fig. 2C).FIGURE 2Expression patterns of mouse SASPase mRNA. A, Northern blotting. Nylon membranes blotted with total RNA (20 μg) from various tissues of 8-week-old female BALB/c mice were
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