From the University of Manchester, School of Biological Sciences, Wellcome Trust Centre for Cell/Matrix Research, 2.205 Stopford, Manchester M13 9PT, United Kingdom
Received for publication, April 4, 2000, and in revised form, September 25, 2000
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ABSTRACT |
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Constructs of each of the three chains of type VI
collagen were generated and examined in an in vitro
transcription/translation assay supplemented with semipermeabilized
cells. Each of the constructs when used in the in vitro
system was shown to be glycosylated and to undergo intracellular
assembly, the extent of which was determined by the nature of the
C-terminal globular domains. All three chains containing the C1 domain
formed monomers; however, the C2 domain was required for dimer and
tetramer formation. In the case of the full-length Type VI collagen appears to have a ubiquitous distribution
throughout connective tissues, and although it has been suggested that
it links cells and other matrix components, its precise role in the
matrix has still to be determined. Mutations in each of the three genes
of type VI collagen have been linked to Bethlem myopathy, a mild form
of muscle weakness and wasting (1). It is unclear why mutations produce
a muscle disease rather than a more generalized phenotype, but perhaps
type VI collagen has some tissue-specific functions, as well as
interactions that occur in all tissues.
Three different chains have been described for type VI collagen, the
Tissue extraction and biosynthetic studies have demonstrated that
Triple helix formation in collagen involves chain association,
registration, nucleation, and propagation (11). Proper chain selection
and registration is initiated by association of the C-propeptide.
Sequences have been defined for the fibrillar collagens in the
C-propeptide that are essential for chain selection (12), and the NC1
domains of both type VIII and X collagens have been shown to be
critical in chain association (13, 14). By analogy, it is likely that
the C-terminal domains in the We have been interested in understanding what regulates and controls
chain association in type VI collagen and have produced constructs of
the human Generation of Generation of Generation of Generation of
For each of the nine Preparation of Semipermeabilized Cells--
HT-1080 human
fibrosarcoma cells were maintained in Dulbecco's modified Eagle's
medium containing sodium pyruvate (0.11 g/liter) and pyridoxine,
supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin/penicillin, and 10% (v/v) fetal calf serum (Life Technologies, Inc., Paisley, UK). Confluent HT-1080 cells from a
75-cm2 flask were washed with phosphate-buffered saline,
trypsinized using 2.5 mg/ml trypsin for 3 min and then resuspended in 8 ml of ice-cold KHM buffer (100 mM potassium acetate, 20 mM Hepes, pH 7.2, 2 mM magnesium acetate)
containing 300 µg/ml soya bean trypsin inhibitor (Sigma).
Cells were isolated by centrifugation at 280 × g for 3 min and then resuspended in 6 ml of KHM buffer containing 40 µg/ml
digitonin (Calbiochem, Nottingham, UK) and incubated on ice for 5 min.
Permeabilization was terminated by adding 8 ml of KHM buffer, isolating
the cells by centrifugation, resuspending in 11 ml of 50 mM
Hepes, pH 7.2, 90 mM potassium acetate, and incubating on
ice for 10 min. Semipermeabilized cells were isolated by centrifugation
and resuspended in 200 µl of KHM buffer, and endogenous mRNA was
removed by adding CaCl2 to a concentration of 1 mM and monococcal nuclease (Sigma) to 10 µg/ml and
incubating at room temperature for 12 min. After terminating the
reaction by adding EGTA to a concentration of 2 mM,
semipermeabilized cells were pelleted and resuspended in 100 µl of
KHM buffer.
Transcription and in Vitro Translation--
Type VI collagen
cloned cDNA was linearized downstream to the
Translations were carried out using a rabbit reticulocyte lysate system
(Promega, Southampton, UK) for 15 min to 4 h at 30 °C. Each
reaction contained 35 µl of lysate, 50 µg/ml sodium ascorbate, 50 mM KCl, 20 µM amino acids (minus methionine),
25 µCi of [35S]methionine, 40 units of RNase inhibitor,
2.5 µl of transcribed RNA (preheated to 60 °C for 10 min and then
chilled on ice), and 4 × 105 semipermeabilized
HT-1080 cells. Translation was terminated by addition of cycloheximide
to a concentration of 1 mM, and reaction mixtures were
immediately placed on ice. CaCl2 was added to a concentration of 10 mM, and reaction mixtures were digested
with 250 µg/ml proteinase K (Sigma, Poole, UK) for 20 min. Digestion was stopped by adding 10 mM phenylmethylsulfonyl fluoride
(Sigma, Poole, UK) and incubating on ice for 5 min and then
N-ethylmaleimide (Sigma, Poole, UK) was added to a
concentration of 25 mM and incubated on ice for 15 min.
Cells were then washed by addition of KHM buffer and isolated by
centrifugation three times at 14,000 × g for 4 min and
was then resuspended in an appropriate buffer for subsequent digestion,
immunoprecipitation, or direct SDS-PAGE analysis.
Pepsin Digestion--
Immunoprecipitates were resuspended in
digestion buffer and incubated on ice for 10 min. The solution was then
acidified with HCl (100 mM). Pepsin (300 µg/ml; Sigma,
Poole, UK) was added, and samples were digested at 30 °C for 2 h. Digestion was stopped by neutralization with Tris base (100 mM) and addition of SDS-PAGE sample buffer.
