©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Endoplasmic Reticulum-mediated Quality Control of Type I Collagen Production by Cells from Osteogenesis Imperfecta Patients with Mutations in the pro1(I) Chain Carboxyl-terminal Propeptide which Impair Subunit Assembly (*)

Shireen R. Lamandé (1), Steven D. Chessler (2), Suzanne B. Golub (1), Peter H. Byers (2) (3), Danny Chan (1), William G. Cole (§) , David O. Sillence (4), John F. Bateman (1)(¶)

From the (1) Orthopaedic Molecular Biology Research Unit, Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia, the Departments of (2) Pathology and (3) Medicine, University of Washington, Seattle, Washington 98195, and the (4) Department of Genetics, The Children's Hospital, Camperdown, New South Wales 2050, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A heterozygous single base change in exon 49 of COL1A1, which converted the codon for pro1(I) carboxyl-terminal propeptide residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a baby with lethal osteogenesis imperfecta (OI64). The C-propeptide mutations in OI64 and in another lethal osteogenesis imperfecta cell strain (OI26), which has a frameshift mutation altering the sequence of the carboxyl-terminal half of the propeptide (Bateman, J. F., Lamande, S. R., Dahl, H.-H. M., Chan, D., Mascara, T. and Cole, W. G. (1989) J. Biol. Chem. 264, 10960-10964), disturbed procollagen folding and retarded the formation of disulfide-linked trimers. Although assembly was delayed, the presence of slowly migrating, overmodified 1(I) and 2(I) chains indicated that mutant pro1(I) could associate with normal pro1(I) and pro2(I) to form pepsin-resistant triple-helical molecules, a proportion of which were secreted. Further evidence of the aberrant folding of mutant procollagen in OI64 and OI26 was provided by experiments demonstrating that the endoplasmic reticulum resident molecular chaperone BiP, which binds to malfolded proteins, was specifically bound to type I procollagen and was coimmunoprecipitated in the osteogenesis imperfecta cells but not control cells. Experiments with brefeldin A, which inhibits protein export from the endoplasmic reticulum, demonstrated that unassembled mutant pro1(I) chains were selectively degraded within the endoplasmic reticulum resulting in reduced collagen production by the osteogenesis imperfecta cells. This biosynthetic deficiency was reflected in the inability of OI64 and OI26 cells to produce a substantial in vitro collagenous matrix when grown in the continuous presence of ascorbic acid to allow collagen matrix formation. Both these carboxyl-terminal propeptide mutants showed a marked reduction in collagen accumulation to 20% (or less) of control cultures, comparable to the reduced collagen content of tissues from OI26.


INTRODUCTION

The ``brittle-bone'' disease, osteogenesis imperfecta (OI),() results from mutations in the type I collagen genes (COL1A1 and COL1A2) in the vast majority of cases studied to date (1, 2) . The mutations include insertions, deletions, gene rearrangements and, most commonly, point mutations in the triple helical domains of the 1(I) and 2(I) chains. These substitutions interrupt the obligatory Gly- X-Y repeat sequence and compromise the structural integrity of the helix. Mutations outside the helical domain, in the carboxyl-terminal propeptides, are infrequent but demonstrate the critical role of these regions in directing chain assembly, trimerization, and the initiation of helix formation. The severity of OI resulting from carboxyl-terminal propeptide mutations is dependent on the extent to which the mutations compromise procollagen assembly. Two frameshift mutations, one in pro1(I) and the other in pro2(I), completely prevented incorporation of the mutant pro-chains into type I procollagen heterotrimers resulting in a ``functionally null'' allele and consequently relatively mild clinical phenotypes (3, 4) . While the heterozygotes for the COL1A1 mutation had OI type I, patients heterozygous for the COL1A2 frameshift mutation were clinically normal, but the homozygote had a moderate OI type III phenotype. However, four different carboxyl-terminal propeptide mutations which disturbed, but did not prevent molecular assembly, resulted in lethal OI (5, 6, 7) .

In this study we have defined a point mutation which produces a cysteine for tryptophan substitution at amino acid 1312() of the prepro1(I) chain (carboxyl-terminal propeptide residue 94) and causes a lethal form of OI. To further characterize the biosynthetic consequences of carboxyl-terminal propeptide mutations, procollagen molecular assembly, intracellular degradation, secretion, and accumulation in the extracellular matrix were examined in this cell strain (OI64) and also in OI26 cells, where a frameshift mutation produced a change in the amino acid sequence beginning at residue 106 of the carboxyl-terminal propeptide and resulted in a truncated chain (5, 6) . In both cell strains intermolecular carboxyl-terminal propeptide disulfide-bond formation was disturbed, and procollagen assembly was slowed. Furthermore, the endoplasmic reticulum-resident molecular chaperone BiP was induced and bound to type I procollagen in both cell strains. Although mutant pro1(I) chains were degraded within the endoplasmic reticulum, degradation was not complete and some abnormal, overmodified molecules were secreted by both OI cell lines. The formation of a collagenous matrix by the OI cells was evaluated in an in vitro culture system where cells were incubated for extended periods in the presence of ascorbate, leading to the deposition and maturation of an extensive collagen matrix in control cell cultures. As a consequence of defective molecular assembly and increased intracellular degradation both cell strains showed a dramatic collagen matrix deficiency.


