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From the
A monomer of type VI collagen is composed of three different
chains of 140 (
Type VI collagen is a component of a microfilamentous meshwork
(1-4) that is believed to play a significant role in cell-matrix
interactions. Unique among the collagens discovered to date, type VI
collagen constituent chains have significantly different sizes. The
Tissue
extraction and biosynthetic studies have demonstrated that the three
chains occur in stoichiometric proportions in a 1:1:1 ratio
(17-19). In vitro studies have shown that type VI
collagen has a peculiar pathway of intracellular assembly; association
of the three chains in a type VI collagen monomer is followed by the
formation of S-S bonded dimers (6 chains) and tetramers (12
chains) before secretion
(19, 20) .
In an effort to
understand the biology of type VI collagen assembly and cell-matrix
interactions, a model system was initially developed in which murine
NIH/3T3 cells were stably transfected with cDNAs encoding chicken type
VI collagen chains. Cell lines were obtained that constitutively
express the individual chicken chains; no self-association was observed
with
In the present study, NIH/3T3 cells
were successfully cotransfected with the cDNAs (3.15, 3.2, and 8.1
kb)
Total RNA was isolated by
guanidine isothiocyanate extraction
(29) . Electrophoresis of the
RNA was performed on a 0.7% (w/v) agarose gel containing 2.3 M
formaldehyde in MOPS buffer (20 mM MOPS, pH 7.0, 5 mM sodium acetate, and 1 mM EDTA) for 8 h at 150 V using
20-cm-long plates. RNA was then transferred onto nitrocellulose filters
and hybridized with [
The
filters were hybridized at 68 °C overnight in 5
In some instances, before
proceeding with the immunoprecipitation with anti chicken type VI
collagen antibodies, the medium and the cell lysate were preincubated
with AS-72. Immunoprecipitated material was resolved by SDS-PAGE on 6%
(v/v) or 3-10% linear gradient polyacrylamide slab gels using the
buffer system of Laemmli and was exposed to MP hyperfilms (Amersham).
The demonstration that
transfected chicken type VI polypeptides apparently assembled to form
S-S bonded tetramers suggests but does not prove that these
composites are stable and have a proper triple helical conformation in
their collagenous domain. The low methionine content of the triple
helix and the amounts of transfected chicken type VI collagen secreted
by [
In this study, we have constructed cDNAs encoding the chicken
Production of chicken type VI collagen assemblies was
confirmed by preclearing experiments in which the murine type VI
collagen present in cell lysates was specifically and completely
removed, allowing the recovery of pure transfected heterologous chicken
type VI collagen. The preclearing steps could not be as extensive with
cell medium, because in this case also the chicken molecules were
removed. Secreted type VI collagen multimers are larger than those
present in cell lysates (21),
The synthesis of endogenous type VI
collagen in NIH/3T3 cells renders more complex the analysis of
transfected cell lines. In preliminary experiments we have attempted to
stably transfect with chicken type VI collagen cDNAs cell lines that do
not produce endogenous type VI collagen, but we have so far failed to
demonstrate assembly and secretion of transfected chains. It is
possible that cells that already synthesize and secrete their own type
VI collagen also favored the assembly and deposition into the ECM of
the transfected chicken chains. Previously reported cell systems for
analysis of transfection and full expression of type I collagen DNAs
made use of cell lines that retained
(31, 32) or did not
retain
(33) endogenous expression of one of the two chains of
type I collagen. Secretion of trimers of type XII collagen was achieved
in HeLa cells
(34) , which are known to synthesize only low
levels of type IV and type V collagens (35). In these latter studies,
only the formation of a proper triple helix was reported, whereas the
ECM deposition of heterologous recombinant collagens was not
investigated.
One observation we have made frequently is that, in
addition to the correct 8.0-kb
The present model offers a tool for detailed
mutational analysis and the production of recombinant type VI collagen
molecules of predefined composition to clarify the contribution of
their globular non-triple helical domains in cell adhesion and
migration phenomena
(41, 42) . Currently, we are trying
to establish stable cell lines with more efficient expression of
transfected type VI collagen molecules in order to enable a more
thorough structure-function analysis in the complex process of type VI
collagen assembly and deposition.
We thank Francesco Bucciotti for excellent technical
assistance in the preparation of the vectors, Dr. Roberto Perris for
reading the manuscript, and Antonella Moro for typing the manuscript.
