From the Orthopaedic Research Laboratories, University of
Washington, Seattle, Washington 98195
A comparison of noncollagenous matrix proteins
from different types of bovine cartilage by SDS-polyacrylamide gel
electrophoresis showed a prominent 240-kDa component in extracts of
epiphyseal but not tracheal tissue. On amino-terminal sequence
analysis, it gave two sequences. One matched the NH2
terminus of cartilage matrix protein (CMP) as reported for tracheal
cartilage. The other did not match any known protein sequence. Further
analysis of the 240-kDa protein after reduction of disulfides resolved
two bands on SDS-polyacrylamide gel electrophoresis. Isolation and sequence analysis of tryptic peptides confirmed that one was bovine CMP
and the other a CMP homolog. A data base search identified the latter
as matrilin-3, a molecule recently predicted from human and mouse
cDNA sequences (Wagener, R., Kobbe, B., and Paulsson, M. (1997)
FEBS Lett. 413, 129-134). Matrilin-3 and CMP (matrilin-1) were prominent in equimolar amounts in fetal bovine epiphyseal cartilage and absent from adult articular cartilage. Adult tracheal cartilage contained almost exclusively CMP. Although the mechanism of
polymeric assembly is unknown, the matrilin-3 chain appears to
function in the matrix linked to matrilin-1 in the form of disulfide-bonded heteromeric molecules. The results indicate a molecular stoichiometry of
(matrilin-1)2(matrilin-3)2.
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INTRODUCTION |
Cartilage matrix protein
(CMP)1 is most prominent in
tracheal cartilage. It forms insoluble filaments and accumulates in the matrix with increasing tissue maturity (1). The protein is also present
in nasal septum, auricular, xiphisternal, and epiphyseal growth
cartilages (2, 3). The native molecule consists of a disulfide-linked
trimer of three identical 50-kDa chains (1-4). Based on the primary
structure predicted from chicken and human cDNAs, the CMP monomer
consists of two homologous von Willebrand factor type A-like domains
separated by an epidermal growth factor-like domain and a coiled-coil
-helix at the carboxyl terminus (5, 6). The latter is the site of
chain interactions for trimer formation (7, 8) including interchain
disulfide bonds through two cysteines (Cys-458/460 (human)/Cys-455/457
(chick) (8).
The function of CMP is unclear. It binds to type II collagen fibrils
and aggrecan in the extracellular matrix of cartilage (9, 10) and may
be involved in collagen fibrillogenesis (11). During skeletal
development, CMP mRNA was most evident in the upper hypertrophic
and lower proliferative zones of growth plate cartilages (12). This was
confirmed at the protein level by immunomicroscopy of chick epiphyseal
cartilage, which showed CMP concentrated in the matrix around
post-proliferative chondrocytes at the junction of the proliferative
and hypertrophic zones (13).
During a study of matrix proteins in bovine skeletal and tracheal
cartilages, we consistently observed a novel CMP-like molecule that
appeared to be unique to epiphyseal growth cartilage. We here report on
the properties of this protein, including comparative sequence data
with matrilin-1 (CMP) and matrilin-3, the latter a protein so far
described only at the cDNA level.
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MATERIALS AND METHODS |
Preparation of CMP--
Cartilage was dissected from the distal
ends of bovine femurs at three animal ages (fetal, 3 months, and 2 years). Tracheal ring cartilage was obtained from a fetus and a 2-year
steer. Tissue was washed in 0.15 M NaCl, 0.05 M
Tris-HCl, pH 7.4, containing protease inhibitors (2 mM
phenylmethylsulfonyl fluoride, 2 mM EDTA, 10 mM
N-ethylmaleimide, and 5 mM benzamidine HCl) at
4 °C for 24 h, then digested in sequence with chondroitinase
ABC (Sigma; 5 units/g of dry tissue) and Streptomyces
hyaluronidase (Seikagaku Kogyo Co., Tokyo, Japan; 50 turbidity-reducing units/g of dry tissue) as described previously (14).
The residue was extracted with 4 M guanidine HCl, 0.05 M Tris-HCl, pH 7.5, containing the above protease
inhibitors at 4 °C for 24 h. The extracts were dialyzed against
distilled water and freeze-dried.
