From the Institute for Biochemistry and the Center
for Molecular Medicine Cologne Service Laboratory, Medical Faculty,
University of Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, Germany
Received for publication, January 22, 2001, and in revised form, February 12, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Matrilin-4 is the most recently identified member
of the matrilin family of von Willebrand factor A-like domain
containing extracellular matrix adapter proteins. Full-length
matrilin-4 was expressed in 293-EBNA cells, purified using affinity
tags, and subjected to biochemical characterization. The largest
oligomeric form of recombinantly expressed full-length matrilin-4 is a
trimer as shown by electron microscopy, SDS-polyacrylamide gel
electrophoresis, and mass spectrometry. Proteolytically processed
matrilin-4 species were also detected. The cleavage occurs in the short
linker region between the second von Willebrand factor A-like domain
and the coiled-coil domain leading to the release of large fragments
and the formation of dimers and monomers of intact subunits still containing a trimeric coiled-coil. In immunoblots of calvaria extracts
similar degradation products could be detected, indicating that a
related proteolytic processing occurs in vivo. Matrilin-4 was first observed at day 7.5 post-coitum in mouse embryos.
Affinity-purified antibodies detect a broad expression in dense and
loose connective tissue, bone, cartilage, central and peripheral
nervous systems and in association with basement membranes. In the
matrix formed by cultured primary embryonic fibroblasts, matrilin-4 is
found in a filamentous network connecting individual cells.
The matrilins are a novel family of oligomeric extracellular
proteins containing von Willebrand factor A
(vWFA)1-like domains (1). In
mouse, four matrilins exist with matrilin-1, previously referred to as
cartilage matrix protein, being the prototype member. The expression of
matrilin-1 and -3 is restricted to skeletal tissues, whereas the
matrilin-2 and -4 have a broad tissue distribution. It has been
suggested that matrilins act as adapter molecules connecting other
proteins and proteoglycans in the extracellular matrix (2).
Mouse matrilin-4 has the modular structure typical for matrilins and
consists of two vWFA-like domains connected by four epidermal growth
factor (EGF)-like domains and a C-terminal We recombinantly expressed the full-length mouse matrilin-4 in a
mammalian expression system. The recombinant protein was used for
production of a specific antiserum, which allowed immunohistochemical characterization of matrilin-4 expression in the mouse and an analysis
of supramolecular assembly forms in the extracellular matrix formed by
cultured primary embryonic fibroblasts. The full-length protein was
used for structural studies by which the molecular dimensions and
oligomeric state of recombinant matrilin-4 could be determined.
Structural analysis also revealed a unique, conserved cleavage site
used for proteolytic processing of matrilin-4. Extraction of native
matrilin-4 from mouse tissues allowed a characterization of the
naturally occurring oligomers by SDS-PAGE and Western blotting.
Expression and Purification of Recombinant
Matrilin-4--
Murine matrilin-4 cDNA was generated by reverse
transcriptase-polymerase chain reaction on total lung RNA. Suitable
primers introduced a 5'-terminal SpeI and a 3'-terminal
NotI restriction site. The cDNA was inserted into the
expression vector pCEP-Pu (5) downstream of the sequence encoding the
BM-40 signal peptide. The vector contained either an N-terminal
His6-Myc tag downstream of the signal peptide coding
sequence and upstream of a 5' NheI site (6) or a C-terminal
strepII tag (7) downstream of the NotI site.
The recombinant plasmids were introduced into the human embryonic
kidney 293-EBNA cell line (Invitrogen) by transfection with DAC-30TM (Eurogentec). The cells were selected with
puromycin (1 µg/ml) and were transferred to serum-free medium for
harvesting of the recombinant protein. After filtration and
centrifugation (1 h, 10,000 × g), cell culture
supernatant containing the N-terminally His6-Myc-tagged
matrilin-4 was applied to a nickel-nitrilotriacetic acid column (10 ml,
Qiagen). The bound fraction was eluted with 0.25 M
imidazole in 2 M urea, 0.3 M NaCl, 50 mM NaH2PO4, pH 8.0. The
matrilin-4-containing fractions were diluted with an equal volume 2 M urea, 50 mM Tris-HCl, pH 7.4, and
concentrated on a SP HiTrap column (1 ml, Amersham Pharmacia Biotech).
Elution was achieved with 1 M NaCl, 2 M urea,
50 mM Tris-HCl, pH 7.4. The cell culture supernatant
containing the C-terminally strepII-tagged matrilin-4 was dialyzed
against 1 mM EDTA, 0.1 M Tris-HCl, pH 8.0, applied to a streptactin column (3 ml, IBA), and eluted with 2.5 mM desthiobiotin, 1 mM EDTA, 0.1 M
Tris-HCl, pH 8.0.
Preparation of Antibodies to Matrilin-4--
The purified,
N-terminally His6-Myc-tagged matrilin-4 was used to
immunize rabbits. The antiserum obtained was purified by affinity
chromatography on a column containing the C-terminally strepII-tagged
matrilin-4 coupled to CNBr-activated Sepharose (Amersham Pharmacia
Biotech). The specific antibodies were eluted with 0.1 M
glycine, pH 2.5, and the eluate was neutralized with 1 M
Tris-HCl, pH 8.8.
Tissue Extraction--
Mouse calvaria was extracted for 6 h
at 4 °C with 10 volumes (ml/g wet tissue) of 0.1 M NaCl,
50 mM Tris-HCl, pH 7.4, containing 10 mM EDTA
and 2 mM N-ethylmaleimide, and mouse brain each
consecutively with 5 volumes for 1 min at 4 °C with 0.15 M NaCl, 50 mM Tris-HCl, pH 7.4 (TBS), TBS
containing 10 mM EDTA, TBS containing 10 mM EDTA and 4 M urea, and finally 0.1 M Tris-HCl,
pH 7.4, containing 10 mM EDTA and 4 M GdnHCl.
All extraction buffers contained 2 mM phenylmethylsulfonyl fluoride.
SDS-Polyacrylamide Gel Electrophoresis, Immunoblotting, and
Determination of N-terminal Sequences--
SDS-polyacrylamide gel
electrophoresis was performed as described by Laemmli (8). For
immunoblots the proteins were transferred to nitrocellulose and
incubated with the appropriate affinity-purified rabbit antibody
diluted in TBS containing 5% low fat milk powder. Bound antibodies
were detected by luminescence using peroxidase-conjugated swine
anti-rabbit IgG (Dako), 3-aminophthalhydrazide (1.25 mM), p-coumaric acid (225 µM), and 0.01%
H2O2. For N-terminal sequencing, 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
determined in a Applied Biosystems 473A protein sequencer.
