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J Biol Chem, Vol. 275, Issue 6, 3999-4006, February 11, 2000
From the Institute for Biochemistry, Medical Faculty, University of
Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, Germany
and the § Department of Cell and Molecular Biology, Lund
University, S-22100 Lund, Sweden
Matrilin-3 is a recently identified member of the
superfamily of proteins containing von Willebrand factor A-like domains and is able to form hetero-oligomers with matrilin-1 (cartilage matrix
protein) via a C-terminal coiled-coil domain. Full-length matrilin-3
and a fragment lacking the assembly domain were expressed in 293-EBNA
cells, purified, and subjected to biochemical characterization. Recombinantly expressed full-length matrilin-3 occurs as monomers, dimers, trimers, and tetramers, as detected by electron microscopy and
SDS-polyacrylamide gel electrophoresis, whereas matrilin-3, purified
from fetal calf cartilage, forms homotetramers as well as
hetero-oligomers of variable stoichiometry with matrilin-1. In the
matrix formed by cultured chondrosarcoma cells, matrilin-3 is found in
a filamentous, collagen-dependent network connecting cells
and in a collagen-independent pericellular network. Affinity-purified antibodies detect matrilin-3 expression in a variety of mouse cartilaginous tissues, such as sternum, articular, and epiphyseal cartilage, and in the cartilage anlage of developing bones. It is found
both inside the lacunae and in the interterritorial matrix of the
resting, proliferating, hypertrophic, and calcified cartilage zones,
whereas the expression is lower in the superficial articular cartilage.
In trachea and in costal cartilage of adult mice, an expression was
seen in the perichondrium. Furthermore, matrilin-3 is found in bone,
and its expression is, therefore, not restricted to chondroblasts and chondrocytes.
The matrilins constitute a recently discovered family of
noncollagenous proteins (1) belonging to the von Willebrand factor A
(vWFA)1-like domain
superfamily. To date, there are four matrilins known. Matrilin-2 (2, 3)
and matrilin-4 (4, 5) have a broad tissue distribution, whereas the
expression of matrilin-1 (also known as cartilage matrix protein)
(6-8) and matrilin-3 (9-11) is more restricted to skeletal tissues.
The division of the family into two subgroups can also be concluded
from evolutionary studies (1). The descent from a common ancestor and
the divergence through duplication of whole domains indicates the
possibility of the different family members providing similar
functions in different tissues. The at least partially coordinated
expression of matrilin-1 and -3 gains further functional significance
through the recent discovery of hetero-oligomers formed by matrilin-1 and -3 in epiphyseal cartilage of fetal calf femur (12).
Matrilin-3 has most features of the modular structure typical for
matrilins and consists of an N-terminal vWFA-like domain, four EGF-like
domains, and a C-terminal We have recombinantly expressed the full-length mouse matrilin-3 as
well as a truncated version lacking the coiled-coil domain in a
mammalian expression system. The truncated matrilin-3 was used for
production of a specific antiserum that allowed immunohistochemical characterization of matrilin-3 expression and an analysis of assembly forms in the pericellular matrix formed by cultured chondrocytes. The
full-length protein was used for structural studies by which the
molecular dimensions and oligomeric state of recombinant matrilin-3 could be determined. Purification of native matrilin-3 from fetal calf
cartilage allowed a comparison with the naturally occurring hetero-oligomers formed with matrilin-1.
Expression and Purification of Recombinant Matrilin-3--
The
full-length and the truncated matrilin-3 cDNA-construct, lacking
the coiled-coil domain, were generated by polymerase chain reaction on
a murine full-length clone (9). Suitable primers introduced a 5'
terminal SpeI and a 3' terminal NotI restriction site. The digested cDNA-constructs were inserted between the
NheI and NotI site of the expression vector
pCEP-Pu (13), downstream of the sequence encoding the BM-40 signal
peptide. Both cDNA clones were used for transfection of the human
embryonic kidney cell line 293-EBNA (Invitrogen). The cells were
selected with Geneticin (350 µg/ml) and puromycin (1 µg/ml), and
were transferred to serum free medium for harvest of recombinant
protein. After centrifugation (1 h at 10,000 × g),
cell culture supernatant was passed through a column of DEAE-Sepharose
Fast Flow (20 × 2.6 cm, Amersham Pharmacia Biotech). The
flow-through fraction was dialyzed against 2 M urea, 0.01 M EDTA, 0.05 M sodium acetate, pH 4.8, containing 0.1 M NaCl and applied to a column of
SP-Sepharose Fast Flow (10 × 2.6 cm, Amersham Pharmacia Biotech).
