From the Orthopaedic Molecular Biology Research Unit,
Department of Paediatrics, University of Melbourne, Royal Children's
Hospital, Parkville, Victoria 3052, Australia, § Department
of Dermatology and Cutaneous Biology and Department of Biochemistry and
Molecular Pharmocology, Thomas Jefferson University, Philadelphia,
Pennsylvania 19107-5541, ¶ Department of Anatomy and Cell Biology,
University of Melbourne, Parkville, Victoria 3052, Australia, and
Max-Planck-Institut für Biochemie,
82152 Martinsried, Germany
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ABSTRACT |
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Collagen VI is a microfibrillar
protein found in the extracellular matrix of virtually all connective
tissues. Three genetically distinct subunits, the 1(VI),
2(VI),
and
3(VI) chains, associate intracellularly to form triple-helical
monomers, which then assemble into disulfide-bonded dimers and
tetramers before secretion. Although sequence considerations suggest
that collagen VI monomers composed of all three chains are the most
stable isoform, the precise chain composition of collagen VI remains
controversial and alternative assemblies containing only
1(VI) and
2(VI) chains have also been proposed. To address this question
directly and study the role of the
3(VI) chain in assembly, we have
characterized collagen VI biosynthesis and in vitro matrix
formation by a human osteosarcoma cell line (SaOS-2) that is deficient
in
3(VI) production. Northern analysis showed an abundance of
1(VI) and
2(VI) mRNAs, but no detectable
3(VI) mRNA
was apparent in SaOS-2 cells. By day 30 of culture, however, small
amounts of
3(VI) mRNA were detected, although the level of
expression was still much less than
1(VI) and
2(VI). Collagen VI
protein was not detected in SaOS-2 medium or cell layer samples until
day 30 of culture, demonstrating that despite the abundant synthesis of
1(VI) and
2(VI), no stable collagen VI protein was produced
without expression of
3(VI). The
1(VI) and
2(VI) chains
produced in the absence of
3(VI) were non-helical and were largely
retained intracellularly and degraded. The critical role of the
3(VI) chain in collagen VI assembly was directly demonstrated after
stable transfection of SaOS-2 cells with an
3(VI) cDNA
expression construct that lacked 4 of the 10 N-terminal type A
subdomains. The transfected
3(VI) N6-C5 chains associated with
endogenous
1(VI) and
2(VI) and formed collagen VI dimers and
tetramers, which were secreted and deposited into an extensive
network in the extracellular matrix. These data demonstrated that
3(VI) is essential for the formation of stable collagen VI molecules
and subdomains N10-N7 are not required for molecular
assembly.
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INTRODUCTION |
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The collagens, a large protein family, form highly organized supramolecular assemblies in the extracellular space surrounding connective tissue cells. Although many collagen types form well characterized fibrils assembled by lateral aggregation of cross-linked collagen molecules, collagen VI is unique in forming an abundant microfibrillar extracellular network in virtually all connective tissues (see Refs. 1-4 for reviews). Collagen VI has both cell adhesion properties and a diverse range of extracellular matrix protein binding activities, suggesting a critical bridging role between the cell and the pericellular matrix. Thus, although it is likely that collagen VI provides both structural integrity and key matrix regulatory signals, the precise role of collagen VI during extracellular matrix development and remodeling has not been elucidated.
Collagen VI also differs from other collagens in that it is not
secreted as a triple-helical monomer, but assembles intracellularly into antiparallel, overlapping dimers, which then align in a parallel manner to form tetramers that are secreted and aggregate to form extracellular microfibrils (5, 6). The component -chains of collagen
VI (
1(VI),
2(VI), and
3(VI)) are complex multidomain polypeptides comprising short triple helical domains and large globular
N- and C-terminal regions containing subdomains with sequence
similarities to von Willebrand factor type A domains, fibronectin type III domains, Kunitz-type protease inhibitors, and
several other recognized protein motifs (7-9). The
3(VI) chain is
much larger than the homologous
1(VI) and
2(VI) chains with an
extended N-terminal globular domain (7), and multiple alternatively
spliced forms of the
3(VI) and
2(VI) mRNAs give rise to
protein variants, which have been proposed to modulate the organization
of the microfilaments and interactions with cells and other matrix
molecules (10-12).
Collagen VI 1(VI),
2(VI), and
3(VI) chains are commonly
isolated from tissues in near equimolar amounts (3, 13-15) and the
corresponding mRNAs are present in a 1:1:1 ratio in human skin
fibroblast cultures (16, 17) leading to the conclusion that the
collagen VI triple-helical molecule is a heterotrimer containing three
genetically distinct chains. The collagen VI mRNAs can, however, be
differentially regulated. Transforming growth factor-
and
-interferon up-regulate and down-regulate
3(VI) mRNA,
respectively (18, 19), and both
1(VI) and
2(VI) mRNAs are
up-regulated when fibroblasts are grown in a collagen gel (17).