In some experiments, CMP was recovered from the 4 M
guanidine HCl extract of cartilage samples without any prior
chondroitinase ABC or hyaluronidase treatments. The 4 M
guanidine extracts were diluted to 2 M with an equal volume
of distilled water, and CMP was precipitated by adding NaCl to 4 M (i.e. 2 M guanidine HCl, 4 M NaCl). The pellet recovered by centrifugation was
resuspended in and dialyzed against distilled water and freeze-dried.
In addition, an A5 + A6 fraction from a 4 M guanidine HCl
extract of fetal epiphyseal cartilage prepared by associative density
gradient centrifugation in 3.5 M CsCl was a generous gift
from Dr. Lawrence Rosenberg. This fraction, which is enriched in
nonproteoglycan matrix proteins (15), was dialyzed to equilibrium
against 1 M NaCl, 0.05 M Tris-HCl, pH 7.4. Protein was precipitated (30% saturated
(NH4)2SO4). The precipitate was
extracted in 0.5 M acetic acid, and noncollagenous matrix
proteins were recovered in the acid-insoluble fraction.
Column Chromatography--
For molecular sieve chromatography,
matrix proteins were dissolved in 4 M guanidine HCl, 0.05 M Tris-HCl, pH 7.5, heat denatured at 60 °C for 10 min,
and eluted from a column of agarose A5m (170 cm × 1.5 cm,
200-400 mesh; Bio-Rad), in 2 M guanidine HCl, 0.05 M Tris-HCl, pH 7.5, at room temperature.
Gel Electrophoresis and Electroblotting--
Protein fractions
were analyzed by SDS-PAGE (16). For amino-terminal sequence analysis,
resolved protein bands were transblotted to polyvinylidene difluoride
membrane (Bio-Rad) (17) using a MilliBlot-SDE electroblotting apparatus
(Millipore). The membrane was washed thoroughly in ultrapure water
(Millipore Milli-Q) to remove salts, then stained with Coomassie
Brilliant Blue to detect protein bands. Bands were excised for
automated sequence analysis.
For elution of protein bands after SDS-PAGE, strips containing the
bands of interest were cut from the gel after locating them by briefly
staining with Coomassie Blue. The gel strips were destained in 50%
(v/v) methanol in water and homogenized. The proteins were then
extracted in 0.2 M NH4HCO3
containing 0.05% SDS and 0.05 M DTT for 3 h at
60 °C. The extraction step was repeated. Supernatants were combined
and lyophilized. The sample was dissolved in 1 ml of distilled water
and passed through a Bio-Rad 10DG column to remove SDS, DDT, and
NH4HCO3.
Trypsin Digestion--
Eluted CMP chains were heat denatured and
digested with trypsin (Boehringer sequencing grade) at an
enzyme-to-substrate ratio of 1:100 (w/w) in 0.2 M
NH4HCO3 at 37 °C for 24 h. Tryptic
peptides were resolved by reverse-phase HPLC on a C8 column (Brownlee
Aquapore RP-300; 4.6 mm × 25 cm) with a 0-40% linear gradient
of acetonitrile:1-propanol (3:1, v/v) in aqueous 0.1% (v/v)
trifluoroacetic acid over 60 min as described previously (18).
Protein Sequencing--
Amino-terminal sequence analyses were
performed on a Porton 2090E gas-phase microsequencer equipped with
on-line high performance liquid chromatography analysis of
phenylthiohydantoin derivatives.
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RESULTS |
Characterization of CMP from Epiphyseal and Tracheal
Cartilages--
On comparing the matrix proteins extracted from bovine
tracheal and epiphyseal cartilages on SDS-PAGE, 6% gel, both tissues gave a strong band at about 180 kDa (Fig.
1). In addition, a prominent protein band
of apparent Mr = 240,000 was seen in the extract of epiphyseal cartilage but not of tracheal cartilage. After reduction of disulfide bonds, both the 240- and 180-kDa protein bands disappeared and gave rise to a lower, broad band of Mr = 60,000 (Fig. 1). Amino-terminal sequence analysis of this latter
protein from epiphyseal cartilage after transblotting to a
polyvinylidene difluoride membrane gave two distinct sequences. One
sequence, LAPLSRGHLCRTRPTDLVF-, was prominent in the lower half of
the 60-kDa protein band and was identical to the NH2
terminus of the reduced CMP band from bovine tracheal cartilage. The
upper half gave a novel sequence, APMARPGLRRLGTRGPG-, which did not
match any known protein sequence. Further sequence analyses of the
transblotted nonreduced 180- and 240-kDa proteins detected the unknown
protein only in the 240-kDa band, whereas CMP was prominent in both
180- and 240-kDa bands. The yields of the two sequences from the
240-kDa band were in a ratio of about 1:1. The reduced band from
tracheal cartilage showed only the NH2 terminus of CMP on
sequence analysis. Extracts of fetal tracheal cartilage also showed
only the 180-kDa band on SDS-PAGE (not shown).