The bound fraction was eluted with a linear gradient from 0.1 to 1 M NaCl (800 ml) in the same buffer. Matrilin-3, eluted
between 0.2 and 0.5 M NaCl, was concentrated 10-fold by
ultrafiltration (DIAFLO membrane YM10, Amicon) and applied to a gel
filtration column of Sepharose CL-6B (90 × 1.6 cm, Amersham
Pharmacia Biotech) for final purification. The column was equilibrated
in 2 M urea, 0.01 M EDTA, 0.05 M
sodium acetate, pH 4.8, containing 0.75 M NaCl.
The truncated matrilin-3 protein was, after chromatography on
SP-Sepharose Fast Flow, concentrated on a SP HiTrap column (1 ml,
Amersham Pharmacia Biotech). For that purpose, the fractions containing
the truncated protein were diluted with two volumes of 2 M
urea, 0.05 M sodium acetate, pH 4.8, containing 0.1 M NaCl, before application to the column and eluted with 2 M urea, 0.05 M sodium acetate, pH 4.8, containing 1 M NaCl. Final purification was achieved by gel
filtration on a column of Superose 12 (25 ml, Amersham Pharmacia
Biotech) equilibrated in the same buffer.
Preparation of Antibodies to Matrilin-3--
The pure truncated
matrilin-3 fragment was used to immunize a rabbit. The antiserum was
purified by affinity chromatography with the same antigen coupled to
CNBr-activated Sepharose (Amersham Pharmacia Biotech).
Extraction and Purification of Matrilin-3 from Fetal Calf
Cartilage--
A calf fetus at 5-6 months of gestation was obtained
at the local abattoir. Cartilage from the proximal and distal ends of femur and humerus was dispersed using a Polytron homogenizer and extracted overnight in 0.1 M NaCl, 0.05 M
Tris/HCl, pH 7.4, containing 0.01 M EDTA, 2 mM
phenylmethylsulfonyl fluoride and 2 mM
N-ethylmaleimide. After centrifugation, the cartilage
extract (300 ml) was applied to a column of SP-Sepharose Fast Flow
(10 × 2.6 cm, Amersham Pharmacia Biotech). The bound protein was
eluted with a linear gradient of 0.1-1 M NaCl in 0.05 M Tris/HCl, pH 7.4, containing 0.01 M EDTA (300 ml, 60 ml/h). Matrilin-3 eluted between 0.2 and 0.5 M NaCl.
A corresponding pool was applied for final purification to a column of
Sepharose CL-6B (90 × 5 cm, Amersham Pharmacia Biotech)
equilibrated in 0.1 M NaCl, 0.05 M Tris/HCl, pH
7.4, containing 0.01 M EDTA.
SDS-Polyacrylamide Gel Electrophoresis, Immunoblotting, and
Determination of N-terminal Sequences--
SDS-polyacrylamide gel
electrophoresis was performed as described by Laemmli (14). For
immunoblots, the proteins were transferred to nitrocellulose and
incubated with a dilution of the appropriate rabbit antiserum. Bound
antibodies were detected using peroxidase-conjugated swine anti-rabbit
IgG (Dakopats), 3-aminopthalhydrazide (1.25 mM),
p-coumaric acid (225 µM), and 0.01%
H2O2. Reference samples of protein from mouse
sternum were extracted overnight with 0.1 M NaCl, 0.05 M Tris/HCl, pH 7.4, containing 0.01 M EDTA, 2 mM phenylmethylsulfonyl fluoride and 2 mM
N-ethylmaleimide at 4 °C. For sequencing, both
recombinant proteins were subjected to SDS-polyacrylamide gel
electrophoresis and electroblotted to a polyvinylidene difluoride membrane (Immobilon P, Millipore). Protein bands were cut out, and
their N-terminal amino acid sequences were determined in a Applied
Biosystems 473A protein sequencer.
In Situ Hybridization--
Limbs from newborn mice were fixed
over night with 4% paraformaldehyde in phosphate-buffered saline, pH
7.4, at 4 °C, washed overnight with phosphate-buffered saline
(4 °C), dehydrated, and embedded in paraffin. Sections of 7 µm
were cut, mounted on 3-aminopropyltriethoxy-silane treated glass
slides, dewaxed in xylene, and rehydrated. After washing in
phosphate-buffered saline, they were digested with 10 µg/ml
proteinase K, postfixed, and acetylated with 0.25% acetic anhydride.
The sections were hybridized overnight at 55 °C with digoxigenin-labeled riboprobes covering the first 827 nucleotides of the matrilin-3 cDNA (9). After hybridization, the sections were
washed in 50% formamide, 2× SSC for 30 min at 52 °C; digested with
RNase A; and washed once with 2× SSC and twice with 0.2× SSC for 20 min at 52 °C. The immunological detection of the digoxigenin-labeled sections was carried out according to the instructions of the manufacturer (Roche Molecular Biochemicals) with additional use of
polyvinyl alcohol in the detection solution (15).