Likewise,
1(VI) and
2(VI) mRNAs are more abundant than
3(VI) mRNA in corneal fibroblasts (14), in fetal skin (20), and
in fetal skin fibroblasts (20). Although sequence data suggest that
collagen VI monomers composed of all three chains are required to allow
the formation of stable higher order structures (21), the possibility
that these variations at the mRNA level may be reflected in the
formation of alternative collagen VI isoforms with potentially
different functions remains controversial. Assemblies apparently
composed of only
1(VI) and
2(VI) chains (20), or a predominance
of
3(VI) chains (22) have been reported. It is important to
determine the possible chain assembly isoforms to understand not only
the normal biological roles of collagen VI, but also the affects of
altered expression of collagen VI chains in diseases. For example, it
has been proposed that triplication of the COL6A1 and
COL6A2 genes and a subsequent stoichiometric disturbance of
collagen VI chain composition may be responsible for some of the
extracellular matrix manifestations of trisomy 21 (23, 24).
One way to address these questions directly and determine the role of
the 3(VI) in regulating intracellular collagen VI assembly would be
to examine assembly of
1(VI) and
2(VI) in the absence of
3(VI)
chain expression. A mammalian cell line expressing only
1(VI) and
2(VI) would also be an important tool for assessing the role of
helical and N- and C-terminal globular
3(VI) domains in molecular
assembly, microfibril formation, and architecture, and in cell adhesion
and interactions with other matrix molecules, by site-directed
mutagenesis and stable expression. Ideally, to allow questions of
collagen VI matrix deposition and interactions to be examined, the cell
model system should be competent to make collagen VI microfibrils and
capable of forming an extensive cross-linked fibrillar collagen matrix
in vitro containing a "tissue equivalent" composition of
other extracellular matrix components. These characteristics have not
been identified in a cell line to date (25).
In this study, we directly demonstrate the critical role of the
3(VI) chain in collagen VI assembly by characterization of the human
SaOS-2 bone cell line, which is deficient in
3(VI) chain expression
and produces no triple-helical collagen VI. However, the
1(VI) and
2(VI) chains are abundantly expressed by SaOS-2, and we demonstrate
that when low level expression of the endogenous
3(VI) is induced
during long term culture,
3(VI) synthesis rescues the
1(VI) and
2(VI) chains from degradation and allows secretion and deposition of
collagen VI microfibrils within the fibrillar collagen matrix produced
by these cells (26). To explore the
3(VI) domains important in
molecular assembly, SaOS-2 cells were stably transfected with an
3(VI) cDNA construct, which lacked 4 of the 10 N-terminal type A
subdomains. Despite the deletion, the
3(VI) N6-C5 chains retained
the ability to associate with endogenous
1(VI) and
2(VI) and
formed collagen VI dimers and tetramers, which were secreted and
deposited into an extensive network in the extracellular matrix. These
studies provide direct evidence that
1(VI) and
2(VI) chains do
not form stable secreted assemblies and that
3(VI) expression is
essential for the formation of functional collagen VI molecules, and
demonstrate that
3(VI) subdomains N10-N7 are not required for
molecular assembly.
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EXPERIMENTAL PROCEDURES |
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Production of an 3(VI) cDNA Expression Construct--
The
3(VI) expression construct was prepared by ligating previously
characterized partial cDNA clones (7). The 3' end of the
3(VI)
cDNA was excised from clone FM22 by digesting with PflMI, which cuts within the C2 domain, and SalI,
which cleaves within the polylinker of the Bluescript II vector
(Stratagene). This 2.5-kilobase fragment was ligated into the
corresponding sites of F376 to generate a cDNA encoding protein
domains N2-C5. A SacII fragment of this clone was excised
using sites within the triple helix and the Bluescript II polylinker,
and ligated into the SacII-digested FO4 cDNA, producing
a clone encoding domains N8-C5. A fragment coding for domains N6-C5
was excised from the N8-C5 clone by digesting with NheI,
which cuts within the N7 domain, filling in the restriction site
overhang with the Klenow fragment of DNA polymerase I, followed by
cleavage at the 3' NotI polylinker site. This fragment was
ligated into the expression plasmid pRc/AC (27), which had also been
cleaved with NheI, made blunt-ended with Klenow DNA
polymerase I, and then digested with NotI. The resulting
expression construct,
3(VI) N6-C5, encoded the BM-40 signal
sequence, 54 amino acids of the
3(VI) N7 domain, domains N6-C5, and
the 3' untranslated region and polyadenylation sequence, in the vector
pRc/CMV (Invitrogen), which also encodes the neomycin phosphotransferase gene conferring resistance to the antibiotic G418.
