FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1533587775> ?p ?o ?g. }

• W1533587775 endingPage "17865" @default.
• W1533587775 startingPage "17858" @default.
• W1533587775 abstract "The C-propeptides of the proα1(I) and proα2(I) chains of type I collagen are each substituted with a single high-mannose N-linked oligosaccharide. Conservation of this motif among the fibrillar collagens has led to the proposal that the oligosaccharide has structural or functional importance, but a role in collagen biosynthesis has not been unambiguously defined. To examine directly the function of the proα1(I) C-propeptide N-linked oligosaccharide, the acceptor Asn residue was changed to Gln by site-directed mutagenesis. In transfected mouse Mov13 and 3T6 cells, unglycosylated mutant proα1(I) folded and assembled normally into trimeric molecules with proα2(I). In biosynthetic pulse-chase experiments mutant proα1(I) were secreted at the same rate as wild-type chains; however, following secretion, the chains were partitioned differently between the cell layer and medium, with a greater proportion of the mutant proα1(I) being released into the medium. This distribution difference was not eliminated by the inclusion of yeast mannan indicating that the high-mannose oligosaccharide itself was not binding to the matrix or the fibroblast surface after secretion. Subtle alterations in the tertiary structure of unglycosylated C-propeptides may have decreased their affinity for a cell-surface component. Further support for a small conformational change in the mutant C-propeptides came from experiments suggesting that unglycosylated proα1(I) chains were cleaved in vitro by the purified C-proteinase slightly less efficiently than wild-type chains. Mutant and normal proα1(I) were deposited with equal efficiency into the 3T6 cell accumulated matrix, thus the reduced cleavage by C-proteinase and altered distribution in the short pulse-chase experiments were not functionally significant in this in vitro extracellular matrix model system. The C-propeptides of the proα1(I) and proα2(I) chains of type I collagen are each substituted with a single high-mannose N-linked oligosaccharide. Conservation of this motif among the fibrillar collagens has led to the proposal that the oligosaccharide has structural or functional importance, but a role in collagen biosynthesis has not been unambiguously defined. To examine directly the function of the proα1(I) C-propeptide N-linked oligosaccharide, the acceptor Asn residue was changed to Gln by site-directed mutagenesis. In transfected mouse Mov13 and 3T6 cells, unglycosylated mutant proα1(I) folded and assembled normally into trimeric molecules with proα2(I). In biosynthetic pulse-chase experiments mutant proα1(I) were secreted at the same rate as wild-type chains; however, following secretion, the chains were partitioned differently between the cell layer and medium, with a greater proportion of the mutant proα1(I) being released into the medium. This distribution difference was not eliminated by the inclusion of yeast mannan indicating that the high-mannose oligosaccharide itself was not binding to the matrix or the fibroblast surface after secretion. Subtle alterations in the tertiary structure of unglycosylated C-propeptides may have decreased their affinity for a cell-surface component. Further support for a small conformational change in the mutant C-propeptides came from experiments suggesting that unglycosylated proα1(I) chains were cleaved in vitro by the purified C-proteinase slightly less efficiently than wild-type chains. Mutant and normal proα1(I) were deposited with equal efficiency into the 3T6 cell accumulated matrix, thus the reduced cleavage by C-proteinase and altered distribution in the short pulse-chase experiments were not functionally significant in this in vitro extracellular matrix model system. Asparagine-linked (N-linked) glycosylation of proteins begins with the synthesis of a high-mannose carbohydrate chain, Glc3Man9GlcNAc2, attached to a dolicol lipid carrier (for review, see (1Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Google Scholar)). Transfer of the oligosaccharide to an Asn residue in the consensus sequence Asn-X-Ser/Thr (where X is any amino acid(2Kaplan H.A. Welply J.K. Lennarz W.J. Biochim. Biophys. Acta. 1987; 906: 