EXPERIMENTAL PROCEDURES

The Patients

The first infant (OI64), was delivered at 38 weeks gestation and died within 24 h. The ribs contained healing fractures and the long bones showed numerous metaphyseal and diaphyseal fractures but were not as wide and crumpled in appearance as observed in many cases of OI type II (8) . The parents were unrelated and normal. The second patient (OI26) with a previously defined pro1(I) propeptide frameshift mutation (6) also had lethal OI with a strikingly similar radiological appearance (9) . OI and control fibroblast cultures were established from dermal samples obtained at autopsy with parental consent and the approval of the Ethics Committee of this hospital and were maintained as described previously (5, 10) .

Antibodies

Polyclonal antibodies to the carboxyl-terminal propeptides of pro1(I) and pro2(I) (PC), and the carboxyl-terminal (LF-41, 11) and amino-terminal propeptides of pro1(I) (LF-9, 12) were kindly provided by Dr. Burton Goldberg (University of Wisconsin School of Medicine) and Dr. Larry Fisher (National Institute of Dental Research). Rat anti-BiP monoclonal antibody (13) was a generous gift from Dr. David Bole (University of Michigan School of Medicine).

Biosynthetic Labeling and Immunoprecipitation

Procollagens were biosynthetically labeled with 5 µCi/ml L-[5-H]proline (8.5 Ci/mmol, NEN Research Products) for 18 h in medium containing 10% (v/v) dialyzed fetal calf serum and 0.25 m M ascorbic acid. In some experiments the ascorbic acid was replaced with 0.1 m M ,`-dipyridyl to prevent the post-translational modification of proline and lysine residues, or 100 ng/ml brefeldin A (Boehringer Mannheim) was added to prevent transport of secretory proteins out of the endoplasmic reticulum (14) . Cell layer and medium fractions were harvested separately in the presence of the proteinase inhibitors 5 m M EDTA, 10 m M N-ethylmaleimide, and 1 m M phenylmethanesulfonyl fluoride as described previously (5, 10) . Procollagens and collagens were precipitated with 25% saturated (NH)SOand resuspended in 50 m M Tris-HCl, pH 7.5, containing 0.15 M NaCl, and proteinase inhibitors. Aliquots of procollagens were precipitated with 75% (v/v) ethanol and converted to collagen by limited pepsin digestion (100 µg/ml pepsin in 0.5 M acetic acid, 4 °C, 16 h).

For the pulse-chase analysis of procollagen assembly, confluent cultures were incubated overnight in medium containing 10% (v/v) fetal calf serum and 0.25 m M ascorbic acid, preincubated for 2 h in serum-free medium supplemented with 0.25 m M ascorbic acid, then labeled with 250 µCi/ml L-[2,3,4,5-H]proline (114 Ci/mmol, NEN Research Products). After 30 min the medium was removed, the cells were washed with medium containing 10% (v/v) fetal calf serum, 0.25 m M ascorbic acid, and 10 m M proline, and then incubated in this chase medium for up to 6 h. Cell layer procollagens were harvested in the presence of 20 m M iodoacetamide to prevent further disulfide bond formation and were precipitated with 30% (v/v) ethanol (15) . In some experiments 100 ng/ml brefeldin A was included during the preincubation, labeling, and chase periods.

For biosynthetic labeling with [S]methionine, the cells were rinsed in methionine-deficient medium and then incubated for 3 h in methionine-deficient medium supplemented with 100 µCi/ml [S]methionine (>1000 Ci/mmol, Amersham Corp.). Cells were lysed in an ATP-depleting non-ionic detergent buffer (1% Nonidet P-40, 150 m M NaCl, 50 m M Tris, pH 7.5, 1 m M phenylmethanesulfonyl fluoride, 8 units/ml hexokinase (Sigma), 11 m M glucose, and 24 units/ml apyrase (Sigma)). After 30 min on ice, iodoacetamide (30 m M, final) was added to the lysates, and they were centrifuged briefly to remove nuclei and insoluble material. Aliquots of cell extract were diluted with lysis buffer, primary antibody was added, and samples mixed by end-over-end rotation. After 1-2 h, protein G-Sepharose (Pharmacia) was added to the reactions, and they were mixed for several hours more. The beads were washed three times, 30 min each time, first in 50% lysis buffer and 50% NET buffer (0.1% Nonidet P-40, 150 m M NaCl, 1 m M EDTA, 0.02% sodium azide, 50 m M Tris-HCl, pH 7.5), then twice with NET buffer. Precipitated protein complexes were examined by SDS-PAGE. To verify the position of BiP, cell extracts from fibroblasts treated overnight with tunicamycin (5 µg/ml) and labeled as above with [S]methionine were loaded on most gels (16) .

Extraction of Collagen from the Extracellular Matrix

Cell layers from cultures which had been supplemented daily from confluence (day 0) with 0.25 m M ascorbic acid were sequentially extracted with 50 m M Tris-HCl, pH 7.5, containing 0.15 M NaCl and proteinase inhibitors (neutral salt soluble fraction), 0.5 M acetic acid (acetic acid soluble fraction), and by digestion with pepsin (0.1 mg/ml pepsin in 0.5 M acetic acid) as described previously (17) . Cells were released from the extracellular matrix by digestion with bacterial collagenase and trypsin (17) and the cell DNA content quantified using the Hoescht 332558-specific fluorescence procedure (18) .

