(Received for publication, March 20, 1995; and in revised form, May 15, 1995)
From the
The C-propeptides of the pro Asparagine-linked (N-linked) glycosylation of proteins
begins with the synthesis of a high-mannose carbohydrate chain,
Glc The
C-propeptides 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
pro
Figure 1:
Production of the pro
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,
Figure 4:
Electrophoresis of pepsin-digested
collagen produced by Mov13 cells transfected with the control and N-glycosylation mutant constructs. Collagens were labeled with
[
Figure 5:
Electrophoretic analysis of the type I
collagen C-propeptides produced by transfected Mov13 cells.
[
Figure 6:
Processing of the mutant unglycosylated
pro
Figure 7:
Electrophoresis of CNBr peptides of
pepsin-digested collagen produced by 3T6 cells transfected with the N-linked oligosaccharide mutant construct pWTCI-IQA.
[
Figure 8:
Electrophoretic analysis of the
Figure 9:
Secretion of endogenous pro
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).
Figure 10:
Distribution of newly synthesized
To prevent N-glycosylation of the pro The control 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
Normal secretion of mutant
pro Since the unglycosylated
pro 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
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 pro Failure to demonstrate a functional difference between normal and
unglycosylated pro
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1(I) and pro
2(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 pro
1(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 pro
1(I) folded and assembled normally into
trimeric molecules with pro
2(I). In biosynthetic pulse-chase
experiments mutant pro
1(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 pro
1(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 pro
1(I) chains were cleaved in vitro by the purified C-proteinase slightly less efficiently than
wild-type chains. Mutant and normal pro
1(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.
Man
GlcNAc
, 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) .
(
)of the pro
1(I) and
pro
2(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.
1(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 pro
1(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 pro
1(I) chains expressed
in the same transfected cell were directly compared. Unglycosylated
pro
1(I) subunits assembled normally into trimeric molecules with
pro
2(I) and were secreted at the same rate as endogenous
pro
1(I). Molecules containing unglycosylated pro
1(I) chains
were processed by the procollagen C-proteinase and deposited into an in vitro extracellular matrix.
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).
1(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.
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) .
Expression of the Mutant pro
The constructs were transfected into Mov13 cells to assess
the ability of unglycosylated pro1(I) Chain in Mov13
Cells
1(I) chains to fold and assemble
into trimeric helical molecules. Untransfected Mov13 cells synthesize
only the pro
2(I) subunit of type I collagen; transcription of the
endogenous pro
1(I) genes is blocked by a retroviral insertion in
the first intron(29, 38) . Pro
2(I) chains are
unable to fold into homotrimeric molecules and, in the absence of
pro
1(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 pro
1(I) chains which associated with the
endogenous pro
2(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) .
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 pro
1(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.
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 pro
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
[1(I) Chains in Vitro by
Purified Procollagen C-Proteinase
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 pro
1(I), pC
1(I)/pN
1(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]pro
1(I)
and mutant [IQA]pro
1(I) C-propeptides as judged by the
conversion of pro
1(I) chains to the pN
1(I) processing
intermediate. In each experiment, control [IA]pro
1(I)
chains were processed at the same rate as the wild-type pro
1(I)
chains (Fig. 6, a, c, and e), but the
unglycosylated [IQA]pro
1(I) chains were cleaved slightly
more slowly (Fig. 6, b, d, and f).
While the difference in the rate of processing of the mutant
pro
1(I) was small, it was reproducible and was accurately compared
to processing of the wild-type pro
1(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).
1(I) chains in vitro by purified procollagen
C-proteinase. [
H]Proline-labeled wild-type
pro
1(I) chains produced were mixed with either
[
C]proline-labeled control
[IA]pro
1(I) chains (panels a, c, and e) or [
C]proline-labeled mutant
unglycosylated [IQA]pro
1(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 pro
1(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 pro
The mutant construct was transfected into 3T6 cells to
allow the biosynthetic fate of normal endogenous pro1(I) in 3T6
Cells
1(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 pro
1(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).
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.
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 pro
To directly compare the rate of secretion of
endogenous and transfected mutant pro1(I) Lacking the N-Linked
Oligosaccharide
1(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 pro
1(I) chains produced from the transfected genes allowed
the fate of these chains and endogenous wild-type pro
1(I) chains
expressed in the same cell to be compared accurately. Control
transfected [IA]pro
1(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]pro
1(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 pro
1(I) chains.
1(I) and
mutant pro
1(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 pro
1(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.
Mutant Unglycosylated pro
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 [1(I) Chains Are Efficiently
Incorporated into an in Vitro Accumulated Extracellular
Matrix
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
pro
1(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
pro
1(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.
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.
1(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.
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 pro
1(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.
-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 pro
1(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.
1(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) .
1(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) .
(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.
1(I) chains were
processed and incorporated into the insoluble cross-linked matrix at
the same rate as reporter and control pro
1(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.
1(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.
1(I).
We thank Dr. Karl Kadler (University of Manchester)
for his generous gift of purified C-proteinase and for critical
evaluation of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.