©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Type I Collagen pro1(I) COOH-terminal Propeptide N-Linked Oligosaccharide
FUNCTIONAL ANALYSIS BY SITE-DIRECTED MUTAGENESIS (*)

(Received for publication, March 20, 1995; and in revised form, May 15, 1995)

Shireen R. Lamand John F. Bateman (§)

From the Orthopaedic Molecular Biology Research Unit, Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The C-propeptides of the pro1(I) and pro2(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 pro1(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 pro1(I) folded and assembled normally into trimeric molecules with pro2(I). In biosynthetic pulse-chase experiments mutant pro1(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 pro1(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 pro1(I) chains were cleaved in vitro by the purified C-proteinase slightly less efficiently than wild-type chains. Mutant and normal pro1(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.


INTRODUCTION

Asparagine-linked (N-linked) glycosylation of proteins begins with the synthesis of a high-mannose carbohydrate chain, GlcManGlcNAc, attached to a dolicol lipid carrier (for review, see (1) ). Transfer of the oligosaccharide to an Asn residue in the consensus sequence Asn-X-Ser/Thr (where X is any amino acid(2) ) 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(3, 4, 5, 6, 7) . Failure to achieve a native folded structure can severely retard intracellular transport and lead to degradation of the retained proteins(8, 9, 10, 11, 12) . Oligosaccharides also modulate the biological activity of proteins by influencing solubility, protease resistance, and protein-protein interactions and can act as protein targeting signals(13, 14, 15) .

The C-propeptides()of the pro1(I) and pro2(I) subunits of type I collagen contain a single Asn-Ile-Thr consensus sequence for N-linked oligosaccharide addition (16, 17) . 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(18, 19) . 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.

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(20, 21, 22) and normal and impaired extracellular cleavage of the C-propeptide have been reported(20, 21, 23, 24) .

In this study we have used site-directed mutagenesis to remove the pro1(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 (25, 26) and effects on other glycoproteins involved in the collagen biosynthetic pathway. The mutation was introduced into a pro1(I) reporter protein construct with a helical Met-Ile substitution which allowed mutant and normal gene products to be experimentally distinguished and quantified by their different CNBr cleavage patterns(27) . In this way, the biosynthetic fates of normal and mutant pro1(I) chains expressed in the same transfected cell were directly compared. Unglycosylated pro1(I) subunits assembled normally into trimeric molecules with pro2(I) and were secreted at the same rate as endogenous pro1(I). Molecules containing unglycosylated pro1(I) chains were processed by the procollagen C-proteinase and deposited into an in vitro extracellular matrix.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis of the Mouse COL1A1 Gene

To determine the role of the C-propeptide N-linked oligosaccharide in type I collagen biosynthesis, a COL1A1 gene construct was produced in which ()Asn (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 1(I) protein reporter gene construct containing a helical Met-Ile substitution (27) , 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(27) ), the control construct pWTCI-IA (25-kb COL1A1 gene containing the Met-Ile substitution and a silent C-propeptide Met-Ala alteration), and the mutant construct pWTCI-IQA (25-kb COL1A1 gene containing the Met-Ile and Met-Ala substitutions and the Asn-Gln mutation deleting the N-linked oligosaccharide attachment site).


Figure 1: Production of the pro1(I) construct lacking the C-propeptide N-linked oligosaccharide attachment signal. a, nucleotide and deduced amino acid sequence of the wild-type StuI-BstXI fragment and the synthetic oligonucleotides used to produce the control and mutant constructs by site-directed mutagenesis. The control fragment contains substitutions which alter the codon for amino acid 1199 from Met (ATG) to Ala (GCG). Silent base changes in the codons for amino acids 1196 and 1197 introduce a new AccII restriction enzyme site. In addition to these changes the mutant fragment contains base changes which alter the codon for amino acid 1187 within the Asn-X-Thr N-linked oligosaccharide attachment signal (boxed sequences) from Asn (AAC) to Gln (CAA). b, schematic description of the mutagenesis protocol. The wild-type StuI-BstXI fragment (hatchedboxes) of a 2-kb XbaI-EcoRI subclone of the mouse COL1A1 gene was replaced with the control and mutant synthetic oligonucleotides (blackboxes). The altered XhoI-ClaI fragment of the 2-kb subclone was then used to replace the normal fragment of the reporter construct, pWTCI-I. The final step was the reinsertion of the wild-type 4.5-kb ClaI fragment to produce the full-length control (pWTCI-IA) and mutant (pWTCI-IQA) constructs. Restriction enzyme recognition sites are designated B, BstXI; C, ClaI; E, EcoRI; X, XhoI.




