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• W2016643048 abstract "The mammalian bone morphogenetic protein-1 (BMP-1)/Tolloid-related metalloproteinases play key roles in regulating formation of the extracellular matrix (ECM) via biosynthetic processing of various precursor proteins into mature functional enzymes, structural proteins, and proteins involved in initiating the mineralization of hard tissue ECMs. They also have been shown to activate several members of the transforming growth factor-β superfamily, and may serve to coordinate such activation with formation of the ECM in morphogenetic events. Osteoglycin (OGN), a small leucine-rich proteoglycan with unclear functions, is found in cornea, bone, and other tissues, and appears to undergo proteolytic processing in vivo. Here we have successfully generated recombinant OGN and have employed it to demonstrate that a pro-form of OGN is processed to varying extents by all four mammalian BMP-1/Tolloid-like proteinases, to generate a 27-kDa species that corresponds to the major form of OGN found in cornea. Moreover, whereas wild-type mouse embryo fibroblasts (MEFs) produce primarily the processed, mature form of OGN, MEFs homozygous null for genes encoding three of the four mammalian BMP-1/Tolloid-related proteinases produce only unprocessed pro-OGN. Thus, all detectable pro-OGN processing activity in MEFs is accounted for by products of these genes. We also demonstrate that both pro- and mature OGN can regulate type I collagen fibrillogenesis, and that processing of the prodomain by BMP-1 potentiates the ability of OGN to modulate the formation of collagen fibrils. The mammalian bone morphogenetic protein-1 (BMP-1)/Tolloid-related metalloproteinases play key roles in regulating formation of the extracellular matrix (ECM) via biosynthetic processing of various precursor proteins into mature functional enzymes, structural proteins, and proteins involved in initiating the mineralization of hard tissue ECMs. They also have been shown to activate several members of the transforming growth factor-β superfamily, and may serve to coordinate such activation with formation of the ECM in morphogenetic events. Osteoglycin (OGN), a small leucine-rich proteoglycan with unclear functions, is found in cornea, bone, and other tissues, and appears to undergo proteolytic processing in vivo. Here we have successfully generated recombinant OGN and have employed it to demonstrate that a pro-form of OGN is processed to varying extents by all four mammalian BMP-1/Tolloid-like proteinases, to generate a 27-kDa species that corresponds to the major form of OGN found in cornea. Moreover, whereas wild-type mouse embryo fibroblasts (MEFs) produce primarily the processed, mature form of OGN, MEFs homozygous null for genes encoding three of the four mammalian BMP-1/Tolloid-related proteinases produce only unprocessed pro-OGN. Thus, all detectable pro-OGN processing activity in MEFs is accounted for by products of these genes. We also demonstrate that both pro- and mature OGN can regulate type I collagen fibrillogenesis, and that processing of the prodomain by BMP-1 potentiates the ability of OGN to modulate the formation of collagen fibrils. Bone morphogenetic protein-1 (BMP-1) 1The abbreviations used are: BMP, bone morphogenetic protein; mTLD, mammalian Tolloid; mTLL, mammalian Tolloid-like; ECM, extracellular matrix; SLRP, small leucine-rich proteoglycan; OGN, osteoglycin; MEF, mouse embryo fibroblast; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. is the prototype of a class of structurally similar metalloproteinases that play various morphogenetic roles in a broad spectrum of species (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (357) Google Scholar, 2Greenspan D.S. Top. Curr. Chem. 2005; 229 (in press)Google Scholar). There are four mammalian members of this family: BMP-1, mammalian Tolloid (mTLD), and mammalian Tolloid-like 1 and 2 (mTLL-1 and mTLL-2) (3Takahara K. Lyons G.E. Greenspan D.S. J. Biol. Chem. 1994; 269: 32572-32578Abstract Full Text PDF PubMed Google Scholar, 4Takahara K. Brevard R. Hoffman G.G. Suzuki N. Greenspan D.S. Genomics. 