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Fig. 1.
SDS-PAGE of matrix proteins from bovine
cartilages. Matrix proteins were extracted (4 M
guanidine HCl) from 2-yr bovine tracheal cartilage or from fetal bovine
epiphyseal cartilage (see "Materials and Methods" for details).
Samples were run on a 6% polyacrylamide gel (lanes 1 and
3, tracheal cartilage; lanes 2 and 4,
epiphyseal cartilage), with (lanes 3 and 4) and
without (lanes 1 and 2) DTT to cleave disulfide
bonds. COMP, cartilage oligomeric matrix protein;
1(II), type II collagen chain; bands A and
B, upper and lower halves of the reduced 60-kDa
monomer.
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Tryptic Peptide Analysis--
To further characterize the novel
matrix protein from epiphyseal cartilage, protein bands A (the upper
half of the 60-kDa protein) and B (the lower half of the 60-kDa
protein) (Fig. 1) were eluted from excised gel strips and digested with
trypsin, and the digests were resolved by reverse phase-HPLC. Fig.
2 shows the elution profiles for the
trypsin digests of protein bands A and B. The amino-terminal sequences
of individual tryptic peptides are given in Table
I.

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Fig. 2.
Reverse-phase HPLC fractionation of tryptic
peptides from protein bands A and B. Eluted bands A and B were
digested with trypsin (1:100 w/w) in 0.2 M
NH4HCO3 at 37 °C for 24 h. Digests were
eluted from a C8 column (Brownlee Aquapore RP-300; 25 cm × 4.6 mm) by a linear gradient (0-40%) of solvent B in A over 60 min. at a
flow rate of 1 ml/min. Solvent A was 0.1% trifluoroacetic acid (v/v)
in water and solvent B, 0.085% trifluoroacetic acid (v/v) in
acetonitrile/1-propanol (3:1, v/v). Amino-terminal sequences of
peptides T1-T9 (A) and T11-T23 (B) are shown in
Table I.
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Table I
Amino-terminal sequences of tryptic peptides
Protein eluted from band A or B was digested with trypsin and
fractionated by reverse-phase HPLC. Peptides in peaks T1-T9 and
T11-T23 (Fig. 2) were identified by amino-terminal sequence analysis.
Several peaks (T3, T5, T7, T15, and T19) gave two sequences as
indicated. All the peptides of protein B could be matched to fragments
of human CMP establishing that protein B is bovine CMP. Residue numbers
are as reported for human CMP cDNA (6). The peptides recovered from
protein A were all matched by homology to sites in matrilin-3, a novel
CMP-like protein (results shown in Fig. 4).
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Each of the tryptic peptides (T11-T23) from band B could be matched to
a sequence in human CMP confirming that protein B is bovine CMP. Four
tryptic peptides from band A (T6-T9) could also be aligned by sequence
similarity to sites in CMP, but these sequences were clearly less
closely matched (Fig. 3). The findings
indicate that protein A is a distinct gene product but a relative
of CMP (protein B). On searching gene and protein data bases for
similarity, one fragment (T6) that had been derived from band A,
VAIIVTDGRPQDQVNEVA, matched exactly part of a sequence in
the data base predicted by a cDNA cloned at random from a human
lung cDNA library (clone ID 119728; T94707). This latter cDNA
sequence in its entirety was homologous to but distinct from that of
the known human CMP gene (6). Finally, Wagener et al. (19)
recently reported the sequences of human and mouse cDNAs which they
cloned based on human lung clone ID 119728. They named the encoded
protein matrilin-3. On comparing the tryptic peptide sequences from
band A with the mouse and human matrilin-3 sequences, all the fragments could be matched (Fig. 4). The results
provide compelling evidence that the CMP-like protein is bovine
matrilin-3.

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Fig. 3.
Tryptic peptides from proteins A and B
showing sequence similarities. Sequences are aligned with the
corresponding domain in human CMP matched from cDNA data (6). The
results indicate that B is bovine CMP and A is a homolog of CMP.
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Fig. 4.
