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J. Biol. Chem., Vol. 276, Issue 26, 24352-24359, June 29, 2001
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From the Institut de Biologie et Chimie des Protéines, Unite
Mixte de Recherche, Centre National de la Recherche Scientifique 5086, Université Claude Bernard Lyon I,
69367 Lyon Cedex 07, France
Received for publication, February 27, 2001, and in revised form, April 11, 2001
Although the collagen V heterotrimer is known to
be involved in the control of fibril assembly, the role of the
homotrimer in fibrillar organization has not yet been examined. Here,
the production of substantial amounts of recombinant collagen V
homotrimer has allowed a detailed study of its role in homotypic and
heterotypic fibril formation. After removal of terminal regions by
pepsin digestion, both the collagen V heterotrimer and homotrimer
formed thin homotypic fibrils, thus showing that diameter limitation is
at least in part an intrinsic property of the collagen V triple helix.
When mixed with collagen I, however, various complementary approaches
indicated that the collagen V heterotrimer and homotrimer exerted
different effects in heterotypic fibril formation. Unlike the
heterotrimer, which was buried in the fibril interior, the homotrimer
was localized as thin filamentous structures at the surface of wide
collagen I fibrils and did not regulate fibril assembly. Its
localization at the fibril surface suggests that the homotrimer can act
as a molecular linker between collagen fibrils or macromolecules in the
extracellular matrix or both. Thus, depending on their respective
distribution in tissues, the different collagen V isoforms might
fulfill specific biological functions.
Fibrillar collagens, namely types I, II, III, V, and XI, are found
in essentially all connective tissues of most multicellular organisms,
being particularly abundant in bone, cartilage, and skin (1). One of
their prominent functions is to maintain the architecture of tissues
and organs and to confer mechanical strength. This is mainly achieved
through interactions with other extracellular components such as
proteoglycans but also through interactions between collagen molecules
themselves. Indeed, collagen I, the most abundant and well known
collagen, forms fibrils mostly in association with collagen V, a
quantitatively minor collagen, which occupies the inner part of these
fibrils, being almost buried by collagen I (2). Interestingly, this
association between collagens I and V leads to heterotypic fibrils with
controlled diameters, which is of considerable importance in
influencing the functional characteristics of a given tissue. For
example, many studies point to a role for collagen V in the control of collagen fibril diameter in the cornea, which contributes to corneal transparency (3-5).
Three different parameters acting either concomitantly or independently
could account for diameter control by collagen V. First, the amount of
collagen V in tissues is an unquestionable factor. Heterotypic fibril
diameters are greater when collagen V synthesis is prevented, as has
been shown in cellulo using chicken corneal fibroblasts (6).
Second, collagen V, like collagen XI, is unusual in retaining large
parts of its N-terminal procollagen domain during extracellular
processing, unlike other fibrillar collagens, which retain only an
~20-residue N-telopeptide. As a result of its size and flexibility,
the remaining N-terminal part of the mature collagen V molecule could
sterically prevent the growth of heterotypic fibrils (4, 5). For
exemple, transgenic mice lacking the exon encoding the Almost all if not all biochemical and functional studies have
been carried out on the most abundant molecular form found in tissues, the heterotrimer [ Because the collagen V triple helix in itself can modulate heterotypic
fibril growth, and the precise N-terminal cleavage sites of the
different collagen V stoichiometries are still controversial (5, 13,
18, 19), the pepsinized forms of the different molecules were used.
Using a variety of complementary approaches, we show that, unlike the
collagen V heterotrimer, homotrimeric collagen V does not modulate
collagen I fibril formation kinetics and does not decrease fibril
diameter. Furthermore, although homotrimeric collagen V interacts with
collagen I, it is found not within the core but rather at the surface
of the collagen I fibrils. These differences in the behavior of the
collagen V homotrimer and heterotrimer with respect to collagen I give
insights into their possible roles in vivo.
Collagen Purification
Collagen I and V heterotrimers were extracted from embryonic
calf bones by pepsin digestion in 0.5 M acetic acid and 0.2 M NaCl at 4 °C for 20 h. They were then purified by
repeated salt fractionation in acetic acid as previously described (20,
21). Purified collagens were analyzed by 6% SDS-polyacrylamide gel electrophoresis (SDS-PAGE)1
and stained with Coomassie blue, using human placental collagen V
(Sigma) as standard. Collagens were dialyzed against 0.1 M
acetic acid and then lyophilized and stored at Production and purification of the recombinant collagen V homotrimer
were performed as previously described (14). Briefly, 293-EBNA cells
were transfected by electroporation with an expression vector
containing full-length cDNA encoding the human pro Reconstitution of Collagen V Fibrils
The capacity of the homotrimer to form homotypic fibrils was
assessed by dialyzing the pepsinized preparation against phosphate buffers at different pH values and ionic strengths. With this aim, the
collagen V homotrimer (HomV), the collagen V heterotrimer (HetV), and
the collagen I heterotrimer, as controls, were diluted in 0.1 M acetic acid at 400 µg/ml and dialyzed at 4 °C
overnight against the following buffers: 1) phosphate buffered saline,
pH 7.4 (PBS; 8 mM Na2HPO4, 0.15 mM K2HPO4, 2.6 mM KCl,
0.137 M NaCl); 2) 20 mM
K2HPO4, pH 7.4; 3) 20 mM
K2HPO4 and 140 mM NaCl, pH 7.4; and
4) 20 mM K2HPO4 and 140 mM NaCl, pH 9. Fibril formation was initiated by increasing
the incubation temperature to 34 °C. Collagen aggregates were
negatively stained and analyzed by electron microscopy as described below.