SDS-Polyacrylamide Gel Electrophoresis

Procollagen and collagen chains were resolved on 5% (w/v) polyacrylamide separating gels with a 3.5% (w/v) stacking gel. Sample preparation, electrophoresis conditions, Coomassie Brilliant Blue staining, and fluorography of radioactive gels have been described elsewhere (5, 10) . Coomassie-stained bands were quantitated by densitometry in comparison to standard collagen samples loaded on each gel. The radioactivity in the procollagen and collagen bands was measured by scintillation counting of excised bands (19) . Procollagen chains labeled in culture or produced by cell-free translation of mRNA were also analyzed by two-dimensional gel electrophoresis (20) which resolves the chains on the basis of both charge and size.

Immunoblotting

Proteins were electrophoretically transferred from 5% (w/v) SDS-polyacrylamide gels to nitrocellulose filters (BA85, Schleicher and Schuell). Bound LF-41 antibody was detected using horseradish peroxidase-conjugated protein A (Bio-Rad) and an enhanced chemiluminescence kit (ECL, Amersham Corp.).

Detection of the OI64 RNA Mutation by Chemical Cleavage of mRNA cDNA Heteroduplexes

Total RNA was purified from OI64 fibroblasts (21) and hybridized to a 672-bp XhoI- EcoRI fragment of the 1(I) cDNA clone Hf677 (22) which had been end-labeled with [-P]dCTP (3000 Ci/mmol, Amersham Corp.). Heteroduplex formation, hydroxylamine and piperidine treatment, and electrophoretic analysis were performed as described previously (6, 23) .

Preparation of Genomic DNA, PCR Amplification, and Direct Sequencing

Genomic DNA was purified from cultured OI64 fibroblasts and whole blood from the parents (24) and the region of the COL1A1 gene containing the mutation amplified through 30 cycles by the PCR using the primers 5`-TACTGGATTGACCCCAACCA-3` and 5`-TGGTAGGTGATGTTCTGGGA-3` and Taq polymerase (Perkin-Elmer Cetus). The 575-bp amplification product was gel purified and sequenced directly using the 5` PCR primer (25) .

Quantitation of pro1(I) and pro2(I) mRNA Ratios

OI64 and control fibroblasts were grown to confluence then supplemented daily with 0.25 m M ascorbic acid for 3 days to maximize type I collagen mRNA levels (26) . Total RNA was extracted and dilutions dotted onto duplicate nitrocellulose membranes. cDNA probes for 1(I) (Hf677, 22) and 2(I) (Hf32, 27) were labeled with [-P]dCTP (3000 Ci/mmol, Amersham Corp.) by nick translation and the filters hybridized, washed, and specific hybridization quantified as described previously (28) .

Quantitation of Normal and Mutant pro1(I) mRNA Transcripts

The mutation deleted a RsaI site which allowed PCR products derived from the normal and mutant alleles to be discriminated. Total RNA from two separate preparations was amplified by 25 PCR cycles using the same primers and conditions used for the genomic DNA PCR. Two aliquots from each reaction were diluted 1:10 in fresh PCR buffer and subjected to a final cycle in the presence of [-P]dCTP. The products were digested with RsaI, separated on an 8% non-denaturing polyacrylamide gel, and the radioactivity in each band quantitated by excision and scintillation counting. The final cycle eliminated heteroduplexes which are resistant to restriction enzyme digestion and commonly form during later PCR cycles when amplification has plateaued (29) .

Cell-free Translation

Cell-free translation of control and OI26 mRNA was performed in a rabbit reticulocyte lysate system (Promega) labeling with L-[2,3,4,5-H]proline.


RESULTS

Retarded Chain Association of Type I Procollagen

When unhydroxylated procollagens, produced by labeling cells in the presence of ,`-dipyridyl, were analyzed without reduction, the pro1(I) and pro2(I) chains in the control were linked by disulfide bonds and migrated near the top of the gel (Fig. 1, lane 2). In the OI26 and OI64 samples, there was an additional band which had a similar migration to reduced pro1(I) chains (Fig. 1, lanes 4 and 6). This band was identified as a pro(I) chain by immunoblotting with LF-41, an antibody which specifically recognizes the last 21 amino acids at the carboxyl terminus of pro1(I) (Fig. 1, lane 10). This suggested that, as in OI26 where an 1(I) carboxyl-terminal propeptide mutation had been characterized previously, pro1(I) chains from OI64 contained a mutation which interfered with chain association and the formation of interchain disulfide bonds. Unreduced pro1(I) monomers migrated faster than fully reduced pro1(I) indicating the presence of internal disulfide bonds (30) .


