From the Cell and Matrix Biology Research Unit,
Department of Paediatrics, University of Melbourne, and the Murdoch
Childrens Research Institute, Royal Children's Hospital,
Parkville, Victoria 3052, Australia, the § Section for
Connective Tissue Biology, Department of Cell and Molecular Biology,
Lund University, S-221 00 Lund, Sweden, and the ¶ Shriners
Hospital Research Facility, Portland, Oregon 97201
Received for publication, September 7, 2000, and in revised form, October 6, 2000
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ABSTRACT |
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Collagen VI assembly is unique within the
collagen superfamily in that the Although collagen VI is expressed in virtually all connective
tissues, where it forms a complex and extensive microfibrillar network
(see Refs. 1 and 2 for reviews), a definition of the precise functional
roles of the collagen VI microfibrils in development and matrix
architecture has been elusive. Even though the cell adhesion and
extracellular matrix protein binding capabilities of collagen VI have
suggested that it may play an important role in the interconnection
between the cell and the structural scaffolding of the extracellular
matrix, it is only recently that one such critical tissue-specific role
in muscle has been demonstrated by the detection of mutations in
collagen VI genes in Bethlem myopathy (3-6) and by the generation of a
myopathy by targeted inactivation of Col6a1 in mice (7).
Collagen VI is composed of three genetically distinct The precise protein interactions initiating and regulating formation of
the unique collagen VI microfibril supramolecular assemblies are not
known, but elegant structural studies have identified the organization
of assembly intermediates, providing a model for a hierarchical
assembly process unlike that of any other members of the collagen
family (12, 13). Following heterotrimeric assembly of the It is likely that numerous motifs within the collagen VI To examine systematically the role of the Production of
The
To facilitate production of the
To ensure that no errors had been introduced during PCR and cloning,
all constructs were transcribed and translated in vitro (TNT®, Promega), and the regions generated by PCR were
sequenced (AmpliCycleTM, PerkinElmer Life Sciences) (data not shown).
Cell Culture and Transfections--
The SaOS-2 human
osteosarcoma cell line (ATCC HTB-85), which expresses Northern Blot Analysis--
RNA was isolated from cultured
SaOS-2 cells using RNeasyTM (Qiagen). Total RNA (5 µg) was analyzed
under denaturing conditions on 1% agarose gels and transferred to
nitrocellulose membranes (17). Filters were hybridized to
[ Collagen VI Biosynthetic Labeling and Analysis--
SaOS-2 cells
were grown to confluence in 10-cm2 dishes, incubated
overnight in the presence of 0.25 mM sodium ascorbate, and then biosynthetically labeled for 18 h with 100 µCi/ml
[35S]methionine (Tran35S-labelTM 1032 Ci/mmol, ICN Pharmaceuticals, Inc.) in 750 µl of methionine-free and
serum-free Dulbecco's modified Eagle's medium containing 0.25 mM sodium ascorbate. The medium was removed, Nonidet P-40
was added to a final concentration of 1%, and protease inhibitors were
added to the following final concentrations: 1 mM 4-(2
aminoethyl)-benzenesulfonyl-fluoride, 1 mM
phenylmethylsulfonyl fluoride, 20 mM
N-ethylmaleimide, and 5 mM EDTA. The cell layer
was solubilized in 50 mM Tris/HCl, pH 7.5, containing 5 mM EDTA, 0.15 M NaCl, 1% Nonidet P-40, 1 mM 4-(2 aminoethyl)-benzenesulfonyl-fluoride, 1 mM phenylmethylsulfonyl fluoride, and 20 mM
N-ethylmaleimide. Cell lysates and medium samples were
clarified by centrifugation and the supernatants precleared with 100 µl of 20% protein A-Sepharose (Amersham Pharmacia Biotech) at
4 °C for 2 h. Collagen VI in the supernatants was immunoprecipitated overnight at 4 °C using either the Transmission Electron Microscopy--
SaOS-2 cells were grown to
confluence and then supplemented daily for 15 days with 0.25 mM sodium ascorbate. Cell layers were washed with
phosphate-buffered saline (PBS), fixed in 0.15 M sodium cacodylate, pH 7.3, containing 1.5% paraformaldehyde (w/v), 1.5% gluteraldehyde (v/v), and 0.1% (w/v) tannic acid for 90 min, rinsed in
0.15 M sodium cacodylate, pH 7.3, post-fixed in 1% (v/v)
osmium tetroxide for 1 h, washed with cacodylate buffer,
dehydrated in a graded series of ethanol to 100%, and then embedded in
Spurr's epoxy and sectioned (50 nm).