In Vitro Fibrillogenesis Kinetics
Stock solutions of collagens I and V (HetV and HomV) diluted to
400 µg/ml in 0.1 M acetic acid were centrifuged at
5000 × g for 10 min to remove any large molecular
aggregates, and then supernatants were kept at 4 °C until use. For
the mixing experiments, the collagen I concentration was held constant
(200 µg/ml), whereas the collagen V concentration (HomV and HetV) was
varied from 22 µg/ml (10% of total collagen concentration) to 200 µg/ml (50% of total collagen concentration). Samples were then
dialyzed against PBS, pH 7.2, at 4 °C overnight and then transferred
to thermostatted quartz microcuvettes. Cuvettes were placed in a
spectrophotometer (DU 640; Beckman Instruments) and connected to a
water bath at 34 °C, and absorbance at 315 nm was recorded.
Each experiment was repeated at least three times.
Analysis of Fibril Composition by Cosedimentation
After fibrillogenesis experiments, samples were centrifuged at
low speed (5000 × g, 15 min, 4 °C) to pellet
fibrils. Supernatants and pellets were analyzed by 6% SDS-PAGE
followed by Coomassie blue staining. The presence of collagens I and V
in the pellets was interpreted as cosedimentation of collagen V with
collagen I fibrils.
Light Scattering
Dynamic light scattering experiments were carried out with a
Malvern 4700 spectrometer (Malvern Instruments) using a Siemens He-Ne
laser (wavelength, 633 nm; power, 40 mW) and a 256-channel correlator.
Solutions of collagen V were analyzed in 0.5 M acetic acid,
in the concentration range of 0.2-0.5 mg/ml, after centrifugation at
4 °C for 30 min at 5000 × g. Further measurements
were made on the same samples after overnight dialysis at 4 °C
against PBS, without additional centrifugation. All measurements were
made in the temperature range of 12-15 °C and in the angular range of 30-120°. For a mixed population, the relative contributions from
the different-size subpopulations vary with scattering angle. To
facilitate comparison, data presented here are for a scattering angle
of 90°. Population distributions were fitted to the experimental correlation curves using the Contin program provided by the
manufacturers. In general, slowly diffusing species (small values of
the diffusion coefficient (D20,w))
correspond to high-molecular weight aggregates.
Electron Microscopy
Negative Staining and Fibril Diameter Measurement--
After
fibril formation, drops of the different samples were placed onto
Formvar-carbon grids, rinsed with distilled water, and then negatively
stained with 2% phosphotungstic acid, pH 7.4. Stained aggregates were
examined using a Philips CM 120 transmission electron microscope at the
Centre de Microscopie Appliqué à la Biologie et à la
Géologie (Université Claude Bernard, Villeurbanne, France).
For each sample, >80 fibril diameters were measured directly from
calibrated electron micrographs, and values were plotted as histograms.
Immunogold Labeling--
Rabbit polyclonal antibodies against
human pepsinized collagens I, II, and V used for this study were
purchased from Novotec. All these antibodies have previously been
characterized (22) and have been shown to recognize collagen
type-specific helical epitopes.
After fibrillogenesis, fibrils were centrifuged at 6000 × g for 15 min, and labeling experiments were carried out on
the pellet after homogenization in 600 µl of PBS. Drops of fibril
suspension were then transferred to Formvar-carbon-coated grids and
fixed in 4% paraformaldehyde in PBS for 15 min. When indicated,
samples were treated with 0.1 M acetic acid for 20 min to
partially disrupt the fibrillar structure and thus to facilitate
immunodetection of buried collagens. All samples were then quenched in
sodium borohydride (500 µg/ml) for 60 min to reduce any free
aldehydes, washed several times in PBS, and incubated with 1% bovine
serum albumin in PBS. Grids were incubated with specific antibodies for
2 h at room temperature, followed by a 1 h incubation with 10-nm
colloidal gold particles coated with goat anti-rabbit IgG (British Bio
Cell International). Collagen aggregates were then stained and viewed
as described above.