Cell Culture and Transfection--
A human osteosarcoma (SaOS-2)
cell line (28, 29) (ATCC HTB-85), mouse NIH/3T3 (ATCC CRL-1658) and 3T6
fibroblasts (ATCC CCL-96), and a rat osteogenic sarcoma (UMR 106) cell
line (30) (ATCC CRL-1661) were obtained from American Type Culture
Collection. Mouse Mov-13 cells (31) were provided by Dr. R. Jaenisch
(Whitehead Institute for Biomedical Research, Cambridge, MA). A mouse
osteoblastic cell line, MC-3T3 E1 (32) was obtained from Dr. H. Kodama
(Tohoku Dental College, Kohriyama, Japan). Human dermal fibroblasts
were established from skin biopsies (33). Cell cultures were maintained in Dulbecco's modified Eagle's medium
(DMEM)1 containing 10% (v/v)
fetal calf serum as described previously (33). From confluence (day 0),
the cell cultures were grown in DMEM containing 10% (v/v) fetal calf
serum, and supplemented daily with sodium ascorbate to a final
concentration of 0.25 mM (34). SaOS-2 cells were
transfected with the 3(VI) N6-C5 cDNA expression construct
using LipofectAMINE reagent (Life Technologies, Inc.) according to the
manufacturer's protocol. Stably transfected cells were selected in
growth medium containing 500 µg/ml G418 (Life Technologies, Inc.).
Individual G418-resistant colonies were isolated and expanded into cell
lines. G418 was removed from the culture medium after the fourth
passage.
Northern Blot Analysis--
Total RNA was prepared by the
guanidine hydrochloride extraction method of Wake and Mercer (35). RNA
samples were analyzed under denaturing conditions on 0.8% (w/v)
agarose gels and transferred to nitrocellulose or nylon filters (36).
Filters were hybridized to [-32P]dCTP-labeled
1(VI),
2(VI), and
3(VI) cDNA probes (P18, P1, and P24;
Ref. 21), washed and specific hybridization visualized by
autoradiography (36).
Antibodies--
Antibodies specifically recognizing collagen VI
1(VI) (37) and
3(VI) chains (38) have been described previously.
Antibodies used for immunoprecipitations were raised against
pepsin-digested collagen VI and, although they both recognized all
three collagen VI subunits on Western blots, one precipitated only
assemblies of
1(VI),
2(VI), and
3(VI) chains (Life
Technologies, Inc.), and the other precipitated both assembled and
individual, unassociated
1(VI),
2(VI), and
3(VI) chains
(39).
Collagen Biosynthetic Labeling-- Skin fibroblasts and SaOS-2 cells in 10-cm2 culture dishes were grown to confluence, supplemented with 0.25 mM sodium ascorbate for 24 h, then biosynthetically labeled for 18 h with 100 µCi/ml [35S]methionine (Tran35S-labelTM 1032 Ci/mmol, ICN Pharmaceuticals, Inc.) in methionine-free and serum-free DMEM containing 0.25 mM sodium ascorbate. The medium was removed and added to an equal volume of 2× NET buffer (NET buffer is 50 mM Tris-HCl, pH 7.5 containing 1 mM EDTA, 150 mM NaCl, and 0.1% Nonidet P-40). Protease inhibitors were included at the following final concentrations: 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 mM N-ethylmaleimide (NEM), and 5 mM EDTA. The cell layer was solubilized in 50 mM Tris/HCl, pH 7.5 containing 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM PMSF and 20 mM NEM. Cell lysates and medium samples were clarified by centrifugation and precleared with 30 µl of anti-fibronectin antibody (Life Technologies, Inc.) and 100 µl of 20% Protein A-Sepharose (Amersham Pharmacia Biotech) at 4 °C for 2 h. Supernatants were incubated with 5 µl of collagen VI antiserum (Life Technologies, Inc.) and 100 µl of 20% Protein A-Sepharose for 18 h at 4 °C, and the immunoprecipitated complexes washed two times for 30 min each with fresh NET buffer, then once with 10 mM Tris-HCl, pH 7.5, containing 0.1% Nonidet P-40.
For pulse-chase experiments, confluent SaOS-2 cells were incubated overnight in medium containing 10% fetal calf serum and 0.25 mM sodium ascorbate, preincubated for 30 min in methionine-free and serum-free DMEM supplemented with 0.25 mM sodium ascorbate, then pulse-labeled for 1 h with 100 µCi/ml [35S]methionine. The labeling medium was removed, after which the cells were washed with serum-free DMEM containing 50 mM cold methionine and 0.25 mM sodium ascorbate, and then incubated in this chase medium for up to 24 h. Medium and cell layer fractions were harvested as above and the collagen VI immunoprecipitated with 5 µl of the collagen VI antiserum, which precipitates both assembled and unassociatedPepsin Digestion-- Immunoprecipitated collagen VI was resuspended in 0.5 M acetic acid containing 100 µg/ml pepsin and incubated at 4 °C for 16 h. Digestion was terminated by lyophilization.