Negative Staining Electron Microscopy--
Confluent SaOS-2
cultures in 80-cm2 flasks were incubated for 18 h in
10 ml of serum-free medium containing 0.25 mM sodium ascorbate. The medium was collected, the protease inhibitors described earlier were added, and sodium azide was included at a final
concentration of 0.1% (w/v). To examine collagen VI that had been
deposited into the extracellular matrix, confluent SaOS-2 cells were
grown in the continuous presence of 0.25 mM sodium
ascorbate for 15 days. Cell layers were washed with 50 mM
Tris/HCl, pH 7.4, containing 0.15 M NaCl, scraped into 5 ml
of this buffer, and then mildly disrupted using a Teflon homogenizer.
Culture medium and homogenized matrix samples were clarified by
centrifugation, adsorbed onto carbon-coated grids for 1 min, washed
with water, and stained with 0.75% uranyl formate. The grids were
rendered hydrophilic by glow discharge in air, and samples were
observed in a Jeol 1200 EX electron microscope operated at 60 kV
accelerating voltage.
Indirect Immunofluorescence--
SaOS-2 cells were grown to
confluence in 8-well chamber glass slides (Nunc) and then supplemented
daily for 15 days with 0.25 mM sodium ascorbate. Cell
layers were washed with PBS, fixed with 3.7% (v/v) formaldehyde at
room temperature for 10 min and then air-dried. Slides were
preincubated with 5% (v/v) fetal calf serum in PBS for 1 h at
room temperature and then incubated with either the collagen VI
antibody 3C4 in PBS or PBS alone for 1 h at room temperature.
Bound antibody was detected using fluorescein
isothiocyanate-conjugated sheep anti-mouse Ig (Silenus). Slides
were mounted in FluorSaveTM reagent (Calbiochem) and then viewed and
photographed using a Zeiss fluorescence microscope.
Collagen VI Intracellular Assembly in Transfected SaOS-2
Cells--
To examine the contribution of the
Secretion of all three collagen VI chains by each of the transfected
cell lines provided indirect evidence that heterotrimers containing the
shortened Collagen VI Microfibril Formation--
To determine whether
collagen VI tetramers containing shortened
To formally exclude the possibility that the efficiency of tetramer
aggregation was dependent on the concentration of tetramers in the
medium, the levels of
To determine which cell lines were able to incorporate collagen VI
tetramers into an extracellular matrix, transfected SaOS-2 cells were
grown for 15 days post-confluence in the presence of sodium ascorbate,
stained with collagen VI antibodies, and examined by fluorescence
microscopy. A collagen VI matrix was present in cells expressing
In this study, we investigated the contribution of The mechanism of collagen VI heterotrimer association has not been
elucidated, but our studies clearly exclude a role for The subsequent complex hierarchical process of collagen VI
intracellular assembly, the formation of dimers and tetramers, does
critically involve the participation of collagen VI triple-helix sequences. Ultrastructural and binding studies predict that a specific
interaction between the C-terminal globular domain of one monomer and
the triple helix of the adjacent overlapping antiparallel monomer is
involved in dimer formation, an interaction that is stabilized by
disulfide bonding (12, 13, 28). Tetramers are thought to be stabilized
by disulfide bonds between the N-terminal ends of the triple helix of
the dimers (12, 13). Furthermore, collagen VI heterotrimers, containing
deletions that disturb the structure of the helix, are unable to
assemble further into disulfide-bonded dimers and tetramers, and
molecules containing the mutant chain are not secreted (5).
The structural models proposed for the supramolecular organization of
collagen VI tetramers into microfibrils (9, 12, 13) provide us with a
basis for understanding the importance of the type A domains in
microfibril formation. The lateral association of dimers into tetramers
generates a structure in which the type A domains from the N termini of
all three There are several other examples of interactions between A domains,
either within the same molecule or between different extracellular matrix components, suggesting that these protein modules may
play an important role in matrix architecture and assembly. Matrilin 1 contains two vWF type A-like domains, which have been shown to be
involved in the assembly of matrilin oligomers and in the formation of
matrilin-1 filamentous networks, suggesting that interactions between
matrilin-1 type A domains can occur (30). Interaction between
type A domains from different matrix proteins have been demonstrated
for the A1 and A3 domains of vWF, both of which associate with the
globular regions of chicken collagen VI microfibrils to promote the
adhesion of platelets to collagen VI at high shear forces (31).