161-173Google Scholar)) occurs co-translationally during translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum. Processing of the oligosaccharide begins in the endoplasmic reticulum, and continues as the protein is transported through the Golgi, to produce a wide variety of oligosaccharide structures. The roles of these carbohydrate groups are not fully understood. Their contribution to glycoprotein function is variable and inherent to a given protein; however, some general principles are emerging. N-Linked oligosaccharide attachment is required for proper folding and oligomerization of many secreted and cell surface glycoproteins(3Tifft C.J. Proia R.L. Camerini-Otero R.D. J. Biol. Chem. 1992; 267: 3268-3273Google Scholar, 4Matzuk M.M. Boime I. J. Cell Biol. 1988; 106: 1049-1059Google Scholar, 5Singh I. Doms R.W. Wagner K.R. Helenius A. EMBO J. 1990; 9: 631-639Google Scholar, 6Ng D.T.W. Hiebert S.W. Lamb R.A. Mol. Cell. Biol. 1990; 10: 1989-2001Google Scholar, 7Taylor A.K. Wall R. Mol. Cell. Biol. 1988; 8: 4197-4203Google Scholar). Failure to achieve a native folded structure can severely retard intracellular transport and lead to degradation of the retained proteins(8Hurtley S.M. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Google Scholar, 9Lodish H.F. J. Biol. Chem. 1988; 263: 2107-2110Google Scholar, 10Gething M. McCammon K. Sambrook J. Cell. 1986; 46: 939-950Google Scholar, 11Klausner R.D. Sitia R. Cell. 1990; 62: 611-614Google Scholar, 12Lippincott-Schwartz J. Bonifacino J.S. Yuan L.C. Klausner R.D. Cell. 1988; 54: 209-220Google Scholar). Oligosaccharides also modulate the biological activity of proteins by influencing solubility, protease resistance, and protein-protein interactions and can act as protein targeting signals(13Olden K. Parent J.B. White S.L. Biochim. Biophys. Acta. 1982; 650: 209-232Google Scholar, 14Rademacher T.W. Parekh R.B. Dwek R.A. Annu. Rev. Biochem. 1988; 57: 785-838Google Scholar, 15Paulson J.C. Trends Biochem. Sci. 1989; 14: 272-276Google Scholar). The C-propeptides1 1The abbreviations used are: C-propeptidecarboxyl-terminal propeptideC-proteinasecarboxyl-terminal proteinasepNprocollagen processing intermediate retaining the amino-terminal propeptidepCprocollagen processing intermediate retaining the carboxylterminal propeptidekbkilobase(s)PAGEpolyacrylamide gel electrophoresis. of the proα1(I) and proα2(I) subunits of type I collagen contain a single Asn-Ile-Thr consensus sequence for N-linked oligosaccharide addition (16Bernard M.P. Chu M. Myers J.C. Ramirez F. Eikenberry E.F. Prockop D.J. Biochemistry. 1983; 22: 5213-5223Google Scholar, 17Bernard M.P. Myers J.C. Chu M.-L. Ramirez F. Eikenberry E.F. Prockop D.J. Biochemistry. 1983; 22: 1139-1145Google Scholar). In chick tendon fibroblasts, the Asn residue is substituted with a high-mannose carbohydrate group consisting of 9-13 mannose and 2 N-acetylglucosamine residues(18Olsen B.R. Guzman N.A. Engel J. Condit C. Aase S. Biochemistry. 1977; 16: 3030-3036Google Scholar, 19Clarke C.C. J. Biol. Chem. 1979; 254: 10798-10802Google Scholar). The attachment signal is highly conserved between species and within the fibrillar group of collagens (types I, II, III, V, and XI) suggesting that the N-linked oligosaccharide may play a common essential structural or functional role in procollagen biosynthesis. carboxyl-terminal propeptide carboxyl-terminal proteinase procollagen processing intermediate retaining the amino-terminal propeptide procollagen processing intermediate retaining the carboxylterminal propeptide kilobase(s) polyacrylamide gel electrophoresis. Several studies have addressed the role of the N-linked carbohydrate chain on the type I collagen C-propeptides using tunicamycin to inhibit the synthesis of the lipid-linked carbohydrate donor and thus prevent N-glycosylation. However, the results have been contradictory; both normal and impaired secretion(20Duksin D. Bornstein P. J. Biol. Chem. 1977; 252: 955-962Google Scholar, 21Tanzer M.L. Rowland F.N. Murray L.W. Kaplan J. Biochim. Biophys. Acta. 1977; 500: 187-196Google Scholar, 22Housley T.J. Rowland F.N. Ledger P.W. Kaplan J. Tanzer M.L. J. Biol. Chem. 1980; 255: 121-128Google Scholar) and normal and impaired extracellular cleavage of the C-propeptide have been reported(20Duksin D. Bornstein P. J. Biol. Chem. 1977; 252: 955-962Google Scholar, 21Tanzer M.L. Rowland F.N. Murray L.W. Kaplan J. Biochim. Biophys. Acta. 1977; 500: 187-196Google Scholar, 23Duksin D. Davidson J.M. Bornstein P. Arch. Biochem. Biophys. 