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INTRODUCTION |
Extracellular matrix provides physical support to the cells,
delineates pathways for cell migration during differentiation and
tissue regeneration, and provides the necessary milieu for the normal
cell metabolism and development. Collagen fibers and proteoglycan
aggregates provide the structural basis for matrix architecture.
Noncollagenous proteins modulate the organization of these elements,
form collagen-associated or independent networks, and are parts of cell
migratory pathways.
The matrix molecules share homologous modules, protein domains of
common evolutionary origin, but a great functional variability of the
homologous modules in different proteins has been observed. The
recently discovered matrilins (for a review, see Ref. 1) are typical
modular proteins belonging to the superfamily with von Willebrand
factor type A-like (vWFA)1
modules. Members of the matrilin family are found in a wide variety of
extracellular matrices. Matrilin-1, formerly called cartilage matrix
protein, and matrilin-3 (2, 3) are abundant in cartilage, while
matrilin-2 (4) and matrilin-4 (5) show a broader tissue distribution.
Thus, all forms of connective tissue appear to contain at least one
form of matrilin, indicating a general and important function for this
protein family.
Matrilin-2 was found to contain the same protein modules in the same
order as matrilin-1 (4). The precursor protein in mouse is 956 amino
acids long and consists of a putative signal peptide, two vWFA domains
connected by 10 epidermal growth factor-like modules, a potential
oligomerization domain, and a unique segment. The ability of the 38 C-terminal amino acid moieties to form an
-helical coiled-coil was
shown by Pan and Beck (6). Matrilin-2 mRNA was detected by filter
hybridization in a variety of mouse organs including calvaria, uterus,
heart, and brain as well as fibroblast and osteoblast cell lines. A
group of 120-150-kDa bands was, after reduction, recognized
specifically with an antiserum against the matrilin-2-glutathione
S-transferase fusion protein in media of the
matrilin-2-expressing cell lines. Immunolocalization of matrilin-2 in
developing skeletal elements showed reactivity in the perichondrium and
the osteoblast layer of trabecular bone.
In order to gain a better understanding of the potential function of
matrilin-2, we have determined the spatial expression of the gene by
radioactive in situ hybridization. A new antiserum with a
higher titer to the native matrilin-2 was raised using, as an antigen,
matrilin-2 expressed in a eukaryotic cell line, and the protein was
immunolocalized in mouse tissues. Furthermore, matrilin-2 was purified
from media of cells overexpressing the full-length protein and
visualized by electron microscopy to provide information on the
molecular dimensions and oligomeric structure of the protein. The
structural information was extended by SDS-PAGE analysis of the intact
protein and the reduced subunits. Posttranslational modification of the
protein and alternative splicing of the mRNA were also
characterized. Finally, formation of an extracellular network in
cultures of cells expressing matrilin-2 was demonstrated by indirect
immunofluorescence. The potential function and suggested molecular
architecture of the protein is discussed.
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MATERIALS AND METHODS |
Mouse Strains and Cell Cultures
BALB/c or NMRI mouse strains were used for RNA or protein
analysis, respectively. The mouse fibroblastic cell lines WEHI 164 and
NIH 3T3, the rat osteogenic sarcoma UMR-106, and the small intestine
epithelial IEC-6 cell lines were obtained from the American Type
Culture Collection (Rockville, MD). The mouse C2/7 cells with skeletal
muscle characteristics (7), the smooth muscle-like cell 9E11G (8), the
keratinocyte carcinoma PVD(A)I (9), the rat Schwann cell line RN22
(10), and the Swarm rat chondrosarcoma cell line (RCSC) (11) were
obtained from the laboratories of origin. The mouse immortalized
endothelial cells m1END, derived from mesenteric lymph nodes (12), and
the SV40-transformed lymphoid vascular endothelial cell SVEC (13) were
provided by L. Sorokin and R. Hallmann (Erlangen); smooth muscle cells
(SMC) from rat aorta were cultured by F. Michaelsen (Cologne), using
standard methods (14). Unless recommended otherwise by the supplier, the cell lines were cultivated in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (Life Technologies, Inc.), and
utilized for RNA and protein analyses.
RNA Preparation and Analysis
Total RNA was prepared from guanidinium thiocyanate extracts of
various cell lines using the RNA isolation kit of Stratagene. For RNA
blot analysis, 7-µg aliquots were electrophoresed, blotted to Hybond
N filter (Amersham Pharmacia Biotech) and hybridized consecutively with
pCRP12 cDNA (4) and chicken 27 S rRNA gene fragment. For the study
of RNA alternative splicing, total RNA was reverse transcribed with
Moloney murine leukemia virus reverse transcriptase (Life Technologies)
using oligo(dT) primer. Nested polymerase chain reactions were
performed using first the distal primers 5'-GGACGGGCTCAGGATGA-3' and
5'-CTGTATCTCAGGCGATTTTC-3' and then the proximal primer pair
5'-CATTGACAAGCATCTCTTCT-3' and 5'-TTTGTGTAGACCGTGAAAGA-3' flanking
the unique region of mouse matrilin-2.
In Situ Hybridization
Preparation of Tissue Sections--
For paraffin embedding,
tissue specimens were fixed overnight in 95% ethanol, 1% acetic acid;
dehydrated in ethanol; cleared in xylol; and embedded in low melting
point paraffin (Paraplast, Sigma). Sections of 7-9 µm were cut and
mounted on poly-L-lysine-coated slides. For cryostat
sections, specimens were fixed overnight in 4% paraformaldehyde, 8 mM NaHPO4, 0.15 M NaCl, pH 7.4;
decalcified, if necessary, in 15% EDTA, 2% paraformaldehyde, 4 mM NaHPO4, pH 7.4; and embedded in Tissue-Tek
O.C.T. compound (Sakura Finetek Europe). 12-µm sections were cut.