Further evidence for the role of Our emerging understanding of collagen VI assembly is that the 1(VI),
2(VI), and
3(VI)
chains associate intracellularly to form triple helical monomers, and
then dimers and tetramers, which are secreted from the cell. Secreted
tetramers associate end-to-end to form the distinctive extracellular
microfibrils that are found in virtually all connective tissues.
Although the precise protein interactions involved in this process are
unknown, the N-terminal globular regions, which are composed of
multiple copies of von Willebrand factor type A-like domains, are
likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these
subdomains in collagen VI intracellular and extracellular assembly,
3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell
lines that express
3(VI) chains with N-terminal globular domains
containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as
the complete triple helix and C-terminal globular domain (C1-C5). All
of these transfected
3(VI) chains were able to associate with
endogenous
1(VI) and
2(VI) to form collagen VI monomers, dimers,
and tetramers, which were secreted. Importantly, cells that expressed
3(VI) chains containing the N5 subdomain,
3(VI) N9-C5, N6-C5, and
N5-C5, formed microfibrils and deposited a collagen VI matrix. In
contrast, cells that expressed the shorter
3(VI) chains, N4-C5,
N3-C5, and N1-C5, were severely compromised in their ability to form
end-to-end tetramer assemblies and failed to deposit a collagen VI
matrix. These data demonstrate that the
3(VI) N5 module is critical
for microfibril formation, thus identifying a functional role for a
specific type A subdomain in collagen VI assembly.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain
subunits,
1(VI),
2(VI), and
3(VI), each of which contain a
relatively short triple helix and N- and C-terminal globular regions.
Recent studies have demonstrated that initial association of all three
chains into a collagen VI triple helical heterotrimer is a prerequisite
for further intracellular assembly, stability and secretion (8). The
1(VI) and
2(VI) chains are similar in size (1009 and 998 amino
acids, respectively) and contain one N- and two C-terminal subdomains
of approximately 200 amino acids that show homology to the type A
domains of von Willebrand factor (vWF)1 (9). In contrast, the
3(VI) chain is much larger (up to 3149 amino acids), containing 6 to
10 N-terminal vWF type A-like subdomains (depending on alternative
splicing), two C-terminal vWF type A-like subdomains, and subdomains
similar to type III fibronectin repeats, and Kunitz protease inhibitors
(10, 11).
1(VI),
2(VI), and
3(VI) chains, these collagen VI monomers form higher
order structures intracellularly by aligning in an anti-parallel manner
with a 30-nm stagger, first to form dimers and then tetramers by
lateral association of dimers. After secretion, the tetramers associate
end-to-end to form microfibrils with a distinctive 100-nm periodicity,
comprising beaded globular domains separated by short triple helical
regions (12, 13). However, the critically important questions of what
protein structures and interactions drive these complex and specific
associations during collagen VI assembly have not been addressed in detail.
-chains
contribute to the final microfibril structure and to the development
of its heterotypic interactions with other matrix components.
The type A subdomains present in all three collagen VI subunits, and
found in a number of other extracellular proteins including integrins,
matrilins, complement components, and collagens VII, XII, and XIV
(14-16), are likely to play an important role, as they have been shown
to be commonly involved in specific molecular interactions (14). In
particular, it seems probable that the N terminus of the
3(VI) chain
may be the crucial domain involved in tetramer-tetramer association
because its multiple type A modules would be exposed at both ends of
the tetramers during microfibril formation.