We thank also Dr. Beat Trüeb, Eidgenössische Technische
Hochschule, Zurich, Switzerland for supplying us with clones of chicken
Volume 270,
Number 22,
Issue of June 2, pp. 13105-13111, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1), 130 (2), and 250-350 kDa (3).
Monomers assemble into dimers (6 chains) and tetramers (12 chains) that
are stabilized by disulfide bonds and, once associated one to another,
give rise to a microfilamentous network in close apposition with cell
surfaces and banded collagen fibers. We have derived murine NIH/3T3
cell lines that were transfected with the cDNAs for the three chains
and that constitutively expressed chicken type VI collagen.
Cotransfection was efficient because, in three out of six isolated cell
lines, all chicken chains were expressed. Southern blotting
demonstrated that several copies of each cDNA were integrated
approximately in equal number. Expression of the three polypeptide
chains was consistent with the levels of the respective mRNAs. The
three chicken chains assembled by disulfide bonding to form correctly
folded triple helical aggregated composites with sizes corresponding to
type VI collagen monomers, dimers, and tetramers. These functional
recombinant assemblies were secreted and became incorporated into the
extracellular matrix, where they formed an extensive fibrillar network.
1(VI) and 2(VI) chains are about 130-140 kDa; the
3(VI) chain is much larger, ranging from 250 to 350
kDa
(5, 6, 7, 8) . Cloning and sequencing
of the cDNAs for the chicken
(9, 10, 11) and
human
(12, 13) chains have shown that 1(VI) and
2(VI) chains consist of a short collagenous sequence flanked at
the N and C termini by one and two type A modules, respectively. A
major portion of the additional sequences of the 3(VI) chain
consists of eight
(11, 13) or nine
(14, 15) type A modules. Furthermore, the 3(VI) chain differs
from the other two chains due to the presence of unique additional
sequences at the C terminus
(13, 16) .
1(VI) nor with 2(VI) chains, which were secreted as
single polypeptides. Instead, chimeric chicken/murine type VI collagen
molecules were detected in cell lines transfected with the chicken
3(VI) cDNA
(21) .
()
coding for the three chicken type VI
collagen chains and for a selectable marker. Stable coexpression of all
three chains was obtained, and the three transfected chains were
assembled into pepsin-resistant type VI collagen. These molecules were
secreted as monomers and higher order multimers of the proper size and
became deposited into the extracellular matrix (ECM). This approach
demonstrates for the first time the production of a recombinant
nonfibrillar collagen composed of three genetically different chains.
Antibodies
The following rabbit polyclonal
antisera were used: AS-5, specific for chicken type VI
collagen
(19) ; AS-72, specific for murine type VI collagen; and
AS-46 and AS-47, specific for the chicken 1(VI) and 2(VI)
chains, respectively.
()
Tissue culture
supernatants of the mAbs 111A3, 116A8, 192C2, and 76G11, specific for
the chicken 3(VI) chain, and mAb 108F3, specific for the chicken
1(VI) chain (22),
()
were used; ascites of
the antifibronectin mAb HB91 (ATCC) was also used.
Expression Vectors
Construction of the 1(VI)
full-length cDNA (3.15 kb) and insertion into the SV40 early
replacement vector pSV23 plus
(23) was described
previously
(24) . The 2(VI) full-length cDNA (3.2 kb) was
constructed from four partial cDNA clones (7a, 7u, 4a0, and 2a0)
obtained from Dr. B. Trüeb (Zurich, Switzerland). The clones were
digested with XbaI and AccI (7a), AccI and
EcoRI (7u), EcoRI (4a0), or EcoRI and
BglII (2a0) and were ligated in the M13mp19 vector. An
XbaI-NheI full fragment was then excised from M13 and
inserted into the pSV23 plasmid. The construction of 3(VI) cDNA
(8.1 kb) required the use of four cDNA clones that were ligated two by
two (pB118 with pB4 and pB32 with pB72) in the M13 mp 18 vector. The
two constructs were then excised from M13 and ligated into pSV23
digested with SmaI. The pB118 cDNA clone used for this
construction lacked the modules A8 and A6, which are coded by
individual exons that can undergo alternative splicing (14). A
schematic drawing of these steps is shown in Fig. 1.