Endoglycosidase H Digestion--
Isolated cells were resuspended
in 100 mM Tris-HCl, pH 8.0, 1% SDS, 1%
Immunoprecipitation--
Isolated cells were resuspended in 1 ml
of NET buffer (50 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 5 mM EDTA, 0.25% (w/v) gelatin, 0.05% (v/v) Nonidet
P-40, and 0.02% (w/v) sodium azide) and incubated on ice for 30 min.
Samples were centrifuged at 14,000 × g for 10 min to
remove insoluble material and then precleared at 4 °C for 1 h
with 40 µl (10% (v/v)) of protein A-Sepharose. After centrifugation at 800 × g for 3 min, the supernatant was
immunoprecipitated at 4 °C for 16 h with 20 µl of type VI
collagen polyclonal antibody VIA or VIB (kindly donated by
Dr. S. Ayad) (18). Samples were then incubated at 4 °C for 2 h with 80 µl (10% (v/v)) of protein A-Sepharose before the
immunoprecipitates were isolated by centrifuging at 800 × g for 3 min. Immunoprecipitates were washed in 1 ml of NET
buffer without gelatin or Nonidet P-40 and isolated three times and
then resuspended in SDS-PAGE sample buffer.
SDS-PAGE Analysis--
Samples were resuspended in SDS-PAGE
sample buffer in the presence or absence of 5% Determining Chain Composition of Assemblies--
Directly after
electrophoresis, individual assemblies were excised from a composite
gel and incubated in 125 mM Tris-HCl, pH 6.8, 2% SDS, and
5% Homology Modeling--
The amino acid sequence of the C2 domain
from the
All molecular modeling was performed on an R10,000 O2
Silicon graphics work station using QUANTA® and CHARMm
23.2® programs. The three-dimensional model of the C2
domain from the
The C2 domain was energy minimized using steepest descents followed by
the conjugate gradient algorithm to convergence removing bad steric and
electrostatic contacts. The Protein Health module in QUANTA was used to
check the integrity of the model using a Ramachandran plot and to
identify buried hydrophilic or exposed hydrophobic residues and close contacts.
Translocation and Glycosylation of Products--
Short-length
constructs consisting of the N1 domain, helical region, and C1 domain
of each of the three
All three short-length Intracellular Assembly of Type VI Collagen--
Type VI collagen
monomers undergo intracellular assembly into disulfide-linked dimers
and tetramers prior to secretion. These assemblies are too large to
monitor by SDS-PAGE gels, so low percentage agarose/acrylamide gels
were used to examine formation of monomers, dimers, and tetramers.
After transcription/translation of constructs coding for each
Comparison of the products from transcription/translation of constructs
containing the N1 and C1 of each chain showed that material
corresponding to monomers (molecular mass about 330,000) could be
detected for each chain (Fig. 3, lanes 2-4). When
constructs containing both the C1 and C2 domains were tested,
additional bands were evident. In the case of the
That these assemblies were glycosylated and formed from triple-helical
monomers was shown by treatment with endoglycosidase H or pepsin,
respectively (Fig. 5). Treatment of the
immunoprecipitate from a full-length
To look more closely at the rate of formation of intracellular
assemblies, the full-length
Using a full-length
Cotranslating equal amounts of RNA derived from the Rate of Formation of Dimers and Tetramers--
To follow the time
course of formation of dimers and tetramers, agarose/acrylamide
composite gels were scanned, and band intensity was determined by
densitometry and then plotted against time (Fig. 7, A-D). These plots clearly
showed that for the full-length
To examine how the The semipermeabilized cells in conjunction with in
vitro transcription/translation have been shown to recapitulate
the early events of protein folding and assembly of multisubunit
proteins destined for secretion (12, 14-16). The present results have shown for the first time that the A variety of studies have shown the importance of C-terminal
interactions in chain selection and helix formation (11, 12, 14). In
the case of type VI collagen, the implications from a number of studies
were that Structural studies have shown A-domains to be composed of a central
Sequence studies have shown that the C1 domains of the Of the A-domains for which there is high resolution structural data,
the C2 domain of the 2(VI) chain,
monomers, dimers, and tetramers formed in a time-dependent
manner. Although the splice variant
2(VI)C2a could form monomers, it
was unable to form dimers and tetramers. Similar results to the
2(VI) chain were found for the full-length
1(VI) chain, although
assembly was at a slower rate. In the case of the
3(VI) chain
containing both C1 and C2 domains only monomers were observed. Addition
of the C3, C4, and C5 did not change this pattern. Homology modeling
suggested that a 10-amino acid insertion in the C2 domain of the
3(VI) chain may interfere with dimer formation. A near full-length
construct of the
3(VI) chain only formed monomers but was shown to
facilitate tetramer formation in cotranslation experiments.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(VI),
2(VI), and
3(VI) (2-5). Each chain contains a short
triple-helical region and globular extensions at the N and C terminus.
The majority of noncollagenous domains have homology to von Willebrand
factor A-domains, and these are found at both the N and C termini of
all three chains. In the case of the
3(VI) chain, ten such domains
(N1 to N10) may be found at the N terminus (2-5). In contrast to most
other collagens, type VI collagen undergoes some polymerization prior
to secretion. Intracellularly, triple-helical monomers align in an
antiparallel manner with the C-terminal globular domains attached to
the adjacent helix by disulfide bonds. These dimers then align with
their ends in register to form tetramers, which are the secreted form
of the molecule. End to end accretion of tetramers leads to microfibril
formation in the extracellular matrix (6).