3(VI) N-terminal type A
subdomains, N9 to N1, in collagen VI intracellular tetramer assembly
and extracellular microfibril formation
3(VI) cDNA constructs with sequential deletions of the N-terminal type A modules were stably
transfected into SaOS-2 cells, producing a range of cell lines that
express
3(VI) chains with N-terminal domains containing modules
N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete
triple helix and C-terminal globular domain. Our data show that
3(VI) subdomains N2 to N9 are not required for intracellular heterotrimer assembly or for the formation of disulfide-bonded dimers
and tetramers. Ultrastructural examination of the secreted collagen VI
demonstrated that tetramers were able to associate end-to-end to form
microfibrils in cells expressing
3(VI) chains containing the
N-terminal N9-N1, N6-N1, and N5-N1 modules. Significantly, however, the
efficiency of tetramer-tetramer association was reduced in cells
expressing
3(VI) chains with N-terminal domains lacking the N5
module. These data provide the first evidence that an
3(VI) N-terminal subdomain (N5) plays a critical role in the interactions between collagen VI tetramers leading to the formation of the microfibrillar network.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3(VI) cDNA Expression Constructs--
All
constructs were cloned in the mammalian expression vector pRc/CMV
(Invitrogen), which contains the neomycin phosphotransferase gene
conferring resistance to G418. Production and stable expression of the
3(VI) N6-C5 construct, encoding the BM40 signal sequence and the
3(VI) protein domains N6 to C5 (Fig. 1), have been described previously (8). Constructs encoding
3(VI) domains N5-C5, N4-C5, and
N1-C5 were generated by PCR using the
3(VI) N6-C5 cDNA as a
template. Forward primers were designed to anneal at the 5' end of
subdomains N5, N4, and N1, beginning at nucleotides 2710 (domains
N9-C5, ATG at base 256 (10)), 3317, and 5131, respectively, and each
contained an NheI site at the 5' end to allow cloning of the
PCR product into the NheI site at the 3' end of the BM40 signal sequence of pRc/CMV
3(VI) N6-C5 (8). The downstream primer in
each reaction corresponded to nucleotides 5748-5771 near the 5' end of
the triple helix and included the SacII site at base 5757. PCR was performed using the proof-reading polymerase Pfu
(Stratagene). The resultant fragments were digested with
NheI and SacII and used to replace the
corresponding fragment of the
3(VI) N6-C5 construct.
3(VI) N3-C5 construct was generated by digesting
3(VI) N6-C5
with NheI and Bpu1 1021 (recognition site at base
3778). The restriction enzyme-generated overhangs were filled in using Klenow polymerase (17), and the resulting blunt ends were religated. The Bpu1 1021 site is in the N4 subdomain, and therefore the
3(VI) N3-C5 construct contains 151 nucleotides of N4 at the 5' end.
3(VI) N9-C5 construct, an
NheI site within the N9 subdomain cDNA was deleted using
strand overlap extension PCR (18) to introduce a silent T to C
substitution at position 747. Primers A and B were used to amplify
bases 331-755 using the cDNA clone FO19 (10) as a template, and
primers C and D to amplify bases 735-2097. Primers B and C
overlapped, and both contained the single nucleotide
substitution. To allow cloning, primer A contained a NheI
site at the 5' end. These amplification products were gel-purified, and
100 ng of each used was as the template in a second round of
overlapping PCR with primers A and D. The resulting PCR product was
digested with NheI and cloned into the NheI site
of
3(VI) N6-C5 to generate the
3(VI) N9-C5 construct.
1(VI) and
2(VI) mRNA but not
3(VI) mRNA (8), was maintained in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum
as described previously (19). Cells were transfected with the
3(VI)
expression constructs using LipofectAMINETM reagent (Life
Technologies, Inc.). Stably transfected cells were selected in medium
containing 500 µg/ml G418 (Life Technologies, Inc.) and individual
clones isolated and expanded into cell lines.
-32P]dCTP-labeled
1(VI),
2(VI), and
3(VI)
cDNA probes (P18, P1, and P24 (20)) and washed, and specific
hybridization was visualized by autoradiography.
3(VI)
monoclonal antibody 3C4 obtained from Dr E. Engvall (21) or a
polyclonal collagen VI antibody (Life Technologies, Inc.) and 100 µl
of 20% protein A-Sepharose. Immunoprecipitated complexes were washed twice with 50 mM Tris/HCl, pH 7.5, containing 0.15 M NaCl, 5 mM EDTA, and 0.1% Nonidet P-40 for
30 min each. Immunoprecipitated collagen VI was eluted into gel loading
buffer at 65 °C for 15 min and analyzed following reduction with 25 mM dithiothreitol by SDS-polyacrylamide gel electrophoresis
on 5% (w/v) polyacrylamide gels. Collagen VI triple helical monomers,
dimers, and tetramers were analyzed on 2.4% (w/v) acrylamide/0.5%
(w/v)-agarose composite gels under nonreducing conditions (5, 6, 8).
