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• W2029957965 abstract "Tartrate-resistant acid phosphatase (TRAP) is a metallophosphoesterase participating in osteoclast-mediated bone turnover. Activation of TRAP is associated with the redox state of the di-iron metal center as well as with limited proteolytic cleavage in an exposed loop domain. The cysteine proteinases cathepsin B, L, K, and S as well as the matrix metalloproteinase-2, -9, -13, and -14 are expressed by osteoclasts and/or other bone cells and have been implicated in the turnover of bone and cartilage. To identify proteases that could act as activators of TRAP in bone, we report here that cathepsins K and L, in contrast to the matrix metalloproteinases, efficiently cleaved and activated recombinant TRAP in vitro. Activation of TRAP by cathepsin K/L was because of increases in catalytic activity, substrate affinity, and sensitivity to reductants. Processing by cathepsin K occurred sequentially by an initial excision of the loop peptide Gly143–Gly160 followed by the removal of a Val161–Ala162 dipeptide at the N terminus of the C-terminal 16-kDa TRAP subunit. Cathepsin L initially released a shorter Gln151–Gly160 peptide and completed processing at Ser145 or Gly143 at the C terminus of the N-terminal 23-kDa TRAP subunit and at Arg163 at the N terminus of the C-terminal 16-kDa TRAP subunit. Mutation of Ser145 to Ala partly mimicked the effect of proteolysis on catalytic activity, identifying Ser145 as well as Asp146 (Funhoff, E. G., Ljusberg, J., Wang, Y., Andersson, G., and Averill, B. A. (2001) Biochemistry 40, 11614–11622) as repressive amino acids of the loop region to maintain the TRAP enzyme in a catalytically latent state. The C-terminal sequence of TRAP isolated from rat bone was consistent with cathepsin K-mediated processing in vivo. Moreover, cathepsin K, but not cathepsin L, co-localized with TRAP in osteoclast-resorptive compartments, supporting a role for cathepsin K in the extracellular processing of monomeric TRAP in the resorption lacuna. Tartrate-resistant acid phosphatase (TRAP) is a metallophosphoesterase participating in osteoclast-mediated bone turnover. Activation of TRAP is associated with the redox state of the di-iron metal center as well as with limited proteolytic cleavage in an exposed loop domain. The cysteine proteinases cathepsin B, L, K, and S as well as the matrix metalloproteinase-2, -9, -13, and -14 are expressed by osteoclasts and/or other bone cells and have been implicated in the turnover of bone and cartilage. To identify proteases that could act as activators of TRAP in bone, we report here that cathepsins K and L, in contrast to the matrix metalloproteinases, efficiently cleaved and activated recombinant TRAP in vitro. Activation of TRAP by cathepsin K/L was because of increases in catalytic activity, substrate affinity, and sensitivity to reductants. Processing by cathepsin K occurred sequentially by an initial excision of the loop peptide Gly143–Gly160 followed by the removal of a Val161–Ala162 dipeptide at the N terminus of the C-terminal 16-kDa TRAP subunit. Cathepsin L initially released a shorter Gln151–Gly160 peptide and completed processing at Ser145 or Gly143 at the C terminus of the N-terminal 23-kDa TRAP subunit and at Arg163 at the N terminus of the C-terminal 16-kDa TRAP subunit. Mutation of Ser145 to Ala partly mimicked the effect of proteolysis on catalytic activity, identifying Ser145 as well as Asp146 (Funhoff, E. G., Ljusberg, J., Wang, Y., Andersson, G., and Averill, B. A. (2001) Biochemistry 40, 11614–11622) as repressive amino acids of the loop region to maintain the TRAP enzyme in a catalytically latent state. The C-terminal sequence of TRAP isolated from rat bone was consistent with cathepsin K-mediated processing in vivo. Moreover, cathepsin K, but not cathepsin L, co-localized with TRAP in osteoclast-resorptive compartments, supporting a role for cathepsin K in the extracellular processing of monomeric TRAP in the resorption lacuna. Tartrate-resistant acid phosphatase (TRAP), 1The abbreviations used are: TRAP, tartrate-resistant acid phosphatase; pNPP, para-nitrophenyl phosphate; PVDF, polyvinylidene difluoride; MMP, matrix metalloproteinase; PAP, purple acid