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. Lane1, &cjs0625;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.




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 (lane3) and the mutant construct pWTCI-IQA (lane4) were identical to the wild-type gene pWTCI (lane2). The 2.1 kb band present in correctly reassembled constructs is marked. Lane1, HindIII molecular weight markers. HindIII restriction maps of constructs containing the ClaI fragment (shadedbox) in the correct orientation (b) and reverse orientation (c) are shown. Restriction enzyme recognition sites are designated C, ClaI; H, HindIII.



Cell Culture and Transfection

Mouse Mov13 (29) and 3T6 cells (American Type Culture Collection, CCL-96) were grown in culture as described for human skin fibroblasts (30) Cells were co-transfected with the COL1A1 gene constructs and pSV2neo(31) , neomycin-resistant transfected cells were selected in G418, and individual colonies isolated and expanded into cell lines as described previously(27) . G418 was removed from the culture medium after the fourth passage.

Collagen Biosynthetic Labeling

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-H]proline (8.5 Ci/mmol, NEN Research Products) or 5 µCi of L-[C]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-[C]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(30, 32) . 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.

Extraction of Collagen from the Extracellular Matrix

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(33) .

CNBr Peptide Mapping

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(34) . After cleavage the samples were diluted with water and freeze-dried.

Collagenase Digestion

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 CaCl, and 3.5 mMN-ethylmaleimide. Digestion was terminated by lyophilization.

Cleavage with Procollagen C-proteinase

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-H]proline (8.5 Ci/mmol; NEN Research Products) or 5 µCi of L-[C]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(35) . 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 CaCl. Digestion was terminated by the addition of SDS-PAGE sample buffer.

SDS-Polyacrylamide Gel Electrophoresis

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(30, 32) . The radioactivity in the collagen bands was quantified by excision and scintillation counting(36) .

Immunoblotting

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 pro1(I) chain (LF-41(37) ) 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).

Quantitation of Transfected Gene Expression in 3T6 Cells

The Met-Ile marker substitution in the helical domain allowed the transfected gene products to be distinguished from endogenous 1(I) and quantified in processed chains by the altered CNBr cleavage pattern(27) . The functionally neutral substitution removed the CNBr cleavage site between the CB7 and CB6 peptides. The level of expression of the transfected 1(I) genes containing the [Ile]1(I) substitution was calculated using a previously determined formula which takes into account the low level of uncleaved CB7-6 contributed by the endogenous 1(I)(27) .


RESULTS

Expression of the Mutant pro1(I) Chain in Mov13 Cells

The constructs were transfected into Mov13 cells to assess the ability of unglycosylated pro1(I) chains to fold and assemble into trimeric helical molecules. Untransfected Mov13 cells synthesize only the pro2(I) subunit of type I collagen; transcription of the endogenous pro1(I) genes is blocked by a retroviral insertion in the first intron(29, 38) . Pro2(I) chains are unable to fold into homotrimeric molecules and, in the absence of pro1(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 pro1(I) chains which associated with the endogenous pro2(I), folded into pepsin-resistant molecules, and were efficiently secreted (Fig. 4). There was no evidence of slowly migrating 1(I) or 2(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(30, 32, 39) .


Figure 4: Electrophoresis of pepsin-digested collagen produced by Mov13 cells transfected with the control and N-glycosylation mutant constructs. Collagens were labeled with [H]proline and pepsin-digested samples from the cell layer (C) and medium (M) analyzed without reduction on 5% polyacrylamide gels. a, collagens produced by the untransfected parental cell line Mov13 (lanes1 and 2), cells transfected with the reporter construct pWTCI-I (lanes3 and 4), and four clonal cell lines transfected with the control construct pWTCI-IA (lanes5-12) are shown. b, collagens produced by six clonal cell lines transfected with the N-linked oligosaccharide mutant construct pWTCI-IQA (lanes5-16) are compared with those produced by untransfected Mov13 cells (lanes1 and 2) and cells transfected with the reporter gene construct (lanes3 and 4). The migration positions of the type I collagen 1(I) and 2(I) chains are indicated.



Demonstration of the Protein Defect

To directly demonstrate that the mutation altering the N-glycosylation consensus sequence prevented propeptide glycosylation, [H]proline-labeled procollagens were digested with bacterial collagenase to degrade the collagen -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 pro1(I) C-propeptides migrated as a doublet of a minor and major species (Fig. 5a, lanes2 and 3; b, lanes1 and 2). The minor, faster migrating band in each lane was also present in samples not digested with collagenase (Fig. 5a, lane1) and is probably 1(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 1(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, lane4; blane3). 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.