Hybridization--
For pretreatment, in situ
hybridization, and washing, the protocol as outlined by Hofstetter
et al. (15) was used. Briefly, sections were deparaffinized
and rehydrated, if necessary, and then digested with proteinase K (1 µg/ml), postfixed, and acetylated in 0.25% acetic anhydride.
Riboprobes were labeled with [35S]CTP and hydrolyzed to
150-nucleotide average length. The sections were hybridized for 12-16
h at 53 °C with riboprobes at a final activity of 1-4 × 107 cpm/ml, depending on their length. After hybridization,
the tissue sections were washed at 53 °C in 50% formamide, 2 × SSC, 1 mM EDTA, 10 mM dithiothreitol;
treated with RNase T1 (1 unit/ml); and washed again at 53 °C in 50%
formamide, 0.2× SSC, 1 mM EDTA. The slides were dehydrated
and dipped in LM1 photoemulsion (Amersham Pharmacia Biotech).
Autoradiography was performed for 5-10 days, and sections were
counterstained in Mayer's hematoxylin (Merck).
Expression and Purification of Recombinant Matrilin-2
Partially overlapping cDNA fragments in the mouse matrilin-2
clones pCRP207, pCRP190, and pCRP12 (4) were combined to full-length cDNA using suitable restriction enzymes. One AflII site
was inserted immediately upstream of the first AUG codon by polymerase
chain reaction. After digestion with AflII and
NotI, a 3.3-kilobase pair cDNA fragment was inserted
into the expression vector pCEP-Pu (16), cleaved previously with
AflII and NotI. The resulting clone, pCEP-Mtr2,
encoded the full-length matrilin-2 precursor, including the secretion
signal peptide.
The recombinant plasmid was introduced into the human embryonic kidney
cell line 293-EBNA (Invitrogen), which constitutively expresses the
EBNA-1 gene product from Epstein-Barr virus. The transfected cells were
selected with 1 µg/ml puromycin and grown to confluency. Secretion of
matrilin-2 into the culture medium was verified by SDS-PAGE and
immunoblotting, using antiserum against a matrilin-2-glutathione
S-transferase fusion peptide (4). Serum-free culture medium
was dialyzed against 2 M urea, 50 mM Tris-HCl,
pH 8.6, and applied to a DEAE-Sepharose-FF column. The bound proteins
were eluted with a linear gradient of 0.05-0.4 M NaCl.
Fractions eluted between 0.1 and 0.2 M NaCl were pooled, and matrilin-2 was further purified by gel filtration through a
Sepharose CL-4B column equilibrated in 2 M urea, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4. The final
purification was achieved on a Heparin-Sepharose column equilibrated in
the same buffer. Matrilin-2 bound exclusively to heparin and was eluted
at about 0.3 M NaCl.
Another cDNA fragment encoding the 10 EGF-like modules and the
vWFA2 module of mouse matrilin-2 was inserted into the
NheI-NotI sites of the pCEP-Pu vector,
downstream of the secretion signal sequence of BM40 (16). The cells
were transfected, and the selection and collection of media followed as
mentioned above. The serum-free medium was diluted 3-fold with 50 mM Tris-HCl, pH 7.4, and applied to a Q-Sepharose-FF
column. The bound proteins were eluted with a linear gradient of
0.05-0.4 M NaCl. Fractions containing the matrilin-2
fragment were refractionated on a Mono-Q fast protein liquid
chromatography column and were apparently free of contaminants. The
purified matrilin-2 fragment was used to immunize rabbits. The
antiserum was purified by affinity adsorption to the antigen.
Immunoblotting of Cell Culture Media and Mouse Organ Extracts
Cell cultures were grown to confluency in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. The cell layers were washed and cultured for 48 h without serum, and the medium was harvested. Several mouse organs were homogenized using a
Polytron homogenizer and extracted with 0.25 M NaCl, 50 mM Tris-HCl, pH 7.4, containing as protease inhibitors 10 mM EDTA, 2 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride. After centrifugation,
aliquots of the supernatant were analyzed. For immunoblotting, samples were submitted to SDS-polyacrylamide gel electrophoresis according to
the protocol of Laemmli (17), using gradient gels of 4-15% polyacrylamide. Proteins were transferred electrophoretically to a
nitrocellulose filter and developed with affinity-purified antiserum to
matrilin-2, followed by peroxidase-conjugated swine anti-rabbit IgG
(DAKO) and the ECL chemiluminescence procedure (Amersham Pharmacia
Biotech) as suggested by the suppliers.
Immunohistochemistry and Immunofluorescence of Cell Layers
Immunohistochemistry was performed as described previously (18),
using the affinity-purified anti-matrilin-2 antiserum together with a
swine anti-rabbit IgG-peroxidase complex and 3-amino-9-ethylcarbazole as substrate on unfixed cryosections from adult and newborn mouse. For
immunofluorescence of cell cultures, cells were plated onto plastic
chamber slides, and after reaching confluency they were fixed in 2%
paraformaldehyde in phosphate-buffered saline for 10 min. In some
experiments, cells were permeabilized by treatment with 10% Nonidet
P-40 in phosphate-buffered saline for 10 min. Nonspecific antibody
binding was blocked by incubation with 1% (w/v) bovine serum albumin
in phosphate-buffered saline for 1 h. The cells were treated with
the affinity-purified antibody to matrilin-2 for 1 h followed by
CyTM3-conjugated affinity-pure goat anti-rabbit IgG
(Jackson ImmunoResearch Laboratories). Pictures were taken with a
Zeiss Axiophot microscope equipped with a fluorescence source.