In Situ Hybridization--
Tissues from newborn mice were fixed
overnight with 4% paraformaldehyde in phosphate-buffered saline (PBS),
pH 7.4, at 4 °C, washed overnight with PBS (4 °C), dehydrated,
and embedded in paraffin. Sections of 7 µm were cut, mounted on
3-aminopropyltriethoxysilane-treated glass slides, dewaxed in
xylene, and rehydrated. After washing in PBS they were digested with
110 milliunits/ml proteinase K, postfixed, and acetylated with 0.25%
acetic anhydride. The sections were hybridized overnight at 50 °C
with digoxigenin-labeled riboprobes covering the nucleotides 9-704 of
the matrilin-4 cDNA (3). After hybridization, the sections were
washed in 50% formamide, 2× SSC (0.3 M NaCl containing
0.03 sodium citrate) for 30 min at 50 °C, digested with RNase A,
washed once with 2× SSC and twice with 0.2× SSC for 20 min at
50 °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 (9).
Immunohistochemistry--
Immunohistochemistry was performed on
frozen and paraffin-embedded sections of fetal, newborn, and 6-week-old
mice. The tissues were prefixed, and the tissues from 6-week-old mice
were demineralized with 5% HNO3 overnight at 4 °C.
After demineralization the tissues were transferred for 24 h into
5% Na2SO4. Deparaffinization ensued through
incubation for 30 min in Rotihistol (Carl Roth GmbH, Karlsruhe, Germany) at 52 °C. After rehydration, the sections were digested with hyaluronidase (500 units/ml PBS, pH 5-6; Sigma) at 37 °C for
30 min followed by proteinase K (5.5 milliunits/ml, in 1 mM EDTA, 10 mM Tris-HCl, pH 7.5; Sigma) at 37 °C for 5-10
min. They were briefly washed with PBS and postfixed with 4%
formaldehyde in PBS for 15 min. After three washing steps, the sections
were blocked for 1 h with 1% (w/v) bovine serum albumin in TBS
and incubated with the affinity-purified matrilin-4 antibodies
overnight at 4 °C. The primary antibodies were visualized by
consecutive treatment of the sections for 1 h with
biotin-SP-conjugated goat anti-rabbit IgG (Dianova) and alkaline
phosphatase-conjugated streptavidin (Dianova). All antibodies and the
alkaline phosphatase-conjugated streptavidin were diluted in 1% (w/v)
bovine serum albumin in TBS, and the slides were developed with fast
red TR/naphthol (Sigma). Immunofluorescence and peroxidase staining was
performed as described previously (10).
Immunofluorescence Microscopy of Cell Cultures--
Cells were
plated onto glass or permanox chamber slides, and after reaching
confluence cells were fixed in 4% formaldehyde in PBS. Nonspecific
antibody binding was blocked by incubation with 1% (w/v) bovine serum
albumin in TBS, and immunolabeling was done by incubation with
affinity-purified antibodies to matrilin-4 for 1 h followed by
CyTM3-conjugated affinity-pure goat anti-rabbit IgG (Jackson
ImmunoResearch Laboratories).
Electron Microscopy--
Negative staining was performed as
described previously (11, 12) using non-reduced or reduced samples of
C-terminally strepII-tagged matrilin-4 (5-10 µg/ml) stained with
0.75% uranyl formate. Reduction was carried out for 2 h at
20 °C using dithiothreitol (DTT) at concentrations between 0.05 and
10 mM. The reaction was stopped by alkylation with a 3-fold
excess of N-ethylmaleimide for 1 h at 20 °C prior to
analysis by SDS-PAGE. The sample that showed quantitative conversion to
the monomeric form at the lowest DTT concentration was subjected to
electron microscopy.
In Gel Digestion of Proteins Separated by SDS-PAGE--
Cut-out
gel bands were reduced with 20 mM DTT for 15 min at
37 °C and alkylated with 50 mM iodoacetamide for 15 min
at 37 °C in 50 mM ammonium bicarbonate, pH 8.0. The in
gel digestion with trypsin or endoprotease Glu-C was carried out
overnight at an enzyme concentration of 12.5 ng/µl in 50 mM ammonium bicarbonate buffer, pH 8.0, at 37 °C.
Subsequent elution was performed in two steps with 0.1%
trifluoroacetic acid/acetonitrile (2:3) for a total of 30 h. The
supernatants were pooled and dried in a vacuum centrifuge, and the
peptides were dissolved in 5% formic acid.
Mass Spectrometry--
MALDI-TOF mass spectrometry was carried
out in a Bruker Reflex III mass spectrometer equipped with a 26-sample
SCOUT source and video system, a nitrogen UV laser (
Electrospray mass spectrometry was carried out with a Micromass Q-TOF
II instrument (Micromass, Manchester, UK). The samples were introduced
by nano-ESI. Prior to the analysis the samples were desalted and
concentrated using home-built microcolumns consisting of ~2.5 µl of
C18-reversed phase material (ODS-AQ, 120 Å, 50 µm; YMC Europe,
Schermbeck, Germany) in a gel-loader tip (Biozym, Hessisch-Oldendorf,
Germany). The adsorbed peptides were washed with 5% formic acid and
subsequently eluted with 3 µl of 5% formic acid/methanol (1:1)
directly into a metal-coated nanospray capillary (Protana A/S, Odense,
Denmark). Spray voltage was adjusted between 2700 and 3000 V, and block
temperature was set to 30 °C. For MS experiments collision energy
was set to 12 V, whereas for MS/MS experiments this was raised to a
value between 25 and 40 V depending on size and charge state of the
precursor ion to obtain optimal fragment ion spectra. Calibration was
carried out between m/z 400 and 2500 using 40 mM
H3PO4.
Recombinant Expression and Purification of Matrilin-4--
cDNAs
encoding the sequence of mature mouse matrilin-4 were cloned into
vectors carrying nucleotide sequences coding for either an N-terminal
His6-Myc tag (HM4) or a C-terminal strepII tag (M4S). The
use of different tags at different positions allowed the evaluation of
their potential effects on the properties of the recombinant protein.
The constructs were inserted into the pCEP-Pu vector utilizing the
secretion signal sequence of BM-40 (5). The recombinant plasmids were
introduced into human embryonic kidney 293-EBNA cells and maintained in
an episomal form. The secreted matrilin-4 protein constructs were
purified from the cell culture medium by affinity chromatography on
either a streptavidin or a Ni2+ column. The correct usage
of the predicted signal peptide cleavage site was confirmed by
N-terminal protein sequencing of the purified recombinant
strepII-tagged matrilin-4.