Figure 5: Electrophoretic analysis of the type I collagen C-propeptides produced by transfected Mov13 cells. [H]Proline-labeled medium procollagens were digested with bacterial collagenase (+) and the remaining proteins analyzed on 12.5% polyacrylamide gels in the presence of 10 mM DTT. a, 1(I) C-propeptides produced by Mov13 cells transfected with the reporter construct pWTCI-I (lanes1 and 2), the control construct pWTCI-IA (lane3), and the mutant gene pWTCI-IQA (lane4) were detected by fluorography. b, 1(I) C-propeptides detected by immunoblotting with LF-41, an antibody directed against the most COOH-terminal 21 amino acids; lane1, wild-type C-propeptides; lane2, control; lane3, mutant. c, 1(I) C-propeptides detected with the lectin GNA which identifies high-mannose oligosaccharide groups; lane1, wild-type C-propeptides; lane2, control; lane3, mutant.



Processing of Mutant pro1(I) Chains in Vitro by Purified Procollagen C-Proteinase

Several studies have reported impaired C-propeptide processing in cultures treated with tunicamycin, a general inhibitor of N-linked glycosylation(20, 23, 24) . 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 [C]proline (Mov13-Ile4) or [H]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 pro1(I), pC1(I)/pN1(I), and 1(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]pro1(I) and mutant [IQA]pro1(I) C-propeptides as judged by the conversion of pro1(I) chains to the pN1(I) processing intermediate. In each experiment, control [IA]pro1(I) chains were processed at the same rate as the wild-type pro1(I) chains (Fig. 6, a, c, and e), but the unglycosylated [IQA]pro1(I) chains were cleaved slightly more slowly (Fig. 6, b, d, and f). While the difference in the rate of processing of the mutant pro1(I) was small, it was reproducible and was accurately compared to processing of the wild-type pro1(I) by digesting mutant and normal procollagens, differentially labeled with C and H, 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).


Figure 6: Processing of the mutant unglycosylated pro1(I) chains in vitro by purified procollagen C-proteinase. [H]Proline-labeled wild-type pro1(I) chains produced were mixed with either [C]proline-labeled control [IA]pro1(I) chains (panels a, c, and e) or [C]proline-labeled mutant unglycosylated [IQA]pro1(I) chains (panels b, d, and f). The procollagens were incubated for up to 120 min with purified C-proteinase, analyzed by SDS-PAGE under reducing conditions, and the percentage of pro1(I) chains remaining at each point determined by excision of the bands and dual-label scintillation counting. The results of three independent experiments are shown.



Expression of the Mutant pro1(I) in 3T6 Cells

The mutant construct was transfected into 3T6 cells to allow the biosynthetic fate of normal endogenous pro1(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 Met-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(27) ). 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 pro1(I) C-propeptide in the medium of 3T6 cells transfected with the mutant construct (Fig. 8, lanes4 and 5). Only the larger, glycosylated form was present in untransfected 3T6 cells and cells transfected with the control gene (Fig. 8, lanes1-3).


Figure 7: Electrophoresis of CNBr peptides of pepsin-digested collagen produced by 3T6 cells transfected with the N-linked oligosaccharide mutant construct pWTCI-IQA. [H]Proline-labeled CNBr peptides were prepared from the cell culture medium and resolved of 12.5% polyacrylamide gels. [H]Proline incorporation into the CNBr peptides was determined by scintillation counting and the percentage of the total 1(I) chains that were mutant calculated using the formula described under ``Experimental Procedures.'' The percentage mutant chains is indicated below the lanes. Lanes6 and 11, collagens produced by untransfected 3T6 cells. The migration positions of the wild-type peptides 1(I)-CB6 and 1(I)-CB7 and the composite CB7-6 peptide produced from the mutant gene are indicated.




Figure 8: Electrophoretic analysis of the 1(I) C-propeptides produced by transfected 3T6 cells. Medium procollagens were purified and analyzed under reducing conditions on 12.5% acrylamide gels. The 1(I) C-propeptides were detected by immunoblotting with the polyclonal antibody LF-41. Lane1, untransfected 3T6 cells; lane2, 3T6 cells transfected with the reporter construct pWTCI-I; lane3, 3T6 cells transfected with the control construct pWTCI-IA. Lanes4 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 1(I) chains synthesized by these cells.