1(VI),
2(VI), and
3(VI) chains occur in stoichiometric proportions in a 1:1:1 ratio (6-8). These early observations have been
confirmed by using cell lines deficient in one of the chains (9) and by
examining secretion and assembly of recombinant type VI collagen (10).
In the case of recombinant expression studies, it was shown that, when
cell lines were stably transfected with either the
1(VI) or
2(VI)
chain, only single polypeptides were secreted. Cells transfected with
all three chains, however, produced a pepsin-resistant type VI collagen
(10). The human osteosarcoma cell line (SaOS-2), under certain growth
conditions, is deficient in
3(VI), and although these cells have an
abundance of
1(VI) and
2(VI) mRNAs, collagen VI was not
detected in the medium by 4 weeks of culture (9). Stable transfection
of these cells with the
3(VI) cDNA enabled formation of type VI
collagen dimers and tetramers, which were secreted and deposited into
an extensive network (9). These results suggest that the
3(VI) chain
contains the sequences that enable chains to associate in the
endoplasmic reticulum and form a triple helix.
1(VI),
2(VI), and
3(VI) chains
of type VI collagen play essential roles in chain association and selection.
1(VI),
2(VI), and
3(VI) chains, which contain the N1
and C1 with and without the C2 domains, and a near full-length
3(VI)
chain and have used them in an in vitro
transcription/translation system supplemented with semipermeabilized
cells. This system has been used successfully to show correct folding
of correctly aligned triple helices (15), to identify molecular
recognition sequences that determine chain association (16), and to
highlight the importance of C-terminal (NC1) sequences to short chain
collagens (14). Our results show that the recombinant
1(VI) and
2(VI) chains can, indeed, form triple-helical monomers, and
subsequently dimers and tetramers when the C2 domains were present, but
not when only the C1 domain was present. Interestingly, the
3(VI) chains could form monomers but were unable to form dimers and tetramers, although they were able to facilitate dimer and tetramer assembly in cotranslation experiments.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Chain Constructs--
Nine
-chain cDNA
constructs were generated by reverse transcription followed by overlap
PCR1 (Fig.
1). Human placental total RNA (1 µg)
was reverse transcribed with an oligo(dT) primer for 90 min
using an Expand reverse transcription kit (Roche Diagnostics, Lewes,
United Kingdom), according to the manufacturers instructions. Overlap
extension PCR was performed using an Expand high fidelity PCR kit
(Roche Diagnostics, Lewes, UK). Reaction mixtures (50 µl) consisted
of ~10 ng of template cDNA, reaction buffer containing 1.5 mM MgCl2, 200 µM dNTPs, 50 pmol
of forward and reverse primers, and 2.6 units of DNA polymerase mixture
(thermostable Taq and Pwo). The reaction mixture
was incubated for 3 min at 94 °C, immediately followed by 25 cycles
of 1 min at 94 °C, 2 min at 60 °C, and 2 min at 72 °C and then
incubated for 7 min at 72 °C. Each forward
-chain primer
represented a chimera of the last 19 bases at the 3' end of the signal
peptide sequence (CAGGCTCATTCAGGCTGGT), and the remainder were based on the start of the
-chain construct. In the case of the
1(VI) and
2(VI) chain sequences, the initial ATG start codon was omitted from
each primer sequence. Each reverse
-chain primer contained either
the endogenous or inserted stop codon (shown in below in bold
face).
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Fig. 1.
Recombinant -chain
cDNAs used in this study. Nine type VI collagen
-chain
constructs were generated, each containing the same signal peptide
sequence, designated SP. N- and C-terminal domains are shown
as boxes, whereas the helical region is represented by
hatched lines. The estimated molecular size of each
translation product is given, together with the number of base pairs
(bp) of each construct, shown in brackets.
1(VI) Chain Constructs--
The
1N1
forward primer (5'-CAGGCTCATTCAGGCTGGTAGGGCGGCCCGTGCTCTGC-3') (bases 52 to 70; see Ref. 4) and
1C1 reverse primer (5'-GGGCAGGTGTACTATGGACACTTC-3') (bases 2487 to 2510; see Ref. 4) were used to amplify an expected fragment of 2458 base pairs
(
1C1; Fig. 1), whereas the
1N1 forward primer and
1C2 reverse
primer (5'-GCAGGGTGGGCTAGCCCAGCGCC-3') (bases 3123 to
3145; see Ref. 4) generated an expected fragment of 3093 base pairs
(
1C2; Fig. 1).
2(VI) Chain Constructs--
The
2N1
forward primer (5'-CAGGCTCATTCAGGCTGGTCTCCAGGGCACCTGCTCCG-3')
(bases 25 to 43; see Ref. 4) and
2C1 reverse primer (5'-CACGGACAGCTATGTTTGGCAGGG-3') (bases 2467 to 2490; see Ref. 4) were used to amplify an expected fragment of 2465 base pairs
(
2C1; Fig. 1), and the
2N1 forward primer and
2C2 reverse primer (5'-CGGCGGCGCTAGCAGATCCAGCGG-3') (bases 3063 to 3086;
see Ref. 4) amplified a product of 3061 base pairs (
2C2; Fig. 1),
whereas the
2N1 forward primer and
2C2a reverse primer (5'-GCCCGGGCTTTAGCAGAGGGCCAGCGCGGTCTCGGGGT-3') (bases
3040 to 3077; see Ref. 21) generated an expected fragment of
2759 base pairs (
2C2a; Fig. 1).