Alignment of tryptic peptide sequences from
protein A with mouse and human matrilin-3. The amino-terminal
sequences of the peptides are aligned with their predicted sites of
origin in matrilin-3 by comparison with mouse and human matrilin-3
cDNA sequences (19). A dash indicates residues identical
to the mouse sequence. Gaps were introduced to maximize the homology of
the NH2-terminal sequence. Residue numbering is based on
mouse matrilin-3 from its cDNA (19).
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Characterization of Matrilin-3--
Fig.
5 compares the guanidine-extracted matrix
proteins from fetal, 3-month calf, and 2-year steer cartilages by
SDS-PAGE. The staining patterns (180- and 240-kDa proteins) indicate
that CMP (matrilin-1) and matrilin-3 were the dominant proteins in this
noncollagenous matrix pool from fetal cartilage. Based on tissue and
recovered fraction weights and the staining intensities of protein
bands on SDS-PAGE, the two matrilin proteins together accounted for at
least 5% of the fetal tissue dry weight. Both protein subunits were
still detected but in low yield in 3-month calf articular cartilage,
but were essentially absent from adult (2-year) articular
cartilage.

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Fig. 5.
SDS-PAGE, 7.5% gel, of matrilins from
epiphyseal and articular cartilages. Tissue extracts (4 M guanidine HCl) were pretreated with chondroitinase ABC
and Streptomyces hyaluronidase. Lane 1, fetal
cartilage; lane 2, 3-month calf cartilage; lane
3, 2-year articular cartilage. Loads were equal based on original
tissue weight.
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Fig. 6 shows the elution profile on
Bio-Rad A5m molecular sieve chromatography and analysis of resulting
fractions by SDS-PAGE of the matrix protein fraction from fetal bovine
epiphyseal cartilage. The results show that the 180-kDa
disulfide-bonded oligomer gave a single CMP band after disulfide
cleavage, and the 240-kDa oligomer gave a doublet (Fig. 6, panel
C). On protein sequence analysis the upper band of the doublet
gave APMARPGLRRLGTRGPG-, the matrilin-3 amino-terminal sequence, and
the lower band gave the CMP NH2 terminus. From the results,
we conclude that the 180-kDa band is the known homotrimer of CMP (4).
The previously undescribed 240-kDa protein is an equimolar mixture of
CMP (matrilin-1) and matrilin-3. In addition to being present in the
240-kDa component linked to matrilin-1, matrilin-3 was detected in
molecular sieve column fractions that contained disulfide-bonded high
molecular weight polymers. As shown in Fig. 6, panel B, both
matrilin-1 and matrilin-3 were released on disulfide cleavage of the
pooled high molecular weight material in about 1:1 ratio.

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Fig. 6.
Fractionation of native matrilin oligomers by
agarose A5m molecular sieve chromatography. The protein sample (25 mg), prepared from 4 M guanidine HCl extract of fetal
epiphyseal cartilage by CsCl density gradient centrifugation and
(NH4)2SO4 precipitation (see
"Materials and Methods"), was eluted from an agarose A5 m column
(Bio-Rad Laboratories; 200-400 mesh; 1.5 × 170 cm) in 2 M
guanidine HCl, 0.05 M Tris-HCl, pH 7.5, 5.7 ml/h collecting
1.9-ml fractions. Aliquots of collected fractions were desalted and
analyzed by SDS-PAGE, 7.5% gel, without (A) or with
(B and C) disulfide-cleavage by DTT. Lane
T, starting material. Fractions 77-81 contained
primarily the 180-kDa CMP homotrimer (panels A and
C). Fractions 63-73 contained primarily the
240-kDa matrilin-1/matrilin-3 heteromer (panels A-C).
Fractions 29-62 contained disulfide-bonded higher molecular
weight matrilin-1/matrilin-3 oligomers (panel B).
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Further evidence for a stoichiometry of 1:1 in the assembly of
matrilins 1 and 3 into heterooligomers was provided by analysis of CNBr
digests of the 240-kDa component. Fig.