Figure 1:
Schematic diagram of the pSV-based
vectors. The construction of chicken 1(VI), 2(VI), and
3(VI) cDNA is shown. The cDNA clones used for the construction
were described previously (9, 10, 14, 24). Polylinker restriction sites
are within parentheses. A, AccI; B,
BamHI; Bg, BglII; C, ClaI;
E, EcoRI; H, HindIII, K,
KpnI; N, NheI; P, PstI;
S, SalI; Sm, SmaI; X,
XbaI. P, t, and pA indicate the
SV40 early promoter, the small-t intron, and the polyadenylation site,
respectively.
Cell Culture, DNA Transfection, and Cell Line Selection
and Identification
NIH/3T3 cells were grown in Dulbecco's
modified minimum essential medium supplemented with 10% fetal calf
serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 100 µg/ml gentamycin. DNA transfections were
performed using the calcium phosphate coprecipitation procedure of
Graham and van der Eb
(25) as modified by Chen and
Okayama
(26) , and DNA was purified by double sedimentation in a
cesium chloride gradient. In a typical experiment, 6 10
cells plated 24 h earlier in a 60-mm Petri dish were
cotransfected for 20 h at 37 °C with 1.5 µg each of supercoiled
DNA of pSV23-1(VI) and -2(VI) and 3 µg of supercoiled DNA
of pSV23-3(VI) plus 40 ng of a plasmid containing the neo gene (pWLneo, Stratagene, La Jolla, CA). The cells were maintained
overnight in Dulbecco's modified minimum essential medium
supplemented with 10% fetal calf serum in a 3% CO
atmosphere, and the following day the medium was changed and the
CO concentration was raised to 5%. Selection was carried
out in 500 ng/ml of G418 (Sigma), and individual colonies were isolated
and grown into separate cell lines.
Northern and Southern Blotting
High molecular
weight DNA was isolated from transfected cell lines following
established procedures
(27) , and 3 µg were digested to
completion with restriction enzymes. The fragments were resolved on a
0.7% (w/v) agarose gel, transferred onto Hybond nylon membrane
(Amersham International, Buckinghamshire, UK), and hybridized to cDNA
probes of the 1(VI), 2(VI), and 3(VI) chains labeled
with [-P]dCTP (Amersham) by the random
primed oligolabeling method
(28) .
-P]dCTP-labeled cDNA
probes specific for the different type VI collagen chains.
NaCl/phosphate/EDTA (180 mM NaCl, 10 mM phosphate, 1
mM EDTA, pH 7.7) (buffer A) containing 5
Denhardt's solution, 0.5% (w/v) SDS, and 100 µg/ml salmon
sperm DNA. After washing in 2
NaCl/phosphate/EDTA for 5 min at
room temperature, in 1
buffer A plus 1% SDS for 30 min at 65
°C, and in 0.1
NaCl/phosphate/EDTA for 30 min at room
temperature, the filters were exposed to
-max Hyperfilms
(Amersham).
Metabolic Labeling and Immunoprecipitation
Cells
were metabolically labeled in methionine-free Dulbecco's modified
minimum essential medium containing 1% dialyzed fetal calf serum.
Labeling with [S]methionine (800 Ci/mmol,
Amersham) at 100-300 µCi/ml was carried out in the presence
or the absence of 50 µg/ml ascorbate. After 3-4 h the cell
layer was solubilized for 30 min at 0 °C in extraction buffer with
the following final concentrations: 20 mM Tris-HCl, pH 7.6,
100 mM NaCl, 1% (w/v) Nonidet P-40, 0.5% (w/v) sodium
deoxycholate, 0.1% (w/v) SDS, 25 mM EDTA, 2 mM
phenylmethylsulfonyl fluoride, 5 mMN-ethylmaleimide,
and 1 mMpara-aminobenzamidine. When needed, the
medium was brought to extraction buffer. The medium and the cell lysate
were clarified by centrifugation at 10,000
g and used
for immunoprecipitation. The samples were first precleared by
incubation with 2 µl of antifibronectin mAb (HB91) followed by
incubation with 100 µl protein A-Sepharose (Pharmacia Biotech Inc.,
Uppsala, Sweden) for 1 h at 0 °C. After centrifugation for 15 min
at 3,000
g, 2 µl of AS-5 or 100 µl of mAbs
against the chicken
3(VI) chain and 50 µl of protein
A-Sepharose were added to the supernatant, and the incubation proceeded
for 4-6 h. The samples were centrifuged, and the precipitates
were washed extensively in extraction buffer prior to dissolving in
Laemmli sample buffer in the presence or in the absence of 5% (v/v)
2--mercaptoethanol
(30) .