Secretion Kinetics of pro1(I) Lacking the N-Linked Oligosaccharide

To directly compare the rate of secretion of endogenous and transfected mutant pro1(I) chains, cells were pulse-labeled with [C]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 Met-Ile substitution in the pro1(I) chains produced from the transfected genes allowed the fate of these chains and endogenous wild-type pro1(I) chains expressed in the same cell to be compared accurately. Control transfected [IA]pro1(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 1(I) chains remained associated with the cell layer and probably represented secreted molecules which had been deposited into the extracellular matrix. In contrast, from 45 min of chase a greater proportion of the mutant [IQA]pro1(I) chains had been released into the medium compared to the endogenous chain (Fig. 9b). To determine if this resulted from an increase in the rate of intracellular trafficking or reflected a difference of the properties of the chains in the extracellular environment, secretion during the first hour of chase was re-examined (Fig. 9c). These data were the average of determinations on quadruplicate cultures and demonstrated that there was no difference in the rate of secretion of mutant unglycosylated and endogenous pro1(I) chains.


Figure 9: Secretion of endogenous pro1(I) and mutant pro1(I) chains lacking the N-linked oligosaccharide. Cells were pulse-labeled with [C]proline for 1 h and the radiolabeled collagen chased for up to 4 h in the presence of excess cold proline. At each point the pepsin-resistant cell layer and medium collagens were cleaved with CNBr and resolved on 10% polyacrylamide gels. [C]Proline incorporation into the CNBr peptides was determined by scintillation counting. The proportion of endogenous and transfected pro1(I) gene products which had been secreted into the medium was calculated using the formula described under ``Experimental Procedures.'' a, secretion of endogenous and control 1(I) chains by 3T6-IA5 cells. b and c, secretion of endogenous and mutant 1(I) chains by 3T6-IQA15 cells. In a and b each point represents the average of determinations on duplicate cultures. Quadruplicate cultures were analyzed in c.



It seemed possible that the high-mannose oligosaccharide was interacting with a cell surface or pericellular matrix component and that loss of this interaction may have allowed mutant chains to be more readily released into the medium. To test this 1 mg/ml yeast mannan was included during the preincubation, labeling, and chase periods to compete with the high-mannose oligosaccharide for extracellular binding sites. The presence of mannan did not alter the distribution of normal or mutant chains in these experiments (data not shown).

Mutant Unglycosylated pro1(I) Chains Are Efficiently Incorporated into an in Vitro Accumulated Extracellular Matrix

The different partitioning of mutant and wild-type chains between the cell layer and medium seen in the short pulse-labeling experiments may have been caused by a reduction in the efficiency of deposition of the unglycosylated chains into the extracellular matrix, reflecting the reduced C-propeptide processing suggested by the in vitro experiments. To test this, 3T6 cells transfected with the reporter gene (3T6-Ile21), the control construct (3T6-IA5), and the oligosaccharide attachment mutant construct (3T6-IQA15) were grown to confluence and then supplemented daily with ascorbic acid for 3 days to allow a collagenous matrix to develop. The cells were labeled for 18 h with [H]proline and the percentage of newly synthesized 1(I) chains produced from the transfected gene in the medium, the neutral salt-soluble cell layer and the mature pepsin-extracted matrix fractions determined. Expression of the transfected gene was different in each cell line; 59% of the pro1(I) chains in the medium of 3T6-IQA15 were synthesized from the transfected gene compared to 46 and 39% in 3T6-Ile21 and 3T6-IA5, respectively. To account for this and allow deposition by these three cell lines to be directly compared, the data were corrected so that the level of transfected gene expression in the medium of each cell line was the same. When compared in this way to the reporter and control pro1(I) chains, loss of the N-linked oligosaccharide had no effect on the pattern of deposition into the cell culture extracellular matrix (Fig. 10). The apparent reduction in the proportion of marker [Ile]1(I) chains in the neutral salt and pepsin-extracted fractions can be accounted for by a systematic error in the formula used to calculate the percentage of marker chains. Collagen deposited into the insoluble matrix becomes increasingly cross-linked in these cultures, and some of this collagen is cross-linked via lysine 930 in the CB6 peptide(40) . CNBr digestion will release the wild-type CB7 from these high molecular weight components, but not the composite CB7-6 from the marker chains. The recovery of [Ile]1(I) marker chain peptides is thus reduced relative to wild-type peptides.