Figure 1: Identification of undisulfide-bonded pro1(I) chains in OI26 and OI64 cells. Fibroblast cultures were labeled with [H]proline for 18 h in the presence of ,`-dipyrridyl, and the unhydroxylated procollagens from control ( C), OI26, and OI64 cultures were examined on 5% SDS-polyacrylamide gels either with (+) or without (-) reduction with 10 m M dithiothreitol ( DTT). [H]Proline-labeled proteins ( lanes 1-6) were detected by fluorography and quantitated by scintillation counting as described under ``Experimental Procedures.'' Lanes 2, 4, and 6 contain twice the sample loading as lanes 1, 3, and 5. Proteins were transferred to nitrocellulose membranes and pro1(I) chains detected using the antibody LF-41 which recognizes the last 21 amino acids of the pro1(I) carboxyl-terminal propeptide ( lanes 7-10). The migration positions of the reduced unhydroxylated pro1(I) and pro2(I) chains are indicated. Electrophoretic transfer of the large disulfide-bonded procollagen molecules is inefficient, and this species is therefore not routinely detected by immunoblotting.



Molecular Characterization of a Carboxyl-terminal Propeptide Mutation in OI64

The mutation was initially detected and localized by chemical modification and cleavage of heteroduplexes formed between mRNA extracted from OI64 fibroblasts and a control 1(I) 672 bp XhoI- EcoRI cDNA probe which spanned the majority of the carboxyl-terminal propeptide. Hydroxylamine/piperidine treatment resulted in cleavage of the probe and the production of a 251-bp fragment (data not shown) indicating the presence of a mismatched C in the probe in the region coding for prepro1(I) tryptophan 1312 (carboxyl-terminal propeptide residue 94).

To define the mutation, genomic DNA from the patient and from the parents was amplified by the PCR and sequenced directly. The patient was heterozygous for a single base change in exon 49 that converted the codon for amino acid 1312 from tryptophan (TGG) to cysteine (TGT). The mutation was not present in either of the phenotypically normal parents (data not shown).

Quantitation of OI64 Type I Collagen mRNA Transcripts

Because the mutation deleted a RsaI restriction site in cDNA we could assess the effect of the mutation on mRNA expression. The normal transcript represented an average of 54% of the total 1(I) mRNA indicating that the mutation did not measurably affect steady-state mRNA levels. This was further confirmed by comparing the levels of pro1(I) and pro2(I) mRNA. In control cells the 1(I)/2(I) ratio was 1.47 ± 0.08 (mean ± S.D., n = 8) compared with 1.46 in OI64 cells (average of determinations on mRNA preparations from two separate cultures). These values were not corrected for the probe size and specific activity.

Analysis of Interchain Disulfide Bond Formation in OI64 and OI26

To characterize the effect of the point mutation in OI64 and a frameshift mutation in OI26 (6) on interchain disulfide bond formation and molecular assembly, control, OI64, and OI26 cells were pulse-labeled for 30 min and then chased for up to 6 h prior to analysis of the procollagen by SDS-PAGE under non-reducing conditions. In control cells almost all of the pro1(I) chains were incorporated into disulfide-linked trimers by 40 min of chase (Fig. 2 a). In contrast, monomeric pro1(I) chains were present in OI64 cells for more than 120 min and in OI26 cells even after 6 h of chase (Fig. 2, c and d) indicating that the mutations slowed the formation of interchain disulfide bonds. In control cells monomeric pro2(I) chains disappeared more slowly than free pro1(I) chains, after 1-2 h of chase, suggesting that they were synthesized in excess (Fig. 2 a).


Figure 2: Delayed procollagen assembly in OI26 and OI64 cells. Control, OI26, and OI64 fibroblasts were pulse-labeled for 30 min with [H]proline then chased in proline-supplemented medium for up to 360 min. Cell layer procollagens were separated without reduction on 5% SDS-polyacrylamide gels. a, control cells; c, OI26; d, OI64. In each case lanes 1-7 show procollagens from cells chased for 0, 20, 40, 60, 120, 240, and 360 min, respectively. The migration positions of pro1(I) and pro2(I) chains and the disulfide-bonded pro chain dimers and trimers are indicated. The protein migrating above pro2(I) ( arrow in a)) is unknown. b, control cells were preincubated, pulse labeled, and chased in the presence of brefeldin A which prevents protein transport out of the endoplasmic reticulum.



Intracellular Accumulation of Excess pro2(I) Chains in Brefeldin A-treated Cells

The excess synthesis of pro2(I) chains relative to pro1(I) was highlighted when brefeldin A was included during the preincubation, pulse labeling, and chase periods. Brefeldin A is a fungal metabolite which inhibits protein secretion by causing the cis-Golgi to fuse with the endoplasmic reticulum, preventing further protein transport (14) . While pro1(I) chain disulfide-bonding may have been slightly slowed in the presence of brefeldin A, incorporation of pro1(I) monomers into disulfide-bonded trimers was complete by 60 min of chase (Fig. 2 b), and the most obvious effect of brefeldin A was to almost completely prevent the disappearance of undisulfide-bonded pro2(I) chains. These chains migrated more slowly with increasing chase time consistent with the proposal that they were excess non-helical subunits and continued to be substrates for post-translational enzymatic hydroxylation and glycosylation (31) .