Solid-phase Binding Assay
Collagen I and V stock solutions (2 mg/ml) were diluted to 500 µg/ml and dialyzed against PBS. A 20-fold molar excess of
N-hydroxysuccinimido-biotin (Sigma) was incubated with the
different collagen samples for 3 h at room temperature.
Biotinylation was then stopped by adding a 50 mM final
concentration of Tris-HCl, pH 7.4. Samples were then dialyzed against
PBS to remove free N-hydroxysuccinimido-biotin.
Collagen I (0.25 µg/well) was coated onto microtiter plates (96 wells; Greiner) in 50 mM Tris-HCl and 150 mM
NaCl, pH 7.5, overnight at 4 °C. The wells were then blocked with
1% bovine serum albumin in PBS for 1 h. Serial concentrations of
biotinylated collagen V homotrimer and heterotrimer (5-40 µg/ml)
diluted in PBS containing 1% bovine serum albumin and 0.04% Tween 20 were then incubated with immobilized collagen I for 3 h at room
temperature. For inhibition binding assays, collagen V (HetV or HomV)
at 7.5 µg/ml was preincubated for 2 h at room temperature with
soluble collagen I at different concentrations (25-500 µg/ml) before
addition to collagen I-coated wells. Bound ligands were detected with
extravidin-peroxidase conjugate (Sigma) using
2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) as the
chromogenic substrate. Absorbance was measured at 405 nm.
Thrombin Digestion
To determine whether collagen V (HetV and HomV) is buried within
the reconstituted fibrils and is thus protected from proteolytic digestion, samples from fibrillogenesis experiments were submitted to
thrombin digestion. This neutral protease has been shown to cleave
specifically both collagen V chains at 37 °C but not collagen I
chains (23). After in vitro fibrillogenesis experiments,
collagen alone and mixtures (ratio 1:1) were centrifuged at 5000 × g for 10 min at 4 °C to pellet fibrils. Pellets, or
supernatants when indicated, were subsequently submitted to thrombin
digestion (human thrombin; Roche Molecular Biochemicals). In
preliminary experiments, different conditions of thrombin digestion
were assayed to obtain complete digestion of reconstituted collagen V. Incubation for 5 h at 37 °C with a 1:2 enzyme:substrate ratio
was found to be optimal. Thrombin digestion products were analyzed by
6% SDS-PAGE with Coomassie blue staining. The ratio between collagens
I and V was determined by scanning densitometry (Molecular Dynamics).
Purification of Collagens
Collagen I and V heterotrimers were extracted from embryonic calf
bones by pepsin digestion and then isolated by repeated differential
salt precipitation. Both heterotrimeric and homotrimeric forms of
collagen I are present in calf bones, as previously shown (21), but
only the major form of collagen I, the heterotrimer [ The collagen V homotrimer used for fibrillogenesis experiments was
obtained as a recombinant protein. As shown previously (14), the
protein is produced as a trimeric molecule migrating at 250 kDa. This
includes the main triple helix and the complete N-propeptidic domain,
whereas the C-propeptide is rapidly cleaved in the transfected cell
medium. The purified recombinant homotrimer was pepsinized to remove
essentially all but the main triple-helical region of the molecule. The
migration of the pepsinized recombinant Fibril Formation Competence of Collagen V
The capacity of the recombinant homotrimer to form fibrils
in vitro was investigated. In parallel, similar experiments
were carried out with collagen V and I heterotrimers extracted and purified from bovine bones. To optimize fibril formation, several phosphate buffers were tested at different pH values and ionic strengths as indicated under "Materials and Methods." After
dialysis, electron microscopic observations of the samples showed that
the collagen V homotrimer was unable to associate into banded fibrils regardless of which buffer was used. As illustrated with PBS, although
collagen I assembled into large banded fibrils (Fig. 2A), the collagen V
heterotrimer and homotrimer associated into thin fibrils (Fig. 2,
B and C). Fibrils formed from the heterotrimer occasionally showed a visible cross-striation, whereas the homotrimer aggregated only into a homogenous, loosely formed filamentous network.
A broad diameter distribution was observed for collagen I homotypic
fibrils, ranging from 40 to >250 nm, whereas collagen V homotrimer and
heterotrimer fibril diameters exhibited narrow distributions (Fig.
2D).
Control of Heterotypic Fibril Formation by Collagen V Is
Determined by Chain Stoichiometry*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2(V) chain
N-telopeptide present highly disorganized heterotypic fibrils (7). As a
consequence, homozygous animals suffer from spinal deformities, and
most die within 3 weeks of asphyxiation. The third parameter is the
nature of the collagen V triple helix itself. Even when deprived of its N- and C-terminal nonhelical regions, collagen V is not able to form
thick fibrils in vitro under physiological conditions,
indicating that the helical structure of collagen V intrisically plays
a part in preventing fibril growth (4, 8).
1(V)]22(V).