Recombinant Expression of Matrilin-4 Yields Trimers, Dimers, and
Monomers--
SDS-PAGE analysis of both the N- and C-terminally tagged
proteins showed the presence of trimers, dimers, and monomers,
indicating that the tags do not influence the oligomerization (Fig.
1). The His6-Myc tag and the
strepII tag contribute 5.5 and 1.3 kDa, respectively, to the mass of
each subunit. Without reduction, His6-Myc-tagged matrilin-4
gave three strong bands with apparent molecular weights of 235 (t),2 155 (d + cc), and 66 kDa (m Proteolytic Processing of Recombinant Matrilin-4--
The
consistent occurrence of matrilin-4 as a mixture of trimers, dimers,
and monomers could be due either to an incomplete assembly of a triple
coiled-coil or to cleavage of subunits close to the assembly domain,
after formation of the trimer, yielding dimeric and monomeric
fragments. Direct SDS-PAGE and Western blot analysis of non-purified
culture media from 293-EBNA cells transfected with N- and C-terminally
tagged matrilin-4 (Fig. 3) provided an indication of a C-terminal processing of matrilin-4. Bands that correspond to intact trimers as well as dimers and monomers with one
and two additional coiled-coil strands, respectively, were observed
(Fig. 3, lanes 1 and 2). Also truncated monomers
lacking the coiled-coil domain of the N-terminally
His6-Myc-tagged and C-terminally strepII-tagged matrilin-4
could be detected (Figs. 3 and 4).
Furthermore, the C-terminal processing was confirmed by Western blot
analysis of reduced samples from cell culture supernatant of 293-EBNA
cells expressing N-terminally His6-Myc-tagged matrilin-4
(Fig. 3, lanes 3 and 4). Both the unprocessed and
the C-terminally processed matrilin-4 subunits were detectable by the
antiserum against matrilin-4.
We used MALDI-TOF mass spectrometry to confirm the C-terminal
processing by analysis of the fragments released upon cleavage. Partial
reduction of the strepII-tagged matrilin-4 on the target yielded new
peaks at 146 (d) and 80 kDa (m + cc) in addition to the 72.9 kDa
(m) fully reduced monomer (Fig. 2B). This results from the
loss of 7-kDa fragments from the 153-kDa (d + cc) and the 87-kDa (m + 2cc) molecule ions, respectively (Fig. 2A). After complete
reduction, in addition to the fully reduced monomer of 72.9 kDa, a
molecule ion of 6944.8 Da (cc) was detected (Fig. 2C,
inset), presumably resulting from the release of a
disulfide-bonded fragment. The mass of this fragment corresponds to the
C-terminal end of the recombinant matrilin-4 starting with the amino
acid residue Gly572 (3) and lacking the very
C-terminal lysine residue, with the theoretical mass of 6943.7 Da (Fig.
4). Matrilin-4 fragments without the coiled-coil domain could not be
detected as they do not bind to the streptactin column.
SDS-PAGE of recombinant strepII-tagged matrilin-4 without prior
reduction shows a band with an apparent molecular mass of ~21 kDa
(3cc) that is lost upon reduction (Fig. 1). Isolation of this band
followed by in gel trypsin digestion and MALDI-TOF analysis revealed
the presence of a tryptic fragment of a mass of 2552.2 Da corresponding
to the sequence Gly572 to Arg594, indicating a
cleavage between amino acid residues Glu571 and
Gly572 in the region between the second vWFA-like domain
and the coiled-coil domain (Fig. 4). Under reducing conditions a 7-kDa
(cc) fragment of strepII-tagged matrilin-4 was isolated by SDS-PAGE.
Subsequent in gel digestion with trypsin and endoprotease Glu-C
followed by ESI Q-TOF mass spectrometry yielded two sequences,
572GIGATE and AAAWSHPQFE, originating from the
N-terminal end and the C-terminal strepII tag of the 7-kDa (cc)
fragment, respectively (Fig. 4), supporting that the fragment results
from a proteolytic cleavage between the second vWFA-like domain and the
two cysteine residues stabilizing the coiled-coil. In addition, the
localization of the cleavage site between residues Glu571
and Gly572 was unequivocally confirmed by Edman sequencing
of the 21-kDa (3cc) band (Fig. 1) which gave
572GIG(A/H)TELRS (Fig. 4). A cleavage at this point
releases a matrilin-4 subunit, less its coiled-coil domain, which
remains disulfide-bonded to the assembly domains from the other
matrilin-4 chains (Fig. 4).
Electron Microscopy--
The purified strepII-tagged full-length
matrilin-4 was submitted to electron microscopy after negative staining
with uranyl formate (Fig. 5). The protein
particles were heterogeneous in size, and a closer examination of
single particles revealed that all species from monomer to trimer were
present in the sample, in agreement with the results from SDS-PAGE and
MALDI-TOF analysis. 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 other matrilins (2, 10, 11). This indicates assembly of the oligomers via the C-terminal coiled-coil domain. A compact structure was seen at the center of the particles, representing the C-terminal, globular vWFA-like domains. These are
connected to the extending N-terminal vWFA-like domains (diameter 5 ± 1 nm) by a well resolved stalk made up from the four EGF-like repeats. The center-to-center distance between the vWFA-domains is
14 ± 2 nm. After mild reduction with 5 mM DTT, the
monomers released show two globular units, representing vWFA-like
domains, connected by the flexible rod of EGF-like repeats (results not shown). Such reduced monomers have a shape similar to subunits found in
non-reduced samples with the same center-to-center distance between the
globular domains.
Matrilin-4 Is Immunologically Distinct from Other
Matrilins--
Purified, N-terminally His6-Myc-tagged
matrilin-4 was used for the immunization of a rabbit. The resulting
antiserum was affinity-purified by adsorption to a column of
CNBr-Sepharose to which strepII-tagged matrilin-4 has been coupled. By
this procedure antibodies specific for matrilin-4 were obtained that
lacked reactivity to the tags. The specificity was verified by SDS-PAGE
of culture media from 293-EBNA cells expressing N- and C-terminally
tagged matrilin-4 followed by immunoblotting (Fig. 3). It was further
shown that these antibodies do not cross-react with other matrilins in
immunoblots (Fig. 6A).
Characterization of Matrilin-4 Extracted from Mouse
Tissues--
To study the structure of tissue-derived matrilin-4 and
to identify similarities or differences to the recombinantly expressed protein, extracts from several murine tissues were analyzed by non-reducing SDS-PAGE and immunoblotting using the affinity-purified antiserum against matrilin-4. Among these tissues, sternum, calvaria, cerebellum, medulla oblongata, spinal cord, cortex, lung, and muscle
were positive, whereas heart, testis, colon, and adrenal gland were
negative for matrilin-4 (results not shown). In an extract of murine
calvaria, a major band with an apparent molecular mass of 200 kDa,
representing the trimeric form of matrilin-4, was detected (Fig.