Intracellular accumulation of pro2(I) chains was also observed when control, OI64, and OI26 cells were continuously labeled for 18 h in the presence of brefeldin A. About 25% of the total pro2(I) chains from control cells migrated as undisulfide-bonded monomers when exit from the endoplasmic reticulum was blocked by brefeldin A (Fig. 3, lanes 1 and 2); however, in brefeldin A-treated OI64 cells, undisulfide-bonded pro2(I) chains represented a larger proportion, about 40%, of the total pro2(I) (Fig. 3, lanes 3 and 4). In contrast, brefeldin A-treated OI64 cells did not accumulate extra undisulfide-bonded pro1(I) chains; approximately 10% of the pro1(I) chains migrated as monomers in both ,`-dipyridyl and brefeldin A-treated OI64 cells (Fig. 1, lanes 5 and 6; Fig. 3, lanes 3 and 4). OI26 cells exhibited a similar pattern of intracellular procollagen chain accumulation. Brefeldin A-treated OI26 cells accumulated excess undisulfide-bonded pro2(I) chains to a greater extent than control cells (Fig. 3, lanes 5 and 6), while the proportion of undisulfide-bonded pro1(I) chains was not increased compared with ,`-dipyridyl-treated OI26 cells (Fig. 1, lanes 3 and 4; Fig. 3, lanes 5 and 6). This pattern of additional pro2(I) accumulation by brefeldin A-treated OI26 and OI64 cells was consistent in three separate experiments.


Figure 3: Electrophoretic analysis of intracellular procollagens from brefeldin A-treated fibroblasts. Control ( C), OI26, and OI64 fibroblasts were labeled with [H]proline for 18 h in the presence of brefeldin A to prevent protein transport out of the endoplasmic reticulum, as described under ``Experimental Procedures.'' Intracellular procollagens were analyzed with (+) and without (-) reduction with 10 m M dithiothreitol ( DTT) on 5% SDS-polyacrylamide gels. [H]Proline-labeled proteins were detected by fluorography and quantitated by scintillation counting as described under ``Experimental Procedures.'' Lanes 2, 4, and 6 contain twice the sample loading as lanes 1, 3, and 5. The migration positions of the reduced pro1(I) and pro2(I) chains are shown.



Selective Intracellular Degradation of Mutant pro1(I) Chains within the Endoplasmic Reticulum

The extra accumulation of excess, monomeric pro2(I) chains in brefeldin A-treated OI26 and OI64 cells when compared to control cells suggested that relatively fewer pro1(I) chains were available for assembly with pro2(I) subunits in the OI cells probably because of increased intracellular pro1(I) degradation. The frameshift mutation in OI26 results in the synthesis of a more basic pro1(I) chain which is 37 amino acids shorter than normal. Mutant and normal OI26 pro1(I) chains were resolved by two-dimensional gel electrophoresis, which separates proteins on the basis of both charge and size, and this allowed the intracellular fate of these chains to be directly compared (Fig. 4). The identity of pro1(I) chains was confirmed by immunoblotting (data not shown). Mutant and normal pro1(I) chains were almost equally abundant in samples produced by cell-free translation of fibroblast mRNA (Fig. 4 b) indicating that the steady-state levels of normal and mutant mRNA were similar. In contrast, the mutant chain was significantly reduced relative to normal pro1(I) in cells labeled for 18 h in the presence of ,`-dipyridyl (Fig. 4 d), demonstrating that it was selectively degraded. Degradation of the mutant pro1(I) chain was not inhibited by brefeldin A (Fig. 4 f) consistent with an endoplasmic reticulum-localized degradation pathway.


Figure 4: Two-dimensional gel electrophoresis of control and OI26 procollagens. [H]Proline-labeled procollagens were produced by cell-free translation of control ( a) and OI26 ( b) fibroblast mRNA, and by incubation with ,`-dipyridyl ( c and d) or brefeldin A ( e and f). Pro1(I) and pro2(I) chains are designated in a by 1 and 2, respectively. The arrowhead indicates the migration position of the mutant, more basic pro1(I) chain produced by OI26 cells and by cell-free translation.



Normal and abnormal OI64 pro1(I) carboxyl-terminal propeptides could not be resolved on two-dimensional isoelectric focusing gels (data not shown) so it was not possible to determine the relative proportions of normal and mutant pro1(I) chains remaining in the cells in these experiments.

Coprecipitation of BiP and Type I Procollagen

The endoplasmic reticulum-resident protein BiP has been shown to bind to malfolded secretory proteins (32) . When type I procollagen was immunoprecipitated with an antibody to its carboxyl-terminal propeptide (that recognizes both normal and abnormal procollagen molecules), BiP coprecipitated in extracts from OI64 and OI26 cells but not from control cells (Fig. 5), demonstrating BiP binding to the abnormal procollagen produced by the OI cells. BiP has ATPase activity and, in vitro, dissociates from proteins to which it is bound in the presence of ATP and Mg(33, 34) . When immunoprecipitated type I procollagen from OI64 and OI26 cells was incubated with ATP, BiP was released from the precipitated protein complex, (Fig. 5, a, d, and e), verifying the specificity of BiP-pro chain binding in both OI26 and OI64. BiP synthesis increased in both OI64 and OI26 cells after the addition of ascorbate to the culture medium. After 20-24 h of ascorbate treatment, BiP protein content was substantially greater in OI64 and OI26 cells than in control cells (data not shown).