However, two other different molecules exist, composed strictly of
collagen V chains, making the study of the role of collagen V in fibril
assembly more complex. The heterotrimeric form,
1(V)2(V)3(V), only found in placenta (9), remains poorly
studied, although the recent determination of the primary structure of
the human 3(V) chain will facilitate further research (10). The
homotrimeric form [1(V)]3 was first observed in cell
cultures and is likely to be present in embryonic tissues, although in
too small amounts to be purified (11-13). Recently, however, using
recombinant technology, we have reported the large-scale production and
characterization of the collagen V homotrimer (14). Whether the
homotrimer can interact with collagen I, as does the main isotype
[1(V)]22(V), is an intriguing question. Clearly,
collagen V is fundamental not only for fibril diameter regulation but
also for the integrity of connective tissues. This has been suggested
by observations on Ehlers-Danlos syndrome patients bearing
COL5A1 or COL5A2 gene mutations (15-17).
Surprisingly, the tissues affected by the mutations are mainly those
with relatively low amounts of collagen V (skin and tendon).
Considering that different collagen V isoforms are present in tissues,
one possible explanation is that a given minor stoichiometry might
fulfill a specific role in matrix assembly. To examine this
possibility, we decided to undertake a comprehensive comparative study
of in vitro fibril formation by collagen I in the presence
of the heterotrimeric or homotrimeric form of collagen V.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C. For
fibrillogenesis studies, stock solutions of collagens I and V were
solubilized in 0.1 M acetic acid at a concentration of 2 mg/ml.
1(V) chain.
The homotrimer was recovered from transfected cell medium by dialysis
against 50 mM Tris-HCl, pH 8.6. The precipitate containing the recombinant protein was then resuspended in 50 mM
Tris-HCl, pH 7.4, and 150 mM NaCl, dialyzed against 0.5 M acetic acid and 0.2 M NaCl, and then digested
with pepsin. The digestion product was dialyzed against 0.1 M acetic acid and stored at 20 °C. The concentration
of the pepsinized collagen V homotrimer was determined after hydrolysis
under vacuum (6 N HCl, 115 °C, 24 h) on an amino acid analyzer (Beckman Instruments).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(I)]22(I), was used here. The collagen I
heterotrimer and homotrimer precipitated selectively at 0.7 and
0.9 M NaCl, respectively (Fig.
1A, lanes 1 and 2).
As expected, the collagen V heterotrimeric form
[1(V)]22(V), referred to as HetV, was recovered in
the 1.2 M NaCl precipitate (Fig. 1A, lane
3).
View larger version (27K):
[in a new window]
Fig. 1.
SDS-PAGE analysis of purified
collagens I and V. A, collagens were extracted from
bovine bones with pepsin and purified by repeated salt fractionation.
Lane 1, the 0.7 M NaCl precipitate contained the
collagen I heterotrimer [ 1(I)]22(I); lane
2, the 0.9 M NaCl precipitate contained the collagen I
homotrimer [1(I)]3; lane 3, the 1.2 M NaCl precipitate contained the collagen V heterotrimer
[1(V)]22(V). B, the collagen V
homotrimer was produced in 293-EBNA cells transfected with cDNA
coding for the human pro-1(V) chain. Lane 1, transfected
cell medium; lane 2, purified recombinant collagen V
homotrimer; lane 3, pepsinized recombinant collagen V
homotrimer. Migration positions of protein standards are indicated in
kilodaltons. Proteins were separated on 6% acrylamide gels under
reducing conditions and then stained with Coomassie blue.
1(V) chain corresponds to
that of the 1(V) chain from pepsinized human placental collagen V
(Fig. 1B).
View larger version (68K):
[in a new window]
Fig. 2.
Fibril formation competence of collagen
V. A-C, negative staining of collagen fibrils formed
in PBS. A, collagen I; B, collagen V
heterotrimer; C, collagen V homotrimer. Scale
bar, 500 nm. D, distribution of diameters of fibrils
formed by collagen I (gray bars), collagen V heterotrimer
(black bars), and collagen V homotrimer (white
bars).
In Vitro Fibrillogenesis Kinetics
Collagen V Homotrimer and Heterotrimer Alone--
As shown above
(Fig. 2, B and C), both the collagen V
heterotrimer and homotrimer were found to form thin fibrils when
examined after incubation for 3 h at 34 °C. Surprisingly,
neither showed an increase in turbidity during the incubation period
(Fig. 3A). We noticed,
however, that the turbidity was already elevated at the beginning of
the incubation, after overnight dialysis against PBS at 4 °C. This
suggested that assembly into the observed thin fibrils had already
occurred before raising the temperature to 34 °C. To study the state
of aggregation of these collagens both before and after dialysis into
PBS, dynamic light-scattering experiments were carried out. When
examined in 0.5 M acetic acid, dynamic light scattering
from a solution of the collagen V heterotrimer was consistent with a
mixture of mostly (>70%) monomers
(D20,w = 10.6 ± 2.6 × 10
8 cm2/s; Ref. 24) with a small
amount (<30%) in aggregate form
(D20,w = 0.3 ± 0.1 × 108 cm/s), as illustrated in Fig.