Immunohistochemistry--
Immunohistochemistry was performed on
cryosections frozen on dry ice in Tissue-Tek® (Miles, Inc.) as well as
on paraffin-embedded sections of fetal, newborn and 6-week-old mice.
The cryosections were used either unfixed or fixed with 1%
paraformaldehyde for 10 min., treated for 1 h with 0.04 units/ml
chondroitinase ABC (Sigma), and incubated twice for 20 min in 1%
H2O2. Immunolabeling was done by consecutive
treatment of the sections for 1 h with affinity-purified
antibodies to matrilin-3 and peroxidase conjugated swine anti-rabbit
IgG. Both antibodies were diluted in 1% (w/v) bovine serum albumin in
Tris-buffered saline and the slides developed with
3-amino-9-ethylcarbazole. The paraffin-embedded tissues were prefixed
and demineralized after fixation in 0.6 M EDTA,
Tris-buffered saline, pH 8.0, for 21 days. Deparaffinization ensued
through incubation for 30 min in rotihistol (Carl Roth GmbH, Karlsruhe, Germany) at 52 °C. After rehydration, immunostaining was performed as described above, but with the incubation time with the first antibody increased to 18 h. For immunofluorescence detection, a
CyTM3-conjugated affinity-purified goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) was used. Nonspecific antibody binding was
blocked by incubation for 1 h with 5% (w/v) normal goat serum in
Tris-buffered saline.
Cell Culture of Chondrosarcoma Cells--
The Swarm rat
chondrosarcoma cell line (16) was obtained from Dr. J. Kimura (Henry
Ford Hospital, Detroit, MI). The immortalized cells were cultured on
plastic chamber slides in Ham's F-12 nutrient mixture supplemented
with 10% fetal calf serum, 50 units/ml penicillin, 50 units/ml
streptomycin, and, if desired, 50 µg/ml L-ascorbic acid.
Electron Microscopy--
Electron microscopy was performed as
described previously (3, 17) using matrilin samples with typical
concentrations of 5-10 µg/ml in Tris-buffered saline that were
stained with 0.75% uranyl formate.
Mass Spectrometry--
Sample preparation for MALDI-TOF mass
spectrometry was performed as described (18). As a matrix,
Recombinant Expression of Matrilin-3 Constructs--
A cDNA
fragment encoding the sequence of mature mouse matrilin-3 and a
fragment lacking the oligomerization domain were inserted into the
pCEP-Pu vector utilizing the secretion signal sequence of BM-40 (13).
The recombinant plasmids were introduced into 293-EBNA cells and stably
maintained in an episomal form. The secreted matrilin-3 proteins were
purified from the medium (Fig. 1), and
their identities were confirmed by N-terminal sequencing. Surprisingly,
the first eight amino acid residues encoded by the cDNA-constructs
were missing, presumably due to the cleavage at the potential furin
protease cleavage site Arg-Leu-Ala-Arg (amino acids 31-34). The
resulting N-terminal sequence of the mature protein starts with
Ala35-Ser-Val. The purified protein lacking the
oligomerization domain was used to immunize rabbits. After affinity
purification, the antiserum reacted specifically with matrilin-3 (Fig.
1).
Recombinantly Expressed Matrilin-3 Forms Oligomers--
Purified
recombinant full-length matrilin-3 was analyzed by SDS-PAGE under both
reducing and nonreducing conditions (Fig. 1). Without reduction four
bands representing the tetra-, tri-, di-, and monomeric forms of
matrilin-3 were detected. The purified full-length matrilin-3 was
submitted to electron microscopy after negative staining with uranyl
formate (Fig. 2A). The stained
particles were heterogeneous in size, and a closer examination of
single particles revealed that all species from monomer to tetramer
were present in the sample. At high magnification, it was seen that in
oligomers all subunits are joined at a single point in a manner reminiscent of the bouquet-like structure known from matrilin-1 (19)
and matrilin-2 (3), but different in that in matrilin-3 a stalk made up
from the four EGF-repeats is clearly resolved. Monomers have either a
stretched tadpole-like or a collapsed shape.
Purification of Native Matrilin-1/Matrilin-3 Hetero-oligomers from
Fetal Calf Cartilage--
Preliminary immunoblot results revealed that
expression and/or solubility of matrilin-3 is much higher in fetal than
in adult tissues (not shown). Therefore, fetal calf cartilage was
extracted to obtain the tissue form of matrilin-3. Extraction under
nondenaturing conditions, followed by chromatographic purification,
yielded preparations highly enriched in matrilin-3 but also containing considerable amounts of matrilin-1. SDS-PAGE and immunoblot showed the
presence of different matrilin-1/matrilin-3 hetero-oligomers as well as
of homotrimeric matrilin-1 (Fig. 3).