phosphatase; FITC, fluorescein isothiocyanate; FPLC, fast protein liquid chromatography; rec, recombinant; DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcriptase; qPCR, quantitative PCR. also known as type 5 acid phosphatase (EC 3.1.3.2) or uteroferrin, belongs to the purple acid phosphatase (PAP) subfamily of the non-heme dinuclear metallophosphatases (1.Twitchett M.B. Sykes A.G. Eur. J. Inorg. Chem. 1999; 12: 2105-2115Crossref Google Scholar, 2.Vincent J.B. Averill B.A. FEBS Lett. 1990; 263: 265-268Crossref PubMed Scopus (49) Google Scholar, 3.Vogel A. Spener F. Krebs B. Messerschmidt A. Huber R. Poulos T. Wieghardt K. Handbook of Metalloproteins. John Wiley & Sons, Ltd., Chichester, UK2001: 752-769Google Scholar). The metals of the catalytic center of all PAPs consist of a common ferric ion and a divalent metal cation in an active enzyme, where mammalian PAPs characteristically contain a redox-active iron in the M(II) site (4.Davis J.C. Lin S.S. Averill B.A. Biochemistry. 1981; 20: 4062-4067Crossref PubMed Scopus (78) Google Scholar, 5.Strater N. Klabunde T. Tucker P. Witzel H. Krebs B. Science. 1995; 268: 1489-1492Crossref PubMed Scopus (414) Google Scholar, 6.Schenk G. Ge Y. Carrington L.E. Wynne C.J. Searle I.R. Carroll B.J. Hamilton S. de Jersey J. Arch. Biochem. Biophys. 1999; 370: 183-189Crossref PubMed Scopus (142) Google Scholar). The TRAP enzyme is abundantly expressed by bone-resorbing cells, osteoclasts, and certain subpopulations of monocytes/macrophages and dendritic cells (7.Hayman A.R. Macary P. Lehner P.J. Cox T.M. J. Histochem. Cytochem. 2001; 49: 675-684Crossref PubMed Scopus (77) Google Scholar, 8.Hayman A.R. Bune A.J. Bradley J.R. Rashbass J. Cox T.M. J. Histochem. Cytochem. 2000; 48: 219-228Crossref PubMed Scopus (112) Google Scholar, 9.Ek-Rylander B. Bill P. Norgard M. Nilsson S. Andersson G. J. Biol. Chem. 1991; 266: 24684-24689Abstract Full Text PDF PubMed Google Scholar, 10.Angel N.Z. Walsh N. Forwood M.R. Ostrowski M.C. Cassady A.I. Hume D.A. J. Bone Miner. Res. 2000; 15: 103-110Crossref PubMed Scopus (144) Google Scholar). The precise role of osteoclastic TRAP is not fully understood, but studies on TRAP knock-out mice showed disturbed endochondral ossification with decreased resorptive activity of osteoclasts (11.Hayman A.R. Jones S.J. Boyde A. Foster D. Colledge W.H. Carlton M.B. Evans M.J. Cox T.M. Development (Camb.). 1996; 122: 3151-3162PubMed Google Scholar, 12.Hollberg K. Hultenby K. Hayman A. Cox T. Andersson G. Exp. Cell Res. 2002; 279: 227-238Crossref PubMed Scopus (91) Google Scholar), whereas overexpression of TRAP was associated with increased bone turnover (10.Angel N.Z. Walsh N. Forwood M.R. Ostrowski M.C. Cassady A.I. Hume D.A. J. Bone Miner. Res. 2000; 15: 103-110Crossref PubMed Scopus (144) Google Scholar). Different functions have been suggested for TRAP, e.g. as an osteopontin phosphatase (13.Suter A. Everts V. Boyde A. Jones S.J. Lullmann-Rauch R. Hartmann D. Hayman A.R. Cox T.M. Evans M.J. Meister T. von Figura K. Saftig P. Development (Camb.). 2001; 128: 4899-4910PubMed Google Scholar, 14.Ek-Rylander B. Flores M. Wendel M. Heinegard D. Andersson G. J. Biol. Chem. 1994; 269: 14853-14856Abstract Full Text PDF PubMed Google Scholar, 15.Andersson G. Ek-Rylander B. Hollberg K. Ljusberg-Sjolander J. Lang P. Norgard M. Wang Y. Zhang S.J. J. Bone Miner. Res. 2003; 18: 1912-1915Crossref PubMed Scopus (60) Google Scholar), generation of reactive oxygen species (16.Kaija H. Alatalo S.L. Halleen J.M. Lindqvist Y. Schneider G. Vaananen H.K. Vihko P. Biochem. Biophys. Res. Commun. 2002; 292: 128-132Crossref PubMed Scopus (53) Google Scholar, 17.Sibille J.C. Doi K. Aisen P. J. Biol. Chem. 1987; 262: 59-62Abstract Full Text PDF PubMed Google Scholar, 18.Hayman A.R. Cox T.M. J. Biol. Chem. 1994; 269: 1294-1300Abstract Full Text PDF PubMed Google Scholar, 19.Halleen J.M. Raisanen S. Salo J.J. Reddy S.V. Roodman G.D. Hentunen T.A. Lehenkari P.P. Kaija H. Vihko P. Vaananen H.K. J. Biol. Chem. 1999; 274: 22907-22910Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), iron transport (20.Buhi W.C. Ducsay C.A. Bazer F.W. Roberts R.M. J. Biol. Chem. 1982; 257: 1712-1723Abstract Full Text PDF PubMed Google Scholar, 21.Ducsay C.A. Buhi W.C. Bazer F.W. Roberts R.M. Combs G.E. J. Anim. Sci. 1984; 59: 1303-1308Crossref PubMed Scopus (28) Google Scholar, 22.Roberts R.M. Raub T.J. Bazer F.W. Fed. Proc. 1986; 45: 2513-2518PubMed Google Scholar, 23.Saunders P.T. Renegar R.H. Raub T.J. Baumbach G.A. Atkinson P.H. Bazer F.W. Roberts R.M. J. Biol. Chem. 1985; 260: 3658-3665Abstract Full Text PDF PubMed Google Scholar, 24.Naseri J.I. Truong N.T. Horentrup J. Kuballa P. Vogel A. Rompel A. Spener F. Krebs B. Arch. Biochem. Biophys. 