Extraction of Collagen VI from the in Vitro Accumulated Extracellular Matrix-- At the appropriate time points, the matrix was harvested by rinsing the cell layer twice with phosphate-buffered saline and scraping it into 20 mM sodium phosphate buffer, pH 7.4 containing fresh protease inhibitors (20 mM NEM, 1 mM PMSF, 25 mM EDTA). The insoluble matrix was collected by centrifugation (10,000 × g, 10 min) and rinsed briefly in cold distilled water containing protease inhibitors. The pellet was solubilized in 50 mM Tris-HCl, pH 7.5 containing 0.5% (w/v) SDS and 8 M urea by heating at 80 °C for 5 min.
SDS-PAGE and Immunoblotting-- Collagen VI chains were analyzed by SDS-PAGE on 5% (w/v) polyacrylamide gels and pepsinized type VI collagen on 7.5% (w/v) polyacrylamide gels along with [14C]methylated protein molecular weight markers (Amersham Pharmacia Biotech). Where indicated, samples were reduced before electrophoresis by the addition of dithiothreitol to a final concentration of 25 mM. Collagen VI dimers and tetramers were analyzed on 2.4% (w/v) acrylamide/0.5% (w/v) agarose composite gels (40) under non-reducing conditions. Molecular size standards were the reduced (230 kDa and 400 kDa) and unreduced (900 kDa) forms of laminin (Boehringer Mannheim). Radioactively labeled proteins were detected by fluorography (33, 41) or imaged using a PhosphorImager (Molecular Dynamics, STORMTM).
For immunoblotting, collagen chains resolved by SDS-PAGE were electrophoretically transferred to nitrocellulose filters. Bound antibodies were detected using horseradish peroxidase-conjugated Protein A (Bio-Rad) and an enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech).Indirect Immunofluorescence-- SaOS-2 cells were grown in eight-well chamber glass slides (Nunc Inc.) under culture conditions described above, washed with phosphate-buffered saline, fixed with ice-cold methanol for 10 min, then air-dried. Slides were incubated with either primary collagen VI antibody (Life Technologies, Inc.) or non-immune rabbit serum for 1 h at room temperature and bound antibody detected using fluorescein-conjugated AffiniPure donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories, Inc.). Slides were mounted in FluorSaveTM reagent (Calbiochem) then viewed and photographed using a Zeiss fluorescence microscope.
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RESULTS |
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Collagen VI mRNA Expression in Cell Lines--
A range of cell
lines capable of producing type I collagen and other extracellular
matrix components were screened for collagen VI mRNA expression by
Northern blotting. Two cell lines, NIH/3T3 and MC-3T3 E1, produced
trace quantities of 1(VI),
2(VI), and
3(VI) mRNAs; Mov13
and 3T6 synthesized trace amounts of
1(VI) and
2(VI) mRNA;
and UMR 106 did not produce detectable levels of any collagen VI
subunit mRNA (data not shown). Primary cultures of dermal
fibroblasts, however, expressed abundant
1(VI),
2(VI), and
3(VI) mRNA (Fig. 1, b,
d, and f, lane 1), reflecting the high
level of expression of collagen VI in skin (16). Confluent SaOS-2 cells
also produced
1(VI) and
2(VI) mRNA, at levels comparable to
that of skin fibroblasts, but were totally deficient in
3(VI) transcription (Fig. 1f, lane 2) (14). The SaOS-2
cell line was thus the only cell line examined that expressed
1(VI)
and
2(VI) mRNAs at relatively high levels, making it a
potentially valuable tool for the analysis of collagen VI assembly in
the absence of
3(VI), and subsequent stable transfection and
expression of
3(VI) chains.
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SaOS-2 Collagen VI mRNA Expression during Long Term Culture
with Ascorbic Acid--
SaOS-2 bone cells can be induced to lay down
an extensive type I collagen matrix, which can mineralize in
vitro by extended culture in the presence of sodium ascorbate
(26). Ascorbate is an essential cofactor for collagen hydroxylation and
subsequent helix formation and secretion, and this technique has been
used extensively to stimulate collagen matrix deposition and generate in vitro "tissue equivalent" extracellular matrices (34,
42). Because during this ascorbate-induced matrix formation in
vitro, major changes in collagen gene expression have been
reported (34), it was of importance to see if 3(VI) gene expression
was influenced by long term culture and type I collagen matrix
deposition. The changes in mRNA expression of the three
-chains
over a 30-day culture period with sodium ascorbate is shown in Fig. 1.