Figure 5: BiP binds type I procollagen in OI26 and OI64 cells. OI26, OI64, and control cells ( C) were treated overnight with ascorbate, labeled for 3 h with [S]methionine, harvested in ATP-depleting lysis buffer, and analyzed by SDS-PAGE under reducing ( a and b) and non-reducing ( c and d) conditions. a, OI26 cell extracts were immunoprecipitated with a polyclonal antibody to the carboxyl termini of pro1(I) and pro2(I) ( PC). The right lane shows the effect of ATP on the binding of BiP to pro chains from OI26. Between washes, the precipitated protein complexes were incubated for 30 min at 23 °C in NET buffer without EDTA and with either 10 m M MgClalone (-) or with 10 m M MgCland 4 m M ATP (+). ATP specifically disrupted BiP-procollagen chain binding. b, OI64 cell extracts immunoprecipitated with antibodies to the carboxyl-terminal and amino-terminal propeptides of type I procollagen. BiP coprecipitated with type I procollagen in lysates from OI64 cells. c, OI26 proteins were precipitated with either the monoclonal antibody to BiP ( B), or the polyclonal antibody to the 1(I) and 2(I) carboxyl-terminal propeptides ( PC). The type I pro chains that coprecipitated with BiP from OI26 cells migrated as monomers under non-reducing conditions. d, OI64 proteins were immunoprecipitated, washed, and treated with (+) or without ( -) 4 m M ATP as in a. e, a longer exposure of the region of the gel containing the coprecipitated proteins. BiP was bound to type I procollagen molecules from OI64 cells and was released by treatment with ATP. The protein that coprecipitated with type I procollagen from control cells is unknown. The positions of the type I pro chains, BiP, type I procollagen trimers , and fibronectin ( FN) are indicated.



Secretion of Mutant pro1(I) Chains by OI64 and OI26 Fibroblasts

To further compare the fate of pro1(I) chains with mutations in the carboxyl-terminal propeptide domain, collagens from OI26 and OI64 were biosynthetically labeled with [H]proline and aliquots from the medium were examined for the presence of undisulfide-linked pro1(I) chains. Some undisulfide-linked pro1(I) chains were seen in the medium of OI26 cells (Fig. 6 a, lane 2); however, OI64 cells did not secrete undisulfide-bonded pro1(I) (Fig. 6 a, lane 3). The presence of slowly migrating 1(I), pN2(I), and 2(I) chains in the medium of both cell stains (Fig. 6 b, lanes 2 and 4) confirmed that some procollagen molecules containing mutant chains were secreted. No undisulfide-bonded pro2(I) chains were secreted by either control or OI cells (Fig. 6 a) indicating that the excess pro2(I) chains synthesized were degraded within the cell.


Figure 6: Mutant pro1(I) chains are secreted and deposited into the extracellular matrix by OI26 and OI64 fibroblasts. a, control, OI26 , and OI64 fibroblasts were labeled with [H]proline for 18 h and procollagens prepared as described under ``Experimental Procedures .'' Samples from the medium were analyzed without reduction on 5% SDS-polyacrylamide gels. Undisulfide-bonded pro1(I) chains were present in the medium from OI26 cells ( lane 2) but were not secreted by control ( lane 1) or OI64 fibroblasts ( lane 3). The migration positions of pro1(I), pN1(I), 1(I), pN2(I) , and 2(I) chains are indicated. The expected migration position of pro2(I) chains is shown by the arrow. b, control, OI26 , and OI64 cells were grown from confluence(day 0) in the presence of ascorbic acid and labeled with [H]proline for 18 h on day 8. Labeled collagens from the medium, neutral salt, and pepsin extracted fractions were analyzed by SDS-PAGE and detected by fluorography (see ``Experimental Procedures'' for details). Slowly migrating, overmodified 1(I) and 2(I) chains were present in the medium and neutral salt soluble fractions, as well as the mature pepsin extracted matrix from OI26 ( lanes 2, 6, and 10) and OI64 ( lanes 4, 8, and 12). Collagens from the medium, neutral salt soluble fraction , and pepsin -extracted matrix of control cells were loaded in alternate lanes to facilitate comparison of chain migrations ( lanes 1, 3, 5, 7, 9, and 11)



The Effect of Carboxyl-terminal Propeptide Mutations on Matrix Formation by OI64 and OI26 Fibroblasts

When cultured in the continuous presence of ascorbic acid human fibroblasts accumulate an extensive, insoluble, collagenous matrix (26) . This culture system was exploited to examine and compare the effects of these 1(I) carboxyl-terminal propeptide mutations on the deposition and maturation of an in vitro extracellular matrix. The matrix deposited by control and OI cells grown for up to 12 days post-confluence was sequentially extracted with a neutral salt buffer to remove the newly synthesized soluble collagens, acetic acid to extract the acid-labile cross-linked collagens, and pepsin to solubilize the more mature, covalently cross-linked collagen matrix. The total collagen accumulated in these three extracted fractions was related to the DNA content of the cells and is shown in Fig. 7. Both OI26 and OI64 showed a striking deficiency in matrix production. Slowly migrating, overmodified 1(I) and 2(I) chains were apparent, not only in the readily soluble fraction (Fig. 6 b, lanes 6 and 8), but also in the mature, pepsin-extracted matrix (Fig. 6 b, lanes 10 and 12). The distribution of the collagen among the neutral salt, acetic acid, and pepsin extracts was similar in all three cultures with 80-90% of the total collagen present in the pepsin extracted fraction (data not shown).