7A shows the products of CNBr
digestion of pooled material containing primarily 240 kDa from
molecular sieve fractions 63-73 (Fig. 6) run on SDS-PAGE. The apparent
size was reduced from 240 to 210 kDa (lanes 1 and 2) when run without disulfide cleavage. Amino-terminal
sequencing of the 210-kDa product transblotted to a polyvinylidene
difluoride membrane gave four amino-terminal sequences in about 1:1:1:1
ratio matchable to matrilins 1 and 3: matrilin-1,
26Leu-Ala-Pro-Leu-Ser-Arg-Gly-His-Leu-(Cys)-Arg-Thr-Arg-Pro-Thr-; matrilin-1,
407Phe-Ala-Val-Gly-Val-Gly-Asn-Ala-Val-Glu-Asp-Glu-Leu-Arg-Glu-; matrilin-3,
33Ala-Arg-Pro-Gly-Leu-Arg-Arg-Leu-Gly-Thr-Arg-Gly-Pro-Gly-;
matrilin-3, Ala-Ser-Glu-Pro-Leu-Asp-Glu-His-Val-Phe-Tyr-Val-Glu-Thr-Tyr-.

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Fig. 7.
SDS-PAGE (A) and schematics
(B) of CNBr-derived matrilin oligomers. Material
recovered from Bio-Gel A5m-pooled fractions 63-73 (Fig. 6) was treated
with CNBr in 70% formic acid under N2 at room temperature
for 24 h. A, digests were dried and analyzed by
SDS-PAGE, 6% gel, without DDT reduction (lanes 1 and
2) and by SDS-PAGE, 12% gel, with DTT reduction
(lanes 3 and 4) of disulfide bonds. Lanes
1 and 3, 240-kDa component from Bio-Gel A5m pools
63-73 (Fig. 6); lanes 2 and 4, CNBr digest of
the same material. Amino-terminal sequences from the transblotted bands
are shown. The Met119-Thr120 bond was cleaved
by CNBr in low yield (less than 30%), explaining why the sequence
beginning at Thr120 was not prominent in the 210-kDa band.
Residue numbering is based on human matrilin-1 (6) and mouse matrilin-3
(19) cDNAs. Migration positions and molecular weights
(×10 3) of protein standards are shown on the
left. B, upper part shows the modular
structures of matrilin-1 and matrilin-3 monomers (19); lower
part shows a model of the CNBr-derived matrilin-1/matrilin-3
tetrameric 210-kDa protein. Cysteine residues shown involved in intra-
and inter-chain disulfide-bonding in matrilin-3 are predicted from
known matrilin-1 sites (8).
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Two were derived from matrilin-1 and two from matrilin-3. On sequencing
of bands recovered from the CNBr digestion after reduction of disulfide
bonds with DTT, all the major CNBr peptides of matrilin-1 and -3 were
identified (Fig. 7A, lanes 3 and 4).
Based on the methionine distributions from human and mouse matrilin-1
and-3 cDNA data (6, 19), the bovine protein sequences (Table I and
Fig. 4), and reported intrachain disulfide positions on matrilin-1 (8),
the four polypeptide fragments comprising the 210-kDa CNBr cleavage
product were as follows (Fig. 7B). An about 320-amino acid
polypeptide from the amino terminus of matrilin-1 was linked to a
90-residue sequence from the carboxyl terminus of matrilin-1 by an
intrachain disulfide bond (Cys-265/452) (8). From matrilin-3, an
82-residue segment from the amino terminus was linked to a 253-residue
segment from the carboxyl terminus by an intrachain disulfide bond
(Cys-72/258). Finally, the two pairs of peptides from matrilin-1 and
matrilin-3 were covalently linked by interchain disulfide bonds
(Cys-458/460 (matrilin-1), Cys-441/443 (matrilin-3)) in the
carboxyl-terminal fragments (8).
The interchain disulfide bonds responsible for the 180- and 240-kDa
oligomers were readily cleaved by 1 mM DTT. However, the mobility of the resulting matrilin-1 and matrilin-3 monomers on SDS-PAGE varied significantly depending on the concentration of the
reducing agent (Fig. 8). The two protein
monomers were best resolved after reduction of cystine residues and
alkylation with iodoacetate prior to electrophoresis. Alkylated
matrilin-3 monomer (apparent Mr = 81,000) ran
far more slowly than the reduced but not alkylated product; in
contrast, the shift in mobility between alkylated (apparent
Mr = 62,000) and nonalkylated CMP (matrilin-1) monomers was less dramatic (Fig. 8, lanes 5 and
6). These findings can be explained by the higher content of
cysteine residues, 28 versus 12, in matrilin-3 than in
matrilin-1 evident from their sequences (6, 19).

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Fig. 8.
Effect of DTT concentration and
cysteine alkylation on matrilin mobility on SDS-PAGE, 7.5% gel.