Analysis of Posttranslational Modifications
The potential substitution with sulfated glycosaminoglycans was
determined by metabolic labeling. 293-EBNA cells transfected with
pCEP-Mtr2 were grown in serum- and sulfate-free minimal essential medium containing 50 µCi/ml [35S]sulfate (Amersham
Pharmacia Biotech). After a 48-h labeling period, media were harvested
and precipitated with trichloroacetic acid (final concentration 12%).
The radiolabeled proteins were separated by SDS-PAGE, and radioactive
bands were visualized by fluorography after treatment with 1 M sodium salicylate.
Chondroitinase ABC and Heparitinase Digestions--
Cell culture
media from 293-EBNA cells transfected with pCEP-Mtr2 were incubated
with 0.7 milliunits/µl heparitinase I (Sigma; from
Flavobacterium heparinum) and 1.7 milliunits/µl
chondroitinase ABC (Sigma) for 7 h at 37 °C. Aliquots of the
digested and control media were analyzed after SDS-PAGE and
immunoblotting with specific antiserum to matrilin-2.
To test for the presence of N-glycosidically linked
oligosaccharides, 293-EBNA cells transfected with pCEP-Mtr2 were grown in serum-free Dulbecco's modified Eagle's medium for 48 h in the presence of tunicamycin (Sigma) at 0.5 mg/ml. Media from
tunicamycin-treated and control cells were analyzed after SDS-PAGE and
immunoblotting with specific antiserum to matrilin-2. Parallel blots
were developed with antibodies to nidogen, which is endogenously
produced by the 293-EBNA cells and served as a positive control of
better known glycosylation (not shown).
N-Glycosidase F Digestion--
Purified matrilin-2 in incubation
buffer containing 50 mM Tris-HCl, pH 7.4, 50 mM
sodium chloride, 0.5% Nonidet P-40, and 0.1% SDS was denatured by
heating at 100 °C for 2 min. After denaturation, the protein was
incubated with 0.3 units of N-glycosidase F (Roche Molecular
Biochemicals) per mg of protein for 20 h at 37 °C. The control
sample was treated similarly without adding N-glycosidase F. The digested and control samples were analyzed by 4-15% SDS-PAGE and
stained with silver nitrate.
Electron Microscopy
Purified matrilin-2 (10 µg/ml) was adsorbed to a 400-mesh
carbon-coated copper grid, which was rendered hydrophilic by glow discharge at low pressure in air. The grid was immediately blotted, washed with two drops of water, and stained with 0.75% uranyl formate
for 15 s. Samples were observed in a Jeol 1200 EX transmission electron microscope operated at 60-kV accelerating voltage and × 75,000 magnification. Images were recorded on Kodak ESTAR Thick Base
4489 plates without preirradiation at a dose of typically 2000 electrons/nm2. Evaluation of the data from electron
micrographs was done as described previously (19).
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RESULTS |
Matrilin-2 Is Deposited in All Forms of Connective Tissue, Some
Types of Smooth Muscle, and a Few Epithelia--
A cDNA fragment
encoding the EGF-like modules and the second vWFA domain of mouse
matrilin-2 was inserted into the pCEP-Pu vector, utilizing the
secretion signal sequence of BM40 (16). The recombinant plasmid was
introduced into the 293-EBNA cell line, where it was stably maintained
in episomal form. The matrilin-2 fragment, secreted into the tissue
culture medium, was purified and used to immunize rabbits. The
antiserum, after affinity purification, specifically reacts with
matrilin-2 and does not show any cross-reactivity (see Fig.
5A).
Matrilin-2 was localized immunohistochemically in cryostat sections of
adult and newborn mice (Fig. 1). The
protein is most abundant in dense connective tissue, including tendon,
ligaments, perichondrium, periosteum, dura mater, epineurium (Fig. 1,
G-I); perimysium of skeletal, heart, and smooth muscle
(Fig. 1, B, C, E, and G);
submucosa of alimentary canal (Fig. 1, E and F);
the reticular layer of dermis (Fig. 1, A and G);
spleen capsule; and the annulus fibrosus, chordae tendineae, and valves
of heart (not shown). In loose connective tissue, local concentration
of the protein is not as high. It is most abundant in the papillary
layer of dermis (Fig. 1A) and spleen trabeculae (not shown).
It is less abundant, but detectable, in the lamina propria of
alimentary canal and the tunica adventitia of blood vessels and
respiratory tract (not shown in detail). Matrilin-2 is also detectable
to variable extents in specialized connective tissue, including the zones of proliferation and hypertrophy in epiphyseal cartilage (Fig.
1G), elastic cartilage of the ear (not shown),
fibrocartilage in the annulus fibrosus of the intervertebral disc (Fig.
1, H-I), and bone, where it lines the marrow cavities. The
protein was abundant in the myometrium (not shown) and was also
detectable between muscularis mucosae and muscularis externa of the
alimentary canal, possibly associated with the nervous plexus (Fig.
1E). The amount of the protein was above the detectability
threshold in a few specialized epithelia, e.g. the
sublingual gland in the newborn head and the lens epithelium or
underlying basement membrane of day 16.5 embryos (not shown). In
nervous tissue, matrilin-2 was observed in the dura and pia mater of
brain and spinal cord as well as the perineurium of peripheral nerves
(Fig. 1H).

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Fig. 1.
Immunohistochemistry of mouse tissue.
Sections from newborn (A, F, G,
H, I, and J) and adult
(B-E) animals were made, and matrilin-2 (A-I)
or matrilin-1 (J) was detected with affinity-purified
antisera. A, in skin from the leg, strong immunostaining was
observed in the papillary layer (pl), and somewhat weaker
staining was seen in the reticular layer (rl) of dermis and
at the base of hair papillae (hp). ep, epidermis.