1978; 185: 326-332Google Scholar, 24Duksin D. Mahoney W.C. J. Biol. Chem. 1982; 257: 3105-3109Google Scholar). In this study we have used site-directed mutagenesis to remove the proα1(I) C-propeptide N-linked oligosaccharide attachment site. This approach overcomes the problems associated with the general glycosylation inhibitor tunicamycin such as the variable reduction of protein synthesis by different preparations (25Mahoney W.C. Duksin D. J. Biol. Chem. 1979; 254: 6572-6576Google Scholar, 26Elbein A.D. Annu. Rev Biochem. 1987; 56: 497-534Google Scholar) and effects on other glycoproteins involved in the collagen biosynthetic pathway. The mutation was introduced into a proα1(I) reporter protein construct with a helical Met822-Ile substitution which allowed mutant and normal gene products to be experimentally distinguished and quantified by their different CNBr cleavage patterns(27Fenton S.P. Lamande S.R. Hannagan M. Stacey A. Jaenisch R. Bateman J.F. Biochim. Biophys. Acta. 1993; 1216: 469-474Google Scholar). In this way, the biosynthetic fates of normal and mutant proα1(I) chains expressed in the same transfected cell were directly compared. Unglycosylated proα1(I) subunits assembled normally into trimeric molecules with proα2(I) and were secreted at the same rate as endogenous proα1(I). Molecules containing unglycosylated proα1(I) chains were processed by the procollagen C-proteinase and deposited into an in vitro extracellular matrix. To determine the role of the C-propeptide N-linked oligosaccharide in type I collagen biosynthesis, a COL1A1 gene construct was produced in which Asn1187 2Amino acids are numbered from the translation start site of pre-proα1(I). (28), within the Asn-Ile-Thr attachment recognition sequence, was changed to Gln. The mutagenesis protocol is summarized in Fig. 1. Two COL1A1 gene constructs were produced. In the control construct, to act as a protein marker and allow discrimination of the mutant and wild-type C-propeptides by CNBr mapping, amino acid 1199 (C-propeptide residue 159) was changed from Met (ATG) to Ala (GCG). Two silent base changes introduced an AccII site (Fig. 1a). In addition to these changes, the C-propeptide N-linked oligosaccharide attachment signal was removed in the mutant construct by changing amino acid 1187 (C-propeptide residue 147) from Asn (AAC) to Gln (CAA). The substituting marker and mutant amino acids were chosen because they were predicted to have a minimal effect on the secondary structure of the region based on computerized sequence analysis (PepPlot, Genetics Computer Group). Synthetic StuI-BstXI DNA fragments containing the sequence changes (Fig. 1a) were ligated into a 2.0-kb XbaI-EcoRI genomic subclone (Fig. 1b). Plasmids containing the additional AccII site (Fig. 2) were selected and the modified region sequenced (data not shown). The 1.4-kb XhoI-ClaI fragment from selected clones was used to replace the normal fragment of pWTCI-I, an α1(I) protein reporter gene construct containing a helical Met822-Ile substitution (27Fenton S.P. Lamande S.R. Hannagan M. Stacey A. Jaenisch R. Bateman J.F. Biochim. Biophys. Acta. 1993; 1216: 469-474Google Scholar), and the wild-type 4.5-kb ClaI fragment reinserted (Fig. 1b). The orientation of the ClaI fragment was determined by digestion with HindIII (Fig. 3). The final reassembled genes, named to indicate the amino acid substitutions in the protein products, were the reporter construct pWTCI-I (25-kb COL1A1 gene containing the Met-Ile substitution at amino acid 822 of the triple helix(27Fenton S.P. Lamande S.R. Hannagan M. Stacey A. Jaenisch R. Bateman J.F. Biochim. Biophys. Acta. 1993; 1216: 469-474Google Scholar)), the control construct pWTCI-IA (25-kb COL1A1 gene containing the Met822-Ile substitution and a silent C-propeptide Met1199-Ala alteration), and the mutant construct pWTCI-IQA (25-kb COL1A1 gene containing the Met822-Ile and Met1199-Ala substitutions and the Asn1187-Gln mutation deleting the N-linked oligosaccharide attachment site).Figure 2:Restriction enzyme mapping of the 2-kb XbaI-EcoRI fragment in pUC19 containing the sequence changes. a, AccII digestion of wild-type (lane 