Radioactively labeled proteins were detected by fluorography (19) or
imaged using a PhosphorImager (Molecular Dynamics, STORMTM).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3(VI) N-terminal
globular subdomains to collagen VI intracellular assembly, microfibril formation, and matrix deposition, we stably transfected SaOS-2 cells,
which express
1(VI) and
2(VI) mRNA and protein but no
3(VI) mRNA (8), with six
3(VI) cDNA constructs
from which the N-terminal globular domains N9 to N1 were progressively
deleted. As a result of alternative splicing, more than 95% of the
3(VI) mRNAs found in cells and tissues lack the N10 subdomain
(11); therefore, the
3(VI) N9-C5 construct was used as the
"full-length" control in this study. Individual cell clones
transfected with either the control construct (
3(VI) N9-C5) or the
shorter constructs (
3(VI) N6-C5, N5-C5, N4-C5, N3-C5, and N1-C5)
(Fig. 1) were selected in medium
containing G418 and screened for expression of
3(VI) mRNA by
Northern blotting (data not shown). Cell lines were metabolically labeled with [35S]methionine and cell layer and medium
fractions immunoprecipitated with collagen VI antibodies. The
immunoprecipitated material was electrophoresed on 5% polyacrylamide
gels under reducing conditions to visualize the individual collagen VI
chains. All transfected cell lines expressed
3(VI) protein that
migrated with an apparent molecular mass in good agreement with the
predicted size of each of the shorter chains (Fig.
2) when compared with the control
3(VI) N9-C5 (Fig. 2, lanes 11 and 12). Because
the collagen VI antibodies used for immunoprecipitation were specific
for the
3(VI) chain (N9, N6, N5, N4, and N3 constructs), or
assemblies of all three chains (N1 construct), co-immunoprecipitation
of
1(VI) and
2(VI) chains in all of the cell lines indicated that the shortened
3(VI) chains had formed collagen VI heterotrimers that
were able to be secreted. Collagen VI found in the medium had a slower
electrophoretic mobility compared with that isolated from the cell
layer fraction. This is a consistent finding in both transfected SaOS-2
cells and primary human fibroblasts (5, 6, 8), and although the reason
for this size difference is not known, it may reflect differences in
glycosylation between secreted collagen VI and the more recently
synthesized intracellular collagen VI.
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Fig. 1.
Structure of the collagen VI subunits
and 3(VI) expression constructs.
A, diagram of collagen
1(VI),
2(VI), and
3(VI)
chains showing the triple helix and numbered C- and
N-terminal subdomains. The shaded type A subdomains, N10,
N9, and N7 in
3(VI) and C2 in
2(VI), undergo alternative
splicing. B, diagram of the
3(VI) cDNA expression
constructs. Deletions were made from the N terminus leaving the triple
helix and C-terminal domains intact.
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Fig. 2.
Electrophoretic analysis of collagen VI.
Transfected SaOS-2 cells were metabolically labeled for 18 h with
[35S]methionine, and collagen VI in the cell
(C) and medium (M) fractions were
immunoprecipitated and resolved on 5% polyacrylamide gels under
reducing conditions. Collagen VI 1(VI),
2(VI), and
3(VI)
subunits are indicated on the right, and the migration
position of the 14C-methylated 200-kDa standard is shown on
the left.
3(VI) chains were able to assemble further into tetramers,
the normal secreted form of collagen VI (22). Tetramer formation was
demonstrated directly by composite agarose/acrylamide gel
electrophoresis under nonreducing conditions (Fig.
3). The predominant form of collagen VI
secreted by cells expressing control
3(VI) N9-C5 chains migrated
with a molecular mass of approximately 2000 kDa (Fig. 3, lane
12) as expected for collagen VI tetramers. Comparable results were
seen in cultures expressing each of the shortened
3(VI) chains (Fig.
3, lanes 1-10), clearly demonstrating that the
3(VI)
N-terminal subdomains N9-N2 are not required for collagen VI monomer,
dimer, and tetramer formation and secretion.
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Fig. 3.
Analysis of collagen VI tetramer
assembly. Transfected SaOS-2 cells were labeled biosynthetically
for 18 h, and collagen VI was immunoprecipitated from the cell
(C) and medium (M) fractions electrophoresed on
2.4% (w/v) acrylamide, 0.5% (w/v) agarose composite gels under
nonreducing conditions to visualize collagen VI tetramers. Collagen VI
tetramers are indicted on the right, and the migration
position of the unreduced laminin marker (900 kDa) is shown on the
left.
3(VI) chains could
self-associate to form end-to-end assemblies of tetramers, secreted
collagen VI was examined by negative staining electron microscopy.
Medium from cells expressing
3(VI) N9-C5,
3(VI) N6-C5, and
3(VI) N5-C5 (Fig. 4A)
contained microfibrils composed of multiple tetramers as well as single
tetramers. In contrast, the majority of the collagen VI in medium from
cells expressing the shorter
3(VI) chains,
3(VI) N4-C5,
3(VI)
N3-C5, and
3(VI) N1-C5 (Fig. 4B), was present as single
tetramers. Microfibril formation in multiple clonal cell lines
expressing the control and shortened
3(VI) chains was quantified by
analysis of a large number of micrograph fields (Fig.