1996; 34: 157-165Crossref PubMed Scopus (84) Google Scholar, 5Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W.Y. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (233) Google Scholar). BMP-1 and mTLD are encoded by alternatively spliced mRNAs of the same gene (3Takahara K. Lyons G.E. Greenspan D.S. J. Biol. Chem. 1994; 269: 32572-32578Abstract Full Text PDF PubMed Google Scholar), whereas mTLL-1 and -2 are genetically distinct (4Takahara K. Brevard R. Hoffman G.G. Suzuki N. Greenspan D.S. Genomics. 1996; 34: 157-165Crossref PubMed Scopus (84) Google Scholar, 5Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W.Y. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (233) Google Scholar). These proteinases play key roles in regulating formation of mammalian extracellular matrix (ECM), via biosynthetic processing of precursor proteins to form mature, functional matrix components. In the case of collagen fibers, this includes processing of the C-propeptides of procollagens I–III to yield the major fibrous components of ECM (5Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W.Y. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (233) Google Scholar, 6Kessler E. Takahara K. Biniaminov L. Brusel M. Greenspan D.S. Science. 1996; 271: 360-362Crossref PubMed Scopus (458) Google Scholar, 7Li S. Sieron A.L. Fertala A. Hojima Y. Arnold W.V. Prockop D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5127-5130Crossref PubMed Scopus (202) Google Scholar, 8Suzuki N. Labosky P.A. Furuta Y. Hargett L. Dunn R. Fogo A.B. Takahara K. Peters D.M. Greenspan D.S. Hogan B.L. Development. 1996; 122: 3587-3595Crossref PubMed Google Scholar, 9Pappano W.N. Steiglitz B.M. Scott I.C. Keene D.R. Greenspan D.S. Mol. Cell. Biol. 2003; 23: 4428-4438Crossref PubMed Scopus (105) Google Scholar); proteolytic activation of the enzyme lysyl oxidase (10Uzel M.I. Scott I.C. Babakhanlou-Chase H. Palamakumbura A.H. Pappano W.N. Hong H.-H. Greenspan D.S. Trackman P.C. J. Biol. Chem. 2001; 276: 22537-22543Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), which is necessary to the formation of covalent cross-links in collagen and elastic fibers (11Kagan H. Trackman P.C. Am. J. Respir. Cell Mol. Biol. 1991; 5: 206-210Crossref PubMed Scopus (281) Google Scholar); and processing of NH2-terminal globular domains, or in some cases C-propeptides, of minor fibrillar procollagen V and XI chains (12Imamura Y. Steiglitz B.M. Greenspan D.S. J. Biol. Chem. 1998; 42: 27511-27517Abstract Full Text Full Text PDF Scopus (95) Google Scholar, 13Unsöld C. Pappanoo W.N. Imamura Y. Steiglitz B.M. Greenspan D.S. J. Biol. Chem. 2002; 277: 5596-5602Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 14Medeck R.J. Sosa S. Morris N. Oxford J.T. Biochem. J. 2003; 376: 361-368Crossref PubMed Google Scholar) to yield type V and XI monomers. Such monomers are incorporated into collagen types I and II fibrils, respectively, and appear to control the shapes and diameters of the resultant heterotypic collagen fibrils (15Birk D.E. Fitch J.M. Babiarz J.P. Linsenmayer T.G. J. Cell Biol. 1988; 106: 999-1008Crossref PubMed Scopus (297) Google Scholar, 16Birk D.E. Fitch J.M. Babiarz J.P. Doane K.J. Linsenmayer T.F. J. Cell Sci. 1990; 95: 649-657Crossref PubMed Google Scholar, 17Toriello H.V. Glover T.W. Takahara K. Byers P.H. Miller D.E. Higgins J.V. Geenspan D.S. Nat. Genet. 1996; 13: 361-365Crossref PubMed Scopus (109) Google Scholar, 18Wenstrup R.J. Langland G.T. Willing M.C. D-Souza V.N. Cole W.G. Hum. Mol. Genet. 1996; 5: 1733-1736Crossref PubMed Scopus (81) Google Scholar). Members of the same small group of proteinases also process precursors for laminin 5 (19Amano S. Scott I.C. Takahara K. Koch M. Champliaud M.-F. Gerecke D.R. Keene D.R. Hudson D.L. Nishiyama T. Lee S. Greenspan D.S. Burgeson R.E. J. Biol. Chem. 2000; 275: 22728-22735Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 20Veitch D.P. Nokelainen P. McGowan K.A. Nguyen T.