Reduction of the oligomeric forms resulted in two bands with apparent
masses of 66 and 57 kDa (Fig. 3). These were by immunoblotting
identified as matrilin-3 and matrilin-1, respectively (Fig. 3), in
agreement with the recent assignment of Wu and Eyre (12) by N-terminal protein sequencing. MALDI-TOF mass spectrometry gave masses of 53.0 and
49.3 kDa for the subunits of matrilin-1 and -3, respectively (results
not shown). Apparently the matrilin subunits show an anomalous behavior
in SDS-PAGE, as earlier noted for matrilin-1 (20). In nonreducing
SDS-PAGE, four bands of differing intensity could be distinguished in
the range of 170-220 kDa. In addition, two weak bands were present at
around 160 kDa, of which the upper band reacted with antibodies to both
matrilin-1 and -3 and the lower only with antibodies to matrilin-1
(Fig. 3). These bands probably represent heterotrimeric
matrilin-1/matrilin-3 and homotrimeric matrilin-1. A sample that had
been chromatographically enriched for the three largest oligomers,
presumably tetramers, was further investigated with two-dimensional
SDS-PAGE using nonreducing conditions in the first dimension and
reducing in the second (Fig. 4). The oligomer with the highest apparent Mr was found
to be composed only of matrilin-3 subunits, whereas the two forms of
higher mobility contain both matrilin-3 and matrilin-1. The complex
band pattern detected in SDS-PAGE under nonreducing conditions (Fig. 3)
is in contrast to the recently published results (12) that point to the
existence of only heterotetramers with a two and two stoichiometry.
MALDI-TOF mass spectroscopy of an nonreduced sample of the same
preparation as shown in Fig. 3 yielded two broad molecule ion peaks at
around 156 and 202 kDa, indicating the presence of both trimeric and
tetrameric populations (results not shown). A resolution of the
different species within those peaks could not be achieved. In electron
microscopy, tetrameric and, less often, trimeric molecules were seen
that frequently displayed the bouquet-like shape typical for matrilins
(Fig. 2B). In addition to compact trimeric particles,
presumably representing matrilin-1 homotrimers, and tetrameric
particles with clearly visible stalks, closely resembling the
recombinant matrilin-3 homo-oligomers, particles were seen that,
judging from their morphology, could represent matrilin-1/matrilin-3
hetero-oligomers of varying stochiometries. Furthermore, trimeric and
tetrameric particles were seen in which the globular domains show an
almost linear arrangement.
Matrilin-3 Forms Extracellular Filamentous Networks in Cell
Culture--
In order to study the extracellular assembly forms of
matrilin-3, the matrix produced by cultured Swarm rat chondrosarcoma cells was analyzed by immunofluorescence microscopy. When ascorbate was
present in the cell culture medium, matrilin-3 was detected in an
extensive, fibrillar network connecting cells over a distance of
several cell diameters (Fig.
5A). These extended fibrils
could not be detected in the absence of ascorbate (Fig. 5B),
conditions known to inhibit collagen secretion (21). Under those
conditions, matrilin-3 was seen on the surface of individual cells or
in network structures connecting adjacent cells (Fig. 5B).
Such collagen-independent fibrils were formed at cell contacts that
develop immediately after mitosis (Fig. 5C).
Matrilin-3 Is Expressed in Dense Connective Tissue during Growth
and Remodeling--
Immunohistochemistry was performed on sections of
mouse embryos and of tissues from newborn and 6-week-old mice. The
earliest expression of matrilin-3 could be detected in a day 12.5 postcoitum mouse embryo in the cartilage anlage of the developing bones
(not shown). At day 14.5 postcoitum, the primordial skeleton
(e.g. rib cartilage, vertebral bodies, and the cartilage
primordium of the legs) showed strong staining for matrilin-3 (Fig.
6A). At birth, matrilin-3 is
present in the developing occipital bones and in the bones of the nasal
cavity, whereas no expression was detected in maxilla and mandible
(Fig. 6B). Matrilin-3 was also detected in the manubrium and
corpus of sternum (Fig. 6C), as well as in the cartilage
plates of trachea (Fig. 6D). In the tail of newborn mice,
the cartilage primordium of the vertebral bodies showed a strong
expression, in contrast to a weaker one in the annulus fibrosus and
none in the nucleus pulposus (Fig. 6E). In long bones of
newborn mice, e.g. the tibia, matrilin-3 is broadly expressed in the epiphysis and could be detected in both the
territorial and interterritorial matrix of reserve, proliferating and
hypertrophic cartilage (Fig. 6F). In the hypertrophic
cartilage, the signal is weaker and in part missing in the lower zone.
Matrilin-3 was also detected in the ossified portion of the epiphysis,
whereas no signal was seen in the articular cartilage. The strong
expression in the epiphyseal growth plate persisted in long bones of
6-week-old mice (Fig. 6G). At this stage, matrilin-3 was
also detected in peripheral and deeper portions of the articular
cartilage and in the interior of the lateral meniscus. In addition to
cartilaginous tissues, matrilin-3 was present in the osteoid around
osteoblasts attached to bone trabeculae in the subchondral bone (Fig.
