Post-translational Modification of Bone Morphogenetic Protein-1 Is Required for Secretion and Stability of the Protein
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m207342200
ISSN1083-351X
AutoresLaure Garrigue‐Antar, Nichola Hartigan, Karl E. Kadler,
Tópico(s)Orthopaedic implants and arthroplasty
ResumoBone morphogenetic protein (BMP)-1 is a glycosylated metalloproteinase that is fundamental to the synthesis of a normal extracellular matrix because it cleaves type I procollagen, as well as other precursor proteins. Sequence analysis suggests that BMP-1 has six potential N-linked glycosylation sites (i.e. NXS/T) namely: Asn91 (prodomain), Asn142 (metalloproteinase domain), Asn332 and Asn363 (CUB1 domain), Asn599 (CUB3 domain), and Asn726 in the C-terminal-specific domain. In this study we showed that all these sites are N-glycosylated with complex-type oligosaccharides containing sialic acid, except Asn726 presumably because proline occurs immediately C-terminal of threonine in the consensus sequence. Recombinant BMP-1 molecules lacking all glycosylation sites or the three CUB-specific sites were not secreted. BMP-1 lacking CUB glycosylation was translocated to the proteasome for degradation. BMP-1 molecules lacking individual glycosylation sites were efficiently secreted and exhibited full procollagen C-proteinase activity, but N332Q and N599Q exhibited a slower rate of cleavage. BMP-1 molecules lacking any one of the CUB-specific glycosylation sites were sensitive to thermal denaturation. The study showed that the glycosylation sites in the CUB domains of BMP-1 are important for secretion and stability of the molecule. Bone morphogenetic protein (BMP)-1 is a glycosylated metalloproteinase that is fundamental to the synthesis of a normal extracellular matrix because it cleaves type I procollagen, as well as other precursor proteins. Sequence analysis suggests that BMP-1 has six potential N-linked glycosylation sites (i.e. NXS/T) namely: Asn91 (prodomain), Asn142 (metalloproteinase domain), Asn332 and Asn363 (CUB1 domain), Asn599 (CUB3 domain), and Asn726 in the C-terminal-specific domain. In this study we showed that all these sites are N-glycosylated with complex-type oligosaccharides containing sialic acid, except Asn726 presumably because proline occurs immediately C-terminal of threonine in the consensus sequence. Recombinant BMP-1 molecules lacking all glycosylation sites or the three CUB-specific sites were not secreted. BMP-1 lacking CUB glycosylation was translocated to the proteasome for degradation. BMP-1 molecules lacking individual glycosylation sites were efficiently secreted and exhibited full procollagen C-proteinase activity, but N332Q and N599Q exhibited a slower rate of cleavage. BMP-1 molecules lacking any one of the CUB-specific glycosylation sites were sensitive to thermal denaturation. The study showed that the glycosylation sites in the CUB domains of BMP-1 are important for secretion and stability of the molecule. bone morphogenetic protein-1 bone morphogenetic protein-1 containing a FLAG tag at the C terminus procollagen C proteinase complement subcomponents C1r/C1s Uegf andbone morphogenetic protein-1 endoplasmic reticulum boar seminal plasma protein bovine acidic seminal fluid protein Dulbecco's modified Eagle's medium endopeptidase F/N-glycosidase F endoglycosidase H phosphate-buffered saline Bone morphogenetic protein-1 (BMP-1),1 also known as procollagen C-proteinase-1 (PCP-1) was first identified in osteogenic extracts of bone (1Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3363) Google Scholar). BMP-1 is a smaller splice variant of mammalian tolloid, which is the vertebrate ortholog of tolloid, inDrosophila. BMP-1, mammalian tolloid, and two highly homologous relatives tolloid-like 1 and 2 constitute the tolloid clade of astacin-like metalloproteinases that have important functions in development and extracellular matrix formation (2Scott 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. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (233) Google Scholar). BMP-1 cleaves fibrillar procollagen type I, II, III (3Hojima Y. van der Rest M. Prockop D.J. J. Biol. Chem. 1985; 260: 15996-16003Abstract Full Text PDF PubMed Google Scholar, 4Kessler E. Adar R. Goldberg B. Niece R. Collagen Relat. Res. 1986; 6: 249-266Crossref PubMed Scopus (47) Google Scholar), and V (5Imamura Y. Steiglitz B.M. Greenspan D.S. J. Biol. Chem. 1998; 273: 27511-27517Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 6Kessler E. Fichard A. Chanut-Delalande H. Brusel M. Ruggiero F. J. Biol. 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Bmp1homozygous null mice are perinatal lethal, with defects in ventral body wall closure and collagen fibrillogenesis (14Suzuki 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), which illustrates the importance of BMP-1 in tissue assembly and development. BMP-1 consists of a prodomain that is cleaved by a furin-like enzyme in the trans-Golgi network, 2M. Leighton and K. E. Kadler, manuscript in preparation. an astacin-like zinc metalloproteinase domain (15Dumermuth E. Sterchi E.E. Jiang W.P. Wolz R.L. Bond J.S. Flannery A.V. Beynon R.J. J. Biol. Chem. 1991; 266: 21381-21385Abstract Full Text PDF PubMed Google Scholar), one epidermal growth factor-like domain, and three CUB domains. In other proteins, CUB domains mediate protein-protein interactions (16Bork P. Beckmann G. J. Mol. Biol. 1993; 231: 539-545Crossref PubMed Scopus (523) Google Scholar). BMP-1 purified from mouse fibroblasts culture medium has been shown to be N-glycosylated (17Kessler E. Takahara K. Biniaminov L. Brusel M. Greenspan D.S. Science. 1996; 271: 360-362Crossref PubMed Scopus (458) Google Scholar). Sequence analysis reveals six potential N-glycosylation sites, one of which is located in the prodomain and five in the mature (active) molecule. However, no information is available on the structure and function of the glycosylation sites. Recent studies indicate that post-translational modification of proteins can have multiple roles including regulation of intracellular trafficking (18Martina J.A. Daniotti J.L. Maccioni H.J. J. Biol. Chem. 1998; 273: 3725-3731Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), stabilization of folded domains (19Yasuda Y. Ikeda S. Sakai H. Tsukuba T. Okamoto K. Nishishita K. Akamine A. Kato Y. Yamamoto K. Eur. J. Biochem. 1999; 266: 383-391Crossref PubMed Scopus (25) Google Scholar,20Mimura Y. Sondermann P. Ghirlando R. Lund J. Young S.P. Goodall M. Jefferis R. J. Biol. Chem. 2001; 276: 45539-45547Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), protection from proteolytic degradation of the core protein (21Sareneva T. Pirhonen J. Cantell K. Julkunen I. Biochem. J. 1995; 308: 9-14Crossref PubMed Scopus (88) Google Scholar), and modulation of enzyme/hormone activities (22Kadowaki T. Tsukuba T. Bertenshaw G.P. Bond J.S. J. Biol. Chem. 2000; 275: 25577-25584Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 23Lin X. Koelsch G., Wu, S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (741) Google Scholar, 24Charlwood J. Dingwall C. Matico R. Hussain I. Johanson K. Moore S. Powell D.J. Skehel J.M. Ratcliffe S. Clarke B. Trill J. Sweitzer S. Camilleri P. J. Biol. Chem. 2001; 276: 16739-16748Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Furthermore, high variability exists in the functions of N-glycan chains of proteins, which makes it difficult to predict with any confidence the functions of oligosaccharides on proteins (reviewed by Ref. 25Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (5000) Google Scholar). In this study, we expressed FLAG-tagged BMP-1 in two different mammalian systems (HT1080 and 293-EBNA cells) and identified the type of glycosylation on BMP-1. By site-directed mutagenesis, we also established the role of the N-glycosylation in folding, secretion, and C-proteinase activity of BMP-1. PCR