2), control (lane 3) and oligosaccharide-attachment mutant (lane 4) plasmids. The introduction of the AccII site in the control and mutant plasmids results in cleavage of the 1334-base pair wild-type fragment to produce fragments of 680 and 654 base pair. Lane 1, ∅XI74 HaeIII molecular weight markers. b, the locations of the AccII sites in the wild-type, control, and mutant constructs are shown schematically. The arrow indicates the AccII site introduced by site-directed mutagenesis. Restriction enzyme recognition sites are designated A, AccII; E, EcoRI; X, XbaI.View Large Image Figure ViewerDownload (PPT)Figure 3:HindIII restriction enzyme mapping of the control, pWTCI-IA, and mutant, pWTCI-IQA, genomic constructs. Reassembled gene constructs were screened by HindIII digestion to ensure that the 4.5-kb ClaI fragment was present in the correct orientation. a, bands generated by HindIII digestion of the control construct pWTCI-IA (lane 3) and the mutant construct pWTCI-IQA (lane 4) were identical to the wild-type gene pWTCI (lane 2). The 2.1 kb band present in correctly reassembled constructs is marked. Lane 1, γ HindIII molecular weight markers. HindIII restriction maps of constructs containing the ClaI fragment (shaded box) in the correct orientation (b) and reverse orientation (c) are shown. Restriction enzyme recognition sites are designated C, ClaI; H, HindIII.View Large Image Figure ViewerDownload (PPT) Mouse Mov13 (29Schnieke A. Harbers K. Jaenisch R. Nature. 1983; 304: 315-320Google Scholar) and 3T6 cells (American Type Culture Collection, CCL-96) were grown in culture as described for human skin fibroblasts (30Bateman J.F. Mascara T. Chan D. Cole W.G. Biochem. J. 1984; 217: 103-115Google Scholar) Cells were co-transfected with the COL1A1 gene constructs and pSV2neo(31Southern P.J. Berg P. J. Mol. Appl. Genet. 1982; 1: 327-341Google Scholar), neomycin-resistant transfected cells were selected in G418, and individual colonies isolated and expanded into cell lines as described previously(27Fenton S.P. Lamande S.R. Hannagan M. Stacey A. Jaenisch R. Bateman J.F. Biochim. Biophys. Acta. 1993; 1216: 469-474Google Scholar). G418 was removed from the culture medium after the fourth passage. Cells were grown to confluence in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum and then supplemented daily with 0.25 mM ascorbic acid. Procollagens were biosynthetically labeled routinely at 1-2 days post-visual confluence and at other relevant times during long term culture experiments. The medium was removed and replaced with 9.9 ml of Dulbecco's modified Eagle's medium containing 10% (v/v) dialyzed fetal calf serum and 0.25 mM ascorbic acid. After 4 h, 0.1 ml of Dulbecco's modified Eagle's medium containing 50 μCi of L-[5-3H]proline (8.5 Ci/mmol, NEN Research Products) or 5 μCi of L-[14C]proline (284.6 mCi/mmol, NEN Research Products) was added to the medium, and the incubation was continued for a further 18 h. The final concentration of proline in the medium was 0.1 mM. To examine procollagen secretion kinetics, cells were labeled for 1 h with 5 μCi L-[14C]proline, the labeling stopped by the addition of proline to a final concentration of 50 mM, and the radioactive collagen chased for up to 4 h. In some experiments 1 mg/ml yeast mannan (Sigma) was included throughout the preincubation, labeling, and chase periods. Following incubation, the cell layer and medium fractions were treated separately as described previously(30Bateman J.F. Mascara T. Chan D. Cole W.G. Biochem. J. 1984; 217: 103-115Google Scholar, 32Bateman J.F. Chan D. Mascara T. Rogers J.G. Cole W.G. Biochem. J. 1986; 240: 699-708Google Scholar). Briefly, after disruption of the cell layer by sonication, procollagens and collagens were precipitated from the cell and medium fractions with ammonium sulfate at 25% saturation. The precipitate was redissolved in 2 ml of 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl and the protease inhibitors 5 mM EDTA, 10 mMN-ethylmaleimide, and 1 mM phenylmethanesulfonyl fluoride. Aliquots of procollagens were precipitated with 75% ethanol and subjected to limited pepsin digestion (100 μg/ml pepsin in 0.5 M acetic acid, 4°C, 16 h) to remove noncollagen sequences. Cell layers from cultures which had been supplemented daily from confluence (day 0) with 0.25 mM ascorbic acid were