2004; 432: 25-36Crossref PubMed Scopus (10) Google Scholar), and as a growth/differentiation factor for hematopoietic (25.Bazer F.W. Worthington-White D. Fliss M.F. Gross S. Exp. Hematol. 1991; 19: 910-915PubMed Google Scholar) and osteoblastic (26.Sheu T.J. Schwarz E.M. Martinez D.A. O'Keefe R.J. Rosier R.N. Zuscik M.J. Puzas J.E. J. Biol. Chem. 2003; 278: 438-443Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) cells. Mammalian PAPs are synthesized as 35–37-kDa monomers but are commonly isolated from tissues as proteolytically cleaved two-subunit forms consisting of a 23-kDa N-terminal domain disulfide-linked to a 16-kDa C-terminal domain. The monomeric form exhibits properties of a proenzyme with low phosphatase activity that is converted to a high activity, two-subunit form upon proteolytic cleavage in the intervening loop domain with either serine proteases, e.g. trypsin or chymotrypsin (27.Orlando J.L. Zirino T. Quirk B.J. Averill B.A. Biochemistry. 1993; 32: 8120-8129Crossref PubMed Scopus (35) Google Scholar), or members of the cathepsin family (28.Ljusberg J. Ek-Rylander B. Andersson G. Biochem. J. 1999; 343: 63-69Crossref PubMed Scopus (99) Google Scholar, 29.Funhoff E.G. Klaassen C.H. Samyn B. Van Beeumen J. Averill B.A. Chembiochem. 2001; 2: 355-363Crossref PubMed Scopus (45) Google Scholar). Mutagenesis studies suggested that proteolysis removes or alters repressive interactions between loop amino acids and active site residues because replacement of Asp146 of the exposed loop domain with Ala resulted in activation of unproteolyzed TRAP (30.Funhoff E.G. Ljusberg J. Wang Y. Andersson G. Averill B.A. Biochemistry. 2001; 40: 11614-11622Crossref PubMed Scopus (53) Google Scholar). Several lines of evidence indicate a role for cathepsin K in bone resorption. Cathepsin K is highly expressed in osteoclasts near the ruffled border membrane and has been shown to participate in osteoclast-mediated degradation of the sub-osteoclastic collagenous bone matrix (31.Bossard M.J. Tomaszek T.A. Thompson S.K. Amegadzie B.Y. Hanning C.R. Jones C. Kurdyla J.T. McNulty D.E. Drake F.H. Gowen M. Levy M.A. J. Biol. Chem. 1996; 271: 12517-12524Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 32.Littlewood-Evans A. Kokubo T. Ishibashi O. Inaoka T. Wlodarski B. Gallagher J.A. Bilbe G. Bone (NY). 1997; 20: 81-86Crossref PubMed Scopus (178) Google Scholar, 33.Votta B.J. Levy M.A. Badger A. Bradbeer J. Dodds R.A. James I.E. Thompson S. Bossard M.J. Carr T. Connor J.R. Tomaszek T.A. Szewczuk L. Drake F.H. Veber D.F. Gowen M. J. Bone Miner. Res. 1997; 12: 1396-1406Crossref PubMed Scopus (165) Google Scholar, 34.Almeida P.C. Nantes I.L. Chagas J.R. Rizzi C.C. Faljoni-Alario A. Carmona E. Juliano L. Nader H.B. Tersariol I.L. J. Biol. Chem. 2001; 276: 944-951Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 35.Soderstrom M. Salminen H. Glumoff V. Kirschke H. Aro H. Vuorio E. Biochim. Biophys. Acta. 1999; 1446: 35-46Crossref PubMed Scopus (64) Google Scholar, 36.Dodds R.A. James I.E. Rieman D. Ahern R. Hwang S.M. Connor J.R. Thompson S.D. Veber D.F. Drake F.H. Holmes S. Lark M.W. Gowen M. J. Bone Miner. Res. 2001; 16: 478-486Crossref PubMed Scopus (71) Google Scholar). Cathepsin K-overexpressing mice showed an increased turnover of metaphyseal trabecular bone (37.Kiviranta R. Morko J. Uusitalo H. Aro H.T. Vuorio E. Rantakokko J. J. Bone Miner. Res. 2001; 16: 1444-1452Crossref PubMed Scopus (120) Google Scholar), whereas cathepsin K knock-out mice displayed an osteopetrotic phenotype because of impaired matrix degradation (38.Gowen M. Lazner F. Dodds R. Kapadia R. Feild J. Tavaria M. Bertoncello I. Drake F. Zavarselk S. Tellis I. Hertzog P. Debouck C. Kola I. J. Bone Miner. Res. 1999; 14: 1654-1663Crossref PubMed Scopus (424) Google Scholar, 39.Saftig P. Hunziker E. Wehmeyer O. Jones S. Boyde A. Rommerskirch W. Moritz J.D. Schu P. von Figura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13453-13458Crossref PubMed Scopus (759) Google Scholar, 40.Kiviranta R. Morko J. Alatalo S.L. Nicamhlaoibh R. Risteli J. Laitala-Leinonen T. Vuorio E. Bone (NY). 