Steady-state levels of
1(VI) mRNA and
2(VI) mRNA remained
constant over the first 10-12 days of culture (Fig. 1, b
and d, lanes 2-6), but diminished significantly
between days 15 and 30 (lanes 7 and 8). Although
the steady state levels of
3(VI) mRNA remained below the level
of detection under identical analytical conditions during the entire
30-day culture period (Fig. 1f), overexposure of the Northern blots demonstrated that low level
3(VI) expression is gradually induced during the extended culture period (Fig.
1g), with the highest levels of expression at days 15-30
(Fig. 1g, lanes 7 and 8).
Collagen VI Matrix Deposition in SaOS-2 Cultures--
Collagen VI
matrix deposition during the 30-day culture period was assessed by
immunoblotting of urea-extracted matrix using chain-specific antibodies
(Fig. 2). Skin fibroblasts, which have steady state levels of 1(VI) and
2(VI) mRNA similar to those in the SaOS-2 cells, produced an extensive collagen VI matrix by 8 days
of culture (Fig. 2, a-c, lane 1). In contrast,
1(VI) and
2(VI) chains were not detected in the SaOS-2
extracellular matrix until day 15 (Fig. 2, b and
c, lanes 2-6), despite the abundant synthesis of
1(VI) and
2(VI) mRNA. However, from day 15, which coincided
with the induction of expression of
3(VI) mRNA, the SaOS-2 cells
did produce a small amount of collagen VI matrix, which was detected at
day 15 with just the
1(VI) antibody (Fig. 2c, lanes
7), but by day 30 was detected by both the
1(VI) and
3(VI)
antibodies (Fig. 2, b and c, lane 8).
The appearance of collagen VI chains in the matrix only after low level
expression of
3(VI) mRNA, suggested that the
1(VI) and
2(VI) chains were unable to associate into collagen VI microfibrils
without the co-expression of
3(VI).
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Fate of the 1(VI) and
2(VI) Polypeptides Synthesized by
SaOS-2--
The demonstration of
1(VI) and
2(VI) mRNA and
the inability to detect collagen VI matrix formation in the absence of
the
3(VI) provided strong evidence that the
3(VI) chain was
essential for molecular assembly and matrix formation. To determine if
the
1(VI) and
2(VI) chains were retained within the cell and
degraded, or were secreted as individual polypeptide chains or as
stable triple helical assemblies, collagen VI chains synthesized during a 1-h pulse-labeling were chased for up to 24 h,
immunoprecipitated, and examined by both reducing and non-reducing
electrophoresis. Immediately after the pulse-labeling
1(VI) and
2(VI) were abundant intracellularly (Fig.
3a, lane 1), and a
proportion of these chains had associated into higher order
disulfide-bonded structures (Fig. 3b, lane 1).
These assemblies migrated with the molecular weight expected of
-chain dimers and most likely represent disulfide-bonded
1(VI)
and
2(VI) homo- or heterodimers. Because
3(VI) mRNA is not
detectable in SaOS-2 cells at confluence (Fig. 1f,
lane 2), the feint band migrating above
1(VI) and
2(VI) is most probably fibronectin, which has not been completely
removed before immunoprecipitation with the collagen VI antibody (20).
The intracellular
1(VI) and
2(VI) diminished gradually during the
24-h chase period (Fig. 3a, lanes 2-6) with the
concomitant appearance of a small proportion of unassociated chains and
disulfide-bonded
1(VI) and
2(VI) dimers in the medium (Fig. 3,
c and d). However, when the amount of total
intracellular and secreted collagen VI remaining at 24 h was
compared with that synthesized during the 1-h labeling, it was clear
that the
(VI) and
2(VI) chains were not efficiently secreted and
the vast majority were retained intracellularly and degraded. When the
SaOS-2 intracellular and extracellular
1(VI) and
2(VI) chains
were digested with pepsin, they were completely degraded, clearly
demonstrating that these chains have not formed helical assemblies
(data not shown).
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Stable Expression of the 3(VI) N6-C5 Construct in SaOS-2
Cells--
To confirm the essential role of the
3(VI) chain in
collagen VI assembly and secretion and explore the importance of the alternatively spliced N-terminal domains in microfibril formation, SaOS-2 cells were transfected with the
3(VI) N6-C5 expression construct (Fig. 4). Individual, stably
transfected clones were selected in medium containing G418 and then
screened for expression of
3(VI) mRNA by Northern blot. Two cell
lines expressing significant amounts of
3(VI) mRNA as well as
endogenous
1(VI) and
2(VI) mRNAs were obtained (data not
shown). The cell line expressing the highest level of
3(VI)
mRNA, at levels approximately equal to
1(VI) and
2(VI)
mRNAs, was used in subsequent experiments.