3(VI) Chain Constructs--
The
3N1
forward primer
(5'-CAGGCTCATTCAGGCTGGTGCTTGTAATCTGGATGTGATTCTGGGG-3') (bases 5131 to
5157; see Ref. 5) and
3C1 reverse primer
(5'-CCACAGGATGGCTAGATGTTGCAGATG-3') (bases 7398 to 7424; see
Ref. 5) generated a fragment of 2293 base pairs (
3C1; Fig. 1). The
3N1 forward primer and
3C2 reverse primer
(5'-GGTTACAGGCTATGTTGTGGTGG-3') (bases 8282 to 8304; see
Ref. 5) amplified a product of 3173 base pairs (
3C2; Fig. 1),
whereas the
3N1 forward primer and
3C5 reverse primer
(5'-CGCTTAGGTTCCCATCACACTG-3') (bases 9144 to
9165; see Ref. 5) were used to generate an expected fragment of 4034 base pairs (
3C5; Fig. 1). A
3N8 forward primer (5'-CAGGCTCATTCAGGCTGGCTGGTTCATTGAAGTCAACAAGAGAG-3') (bases 944 to 964; see Ref. 5) and
3C5 reverse primer were used to generate a
fragment of 8221 base pairs (
3N8; Fig. 1).
-chain constructs, a signal peptide sequence of
the
1(VIII) collagen chain and ~200 base pairs of a 5'-untranslated sequence, known to promote efficient translation using
semipermeabilized cells (14), was generated by overlap extension PCR
from clone pBlu-Hal8. The signal peptide forward primer
(5'-CGGGCACACTAAGAGTCAGC-3'), corresponding to the 5' end of the
untranslated region, was used in each case, whereas the signal peptide
reverse primer (5'-
-chain sequence-ACCAGCCTGAATGAGCCTG-3') represented a chimera consisting of between 9 and 34 bases of the 5'
end of a specific
-chain construct and the last 19 bases at the 3'
end of the signal peptide sequence. The signal peptide primers
generated an expected fragment of ~300 base pairs for each
-chain.
Purified PCR products from an
-chain construct reaction and a
corresponding signal peptide reaction were combined in a second round
overlap extension PCR. Reactions were performed in the absence of
primers for six thermal cycles of 30 s at 94 °C, 1 min at
55 °C, and 30 s at 72 °C, to allow self-priming of the
product overlap regions. Subsequent addition of the signal peptide
forward primer and the appropriate
-chain reverse primer for 25 cycles of 1 min at 94 °C, 1 min at 60 °C, and 2 min at 72 °C
allowed amplification of the
-chain construct. Each
-chain construct was purified and cloned into TA vector pGEM (Promega, Southampton, UK), except for the
3N8 chain construct, which was cloned using an Expand cloning kit (Roche Diagnostics, Lewes, UK). The
sequence identity and reading frame integrity were confirmed by
dye-terminator automated sequencing.
-chain insert using
SpeI (for
1C1,
2C1,
2C2,
3C1,
3C2,
3C5,
and
3N8) and PvuI (for
1C2 and
2C2a). Transcription
was performed for 4 h at 37 °C in a reaction mixture containing
100 units of T7 RNA polymerase (for
1C1,
2C1,
2C2,
3C1,
3C2,
3C5, and
3N8) or SP6 RNA polymerase (for
1C2 and
2C2a) (Promega, Southampton, UK), 1× transcription buffer, 10 mM dithiothreitol, 40 units of RNase inhibitor, and 3 mM NTPs (Roche Diagnostics, Lewes, UK). RNA was purified
using spin columns (Qiagen, Crawley, UK) and eluted in 50 µl of
RNase-free water containing 1 mM dithiothreitol and 20 units of RNase inhibitor.
-mercaptoethanol and boiled for 5 min. Samples were centrifuged at
14,000 × g for 5 min to remove insoluble material and
then an equal volume of 150 mM sodium citrate, pH 5.5, was added and incubated with 1 unit of endoglycosidase H (Roche
Diagnostics, Lewes, UK) at 37 °C for 16 h. The reaction was
terminated by adding SDS-PAGE sample buffer. Immunoprecipitates were
treated in an identical manner except that no reducing agent was used.
-mercaptoethanol and
boiled for 5 min prior to analysis (17). Type VI collagen chains were
analyzed using 8% (w/v) polyacrylamide gels, whereas monomers, dimers, and tetramers were analyzed using 3% (w/v) polyacrylamide 0.4% (w/v)
agarose composite gels under nonreducing conditions. Gels were fixed,
dried under vacuum, and imaged using a Fujix BAS 2000 phosphorimager.
-mercaptoethanol at 4 °C for 16 h. Gel slices and the
solution containing SDS-PAGE sample buffer were then loaded onto an 8%
SDS-PAGE gel, and the protein components were electroeluted and
resolved. The gel was fixed, dried under vacuum, and imaged using a
Fujix BAS 2000 phosphorimager.