5). The occurrence of
"microfibrils" containing 1-7 tetramers is shown as a
percentage of the total number of microfibrils. In cells expressing
3(VI) N9-C5, N6-C5, and N5-C5, 70-80% of the microfibrils
contained 2-7 tetramers and only 20-30% were single tetramers (Fig.
5, A-C). In striking contrast, in medium from cells
expressing
3(VI) N4-C5, N3-C5, and N1-C5, more than 70% of all
microfibrils were single tetramers, with only a small number of double
tetramers and virtually no higher aggregations of tetramers (Fig. 5,
D-F). The efficiency of microfibril formation was
comparable in all of the clonal cell lines expressing
3(VI) N9-C5,
N6-C5, and N5-C5 (Fig. 5, A-C) and was reduced in
all of the cell lines expressing
3(VI) N4-C5, N3-C5, and N1-C5 (Fig.
5, D-F). These data demonstrate that all of the cells
expressing
3(VI) chains, including the N5 subdomain, are able to
form microfibrils with similar efficiency, whereas those lacking the N5
have compromised multimerization, suggesting that the
3(VI) N5
module is crucial for the interactions between tetramers that lead to
the formation of microfibrils.
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Fig. 4.
Negative staining electron microscopy of
secreted collagen VI. Transfected SaOS-2 cells were incubated for
18 h in serum-free medium containing 0.25 mM sodium
ascorbate. The medium was removed, clarified by centrifugation,
adsorbed onto carbon-coated grids, stained with uranyl formate, and
examined by electron microscopy. Media from cells expressing 3(VI)
N5-C5 (A), N6-C5, and N9-C5 contained collagen VI
tetramers that had associated end-to-end to form microfibrils. In media
samples from
3(VI) N1-C5 (B), N3-C5, and N4-C5, the
greater portion of the collagen VI was present as single
tetramers.
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Fig. 5.
Quantitative analysis of collagen VI
tetramer-tetramer association. Collagen VI secreted into the
medium of transfected SaOS-2 cells was visualized by negative staining
electron microscopy, and the ability of the tetramers to associate
end-to-end was quantitated. The occurrence of microfibrils containing
1-7 tetramers is shown as a percentage of the total number of
microfibrils. When available, multiple clonal cell lines expressing
each 3(VI) cDNA construct were analyzed. SaOS-2 cells
expressing: A,
3(VI) N9-C5; B,
3(VI) N6-C5;
C,
3(VI) N5-C5; D,
3(VI) N4-C5;
E,
3(VI) N3-C5; F,
3(VI) N1-C5.
3(VI) mRNA and protein in an
3(VI) N5-C5 cell line (able to form microfibrils) and two
3(VI) N3-C5 cell
lines (not able to form microfibrils) were examined. Northern blot
analysis demonstrated that the
3(VI) N5-C5 cell line (clone 28) contained much less
3(VI) mRNA than either of the two
3(VI) N3-C5 cell lines (clones 19 and 17) (Fig.
6A). The amount of collagen VI
protein produced by these clones was consistent with the level of
expression of
3(VI) mRNA. Small amounts of collagen VI protein were present in the cell and medium fractions of the
3(VI) N5-C5 line (Fig. 6B, lanes 3 and 4), and
substantially larger amounts were produced by the two
3(VI) N3-C5
clones (lanes 5-8). The faint bands in lanes 3 and 4 were clearly identified as collagen VI following a
longer exposure. Because mRNA and protein expression is higher in
the two
3(VI) N3-C5 cell lines than in the
3(VI) N5-C5
line, and end-to-end tetramer assembly is more efficient in the
3(VI) N5-C5 line than in either of the two
3(VI) N3-C5 cell lines
(Fig. 5), it is clear that the efficiency of tetramer aggregation is
not related to the level of collagen VI synthesis and the concentration
of tetramers available for microfibril formation.
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Fig. 6.
Relative collagen VI mRNA and protein
expression in SaOS-2 cells. A, Northern blot analysis
of collagen VI mRNA expression. Total RNA from untransfected SaOS-2
cells (lane 1) and from cells expressing 3(VI) N5-C5
(clone #28, lane 2) or
3(VI) N3-C5 chains
(clones #19 and #17, lanes 3 and
4) was separated on a denaturing agarose gel, transferred to
nitrocellulose, and hybridized simultaneously with
32P-labeled
1(VI),
2(VI), and
3(VI) cDNAs.