-T. Nguyen N.E. Stephenson R. Pappano W.N. Keene D.R. Spong S.M. Greenspan D.S. Findell P.R. Marinkovich M.P. J. Biol. Chem. 2003; 278: 15661-15668Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and type VII collagen (21Rattenholl A. Pappano W.N. Koch M. Keene D.R. Kadler K.E. Sasaki T. Timpl R. Burgeson R.E. Greenspan D.S. Bruckner-Tuderman L. J. Biol. Chem. 2002; 277: 26372-26378Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), both of which are involved in securing epithelia to underlying basement membrane-like structures (22Burgeson R.E. Christiano A.M. Curr. Opin. Cell Biol. 1997; 9: 651-658Crossref PubMed Scopus (233) Google Scholar); and for dentin matrix protein-1 (23Steiglitz B.M. Ayala M. Narayanan K. George A. Greenspan D.S. J. Biol. Chem. 2004; 279: 980-986Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), which is involved in initiating mineralization in the ECM of bones and teeth (24Butler W.T. Eur. J. Oral Sci. 1998; 106: 204-210Crossref PubMed Scopus (185) Google Scholar). The mammalian BMP-1-related proteinases are all capable of activating the TGF-β-like protein growth differentiation factor 8 (also known as myostatin), by freeing it from a non-covalent latent complex with its cleaved prodomain (25Wolfman N. McPherron A.C. Pappano W.N. Davies M.V. Song K. Tomkinson K.N. Wright J.F. Zhao L. Sebald S.M. Greenspan D.S. Lee S.-J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15842-15846Crossref PubMed Scopus (363) Google Scholar). Similarly, BMP-1 and mTLL-1, but not mTLD or mTLL-2, are able to free the TGF-β-like morphogens BMP-2 and BMP-4 from latent complexes with the extracellular antagonist chordin (5Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W.Y. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (233) Google Scholar, 9Pappano W.N. Steiglitz B.M. Scott I.C. Keene D.R. Greenspan D.S. Mol. Cell. Biol. 2003; 23: 4428-4438Crossref PubMed Scopus (105) Google Scholar). Thus, BMP-1-like proteinases may orchestrate formation of the ECM with signaling by various TGF-β-like proteins in morphogenetic and homeostatic events. The small leucine-rich proteoglycans (SLRPs) constitute a small family of secreted proteoglycans/glycoproteins with structurally related core proteins, with 11 members in mammals (26Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1349) Google Scholar, 27Hocking A.M. Shinomura T. McQuillan D.J. Matrix Biol. 1998; 17: 1-19Crossref PubMed Scopus (416) Google Scholar, 28Iozzo R.V. J. Biol. Chem. 1999; 274: 18843-18846Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 29Henry S.P. Takanosu M. Boyd T.C. Mayne P.M. Eberspaecher H. Zhou W. de Crombrugghe B. Höök M. Mayne R. J. Biol. Chem. 2001; 276: 12212-12221Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 30Lorenzo P. Aspberg A. Onnerfjord P. Bayliss M.T. Neame P.J. Heinegard D. J. Biol. Chem. 2001; 276: 12201-12211Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). SLRP core proteins contain a central domain consisting of 6–12 tandem leucine-rich repeats, and flanking NH2- and COOH-terminal domains containing characteristic cysteine clusters. The SLRPs can be divided into three subclasses, based on the intron/exon organization of cognate genes, spacing of the four cysteine residues in NH2-terminal regions, and the number of leucine-rich repeats in the central domains. Class I comprises the chondroitin/dermatan sulfate chain-containing proteoglycans decorin and biglycan, and the glycoprotein asporin; class II comprises the keratan sulfate chain-containing proteoglycans fibromodulin, lumican, keratocan, and osteoadherin, and the glycoprotein PRELP; and class III consists of osteoglycin (also known as mimecan) and epiphycan, both of which carry a glycosaminoglycan chain, and the glycoprotein opticin. Whereas evidence is lacking for processing of precursors to mature forms of class II SLRPs, biglycan is cleaved from a precursor to the mature protein by BMP-1-related proteinases, in vitro and in vivo (31Scott I.C. Imamura Y. Pappano W.N. Troedel J.M. Recklies A.D. Roughley P.J. Greenspan D.S. J. Biol. Chem. 2000; 275: 30504-30511Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Because biglycan appears to be a positive regulator of bone growth (32Xu T. Bianco P. Fisher L.W. Longenecker G. Smith E. Goldstein S. Bonadio J. Boskey A. Heegaard A.-M. Sommer B. Satomura K. Dominguez P. Chengyan Z. Kulkarni A.B. Robey G. Young M.F. Nat. Genet. 