B, in the heart, the auricle (au) is stained more
intensely than the atrium wall (aw). C, in the
ventricles, the connective tissue surrounding some capillaries
(ca) are positive, and staining of the basement membrane
around myoblasts is weak. The interstitial connective tissue
(ct) is strongly reactive. D, in kidney, the
arcuate arteries (aa) show strong staining. E, the esophagus
epithelium (ep) is not stained, but the underlying basement
membrane and mucosa show strong immunoreaction. In the muscularis
externa, connective tissue (ct) between the circular and
longitudinal smooth muscle cell layers shows strong immunostaining,
possibly associated with the nervous plexus. F, in the oral
cavity of a newborn mouse, fibers of periodontal membrane around the
developing incisor (i) show strong staining as well as the
submucosa of hard and soft palate and the lamina propria of the tongue
(to). There is no staining in the epithelial layers
(ep). G, in the ossifying skeleton of the leg,
the perichondrium (pc), periosteum (po), meniscus, synovial capsule, and
ligaments (li) are the most strongly positive areas.
Moderate immunostaining of the cartilage matrix (c) is
enhanced in the lacunae of hypertrophic chondrocytes (hc).
Strong immunoreaction of the developing tendon (te) and
staining in the dermis and perimysium can also be observed in the leg.
H, in a cross-section of the vertebral column, in addition
to the perichondrium (pc), periosteum, and ligaments, note the signal
in the dura mater spinalis (dm) and the intense labeling of
the epi- and perineurium covering the dorsal (dr) and
ventral root fibers as well as the spinal nerve. vb,
vertebral body. I, in longitudinal sections of the vertebral
column, the annulus fibrosus of the intervertebral discs
(ivd) gave the strongest signal for matrilin-2. Tendon
(te), ligaments (li), and dermis are also
immunoreactive. vb, vertebral body. J, in
parallel sections, matrilin-1 was detected in the vertebral bodies
(vb), both in the epiphysis and the calcified metaphysis.
Bar, 0.05 mm (A and C), 0.1 mm
(B), 0.2 mm (E and H), 0.266 mm
(D), and 0.4 mm (F, G, I,
and J).
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The Matrilin-2 Gene Is Transcribed in Fibroblasts, Osteoblasts,
Smooth Muscle Cells, and Some Epithelial Cells--
In order to reveal
where the matrilin-2 mRNA is produced, eventually leading to
extracellular deposition of the protein, we performed in
situ hybridization. Three antisense riboprobes, complementary to
nonoverlapping regions of the matrilin-2 mRNA were hybridized to
cryostat and paraffin sections of 5-10-week-old mouse. The hybridization of the radioactive riboprobe was detected by
autoradiography and the silver grains were visualized in dark field.
Bright field photomicrographs of the same fields helped to identify the
hybridizing tissues in the sections (Fig.
2). Parallel sections were hybridized with sense riboprobes and verified the specificity of hybridization (not shown).

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Fig. 2.
In situ hybridization of
matrilin-2 cRNA to sections of adult mouse organs. Antisense
riboprobes were hybridized to cryostat sections (A-D) or
paraffin sections (E-H) of 5-week-old mice.
A-H, light field micrographs, A'-H'
are corresponding dark field micrographs. In a longitudinal section of
the tail (A and B), mRNA is detected in
dermis fibroblasts, at the base of hair follicle, and in tendon
(te) cells. A, the stratified, keratinized
epithelium reflects the light, but is not truly positive. B,
the gene is also expressed in the vertebral body in the perichondrium
(pc), ligaments (li), and epiphyseal
cartilage, especially the zone of early hypertrophy (hc) and
in cells lining the bone marrow (bm) cavities. C,
the cross-section of uterus shows an overall strong hybridization
signal. Note the accumulation of grains in the perimetrium
(pm), mesentery (me), and blood vessels
(bv). D, in the heart, the hybridization signal increases
from the ventricles toward the atria and is the strongest in the
atrioventricular valves (av), the chorda tendinea
(ct), and the auricle (not shown). pm, papillary
muscle. E, in the oblique section of trachea (tr)
and esophagus (es), the esophageal epithelium, the ciliated
tracheal epithelium, and seromucous glands (gl) in the
submucosa and adventitia give a strong hybridization signal.
c, cartilage. F, in a larger magnification, the
cartilage ring (c) is weakly positive, in comparison with
the signal in the seromucous glands (gl). G, in
the esophagus, the epithelium is more strongly labeled than the
subepithelial connective tissue. H, in colon, the mucosa
(mu), submucosa, and serosa show a strong signal.
me, muscularis externa. Scale bar, 0.1 mm (A, B, and F-H), 0.2 mm
(C and D), or 0.4 mm (E).
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The results of in situ hybridization confirmed and extended
the data obtained by immunohistochemistry. Connective tissue cells are
clearly positive in dense and loose as well as specialized connective
tissue. Dense connective tissue fibroblasts show characteristic accumulation of grains in tendon, ligaments, perichondrium, periosteum (Fig. 2, A and B); cells in the reticular layer
of dermis and at the base of hair papillae (Fig. 2A); and
annulus fibrosus of heart, atrioventricular valve, and chordae
tendineae (Fig. 2D). Loose connective tissue cells also gave
hybridization signals in the adventitia of trachea (Fig. 2,
E and G) and the mesentery cells (Fig.
2C). Matrilin-2 gene expression was observed in epiphyseal cartilage, in the zones of proliferation and early hypertrophy (Fig.
2B), as well as in osteoblasts of the calvaria (not shown).
Muscle cells showed a detectable level of gene expression, albeit not
as high as that in fibroblasts. A strong in situ
hybridization signal was observed in the organs where previous Northern
hybridization (4) showed a high steady state level of matrilin-2
mRNA. The uterus gave an overall strong hybridization signal (Fig.