Migration of
1(VI),
2(VI), and
3(VI) mRNAs are indicated
on the right. B, electrophoretic analysis of
collagen VI protein expression. Untransfected SaOS-2 cells and cells
transfected with the
3(VI) N5-C5 and N3-C5 constructs were labeled
metabolically for 18 h with [35S]methionine and
collagen VI in the cell (C) and medium (M)
fractions immunoprecipitated and electrophoresed on a 5%
polyacrylamide gel under reducing conditions. Collagen VI
1(VI),
2(VI), and
3(VI) subunits are indicated on the
right.
3(VI) N9-C5, N6-C5, and N5-C5 (Fig. 7,
A-C), but in contrast, no collagen VI matrix was seen by
immunofluorescence in cells expressing
3(VI) N4-C5 (Fig.
7D), N3-C5, and N1-C5, where only intracellular staining was
present. Transmission electron microscopy of the accumulated
extracellular matrix revealed an extensive branched microfibrillar
network in cells expressing
3(VI) N9-C5 (Fig.
8B), similar to the highly
branched open meshwork formed by collagen VI in skin (23, 24).
Branching points commonly connected 4-5 tetramers in a star-like
arrangement (Fig. 8B). This extensive microfibrillar network
was absent from the matrix of cells expressing
3(VI) N1-C5 (Fig.
8A); the microfibrils seen in Fig. 8A were
extremely rare and were present in only a small number of fields. To
confirm that these were collagen VI microfibrils, the extracellular
matrices were mildly homogenized in a neutral salt buffer and the
solubilized proteins examined by negative staining electron microscopy.
The branching microfibrils extracted from the matrix of cells
expressing
3(VI) N9-C5 were clearly composed of collagen VI, with
the characteristic globular beads at 100-nm intervals (Fig.
8C). Collagen VI microfibrils were not present in the matrix
extracts of cells expressing
3(VI) N1-C5 (not shown). These
findings, which are in agreement with the quantitative electron
microscopy data, demonstrated that collagen VI tetramers containing
3(VI) chains with only the N4-N1, N3-N1, or N1 modules have lost the
capacity to interact end-to-end to form collagen VI microfibrils. The
minimum requirement for N5-N1 modules identifies the N5 subdomain as
critical for collagen extracellular matrix formation.
View larger version (135K):
[in a new window]
Fig. 7.
Collagen VI in the in vitro
accumulated extracellular matrix. SaOS-2 cells transfected
with 3(VI) N9-C5 (A),
3(VI) N6-C5 (B),
3(VI) N5-C5 (C), and
3(VI) N4-C5 (D) were
grown for 15 days post-confluence in the presence of 0.25 mM sodium ascorbate and stained with a collagen VI
antibody. Bound antibody was detected with fluorescein
isothiocyanate-conjugated sheep anti-mouse Ig. All images are
×40.
View larger version (111K):
[in a new window]
Fig. 8.
Electron microscopy of the SaOS-2 accumulated
extracellular matrix. Transfected SaOS-2 cells were grown for 15 days post-confluence in the presence of 0.25 mM sodium
ascorbate. The cell layer was fixed, stained, and then examined by
transmission electron microscopy. A, microfibrils were
almost completely absent from the matrix of cells expressing 3(VI)
N1-C5; a rare field containing a small number of microfibrils is shown.
B, an extensive branching microfibrillar network
(arrows) was present in the matrix of cells expressing
3(VI) N9-C5. Fibrillar collagen fibrils (arrowheads) were
present in both matrices. The accumulated extracellular matrices were
mildly homogenized in a neutral salt buffer and the solubilized
proteins examined by negative staining electron microscopy.
C, branching collagen VI microfibrils extracted from the
matrix of cells expressing
3(VI) N9-C5.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3(VI)
N-terminal vWF type A-like subdomains N2 to N9 to collagen VI
intracellular assembly, secretion, and microfibril formation. We show
that the
3(VI) subdomains N2 to N9 are not necessary for collagen VI
intracellular triple helical heterotrimer assembly, the subsequent
formation of disulfide-bonded dimers and tetramers, and secretion of
the collagen VI tetramers from the cell. However, the N-terminal
3(VI) type A modules were found to play a critical extracellular
role in the end-to-end association of tetramers to form collagen VI microfibrils. Cells that expressed
3(VI) chains containing the N5
subdomain,
3(VI) N5-C5,
3(VI) N6-C5, and
3(VI) N9-C5, had the
ability to form collagen VI microfibrils and deposit a collagen VI
matrix. In contrast, cells that expressed
3(VI) chains without the
N5 subdomain,
3(VI) N4-C5,
3(VI) N3-C5, and
3(VI) N1-C5, were
severely compromised in their ability to form end-to-end tetramer
assemblies, and failed to deposit a collagen VI matrix. These results
indicate that the
3(VI) N5 module is important for stable
extracellular end-to-end association of tetramers to form collagen VI microfibrils.