Pepsin Digestion and
Cultures of D23/3 cells in Dulbecco's
modified minimum essential medium containing fetal calf serum and
supplemented for 2-3 days with ascorbate (50 µg/ml) were
washed with serum-free medium and then maintained overnight in
serum-free medium. The culture medium was collected, dialyzed against
PBS, and labeled with I
Labeling
I by the chloramine-T method.
Labeled proteins were separated from free iodine by gel filtration and
brought up into 0.5 M acetic acid for treatment with 100
µg/ml pepsin for 3 h at 4 °C. The sample was dialyzed
extensively against PBS, and aliquots were immunoprecipitated with
AS-5, AS-46, and AS-72. Immunoprecipitated material was resolved by
SDS-PAGE on 8% (v/v) polyacrylamide slab gels and exposed to MP
Hyperfilms.
Matrix Deposition
Matrix deposition of transfected
chicken type VI collagen polypeptides was analyzed by indirect
immunofluorescence. NIH/3T3 cell clones expressing the three chicken
type VI collagen chains were cultured on chamber slides (A/S Nunc,
Roskilde, Denmark) until almost confluent, washed with PBS, and fixed
for 30 min at room temperature with iced 4% paraformaldehyde in PBS.
Slides were incubated for 60 min at 4 °C with primary antibodies
diluted in PBS containing 1% BSA in a moist chamber. Subsequent
incubations were with rabbit anti-mouse or goat anti-rabbit
fluorescein- or rhodamine-conjugated antibodies (A/S Dako, Glastrup,
Denmark). Stained slides were mounted in glycerol and examined with an
epifluorescent Leitz microscope using a 25 phase/fluorescence
objective. Slides were photographed with Ilford HP5 400 (Ilford, Ltd.,
Mobberley Cheshire, UK).
Establishment of NIH/3T3 Cell Lines That Coexpress the
Three Chains of Chicken Type VI Collagen
NIH/3T3 cells were
cotransfected with the cDNAs coding for chicken type VI collagen chains
along with the neo gene. Cells were put into selective G418
medium, and, after 14 days, 10 colonies were isolated and 6 grew into
individual stable cell lines. The expression of chicken type VI
collagen chains was investigated by immunostaining permeabilized cells
with antibodies specific for the individual chicken type VI collagen
chains. This screening (data not shown) suggested that three cell lines
(D10/1, D23/3, and D24/1) coexpressed all transfected chains, two cell
lines (D23/2 and D28/1) coexpressed only the 2(VI) and 3(VI)
chains, and one cell line (D26/1) coexpressed the 1(VI) and
3(VI) chains (data not shown). Next, the six cell lines were
metabolically labeled with [S]methionine, and
the cell lysates were immunoprecipitated with AS-5 (Fig. 2). No
material was immunoprecipitated from normal NIH/3T3 cells, confirming
that the antibody was specific for chicken type VI collagen and did not
recognize murine type VI collagen. The immunoprecipitates from the six
transfected cell lines contained several bands of different intensity
and migration. Five cell lines showed bands, prominent in D10/1, D23/2,
and D26/1, at about 240 kDa, consistent with the size expected for the
transfected chicken
3(VI) cDNA. In the D28/1 cell line, the
240-kDa band was absent, whereas a band migrating at about 220 kDa was
immunoprecipitated. A band of similar size also was detected in D24/1
cells. Lower bands migrating in the range of 130-140 kDa as
expected for 1(VI) and/or 2(VI) chains were present in all
six cell lines.
Figure 2:
Identification of polypeptides
immunoprecipitated from cell lines cotransfected with chicken
1(VI), 2(VI), and 3(VI) cDNAs. Cells were metabolically
labeled with 100 µCi/ml [S]methionine for 3
h. Cells were collected, and cell lysates were immunoprecipitated with
AS-5 polyclonal antiserum to chicken type VI collagen and resolved in a
6% polyacrylamide gel under reducing conditions. On the right is the migration of the transfected chicken type VI collagen
chains, and on the left the migration of molecular weight
markers is indicated. In this experiment the preabsorption step with
antifibronectin antibody was not exhaustive, and, consequently, some
fibronectin contaminated the
immunoprecipitates.