products were purified with Qiaquick kits (Qiagen). Plasmids were extracted with Qiaprep spin miniprep kit (Qiagen). Prestained protein molecular weight standards (broad range) were from Bio-Rad. Full-length BMP-1 cDNA (GenBankTM accession number P13497) was cloned from a human placental cDNA library. The cDNA was inserted at theKpnI/XhoI sites of the expression vector pcDNA3 (Invitrogen), thereby placing it under the transcriptional control of a cytomegalovirus promoter. A FLAG tag amino acid sequence (DYKDDDDK) recognized by a mouse monoclonal anti-FLAG M2 antibody (Sigma) was introduced into the BMP-1 sequence (BMP-1F) immediately 5′ of the stop codon. The cDNA encoding FLAG-tagged BMP-1 was subcloned into the episomal expression vector pCEP4 (Invitrogen) and pcDNA3, for heterologous protein expression studies in cultured cells. Previous studies have shown that the FLAG peptide at the C terminus of BMP-1 does not affect the procollagen C-proteinase activity of BMP-1 (2Scott 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. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (233) Google Scholar). Plasmids coding for the mutant BMP-1 proteins were produced by replacing wild-type fragments with the same fragments containing the desired mutations. These were generated by strand overlap PCR as described (26Horton R.M. White B.A. PCR Protocols: Current Methods and Applications. 15. Humana Press Inc., Totowa, NJ1993: 251-261Google Scholar) using Pwo polymerase (Roche Molecular Biochemicals), a forward primer upstream and a reverse primer downstream, of unique restriction sites, respectively, and oligonucleotides containing the desired modification in both orientations (in bold, see below). For each mutation, the restriction sites used and their positions on the nucleotide sequence are indicated. Briefly, a DNA fragment was amplified using a forward primer and the antisense mutant primer and an overlapping fragment was amplified using the sense mutant primer and a downstream reverse primer. Both fragments were gel-purified (Qiagen), mixed, and re-amplified with the Pwo enzyme with the forward and reverse primers. The product was digested by the appropriate enzymes, gel-purified, and introduced in place of the corresponding wild-type fragment in BMP-1F. Mutagenic primers (mutation in bold) and restriction sites used to insert the mutant fragment into the wild-type sequence were as follows: N142Q, 5′-CATTGGGGGACAGTTCACTGGTA-3′, XcmI (position 383)/BlpI (position 913); N332Q, 5′-CAGCACAGGCCAGTTCTCCT-3′, BlpI (position 913)/PmlI (position 1416); N363Q, 5′-GATCATCCTGCAATTCACGTCCCT-3′, BlpI (position 913)/PmlI (position 1416); N599Q, 5′-CCTCACCAAGCTCCAAGGCTCCATCA-3′, BamHI (position 1391)/XhoI (multicloning site of pcDNA3). For N91Q (5′-AAAAGCTGCAGTTCCAGGACAGACTTCTAC-3′) and N726Q (5′-CCCCCCTCGAGTCACTTGTCATCGTCGTCCTTGTAGTCCTGGGGGGTCCGTTGTCTTTTCTGCACT-3′), the mutagenic primers, which also contained a restriction site (underlined, N91Q), PstI (position 290), N726Q,XhoI (multicloning site of pcDNA3), and the FLAG tag (N726Q, italicized), were used in a single PCR reaction with reverse and forward primers, respectively. The two mutated PCR products were digested by PstI (position 251) and ApaI (position 586) (N91Q) or BamHI (position 1391) andXhoI (N726Q). Pwo DNA polymerase was used to minimize base misincorporation during the polymerase chain reactions. DNA sequencing (ABI) was used to verify the mutations, and to ensure that the cDNA clones were error-free. Human fibrosarcoma HT1080 cells (ATCC CCL-121) and human embryonic kidney 293-EBNA cells (ECACC 85120602) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen) (complete DMEM) and for the 293 cells, with 0.25 mg/ml Geneticin (G418, Invitrogen) in a 37 °C incubator with 5% CO2. The recombinant wild-type and mutant BMP-1F proteins were expressed in stably transfected HT1080 cells. Transfections were made with Lipofectin reagent (Roche Molecular Biochemicals) and 4 μg of plasmid/T-25 flask. Cells were grown to ∼50% confluency by overnight incubation in complete DMEM. After 2 rinses with Opti-MEM (Invitrogen), cells were transfected in serum-free Opti-MEM (Invitrogen) following the manufacturer's instructions and returned to the incubator. Twenty-four hours after transfection, medium was replaced by DMEM with 10% serum for a further 24 h. The cells were then trypsinized (Invitrogen) and diluted 1:15 for selection in 0.25 mg/ml Geneticin (G418, Invitrogen). 