1998; 20: 78-82Crossref PubMed Scopus (389) Google Scholar), involvement of the BMP-1-related proteinases in biglycan biosynthesis conforms to other roles of these proteinases in formation of bone, through formation and mineralization of ECM and through activation of TGF-β-like BMPs. Interestingly, the two class III SLRPs, osteoglycin (OGN) (33Funderburgh J.L. Corpuz L.M. Roth M.R. Funderburgh M.L. Tasheva E.S. Conrad G.W. J. Biol. Chem. 1997; 272: 28089-28095Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and epiphycan (34Johnson H.K. Rosenberg L. Choi H.U. Garza S. Höök M. Neame P.J. J. Biol. Chem. 1997; 272: 18709-18717Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) appear to be proteolytically processed in vivo and the cleavage sites have features resembling those of the cleavage sites of known substrates of BMP-1-like proteinases. Epiphycan, also known as PG-Lb, is localized to the ECM of growth plate chondrocytes and may play structural and/or instructional roles in the integrity and development of this tissue (34Johnson H.K. Rosenberg L. Choi H.U. Garza S. Höök M. Neame P.J. J. Biol. Chem. 1997; 272: 18709-18717Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 35Johnson J. Shinomura T. Eberspaecher H. Pinero G. Decrombrugghe B. Höök M. Dev. Dyn. 1999; 216: 499-510Crossref PubMed Google Scholar). OGN is one of the three major keratan sulfate-containing proteoglycans in cornea (33Funderburgh J.L. Corpuz L.M. Roth M.R. Funderburgh M.L. Tasheva E.S. Conrad G.W. J. Biol. Chem. 1997; 272: 28089-28095Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), and may play a role in regulating collagen fibrillogenesis (36Tasheva E.S. Koester A. Paulsen A.Q. Garrett A.S. Boyle D.L. Davidson H.J. Song M. Fox N. Conrad G.W. Mol. Vis. 2002; 8: 407-415PubMed Google Scholar). Here we extend the known range of substrates in general and of SLRP substrates in particular of the BMP-1-like proteinases, by demonstrating that such proteinases play roles in biosynthetic processing of the class III SLRP OGN. Moreover, cleavage by these proteinases is shown to affect the ability of OGN to regulate the fibrillogenesis of type I collagen. Pro-OGN Expression Vector—An 897-bp cDNA fragment encoding full-length pro-OGN was amplified by PCR using an EST clone, image number 5067073 (ATCC), as a template, Pfu DNA polymerase (Stratagene) and two primers, 5′-CGGGATCCATGGAGACTGTGCACTCTACATTTCTC-3′ and 5′-GGAATTCCTTAGAAGTATGACCCTATGGGTAATC-3′. Use of these primers created a BamHI site at the 5′ end and an EcoRI site at the 3′ end of the fragment (restriction site sequences are in boldface), which was inserted between BamHI and EcoRI sites of pBluescript II SK(+) (Stratagene). The full-length pro-OGN cDNA was then used as a template for PCR amplification of full-length pro-OGN sequences to which a His6 tag was fused via a thrombin cleavage site to the NH2 terminus. Primers were 5′-CGCGGATCCTCACCATCATCACCATCACCTGGTTCCGCGTGGATCTGCACCACAGTCGCAGCTGGAC-3′ and 5′-GGAATTCCTTAGAAGTATGACCCTATGGGTAATC-3′. These added a BamHI site to the 5′ end and an EcoRI site to the 3′ end (restriction site sequences are in boldface) of the amplified fragment, which was inserted between BamHI and EcoRI sites of pAcGP67-A (BD Pharmingen). Predicted sequences of the resulting expression vector, pOGN, were confirmed by DNA sequence analysis. Oligonucleotide primers were synthesized by IDT, Inc. Insect Cell Culture—Spodoptera frugiperda 9 (Sf9) cells (obtained from Dr. Mary Estes, Baylor College of Medicine, Houston, TX) were grown and maintained on Grace's insect cell medium (Invitrogen) containing 10% (v/v) fetal bovine serum (Gemini Bio-products), lactalbumin hydrolysate (3.33 mg/liter), yeastolate (3.33 mg/liter), and l-glutamine. Spinner cultures were started at an initial density of 5 × 105 cells/ml with stirring at 80–90 rpm. The spinner cultures were subcultured when the cell density reached ∼3 × 106 cells/ml. Expression and Purification of Recombinant Pro-OGN—Monolayers of exponentially growing Sf9 insect cells (2 × 106 cells in a 60-mm plate) were co-transfected with 5 μg of transfer vector pOGN and 0.5 μg of linearized