2 chain of human type VI collagen was used to probe the
Research Collaboratory for Structural Bioinformatics Protein Data Bank
(PDB) using the advanced BLAST 2.0 search (19). Sequences resulting
from this search were the human von Willebrand factor A3 domain
(vWFA3), the I domain from integrin CR3 (CD11a), and human von
Willebrand factor A1 domain (vWFA1), which are all A-domains. The
sequence with the highest homology was the vWFA3 domain with 22%
identity to the C2 domain, and this structure was used as the starting point for homology modeling. Multalin (20) was initially used to align
the sequences and then the secondary structural elements from the vWFA3
domain and the predicted secondary structure for the C2 domains
(calculated using Predict Protein; see Ref. 21) were overlaid, and the
sequence alignment was improved manually to produce a structure-based
alignment. Most of the secondary structural elements would appear to be
conserved based on the comparison of predicted and actual secondary
structural elements.
2 chain of type VI collagen was built based on the
coordinates of the vWFA3 domain (PDB entry 1ATZ (22)). An homology
model was built by copying the coordinates of the backbone of the vWFA3 domain and coordinates of completely conserved residues in the C2
domain. The remaining side chains were built in the Protein Design
module using the Ponder and Richards' rotamer library. There were
three insertions in the C2 domain sequence compared with the vWFA3
sequence; these were 5 residues in the loop between first
-helix and
the second
-strand, 1 residue in the loop following the third
-helix, and 1 residue in the loop between the fourth
-strand and
the fourth
-helix.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chains of type VI collagen were translated in
the presence of semipermeabilized cells and analyzed on SDS-PAGE gels.
The molecular masses of the translation products of the
1(VI),
2(VI), and
3(VI) chain constructs were predicted to be 85, 86, and 80 kDa, respectively. Products of slightly higher molecular mass,
relative to globular molecular mass markers, were observed (Fig.
2, lanes 1, 3, and
5). These bands were not susceptible to proteinase K
treatment, confirming their translocation into the cellular rough
endoplasmic reticulum (data not shown), and only translated
products derived from proteinase K-treated cells were used in
subsequent analysis.
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Fig. 2.
Glycosylation of translation products.
Short-length constructs of each -chain are shown.
1C1,
2C1,
and
3C1 were translated, and isolated cells were treated with
proteinase K and analyzed directly (lanes 1, 3,
and 5) or treated with endoglycosidase H (lanes
2, 4, and 6). Samples were analyzed by
SDS-PAGE on an 8% gel under reducing conditions and visualized by
phosphorimager analysis. Sizes of globular molecular mass markers are
shown.
-chains were post-translationally
glycosylated (Fig. 2, lanes 2, 4, and
6), as demonstrated by their faster migration after
digestion with endoglycosidase H. The translation product of ~200 kDa
(Fig. 2, lanes 1-6) was derived from the rabbit reticulocyte lysate, because it was translated in the absence of an RNA
template or cells (data not shown).
-chain
for various lengths of time, products were immunoprecipitated using
type VI collagen polyclonal antibody VIA or VIB and analyzed on
nonreducing agarose/acrylamide composite gels. Size estimates of
monomers, dimers, and tetramers formed following in vitro
translation in the presence of semipermeabilized cells were obtained by
comparison to monomers and dimers isolated from labeled
immunoprecipitated cultured fibroblast medium (Fig. 3, lane 1).
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Fig. 3.
Agarose electrophoresis of translated
products of 1C1,
2C1,
and
3C1. Short-length constructs of each
-chain, lacking the C2 domain (
1C1 (lane 2),
2C1
(lane 3), and
3C1 (lane 4), were translated
for 4 h. Translation was terminated by the addition of 1 mM cycloheximide and 25 mM NEM, and isolated
cells were treated with proteinase K. Proteins were immunoprecipitated
with type VI collagen polyclonal antibody VIB, analyzed on a
nonreducing composite 0.4% agarose, 3% polyacrylamide gel, and
visualized by phosphorimager analysis. Molecular mass sizes were
estimated from monomers and dimers immunoprecipitated from
35S-labeled cultured fibroblast medium, as shown in
lane 1.
2(VI) chain, bands
corresponding to both dimers and tetramers were seen after 4 h of
translation (Fig. 4, lane 2).
The C2 domain of this
-chain can undergo alternative splicing to
produce
2C2a; however, when this latter construct was translated for
the same period and analyzed on agarose/acrylamide composite gels, no
dimers were detected (Fig. 4, lane 3). Direct translation of
the full-length
2(VI) chain for 4 h in the absence of cells
failed to reveal any dimers or tetramers (Fig. 4, lane 1),
indicating that the formation of dimers and tetramers was cell-directed.
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Fig. 4.
Formation of 2(VI)
chain cell-free translation assemblies. Three different
2(VI)
chain constructs were translated for 4 h,
2C1 lacking the C2
domain (lane 4),
2C2a containing a variant C2 terminal
domain (lane 3), and
2C2, the full-length
2(VI) chain
containing the C2 domain (lane 2). The
2C2 construct was
also translated for 4 h in the absence of cells (lane
1). Translation was terminated by the addition of 1 mM
cycloheximide and 25 mM NEM, and isolated cells were
treated with proteinase K. Proteins were immunoprecipitated with type
VI collagen polyclonal antibody VIB, analyzed on a nonreducing
composite 0.4% agarose, 3% polyacrylamide gel, and visualized by
phosphorimager analysis. Molecular mass sizes were estimated from
monomers and dimers immunoprecipitated from cultured fibroblast
medium.