6A, lane 4). Two additional bands were detected at 180 and 170 kDa that may represent degradation products or alternative assembly forms of matrilin-4. A direct comparison after
longer exposure of a calvaria extract with recombinant matrilin-4 shows
bands in the tissue extract presumably corresponding to the 150-kDa
dimer (d + cc), the 95-kDa monomer (m + 2cc), and the 67-kDa cleaved
monomer (m
Sequential extraction of mouse brain indicated a differential
solubility of the individual oligomeric forms of matrilin-4 (Fig.
6C). All forms could be solubilized in EDTA-containing
buffer but not in TBS alone, which indicates a contribution to the
anchorage by a divalent cation-dependent mechanism.
Denaturation with urea released large additional amounts of
trimeric matrilin-4 and the 170-kDa form. Further denaturation with
4 M GdnHCl released only the trimeric form of matrilin-4,
indicating that the unprocessed protein is most strongly bound to
insoluble tissue elements.
Matrilin-4 Is Deposited in Loose and Dense Connective Tissue, below
Epithelia, in Smooth Muscle and in Nervous Tissue--
The tissue
distribution of matrilin-4 was studied by immunohistochemistry using
affinity-purified antibodies on cryostat and paraffin-embedded sections
of embryonic, newborn, and 6-week-old mice. The earliest expression of
matrilin-4 was detected by immunofluorescence in the ectoplacental
cone at day 7.5 post-coitum (results not shown). At day 10.25 post-coitum matrilin-4 is present in the heart (Fig.
7A), cephalic mesenchymal
tissue (Fig. 7B), and somites, while at day 14.5 post-coitum it was found in the primordial skeleton and cranial
bones, in the larynx, and in the lung (Fig. 7C). A similar
characterization of the expression of matrilin-1, -2, and -3 in mouse
embryos has been performed earlier (15).
After birth, the protein is abundant in dense connective tissue,
including perichondrium, periosteum, tendon, and ligaments (Fig.
8A). Furthermore, matrilin-4
is highly expressed in cartilage (Fig. 8, A-D and
I). In the knee of a newborn mouse (Fig. 8A), matrilin-4 is present at the joint surface, as well as in the resting,
proliferating, and hypertrophic cartilage, while at 6 weeks (Fig.
8B) it was found in the epiphyseal growth plate and in the
cartilage remnants in the metaphysis but not in the articular cartilage. In loose connective tissue, it is present under stratified squamous epithelia (Fig. 9B'),
in the papillary layer of the dermis (Fig. 8E), in the
smooth muscle layer of the intestine (Fig. 8F), and in the
interalveolar walls of the lung (Fig. 8G). In the esophagus (Fig. 9B') and the dermis (Fig. 8E) matrilin-4
was found associated with the basement membrane. In the eye (Fig.
8H) it is expressed in the cornea and sclera and strongly in
the limbus. Furthermore, matrilin-4 is present in the fibrous skeleton
and in the valves of the heart (Fig. 8, D and I),
in the primordium of the incisors (Fig. 8C), and at lower
levels in the adrenal gland (not shown).
Matrilin-4 is also widely distributed in the central nervous system and
in peripheral nerves. In the adult brain (Fig. 8J), matrilin-4 was observed in the neocortex, striatum, pallidum, thalamus,
cerebellum, pons, medulla oblongata, and in the choroid plexus (results
not shown). It was further present in the spinal cord (Fig.
8D) and in the sympathetic ganglion (results not shown).
The Matrilin-4 Gene Is Transcribed in Fibroblasts, Chondroblasts,
Osteoblasts, Epithelial, Muscle, and Neuronal Cells--
The cellular
origins of matrilin-4 were revealed by in situ hybridization
and the results compared with the information on the deposition of the
protein derived from immunohistochemistry. In sections through a
humeroradial joint (Fig. 9, A and A'), the highest mRNA levels could be detected in the perichondrium and at
the joint surface, in good agreement with to the staining pattern obtained by immunohistochemistry. In the esophagus (Fig. 9,
B and B'), epithelial cells show clear
hybridization signals. Matrilin-4 mRNA was also detected in
Purkinje cells in the cerebellum (Fig. 9, C and
C'). Skeletal muscle cells and osteoblasts showed a weak but
significant level of gene expression (results not shown).
Matrilin-4 Forms an Extracellular Filamentous Network in Cell
Culture--
In order to study the extracellular assembly forms of
matrilin-4, mouse primary embryonic fibroblasts were cultured in the presence of ascorbate and analyzed by immunofluorescence. The filamentous network (Fig. 10) seen was
reminiscent of that detected by antibodies to matrilin-1 (16), -2 (11),
and -3 (10) in other cell culture systems.
Recombinant Matrilin-4 Forms Homotrimers--
SDS-PAGE analysis,
MALDI-TOF mass spectrometry, and electron microscopy showed the
production of matrilin-4 homotrimers in 293-EBNA cells transfected with
matrilin-4 cDNA. In electron microscopy (Fig. 5) the trimeric form
shows similarities to the bouquet-like shape observed for other
matrilins (2, 10, 11) with a compact center from which stalk-like
structures with globular ends extend. Self-interactions of the
vWFA-like domains, as proposed to occur between the A1 and A2 domains
in matrilin-1 and -2 (2, 11), were not visible.
Zhang and Chen (17) used rules based on the nature of the hydrophobic
residues in positions "a" and "d" of the heptad repeat of
coiled-coils in GCN4 leucine zipper mutants (18) to predict that based
on sequence matrilin-4 would form trimers. Our experimental work shows
that this prediction holds true, as is also the case for matrilin-1 and
-3 that according to the same rules should form trimers and tetramers,
respectively. However, the largest assembly form of matrilin-2 is a
tetramer (11) and not a trimer as predicted on the basis of the rules
of Harbury et al. (18). The trimers seen in matrilin-2
preparations are, in analogy with matrilin-4, likely to be degradation
products. In contrast to the tetrameric assembly form of full-length
matrilin-2, the isolated matrilin-2 coiled-coil domain assembles
preferentially into a trimer (19). It appears that prediction of the
oligomeric state of matrilin coiled-coil domains (17) leaves
uncertainties and that experimental analysis is required.