6H) and around osteocytes inside the cancellous bone (Fig.
6I). In the still growing distal portion of the radius,
matrilin-3 was more abundant than in the ossified proximal end, where
an epiphysis is no longer present (not shown). In sternal cartilage
plates, matrilin-3 was present in filamentous structures that start and end at the chondroblasts and are arranged in parallel orientation relative to the ossification front (Fig. 6J). In costal
cartilage, it was deposited in the perichondrium around fibroblast-like
cells and in structures spanning the distance from perichondrium to perichondrium (Fig. 6K). A similar, mainly perichondral
expression was seen in the trachea (Fig. 6L). At no stage of
development was matrilin-3 detected in extraskeletal tissues.
Matrilin-3 mRNA Is Transcribed in Chondrocytes and
Osteoblasts--
In situ hybridization was performed on
paraffin sections of newborn mice. In limbs, the matrilin-3 gene was
transcribed by all chondrocytes with the exception of those at the
articular surface and the lower hypertrophic zone (Fig.
7A). A gradient in
transcription of the matrilin-3 mRNA could be seen, with the strongest signals in the peripheral areas of the proliferative zone,
weaker signals in the central part of the resting cartilage, and the
weakest signals or none in the hypertrophic zone, where, however, most
of the intracellular material is lost during the hybridization
procedure. In the rare cases in which RNA was retained, weak
hybridization signals appeared (Fig. 7A). Matrilin-3 is also transcribed by cells, presumably osteoblasts, that are located as a
monolayer on the endosteal surface and on bone trabeculae, showing that
the matrilin-3 detected in bone by immunohistochemistry is not only a
remainder of the resorbed cartilage but also newly synthesized there
(Fig. 7B). Furthermore, immunoblot analysis of U2-OS cell
cultures (22) showed that this osteoblast-like cell line secretes
matrilin-3 into the medium (results not shown).
Matrilin-3 Is Able to Form Homo- and
Hetero-oligomers--
Matrilin-3 is able to form homo-oligomers, as
detected by both SDS-PAGE (Fig. 1) and electron microscopy (Fig.
2A) of recombinant protein from the supernatants of 293-EBNA
cells transfected with matrilin-3 cDNA. The oligomerization occurs
through the coiled-coil domain, as the construct that lacks this domain
only yields monomers (Fig. 1). In electron microscopy (Fig.
2A), the oligomeric forms show similarities to the
bouquet-like shape of matrilin-1 and -2 (19, 3). However, the center of
the particle accumulates more stain, as the single A-domains in
matrilin-3 are connected to the coiled-coil domain by a stalk formed by
four EGF domains. As each subunit contains only a single A-domain,
self-interactions within the subunit cannot occur, and the arms of
matrilin-3 are accordingly more extended than those of matrilin-1 and
-2 (19, 3). The presence of all oligomeric forms from monomers to
tetramers is surprising, but it has also been seen with matrilin-2,
both when expressed recombinantly and when extracted from tissues (3). Matrilin-1 occurs in mature cartilage mainly as homotrimers (20), but
the formation of homotetramers was described for a matrilin-1 peptide,
covering the coiled-coil domain, where a single arginine residue was
replaced with a glutamine (23). As dimers and monomers of matrilin-3
are seen not only under the denaturing conditions of SDS-PAGE (Fig.
1A) but also in electron microscopy (Fig. 2A), they are not due to deficient closure of disulfide bridges. In contrast, in native preparations from fetal bovine rib cartilage, even
though small amounts of matrilin-1 dimers and monomers could be
detected in SDS-PAGE, only intact, trimeric particles were seen by
electron microscopy (19). Chen et al. (24) recently reported
that deletion of the A2 domain in chicken matrilin-1 led to an
incomplete assembly when this protein was recombinantly expressed in
chick fibroblasts. They proposed that the A2 domain facilitates the
formation of the directly adjacent coiled-coil during the biosynthesis.
This is in agreement with our observation that matrilin-3 subunits that
naturally lack the A2 domain occur in monomeric and dimeric forms as
well as in higher oligomers. In matrilin-2, which also assembles
incompletely, the A2 domain is present, but it is separated from the
coiled-coil by a unique stretch of 75 amino acids in an unknown fold
(2, 3). Possibly, the A2 domain may promote coiled-coil formation only
when directly adjacent to the assembly domain. The incomplete
oligomerization of matrilin-3 may, however, equally well reflect a low
stability of the matrilin-3 trimeric and tetrameric coiled-coils,
caused by the high proportion of imperfect heptad repeats in this
matrilin, and it is also possible that subunits are lost from
completely assembled oligomers through proteolytic cleavage close to
the coiled-coil domain.