293-EBNA cells were transfected with the FLAG-tagged BMP-1 cloned into pCEP4 expression plasmid, following the procedure described above. Selection was applied by adding 0.25 mg/ml hygromycin (Invitrogen). Stably transfected HT1080 or 293-EBNA cells were seeded in 100-mm dishes and grown to confluency. Cells were rinsed three times with phosphate-buffered saline (Invitrogen) and incubated in serum-free DMEM for 24 (HT1080) or 48 h (293-EBNA cells), unless otherwise stated. The tissue culture media were collected, cleared of cell debris by centrifugation at 1600 × g for 10 min, and in the case of HT1080-transfected cells, concentrated to 100 μl using Centriprep-30 and Microcon-10 concentrators (Amicon, Inc.). The samples were used immediately or stored at −80 °C. Recombinant BMP-1 was assayed for procollagen C-proteinase activity using humanl-U-14C-type I procollagen substrate (0.4 μg). 14C-Labeled type I procollagen was obtained and purified from the medium of human skin fibroblasts (27Kadler K.E. Hojima Y. Prockop D.J. J. Biol. Chem. 1987; 262: 15696-15701Abstract Full Text PDF PubMed Google Scholar). Analysis of the cleavage products on SDS gels (7% separating, 3.5% stacking) was performed as described (3Hojima Y. van der Rest M. Prockop D.J. J. Biol. Chem. 1985; 260: 15996-16003Abstract Full Text PDF PubMed Google Scholar, 28Garrigue-Antar L. Barker C. Kadler K.E. J. Biol. Chem. 2001; 276: 26237-26242Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The cleavage products were visualized by exposing dried gels to a phosphorimaging plate (Fuji, type BAS III) for phosphorimaging (Fujix BAS 2000). Bands corresponding to the pro-α1(I) and pN-α2(I) chains of type I procollagen and type I pN-collagen, respectively, were quantified using AIDA 2.0 software. The percentage of cleavage was calculated by multiplying the intensity of the pN-α2(I), corrected for molecular mass, by the initial concentration of procollagen (3Hojima Y. van der Rest M. Prockop D.J. J. Biol. Chem. 1985; 260: 15996-16003Abstract Full Text PDF PubMed Google Scholar, 28Garrigue-Antar L. Barker C. Kadler K.E. J. Biol. Chem. 2001; 276: 26237-26242Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Cells were rinsed once with PBS, and incubated on ice for 15 min with occasional shaking with 500 μl of RIPA buffer (150 mm NaCl, 1% deoxycholate, 0.1% SDS, 10 mm Tris pH 7.6) containing 10 mmEDTA, and protease inhibitor mixture (Roche Molecular Biochemicals). Cells in RIPA buffer were scraped on ice and sonicated. Lysates were subjected to a 5-min centrifugation at 14,000 × g at 4 °C. Supernatants were retained and stored at −80 °C until further analysis. The supernatants or cell lysates were resolved by electrophoresis on a 10% (w/v) SDS-Prosieve gel (Biowhittaker Molecular Applications) under reducing conditions and subjected to Western immunoblotting using the mouse monoclonal M2 antibody (Sigma) directed against the FLAG tag. Secondary antibody (anti-mouse peroxidase-conjugated IgG (Sigma)) was detected by the enhanced chemiluminescence method (SuperSignal West Dura extended duration, Pierce). The levels of BMP-1F were quantified by laser densitometry of enhanced chemiluminescence fluorograms exposed to pre-flashed films. Endopeptidase F/N-glycosidase F (endoF/N), endoglycosidase H (endoH), and neuraminidase were from New England Biolabs, and endo-α-N-acetylgalactosaminidase (O-glycosidase) was from Calbiochem. Culture medium (80 