Two cell lines (D23/3 and D24/1) coexpressing all
three transfected cDNAs were successfully subcloned, and their DNA and
RNA were isolated. To demonstrate the presence of each of the
integrated cDNAs, high molecular weight DNA was digested and analyzed
by Southern blotting. Under the stringent conditions used, no
hybridization with endogenous murine type VI collagen genes was
detected. On the other hand, both cell lines incorporated several
copies of all three chicken cDNAs (Fig. 3).
Figure 3:
Southern blot analysis of D23/3- and
D24/1-transfected cell lines. Each lane was loaded with 10
µg of high molecular weight DNA digested with BclI.
Individual strips were hybridized to
[-P]dCTP-labeled cDNA of full-length
chicken
1(VI) cDNA, 2(VI) cDNA, and clone pB10(3) (11).
On the left the migration of DNA markers is
indicated.
Because the sizes
of the bands were ambiguous in the first series of immunoprecipitation,
Northern blot analysis was carried out to control for the sizes of the
transcripts (Fig. 4). Both subcloned D23/3 and D24/1 cell lines
showed 1(VI) and 2(VI) transcripts of the proper size (about
3.2 kb). The D23/3 cell line had a properly sized 3(VI) chain
transcript of about 8.0 kb, whereas in the D24/1 cell line the
3(VI) transcript was of slightly smaller size (about 7.0 kb). In
addition, hybridization with the 3(VI)-specific probe revealed in
both cell lines a prominent band at about 5.4 kb and, in D23/3 cells,
also a minor signal at about 3.0 kb. It is possible that these smaller
RNAs are transcribed from incorrectly integrated cDNAs or that they are
the result of aberrant splicing.
Figure 4:
Northern blot analysis of D23/3- and
D24/1-transfected cell lines. Each lane was loaded with 5
µg of total RNA. Individual strips were hybridized to
[-P]dCTP-labeled full-length chicken
1(VI) cDNA, 2(VI) cDNA, and clone pB10(3) (11). On the
left the migration of RNA markers is
indicated.
Cell Lines Constitutively Expressing
``Variant'' Chicken
Immunoprecipitation and Northern blot analyses revealed
variant 3(VI)
Chains3(VI) mRNAs and polypeptides in some cell lines. To
further investigate these aberrantly sized chicken 3(VI) chains,
the D28/1, D23/3, and D24/1 cell lines were metabolically labeled, and
the cell lysates were immunoprecipitated with mAbs specific for
different domains of the chicken 3(VI) chain (Fig. 5). In
the D28/1 cell line, AS-5 immunoprecipitated two major bands, one at
about 220 kDa and one at 130 kDa (Fig. 5, lane 2, and
also see Fig. 2, lane 7). Accordingly, the D28/1 cells
expressed a correct 2(VI) mRNA (about 3.2 kb) and an aberrant
3(VI) mRNA of about 7.0 kb (not shown). In addition, the chicken
3(VI)-specific mAbs 192C2 and 116A8 immunoprecipitated
the 220 kDa band nearly exclusively (Fig. 5, lanes 5 and
7), whereas neither mAb 111A3 nor 76G11 immunoprecipitated any
polypeptide from D28/1 cells, suggesting that their epitopes are not
present or not exposed in this smaller 3 (VI) polypeptide.
Instead, a polypeptide of about 220 kDa was recognized by mAb 111A3 in
D24/1 cells (Fig. 5, lane 9, and also see Fig. 2,
lane 6), suggesting that this 3(VI) form differs from the
similarly sized 220-kDa 3(VI) polypeptide of D28/1 cells. mAb
111A3 immunoprecipitated from D23/3 cells a polypeptide at about 240
kDa, which is the size expected for the correct 3(VI) transfected
cDNA (Fig. 5, lane 8, and also see Fig. 2,
lane 4). In this experiment a longer 6% gel was run, and the
chicken and murine 1(VI) and 2(VI) chains could be resolved.