Native matrilins from fetal calf epiphyseal cartilage occur in a
complex set of oligomers with matrilin-1/matrilin-3 hetero-oligomers of
different composition being present together with matrilin-1 and
matrilin-3 (Fig. 3) homo-oligomers. The participation of matrilin-1 subunits, with fewer imperfections in the heptad repeat, may stabilize the coiled-coil in mixed oligomers. Nevertheless, matrilin-3
homo-oligomers occur in situ in cartilage, as could be shown
by two-dimensional SDS-PAGE (Fig. 4). In one-dimensional SDS-PAGE, we
observed at least three bands containing both matrilin-1 and matrilin-3
subunits, representing complexes with a different stoichiometry. This,
as well as the presence of homotetrameric matrilin-3, is in conflict with the recent proposal that hetero-oligomers of matrilin-1 and matrilin-3 subunits from fetal calf epiphyseal cartilage show a strict
two-and-two stoichiometry (12). Electron microscopy (Fig.
2B) supports the results from two-dimensional SDS-PAGE. The
stalk, seen in matrilin-3 subunits, often allows the distinction between these and matrilin-1 subunits. When this criterion is used, a
pattern of homo- and hetero-oligomers of varying stoichiometry emerges
that is equally complex as that seen by SDS-PAGE and MALDI-TOF mass spectrometry.
Evidence for a Role of Matrilin-3 in the Formation of Extracellular
Filamentous Networks--
Immunofluorescence microscopy of the matrix
formed by cultured Swarm rat chondrosarcoma cells shows matrilin-3 in
an extended filamentous network. The filaments have a variable
thickness, often form branches and connect cells over a distance of
several cell diameters (Fig. 5A). The formation of this
network depends on the presence of ascorbate in the culture medium,
implying that it contains also collagenous proteins. In the absence of
ascorbate a matrilin-3-containing network is seen in close association
with cells (Fig. 5B). This network focally connects cells
and sometimes covers the whole chondrocyte (Fig. 5C).
Interestingly, the filaments often connect two neighboring cells that
appear to have recently undergone mitosis. Such connections, if they
occur also in situ, could possibly provide chondrocytes with
spatial information.
Similar results were obtained for the distribution of matrilin-1 in
cultures of chicken chondrocytes, in which a more extended collagen-dependent network (21, 25) and a pericellular,
collagen-independent network were also described (21). Recent studies
revealed functional differences between the two A domains of
matrilin-1. Deletion of the A2 domain leads to an impaired
oligomerization, whereas the lack of either A domain results in an
inability to form collagen-independent filaments. Furthermore, double
point mutations of Asp-22 in the A1 and Asp-255 in the A2 domain
disrupt matrilin-1 network formation (24). As these aspartic acid
residues are part of the metal ion-dependent adhesion site
motif, an adhesion mechanism mediated by this site is very likely.
Matrilin-2 also forms a fibrillar network when expressed by cultured
smooth muscle cells (3), showing this feature to be common to several matrilins.
The matrilin-3 containing filamentous structures found in caudal
sternal cartilage plates (Fig. 6J) could represent an
in vivo counterpart of the fibrils seen in cell culture.
These structures are oriented along the lateral growth axis of the
sternum plate. Matrilin-3 positive, filamentous structures were
detected also in costal and tracheal cartilage, in which, in both
cases, the direction of the fibrils is perpendicular to the
perichondrium (Fig. 6K).
Matrilin-3 Is Specific for Dense Connective Tissue and Expressed in
Zones of Growth or Remodeling--
Matrilin-3 has a tissue
distribution similar to that of matrilin-1. Both proteins are found
almost exclusively in skeletal tissues and apparently sometimes
function together in the form of hetero-oligomers. Matrilin-3 is
strongly expressed in growing skeletal tissues, e.g. the
epiphyseal growth plate, or in bone undergoing growth and remodeling.
In bone, it is not only a remnant found in not yet resorbed calcified
cartilage, but it is actively synthesized by osteoblasts and
osteocytes. In bovine trachea, the matrilin-3 expression is decreased
upon maturation (12), and in 6-week-old mice, the residual expression
is more restricted to the perichondrium (Fig. 6L). In
contrast, matrilin-1 accumulates in bovine trachea, and the highest
amounts are present in adult animals (26).
Even though matrilin-1 and matrilin-3 may occur in the same tissues,
their relative amounts differ during the life span of the animal, and
even though their functions may be related, they may in part fullfil
those at different timepoints in development and maturation. Further
work will be directed at determining the structure, composition, and
function of matrilin-containing filaments and the interactions by which
they are formed.
We thank Dr. Ferenc Deák for critical
reading of the manuscript and Dr. Franz Mayer-Posner (Bruker Daltonik
GmbH, Bremen, Germany) for carrying out MALDI-TOF measurements.