DISCUSSION

The carboxyl-terminal propeptide mutations in OI64 and OI26 dramatically slowed the assembly of pro1(I) chains into disulfide-linked trimers. Procollagen assembly is initiated by interactions between the carboxyl-terminal propeptides, and although the details of the folding pathway and intermediates are not known, the carboxyl-terminal propeptides of individual pro chains fold and form intrachain disulfide bonds before the chains associate and are linked by interchain disulfide bonds (35) . Disulfide bond formation during protein folding involves catalysis by protein disulfide isomerase, the -subunit of prolyl-4-hydroxylase (36, 37) , and the formation and interchange of disulfide bond intermediates (38) . The additional cysteine in the OI64 mutant pro1(I) chain may disturb these folding pathways by inappropriately pairing with other cysteines thus stabilizing non-native folding intermediates and slowing the formation of assembly-competent pro1(I) chains. Mutations which impair carboxyl-terminal propeptide folding and retard chain association may also have an effect on chain selection. Since the retardation of propeptide association will also result in delayed folding of the triple helix, and consequently expose the component -chains to increased post-translational lysyl hydroxylation and hydroxylysyl glycosylation, the presence of overmodified -chains was used to determine the -chain composition of mutant-containing trimers. Slowly migrating, overmodified 1(I) and 2(I) chains were present in the cell layer and the medium of OI64 and OI26 cells, thus it is apparent that although assembly was delayed, mutant pro1(I) could associate with normal pro1(I) and pro2(I) to form pepsin-resistant triple-helical molecules, a proportion of which were secreted.

In OI26, the frameshift mutation altered the amino acid sequence of the carboxyl-terminal portion of the propeptide, but the region containing the cysteines involved in intermolecular disulfide bridges was unaffected (6) . The finding of undisulfide bonded pro1(I) chains in the medium of OI26 provided further evidence for the secretion of mutant chains by this cell line. For secretion these chains have presumably associated and folded into a triple helix (39, 40) . Thus, the correct formation of the intermolecular disulfide bonds which stabilize carboxyl-terminal propeptide association is not a prerequisite for helix formation. Recent studies using site-directed mutagenesis to perturb carboxyl-terminal propeptide disulfide bonding confirm that assembly and secretion can occur in the absence of normal interchain disulfide bonding.() Other non-covalent interactions, possibly involving the telopeptides and the amino-terminal half of the carboxyl-terminal propeptide, must be sufficient for nucleation of helix folding.

The tryptophan to cysteine mutation had no measurable effect on mRNA stability. OI64 fibroblasts produced approximately equal amounts of mutant and normal pro1(I) mRNAs, and the ratio of total pro1/pro2 mRNA was the same as in control cells. Cell-free translation of OI26 mRNA also demonstrated that mutant and normal mRNA was present in equal amounts. It is likely that in both OI64 and OI26 type I collagen was synthesized in equal amounts by both normal and mutant alleles, and the reduction in net collagen production in OI64 to around 30% of control values (28) , and to 50% in OI26 (10) , reflected increased degradation. In OI26 increased intracellular collagen degradation was directly demonstrated (10) .

Selective intracellular degradation is an important ``quality control'' mechanism preventing the accumulation within cells and the secretion of unassembled and malfolded proteins (41) . The pulse-chase procollagen assembly experiment indicated that in control cells pro2(I) chains were synthesized in excess of the amount required for stoichiometric assembly with two pro1(I) chains. Excess monomeric pro2(I) chains are not secreted and are not normally seen within fibroblasts during longer biosynthetic labeling experiments probably because they are continually being degraded and thus represent only a small proportion of the total intracellular pro2(I) pool. However, pro2(I) chain monomers were present in brefeldin A-treated cells which indicated that excess, unassembled pro2(I) are normally transported out of the endoplasmic reticulum before degradation. Similarly, the monomeric pro2(I) chains synthesized by Mov13 cells which do not produce pro1(I) subunits are not secreted (42) but are degraded intracellularly by a mechanism which is sensitive to inhibition by brefeldin A,further confirming a post-endoplasmic reticulum site for pro2(I) degradation.

Two observations led to the view that pro1(I) chains with carboxyl-terminal propeptide mutations are selectively degraded in the endoplasmic reticulum. When degradation of excess pro2(I) chains was inhibited by brefeldin A treatment, unassembled pro2(I) monomers represented a larger proportion of the total pro2(I) in the OI cells than in the control. Since the pro chains were synthesized initially in the correct ratios, the increased accumulation of pro2(I) monomers suggested that fewer pro1(I) chains were available for assembly with pro2(I). However, there was no extra accumulation of undisulfide-bonded pro1(I) chains in brefeldin A-treated OI64 or OI26 cells. This indicated that, in contrast to degradation of the excess pro2(I) chains, degradation of pro1(I) chains was not prevented when transport out of the endoplasmic reticulum was blocked. Direct evidence for an endoplasmic reticulum-localized mechanism which recognizes and selectively degrades pro1(I) chains with COOH-terminal propeptide mutations that interfere with subunit assembly was provided by two-dimensional electrophoresis of OI26 procollagens, where the more basic mutant chains could be resolved from normal pro1(I). The mutant protein was present in similar amounts to the normal pro1(I) in cell-free translation products but was dramatically reduced relative to the normal pro1(I) intracellularly. Mutant pro1(I) was not protected from degradation by brefeldin A treatment, consistent with a degradation pathway located in the endoplasmic reticulum.