2C). Heart was also strongly positive, but with a gradient
toward the regions richer in connective tissue cells, e.g.
the atria, auricle, valves, and chordae tendineae (Fig.
2D).
In addition to connective tissue cells and myoblasts, some epithelia
also showed clearly positive hybridization signals. In paraffin
sections, the secretory epithelium of esophagus, the mucosa, and serosa
of colon as well as the seromucous glands of trachea showed strong
hybridization (Fig. 2, E-H).
Relative Abundance of Matrilin-2 mRNA in Established Cell
Lines--
In some cases it was difficult to determine with certainty
the cell types where the gene expression was observed by in
situ hybridization. For example, smooth muscle cells are in close
association with fibroblasts, and epithelial cells form thin layers in
close proximity to the underlying connective tissue. Therefore, we
examined matrilin-2 mRNA and protein production in homogeneous
cultures of permanent cell lines.
Total RNA samples were isolated from cultured cells, and the relative
amount of matrilin-2 mRNA was estimated by Northern hybridization
(Fig. 3). In all of the cell lines
examined, expression of the gene was observed. We previously
demonstrated that the fibroblastic cell lines L929, WEHI 164, NIH 3T3,
and the rat osteogenic sarcoma UMR-106 expressed the gene (4). In the
present experiment, the mRNA level in NIH 3T3 cells (Fig. 3,
lane 1) was compared with other cell lines. In
two samples, isolated from the rat chondrosarcoma cell line and the
9E11G smooth muscle-like cells, the matrilin-2 mRNA level was
higher than in NIH3T3 cells. The other smooth muscle cell line, SMC,
isolated from rat aorta, and the differentiated skeletal muscle myotube
C2/7 contained less, but significant, amounts of matrilin-2 mRNA,
confirming that the gene can be expressed in cells with myoblast
characteristics. The intestinal epithelial cell line IEC6 and the
Schwannoma cell line RN22 also expressed the gene at a detectable
level. The least amount of matrilin-2 mRNA was found in the
keratinocyte carcinoma PVD(A)I. In a separate experiment, matrilin-2
mRNA was detected in the SVEC endothelial cell line (not shown). In
summary, not only the connective tissue cell types contained matrilin-2
mRNA, but gene expression was also observed in myoblasts and in the
epithelial and endothelial cell lines tested.

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Fig. 3.
Filter hybridization of cell culture RNA
samples. The RNA samples were isolated from the cell lines
indicated at the top. The filter was hybridized
consecutively with matrilin-2 cDNA (MTR2) and a chicken
rDNA fragment (28S). For a description of the cell lines,
see "Materials and Methods."
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Previous analysis indicated that the sequence variability within the
unique region may be a consequence of alternative splicing. Therefore,
we performed reverse transcription-polymerase chain reaction analysis
to determine if there is further mRNA heterogeneity within the
translated region. In the SVEC endothelial and the rat chondrosarcoma
cell lines, alternative splicing affected only the middle third of the
unique module but not the region encoding the coiled coil, the vWFA2,
or EGF-like modules (data not shown). Systematic comparison of the RNA
samples showed that a 57-nucleotide-long region is alternatively
retained or spliced out in all of the 10 cell lines studied (Fig.
4).

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Fig. 4.
Analysis of mouse matrilin-2 mRNA
heterogeneity within the unique region by reverse
transcription-polymerase chain reaction. The analysis was
performed as described under "Materials and Methods." Template RNA
was isolated from rat chondrosarcoma tissue (RCS) or the
mouse and rat cell lines indicated at the top. C,
amplification of the longer segment using pCRP12 cDNA clone (4) as
template. M, pUC12 HaeIII DNA ladder. On the
left, sizes of the two alternative splice products are
indicated. At high concentrations, the two specific polymerase chain
reaction products formed heteroduplexes of lower electrophoretic
mobility (hd).
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The Matrilin-2-specific Antiserum Reacts with Multiple Protein
Bands upon Electrophoresis of Unreduced Samples--
In order to gain
information about the relative amount and presumed oligomeric structure
of matrilin-2, culture media from cell lines and extracts from tissues
were compared by SDS-PAGE and, in part, immunoblotting to the
matrilin-2 expressed by transfected cells (Fig.
5C). The specificity of the
antiserum was assessed by comparison of media from 293-EBNA cells
before and after transfection with the recombinant plasmid pCEP-Mtr2,
encoding the full-length mouse matrilin-2 (Fig. 5A). The
transfected cells secreted into the culture medium matrilin-2, which
was detected by the antiserum as a group of antigenic bands in
nonreducing SDS-PAGE with apparent Mr values
ranging from 70,000 to 500,000. On the basis of the calculated
Mr = 104,300 for the nonmodified matrilin-2
monomer, faster migrating bands must represent degradation products,
and the slower ones may correspond to oligomeric forms. We can conclude that the secretory signal peptide of matrilin-2 was functionally active
in 293-EBNA cells and that matrilin-2 can form oligomers stable enough
to resist denaturing electrophoresis if the sample is not reduced. The
medium from nontransfected 293-EBNA cells did not show any reactivity
with the antiserum, demonstrating the specificity of this reagent.

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Fig. 5.
SDS-PAGE analysis of recombinant matrilin-2
before (A) and after (B) purification
and of matrilin-2 in cell media and tissue extracts
(C). A, media from 293-EBNA cells
either transfected with pCEP-Mtr2 (lane 1) or without
transfection (lane 2) were applied to SDS-PAGE without prior
reduction. Matrilin-2 was detected by immunoblotting using an
affinity-purified antiserum to matrilin-2. B, purified
recombinant matrilin-2 was submitted to SDS-PAGE as in A
without ( SH) or after (+SH) sample reduction,
and the gel was stained with Coomassie Brilliant Blue. C,
media from 293-EBNA cells transfected with pCEP-Mtr2 and a variety of
cell lines as well as extracts from mouse skin and uterus were
submitted to SDS-PAGE and immunoblotting as in A. Nonreduced
samples were run on 4-15% polyacrylamide gels; reduced ones were
analyzed on 10% polyacrylamide gels. fn, the position of
fibronectin.