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The 3(VI) N6-C5 Chain Rescues the Endogenous
1(VI) and
2(VI) and Restores Collagen VI Assembly and Secretion--
To
determine if the
3(VI) N6-C5 chain was able to associate with
endogenous
1(VI) and
2(VI), cells were metabolically labeled for
18 h with [35S]methionine, and the collagen VI in
the cell and medium immunoprecipitated with an antibody that
precipitates only collagen VI assemblies and not the unassociated
polypeptide chains (Life Technologies, Inc.). No collagen VI was
immunoprecipitated from either the cell or medium fraction of
untransfected SaOS-2 cells (Fig. 5,
lanes 2 and 3), confirming that in the absence of
the
3(VI),
1(VI), and
2(VI) chains did not form triple helical
collagen VI molecules. In contrast, the
3(VI) N6-C5 chain produced
by the transfected SaOS-2 cells associated with the endogenous
1(VI)
and
2(VI), rescued them from intracellular degradation, and formed
collagen VI assemblies which were efficiently secreted (Fig.
5, lanes 4 and 5).
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Collagen VI Molecules Containing N-terminally Deleted 3(VI)
Chains Form Disulfide-bonded Tetramers--
The normal pathway of
collagen VI assembly involves the intracellular assembly of triple
helical monomers (3 chains) to form disulfide-bonded dimers (6 chains),
and then tetramers (12 chains). To determine if N-terminal
3(VI)
domains are required for this higher order assembly, the collagen VI
synthesized by SaOS-2 cells transfected with
3(VI) N6-C5 was
analyzed on non-reducing acrylamide/agarose composite gels. The
predominant form of collagen VI secreted by human fibroblasts migrated
with a molecular mass of about 2,000 kDa, as expected for collagen VI
tetramers (Fig. 6, lane 4). A small proportion of the collagen VI was secreted as dimers, and a band
migrating at around 4,000 kDa suggested that some tetramers had formed
end-to-end associations (Fig. 6, lane 4). The same pattern
of predominantly collagen VI tetramers with a small proportion of
dimers and some higher order assembly, was seen in the medium of the
transfected SaOS-2 cells (Fig. 6, lane 2), clearly
demonstrating that
3(VI) domains N10-N7 are not required for
intracellular monomer, dimer, or tetramer assembly. Rescue of the
endogenous
1(VI) and
2(VI) chains by
3(VI) N6-C5, and the
efficient secretion of collagen VI tetramers, strongly suggested that
these chains had folded to form a stable triple helix. This was
directly demonstrated by the presence of pepsin-resistant forms of
collagen VI in both the cell and medium of SaOS-2 cells transfected
with the
3(VI) N6-C5 construct (data not shown).
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3(VI) Domains N10-N7 Are Not Required for Assembly of Collagen
VI Microfibrils--
To determine if collagen VI tetramers
containing the
3(VI) N6-C5 chain were incorporated into a
microfibrillar matrix, SaOS-2 cell cultures were grown in the presence
of sodium ascorbate, stained with a collagen VI antibody and examined
by immunofluorescence microscopy. By day 12 of culture, untransfected
SaOS-2 cells had deposited only minute amounts of collagen VI into
their extracellular matrix (Fig.
7a) consistent with the low
level of
3(VI) mRNA expression detected at this time (Fig.
1g). In contrast, SaOS-2 cells expressing
3(VI) N6-C5
chains had deposited an extensive collagen VI network (Fig.
7b) indicating that this shortened
3(VI) chain contained
all the domains required, not only for monomer, dimer, and tetramer
assembly, but also for the end-to-end association of tetramers to form
matrix microfibrils.
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DISCUSSION |
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The experiments presented here provide the first direct evidence
for the crucial role of the 3(VI) chain in collagen VI molecular assembly and matrix formation and demonstrate that the functional collagen VI triple-helical monomer is a heterotrimer which contains
3(VI) chains as well as
1(VI) and
2(VI), most probably in the ratio of 1:1:1. Our data demonstrate that the human osteosarcoma cell
line, SaOS-2 does not synthesize
3(VI) in short term culture, but
produces abundant
1(VI) and
2(VI), which are unable to form helical assemblies and are unstable. Furthermore, long term culture studies where the synthesis of
3(VI) was induced, demonstrating that
the cells have all the necessary cellular machinery to correctly assemble and secrete collagen molecules composed of
1(VI),
2(VI), and
3(VI) chains, and deposit collagen VI into the extracellular matrix.
These data showing the central role of the 3(VI) chain in allowing
stable protein formation support previous predictions from sequence
analyses that collagen VI molecules composed of all three chains are
more stable than other assembly alternatives (21), and are consistent
with studies demonstrating that when a specific reduction in the
steady-state level of
3(VI) mRNA, but not
1(VI) or
2(VI)
mRNA is induced by
-interferon treatment of skin fibroblasts,
all three collagen VI subunits are secreted in reduced amounts (19).