BaculoGold virus DNA (BD Pharmingen) in transfection buffer (BD Pharmingen). Recombinant baculoviruses, vOGN, were selected and purified three times by plaque assay. Positive colonies were confirmed by purification of secreted pro-OGN and identification via Western blot using affinity purified polyclonal rabbit antibodies raised against a synthetic peptide derived from the COOH-terminal region of murine OGN (amino acid 255–298). Positive colonies were used to propagate high titer stocks (108–109 plaque forming units/ml), which were stored at 4 °C. For a scale-up of protein production, Sf9 insect cells were maintained in spinner flasks (Bellco Glass, Inc.) and the recombinant vOGN virus stocks were used at a multiplicity of infection of 10 to infect Sf9 insect cells at a density of 3 × 106 cells/ml. At 1 h post-infection, recombinant virus-infected cells were harvested and resuspended in serum-free medium (BD Pharmingen). At 96 h post-infection conditioned medium was collected, centrifuged, and filtered using a 0.2-μm membrane filter. The supernatant was then applied to the Labscale TFF system (Millipore, Co.) with the Pellicon XL module (Biomax 10) for concentration of the sample volume and buffer change to 20 mm Tris, pH 8.0. The sample was then applied to a 5-ml High Trap metal chelating column charged with Ni2+ (Amersham Biosciences), which was washed with 30 mm imidazole buffer containing 20 mm Tris, pH 8.0, 0.5 m NaCl, and 0.02% CHAPS, after which bound protein was eluted from the resin with the same buffer containing 100 mm imidazole. Eluted fractions were analyzed by SDS-PAGE, and the peak fractions were pooled and concentrated by Centricon-10 spin columns (Amicon). The mass of purified protein was determined by MALDI-TOF mass spectrometry (Tufts University, Protein chemistry facility). Prior to analysis the proteins were dialyzed into 10 mm NH4HCO3. SDS-PAGE and Western Blotting—SDS-PAGE was performed with 10 (Figs. 1 and 2), 15 (Figs. 4 and 5), and 12% (Fig. 6) polyacrylamide gels with 4% stacking gels and the Laemmli buffer system (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). For Western blots, proteins separated on SDS-PAGE were transferred to nitrocellulose membranes and probed with affinity purified polyclonal rabbit antibodies raised against a synthetic peptide derived from the COOH-terminal region of murine OGN (amino acids 255–298). Horse-radish peroxidase-conjugated goat anti-rabbit affinity purified immunoglobulin G (heavy and light chains) (Bio-Rad) was used as the secondary antibody, and SuperSignal chemiluminescent substrate (Pierce) was used for signal detection. Prestained broad-range marker proteins (Bio-Rad) were used as molecular mass standards. A monoclonal mouse antibody (anti-Penta His, Qiagen) and horseradish peroxidase-conjugated goat anti-mouse affinity purified immunoglobulin G (Bio-Rad) were used as first and secondary antibodies, respectively, for detecting the recombinant pro-OGN His tag.Fig. 2Mass spectrometry and peptide N-glycosidase F digestion of recombinant OGN.A, the mass of recombinant OGN, dialyzed overnight against 10 mm NH4HCO3 at 4 °C, was determined by MALDI-TOF mass spectrometry. B, recombinant OGN was analyzed by SDS-PAGE and Coomassie Blue staining without further treatment (lane 2) or after incubation with peptide N-glycosidase F (lane 3). N-Glycosidase F enzyme appears as a band of higher mobility than OGN in lanes 3 and 4, (marked with an asterisk).View Large Image Figure ViewerDownload (PPT)Fig. 4BMP-1 cleaves pro-OGN to produce two detectable fragments. Electrophoretic patterns on an SDS-PAGE gel are compared for recombinant pro-OGN incubated in the absence (–) or presence (+) of BMP-1. MW denotes molecular mass markers, with approximate sizes marked in kDa. Pro-OGN, Mat, and Propep denote pro-OGN, mature OGN, or the cleaved OGN prodomain, respectively. Bands were visualized by staining with Coomassie Blue.View Large Image Figure ViewerDownload (PPT)Fig. 5Cleavage assays of recombinant pro-OGN incubated separately with the four mammalian BMP-1-related proteinases. Western blots are shown of recombinant pro-OGN incubated alone (No enzyme), or in