Figure 10: Distribution of newly synthesized 1(I) chains in the matrix produced by transfected 3T6 cells. Cells were grown from confluence (day 0) in the presence of 0.25 mM ascorbic acid. On day 3 the cells were labeled with [H]proline for 18 h. Radiolabeled collagens released into the medium or deposited into the neutral-salt and pepsin-extracted fractions were digested with CNBr, analyzed on 10% polyacrylamide gels, and detected by fluorography. [H]Proline incorporation into the CNBr peptides was determined by scintillation counting. The percentage of the total newly synthesized 1(I) chains in each fraction which were transfected chains containing the Met-Ile reporter substitution ([Ile]1(I) chains) was calculated using the formula described under ``Experimental Procedures.'' Each point represents the average of determinations on three separate culture dishes. The data were corrected so that the transfected gene expression in the medium of each cell line was the same. 3T6-I21, cells transfected with the reporter construct pWTCI-I; 3T6-IA5, cells transfected with the control construct pWTCI-IA; 3T6-IQA15, cells transfected with the mutant gene pWTCI-IQA.




DISCUSSION

To prevent N-glycosylation of the pro1(I) C-propeptide and determine the importance of the oligosaccharide in procollagen biosynthesis, the acceptor Asn residue in the Asn-Ile-Thr consensus sequence was changed to Gln by site-directed mutagenesis. The mutation was introduced into the 25-kb COL1A1 reporter construct, pWTCI-I(27) , to allow the protein to be easily detected and quantitated at all stages of procollagen biosynthesis. The different CNBr cleavage pattern of normal and mutant chains made it possible to detect expression and subunit assembly and facilitated direct comparisons of the rate of secretion and the efficiency of deposition into the extracellular matrix. Without the helical marker, normal and mutant chains would have been indistinguishable in processed collagen molecules.

The control 1(I) C-propeptides synthesized by Mov13 cells reacted with the lectin GNA which specifically recognizes terminal mannose residues linked to mannose(41) . This indicated the presence of a high-mannose or hybrid type oligosaccharide structure and is consistent with the characterization of high-mannose oligosaccharides on the C-propeptides secreted by chick tendon cells(18, 19) . As expected, Mov13 cells transfected with the mutant construct synthesized 1(I) C-propeptides which migrated faster on SDS-PAGE and failed to react with GNA. Only the larger form of the pro1(I) C-propeptide was seen in control transfected Mov13 cells and untransfected 3T6 cells confirming that 3T6 cells also N-glycosylated the C-propeptide and indicating that each chain was normally substituted with a carbohydrate group.

Procollagen assembly is a poorly understood process initiated by interactions between the three COOH-terminal propeptides. Individual C-propeptides fold and intrachain disulfide bonds form prior to chain association and stabilization by interchain disulfide bridges(42) . Thus, folding of the C-propeptide domain into the correct structure is critical for efficient molecular assembly. C-propeptide mutations which slow chain association and disulfide bond formation cause the component chains to be exposed for longer than normal to the enzymes which post-translationally hydroxylate and glycosylate lysine residues and result in increased levels of these modifications(39, 43, 44) . Overmodified -chains migrate more slowly on SDS-PAGE, and so electrophoretic migration is a sensitive measure of the efficiency of C-propeptide folding, subunit association, and helix formation. The presence of only normally migrating -chains in Mov13 cells, and in 3T6 cells synthesizing both normal and mutant pro1(I) chains, demonstrated that neither folding of the C-propeptide domain or the ability of the molecules to oligomerize were affected by the elimination of the N-linked carbohydrate.

Normal secretion of mutant pro1(I) was also consistent with the proposal that unglycosylated chains were able to fold properly. Unglycosylated proteins are commonly retained in the endoplasmic reticulum, and this is thought to be a consequence of misfolding and failure to achieve a native structure(3, 15, 26, 45) . The reported inhibition of procollagen secretion by tunicamycin (21, 22) may have been due to secondary effects of the antibiotic. Some preparations of tunicamycin have cytotoxic effects (46) or produce a general reduction in protein synthesis(24, 25) . Tunicamycin may also have slowed procollagen secretion by inhibiting the activity of intracellular glycoprotein enzymes involved in collagen biosynthesis, such as the prolyl and lysyl hydroxylases. Prevention of proline hydroxylation, a post-translational modification necessary for stability of the triple helical domain(47) , slows secretion and leads to the accumulation of procollagen chains in the endoplasmic reticulum(48, 49) .