sequentially extracted with 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl and proteinase inhibitors (neutral salt soluble fraction), and by digestion with pepsin (0.1 mg/ml pepsin in 0.5 M acetic acid) as described previously(33Chan D. Lamande S.R. Cole W.G. Bateman J.F. Biochem. J. 1990; 269: 175-181Google Scholar). Freeze-dried samples of pepsin-digested collagen were resuspended in 100 mM ammonium bicarbonate and incubated at room temperature for 30 min to inactivate the pepsin. CNBr cleavage was performed in 70% (v/v) formic acid containing 50 mg/ml CNBr for 4 h at room temperature as described by Scott and Veis(34Scott P.G. Veis A. Connect. Tiss. Res. 1976; 4: 107-116Google Scholar). After cleavage the samples were diluted with water and freeze-dried. Procollagens were precipitated with 75% ethanol and digested with 30 μg/ml bacterial collagenase (Worthington Biochemicals, CLSPA) for 2 h at 37°C in 50 mM Tris-HCl, pH 7.5 containing 0.15 M NaCl, 5 mM CaCl2, and 3.5 mMN-ethylmaleimide. Digestion was terminated by lyophilization. Purified C-proteinase was a gift from Dr. Karl E. Kadler (School of Biological Sciences, University of Manchester, United Kingdom). Procollagen substrate was purified from the medium of cells which had been biosynthetically labeled for 18 h with either 50 μCi of L-[5-3H]proline (8.5 Ci/mmol; NEN Research Products) or 5 μCi of L-[14C]proline (284.6 mCi/mmol; NEN Research Products). L-Arginine (50 mM) was added to the cultures to prevent cleavage of the COOH-terminal propeptide by the C-proteinase present in the medium(35Leung M.K.K. Fessler L.I. Greenberg D.B. Fessler J.H. J. Biol. Chem. 1979; 254: 224-232Google Scholar). Procollagens were precipitated with ammonium sulfate at 25% saturation, resuspended in neutral-salt buffer, and reprecipitated with 18% (v/v) ethanol. Procollagens were incubated with C-proteinase at 34°C in 50 mM Tris-HCl, pH 7.5, containing 0.12 M NaCl and 5 mM CaCl2. Digestion was terminated by the addition of SDS-PAGE sample buffer. Collagen chains were resolved on 5% (w/v) polyacrylamide separating gels with a 3.5% (w/v) stacking gel. Collagen CNBr peptides and C-propeptides were analyzed on 10 or 12.5% (w/v) polyacrylamide gels. Sample preparation, electrophoresis conditions, and fluorography of radioactive gels have been described elsewhere(30Bateman J.F. Mascara T. Chan D. Cole W.G. Biochem. J. 1984; 217: 103-115Google Scholar, 32Bateman J.F. Chan D. Mascara T. Rogers J.G. Cole W.G. Biochem. J. 1986; 240: 699-708Google Scholar). The radioactivity in the collagen bands was quantified by excision and scintillation counting(36Bateman J.F. Harley V. Chan D. Cole W.G. Anal. Biochem. 1988; 168: 171-176Google Scholar). Proteins were electrophoretically transferred from 12.5% (w/v) SDS-polyacrylamide gels to nitrocellulose filters (BA85, Schleicher and Schuell). Blots were probed with an antibody which recognizes the last 21 amino acids of the proα1(I) chain (LF-41(37Fisher L.W. Lindner W. Young M.F. Termine J.D. Connect. Tiss. Res. 1989; 21 (abstr.): 43-48Google Scholar)) which was kindly provided by Dr. Larry Fisher (National Institute of Dental Research). Specific binding was detected using horseradish peroxidase-conjugated Protein A (Bio-Rad) and the color reagent 4-chloro-1-naphthol (Bio-Rad). Terminal mannose residues on the C-propeptide N-linked oligosaccharide were detected using the lectin Galanthus nivalis agglutinin (GNA, DIG Glycan Differentiation Kit, Boehringer Mannheim). The Met822-Ile marker substitution in the helical domain allowed the transfected gene products to be distinguished from endogenous α1(I) and quantified in processed chains by the altered CNBr cleavage pattern(27Fenton S.P. Lamande S.R. Hannagan M. Stacey A. Jaenisch R. Bateman J.F. Biochim. Biophys. Acta. 1993; 1216: 469-474Google Scholar). The functionally neutral substitution removed the CNBr cleavage site between the CB7 and CB6 peptides. The level of expression of the transfected α1(I) genes containing the [Ile822]α1(I) substitution was calculated using a previously determined formula which takes into account the low level of uncleaved CB7-6 contributed by the endogenous α1(I)(27Fenton S.P. Lamande S.R. Hannagan M. Stacey A. Jaenisch R. Bateman J.F. Biochim. Biophys. Acta. 1993; 1216: 469-474Google Scholar). The constructs were transfected into Mov13 cells to assess the ability of unglycosylated proα1(I) chains to fold and assemble into trimeric helical molecules. Untransfected Mov13 cells synthesize only the proα2(I) subunit of type I collagen; transcription of the endogenous proα1(I) genes is blocked by a retroviral insertion in the first intron(29Schnieke A. Harbers K. Jaenisch R. Nature. 