the presence of BMP-1 (BMP1), mTLD (TLD), mTLL-1 (TLL1), or mTLL-2 (TLL2). S.M. denotes starting material. Immunoblots employed either antibodies directed against the NH2-terminal His tag (α His), or antibodies directed against sequences within mature OGN (α OGN). α His antibodies detected only 39-kDa pro-OGN, whereas α OGN antibodies detected both 39-kDa pro-OGN and the 27-kDa OGN cleavage product.View Large Image Figure ViewerDownload (PPT)Fig. 6Absence of proteolytic processing of pro-OGN in cultures of MEF from embryos doubly homozygous null for the Bmp1 and Tll1 genes. Western blot analysis was employed, using antibodies specific for sequences within the mature region of OGN, to monitor pro-OGN processing within cultures of wild-type (WT) MEF or MEF doubly homozygous null (Bmp1/Tll1 null) for the Bmp1 and Tll1 genes. Cultures were either treated (+) or not treated (–) with 2 ng/ml TGF-β. α OGN antibodies detected the 39-kDa pro-OGN and the 27-kDa OGN cleavage products in TGF-β-treated WT MEF cultures, but only detected 39-kDa pro-OGN in TGF-β-treated Bmp1/Tll1 null MEF cultures.View Large Image Figure ViewerDownload (PPT) Peptide N-glycosidase F Digestion—Purified recombinant pro-OGN was denatured with 0.5% SDS, 1% 2-mercaptoethanol for 10 min at 100 °C and was then treated with 25 units of peptide N-glycosidase F (New England Biolabs) in 50 mm sodium phosphate, pH 7.5, 1% Nonidet P-40 at 37 °C for 16 h. After incubation, samples were immediately subjected to SDS-PAGE analysis. Thrombin Cleavage and Purification of Non-tagged Pro-OGN—Purified recombinant pro-OGN was treated with 1 unit of biotinylated thrombin (Novagen) in 20 mm Tris-HCl, pH 8.4, 150 mm NaCl, 0.25 mm CaCl2 at room temperature for 16 h. After incubation, samples were sequentially subjected to streptavidin-agarose column and Ni2+-charged metal chelating column chromatography to remove the biotinylated thrombin and the cleaved His tag, respectively. Cleavage of the His6 tag was confirmed by SDS-PAGE and Western blot analysis, using anti-His antibody. Prior to use in experiments non-tagged proteins were dialyzed into phosphate-buffered saline. Collagen Fibrillogenesis Assay—Fibrillogenesis assays were performed as described previously (38Birk D.E. Silver F.H. Arch. Biochem. Biophys. 1984; 235: 178-185Crossref PubMed Scopus (82) Google Scholar). Briefly, at 4 °C, stock solutions (1–2 mg ml–1) of bovine dermal type I collagen (VITROGEN, Palo Alto, CA) were neutralized with 10× phosphate-buffered saline and brought to a concentration of 0.5 mg ml–1 with 1× phosphate-buffered saline, pH 7.2. Recombinant protein was added to aliquots of the mixture at 1–60 μg ml–1, samples were transferred to a 96-well plate in a SpectraMax Plus 384 Microplate Spectrophotometer (Molecular Devices Co.), and assays were performed at 37 °C. In Vitro Enzyme Assays—500 ng of recombinant pro-OGN was incubated alone or in combination with 15 ng of recombinant BMP-1, mTLD, mTLL-1, or mTLL-2, containing COOH-terminal FLAG epitopes, in 20 μl total volume of 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10 mm CaCl2, and incubated 20 h at 37 °C. Reactions were stopped by addition of 5× SDS-PAGE sample buffer (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) containing 1% 2-mercaptoethanol, and boiling of samples for 5 min. 50 ng of proteins per sample were subjected to SDS-PAGE and subsequent Western blot analysis, as described above. Amino Acid Sequence Analysis—2 μg of purified recombinant pro-OGN was incubated with 50 ng of BMP-1 at 37 °C for 20 h, and the reaction was quenched and prepared for SDS-PAGE as above. Products were resolved by SDS-PAGE on a 12% polyacrylamide gel and electrotransferred to Sequi-Blot polyvinylidene difluoride membrane (BioRad). Proteins were revealed with 0.025% Coomassie Brilliant Blue R-250, and NH2-terminal amino acid sequences were determined by automated Edman degradation at the Harvard University Microchemistry Facility using a PerkinElmer/Applied Biosystems Division Procise 494 HT Protein Sequencing System. Mouse Embryo Fibroblasts (MEFs)—MEFs were isolated from 13.5-day post-conception embryos as previously described (8Suzuki N. Labosky P.A. Furuta Y. Hargett L. Dunn R. Fogo A.B. Takahara K. Peters D.M. Greenspan D.S. Hogan B.L. Development. 