Previous studies showed that the coiled-coil domain of matrilin-1 folds
autonomously (20), even though the second vWFA-like domain possibly
also plays a role (21). Matrilin-3, which lacks a second vWFA-like
domain, and matrilin-2, which carries a unique segment adjacent to the
coiled-coil, form tetramers, whereas matrilin-1 and -4 form trimers. In
contrast, hetero-oligomers of matrilin-1 and -3 occur as both trimers
and tetramers (11, 13, 17). Therefore, the oligomerization may also be
influenced by the distance between the second vWFA-like domain and the
coiled-coil domain.
Matrilin-4 Dimers and Monomers Are Formed by Cleavage of
Trimers--
Supernatants of matrilin-4 transfected 293-EBNA cells
contain a complex mixture of different oligomeric forms. Such
heterogeneity was also seen for matrilin-2 and -3, both when expressed
recombinantly and when extracted from tissues (10, 11, 17), although
matrilin-1 appears more homogenous in tissues (Fig. 6) (22). It
has been suggested that the heterogeneity could result either from a
proteolytic processing (10, 11) or from an imperfect oligomerization
(10, 17). Proteolytic degradation products of matrilin-4 could be demonstrated by mass spectrometry, Edman sequencing, and SDS-PAGE analysis. A cleavage site could be identified that is located between
the second vWFA-like domain and the coiled-coil domain, leading to the
formation of truncated forms that are trimeric with respect to their
coiled-coil domains but lack the more N-terminal parts of the subunit.
Only dimers and monomers containing the intact trimeric coiled-coil
were detected by MALDI-TOF analysis (Fig. 3), which excludes imperfect
oligomerization as the cause for heterogeneity. The demonstration of
N-terminally truncated but trimeric matrilin-4 directly shows that the
oligomerization is achieved by the C-terminal coiled-coil domain.
Further studies will be needed to determine when and where the
processing occurs in vivo and the nature of the protease involved.
Matrilin-4 Extracted from Tissues Shows a Degradation Pattern
Reminiscent of Recombinant Matrilin-4--
The pattern of degradation
products present in recombinant matrilin-4 shows similarities to that
seen for matrilin-4 extracted from tissues (Fig. 6B),
indicating that proteolytic processing occurs also in vivo.
The two glutamic acid residues N-terminal to the cleavage site of
matrilin-4 are conserved in all matrilins, indicating that this
cleavage site may be used also in other matrilins. For matrilin-2 it
was shown that the heterogeneous band pattern seen in SDS-PAGE
is not due to differential substitution with glycosaminoglycans or
N-linked oligosaccharides (11).
Oligomeric forms of matrilins will be multivalent and able to interact
with and connect multiple other extracellular matrix molecules. A
proteolytic cleavage of one or two subunits would release interaction
partners from the supramolecular complex formed and could modulate the
overall binding properties of matrilins. This is supported by the fact
that the larger forms of matrilin-4 require more strongly denaturing
conditions for their extraction from tissues (Fig. 6). Still a
proportion of matrilin-4 molecules could be extracted with
EDTA-containing buffer alone, indicating a cation-dependent
anchorage in the extracellular matrix. A similar behavior has been
described for matrilin-1 (2). The vWFA-like domains of matrilins
contain the metal ion-dependent adhesion site sequence
motif, and the fact that at least some interactions of matrilin-1 and
-4 can be stabilized by divalent cations indicates that this motif is
indeed functional.
Proteolytic cleavage of extracellular matrix molecules is a common
mechanism for the regulation of the matrix architecture during
remodeling, and perturbations in the balance of proteolysis and
de novo protein synthesis may form a basis for pathological changes in tissue structure and function. Furthermore, degradation products may have a function different from that of the unprocessed protein, e.g. endostatin, a fragment of the NC1 domain of
collagen type XVIII, acts as an angiogenesis inhibitor (23). It remains to be investigated if fragments released by degradation of matrilins may have such independent new functions.
Matrilin-4 Is the Most Widespread Member of the Matrilin
Family--
Matrilin-4 is present in highest amounts in tissues
derived from mesenchymal cells, but additionally in situ
hybridization showed transcription in epithelial and neuronal cells,
which have developed from endoderm and neuroectoderm, respectively. In
contrast to matrilin-2 and matrilin-1 and -3 that have a complementary tissue distribution, matrilin-4 is found in most locations where one of
the other matrilins is expressed. Similar to matrilin-2, it is present
in a variety of non-skeletal tissues, but in contrast matrilin-4 is
broadly expressed in cartilage where the matrilin-2 expression is
limited to the hypertrophic zone of the growth plate. Furthermore,
matrilin-4 is present in the surface layer of the developing articular
cartilage, where no other matrilin has been detected. The partial
overlap in the spatial expression of matrilins may allow a functional
redundancy that could explain the lack of an overt phenotype seen when
the matrilin-1 gene is interrupted (14, 24).
Matrilin-4 Forms an Extended Extracellular
Network--
Immunofluorescence microscopy of the matrix formed by
cultured primary embryonic fibroblasts shows matrilin-4 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. 10). Similar results were obtained for matrilin-1 in
cultures of chicken chondrocytes, where a more extended
collagen-dependent (16, 25) and a pericellular,
collagen-independent network was described (16). Matrilin-2 and -3 also
form fibrillar networks when expressed by cultured rat aorta smooth
muscle (11) or rat chondrosarcoma cells (10), indicating mechanisms of
supramolecular assembly common to all matrilins.