1983; 304: 315-320Google Scholar, 38Harbers K. Kuehn M. Delius H. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1504-1508Google Scholar). Proα2(I) chains are unable to fold into homotrimeric molecules and, in the absence of proα1(I), are degraded intracellularly. Mov13 cells stably transfected with either the control construct, pWTCI-IA (Mov13-IA7-Mov13-IA10), or the mutant gene, pWTCI-IQA (Mov13-IQA1, 3, 4, 7, 8, 9), synthesized proα1(I) chains which associated with the endogenous proα2(I), folded into pepsin-resistant molecules, and were efficiently secreted (Fig. 4). There was no evidence of slowly migrating α1(I) or α2(I) chains like those seen in osteogenesis imperfecta patients where mutations which perturb chain association or slow the folding of the triple helix led to excess post-translational hydroxylation and glycosylation of lysine residues resulting in slow electrophoretic migration of the chains(30Bateman J.F. Mascara T. Chan D. Cole W.G. Biochem. J. 1984; 217: 103-115Google Scholar, 32Bateman J.F. Chan D. Mascara T. Rogers J.G. Cole W.G. Biochem. J. 1986; 240: 699-708Google Scholar, 39Chessler S.D. Wallis G.A. Byers P.H. J. Biol. Chem. 1993; 268: 18218-18225Google Scholar). To directly demonstrate that the mutation altering the N-glycosylation consensus sequence prevented propeptide glycosylation, [3H]proline-labeled procollagens were digested with bacterial collagenase to degrade the collagen α-chains and the released C-propeptides compared electrophoretically (Fig. 5a). The identity of the propeptides was confirmed by immunoblotting with a specific C-propeptide antibody (Fig. 5b). The control proα1(I) C-propeptides migrated as a doublet of a minor and major species (Fig. 5a, lanes 2 and 3; b, lanes 1 and 2). The minor, faster migrating band in each lane was also present in samples not digested with collagenase (Fig. 5a, lane 1) and is probably α1(I) C-propeptide released by cleavage with the endogenous procollagen C-proteinase present in the culture medium. The major species of C-propeptide, produced by bacterial collagenase digestion, migrated more slowly because they retained the α1(I) telopeptide domain. The migration of control C-propeptides as sharp bands suggested that all the propeptides were N-glycosylated and homogeneous in the size of the carbohydrate substitutions. The mutant C-propeptide doublet migrated significantly faster than control propeptides (Fig. 5a, lane 4; b lane 3). Failure of the mutant propeptides to bind the lectin GNA which recognized the control C-propeptides (Fig. 5c) indicated that the mutant C-propeptide migrated faster because it lacked the N-linked carbohydrate unit. Several studies have reported impaired C-propeptide processing in cultures treated with tunicamycin, a general inhibitor of N-linked glycosylation(20Duksin D. Bornstein P. J. Biol. Chem. 1977; 252: 955-962Google Scholar, 23Duksin D. Davidson J.M. Bornstein P. Arch. Biochem. Biophys. 1978; 185: 326-332Google Scholar, 24Duksin D. Mahoney W.C. J. Biol. Chem. 1982; 257: 3105-3109Google Scholar). This could be due to changes in the structure or function of either the C-proteinase or the procollagen substrate. To determine if unglycosylated procollagen is a substrate for the N-glycosylated C-proteinase, procollagens produced by cells labeled with either [14C]proline (Mov13-Ile4) or [3H]proline (Mov13-IA8 and Mov13-IQA3) were isolated and incubated for varying periods of time with purified C-proteinase. To eliminate experimental variation due to contaminants in the procollagen samples, the procollagens were mixed and cleavage of wild-type and control or mutant C-propeptides directly compared. Aliquots were examined by electrophoresis under reducing conditions and the proportion of proα1(I), pCα1(I)/pNα1(I), and α1(I) chains present determined by dual-label counting of the excised bands. Results of three separate experiments are shown in Fig. 6. The C-proteinase was able to cleave both control [IA]proα1(I) and