1996; 122: 3587-3595Crossref PubMed Google Scholar). Cells were maintained in growth medium consisting of Dulbecco's modified Eagle's medium, 1 mm L-glutamine, 10 IU/ml penicillin/streptomycin, and 10% fetal bovine serum, and were immortalized by routine serial passage. To detect endogenous OGN protein in MEFs, wild-type or Bmp1/Tll1 doubly homozygous null cells at 80% confluence were washed twice with phosphate-buffered saline, and incubated in serum-free Dulbecco's modified Eagle's medium for 15 min at 37 °C. Cells were then treated with or without 2 ng/ml TGF-β1 (R&D Systems) in serum-free Dulbecco's modified Eagle's medium for 48 h. Proteins in conditioned media were ethanol precipitated and resuspended in 0.5% SDS, 1% 2-mercaptoethanol. Deglycosylation of precipitated media proteins with β1→4 galactosidase was performed according to the manufacturer's protocol (Sigma). Expression, Purification, and Characterization of Recombinant OGN—The murine full-length pre-OGN protein contains 298 amino acid residues that include a signal peptide, six cysteine residues; four within the NH2- and two within the COOH-terminal domains, respectively; six leucine-rich repeats in the central domain, and a single potential N-glycosylation site at amino acid residue 258 (Fig. 1A). To generate recombinant OGN using a baculovirus expression system, a transfer vector, pOGN, was constructed using a cDNA encoding the core protein of murine OGN, such that a fusion protein would be produced corresponding to the acidic glycoprotein gp67 signal sequence, followed by six consecutive histidine residues, a thrombin cleavage site, and the full OGN coding sequence (Fig. 1B). Recombinant baculovirus vOGN was then generated by homologous recombination between transfer vector pOGN and linearized baculoGold virus DNA. Sf9 cells infected with vOGN were incubated in serum-free medium and 96 h later culture medium was harvested and recombinant His-tagged protein was purified by a single passage over a nickel chelating column. The concentration of purified samples was estimated using the theoretical molar extinction coefficient (39Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar) and analyzed by SDS-PAGE and staining with Coomassie Blue (Fig. 1C). Recombinant protein was predominantly eluted from the nickel column at 100 mm imidazole and no contaminating protein bands were visualized in this fraction via SDS-PAGE, indicating a high degree of purity for the sample. The yield of recombinant OGN was ∼5 mg/3.5 × 109 cells per 96 h. Identity of the recombinant protein was confirmed by Western blot analysis (Fig. 1D) using a polyclonal antiserum specific for mouse OGN, with detection of a single band of recombinant OGN (Fig. 1D, lane 3), and no signal detected from samples derived from uninfected control cells (Fig. 1D, lane 1) or from control cells infected with wild-type baculovirus AcNMPV (Fig. 1D, lane 2). Recombinant OGN appeared on SDS-PAGE, under reducing conditions as a single ∼39-kDa band (Figs. 1C and 2), whereas the expected mass of murine OGN is 33,245 Da, based on the primary amino acid sequence. MALDI-TOF MS yielded a peak with a mass of 34,557 Da for recombinant OGN (Fig. 2A). One possible explanation for the mass difference between MALDI-TOF MS results and the expected mass might have been glycosylation of recombinant OGN with the N-linked high mannose oligosaccharides characteristic of insect cells. To test this possibility, recombinant OGN was incubated with peptide N-glycosidase F, which cleaves between the innermost N-acetylglucosamine and asparagine residues of N-linked high mannose oligosaccharides. Upon enzyme digestion, OGN protein underwent a shift to a higher mobility form (Fig. 2B, lanes 2 and 3). Thus, the re" @default.
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• W2016643048 title "Bone Morphogenetic Protein-1/Tolloid-related Metalloproteinases Process Osteoglycin and Enhance Its Ability to Regulate Collagen Fibrillogenesis" @default.
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