The Matrilin Family Consists of Four Members--
Matrilin-4 is in
all probability the final member of the matrilin family. No further
members have been detected in the sequence data bases including those
provided by the human genome project. This is supported by phylogenetic
studies showing that each of the four matrilin genes is located in a
gene cluster together with members of other tetralog protein families
such as the syndecans (26). Each member of the matrilin family has in
the meantime been characterized with regard to structure and
distribution in embryonal and mature tissues, which allows some
conclusions about the overall role of the matrilin family. The
widespread distribution of matrilins with at least one family member
being present in nearly every tissue and at very different time points
of development indicates an important role in the structure and
function of an extracellular matrix. A common feature of the matrilins
is the ability to participate in the formation of a filamentous network but probably also to bind ligands adjacent to or within those filaments. This is facilitated by the oligomeric structure, which enables the simultaneous interaction with multiple ligands. Ligand binding affinity could be modified by altering cooperativity through proteolytic processing, an event that may not be restricted to matrilin-4. The broad tissue distribution of matrilin-4, which nearly
covers the expression domains of all other matrilins, makes it likely
that matrilin-4 is the most versatile member of the family. Therefore,
our further work will focus on matrilin-4 in the hope that detailed
studies of this matrilin will enhance the general understanding of
matrilin structure and function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical coiled-coil domain predicted to mediate oligomerization (3). In addition, a unique
splice variant, which does not contain the N-terminal vWFA-like domain,
was identified in mouse (3). Matrilin-4, like matrilin-1, lacks the
stretch of frequently positively charged amino acids found in other
matrilins between the signal peptide cleavage site and the vWFA-like
domain at the N-terminal end of the mature protein. The mouse
matrilin-4 precursor consists of 624 amino acid residues, and after
cleavage of the 21-amino acid signal peptide a mature protein with a
predicted minimal mass of 66.4 kDa is formed (3). Human
matrilin-4 is highly homologous to the mouse protein with an overall
identity of 91% and a maximum identity of 97% in the second vWFA-like
domain. Due to a mutation in the splice donor site of the third intron
of the human gene, the exon, which corresponds to that specifying the
first EGF-like domain in mouse, is not expressed in man. Instead of the
four EGF-like domains present in mouse, the human matrilin-4 contains three, two, or one EGF-like domains, probably depending on a
differential usage of splice acceptor sites in the exons coding for
EGF-like domains (4). When matrilin-4 expression was studied in the mouse by Northern hybridization, mRNA could be detected in lung, sternum, brain, kidney, and heart (3). In the human, matrilin-4 expression was demonstrated by reverse transcriptase-polymerase chain
reaction in lung and placenta, as well as in the embryonic kidney cell
line 293-EBNA and in WI-26 fibroblasts (4). The broad tissue
distribution is reminiscent of that of matrilin-2, and phylogenetic
analyses show (1) that matrilin-4 and matrilin-2 descend from a common
ancestor, further indicating a close relationship.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
max = 337 nm), and a dual channel plate detector (Bruker Daltonik, Bremen,
Germany). 1 µl of the sample solution was placed on the target, and 1 µl of a freshly prepared saturated solution of
-cyano-4-hydroxycinnamic acid (Aldrich) in
acetonitrile/H2O (2:1) with 0.1% trifluoroacetic acid was
added. For MALDI-TOF mass spectrometry of intact proteins, sinapinic
acid (Aldrich) was used as matrix. When required, the protein was
reduced with 0.01 M DTT 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 Reflex III. Calibration was
based upon the Mr of recombinant protein A
(Repligen) of 44,610.3 and bovine serum albumin dimer (Sigma) of
132,859.0, respectively. Between 50 and 500 single laser shots were
summed into an accumulated spectrum. External calibration was carried
out using a mixture of six synthetic peptides with molecular masses
between 1046 and 2466 Da as well as the protonated dimer of the matrix
(379 Da).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cc), respectively, and fainter bands at 87 (m +2 cc) and
73 kDa (m). Due to incomplete closure of disulfide bonds minor amounts
of uncleaved, free monomers are often seen in matrilin samples. The
strepII-tagged matrilin-4 gave strong bands with apparent molecular
weights of 210 (t), 150 (d + cc), and 95 kDa (m + 2cc), respectively,
and fainter bands at lower molecular weights. The minor bands seen
between 65 and 80 kDa most likely represent a mixture of intact
monomers with proteolytically processed monomers. The band at 21 kDa is a disulfide-bonded complex of three coiled-coil region fragments (see
also Fig. 4). Reduction of the strepII-tagged matrilin-4 yielded a
strong band with an apparent molecular mass of 67 kDa (m) (Fig. 1).
MALDI-TOF mass spectrometry of non-reduced, strepII-tagged matrilin-4
resulted in three single charged molecule ion peaks at 219 (t), 153 (d + cc), and 87 kDa (m + 2cc) (Fig.
2A). After complete reduction,
one single molecule ion peak was detected at 72.9 kDa (m) (Fig.
2C). The theoretical molecular mass of the strepII-tagged, mature matrilin-4 is 67.6 kDa, indicating that 7% of
the mass of the recombinantly expressed protein was contributed by
post-translational modifications. The larger species observed without
prior reduction presumably represent trimers and modified dimers and
monomers of the 72.9-kDa subunit.
View larger version (44K):
[in a new window]
Fig. 1.
SDS-PAGE of recombinant matrilin-4
proteins. N-terminally His6-Myc-tagged
(HM4, lane 1) and C-terminally strepII-tagged
matrilin-4 (M4S, lanes 2 and 3) were submitted to
SDS-PAGE without (lanes 1 and 2) or with (lane 3)
prior reduction and stained with silver (lane 1) or
Coomassie Brilliant Blue (lanes 2 and 3). Electrophoresis
was done on gels containing 4-16% (lane 1) or 4-12%
polyacrylamide (lanes 2 and 3). For nomenclature see also
Footnote 2 and Fig. 4.
View larger version (18K):
[in a new window]
Fig. 2.
MALDI-TOF spectra of purified recombinant
C-terminally strepII-tagged matrilin-4 under non-reducing
(A), partially reducing (B), and
fully reducing (C) conditions. B and
C, the sample was reduced on target with 10 mM
DTT. The inset in C shows the low molecular
weight range. M2+ designates the 2-fold positively charged
molecule ion species. a.i., arbitrary intensity.
m/z, mass per charge. For nomenclature see also Footnote 2 and Fig. 4.
View larger version (44K):
[in a new window]
Fig. 3.
Immunoblot analysis of recombinant matrilin-4
proteins. Cell culture media of 293-EBNA cells transfected with
C-terminally strepII-tagged (M4S, lane 1) or
N-terminally His6-Myc-tagged matrilin-4 (HM4,
lanes 2-4) were submitted to SDS-PAGE, transferred to
nitrocellulose, and developed with either the affinity-purified
antiserum against matrilin-4 (lanes 1-3) or an monoclonal
antibody against the N-terminal Myc tag (lane 4).
Lanes 3 and 4, the samples were reduced prior to
electrophoresis. Electrophoresis was done on gels containing 4-15
(lane 1 and 2) or 4-8% (lane 3 and 4)
polyacrylamide. For nomenclature see also Footnote 2 and Fig. 4. Note
that the band patterns in this immunoblot cannot be directly compared
with those in Fig. 1, because in Fig. 1 certain fragments had been
enriched in the affinity chromatography step by virtue of carrying
either an N- or C-terminal tag, and detection was done by protein
staining.
View larger version (49K):
[in a new window]
Fig. 4.
Proteolytic processing of matrilin-4.
Schematic representation of the forms of matrilin-4 identified by mass
spectrometry and SDS-PAGE of recombinant proteins. cc,
coiled-coil region. The underlined sequence was confirmed by
N-terminal Edman degradation, and the strepII tag sequence is given in
italics. Bold letters indicate sequences
confirmed by ESI Q-TOF.