*
This work was supported by a grant from the Köln
Fortune program/Faculty of Medicine, University of Cologne.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.:
49-221-478-6990; Fax: 49-221-478-6977; E-mail:
Raimund.Wagener@uni-koeln.de.
The abbreviations used are:
vWFA, von Willebrand
factor A;
PAGE, polyacrylamide gel electrophoresis;
MALDI-TOF, matrix-assisted laser desorption ionization/time of flight.
Molecular Structure and Tissue Distribution of Matrilin-3, a
Filament-forming Extracellular Matrix Protein Expressed during
Skeletal Development*
,,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical coiled-coil oligomerization
domain (9-11), but it lacks the second vWFA-like domain that is
present in all the other matrilins. Similarly, a unique mouse
matrilin-4 splice variant lacking the N-terminal vWFA-like domain was
recently identified (4). In addition, matrilin-3 possesses a domain
with a high content of positively charged amino acids between the
N-terminal vWFA-like domain and the signal peptide cleavage site. The
mouse matrilin-3 precursor consists of 481 amino acid residues and
after cleavage of the 27-amino acid signal peptide a mature protein
with a predicted minimal molecular mass of 48.9 kDa is formed (9).
Matrilin-3 from human and chicken are highly homologous to the mouse
protein and differ mainly in the N-terminal positively charged domain, which is longer in human and shorter in chicken. Additionally, all four
EGF-like domains of human and mouse matrilin-3 contain an insertion of
a single aspartic acid residue not found in the corresponding chicken
sequences. Matrilin-3 expression was so far studied by Northern
hybridization, and mRNA could be demonstrated in femur, sternum,
and vertebrae (9, 10). In the case of chick sternum, the results were
extended by in situ hybridization, in which the signal was
more pronounced at the periphery than in the center of the cartilage
(10).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cyano-4-hydroxycinnamic acid (Sigma) was used. When desired, the
protein was reduced with 0.01 M dithiothreitol on the
target for 1 h at 37 °C. Cations were detected and analyzed
utilizing the high mass detector in the linear mode of a Bruker Biflex
III. Calibration of the time of flight analyzer was based upon the
Mr of recombinant protein A (Repligen) of
44,610.3 and the Mr of bovine serum albumin
dimer (Sigma) of 132,859.0.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (70K):
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Fig. 1.
SDS-PAGE analysis of recombinant matrilin-3
proteins and demonstration of the specificity of the matrilin-3
antiserum by immunoblot. Full-length matrilin-3 (M3)
(lanes 1 and 3) and matrilin-3 lacking the
coiled-coil domain (M3 
) (lanes 2 and
4) were submitted to SDS-PAGE without (SH)
(lanes 1 and 2) and with (+SH)
(lanes 3 and 4) prior reduction and stained with
Coomassie Brilliant Blue. Asterisks mark the different
oligomeric forms of nonreduced matrilin-3. Proteins extracted from
sternum were submitted to SDS-PAGE, blotted to nitrocellulose, stained
with Ponceau S (lane 5), and developed with the
affinity-purified matrilin-3 antiserum (lane 6). All samples
were separated on 4-15% polyacrylamide gels. Molecular mass of marker
proteins (right) is given in kDa.
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Fig. 2.
Negative stain electron microscopy of
recombinant matrilin-3 (A) and a matrilin-1/matrilin-3
mixture purified from fetal calf cartilage (B).
Overviews (top) and selected particles at higher
magnification (bottom) show the heterogeneity in subunit
number. The bar corresponds to 100 nm for the overviews
(top) and 25 nm for the enlarged single molecules
(bottom).
View larger version (46K):
[in a new window]
Fig. 3.
Analysis of matrilin-1 and matrilin-3 from
fetal calf cartilage by SDS-PAGE and immunoblotting. Purified
material was applied to SDS-PAGE gels under nonreducing
( SH) (lanes 1-3) and reducing (+SH)
(lanes 4-6) conditions. Lanes 1 and 4 were stained with Coomassie Brilliant Blue and silver nitrate,
respectively. Matrilins were detected by immunoblotting using antisera
specific for matrilin-3 (M3) (lanes 2 and
5) and matrilin-1 (M1) (lanes 3 and
6), respectively. Electrophoresis was performed in 4.5%
(lanes 1-3), 4-15% (lane 4), and 7.5%
(lanes 5 and 6) polyacrylamide gels. Molecular
mass of the marker (left and right) proteins is
given in kDa.
View larger version (74K):
[in a new window]
Fig. 4.
Analysis of matrilin-1/matrilin-3 oligomers
from fetal calf cartilage by two-dimensional SDS-PAGE. High
molecular mass matrilin-1/matrilin-3 complexes were enriched by gel
filtration and separated in a 4% polyacrylamide gel under nonreducing
conditions (top). The lane was cut out and applied
horizontally to a 7.5% gel run under reducing conditions
(bottom). Arrows indicate the direction of
electrophoresis. The gels were stained with Coomassie Brilliant Blue.