2005; 36: 159-172Crossref PubMed Scopus (141) Google Scholar). Besides cathepsin K, also cathepsins B, H, L, and S are expressed by osteoclasts and could participate in bone resorption (35.Soderstrom M. Salminen H. Glumoff V. Kirschke H. Aro H. Vuorio E. Biochim. Biophys. Acta. 1999; 1446: 35-46Crossref PubMed Scopus (64) Google Scholar, 41.Everts V. Delaisse J.M. Korper W. Beertsen W. J. Bone Miner. Res. 1998; 13: 1420-1430Crossref PubMed Scopus (107) Google Scholar, 42.Delaisse J.M. Ledent P. Vaes G. Biochem. J. 1991; 279: 167-174Crossref PubMed Scopus (127) Google Scholar, 43.Furuyama N. Fujisawa Y. Biochem. Biophys. Res. Commun. 2000; 272: 116-124Crossref PubMed Scopus (23) Google Scholar). Deletion of the cathepsin L gene was associated with reduction of trabecular bone and a diminished resorptive response following ovariectomy (44.Potts W. Bowyer J. Jones H. Tucker D. Freemont A.J. Millest A. Martin C. Vernon W. Neerunjun D. Slynn G. Harper F. Maciewicz R. Int. J. Exp. Pathol. 2004; 85: 85-96Crossref PubMed Scopus (64) Google Scholar). In addition to cathepsins, different matrix metalloproteinases (MMPs), e.g. MMP-2, -9, -13, and -14, have been implicated in various aspects of skeletal development and turnover (45.Delaisse J.M. Andersen T.L. Engsig M.T. Henriksen K. Troen T. Blavier L. Microsc. Res. Tech. 2003; 61: 504-513Crossref PubMed Scopus (248) Google Scholar), including processes such as osteoclast recruitment and migration (46.Blavier L. Delaisse J.M. J. Cell Sci. 1995; 108: 3649-3659PubMed Google Scholar, 47.Engsig M.T. Chen Q.J. Vu T.H. Pedersen A.C. Therkidsen B. Lund L.R. Henriksen K. Lenhard T. Foged N.T. Werb Z. Delaisse J.M. J. Cell Biol. 2000; 151: 879-890Crossref PubMed Scopus (491) Google Scholar, 48.Karsdal M.A. Fjording M.S. Foged N.T. Delaisse J.M. Lochter A. J. Biol. Chem. 2001; 276: 39350-39358Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 49.Sato T. Foged N.T. Delaisse J.M. J. Bone Miner. Res. 1998; 13: 59-66Crossref PubMed Scopus (93) Google Scholar), post-osteoclastic cleaning of the resorption lacuna (50.Everts V. Delaisse J.M. Korper W. Jansen D.C. Tigchelaar-Gutter W. Saftig P. Beertsen W. J. Bone Miner. Res. 2002; 17: 77-90Crossref PubMed Scopus (278) Google Scholar), and osteoblast survival (51.Vu T.H. Shipley J.M. Bergers G. Berger J.E. Helms J.A. Hanahan D. Shapiro S.D. Senior R.M. Werb Z. Cell. 1998; 93: 411-422Abstract Full Text Full Text PDF PubMed Scopus (1505) Google Scholar). In this study, several members of the cathepsin and MMP families with known association with the functional activities of osteoclasts were screened for cleavage and activation of rat recombinant TRAP, identifying cathepsins K and L as efficient activators of TRAP. In order to better understand the structural basis for proteolytic activation of mammalian PAPs, we also aimed to define the proteolytic cleavage sites for cathepsin L and K in the repressive loop domain. Finally, the in situ distribution of the cathepsins K and L was compared with that of TRAP in osteoclasts and resorptive subcompartments to address the potential physiological relevance of the in vitro observations. Proteases were supplied from the following sources: cathepsin L (human liver) and cathepsin S from Calbiochem; cathepsin B and MMP-2 from Anawa Trading SA, (Wangen/Zurich, Switzerland); MMP-9l, Roche Applied Science; MMP-13 and MMP-14, Invitek (Berlin, Germany). Human mature cathepsin K was generated as described previously (31.Bossard M.J. Tomaszek T.A. Thompson S.K. Amegadzie B.Y. Hanning C.R. Jones C. Kurdyla J.T. McNulty D.E. Drake F.H. Gowen M. Levy M.A. J. Biol. Chem. 1996; 271: 12517-12524Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). Antibodies came from the following sources: alkaline phosphatase-conjugated goat anti-rabbit IgG from Sigma; horseradish peroxidase-conjugated goat anti-rabbit IgG from Calbiochem; biotinylated rabbit anti-goat IgG and swine anti-rabbit IgG from Dako (Glostrup, Denmark); FITC-conjugated donkey anti-rabbit IgG, Cy3-conjugated donkey anti-rabbit IgG, and Cy3-conjugated donkey anti-goat IgG from Jackson ImmunoResearch (West Grove, PA). Goat anti-mouse cathepsin L was purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Rabbit anti-rat cathepsin K (52.Kamiya T. Kobayashi Y. Kanaoka K. Nakashima T. Kato Y. Mizuno A. Sakai H. J. Biochem. (Tokyo). 