In this gel it was evident that both chicken and murine bands were
coimmunoprecipitated in D23/3 and D24/1 cells. In addition, mAb 111A3
was able to immunoprecipitate from both D23/3 and D24/1 cell lines a
heavily labeled polypeptide of about 160 kDa that migrated slightly
above the 1(VI) and 2(VI) chains (Fig. 5, lanes 8 and 9). A similarly sized polypeptide was
immunoprecipitated by mAbs 111A3 and 76G11 also from a cell line
(C11B7) transfected only with the 3(VI) cDNA (Fig. 5,
lanes 10 and 12)
(21) . This set of
immunoprecipitations indicates that there is a certain degree of
heterogeneity in the nature of the 3(VI) chains produced by
different cell lines.
Figure 5:
Polypeptides immunoprecipitated from
transfected cell lines by chicken 3(VI) chain-specific monoclonal
antibodies. D28/1, D23/3, D24/1, and C11B7 (24) cells were
metabolically labeled for 3 h with 100 µCi of
[S]methionine, and cell lysates were
immunoprecipitated with mAbs to chicken
3(VI) collagen chain
(111A3, 192C2, 76G11, and 116A8), with preimmune serum (NRS,
lane 3), or with polyclonal antiserum against chicken type VI
collagen (AS-5, lane 2) and resolved in a 6% (w/v)
polyacrylamide gel under reducing conditions. The arrowheads indicate the migration of the transfected chicken type VI collagen
chains, and the dots indicate the migration of the 160-kDa
truncated 3(VI) form. On the left the migration of
molecular weight markers is shown. Anti-FN, mAb HB91 against
fibronectin.
Assembly, Secretion, and Matrix Deposition of Transfected
Chicken Type VI Collagen
Type VI collagen chains usually form
stable (1(VI), 2(VI), and 3(VI)) monomers that assemble
intracellularly to give rise to S-S bonded dimers (6 chains) and
tetramers (12 chains). To study the assembly of the transfected chicken
chains, we first assessed that extensive and repeated
immunoprecipitations depleted all of the murine chains from D23/3 cell
lysates, leaving behind polypeptides that could be specifically
immunoprecipitated with AS-5 (Fig. 6A, lane 2)
but not with AS-72 (Fig. 6A, lane 3). D23/3
cells were selected for these experiments because they synthesize type
VI chains of the correct size. Next, in a similar type of experiment we
evaluated the extent of assembly of chicken type VI collagen chains in
cell lysates. Aliquots of the immunoprecipitate were run in SDS-PAGE in
the absence of 2--mercaptoethanol, and, under these conditions,
most of the material consisted of larger forms that barely entered the
6% separating gel (Fig. 6B, lane 5).
Figure 6:
Assembly and secretion of transfected type
VI collagen. A, preclearing with AS-72. Cell lysates of
metabolically labeled D23/3 were subjected to five cycles of
immunoprecipitations with AS-72. After these extensive preclearing
steps, the supernatant was aliquoted and immunoprecipitated with
preimmune serum (NRS, lane 1), AS-5 (lane
2), or AS-72 (lane 3). B, assembly. Aliquots of
the cell lysate immunoprecipitated with AS-5 as in lane 2 and
were resolved under reducing (lane 4) or nonreducing (lane
5) conditions. A dotted line indicates the top of the
separating gel. The migration of the transfected chicken chains and of
molecular weight markers is shown. C, secretion. Cell medium
of D23/3 cells metabolically labeled for 4 h with 300 µCi of
[S]methionine in the presence of 50 µg/ml
ascorbic acid was precleared with one immunoprecipitation cycle of
AS-72. The supernatant was immunoprecipitated with AS-5 (lanes 6 and 9), followed by AS-72 (lane 7). An aliquot
of cell medium was immunoprecipitated with HB91 antifibronectin mAb
(lane 8), and the migration of fibronectin served as a marker
in the nonreduced gel. The immunoprecipitates were analyzed in a
3-10% polyacrylamide linear gradient without stacking gel under
reducing or nonreducing conditions. A dotted line indicates
the top of the gel. FN-M and FN-D indicate the
migration of monomers and dimers of fibronectin, respectively.
VI-M, VI-D, and VI-T indicate the migration
of type VI collagen monomers, dimers, and tetramers, respectively.
1(VI)c, 2(V)c, 3(VI)c,
1(VI)m, 2(VI)m, and 3(VI)m indicate the migration of the chicken (c) and murine
(m) chains of type VI collagen.