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The relative amounts of the different forms of matrilin-2, resolved by
electrophoresis, differ somewhat between the crude media of transfected
cells and preparations chromatographically purified from this source
(Fig. 5B). The reason for this difference is that during
purification we removed degradation fragments and enriched slightly for
the oligomeric forms. Reduction of the purified material yielded
several closely spaced bands with apparent Mr values between 100,000 and 130,000. The size heterogeneity was even
more apparent when nonreduced samples from media of a variety of cell
lines as well as extracts of skin and uterus were analyzed by
immunoblotting (Fig. 5C). All the cell lines tested secreted detectable amounts of matrilin-2, with the exception of the epithelial cell line IEC6. Because the IEC6 cells showed production of matrilin-2 mRNA, we need to assume that the mRNA is translated and/or the protein is secreted with a very low efficiency in that cell line. While
the 293-EBNA cells that had been transfected with pCEP-Mtr2 produced
four groups of bands that may represent monomers, dimers, trimers, and
tetramers, most cell lines secreted mainly the smallest and largest
components. Extracts of skin and uterus showed, in addition, a relative
abundance of the potential trimers. The analysis was, however,
complicated by the presence of discrete differences in the
electrophoretic mobility of corresponding matrilin-2 bands between
sources (Fig. 5C).
Analysis of Posttranslational Modifications in
Matrilin-2--
While the presence of matrilin-2 oligomers of variable
sizes can be explained by source-specific differences in assembly, the
multiple size of monomers indicated that, in addition to this, posttranslational processing occurs, possibly in a tissue-specific manner. A set of experiments was designed to explore this possibility (Fig. 6). The most obvious cause for the
heterogeneity would be proteolysis after secretion into the culture
medium. Samples of medium were, therefore, harvested at different times
after medium change, frozen, and analyzed by immunoblotting (Fig.
6A). The band patterns of matrilin-2 obtained from medium
kept with the cells at 37 °C for different periods of time show
great similarities. With the exception of degradation fragments, which
are clearly smaller than monomers, all components could be observed
already after 8 h, indicating that proteolysis in the medium or in
the intercellular compartment of tissues is not the major cause of heterogeneity. The size differences could also be due to a variable substitution with glycosaminoglycans or oligosaccharides. To test for
the presence of sulfated glycosaminoglycans, cultures of wild type and
matrilin-2-transfected 293-EBNA cells were, therefore, labeled with
[35S]sulfate, and the media were analyzed by SDS-PAGE
followed by autofluorography (Fig. 6B). The labeling
patterns seen with the two sets of cultures showed considerable
similarities, and, most importantly, only minor amounts of label were
detected in the position of matrilin-2 monomers, which were shown by
parallel immunoblotting to be the major matrilin-2 species in the
sample. Further, media from matrilin-2-transfected 293-EBNA cells were digested with the glycosaminoglycan-degrading enzymes
chondroitinase ABC and heparitinase under standard conditions (Fig.
6C). The treated samples appeared unchanged when compared
with untreated samples by immunoblotting for matrilin-2, which provided
further evidence against a substitution with chondroitin/dermatan
sulfate or heparan sulfate. The potential presence of
N-linked oligosaccharides was analyzed in two experiments.
In one approach, parallel cultures of matrilin-2-transfected 293-EBNA
cells were treated or not treated with the inhibitor tunicamycin (Fig.
6, D and E). SDS-PAGE and immunoblot analysis of
the media showed small but significant increases in the mobility of
matrilin-2 bands, which were, because of the better resolution, more
easily seen by electrophoresis after reduction (Fig. 6E).
Treatment of purified matrilin-2 with N-glycosidase F gave
similarly small, but clearly detectable, decreases in the apparent
Mr as visualized by SDS-PAGE (Fig.
6F). Taken together, these results show that the recombinant
matrilin-2 carries N-linked oligosaccharides, but the extent
of this glycosylation is not sufficient to explain the heterogeneity
observed both with recombinantly expressed matrilin-2 and with
matrilin-2 derived from cell lines and tissues. Neither tunicamycin
treatment nor digestion with N-glycosidase F decreased the
heterogeneity of recombinant matrilin-2, a finding that points to
another underlying cause.

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Fig. 6.
Analysis of the heterogeneity of matrilin-2
by SDS-PAGE. A, accumulation of matrilin-2 in cell
culture media. Samples were harvested from 293-EBNA cells transfected
with pCEP-Mtr2 8, 24, and 48 h after medium change. Aliquots were
applied to a 4-15% SDS-PAGE without prior reduction. Matrilin-2 was
detected by immunoblotting using an affinity-purified antiserum.
B, electrophoretic analysis of sulfate-labeled products
secreted into the medium. 293-EBNA cells either transfected with
pCEP-Mtr2 (lane 2) or without transfection (lane
3) were cultured in the presence of 50 µCi/ml
[35S]sulfate for 48 h, and aliquots of the media
were applied to a 4-15% SDS-PAGE without prior reduction. The
radioactive macromolecules were detected by autofluorography. In
parallel, a lane containing medium from the transfected
cells was blotted and reacted with the matrilin-2 antiserum (lane
1). C, media from matrilin-2-transfected cells were
digested with heparitinase (lane 1), not digested
(lane 2), or digested with chondroitinase ABC (lane
3). The samples were applied to gel electrophoresis and developed
by immunoblotting as in A. D and E,
293-EBNA cells transfected with pCEP-Mtr2 were cultured in the presence
(+) or absence ( ) of 0.5 mg/ml tunicamycin, and aliquots of media
were applied to either 4-15% SDS-PAGE without prior reduction
(D) or 6% SDS-PAGE after reduction (E).