Since the unglycosylated pro1(I) chains were cleaved by the purified C-proteinase only slightly less efficiently than wild-type chains, the almost complete lack of C-propeptide processing in fibroblasts treated with tunicamycin (20) probably reflected the importance of N-glycosylation for the secretion or normal activity of the procollagen C-proteinase or its enhancer glycoprotein(50) . This conclusion is supported by the finding that medium from chick tendon cells cultured with tunicamycin lacked C-proteinase activity(23) .

The mechanism responsible for the different partitioning of normal and mutant chains between the cell layer and medium in the pulse-chase secretion experiments remains to be clarified. The difference was not eliminated by the inclusion of yeast mannan indicating that the high-mannose oligosaccharide itself was not binding to the matrix or to the fibroblast surface after secretion. However, this experiment did not exclude the possibility that alterations in the tertiary conformation of the unglycosylated C-propeptide decrease binding of other motifs to a cell surface or matrix component. Several studies have demonstrated specific cell surface-C-propeptide interactions but the molecular basis of these interactions has not yet been determined. The C-propeptides of type I collagen have been immunolocalized to the cell surface (51) and have been shown to interact specifically with a cell surface integrin (52) and with sphingomyelin, a major lipid component of the plasma membrane(53) . Exogenously added type I collagen C-propeptides can be internalized by human lung fibroblasts, and a proportion becomes associated with the nucleus where they are thought to mediate their observed inhibitory effect on procollagen gene transcription(54) . Interactions between procollagen molecules, and between procollagen and cell surface components could also be initiated intracellularly and not be susceptible to competition from the exogenously added yeast mannan. For example, interactions between the propeptides and the secretory vesicle membrane have been proposed to be important in the development of the ordered segment-long-spacing procollagen aggregates which are secreted by tendon cells(55) . If procollagen-cell membrane interactions are important in the formation of fibril assembly intermediates as has been suggested(55, 56) , then a reduction in the strength of these interactions may have led to impaired deposition of the unglycosylated chains into the extracellular matrix.

3T6 cells grown in the continuous presence of ascorbic acid accumulate a highly cross-linked collagenous extracellular matrix(40) . In this in vitro model, unglycosylated pro1(I) chains were processed and incorporated into the insoluble cross-linked matrix at the same rate as reporter and control pro1(I) chains. Reduced processing would be expected to result in less efficient deposition of the mutant into the extracellular matrix. C-propeptide processing is the rate-limiting step in the formation of collagen fibrils in vitro(57) and in this same 3T6 culture system, a mutation which deleted the C-propeptide cleavage site completely prevented deposition of those chains into the accumulated matrix.()In the presence of a pre-existing collagen matrix, efficient processing and matrix deposition of the unglycosylated procollagens masked the difference in the distribution of normal and mutant chains observed in the short pulse-labeling experiments. Thus, although it is tempting to speculate that the reduction in processing of unglycosylated C-propeptides seen in the in vitro experiments may be reproduced in cell culture under certain conditions and may be responsible for the distribution difference, the difference is not measurable in this cell culture extracellular matrix model system, and loss of the oligosaccharide does not prevent alignment of procollagen molecules and fibril assembly in this system.

Failure to demonstrate a functional difference between normal and unglycosylated pro1(I) chains in the biosynthetic events of subunit assembly, secretion, propeptide processing, and matrix deposition was surprising in light of the conservation of the N-linked glycosylation site among the fibrillar collagen subunits(58) . What, then, is the role of the high mannose carbohydrate? Cleaved type I collagen C-propeptides have recently been shown to be endocytosed by liver endothelial cells via the mannose receptor(59) . This pathway is the major route of uptake and subsequent degradation of circulating C-propeptide. It seems likely that the high mannose N-linked oligosaccharide has been conserved because it functions as a targeting signal which directs the clearance of cleaved C-propeptides.


FOOTNOTES

*
This work was supported by grants from the National Health and Medical Research Council of Australia and the Royal Children's Hospital Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia. Fax: 61-3-345-6668.

The abbreviations used are: C-propeptide, carboxyl-terminal propeptide; C-proteinase, carboxyl-terminal proteinase; pN, procollagen processing intermediate retaining the amino-terminal propeptide; pC, procollagen processing intermediate retaining the carboxylterminal propeptide; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis.

Amino acids are numbered from the translation start site of pre-pro1(I).

S. P. Fenton and J. F. Bateman, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Karl Kadler (University of Manchester) for his generous gift of purified C-proteinase and for critical evaluation of the manuscript.


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