Secretion of chicken type VI collagen assemblies was studied in
D23/3 cells labeled in the presence of ascorbate. The cell medium was
immunoprecipitated with AS-5, and the immunoprecipitate was run under
reducing and nonreducing conditions in a 3-10% linear gradient
SDS-PAGE without stacking gel in order to resolve also the larger
assembled composites (Fig. 6C). In the nonreduced gel,
the major polypeptide of 240 kDa corresponding to the chicken
3(VI) chain and other bands in the range of 130-140 kDa were
strongly decreased. Instead, a larger form (about 500 kDa) slightly
above the fibronectin dimer and more heavily labeled higher bands were
detected. The sizes of these disulfide-bonded aggregates are similar to
the sizes of properly assembled type VI monomers (about 500 kDa),
dimers (about 1,000 kDa), and tetramers (about 2,000 kDa). To avoid the
extensive immunodepletion of the murine type VI collagen that could
eliminate coassociated chicken type VI collagen forming the higher
order structures present in the cell medium, the preclearing steps with
AS-72 were not very extensive. As a consequence, the pattern of chains
immunoprecipitated from the cell medium was more complex than the
pattern of chains immunoprecipitated from the cell lysate (see
Fig. 6B), especially in the range of 130-140 kDa.
In this case, when the supernatant from the first immunoprecipitation
with AS-5 was subjected to a second immunoprecipitation with AS-72,
some murine type VI collagen could still be revealed
(Fig. 6C, lane 7).
S]methionine-labeled D23/3 cells prevented
the elucidation of this matter, because no polypeptides corresponding
to the pepsin form of type VI collagen could be detected. Therefore,
serum-free medium from ascorbate-treated D23/3 cells was collected,
labeled with
I, brought to 0.5 M acetic acid,
and digested with pepsin. This analysis was facilitated by the notion
that chicken and murine
1(VI) pepsinized chains migrate at
distinct positions in SDS-PAGE
(18) . As shown in Fig. 7,
not only AS-5 (lane 2), which recognizes all three chicken
chains, but also AS-46 (lane 4), which is specific for only
the chicken 1(VI) chain, immunoprecipitated three pepsin-resistant
fragments. With both antisera, the mobility of the 1(VI) band and
also of the 3 (VI) band was slower compared with the mobility of
the bands detected when the sample was immunoprecipitated with AS-72
(Fig. 7, lane 3). Although a low contamination of murine
chains also was present in lanes 2 and 4 (Fig. 7), these results demonstrate that recombinant chicken
type VI collagen can form stable triple helices.
Figure 7:
Polypeptides immunoprecipitated from
pepsin-digested D23/3-secreted material. Type VI collagen was
immunoprecipitated with normal rabbit serum (NRS, lane
1), AS-5 (lane 2), AS-72 (lane 3), or AS-46
(lane 4). On the left the migration of molecular
weight markers is indicated.
The ability of
D23/3 cells to incorporate the secreted recombinant chicken type VI
collagen into their ECM was investigated by staining D23/3 cells with
different combinations of polyclonal antisera and mAbs that react with
chicken or murine type VI collagen and with fibronectin. The results
are shown in Fig. 8. Cells incubated with preimmune serum or an
unrelated mAb showed only background fluorescence (not shown). Also
negative were untransfected NIH/3T3 cells incubated with AS-5
(Fig. 8b). In contrast, strong immunofluorescence in a
fibrillar pattern was detected when D23/3 cells were incubated with the
AS-5 or with antibodies specific for each of the transfected chicken
chains (Fig. 8, c, d, e, and
g). This staining pattern was superimposable with the pattern
of endogenous murine type VI collagen (Fig. 8, f and
h). The incorporation of heterologous chicken type VI collagen
into the ECM of NIH/3T3 cells indicates that transfected chicken type
VI collagen assemblies are competent for matrix formation.
Figure 8:
Immunofluorescence staining of D23/3
cells. Cells were cultured in medium containing ascorbic acid for 4
days and then fixed and stained with single antibodies or combinations
(e and f or g and h) of various
antibodies. NIH/3T3 cells were stained with AS-5 polyclonal antiserum
specific for the chicken type VI collagen (b). D23 cells were
stained with AS-72 anti murine type VI collagen (a and
h), mAb 111A3 anti chicken 3(VI) chain (c and
g), or AS-47 anti chicken 2(VI) chains (d).