Matrilin-2 was detected in the samples by immunoblot analysis.
F, purified recombinant matrilin-2 was digested (+) or not
digested ( ) with N-glycosidase F, applied to a 4-15%
SDS-PAGE, and stained with silver nitrate.
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Electron Microscopy Reveals That Matrilin-2 Occurs as a Mixture of
Monomers and Oligomers--
Purified, recombinant matrilin-2 (Fig.
5B) was negatively stained with uranyl formate to produce
high resolution images by electron microscopy. Fields of stained
molecules showed particles of heterogenous size (Fig.
7, top), and close examination
of single particles revealed that all species from monomers to
tetramers were present in the sample. At high magnification it was seen that, irrespective of the number of subunits within a molecule, all
subunits are joined at a single point (Fig. 7, bottom),
which presumably represents the coiled-coil
-helix assembled from
the C-terminal domains. In most cases, the subunit is seen as a looped structure with a more heavily stained hole in the middle and
frequently, but not always, carrying most mass in the periphery. The
average diameter of the loops was 8 nm. Occasionally particles were
seen where the loop was opened into a flexible rod, better representing the tandem array of domains making up the subunits.

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Fig. 7.
Negative stain electron microscopy of
purified recombinant matrilin-2. An overview (top) and
selected particles at higher magnification (bottom) show the
heterogeneity in oligomerization and illustrate the presence of
subunits in a looped (particles 1, 2,
4, and 6 from the left) or open
(particles 3 and 5 from the
left) conformation. The bar corresponds to 100 nm
for the overview (top) and 25 nm for the enlarged single
molecules (bottom).
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The Protein Forms an Extracellular Filamentous Network in Cell
Culture--
In order to study the extracellular assembly forms of
matrilin-2, the pericellular matrix of cultured primary smooth muscle cells from rat aorta was analyzed in immunofluorescence microscopy with
specific antibodies against matrilin-2 (Fig.
8). Matrilin-2 was detected in an
extensive, branched fibrillar network. The network may be connected to
the cell surface, but in experiments where the cells were permeabilized
(Fig. 8B), and thereby better outlined through the staining
of the intracellular pool of immunoreactive matrilin-2, it was clear
that the fibrils were not limited to the cell surface but extended over
and beyond cells.

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Fig. 8.
Immunofluorescence microscopy of
matrilin-2-containing assemblies in the pericellular matrix of cultured
primary rat aorta smooth muscle cells. Confluent cell layers were
fixed in 2% paraformaldehyde, and matrilin-2 was detected with
affinity-purified antibodies without (A) and with
(B) permeabilization with 10% Nonidet P-40. An extensive
network of matrilin-2-containing filaments is seen extracellularly, and
after permeabilization an intracellular precursor pool is also
detected. Bar, 20 µm.
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DISCUSSION |
The Matrilin-2 Gene Is Transcribed in Fibroblasts, Myocytes, and
Epithelial Cells, but the Protein Is Transported to Connective Tissue
Structures--
During our previous work, matrilin-2 mRNA was
found in skin of the tail, calvaria, heart, uterus, and brain (4). From
the wide distribution of the transcript, expression of the gene in cell
types common to many organs was presumed. In situ
hybridization and Northern blotting data represented here confirmed
gene expression in connective tissue cells but extended the expression
pattern to muscle and epithelial cells. Most of the expressor cell
types are of mesodermal origin, but the epithelia of trachea,
esophagus, and intestine, which showed strong in situ
hybridization signals, develop from endoderm. Expression of the
matrilin-2 gene in cells of various developmental lineages predicts a
complex mechanism for regulation of gene expression.
In some cases, synthesis of the matrilin-2 mRNA was found in
epithelial cells, but deposition of the protein was observed only in
subepithelial connective tissue. It is likely that the matrilin-2
mRNA is translated in epithelial cells and that the protein is
specifically transported to the subepithelial regions. It is known that
some components of basement membranes are made by epithelial cells;
others are contributions from the underlying connective tissue
fibroblasts. Nidogen is exclusively made and the
1 and
2 chains
of collagen IV are predominantly produced by dermal fibroblasts, but
all three chains of laminin-1 can be expressed by keratinocytes,
especially at the beginning of coculturing (20). In the case of
matrilin-2, the possibility cannot be excluded that epithelial cells
synthesize mRNA, but translation or protein transport is blocked.
The absence of matrilin-2 from media of the IEC6 intestinal epithelial
cell line (Fig. 5C) supports this alternative, but this cell
line might also not be representative for the differentiation state of
epithelial cells in vivo.
Matrilin-2 Displays an Unusual Extent of Structural
Heterogeneity--
In preparations of recombinant matrilin-2, at least
three bands of reduced monomers can be resolved by SDS-PAGE (Fig.
6E). The size differences observed between matrilin-2 from
different sources appear too large to be explained by these three bands occurring in variable proportions. In studies using the recombinant matrilin-2, we have excluded extracellular proteolysis and substitution with glycosaminoglycans or N-linked oligosaccharides as the
major source of heterogeneity. Since the recombinant matrilin-2 is
derived from cloned cDNA, alternative splicing may also be
excluded. Remaining possibilities are unconventional forms of
substitution and/or processing by intracellular or cell surface-bound
proteases during biosynthesis and secretion. Further studies will be
needed to evaluate these alternatives and to determine if the
processing has functional consequences.