mutant [IQA]proα1(I) C-propeptides as judged by the conversion of proα1(I) chains to the pNα1(I) processing intermediate. In each experiment, control [IA]proα1(I) chains were processed at the same rate as the wild-type proα1(I) chains (Fig. 6, a, c, and e), but the unglycosylated [IQA]proα1(I) chains were cleaved slightly more slowly (Fig. 6, b, d, and f). While the difference in the rate of processing of the mutant proα1(I) was small, it was reproducible and was accurately compared to processing of the wild-type proα1(I) by digesting mutant and normal procollagens, differentially labeled with 14C and 3H, in the same tube. The appearance of intact cleaved C-propeptides (data not shown) was an indication that the observed C-propeptide processing was specific. No nonspecific cleavage was seen when the procollagens were incubated for 2 h in the absence of the C-proteinase (data not shown). The mutant construct was transfected into 3T6 cells to allow the biosynthetic fate of normal endogenous proα1(I) and unglycosylated mutant chains to be compared directly. Individual G418-resistant colonies were expanded into cell lines and analyzed separately. Endogenous and transfected gene products could be discriminated and quantified in these cells because the C-propeptide changes were introduced into a reporter construct containing a functionally neutral Met822-Ile substitution which deletes the CNBr cleavage site between the CB7 and CB6 peptides. Expression of transfected mutant gene products was readily detected after CNBr cleavage of pepsin-digested collagen by the increased relative intensity of the CB7-6 peptide (Fig. 7(27Fenton S.P. Lamande S.R. Hannagan M. Stacey A. Jaenisch R. Bateman J.F. Biochim. Biophys. Acta. 1993; 1216: 469-474Google Scholar)). As in the transfected Mov13 cells, there was no evidence of slowly migrating overmodified peptides (Fig. 7). Immunoblotting with antibody LF-41 detected the glycosylated form and a smaller unglycosylated form of the proα1(I) C-propeptide in the medium of 3T6 cells transfected with the mutant construct (Fig. 8, lanes 4 and 5). Only the larger, glycosylated form was present in untransfected 3T6 cells and cells transfected with the control gene (Fig. 8, lanes 1-3).Figure 8:Electrophoretic analysis of the α1(I) C-propeptides produced by transfected 3T6 cells. Medium procollagens were purified and analyzed under reducing conditions on 12.5% acrylamide gels. The α1(I) C-propeptides were detected by immunoblotting with the polyclonal antibody LF-41. Lane 1, untransfected 3T6 cells; lane 2, 3T6 cells transfected with the reporter construct pWTCI-I; lane 3, 3T6 cells transfected with the control construct pWTCI-IA. Lanes 4 and 5, C-propeptides produced by two cell lines transfected with the N-linked oligosaccharide mutant construct pWTCI-IQA. Mutant, unglycosylated chains represented 15 and 60%, respectively, of the total α1(I) chains synthesized by these cells.View Large Image Figure ViewerDownload (PPT) To directly compare the rate of secretion of endogenous and transfected mutant proα1(I) chains, cells were pulse-labeled with [14C]proline for 1 h and the labeled collagens chased for up to 4 h in the presence of excess cold proline. The presence of the marker Met822-Ile substitution in the proα1(I) chains produced from the transfected genes allowed the fate of these chains and endogenous wild-type proα1(I) chains expressed in the same cell to be compared accurately. Control transfected [IA]proα1(I) chains were secreted at the same rate as endogenous wild-type chains; at all time points the proportion of endogenous and transfected control chains which had been released into the medium was the same (Fig. 9a). Around 20% of the total α1(I) chains remained associat" @default.
• W1533587775 created "2016-06-24" @default.
• W1533587775 creator A5027780831 @default.
• W1533587775 creator A5081058369 @default.
• W1533587775 date "1995-07-01" @default.
• W1533587775 modified "2023-09-28" @default.
• W1533587775 title "The Type I Collagen proα1(I) COOH-terminal Propeptide N-Linked Oligosaccharide" @default.
• W1533587775 cites W1482044739 @default.
• W1533587775 cites W1501622884 @default.
• W1533587775 cites W1515021757 @default.
• W1533587775 cites W1523240427 @default.
• W1533587775 cites W1523963881 @default.
• W1533587775 cites W1539808460 @default.