Furthermore, the deposition of collagen VI in the developing mouse
extracellular matrix also correlates with expression of the
3(VI)
gene, irrespective of the earlier expression of the
1(VI) and
2(VI) chains (43).
Variations in the chain composition of triple-helical collagen VI
molecules have, however, been described. In the most comprehensive study (20), post-confluent fetal bovine skin fibroblasts secreted pepsin-resistant collagen VI molecules apparently comprised
predominately of 1(VI) and
2(VI) chains. Fetal skin extracts also
contained an excess of
1(VI) and
2(VI) chains (20). Difficulties
in the interpretation of these experiments arise from the variable nature of pepsin digestion and the sensitivity of the
3(VI) globular domains to proteolytic degradation, both of which tend to give the
impression of an overabundance of
1(VI) and
2(VI) chains. Collagen VI
1(VI),
2(VI), and
3(VI) chains are each degraded to different sized polypeptides by pepsin, and further heterogeneity can arise by non-uniform cleavage within each chain (3). We commonly
see only two bands after pepsin digestion of collagen VI
immunoprecipitated from human fibroblast cultures, even though the
3(VI) chain is a major component before pepsin
digestion.2 These bands show
gel migrations consistent with the major pepsin-resistant forms of the
1(VI) and
2(VI) chains. This phenomenon has also been observed
when examining collagen VI extracted from nucleus pulposis. Native
collagen VI from this tissue shows a 1:1:1 ratio of the three chains,
but the
3(VI) chain is apparently much less abundant after pepsin
digestion (40). It seems likely, then, that variable pepsin digestion
commonly results in forms of
3(VI) that comigrate with
pepsin-resistant
1(VI) and/or
2(VI). In addition, we find that
the globular domains of native
3(VI) chains are readily removed by
proteases present in tissue culture samples, resulting in the recovery
of only 140-kDa components, and in fact, many early studies on
"intact" collagen VI failed to detect the large >200-kDa form of
the
3(VI) chain because of its lability (3). To prevent this
proteolysis and allow recovery of intact
3(VI) chains, samples must
be analyzed rapidly after extraction in the presence of fresh protease
inhibitors, and not stored before purification, even for short
periods.2 Examination of collagen VI biosynthesis and
assembly in SaOS-2 cells overcomes these problems of variable
proteolysis, which make it difficult to accurately determine the
molecular formula of collagen VI in both intact and pepsin-digested
samples, and demonstrated directly that no triple-helical collagen VI
assemblies were produced when the
3(VI) chain was not expressed.
This situation is analogous to that of collagen IX where, in mice,
inactivation of the Col9a1 gene, which encodes the
1(IX)
chain, leads to a functional knock-out of all the collagen IX chains,
even though mRNAs for
2(IX) and
3(IX) are transcribed
normally (44). The
1(IX) chain thus plays a pivotal role in the
assembly of stable triple-helical collagen IX, comparable to the
central role of the
3(VI) in collagen VI assembly demonstrated in
this study.
In the absence of 3(VI), the
1(VI) and
2(VI) chains produced
by SaOS-2 cells are largely retained within the cell as unassembled polypeptides and are degraded. In contrast, when transfected with either
1(VI) or
2(VI) cDNAs, 293 cells efficiently secreted these chains as non-helical polypeptide monomers or homodimers (37),
indicating that cells which do not produce endogenous collagen VI may
lack the quality control mechanisms present in SaOS-2 that normally
prevent secretion of unassembled chains (25). The mechanism of
intracellular degradation of the unassembled
1(VI) and
2(VI) was
not determined, but for other unassembled normal collagen species, such
as the pro
2(I) chain in Mov-13 cells and human fibroblasts, this
degradation occurs predominantly via a post-endoplasmic reticulum
non-lysosomal mechanism (45), similar to that described for basal
collagen degradation (46, 47). In contrast, structurally abnormal
malfolded collagens appear to be rapidly degraded by endoplasmic
reticulum-mediated quality control processes (45). A small proportion
of the
1(VI) and
2(VI) were secreted as monomeric peptides or
disulfidebonded dimers, but these were pepsin-sensitive, thus not in a
stable helical configuration and unable to play a structural role in the matrix. Intact collagen VI microfibrils are susceptible to degradation by serine proteinases and matrix metalloproteinase-9 (MMP-9) (48), and collagen VI assemblies lacking disulfide bonds are
digested by MMP-2 (49). Secreted non-helical, non-disulfide-bonded chains could be expected to be even more susceptible to proteolytic attack (3) and are unlikely to persist in the extracellular matrix.