1998; 123: 752-759Crossref PubMed Scopus (97) Google Scholar) was kindly provided by Dr. Hideaki Sakai, Nagasaki School of Dentistry, Sakamoto, Nagasaki, Japan. Rabbit anti-recombinant rat TRAP antibody (anti-recTRAP) was generated as described previously (53.Ek-Rylander B. Barkhem T. Ljusberg J. Ohman L. Andersson K.K. Andersson G. Biochem. J. 1997; 321: 305-311Crossref PubMed Scopus (74) Google Scholar). Antiserum toward the exposed loop domain in TRAP was produced in rabbits using a synthetic peptide derived from the mouse TRAP sequence (146DDFASQQPKMPRDLGVA(C)162) as the immunogen (54.Lang P. Andersson G. Cell. Mol. Life Sci. 2005; 62: 905-918Crossref PubMed Scopus (34) Google Scholar). Restriction enzymes, DNA purification system, mutation-specific primers, and baculovirus expression system were purchased from Invitrogen. Site-directed Mutagenesis—The full-length 1.4-kb rat tartrate-resistant acid phosphatase cDNA (16.Kaija H. Alatalo S.L. Halleen J.M. Lindqvist Y. Schneider G. Vaananen H.K. Vihko P. Biochem. Biophys. Res. Commun. 2002; 292: 128-132Crossref PubMed Scopus (53) Google Scholar) was cloned into pCMV5. Site-directed mutagenesis was performed using 5′–3′ MORPH site-specific plasmid DNA mutagenesis kit (5 Prime → 3 Prime, Inc., Boulder, CO). Primer used for specific mutation of S145A was 5′-GCTGTGTGGCAATGCAGACGACTTTGTCAG-3′, for Q151A was 5′-GACGACTTTGTCAGCGCGCAGCCTGAAATGCCC-3′, and for Q152A was 5′-GACTTTGTCAGCCAGGCGCCTGAAATGCCCCGAG-3′. The underlined bases indicate changes compared with the wild-type sequence. The primer employed for mutagenesis was phosphorylated by T4 kinase according to the manufacturer's instruction. The recTRAP cDNA mutants were verified by DNA sequence analyses (Cybergene AB, Novum Research Park, Huddinge, Sweden). Construction of Baculovirus Expression Vectors and Recombinant Baculovirus—Wild-type or mutant rat TRAP cDNAs were cloned into baculovirus expression vector pFASTBAC1 using the EcoRI site in the donor plasmid. The correct orientation was determined by PstI cleavage and further confirmed by DNA sequencing. The pFASTBAC1 donor vectors containing wild-type or mutant TRAPs were transformed into DH10Bac cells for homologous recombination with bacmid. Recombinant bacmids were selected on Luria agar plates containing antibiotics (50 μg/ml kanamycin, 7 μg/ml gentamycin, 10 μg/ml tetracycline), 100 μg/ml Bluo-gel (halogenated indolyl-β-galactoside), and 40 μg/ml isopropyl 1-thio-β-d-galactopyranoside and confirmed by PCR. Virus Amplification—Spodoptera frugiperda (Sf9) cells were cultured in SF900 II SFM medium and transfected with purified bacmids using the transfection reagent Cellfectin (Invitrogen). After transfection, the cells were kept and incubated at 27 °C for 3 days. The medium, which constitutes the scale up-1 virus stock, was harvested and stored at 4 °C. This scale up-1 virus stock was used to infect a 25-cm2 flask seeded with 1 × 106 cells, and the culture was incubated at 27 °C for 5 days. The supernatant constitutes the scale up-2. The recombinant virus stock scale up-2 was amplified once more by infection of 100 ml of Sf9 cells seeded at a density of 1 × 106 cells/ml with 3 ml of scale up-2, and after 5 days, high titer virus stock (108 pfu/ml) was harvested. The high titer stock virus was used in protein production. Protein Expression and Purification—Purification of rat bone TRAP and recTRAP expression in Sf9 cells, purification, and protein determination were carried out as described previously (55.Wang Y. Norgard M. Andersson G. Arch. Biochem. Biophys. 