• W1533587775 cites W1540873444 @default.
• W1533587775 cites W1546222819 @default.
• W1533587775 cites W1573653133 @default.
• W1533587775 cites W1573759787 @default.
• W1533587775 cites W1574903262 @default.
• W1533587775 cites W1580985143 @default.
• W1533587775 cites W1599477510 @default.
• W1533587775 cites W1607281048 @default.
• W1533587775 cites W1608156252 @default.
• W1533587775 cites W1641205561 @default.
• W1533587775 cites W1648237278 @default.
• W1533587775 cites W1654463875 @default.
• W1533587775 cites W1780609762 @default.
• W1533587775 cites W1840284621 @default.
• W1533587775 cites W1851592922 @default.
• W1533587775 cites W1856207023 @default.
• W1533587775 cites W1948296685 @default.
• W1533587775 cites W1965030844 @default.
• W1533587775 cites W1965988916 @default.
• W1533587775 cites W1970489088 @default.
• W1533587775 cites W1976981613 @default.
• W1533587775 cites W1987239565 @default.
• W1533587775 cites W1989526931 @default.
• W1533587775 cites W200246087 @default.
• W1533587775 cites W2003292237 @default.
• W1533587775 cites W2014308390 @default.
• W1533587775 cites W2024451651 @default.
• W1533587775 cites W2027109325 @default.
• W1533587775 cites W2030011714 @default.
• W1533587775 cites W2038414337 @default.
• W1533587775 cites W2039847706 @default.
• W1533587775 cites W2043460107 @default.
• W1533587775 cites W2045122282 @default.
• W1533587775 cites W2057589234 @default.
• W1533587775 cites W2063535203 @default.
• W1533587775 cites W2064432213 @default.
• W1533587775 cites W2067369810 @default.
• W1533587775 cites W2080489175 @default.
• W1533587775 cites W2081108449 @default.
• W1533587775 cites W2083183297 @default.
• W1533587775 cites W2090238172 @default.
• W1533587775 cites W2090885889 @default.
• W1533587775 cites W2095082905 @default.
• W1533587775 cites W2138396294 @default.
• W1533587775 cites W2140277678 @default.
• W1533587775 cites W2152708819 @default.
• W1533587775 cites W2172683288 @default.
• W1533587775 cites W2235866438 @default.
• W1533587775 cites W4206701382 @default.
• W1533587775 doi "https://doi.org/10.1074/jbc.270.30.17858" @default.
• W1533587775 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7629088" @default.
• W1533587775 hasPublicationYear "1995" @default.
• W1533587775 type Work @default.
• W1533587775 sameAs 1533587775 @default.
• W1533587775 citedByCount "18" @default.
• W1533587775 countsByYear W15335877752013 @default.
• W1533587775 countsByYear W15335877752014 @default.
• W1533587775 countsByYear W15335877752016 @default.
• W1533587775 countsByYear W15335877752018 @default.
• W1533587775 countsByYear W15335877752021 @default.
• W1533587775 crossrefType "journal-article" @default.
• W1533587775 hasAuthorship W1533587775A5027780831 @default.
• W1533587775 hasAuthorship W1533587775A5081058369 @default.
• W1533587775 hasBestOaLocation W15335877751 @default.
• W1533587775 hasConcept C104317684 @default.
• W1533587775 hasConcept C134018914 @default.
• W1533587775 hasConcept C167625842 @default.
• W1533587775 hasConcept C181199279 @default.
• W1533587775 hasConcept C185592680 @default.
• W1533587775 hasConcept C2777726330 @default.
• W1533587775 hasConcept C2779664074 @default.
• W1533587775 hasConcept C2780644872 @default.
• W1533587775 hasConcept C3018824666 @default.
• W1533587775 hasConcept C41008148 @default.
• W1533587775 hasConcept C55493867 @default.
• W1533587775 hasConcept C71240020 @default.
• W1533587775 hasConcept C76155785 @default.
• W1533587775 hasConcept C7860815 @default.
• W1533587775 hasConcept C86803240 @default.
• W1533587775 hasConceptScore W1533587775C104317684 @default.
• W1533587775 hasConceptScore W1533587775C134018914 @default.
• W1533587775 hasConceptScore W1533587775C167625842 @default.
• W1533587775 hasConceptScore W1533587775C181199279 @default.
• W1533587775 hasConceptScore W1533587775C185592680 @default.
• W1533587775 hasConceptScore W1533587775C2777726330 @default.

• next