2(VI) chain translation with
endoglycosidase H showed that the lower of the two monomer bands was
unglycosylated (Fig. 5, lane 2). Pepsin digestion showed
that triple-helical monomers were formed, and a protease-resistant
fragment, susceptible to endoglycosidase H treatment, was apparent
(Fig. 5, lanes 3 and 4, respectively).
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Fig. 5.
Deglycosylation and pepsin digest of
2C2 translation product. The full-length
2(VI) (
2C2) chain construct was translated for 2 h and then
terminated by the addition of 1 mM cycloheximide and 25 mM NEM. Isolated cells were treated with proteinase K and
analyzed directly (lane 1), subjected to digestion with
endoglycosidase H (lane 2), digested with pepsin (lane
3), or digested with pepsin followed by endoglycosidase H
treatment (lane 4). Proteins were immunoprecipitated with
type VI collagen polyclonal antibody VIB, analyzed on a nonreducing
composite 0.4% agarose, 3% polyacrylamide gel, and visualized by
phosphorimager analysis. Molecular mass sizes were estimated from
monomers and dimers immunoprecipitated from cultured fibroblast medium
and a prestained SDS-PAGE 200-kDa myosin standard.
2(VI) chain construct was translated and
analyzed on composite gels at various time points (Fig.
6B). Assemblies corresponding
approximately to the molecular mass of
2(VI) dimers (660 kDa) were
observed after 2 h (Fig. 6B, lane 6) and to
tetramers (1320 kDa) after 4 h (Fig. 6B, lane
8).
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Fig. 6.
Time course of formation of
1(VI),
2(VI), and
3(VI) assemblies. A, the
full-length
1(VI) (
1C2) was translated for between 15 and 240 min
(lanes 1-8) before translation was terminated by the
addition of 1 mM cycloheximide and 25 mM NEM,
and isolated cells were treated with proteinase K. Proteins were
immunoprecipitated with type VI collagen polyclonal antibody VIB,
analyzed on a nonreducing composite 0.4% agarose, 3% polyacrylamide
gel, and visualized by phosphorimager analysis. Molecular mass sizes
were estimated from monomers and dimers immunoprecipitated from
cultured fibroblast medium. B, the full-length
2(VI)
(
2C2) chain construct was translated for between 15 and 240 min
(lanes 1-8) before translation was terminated by the
addition of 1 mM cycloheximide and disulfide bond formation
stopped by the addition of 25 mM NEM. After treating
isolated cells with proteinase K, proteins were immunoprecipitated with
type VI collagen polyclonal antibody VIB, analyzed on a nonreducing
composite 0.4% agarose, 3% polyacrylamide gel, and visualized by
phosphorimager analysis. Molecular mass sizes were estimated from
monomers and dimers immunoprecipitated from cultured fibroblast medium.
C, the
3(VI) (
3C2) chain construct containing the C2
domain was translated for between 15 and 240 min (lanes
1-8) before translation was terminated by the addition of 1 mM cycloheximide and 25 mM NEM, and isolated
cells were treated with proteinase K. Proteins were immunoprecipitated
with type VI collagen polyclonal antibody VIB, analyzed on a
nonreducing composite 0.4% agarose, 3% polyacrylamide gel, and
visualized by phosphorimager analysis. Molecular mass sizes were
estimated from monomers and dimers immunoprecipitated from cultured
fibroblast medium. D, equal aliquots (1 µl) of
1C2,
2C2, and
3C2 RNA were cotranslated for between 15 and 240 min
(lanes 1-8) before translations were terminated by the
addition of 1 mM cycloheximide and 25 mM NEM,
and isolated cells were treated with proteinase K. Proteins were
immunoprecipitated with type VI collagen polyclonal antibody VIB,
analyzed on a nonreducing composite 0.4% agarose, 3% polyacrylamide
gel, and visualized by phosphorimager analysis. Molecular mass sizes
were estimated from monomers and dimers immunoprecipitated from
cultured fibroblast medium.
1(VI) chain construct, dimers were observed
after 90 min (Fig. 6A, lane 5), but tetramers
were barely visible after 4 h (Fig. 6A, lane
8). Monomers only were observed using the
1(VI) construct
lacking the C2 domain (see above). In the case of the
3(VI)
construct containing C1 and C2, only monomers were observed by 4 h
(Fig. 6C). The addition of the C3, C4, and C5 domains of the
3(VI) chain to this construct made no difference in tetramer
formation (data not shown).
1(VI),
2(VI),
and
3(VI) constructs containing both the C1 and C2 domains produced
assemblies corresponding to monomers, dimers, and tetramers over the
4-h period (Fig. 6D). These assemblies were susceptible to
reducing conditions indicating that they were stabilized by disulfide
bonding (data not shown).
1(VI) and
2(VI) chains there is a
linear increase in monomer with time and that, as this concentration
increases, dimers and tetramers appear. In the case of the
3(VI)
construct containing C1 and C2 domains, although there is an increase
in monomer with time, there is little evidence of dimer and tetramer
formation during this time course.
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Fig. 7.