2005; 435: 147-156Crossref PubMed Scopus (26) Google Scholar). Small Scale Proteolytic Cleavage of TRAP—Proteolytic activation of TRAP was performed with the following proteases and incubation conditions: cathepsin K, L, and B and recTRAP, 7 ng/μl, 4 mol of protease/mol of TRAP, 50 mm NaAc, 1 mm EDTA, pH 5.5, 2 mm DTT; cathepsin S, recTRAP, 7 ng/μl, 4 mol of cathepsin S/mol of TRAP, 50 mm NaAc, 5 mm EDTA, 0.1% Triton X-100, pH 6.5, 2 mm DTT. All proteases were prereduced in 2 mm DTT for 7 min before the addition to the incubation solution, and the protease digestions were performed at 37 °C for 2 h. For MMP-2 and MMP-9, recTRAP, 10 ng/μl, or collagen type IV, 100 ng/μl, as control was incubated with MMP-2, 10 ng/μl (0.6 mol of MMP-2/mol of TRAP), or MMP-9, 20 ng/μl, 4 microunits/μl (0.9 mol of MMP-9/mol of TRAP), at 37 °C for 24 h in 50 mm Tris-HCl, 0.2 m NaCl, 10 mm CaCl2, and 2 mm HgCl2, pH 7.2. For MMP-13 and MMP-14, recTRAP, 10 ng/μl, were incubated with MMP-13, 11.3 ng/μl, 5.7 microunits/μl (2 mol of MMP-13/mol of TRAP), or MMP-14, 11.8 ng/μl, 1.7 microunits/μl (2 mol of MMP-14/mol of TRAP) at 37 °C for 24 h in 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm CaCl2 at pH 7.5. Large Scale Cathepsin K, Cathepsin L, and Trypsin Cleavage of TRAP Prior to FPLC Separation—recTRAP was incubated at 37 °C for 45 min with cathepsin L or cathepsin K with the final concentrations: recTRAP 50 ng/μl; 0.04, 0.2, 0.28, and 1.0 mol of protease/mol of TRAP; 50 mm NaAc, 1 mm EDTA, pH 5.5, 2 mm DTT. recTRAP was digested with trypsin at the final concentrations: recTRAP 35 ng/μl; 0.04, 0.2, 1.0 mol of trypsin/mol of TRAP; 10 mm Tris-HCl, pH 7.5, 20 mm NaCl, 2 mm CaCl2, at room temperature for 45 min. All proteolytic digestions were stopped with the proteinase inhibitor mixture Complete (Roche Applied Science). Phosphatase activity was assayed in 96-well plates (Greiner Labortechnik, Frickenhausen, Germany) with pNPP as substrate in 150 μl of incubation medium, pH 5.8, with final concentrations as follows: 10 mm pNPP, 1 mm ascorbic acid, 0.1 mm Fe(NH4)2(SO4)2, 0.1 m NaAc, 0.15 m KCl, 10 mm disodium tartrate, 0.1% (v/v) Triton X-100. The p-nitrophenol liberated after 1 h of incubation at 37 °C was converted to p-nitrophenolate by addition of 100 μl of 0.3 m NaOH. The absorbance was measured at 405 nm (ϵ = 18.9 mm-1 cm-1) with a Spectramax 250 spectrophotometer (Molecular Devices, Sunnyvale, CA). 1 unit of TRAP activity corresponds to 1 μmol of p-nitrophenol formed per min. Kinetic experiments were performed with p-NPP concentrations varying from 0.2 to 50 mm Km, and Vmax values were calculated by regression analysis of Lineweaver-Burk plots using the Origin software (OriginLab Corp., Northampton, MA). pH optimum was determined in 0.1 m NaAc with pH values ranging from pH 4.0 to 6.7. Sensitivity to reduction was performed with concentrations of sodium ascorbate ranging from 0.5 to 100 mm. SDS-PAGE was run under reducing conditions essentially as described by Laemmli, and proteins were electroblotted to immuno-PVDF membranes (Bio-Rad). General procedure for all membranes was as follows. To minimize nonspecific binding, immunoblots were preincubated for 2 h at room temperature or overnight at 4 °C in 1% Tween 20 in TBS (20 mm Tris, pH 7.5, 500 mm NaCl). The membranes were then incubated with primary antibody for 1 h at room temperature, washed three times for 10 min each in TBST (TBS + 0.05% Tween 20) before incubation with secondary antibody, washed, and developed. Probing and development was as follows. Immunostaining of TRAP was performed with rabbit anti-recTRAP antiserum (diluted ×100) and alkaline phosphatase-conjugated goat anti-rabbit IgG as the secondary antibody (diluted ×500). Development was performed with nitro blue tetrazolium chloride/5-bromo-4-chloroindol-3-yl phosphate p-toluidine salt (Bio-Rad). Separation of proteolytic TRAP fragments was performed according to the methods described previously (56.Igarashi Y. Lee M.Y. Matsuzaki S. J. Chromatogr. B Biomed. Sci. Appl. 2001; 757: 269-276Crossref PubMed Scopus (22) Google Scholar) using an ΔKTApurifier™ 10 FPLC system at 4 °C with a heparin column (flow rate 2 ml/min) equilibrated with 20 mm Tris-HCl, pH 7.2, 0.1 m NaCl, 0.005% Triton X-100 (w/v). TRAP was eluted by a linear gradient of NaCl from 0.1 to 1 m NaCl in 20 mm Tris-Cl, pH 7.2, 0.005% Triton X-100 (w/v) (12.5 column volumes) and collected in 0.75-ml fractions. N-terminal Sequence Analysis—Samples in solution were applied to a Procise HT sequencer (Applied Biosystems) for Edman degradation. C-terminal Sequence