Together these data demonstrated that mutant and excess normal chains were being degraded by separate intracellular pathways. Mutant pro1(I) chains were selectively degraded within the endoplasmic reticulum, while the excess normal pro2(I) was degraded after exiting the endoplasmic reticulum. Two distinct intracellular pathways for collagen degradation have been described previously. Approximately 10-15% of the collagen synthesized by cultured fibroblasts is degraded prior to secretion in a process known as basal degradation (43) , and this intracellular degradation is increased when cells are induced to synthesize structurally abnormal, non-helical collagen by incubation with the proline analogue cis-hydroxyproline (44) . Although both degradation processes occur in the distal region of the secretory pathway, beyond the brefeldin A block (45) , they differ in their sensitivity to lysomotrophic agents; the degradation of cis-hydroxyproline-containing collagen is mediated by lysosomal proteases (43, 44) while basal degradation occurs in an acidic, but non-lysosomal compartment, possibly the trans-Golgi (45, 46) . Continued degradation of the abnormal OI26 and OI64 pro1(I) in the presence of brefeldin A identifies the endoplasmic reticulum as an additional site of collagen degradation and indicates that the mechanisms for degrading unincorporated structurally normal chains and molecules with defective helices may differ from those for disposing of mutant chains which are unable to assemble efficiently.

The mechanisms by which chains or molecules are targeted to different degradation pathways are unclear. One targeting candidate, the endoplasmic reticulum-resident molecular chaperone BiP, bound to the abnormal type I procollagen and was coimmunoprecipitated in OI64 and OI26 but not control cells. BiP was also found to bind to pro chains from three other cell strains in which pro1(I) carboxyl-terminal propeptide mutations interfered with chain association and interchain disulfide bonding (47) . The main function of BiP is thought to be the promotion of intracellular protein assembly (38, 48) as it binds transiently to many secretory proteins before they have folded or assembled into their mature form and remains associated with aberrant, misfolded proteins (12, 32, 48, 49) . In OI26 and OI64 cells, the synthesis of BiP was increased by ascorbate treatment. Ascorbate addition rapidly increases the rate of type I collagen production by human fibroblast cultures (26) . Since BiP levels are elevated when abnormal proteins accumulate in the endoplasmic reticulum (16) it is likely that BiP induction reflected the presence of increased amounts of the mutant protein in ascorbate-treated cells. In contrast to defects in the carboxyl-terminal propeptide, mutations in the triple-helical domain of type I procollagen chains that delay folding of that region do not result in binding of the abnormal molecules to BiP or in increased synthesis of BiP even though the molecules can be quantitatively retained in the endoplasmic reticulum (50) .

The final aspect of procollagen biosynthesis we examined was the effect of the carboxyl-terminal propeptide mutations on collagen matrix formation. When grown for an extended period in the continuous presence of ascorbic acid both OI64 and OI26 cultures showed a marked reduction in collagen accumulation. The deposited collagen was, however, incorporated into a mature, cross-linked matrix since 80-90% was solubilized only after pepsin digestion. The mature in vitro matrix collagen in OI64 and OI26 contained overmodified chains demonstrating that at least some of the secreted mutant-containing molecules were incorporated into the collagen fibrillar matrix. Poor collagen matrix accumulation by OI26 fibroblasts closely resembled the severely reduced matrices in the patients' dermis and bone which contained only about 20% of the normal amount of collagen (10) . Incorporation of overmodified chains into the in vitro matrix was consistent with the increased levels of hydroxylysine in collagen from OI26 dermis and bone (10) . This cell culture extracellular matrix model system thus accurately reflected the in vivo biochemical phenotype of OI26. While no tissues were available from OI64 for analysis, the in vitro data would predict a major deficiency in the collagen content of the tissues.

The pro1(I) carboxyl-terminal propeptide mutations in these patients delay, but do not prevent, the assembly of the mutant pro1(I) into procollagen trimers. The combination of endoplasmic reticulum degradation of mutant pro1(I) chains and further intracellular degradation of malfolded mutant-normal molecules results in a collagen biosynthetic deficiency much more severe than that produced by a null allele. Since collagen matrix deposition is a cooperative process (26) , where a pre-existing matrix induces more efficient collagen deposition, the effect of the collagen deficiency on matrix formation is amplified. In both OI26 and OI64, some mutant-containing trimers escape intracellular degradation, and these post-translationally overmodified collagens are incorporated into the extracellular matrix. The incorporation of these abnormal molecules into collagen fibers may disturb matrix architecture and thus contribute to the severe phenotype.


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.

§
Present address: Div. of Orthopaedics, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.

To whom correspondence should be addressed: Dept. of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia. Fax: 61-3-345-5789.

The abbreviations used are: OI, osteogenesis imperfecta; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); PCR, polymerase chain reaction.

Amino acids are numbered from the translation start site of prepro1(I).

S. R. Lamandé and J. F. Bateman, unpublished data.


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