μl) (unconcentrated in the case of transfected 293-EBNA cells) from transfected HT1080 were incubated with the different glycosidases, in the buffer, detergents, and conditions recommended by the manufacturers. EndoF/N (500 units), endoH (500 units), neuraminidase (50 units), or O-glycosidase (1 milliunit) were used per reaction, which were incubated for 18 h at 37 °C. Digestion byO-glycosidase was preceded by digestion with neuraminidase. Stably transfected HT1080 or 293-EBNA cells expressing BMP-1F were grown to confluency on 100-mm dishes. Cells were preincubated in complete DMEM in the presence of 100 μg/ml castanospermine (Calbiochem), or 5 μg/ml swainsonine (Calbiochem) for 18 h. For tunicamycin (Sigma), cells were preincubated in the presence of 2 μg/ml of the inhibitor, for 5 h. Cells were then rinsed three times with PBS, and reincubated in the presence of the inhibitors for 24 h in serum-free DMEM, and for only 18 h in the case of tunicamycin. Medium was collected and treated as described above. pcDNA3, BMP-1F, and N4Q-transfected HT1080 were plated on glass coverslips in 6-well plates. After 24 h, cells were preincubated with or without 10 mm lactacystin (Calbiochem) for 1 h. Cells were rinsed three times with PBS and further incubated with or without lactacystin at the same concentration in serum-free medium for 4 h. Cells were washed three times with PBS and fixed and permeabilized with cold methanol (−20 °C) for 5 min. Fixed cells were washed three times with PBS and incubated for 20 min at room temperature with anti-FLAG mouse antibody or with anti-calreticulin rabbit antibody (Stressgen) in PBS supplemented with 1 mg/ml bovine serum albumin (Sigma). After washing, cells were incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma) or with rhodamine-conjugated anti-rabbit IgG (Santa Cruz Biotechnology). Cells were washed with PBS and coverslips were mounted in Mowiol 4-88 (Calbiochem) and observed with a Bio-Rad MRC1000 laser confocal microscope. We transfected cultured HT1080 human fibrosarcoma and 293-EBNA human embryonic kidney cells with cDNAs encoding C-terminal flagged BMP-1 (BMP-1F). We chose to express BMP-1 in two different cell types to minimize the risk of cell type-specific post-translational modification. BMP-1F was stably expressed in HT1080 and 293-EBNA cells and the secreted protein was incubated with endoF/N, which cleaves all N-linked structures, regardless of their complexity, by hydrolyzing the asparagine-oligosaccharide bond, or endoH, which cleaves specifically high mannose-type structures. The proteins were also digested with neuraminidase, which removes sialic acids, and O-glycosidase, which specifically cleavesO-linked glycans. Proteins were separated by SDS-PAGE and immunoblotted with anti-FLAG antibody. Fig.1 shows that BMP-1F is correctly processed and secreted in both HT1080 and 293-EBNA cells (lanes 2 and 9). No immunoreactive bands were found in medium from HT1080 cells transfected with pcDNA3 empty vector (lane 1). Complete deglycosylation of BMP-1F by endoF/N produced a decrease in the molecular mass of the BMP-1F molecule from ∼75 to ∼60 kDa (lane 3), strongly suggesting that most, or all, of the 5 potential N-glycosylation sites present in the active form had been stripped of oligosaccharides. Digestion of BMP-1F with endoH did not produce a shift in mobility, suggesting that BMP-1F secreted by HT1080 cells (and 293-EBNA cells, data not shown) did not contain high mannose-type oligosaccharides (lane 4). Neuraminidase treatment (lanes 6 and 10) decreased the molecular mass of BMP-1F by ∼5 kDa, showing that the BMP-1 contained sialylated sugars. However, further treatment withO-glycosidase had no effect (lane 8), suggesting that the BMP-1 lacked O-linked oligosaccharides. To determine the contribution of the complex-type N-linked oligosaccharides to secretion and enzymic activity of BMP-1F, two inhibitors of glycoprotein processing were used: castanospermine, which inhibits endoplasmic reticulum (ER) glucosidases I and II, thereby preventing the removal of the glucose residues of the Glc3Man7–9(GlcNac)2-N-linked glycoprotein (29Elbein A.D. FASEB J. 1991; 5: 3055-3063Crossref PubMed Scopus (350) Google Scholar), and swainsonine, which inhibits Golgi mannosidase II, therefore producing hybrid-type glycosylation (29Elbein A.D. FASEB J. 1991; 5: 3055-3063Crossref PubMed Scopus (350) Google Scholar). To do these experiments, stably transfected HT1080 cells were incubated in the presence and absence of castanospermine or swainsonine, and the secreted BMP-1F was digested with endoF/N and endoH. The digested proteins were examined by Western blotting, using the anti-FLAG antibody (Fig. 2). BMP-1F from cells cultured with castanospermine (lane 5) had the same electrophoretic mobility as BMP-1F from untreated cells (lane 2), whereas BMP-1F secreted in the presence of swainsonine (lane 8) had a faster mobility (∼70 kDa). The levels of secretion of BMP-1F were not affected by the presence of the inhibitors, compared with the control. To characterize the oligosaccharides on BMP-1F secreted in the presence of the inhibitors, the proteins were incubated with endoF/N and endoH enzymes and the products were examined by SDS-PAGE and Western blotting. Whereas BMP-1F secreted in the absence of inhibitors was resistant to endoH (lane 4), BMP-1F secreted in the presence of either inhibitor was sensitive to this enzyme, indicating that the inhibitors had changed the nature of the oligosaccharides. By comparing the electrophoretic mobility of the bands obtained after endoH and endoF/N digestions, it could be observed that BMP-1F secreted in the presence of castanospermine contained only high-mannose structures, because the two digestion products had the same electrophoretic mobility (lanes 6 and 7). In the case of BMP-1F secreted in the presence of swainsonine, the digestion product by endoH (lane 10) migrated slower than the one obtained with endoF/N (lane 9), denoting the presence of both complex-type and high mannose-type structures. To investigate the effects of high mannose- and hybrid-type oligosaccharides on PCP activity of BMP-1, we assayed the enzymes using type I procollagen as substrate. No significant difference of PCP activity was observed between BMP-1F control, BMP-1F containing high-mannose structures (synthesized in the presence of castanospermine), and BMP-1F with hybrid-type glycosylation (synthesized in the presence of swainsonine). Similar results were obtained with the 293-EBNA cells stably transfected with BMP-1F (data not shown). The results showed that complex-type oligosaccharide chains are not required for secretion and PCP activity of BMP-1. To determine the role of N-linked sugars on folding and secretion of BMP-1, stably transfected 293-EBNA cells were incubated in the presence or absence of tunicamycin, which blocks transfer of N-acetylglucosamine onto the lipid carrier dolichol phosphate (30Elbein A.D. Annu. Rev. Biochem. 1987; 56: 497-534Crossref PubMed Google Scholar). Each medium and cell lysate were collected, the proteins were separated by SDS-PAGE and immunoblotted with anti-FLAG antibody (Fig. 3). Whereas BMP-1F was secreted from untreated cells (lane 1), BMP-1F could not be detected in the medium of tunicamycin-treated cells (lane 2). Latent BMP-1F was present in the cell lysates of treated and untreated cells. However, latent BMP-1F in the tunicamycin-treated cells was ∼70 kDa, and therefore smaller than in control samples. The smaller molecular mass confirmed the presence ofN-linked oligosaccharides on BMP-1. Furthermore, the ease of detection of the latent BMP-1F molecule in treated cells indicated that nonglycosylated BMP-1 accumulated within the cells. Although the tunicamycin experiments confirmed the importance of N-glycosylation for BMP-1 secretion, the contribution of individual sites within the molecule could not be