Double immunofluorescence was with mAb 108F3 anti chicken 1(VI)
chain (e) or AS-72 (f) and with mAb 111A3
(g) or AS-72 (h). Fluorescein-conjugated anti-mouse
Ig and rhodamine-conjugated anti-rabbit Ig were used as secondary
reagents.
1(VI), 2(VI), and 3(VI) collagen chains and show that
these three exogenous polypeptides can be constitutively expressed in
murine NIH/3T3 cells in the forms of properly folded pepsin-resistant
type VI collagen monomers and higher order disulfide-bonded multimers.
Cotransfection with a high ratio of the chicken type VI cDNAs
versus the neo selectable marker gene (about 30:1)
resulted in the efficient and stable introduction of all cDNAs; three
out of six selected cell lines expressed all three chains of chicken
type VI collagen. Furthermore, in at least one cell line, heterologous
recombinant chicken type VI collagen was secreted and incorporated into
the ECM. The immunofluorescence pattern obtained with antibodies
specific for the chicken and murine type VI collagen was superimposable
and suggests that molecules made only of chicken or murine chains or
chimeric chicken/murine type VI collagen might coassemble and be
deposited.
and it is likely that chicken
molecules coassemble with secreted murine molecules in larger
assemblies and are more easily removed by the preclearing steps. To
analyze secreted molecules it was necessary to compromise between
detecting a discrete but only partially purified chicken type VI
collagen signal and no signal at all. However, when a more sensitive
I labeling of pepsin-resistant secreted material was
used, polypeptides largely corresponding to chicken type VI collagen
were specifically immunoprecipitated. The resistance to pepsin
digestion demonstrated the triple helical conformation of recombinant
chicken type VI molecules. Antibodies specific for the
1(VI)
chicken chain were able to immunoprecipitate composites including
mainly chicken 2(VI) and 3(VI) chains, indicating minimal
interference from endogenous murine type VI polypeptides in the
formation of chicken type VI monomers. Because the acetic acid
treatment necessary for pepsin digestion disrupts all of the
noncovalent bonds, nearly pure chicken macromolecular assemblies were
immunoprecipitated. On the other hand, chimeric assemblies were more
easily detected in immunoprecipitates made with molecules kept under
native conditions. Therefore, the contribution of some chimeric
murine/chicken molecules (i.e. murine 1(VI) and
2(VI) and chicken 3(VI)) also should be considered in
interpreting the immunodetection of collagen type VI in the deposited
ECM. However, the presence of chimeric assemblies will not undermine
the conclusion that at least a fraction of deposited type VI collagen
is solely of chicken origin.
3(VI) mRNA signal and the 240-kDa
3(VI) polypeptide band, shorter messages in the range of
5.4-7 kb and polypeptides in the range of 160-220 kDa,
which are present together with or instead of the properly sized
molecules, are detected. The 5.4-kb mRNA band has been detected in most
lines, and, because of its frequency, it seems unlikely that this
shorter mRNA is transcribed from incorrectly integrated cDNAs.
Alternatively, the 5.4-kb transcripts and the 160-kDa polypeptides
might be due to aberrant splicing from cryptic splice donor sites of
the 3(VI) cDNA, as has been reported in other experimental
systems
(36, 37, 38, 39) . The inability
of some antibodies to immunoprecipitate the 160-kDa truncated
3(VI) polypeptide could depend on the localization of the
recognized epitopes that are localized in the triple helix (AS-5) or in
the C terminus (mAbs 116A8 and 192C2). This suggests that the truncated
160-kDa polypeptide is comprised of the N-terminal globular end of the
3(VI) chain. The 160-kDa polypeptide does not associate with
1(VI) and 2(VI) chains, and, therefore, it is
likely that this smaller 3(VI) form lacks some sequences/domains
that are involved in the association with the other two chains. It is
noteworthy that a recombinant fragment of the human 3(VI) chain
that includes all of the N-terminal globule and that corresponds to a
large extent to the present 160 kDa polypeptide displayed several
functional interactions, including binding sites for heparin, for
hyaluronan, and also for the triple helical domain of type VI
collagen
(40) .
2(VI) and Dr. Danny Huylebroeck for providing the pSV23 vector.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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