SDS-PAGE revealed also a second level of heterogeneity, with bands
corresponding to various oligomeric species being present in
recombinant matrilin-2 (Fig. 5). This cannot be explained by imperfect
assembly due to overexpression in the transfected 293-EBNA cells, since
similarly variable oligomerization is seen in tissue extracts and in
media from a variety of cell lines (Fig. 5C). Different
assembly forms representing all species from monomers to tetramers are
seen in electron microscopy of samples purified under nondenaturing
conditions (Fig. 7). This proves that the occurrence of multiple bands
in SDS-PAGE is not due to incomplete closure of interchain disulfide
bridges followed by dissociation of the coiled-coil upon treatment with
SDS, but either that coiled-coil
-helices with varying numbers of
protein strands are formed or that matrilin-2 subunits are specifically
proteolytically cleaved at a site close to the coiled-coil region
before or around secretion. Pan and Beck (6) recently investigated the
oligomerization of a synthetic peptide corresponding to the coiled-coil
domain of matrilin-2. By means of chemical cross-linking, they found that a trimer was the preferred species, but even at high cross-linking reagent concentrations, at which a corresponding matrilin-1-related peptide exclusively shows a single band corresponding to a trimeric state (21), the matrilin-2 peptide showed nearly equal amounts of
material running in the position of monomers, dimers, and trimers (6).
Under these conditions, higher molecular mass bands, although of lower
concentrations, which can be interpreted to represent tetramers and
pentamers, were also found. The multiplicity of oligomers could not be
abolished by increasing the ionic strength as it was found for the
matrilin-1 peptide (21). Although these authors argue that the
monomeric and dimeric states observed upon SDS-PAGE might be due to the
limitations of the cross-linking approach, they could not rule out that
indeed different oligomerization states for the matrilin-2 related
peptide are possible. Studies of matrilin-1 derived from tissue sources
show that homooligomerization of the naturally occurring protein leads
to the formation of trimers as the single predominant species (18).
Recent work has, however, demonstrated restrictions in the stringency
of coiled-coil formation among matrilins in showing that a single point
mutation in the coiled-coil domain of matrilin-1 may cause it to form
tetramers instead of trimers (22) and that matrilin-1 occurs in
vivo not only as a homotrimer but also in a heterotetramer
together with matrilin-3 subunits (23). In the case of matrilin-2, it
could be that this lack of stringency leads to the protein occurring in vivo as a mixed set of monomers and oligomers. This
heterogeneity may have functional consequences, since ligand binding
sites will occur in variable copy number within a single molecule, and
differences in oligomerization state may, because of cooperativity,
lead to differences in affinity for macromolecular ligands that may
bind to more than one subunit within a single matrilin-2 molecule. The
possibility cannot be excluded that the oligomers detected by
immunoblot are heterooligomers formed between matrilin-2 and another
matrilin. A candidate would be matrilin-4, which is expressed outside
cartilage as shown by Northern blots that give signals in lung, liver,
brain, sternum, kidney, and heart (5).
Evidence for Self-interaction of vWFA Domains of Matrilin-2 and Its
Potential Role in Fibrillar Network Formation in Cell Culture--
In
electron microscopy using negative stain, the matrilin-2 subunits are
most often seen as loop structures with a diameter of 8 nm, but in a
smaller fraction of the particles they are seen to open into a flexible
rod (Fig. 7). Based on x-ray crystallographic data, we may assume a
diameter of 3.6 nm for vWFA domains and 2.1 nm for EGF-like domains. In
a tandem array, the two A domains together with the 10 EGF-like domains
would have a length of about 28 nm. A circle with a diameter of 8 nm
has a circumference of 25 nm, and the measurements are, therefore,
compatible with a model of the matrilin-2 subunit where a loop is
formed through interactions between the two A domains. In earlier
studies of matrilin-1, we similarly observed a compact structure of the
subunits (18). Domain interactions within the subunit had to be assumed to explain why the length determined from electron micrographs was
considerably smaller than that expected from the dimensions of the
domains in tandem array. Since the A-domains in matrilin-1 are
connected by a single EGF-like domain, substructures within the subunit
could not be resolved, while in the matrilin-2 subunit, where the
A-domains are connected by 10 EGF-like domains, a loop around a heavily
stained hole could be seen. For sterical reasons, it is most likely
that the loop is formed by the EGF-like domains, which are held
together through an interaction between the two A-domains. Indeed,
structures indicating self-interactions between Adomains have been
observed by electron microscopy of von Willebrand factor (24) and of
the N-terminal globule of the
3 chain of type VI collagen (25).
Further, by x-ray crystallography of von Willebrand factor A1-domain a
contact surface was detected between A1-domain pairs, suggesting a
hypothetical mechanism for the regulation of protein assembly and
heterologous ligand binding mediated by homophilic interactions of type
A-domains (26). vWFA domains in those proteins have also been shown to
be involved in other interactions (25, 27).
By immunofluorescence microscopy of the matrix formed by cultured
smooth muscle cells (Fig. 8), we could show that matrilin-2 molecules
assemble into an extracellular fibrillar network, where each fibril may
have a length of several cell diameters and often divides into smaller
branches. In similar studies, matrilin-1 was found in chondrocyte
cultures in close association with collagen II fibers (28), and a
filamentous network, independent of collagen fibers, was also observed
when matrilin-1 was overexpressed using a retroviral system (29).
Matrilin-1 constructs, in which the vWFA1 domain had been deleted,
assembled into trimers but could not form filamentous structures,
thereby implicating the vWFA1 domain as being involved in the
polymerization reaction leading to fibril formation. In analogy, it is
likely that the vWFA domains of matrilin-2 may interact with each other
and that filaments may be formed by this interaction. We do not know at
present the exact molecular composition of the matrilin-2-positive
extracellular filaments, but we are pursuing the study of such
interactions of matrilin-2 with itself and with other extracellular
macromolecules that may form the basis for fibril formation.