assessed. BMP-1 contains 6 potentialN-glycosylation sites (see Fig.4): Asn91 (prodomain), Asn142 (catalytic domain), Asn332 and Asn363 (CUB1 domain), Asn599 (CUB3 domain), and Asn726 (BMP-1 specific domain). To test the function of each site, we generated mutations in the BMP-1 sequence in which each of the asparagine residues was mutated, by site-directed mutagenesis, to glutamine. In addition to the 6 single mutants, we generated the double CUB1 mutant (N332Q and N363Q), as well as a molecule with Asn to Gln mutations of all 3 CUB sites and the C-terminal-specific site (N4Q), and a molecule in which all 6 potential sites were mutated (N6Q). Wild type and mutant constructs were stably transfected into HT1080 cells. Serum-free conditioned medium was analyzed by Western blotting using the anti-FLAG antibody (Fig.5). All the single mutants (panel A, lanes 3–6, 8, and 9) and the double CUB1 mutant (panel B, lane 2) were secreted. The N4Q and N6Q mutants were not secreted (see lanes 10 and 11). The fact that the fully unglycosylated N6Q mutant was not secreted is in agreement with the result obtained with the cells treated with tunicamycin (Fig. 3). Noteworthy, the N4Q and N6Q mutants were not detected in cell lysates (data not shown).Figure 5Expression of wild-type and mutant BMP-1F in HT1080 cells. Vectors containing cDNA-encoding wild-type and mutant BMP-1F were stably expressed in HT1080 cells. When confluent, cells were rinsed and conditioned in serum-free medium for 24 h. Culture supernatants (A and B) and cell lysates (C) were collected as described under "Experimental Procedures." Protein samples were separated by SDS-PAGE (10%) in reducing conditions and detected by Western blot analysis using the anti-FLAG M2 antibody. A and B, the proteins were secreted as mature BMP-1. The N4Q and N6Q (lanes 10 and11) were not detected. C, the protein present in the cell lysate corresponds to the latent form of BMP-1.pcDNA3, medium (lanes 1, panels Aand B) and cell lysate (lane 1, panel C) from cells transfected with the empty vector.View Large Image Figure ViewerDownload (PPT) The single mutants N142Q, N332Q, N363Q, and N599Q (panel A,lanes 4, 5,6, and 8, respectively) migrated faster than the wild-type BMP-1F (lanes 2 and 7), indicating that these glycosylation sites are occupied by oligosaccharides in the wild-type molecule. In contrast, the N726Q mutant (lane 9) was indistinguishable from BMP-1F, suggesting that this site is not glycosylated in BMP-1. The prodomain mutant, N91Q, was secreted as the mature protein, which migrated with the same electrophoretic mobility as BMP-1F. This was in agreement with the fact that BMP-1F is secreted in its active form, and the absence of prodomain glycosylation did not block the cleavage of the prodomain or change the electrophoretic mobility of the mature enzyme. By analyzing the corresponding cell lysates (where BMP-1F exists in the latent form,panel C, lanes 2 and 3), the N91Q mutant was shown to migrate faster than BMP-1F, indicating that the Asn91 site is glycosylated in latent BMP-1. Whereas the N91Q, N142Q, and N363Q mutants were well secreted, the N332Q, N332Q/N363Q, and N599Q mutants were less well secreted, 72 ± 20, 60 ± 15, 86 ± 15% (n = 3) of BMP-1F, respectively. The results showed that theN-glycosylation site at Asn332 and Asn599 are required for efficient secretion of the molecule. All the single mutants exhibited PCP activity, in that they cleaved 14C type I procollagen to completion in 18 h (Fig. 6). In contrast, the double mutant N332Q/N363Q was a weak C-proteinase. Minor differences between the mutants were detected when type I procollagen was incubated with the mutants for 4 h (Fig. 7). No difference was observed between BMP-1F and prodomain N91Q mutant. The single CUB mutants cleaved procollagen slower than BMP-1F. Furthermore, as shown above, the CUB1 double mutant (N332Q/N363Q) was much slower at cle
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