4A. In contrast, the collagen
V homotrimer in acetic acid behaved entirely as a mixture of
aggregates, with three distinct subpopulations
(D20,w = 3.3 ± 1.4 × 108, 0.6 ± 0.2 × 108, and 0.3 ± 0.1 × 108 cm/s). After overnight dialysis against
PBS, both forms of collagen V appeared entirely in aggregate form (Fig.
4B). However, the aggregates formed by the heterotrimer
(D20,w mostly = 0.1-0.3 × 108 cm/s) were significantly larger than
those formed by the homotrimer. In the latter, the diffusion constants
of the two more rapidly diffusing subpopulations
(D20,w = 3.6 ± 0.5 × 108 and 0.9 ± 0.2 × 108 cm/s) were similar to those in acetic
acid (Fig. 4A).
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Mixtures of Collagens I and V-- Fibrillogenesis experiments with mixtures of collagen I and V (HetV and HomV) were carried out by increasing the concentration of collagen V (from 10 to 50% of total collagen) at a fixed concentration of collagen I (200 µg/ml). As expected, the fibrillogenesis kinetics showed that collagen I alone rapidly formed fibrils exhibiting a t1/2 value of 48 min (Fig. 3A). Increasing concentrations of the collagen V heterotrimer led to a delay in the lag phase and a gradual decrease of both the rate of fibril growth and the final plateau levels. In contrast, the profiles obtained with increasing concentrations of the collagen V homotrimer were rigorously the same, except for a constant vertical shift, irrespective of the relative percentage of collagen V (from 10 to 50%). As illustrated for the mixture containing 50% collagen V, both the slowing of fibril formation in the presence of the collagen V heterotrimer and the unchanged profile in the presence of the homotrimer were particularly evident (Fig. 3A). The t1/2 values, to 84 and 52 min for mixtures containing the collagen V heterotrimer and homotrimer, respectively, confirmed that these two molecular forms of collagen V had markedly different effects on fibril formation.
It is noteworthy that the initial turbidity of the sample containing 50% collagen V was particularly elevated compared with the others. This can be accounted for because the overall concentration of collagens in this sample was the highest. Indeed, this value corresponds exactly to the sum of the initial turbidities recorded for collagen I and V heterotrimers alone. However, in the presence of 50% collagen V homotrimer, addition of turbidities recorded for collagens I and V alone at the beginning of fibrillogenesis failed to reach any of the values obtained for the different mixtures. A potential immediate interaction between the homotrimer and collagen I could occur during dialysis against PBS, before increasing the temperature to 34 °C and increasing the initial turbidity values. The differences in the initial turbidities also account for the increase in the respective plateau values (Fig. 3A).
Structural Characteristics of Collagen I and V Fibrils
After fibrillogenesis, fibrils generated were examined by electron microscopy after negative staining (Fig. 3B). As expected from the above data, the collagen V homotrimer formed on its own only poorly structured thin fibrils, and heterotrimeric molecules assembled into narrow and uniform fibrils with almost no periodic striation, whereas collagen I formed large, striated fibrils (Fig. 2). Although fibrils formed from mixtures containing the collagen V heterotrimer or homotrimer almost always showed a visible periodic striation, they clearly differed in their width (Fig. 3B). Indeed, measurement of fibril diameters for each sample demonstrated that addition of the heterotrimer decreased drastically the fibril diameter, whereas the homotrimer did not influence fibril width (Fig. 3C). For instance, addition of 50% collagen V heterotrimer to a constant amount of collagen I reduced the fibril mean diameter to approximately one-third (170.1 ± 58.8 nm for collagen I alone versus 44.4 ± 17.1 nm in the presence of HetV). In contrast, the fibril diameter was almost unaffected when 50% collagen V homotrimer was added to collagen I (127.4 ± 38.4 versus 104 ± 36.5 nm in the presence of HomV). This was in excellent agreement with the fibrillogenesis kinetic experiments (Fig. 3A).
Interaction between Collagens I and V
Because the collagen V homotrimer and heterotrimer influenced both
the formation and the final structure of fibrils differently, it is
possible that interactions of these two molecules with collagen I might
also differ considerably. The first point to elucidate was whether the
collagen V homotrimer was capable of interacting with collagen I. A
preliminary indication was provided by analysis of cosedimentation of
collagen V with collagen I after fibril formation. We were helped by
the fact that after low-speed centrifugation (5000 × g, 15 min, 4 °C), in contrast to the heterotrimer (Fig. 5A, lanes 2 and 7),
aggregates of the collagen V homotrimer did not sediment (Fig.
5A, lanes 4 and 9). Interestingly, after
fibrillogenesis in the presence of collagen I, at least some homotrimer
was subsequently found in the pellet (Fig. 5A, lane 5). A
molecular interaction between collagen I and collagen V homotrimers was
a likely explanation for these results.