View larger version (121K):
[in a new window]
Fig. 5.
Electron microscopy of negatively stained
recombinant strepII-tagged matrilin-4. The overview (A)
and the panels of selected particles (B-D) demonstrate the
heterogeneity in subunit number. The bar corresponds to 100 nm in the overview (A) and to 25 nm in the panels of
selected particles (B-D).
View larger version (63K):
[in a new window]
Fig. 6.
SDS-PAGE and immunoblot of tissue
extracts. Proteins extracted from calvaria (A and
B) or brain (C) of newborn mice and recombinant
C-terminally strepII-tagged matrilin-4 (B) were separated on
4-8% polyacrylamide gels without prior reduction, transferred to
nitrocellulose, and detected with specific antisera. A,
antisera to each of the matrilins were used (M1-4).
C, brain of a newborn mouse (C) was sequentially
extracted with TBS alone (TBS), TBS with 10 mM
EDTA (EDTA), TBS with 4 M urea and 10 mM EDTA (Urea), and TBS with 4 M
GdnHCl (GuHCl). (Cv, calvaria; M4S,
C-terminally, strepII-tagged matrilin-4.) For nomenclature see also
Footnote 2 and Fig. 4. The dashed lines connect bands in
A and B representing the same components.
cc) bands of recombinant strepII-tagged matrilin-4
(Fig. 3 and Fig. 6B), indicating that the cleavage characterized for the recombinant matrilin-4 expressed in 293-EBNA cells may occur also in vivo. The nitrocellulose blots
obtained with calvaria extract were also incubated with
affinity-purified antisera raised against matrilin-1, -2, and -3 (Fig.
6A). All matrilins are expressed in mouse calvaria, but
hetero-oligomers formed between matrilin-4 and another matrilin could
not be identified on the basis of co-migration of immunoreactive bands.
Furthermore, the band patterns obtained for matrilin-1 and -3 (Fig.
6A, lanes 1 and 3) indicate that these
mainly occur as homo-oligomers in murine calvaria, even though
heterotetramers of these matrilins have been unambiguously demonstrated
in bovine cartilage (10, 13). This is in agreement with the recent
result that hetero-oligomers formed between matrilin-1 and -3 cannot be
detected in mouse epiphyseal and tracheal cartilage (14).
View larger version (72K):
[in a new window]
Fig. 7.
Immunohistochemical detection of matrilin-4
in fetal mouse tissues. Matrilin-4 was detected in sections of
mouse embryos from days 10.25 and 14.25 post-coitum. At day
10.25 post-coitum, matrilin-4 was found in the trabeculae of
the heart (A) as well as in the cephalic mesenchymal tissue
(mt) (ne, neuroepithelium). At day 14.25 post-coitum (C), matrilin-4 is present in the
cartilage primordia of the vertebra (vb), sternum
(sn), and the hip (hp), in the anlage of cranial
bones such as the bones of the nasal cavity (nc), Meckels
cartilage (mc), and basisphenoid bone (bb).
Furthermore, it could be detected in the thyroid (td) and
tracheal (tc) cartilages and around the terminal bronchioli
of the lung (lg). Bar, 1.8 (C), 0.08 mm (A and B).
View larger version (121K):
[in a new window]
Fig. 8.
Tissue distribution of matrilin-4.
Immunohistochemistry was performed on paraffin-embedded
(A-E and G-J) or frozen (F) tissue
from newborn (A, C E, and G-I) or 6-week-old
(B and J) mice, or from a day 15.5 post-coitum mouse embryo (F), which were
demineralized if needed (B). Tissues were incubated with an
affinity-purified antiserum against matrilin-4 followed by either
biotin-SP-conjugated goat anti-mouse IgG and alkaline
phosphatase-conjugated streptavidin (A
D and
G-J), or by CyTM-3-conjugated goat anti-rabbit IgG
(E and F). In the knee of a newborn mouse
(A), matrilin-4 was highly expressed throughout the
cartilage including the developing articular surface (as),
the perichondrium (pc), periost (po), and
ligaments (lm). In the epiphyseal cartilage primordium of
the distal femur (fm) and proximal tibia (tb),
matrilin-4 was found in the zone of resting (rt),
proliferating (pl), and hypertrophic (ht)
cartilage. In the knee of a 6-week-old mouse (B), a strong
signal could be detected in the epiphyseal growth plate (gp) and in the
calcified area (ca) of newly synthesized bone. In a
transverse section through the head (C), matrilin-4 was
found in the developing occipital bones (ob), bones of the
nasal cavity (nc), the nasal septum (ns), the
inner and outer mesenchymal layer of hair follicles (hf),
and in the primordium of the upper incisors (ic). In the
thorax (D), matrilin-4 is present in the trachea
(tc), in costal cartilage (cc), in vertebral
bodies (vb) and their transverse processes (tp),
in the manubrium sternum (ms) and processus xiphoideus
(px), in the clavicula (cv), and in the spinal
cord (sc) (os, esophagus). In the skin
(E), matrilin-4 was found in the fibrillar network of the
papillary layer (pl) and associated with the
dermal-epidermal basement membrane (bm). Further signals
were detected in the smooth muscle (sm) layer of the
intestine (F) (lm, lumen) and in the
interalveolar walls (iw) of the lung (G). In the
eye (H), matrilin-4 is present in the cornea (cn)
and sclera (sr) and strongly in the limbus (lb).
In the heart, matrilin-4 was found in the fibrous skeleton
(arrow, D) and in the valves (vv, I). Matrilin-4
is broadly distributed in the brain (C and J),
where it is present in the neocortex (nc), striatum
(st), pallidum (pd), thalamus (tm),
hippocampus (hc), cerebellum (cb), pons
(ps), and medulla oblongata (mo). Bar,
2.8 (C), 2.3 (J), 1.6 (D), 0.7 (H), 0.6 (I), 0.5 (A and
B), 0.3 (G) and 0.075 mm (E and
F).
View larger version (72K):
[in a new window]
Fig. 9.
Comparison of the distribution of matrilin-4
protein and mRNA. Antisense riboprobes labeled with
digoxigenin were hybridized to paraffin-embedded sections of a
humeroradial joint of a newborn mouse (A) and to sections of
esophagus (B) and cerebellum (C) of an adult
mouse. Parallel sections were immunolabeled with affinity-purified
matrilin-4 antibody (A', B', and
C'). In the joint (jt), hybridization is
strongest at the developing articular surface corresponding to strong
signals of the immunofluorescence (hm, humerus;
rd, radius). In the esophagus, mRNA is present in the
epithelial cells (ec), and the protein is associated with
the underlying basement membrane. Hybridization of the cerebellum
showed high transcription levels in the Purkinje cells (pk),
whereas the protein was detected most strongly in the white matter
(wm) and in the granular layer (gl).