3(VI)
N-terminal subdomains distal to N1 in intracellular chain selection and
assembly. Our previous studies demonstrating that two collagen VI
triple-helix mutations, a Bethlem myopathy exon 14-skipping mutation in
COL6A1 and an engineered 202-base pair deletion in the
3(VI) helix, have unimpeded heterotrimer formation (5), indicated
that the N-terminal region of the collagen VI triple helix is not
important for initial chain assembly. These data suggest that the
information for heterotrimer formation is present in the N-terminal N1
subdomain, the C-terminal region of the helix, or the C-terminal
globular domains of
3(VI) and in the other two subunits,
1(VI)
and
2(VI). By analogy with fibrillar collagen trimer assembly (25,
26) and the molecular constraints of the direction of triple-helix
propagation (27), it seems likely that the C-terminal globular domains
play a decisive role in this first event of assembly.
-chains are exposed at both ends of the tetramer. The
model for the junctional complex further predicts that
N-terminal domains of adjacent tetramers overlap in this region,
such that they are available to interact with each other and with the
C-domains or the helical domain of the adjacent tetramer, providing
multiple potential sites for the noncovalent interactions that
stabilize microfibrils (9, 12, 13). Although the N termini in this
junctional complex contain multiple type A subdomains from the
1(VI),
2(VI), and
3(VI) chains, all of which could potentially
function in these interactions, our data demonstrates that the
3(VI)
N5 subdomain is critical for microfibril formation. The
3(VI) chain
is expressed as a number of splice variants in vivo and in
cultured cells in which specific N-terminal modules are deleted from
the protein structure (10, 11, 29). It is of interest to note that the N5 module is present in all of these splice
variants,2 consistent with a
crucial role for N5 in the formation of microfibril assembly competent
collagen VI. Although it is clear that the N5 module is important for
extracellular tetramer association, other subdomains C-terminal to N5,
and/or those within
1(VI) or
2(VI) and/or the N-terminal region
of the triple helix, could contribute to microfibril formation or
stability by providing an interaction partner for N5. A minor role for
other subdomains is also suggested by the small amount of end-to-end
association that occurs between tetramers lacking N5.
3(VI) N-terminal subdomains in the
formation of functional collagen VI comes from studies of a family with
Bethlem myopathy in which the mutation is a glycine to glutamate change
at amino acid 167 of the
3(VI) N2 subdomain (4). Normal levels of
all three chains were detected in the medium of fibroblasts from
affected individuals, suggesting that monomer and tetramer formation
was not impaired (4). This result is consistent with our data showing
that the removal of the entire N2 subdomain, as with the
3(VI) N1-C5
construct, does not affect monomer and tetramer formation and
secretion. The molecular mechanism of how this N2 mutation results in a
clinical phenotype is not known, but it could perturb collagen VI
function by structurally interfering with end-to-end association of
tetramers or, possibly, by modifying interactions of the mutant
collagen VI with other matrix components.
3(VI)
N-terminal domain plays at least two broad roles, in microfibril
assembly and in collagen VI-matrix interactions. The N-terminal domain
of the
3(VI) chain interacts with vWF and heparin in
vitro. The A1 subdomain of vWF interacts with the chicken
3(VI) N8 (31), and heparin binding has been assigned to
3(VI) subdomains N3, N6, and N9 suggesting the potential for binding to heparan sulfate
proteoglycans (32). It seems reasonable to speculate that the specific
role of each A-type subdomain may be related to its location
within the three-dimensional structure of the globular beads of the
tetramer junctional complex. Our data would predict that the N5 domain
must be positioned so that it is exposed on the interacting surface in
intimate contact with the N- and/or C-terminal domains or the
triple helix of the collagen VI in the adjacent tetramer. Other
subdomains may be positioned so as to be available to participate in
interactions with a variety of extracellular matrix proteins.
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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 Institute.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.:
+61-3-9345-6263; Fax: +61-3-9345-7997; E-mail:
lamandes@cryptic.rch.unimelb.edu.au.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M008173200
2 Marie Dziadek, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: vWF, von Willebrand factor; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.
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REFERENCES |
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