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To confirm the interaction between those two collagen types, a solid-phase binding assay was carried out. Both collagen V molecular forms showed strong binding to immobilized collagen I (Fig. 5B). Bovine serum albumin was a weak ligand for the heterotrimer and was almost inactive for the homotrimer, attesting to the binding specificity of collagen V to collagen I. Moreover, addition of a 10-fold excess of soluble collagen I caused >40% inhibition of heterotrimer binding. Concerning the collagen V homotrimer, only 20-30% inhibition was reached in the presence of up to a 30-fold excess of soluble collagen I (Fig. 5C). With both collagen V ligands, addition of a 200-fold excess of soluble collagen I resulted in 85% inhibition of binding. These data show that even if the two collagen V forms had different effects on collagen I fibrillogenesis, they were both able to bind to collagen I.
Localization of Collagen V in Reconstituted Hybrid Fibrils
As previously shown (3, 22, 25), the collagen V heterotrimer is incorporated into heterotypic fibrils in a such way that collagen V molecules are buried within the fibril core. We have investigated whether this holds true for the collagen V homotrimer by routine immunogold-labeling electron microscopy and more innovatively using proteolytic digestion by thrombin.
Immunogold Labeling of Fibrils--
Reconstituted collagen fibrils
with mixtures of collagens I and V (HetV and HomV), in equal ratios,
were examined after immunogold labeling with polyclonal antibodies
specific to the helical domains of collagens I, V, and II (as control).
Collagen I/HetV fibrils reacted positively with antibodies against
collagen I (results not shown) but only weakly with antibodies against
collagen V. When fibrils were disrupted by acetic acidic treatment,
however, gold particles were specifically observed at the site of
fibril disruption (Fig. 6, A
and B). This confirms that the collagen V heterotrimer was
masked by collagen I and thus was mostly incorporated into the fibrils.
Interestingly, when the collagen V homotrimer was mixed with collagen
I, homotrimeric collagen V aggregates were readily detected by
collagen V antibodies, indicating that the homotrimer was likely not
buried within the reconstituted banded fibrils. The untreated large
fibrils were labeled with antibodies against collagen V in the form of
sparse gold particle patches either localized directly at the fibril
surface or associated with loosely formed aggregates (Fig. 6,
C and D) and thin fibrils (Fig. 6E).
In addition, gold particles were also present at the junction of fusing
fibrils (Fig. 6, D and F). Likewise, labeled collagen V homotrimer aggregates were often observed connecting two
banded fibrils, which were seen to fuse further along in the preparation (Fig. 6G). Collagen I antibodies strongly
marked banded fibrils but did not react with small aggregates
bound to their surface, indicating that the latter were composed only
of collagen V molecules (Fig. 6H). No positive reaction was
observed with collagen II antibodies (results not shown).
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Thrombin Digestion--
To confirm our immunolabeling results,
fibrils were digested by thrombin (Fig.
7). Thrombin has been shown to digest
collagen V in specific fragments at 37 °C, whereas collagen I is not
sensitive to the enzyme at this temperature (23). Samples containing
equal starting amounts of collagens I and V (HetV and HomV), as well as
individual collagens, were tested. After the kinetic experiments, samples were centrifuged, and fibril-containing pellets were treated with thrombin. Because collagen V homotrimer aggregates did not precipitate under these conditions, the supernatants were, in this
case, digested instead. The results paralleled the immunogold-labeling data. Complete digestion was obtained for homotypic collagen V fibrils,
whereas those of collagen I were unaffected by thrombin digestion.
After thrombin digestion, the collagen I/HetV fibril electrophoretic
pattern showed faint but clearly visible bands corresponding to
collagen V heterotrimer chains (Fig. 7, lane 6). The
undigested collagen V heterotrimer likely corresponded to that fraction
of collagen V molecules incorporated within the fibrils and thus
protected from proteolytic attack. Quantitation by scanning
densitometry of the 1(V) versus the 1(I) band after thrombin digestion of heterotypic fibrils indicated a ratio of 1:3.6.
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Unlike the heterotrimer, the collagen V homotrimer band completely
disappeared after thrombin digestion of reconstituted fibrils (Fig. 7,
lane 10). This confirmed that the homotrimer can interact with collagen I but most likely without being buried within large collagen I fibrils.
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DISCUSSION
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Fibrillar collagens are generally found in tissues in the form of heterotypic banded fibrils. This coassembly of different collagen types is thought to provide a general mechanism for the control of fibril diameter. Studies on collagens I and V have largely contributed to this view (14), whereas combined in vivo immunocolocalization and in vitro fibrillogenesis studies have also demonstrated that collagens I and III (26), I and II (27), and II and XI (28, 29) can coassemble to form hybrid fibrils. In addition, collagens I and III (30) as well as collagens I and V (31) have been shown to be covalently cross-linked within fibrils isolated from tissues. Genetic diseases and mouse models support the importance of such heterotypic fibrils in tissue properties. Alteration of a single gene encoding one of the fibrillar collagen chains can lead to the appearance in tissues of abnormal fibrils and consequently to connective tissue disorders. The phenotypic consequences of such mutations are now well known and include Ehlers-Danlos syndrome, ostogenesis imperfecta, and chondrodysplasia (32).