Analysis—The sample was treated with phenylisocyanate to block the N terminus and the ϵ-amino group of lysines. The sample was thereafter mounted in the reaction cartridge of a Procise C sequencer instrument (Applied Biosystems). The sequencer was operated essentially according to the manufacturer's recommendations. Briefly, after initial activation of the C terminus with acetic anhydride, the sample was treated with tetrabutylammonium thiocyanate to form the thiohydantoin cyclic structure. Subsequently, this derivative was S-alkylated by bromomethylnaphthalene to yield an alkylated thiohydantoin derivative that is cleaved by trifluoroacetic acid and analyzed by reverse phase (C18) chromatography (detection at 257 nm). This was repeated with exclusion of the acetic anhydride activation step used in the initial cycle only. For testing, myoglobin was used to check the instrument performance, and alkylated thiohydantoin amino acid standards were always run before analysis of sample cycles. At least three cycles were performed for N- and C-terminal sequencing. Reverse Transcriptase-PCR—For RT-PCR, the long bones from 3-week-old Sprague-Dawley rats were immersed in liquid nitrogen immediately after dissection and stored at -70 °C. Total RNA was extracted using ToTALLY RNA™, total RNA isolation kit according to the protocol of the manufacturer (Ambion, Austin, TX). After extraction, the RNA was treated with 5 units of DNase I (Invitrogen) for 30 min at room temperature. The reverse transcriptase reaction was performed with 5 μg of total RNA and 200 units of Superscript™ II reverse transcriptase (Invitrogen) at 42 °C, using (dT)12–18 as primer (Invitrogen), according to the protocol of the manufacturer. In negative controls, reverse transcriptase was excluded from the reaction mixture. PCR amplifications of cathepsin L and cathepsin K were performed on aliquots of cDNA corresponding to 160 ng of total RNA using 2.5 units of TaqDNA polymerase in the presence of 1.5 mm MgCl2 and 1× Q-solution according to the protocol of the manufacturer (Qiagen, Hilden, Germany). The cathepsin L primers were 5′-ACCATGACCCCTTTACTCCTCC-3′ (bases 75–96) and 5′-AAGCCCAGCAAGAACCACAC-3′ (reverse to bases 498 to 479), positions calculated from rat cathepsin L cDNA, available at GenBank™/EMBL Data Bank, accession number NM_013156, corresponding to a product of 424 bp. The cathepsin K primers were 5′-ACGAGAAAGCCCTGAAGAGAGC-3′ (position 734–755) and 5′-TCCGAGCCAAGAGAACATAGCC-3′ (reverse to position 979–958) calculated from rat cathepsin K cDNA available at GenBank™/EMBL Data Bank, accession number AF010306, corresponding to a product of 246 bp. PCR amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from 80 ng of cDNA was performed as above (with the exception of Q-solution) to ensure equal loading of cDNA in the samples, using primers 5′-CTGAACGGGAAGCTCACTGG-3′ and 5′-ATGAGGTCCACCACCCTGTTC-3′ corresponding to positions 735–754 and reverse to position 1048–1029 in the GAPDH cDNA sequence (57.Fort P. Marty L. Piechaczyk M. el Sabrouty S. Dani C. Jeanteur P. Blanchard J.M. Nucleic Acids Res. 1985; 13: 1431-1442Crossref PubMed Scopus (1972) Google Scholar), generating a band of 314 bp. The cDNA was amplified in a GeneAmp PCR system 9700 (PerkinElmer Life Sciences DNA) under the following conditions: 94 °C for 30 s, cathepsin L and cathepsin K at 56 °C, GAPDH 55 °C for 30 s and 72 °C for 30 s. Aliquots corresponding to 20 ng of cDNA of cathepsin L and K and 10 ng of cDNA of GAPDH were removed at cycles 20, 25, 30, and 35. PCR products corresponding to 6 ng of cDNA from cathepsin L and K and 2 ng from GAPDH were electrophoresed on a 1.5% agarose gel containing 0.15 μg/ml ethidium bromide. Real Time qPCR— Real time qPCR was carried out for rat cathepsin K and L and GAPDH, each sample run on three occasions, using the SYBR® green chemistry. Primers were as follows: cathepsin K forward 5′-GTTGTATGTATAACGCCACGGC-3′ (300 nm) corresponding to bp 699–720 and reverse 5′-CTTTCTCGTTCCCCACAGGA-3′ (300 nm) corresponding to bp 756–774 in NM_031560.2 available at PubMed with annealing temperature 57 °C, and for cathepsin L forward 5′-ACATGAGAATGATCCAGCTACACA-3′ (900 nm) corresponding to bp 241–264 and reverse 5′-CATGTCACCGAAGGCGTTCA-3′ (30" @default.