Molecular mass of the marker proteins (top and
right) is given in kDa.
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[in a new window]
Fig. 5.
Immunofluorescence microscopy of
matrilin-3-containing filaments in the extracellular matrix of cultured
Swarm rat chondrosarcoma cells. Cells were cultured in the
presence (A and A') or in the absence (B,
B', and C) of ascorbate. In A, B,
and C, matrilin-3 was detected with affinity-purified
antibodies and a CyTM3-conjugated affinity-pure goat anti-rabbit IgG.
A' and B' show the differential-interference
contrast photomicrographs corresponding to A and
B, respectively. Bars, 40 (A, A', B,
and B') and 5 µm (C)
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Fig. 6.
Tissue distribution of matrilin-3.
Frozen (A-E, L) and paraffin-embedded
(F-K) sections from a day 14.5 postcoitum mouse embryo
(A), newborn (B-F) and 6-week-old
(G-L) animals were incubated with affinity-purified
antibodies against matrilin-3 followed either by peroxidase-conjugated
swine anti-rabbit IgG (A-F) or a CyTM3-conjugated goat
anti-rabbit IgG (G-L). In a day 14.5 postcoitum mouse
fetus, immunostaining was detected in the cartilage primordia of the
vertebral bodies (vb), the dorsal part of ribs
(ri), and the heads of the humerus (hu) and the
femur (fe) (A). In the head, the cartilage
primordia of the supraoccipital (so), basioccipital
(bo), basisphenoid (bs), turbinate
(tu) and hyoid (hy), bones and petrous part of
temporal bone (te) were stained (B). In the
cartilage plates (cp) of corpus sternum and in costal
cartilage (co), strong signals were present (C).
Weaker signals could be detected in the ossification cores
(os) between the cartilage plates. The cricotracheal
cartilage of the trachea (tr) was also positive
(D). Strong signals could be detected in the cartilage
primordia of the vertebral bodies (vb). In the
intervertebral disc, a weak signal could be found in the annulus
fibrosus (af) but not in the nucleus pulposus
(np) (E). The distal end of the tibia shows a
matrilin-3 expression both in the territorial and interterritorial
matrix surrounding resting (rc), proliferating
(pc) and hypertrophic (hc) chondrocytes, weaker
signals in the trabecular calcified bone (cb), and none in
the articular cartilage (ac) (F). Strong
expression could be observed in the epiphyseal growth plate
(gp) and the newly formed trabecular bone (tr) of
tibia. Matrilin-3 is also present in the trabecular meshwork of the
secondary ossification center (so), in the lateral meniscus
(lm), in the subchondral bone of femur (sb), and
at a low level in the peripheral articular cartilage (pa)
(G). The higher magnification of the metaphyseal side of the
growth plate of tibia shows immunostaining around osteoblasts
(ob) attached to bone trabeculae (H). On the
epiphyseal side of the growth plate, matrilin-3 was found on trabecular
surfaces (ts) and around osteocytes (oc) inside
the cancellous bone (I). In adult sternum, matrilin-3 was
observed in filamentous structures (fi) between the lacunae,
which are arranged in a parallel orientation to the ossification front
(J). Strong immunostaining was found in the costal
perichondrium (pe) around fibroblast-like shaped cells and
inside the costal cartilage in structures with transverse orientation
(arrows) (K). In adult trachea, strong
immunostaining was found in the perichondrium
(pe), and only a weak signal was found inside the
tracheal cartilage (tc) (L). Bar, 1.6 mm (A and B), 400 µm (C and
G), 200 µm (D, E, and F), 100 µm
(H), 50 µm (I, K, and L), and 20 µm (J).
View larger version (167K):
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Fig. 7.
In situ hybridization of
matrilin-3 mRNA in the skeleton of newborn mice. Antisense
riboprobes labeled with digoxigenin were hybridized to
paraffin-embedded sections of a knee (A) and tibia
(B) of newborn mice. In tibia (left) and femur
(right), mRNA was detected in most of the cartilaginous
tissues (A). In the periphery of the resting and the
proliferating (pc) cartilage, strong signals were present,
whereas in hypertrophic cartilage (hc) and in the center of
the resting zone, weak or no signals were detected, due in part to a
loss of cellular material during the hybridization procedure. Weak
signals were also found in central areas of the lateral meniscus
(lm). No mRNA could be detected in cells of the
superficial and central parts of the articular cartilage
(A). In the mineralized part of tibia, mRNA was detected
in cells lining bone trabeculae and in endosteal cells (eo)
(B). Bar, 150 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
These authors contributed equally to this work and share the first authorship.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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