This finding that 1(VI) and
2(VI) do not form stable collagen VI
assemblies has important implications for the interpretation of studies
examining collagen VI expression and regulation during development, in
response to growth factors, and in disease. Although we cannot formally
exclude the possibility that stable collagen VI isoforms containing
only
1(VI) and
2(VI) can form under some conditions, our data
indicate that coordinated temporal and spatial expression of the three
-chains will be necessary for collagen VI matrix deposition during
development, and that increased mRNA levels will not necessarily be
reflected in the amount of functional secreted collagen VI. For
example, the COL6A1 and COL6A2 genes, encoding
the
1(VI) and
2(VI) subunits, are both found on chromosome 21, and some studies have suggested that alterations in the extracellular matrix of trisomy 21 skin could be explained by elevated levels of
collagen VI or a stoichiometric disturbance in the chain composition of
collagen VI (23, 24). However, excess
1(VI) and
2(VI) chains in
trisomy 21 cells would most likely not form stable molecules and be
degraded before secretion. Likewise, increased accumulation of collagen
VI in the extracellular matrix of these patients would only result if
3(VI) chains were normally synthesized in excess and collagen VI
assembly was limited by the availability of
1(VI) and
2(VI).
The SaOS-2 cell line is also a powerful model system for stable
transfection and expression of 3(VI) chains modified by
site-directed mutagenesis, and will facilitate studies designed to
define the roles of
3(VI) domains in molecular assembly, microfibril
formation and architecture, cell adhesion, and interactions with other
matrix molecules. The potential of this approach was demonstrated here by the establishment of SaOS-2 cell lines stably expressing the transfected
3(VI) N6-C5 cDNA at high levels. In transfected
cells,
3(VI) chains restored normal collagen VI biosynthesis by
associating with endogenous
1(VI) and
2(VI), rescuing them from
intracellular degradation, and forming collagen VI dimers and tetramers
which were secreted and deposited into the extracellular matrix.
Our experiments also address the unsolved issue of the role of the
3(VI) extended N-terminal domain in collagen VI assembly and
function. The
3(VI) construct used in this study did not contain all
the N-terminal repetitive subdomains, but lacked subdomains N10-N7.
These deleted
3(VI) chains retained the ability to assemble intracellularly into heterotrimeric monomers, and higher
order oligomers. The role of specific motifs in driving assembly
is not known, but by analogy to other collagen types it might be expected that the C-terminal globular domains would play a fundamental role in chain selection, and initiating helix formation (50, 51).
Normal intracellular assembly of the
3(VI) N6-C5 chains is also
consistent with rotary shadowing studies, which have suggested that
dimers are stabilized by interactions between the C-terminal globular
domains of one monomer and the triple helix of the adjacent molecule
(52, 53), whereas the important interactions for tetramer stability are
between the N-terminal regions of the triple helix (52). The formation
of matrix microfibrils involves the end-to-end association of
tetramers, with an overlap of the terminal regions of the tetramers
bringing the N- and C-terminal globular domains into close contact (4,
52, 53). Although the exact nature of the forces stabilizing collagen
VI microfibrils have not been identified, interactions between the N-
and C-terminal globular domains, which have a high affinity for each
other, are thought to be critical (53). The ability of collagen VI
tetramers containing
3(VI) N6-C5 chains to form microfibrils
clearly demonstrates that subdomains N10-N7 are not required for
either intracellular or extracellular collagen VI assembly.
The N-terminal subdomains of the 3(VI) chain, N10-N2, are each
encoded by a separate exon (10, 11, 54). Three of these exons, encoding
subdomains N10, N9, and N7, are known to be alternatively spliced in
both human and chicken, predicting multiple
3(VI) chains, which may
lack one to three subdomains (10, 11, 54). Interestingly, recent
studies characterizing
3(VI) splice variants in the mouse have
demonstrated splicing of additional exons, including exons encoding
subdomains N8 and N5.3 One of
the relatively abundant isoforms found was an
3(VI) N6-C5 chain,
and our data show that this chain assembles normally and is
incorporated into the extracellular matrix. The functional significance
of the
3(VI) splicing events is not known; however, as the
3(VI)
N-terminal region has been shown to bind in vitro to type I
collagen (55) and heparin (38), and subdomains N10-N7 are not required
for assembly, it seems reasonable to speculate that alternative
splicing may modulate collagen VI interactions with other matrix
ligands and allow the formation of microfibrils with tissue-specific
functions.
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FOOTNOTES |
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* This work was supported by grants from the National Health and Medical Research Council of Australia and the Royal Children's Hospital Research Foundation (to J. F. B.) and by National Institutes of Health Grant AR38912 (to M.-L. C.).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. Tel.: 61-3-9345-6367; Fax: 61-3-9345-6668/6367; E-mail: bateman{at}cryptic.rch.unimelb.edu.au.
1 The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis.
2 S. R. Lamandé and J. F. Bateman, unpublished data.
3 M. Dziadek, unpublished data.
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