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• W2029957965 date "2005-08-01" @default.
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• W2029957965 title "Proteolytic Excision of a Repressive Loop Domain in Tartrate-resistant Acid Phosphatase by Cathepsin K in Osteoclasts" @default.
• W2029957965 cites W1486803151 @default.
• W2029957965 cites W1534572318 @default.
• W2029957965 cites W1536296413 @default.
• W2029957965 cites W1545953723 @default.
• W2029957965 cites W1571380461 @default.
• W2029957965 cites W1572561340 @default.
• W2029957965 cites W1580813094 @default.
• W2029957965 cites W1697963672 @default.
• W2029957965 cites W1822169786 @default.
• W2029957965 cites W1930664300 @default.
• W2029957965 cites W1943108778 @default.
• W2029957965 cites W1953148896 @default.
• W2029957965 cites W1969959437 @default.
• W2029957965 cites W1970569189 @default.
• W2029957965 cites W1971000202 @default.
• W2029957965 cites W1973639566 @default.
• W2029957965 cites W1978181825 @default.
• W2029957965 cites W1982485888 @default.
• W2029957965 cites W1984965767 @default.
• W2029957965 cites W1985744266 @default.
• W2029957965 cites W1986441400 @default.
• W2029957965 cites W1991309445 @default.
• W2029957965 cites W1997304859 @default.
• W2029957965 cites W2000983409 @default.
• W2029957965 cites W2004656183 @default.
• W2029957965 cites W2005603603 @default.
• W2029957965 cites W2010750554 @default.
• W2029957965 cites W2015642465 @default.
• W2029957965 cites W2016411107 @default.
• W2029957965 cites W2016749697 @default.
• W2029957965 cites W2017481744 @default.
• W2029957965 cites W2020386376 @default.
• W2029957965 cites W2022866008 @default.
• W2029957965 cites W2023157182 @default.
• W2029957965 cites W2025062557 @default.
• W2029957965 cites W2031900710 @default.
• W2029957965 cites W2032865048 @default.
• W2029957965 cites W2034018882 @default.
• W2029957965 cites W2037504646 @default.
• W2029957965 cites W2038422579 @default.
• W2029957965 cites W2040687623 @default.
• W2029957965 cites W2042303782 @default.
• W2029957965 cites W2048475254 @default.
• W2029957965 cites W2048781270 @default.
• W2029957965 cites W2051819610 @default.
• W2029957965 cites W2055787208 @default.
• W2029957965 cites W2059018472 @default.
• W2029957965 cites W2059129593 @default.
• W2029957965 cites W2062351402 @default.
• W2029957965 cites W2065846481 @default.
• W2029957965 cites W2070239849 @default.
• W2029957965 cites W2077955101 @default.
• W2029957965 cites W2078355796 @default.
• W2029957965 cites W2086201428 @default.
• W2029957965 cites W2089678050 @default.
• W2029957965 cites W2091462414 @default.
• W2029957965 cites W2093909017 @default.
• W2029957965 cites W2094432610 @default.
• W2029957965 cites W2096464236 @default.
• W2029957965 cites W2097354779 @default.
• W2029957965 cites W2099463774 @default.
• W2029957965 cites W2108507447 @default.
• W2029957965 cites W2110236793 @default.
• W2029957965 cites W2124294753 @default.
• W2029957965 cites W2126380442 @default.
• W2029957965 cites W2144207870 @default.
• W2029957965 cites W2147205147 @default.
• W2029957965 cites W2148880214 @default.
• W2029957965 cites W2150494686 @default.
• W2029957965 cites W2153156911 @default.
• W2029957965 cites W2155036500 @default.
• W2029957965 cites W2156270323 @default.
• W2029957965 cites W2161115679 @default.
• W2029957965 cites W2165539758 @default.
• W2029957965 cites W2473802772 @default.
• W2029957965 doi "https://doi.org/10.1074/jbc.m502469200" @default.
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