Bar, 0.7 (C), 0.4 (B), and 0.3 mm
(A).
View larger version (122K):
[in a new window]
Fig. 10.
Immunofluorescence microscopy of
matrilin-4-containing filaments in the extracellular matrix of primary
embryonic mouse fibroblasts. Cells were cultured in the presence
of ascorbate. Matrilin-4 was detected with affinity-purified antibodies
followed by a CyTM3-conjugated affinity-purified goat anti-rabbit IgG.
Bar, 0.14 mm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Ferenc Deák and Neil Smyth for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant WA 1338/2-1 and by Köln Fortune program of the Medical Faculty of the 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: Institute for Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, D-50931 Cologne, Germany. Tel.: 49-221-478-6990; Fax: 49-221-478-6977; E-mail: Raimund.Wagener@uni-koeln.de.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M100587200
2
To allow simple identification of protein
components on the basis of the results of mass spectrometry (Fig. 2)
and protein sequencing (Fig. 4), we introduce a nomenclature where t
represents trimer, d represents dimer, and m represents monomer. These
designations are followed by a cc or a + cc. The
cc
indicates that the coiled-coil region is absent from the subunit, and + cc shows that the fragment carries additional coiled-coil regions bound by disulfide bonds and derived from other cleaved subunits. The digit
before cc refers to the number of these coiled-coil fragments when more
than one is carried. For example m + 2cc means a monomer connected by
disulfide bonds to two additional coiled-coil region fragments thereby
forming a triple coiled-coil
-helix. For a schematic depiction of
this nomenclature see Fig. 4.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: vWFA, von Willebrand factor A; EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF matrix-assisted laser desorption ionization/time-of-flight, ESI Q-TOF, electrospray ionization quadrupole/time-of-flight; DTT, dithiothreitol; PBS, phosphate-buffered saline; MS, mass spectrometry; ESI, electrospray ionization.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Deák, F., Wagener, R., Kiss, I., and Paulsson, M. (1999) Matrix Biol. 18, 55-64[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Hauser, N.,
and Paulsson, M.
(1994)
J. Biol. Chem.
269,
25747-25753 |
3. | Wagener, R., Kobbe, B., and Paulsson, M. (1998) FEBS Lett. 436, 123-127[CrossRef][Medline] [Order article via Infotrieve] |
4. | Wagener, R., Kobbe, B., and Paulsson, M. (1998) FEBS Lett. 438, 165-170[CrossRef][Medline] [Order article via Infotrieve] |
5. | Kohfeldt, E., Maurer, P., Vannahme, C., and Timpl, R. (1997) FEBS Lett. 414, 557-561[CrossRef][Medline] [Order article via Infotrieve] |
6. | Wuttke, M. (2001) Characterization of the Structure and Function of Recombinant and Native Human Bone Sialoprotein. Doctoral dissertation , University of Cologne, Germany |
7. | Smyth, N., Odenthal, U., Merkl, B., and Paulsson, M. (2000) Methods Mol. Biol. 139, 49-57[Medline] [Order article via Infotrieve] |
8. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
9. | De Block, M., and Debrouwer, D. (1993) Anal. Biochem. 215, 86-89[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Klatt, A. R.,
Nitsche, D. P.,
Kobbe, B.,
Mörgelin, M.,
Paulsson, M.,
and Wagener, R.
(2000)
J. Biol. Chem.
275,
3999-4006 |
11. |
Piecha, D.,
Muratoglu, S.,
Mörgelin, M.,
Hauser, N.,
Studer, D.,
Kiss, I.,
Paulsson, M.,
and Deák, F.
(1999)
J. Biol. Chem.
274,
13353-13361 |
12. | Engel, J., and Furthmayr, H. (1987) Methods Enzymol. 145, 3-78[Medline] [Order article via Infotrieve] |
13. |
Wu, J. J.,
and Eyre, D. R.
(1998)
J. Biol. Chem.
273,
17433-17438 |
14. |
Aszódi, A.,
Bateman, J. F.,
Hirsch, E.,
Baranyi, M.,
Hunziker, E. B.,
Hauser, N.,
Bösze, Z.,
and Fässler, R.
(1999)
Mol. Cell. Biol.
19,
7841-7845 |
15. | Segat, D., Frie, C., Nitsche, P. D., Klatt, A. R., Piecha, D., Korpos, E., Deák, F., Wagener, R., Paulsson, M., and Smyth, N. (2000) Matrix Biol. 19, 649-655[CrossRef][Medline] [Order article via Infotrieve] |
16. | Chen, Q., Johnson, D. M., Haudenschild, D. R., Tondravi, M. M., and Goetinck, P. F. (1995) Mol. Biol. Cell 6, 1743-1753[Abstract] |
17. |
Zhang, Y.,
and Chen, Q.
(2000)
J. Biol. Chem.
275,
32628-32634 |
18. | Harbury, P. B., Zhang, T., Kim, P. S., and Alber, T. (1993) Science 262, 1401-1407[Medline] [Order article via Infotrieve] |
19. |
Pan, O. H.,
and Beck, K.
(1998)
J. Biol. Chem.
273,
14205-14209 |
20. | Beck, K., Gambee, J. E., Bohan, C. A., and Bächinger, H. P. (1996) J. Mol. Biol. 256, 909-923[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Chen, Q.,
Zhang, Y.,
Johnson, D. M.,
and Goetinck, P. F.
(1999)
Mol. Biol. Cell
10,
2149-2162 |
22. | Paulsson, M., and Heinegård, D. (1981) Biochem. J. 197, 367-375[Medline] [Order article via Infotrieve] |
23. | Zätterström, U. K., Felbor, U., Fukai, N., and Olsen, B. R. (2000) Cell Struct. Funct. 25, 97-101[CrossRef][Medline] [Order article via Infotrieve] |
24. | Huang, X., Birk, D. E., and Goetinck, P. F. (1999) Dev. Dyn. 216, 434-441[CrossRef][Medline] [Order article via Infotrieve] |
25. | Winterbottom, N., Tondravi, M. M., Harrington, T. L., Klier, F. G., Vertel, B. M., and Goetinck, P. F. (1992) Dev. Dyn. 193, 266-276[Medline] [Order article via Infotrieve] |
26. | Gibson, T. J., and Spring, J. (2000) Biochem. Soc. Trans. 28, 259-264[Medline] [Order article via Infotrieve] |