Although the role of different fibrillar collagen types in
fibrillogenesis is well documented, little is known about the
significance of the different chain stoichiometries that may occur for
a given type of collagen. The present study approaches this critical
question in the case of collagen V and in particular the roles of the
[1(V)]22(V) heterotrimer and
[1(V)]3 homotrimer in fibrillogenesis.
One mechanism of diameter regulation of fibrils in the cornea is based on the persistence of the N-propeptide in mature collagen V molecules, which hampers fibril accretion by steric hindrance (5). However, additional factors have been involved in fibril growth control by collagen V, notably the collagen I:collagen V ratio and properties intrinsic to the triple helix itself. The exact N-terminal cleavage site in the procollagen V heterotrimer is still controversial, but wherever it occurs, a large part of the N-propeptide persists in the mature molecule (5, 13, 18, 33). The presence of this globular domain is clearly an important factor in fibril growth regulation (4, 5). Concerning the homotrimer, the N-terminal processing site is even less well established. The collagen V homotrimer can exist as a fully processed molecule (13, 14), or it can retain a part of the N-propeptide as shown for the heterotrimeric molecule (19). The discrepancies about the exact structures of mature collagen V molecules and the relative insolubility of the pN form of the collagen homotrimer in physiological buffers (14), together with the presumptive role of the triple helix as a regulatory factor, have prompted us to perform our study on pepsinized molecules. Although proteolytic removal of the telopeptides from fibrillar collagen I has been shown to alter the kinetics of in vitro fibrillogenesis, pepsinized collagen is still capable of forming fibrils (34); thus the triple helical region per se contains the information necessary for proper fibril assembly. Moreover, pepsin-treated recombinant collagen II has recently been used for in vitro fibril formation experiments, and the fibrils obtained did not show marked differences from native fibrils (35).
Using classic in vitro fibrillogenesis experiments, we confirmed that the pepsinized collagen V heterotrimer polymerizes on its own into thin fibrils exhibiting, under certain buffer conditions, a periodic striation. In contrast, in vitro fibrillogenesis performed with collagen I alone generated large, striated fibrils. This is in agreement with previous work showing that collagens I and II, the fibrils of which are mostly regulated in vivo by copolymerization with minor collagens, can form much wider fibrils when singly reconstituted in vitro (36, 37). These simple observations argue in favor of an intrinsic property of the triple helix in the control of fibril accretion.
Like the heterotrimer, the collagen V homotrimer polymerized on its own in the form of thin fibrils, although they were unbanded and less organized. Because of its relative scarcity, the collagen V homotrimer cannot be purified from tissues in sufficiently large amounts for physicochemical studies. Therefore, the use of a recombinant homotrimer for these experiments was inevitable. The recombinant homotrimer has previously been well characterized and has been shown to present the molecular features expected for the collagen V homotrimer (14, 38). This molecule is more flexible and less stable than its heterotrimeric counterpart, and these particular features could impede close packing of the molecules and thereby contribute to the formation of loosely formed filaments. It has been reported that the high level of hydroxylysine glycosylation in recombinant collagen II considerably reduced the growth of fibrils formed in vitro (35). The recombinant collagen V homotrimer has previously been shown to contain lower levels of hydroxylysine than expected from the collagen V heterotrimer data (14). However, the fibrils obtained here with the low glycosylated collagen V homotrimer were even thinner than those formed with the tissue-derived heterotrimer, indicating that glycosylation levels are likely not to be a major factor in the control of collagen V fibril morphology.
In contrast to those of collagen I alone and mixtures of both collagen types, fibrillogenesis kinetics of collagen V molecules alone exhibited no increase in turbidity during incubation at 34 °C in PBS. However, electron microscopic observations at the end of the experiments revealed the presence of thin fibrils in both preparations. The initial nonzero turbidity values suggested that collagen V molecules had already aggregated at a low temperature, and this was subsequently confirmed by dynamic light scattering. Indeed, the collagen V homotrimer was in aggregate form even in acetic acid, whereas the heterotrimer was largely monomeric (and hence available for interaction with collagen I). When dialyzed into PBS at a low temperature, the heterotrimer formed large aggregates, consistent with both the relatively high initial turbidity and the electron microscopic data, whereas the homotrimer aggregates were smaller.