Rate of formation of monomers, dimers, and
tetramers. The intensity of type VI collagen assemblies at various
time periods (15 to 240 min) were evaluated by densitometry. The
average densitometry values from three translations were calculated and
then plotted against time. A shows 1C2 translation
assemblies, B shows
2C2 translation assemblies,
C shows
3C2 translation assemblies, and D
shows cotranslation (
1C2/
2C2/
3C2) assemblies.
3(VI) chain influenced type VI collagen assembly
we cotranslated full-length
1(VI) and
2(VI) chains with a near
full-length
3(VI) chain (
3N8; Fig. 1) lacking the N-terminal N10
and N9 domains and compared the rate of formation of monomers, dimers,
and tetramers with those assembled, translating just full-length
2(VI). These translations were performed using the same batch of
semipermeabilized cells. Comparison of the
2(VI) with cotranslation
of
1(VI),
2(VI), and
3(VI) chains appeared to indicate more
tetramer formation in cotranslations compared with single-chain
translation (Fig. 8, compare lanes
8 and 4). That the tetramer in cotranslation
experiments contained all three chains was shown after band isolation
and SDS-PAGE in reduced conditions. Two bands, one corresponding to the
1/
2(VI) and the other to the
3(VI) chain, were evident in the
cotranslation, whereas only one corresponding to the
2(VI) chain was
found in the case of the
2(VI) translation (Fig. 8, see
insets). Translating the
3N8 construct for 4 h
produced only monomers (Fig. 8, lane 1). The influence of
the
3(VI) chain in cotranslation experiments was examined by
quantification of the monomer, dimer, and tetramer bands by
densitometry. This showed that dimers and tetramers were formed at a
quicker rate when all three chains were present (Fig. 9). That the
3(VI) chain was an
important determinant of this increased formation was shown in
cotranslation of both full-length
1(VI) and
2(VI) chains, where
no difference in dimer or tetramer were evident compared with the
individual chains (data not shown).
View larger version (86K):
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Fig. 8.
Homo- and heteroassembly comparison.
Using the same cell batch, translation was performed using the 3N8
chain construct for 240 min (lane 1), the
2C2 chain
construct for various lengths of time (lanes 2-5), and a
cotranslation using equal aliquots (1 µl) of
1C2,
2C2, and
3N8 RNA for the same period (lanes 6-9). Translations
were terminated by the addition of 1 mM cycloheximide and
25 mM NEM, and isolated cells were treated with proteinase
K. Proteins were immunoprecipitated with type VI collagen polyclonal
antibody VIA, analyzed on a nonreducing composite 0.4% agarose, 3%
polyacrylamide gel, and visualized by phosphorimager analysis.
Assemblies corresponding to tetramers, generated from an equivalent
2C2 chain construct translation (as in lane 5) and a
cotranslation (as in lane 9), were excised. Tetramers were
reduced and then separated on an 8% SDS-PAGE gel, shown in boxes
above the corresponding lane.
View larger version (18K):
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Fig. 9.
Rate of homo- and heteroassemblies. The
intensity of type VI collagen homoassemblies, shown in Fig. 8,
lanes 2-5, and heteroassemblies, shown in Fig. 8,
lanes 6-9, were evaluated by densitometry. Heteroassembly
values were adjusted to the methionine content of three 2C2 chain
constructs and then the intensity values were plotted against
time.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(VI) and
2(VI) chains of type
VI collagen can assemble intracellularly into triple-helical monomers
and that dimers and tetramers subsequently form in a time-dependent manner. This is similar to the events that
occur normally in the formation of type VI collagen, where tetramers are subsequently secreted from the cell and aggregate in the
extracellular space end to end to form double-beaded microfibrils. The
finding of defined molecular intermediates in the present study
supports the contention that the semipermeabilized cells allow correct folding and chaperone association to occur and shows that dimer and
tetramer formation occurs early in the secretory pathway of the
cell. It is well known that chain selection and helix formation occur in the endoplasmic reticulum, but once sufficient monomers are
present then dimer and tetramer formation follows.
3(VI) is an important determinant of chain association,
although no information was available about the relative importance of
the C1, C2, C3, C4, or C5 domains of this chain in that process.
Interestingly, this study has shown that each of the three chains of
type VI collagen can form monomers if the C1 domain is present, but the
C2 domain appears to be essential to form higher molecular mass
assemblies. The importance of the C2 domain to dimer and tetramer
formation is shown dramatically for the
2(VI) chain when the
alternatively spliced C2 variant, C2a, was used. When the C2 was
compared with the C2a variant it was clear that whereas constructs
containing the full-length C2 could form dimers and tetramers, those
containing the C2a variant could not. It has been suggested that
production of
2(VI) isoforms may be a mechanism for controlling the
amount of mature type VI collagen (23). The present result would
support this suggestion, as failure to produce dimers would result in
recognition and removal by intracellular proteinases. Interestingly,
the presence of the C2 domain in the
3(VI) chain did not facilitate
dimer and tetramer formation, which suggests that whatever sequence
mediates antiparallel alignment in the C2 domains of the
1(VI) and
2(VI) chains is either not present or obscured in the
3(VI) chain.
-sheet flanked on each side by three
-helices, which is highly
reminiscent of the Rossmann dinucleotide-binding fold (24, 25) The
A-domains in type VI collagen have low (~15%) amino acid similarity
to A-domains in other proteins, such as von Willebrand factor,
complement, and integrin subunits. Identity between certain collagen VI
A-domains is relatively high (47% for N7 and N9) but between others is
much lower (18.5% for N1 and C1 of
3(VI)).