As stated above, the current model for the regulation of collagen I fibrillogenesis by the collagen V heterotrimer is based on the persistence of a large part of the N-propeptide on the mature molecule, which sterically hampers fibril accretion (5). Using a broad panel of different approaches, we have demonstrated that the collagen V triple helix itself is an additional potent factor that regulates the collagen I fibril diameter. We showed that fibrillogenesis kinetics were significantly slowed when increasing amounts of the collagen V heterotrimer were added. In addition, we confirmed the copolymerization of both collagens by immunogold labeling as previously shown by Birk et al. (4). This was supported by two additional approaches. First, solid-phase assays indicated that collagen I and V molecules do interact with each other. Second, thrombin digestion showed that collagen V heterotrimers were protected from proteolytic digestion by surrounding collagen I. Most strikingly, in the presence of excess collagen V, the maximal ratio between incorporated collagen V, corresponding to undigested molecules, and collagen I was estimated to be 1:3.6. This value fits well with the ratio found in human and chicken cornea fibrils, which are particularly regular and narrow (22, 39).
Marked differences were consistently observed between the collagen V heterotrimer and homotrimer. N- and C-terminal collagen V processing, although still controversial, might involve distinct cleavage sites and enzymes (5, 13, 18, 19, 33). Furthermore, the affinity for heparin is also dependent on the stoichiometry (38). Finally, molecular characteristics appear different in terms of flexibility, melting temperature, and diffusion coefficient. In this study we have shown that the collagen V homotrimer can efficiently bind to collagen I molecules in way similar to that of the collagen V heterotrimer. However, in contrast to the collagen V heterotrimer, collagen I fibrillogenesis kinetics were not affected by the addition of increasing amounts of the collagen V homotrimer. Likewise, the diameter of the fibrils obtained did not vary significantly as a function of the proportion of the collagen V homotrimer added. Although cosedimentation of collagen I and V homotrimers occurred after fibrillogenesis, thrombin digestion indicated that no detectable collagen V molecules were incorporated within the collagen I fibrils. Immunolabeling showed that collagen V aggregates were scattered all along the collagen I fibrils. This confirms the competence of this collagen to bind collagen I molecules, as shown by a solid-phase assay, and its accessibility to thrombin digestion. Interestingly, apart from its localization at the banded fibril surface, collagen V homotrimer labeling was often observed at the junction of two fibrils that were fusing in register into larger fibrils.
Altogether, these data indicate that the collagen V homotrimer, unlike
the heterotrimer, does not regulate the diameter of collagen I fibrils.
Its interaction with collagen I and its exposure at the surface of
reconstituted fibrils suggest that its role in vivo could be
more as a bridging agent. In accordance with this hypothesis, earlier
studies have shown that, although collagen V is present in most tissues
as a buried component in collagen I fibrils, some tissues reacted with
collagen V antibodies without unmasking collagen epitopes. Indeed,
collagen V was first immunolocalized as thin filaments in the immediate
vicinity of basement membranes, as observed for smooth muscle cells
(40), the human amion (41), and Bowman's membrane of the chick cornea
(25, 42). From these immunolabeling data, an anchoring function between
basement and stromal matrix was proposed for unmasked collagen V. Unfortunately, the study of the distribution of the two stoichiometries
in tissues has been hampered by the lack of an antibody able to
discriminate [1(V)]3 molecules from
[1(V)]22(V). However, on the basis of our data
suggesting a distinct fibril location depending on collagen V
stoichiometry, we can speculate that the exposed collagen V in tissues
corresponds to the homotrimer.
Collagen V molecules can assemble with a least three different
stoichiometries. In addition, a novel 4(V) chain has recently been
described (43). The possible association of collagen V chains with
collagen XI chains further lengthens the list of collagen V
chain-containing molecules. In view of this molecular diversity, the
question to be addressed is whether this might reflect specific functions in tissues. In the present report, we have shown that distinct stoichiometries of a same collagen type can differentially influence fibril formation, an event of central importance to tissue
architecture and function.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. Hartmann for helpful discussions on the use of collagen antibodies. We also thank M. Courteau for amino acid analysis and A. Bosch for the expert artwork.
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FOOTNOTES |
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* This work was supported by the Association pour la Recherche contre le Cancer and by a program of the European Community (Contract BIO-4-CT96-0537).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.
Recipient of a fellowship from the Ministère de la Recherche.
§ Recipient of a fellowship from the Fondation pour la Recherche Médicale.
¶ To whom correspondence should be addressed: Institut de Biologie et Chimie des Protéines, Unite Mixte de Recherche, Centre National de la Recherche Scientifique 5086, Université Claude Bernard Lyon I, 7 Passage du Vercors, 69367 Lyon Cedex 07, France. Tel.: 33-4-72-72-26-57; Fax: 33-4-72-72-26-02; E-mail: f.ruggiero@ibcp.fr.
Published, JBC Papers in Press, April 19, 2001, DOI 10.1074/jbc.M1011822200
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ABBREVIATIONS |
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The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HomV, collagen V homotrimer; HetV, collagen V heterotrimer; PBS, phosphate-buffered saline; D20, w, diffusion coefficient.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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