1(VI) and
2(VI) chains all have the residues in the metal
ion-dependent adhesion site (MIDAS) sequence conserved, but
this is not the case for C1 of the
3(VI) chain. In the case of the
C2 domain the
1(VI) chain contains
DXSXSTX, whereas the
2(VI)
chain contains DXSXXXD, although interestingly
threonine is replaced by serine in this latter motif. No conservation
of this sequence occurs in the
3(VI) C2 domain. Metal ion
coordination has been one means of generating protein-protein
interactions, because it has been shown that the sixth coordination
position of magnesium in the
-subunit of integrin CR3 is taken up by
a carboxylate oxygen atom in another A-domain (24). However, the A3
domain of human von Willebrand factor binds collagen without metal
binding (25) and highlights that there are other forms of A-domain interactions.
1(VI) chain has most homology to the vWFA3
(Fig. 10). This domain also has a
conserved MIDAS motif, but there is no evidence of metal ion binding,
and this has been shown to bind to the collagen helix. It is
interesting to speculate that binding occurs with the
1(VI) C2
domain and the adjacent, antiparallel type VI triple-helical region
when dimers form. Comparison of the models for the C2 domains of the
1(VI) and
2(VI) with that of the
3(VI) suggest that there may
be subtle changes in secondary folding that impact the ability of this
domain to interact with collagen and hence form dimers (Fig.
11). In particular, there is a 10-amino
acid insertion between the second and third
-strand motifs in
3C2
that may overlap and disrupt interactive residues at the MIDAS site
(see Figs. 10 and 11).
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Fig. 10.
Sequence alignment of C2 domains of the
1(VI),
2(VI), and
3(VI) chains compared with von Willebrand A3
domain. The secondary structure of von Willebrand A3 domain
have been superimposed onto the sequence.
-Helices are colored
yellow, and
-strands are colored blue. For C2
domains the predicted secondary structure has also been superimposed.
Residues involved in the MIDAS motif are shown in
bold.
View larger version (61K):
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Fig. 11.
A cartoon representation of the model
of 2C2.
-Strands are shown as
ribbons, and
-helices are shown as cylinders.
10-Amino acid insertion in the
3C2 domain is sketched in
blue.
The finding that 1(VI) and
2(VI) chains can form monomers,
dimers, and tetramers suggests that one might expect to find these
species secreted from cells and assembled into microfibrils. However,
previous studies have suggested that although chains can be secreted,
all three chains of type VI collagen are required for secretion and
assembly of type VI collagen microfibrils (9, 10).
The literature is strewn with a veritable melange of reports on the
fate of individual chains of type VI collagen. These range from stating
that the 1(VI) is secreted as a chain (26) to its being secreted as
monomers, dimers, and/or tetramers (27). There are reports that the
1(VI) can be found extracellularly in disulfide (28) or nondisulfide
form (29). In SaOS-2 cells, which are deficient in
3(VI) production,
the majority, but not all, of the
1/
2(VI) is apparently retained
in the cell, although chains did appear to get disulfide-bonded (9). In
our hands immunoprecipitation of labeled medium from SaOS-2 cells
showed a distinct monomer on agarose electrophoresis (data not shown). In contrast, following transfection of 293 cells with either
1(VI) or
2(VI) cDNAs, there was efficient secretion of polypeptide monomers and dimers (30).
Although transfection experiments may overload cellular function, it is
surprising that there is so much variation and variability in the fate
of single-chain transcripts or indeed why collagen appears to be able
to avoid the cellular quality control mechanism (31). Early reports
suggested that basal levels of collagen degradation were about 15%
(32) and that as much as 30% of collagen was secreted from the cell in
a nonhelical form (33). These results indicate that up to a third of
unfolded collagen chains may not be removed intracellularly. Although
these results were obtained looking at fibrillar collagens, data on
type VI collagen (26-29) would suggest that a similar level of
secretion occurs for this collagen. The present findings, showing that
homotrimers can form into tetramers, suggests that these correctly
folded assemblies escape quality control detection and are secreted. Their fate in the extracellular space is unknown, but findings with
knockout mice would suggest that these assemblies are not as stable as
heterotrimers, as no microfibrils are detected. Our cotransfection
studies with all three chains of type VI collagen showed an increased
rate of formation of dimers and tetramers and suggests, as previously
reported, that the presence of 3(VI) is an important determinant of
type VI collagen formation.
The system used in the present study folds and assembles protein chains
and shows clearly that, where 1(VI) and/or
2(VI) chains are
present, the chains contain the information to allow formation of
physiological monomers, dimers, and tetramers. We are currently
investigating how these assemblies are handled in the cell and why
homotrimer assemblies do not appear to form recognizable microfibrils.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to the Medical Research Council and Biotechnology and Biological Sciences Research Council for funding this work.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
Manchester, School of Biological Sciences, Wellcome Trust Centre for
Cell/Matrix Research, 2.205 Stopford, Oxford Rd., Manchester M13 9PT,
UK. Tel.: 0161-275-5079; Fax: 0161-275-5078; E-mail: ashuttle@fs1.scg.man.ac.uk.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MIDAS, metal ion-dependent adhesion site; NEM, N-ethylmaleimide.
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