The 240-kDa matrilin heteromer prepared by molecular sieve
chromatography (Fig. 6) was heat denatured (15 min, 60 °C) in 0 mM (lane 1), 1 mM (lane
2), 5 mM (lane 3), 10 mM
(lane 4), and 50 mM (lane 5) DTT
before electrophoresis. An additional sample was reduced with 50 mM DTT and carboxymethylated with iodoacetate (lane
6). The migration positions and molecular weights
(×10 3) of protein standards are shown on the
right.
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DISCUSSION |
Matrilin is the name given to members of an emerging new family of
extracellular matrix proteins (19, 20). The forerunner, matrilin-1, is
well known as CMP and has been extensively characterized (1-13). Only
recently have molecular relatives been recognized, prompting the new
nomenclature. The three known matrilins have molecular features in
common. All three have a signal peptide, either one or two von
Willebrand factor type A-like domain(s), one or more EGF-like
domain(s), and a COOH-terminal coiled-coil
-helix (5, 6, 19-21).
Matrilin-1 (CMP) consists of two von Willebrand factor type A-like
modules connected by one EGF-like module. The matrilin-2 cDNA
predicts two von Willebrand factor type A-like modules separated by ten
consecutive EGF-like modules. Mouse matrilin-2 mRNA was detected
primarily in calvaria, uterus, heart and brain (20). Matrilin-3
cDNA predicts a single von Willebrand factor type A-like module
separated from the COOH-terminal coiled-coil by four EGF-like modules.
Northern blot hybridization showed that matrilin-3 mRNA
tissue expression was restricted to cartilages in the developing chick
and mouse (19, 21).
In the present study, we have characterized a cartilage matrix protein
that is homologous to but not identical to the known CMP gene product
(matrilin-1). The tryptic peptide sequences could be matched to mouse
and human matrilin-3 from cDNA data and indicate that this protein
is bovine matrilin-3. The electrophoresis results show that matrilin-1
and matrilin-3 are equally abundant in fetal epiphyseal cartilage but
absent from mature articular cartilage. Matrilin-1 (CMP) is known to be
a major matrix constituent of tracheal cartilage of any age and to be
absent from adult articular cartilage (1-4). We also conclude from the
present results that tracheal cartilage contains matrilin-1 in the
absence of a significant amount of matrilin-3. Skeletal growth
cartilage, however, appears to be distinguished by containing a
heteromeric form of matrilin molecule that comprises both matrilin-1
and matrilin-3 chains. Matrilin-3, therefore, appears to be
characteristic of cartilages of the growing skeleton. Its function in
the matrix will be important to define.
Matrilin-1 and matrilin-3 differ in cysteine content, with 12 and 28 residues per chain, respectively (6, 19). The changes in mobility of
the matrilin monomers on SDS-PAGE after treatment with increasing
concentrations of DTT (Fig. 8) indicate multiple intrachain disulfides
that differ in their susceptibility to reductive cleavage. Mobility on
SDS-PAGE is not a reliable predictor of molecular size, particularly of
matrilin-3. It has been reported that matrilin-1 behaves larger than
its actual molecular size on SDS-PAGE (4). Similarly, matrilin-3 (Fig.
1) and its largest CNBr-derived fragment (Fig. 7A,
lane 4) ran more slowly relative to reference proteins than
expected from their polypeptide size. After alkylation (Fig. 8), the
exaggerated slowing in mobility of matrilin-3 could reflect the
multiple carboxymethyl cysteine residues altering hydrophobicity and
number of bound SDS molecules (22).
The primary form of CMP (matrilin-1) extracted from cartilage is a
homotrimer of predicted mass 148 kDa (4). This is the 180-kDa band on
SDS-PAGE in Fig. 1. In comparison and based on its elution position on
molecular sieve chromatography, we conclude that the 240-kDa matrilin
protein is a tetramer. Several findings indicate that it contains two
matrilin-1 and two matrilin-3 chains. First, homotrimers of matrilin-1
could be isolated from matrilin-3 by molecular sieve and SDS-PAGE
separations. Matrilin-3-containing oligomers always contained equimolar
matrilin-1. Salt fractionation of the cartilage matrix proteins by
either ammonium sulfate or NaCl also failed to resolve matrilin-3 from
matrilin-1 (data not shown). On CNBr digestion of the 240-kDa protein,
carboxyl-terminal fragments of matrilins-1 and -3 could not be resolved
from the product without breaking disulfide bonds (Fig. 7) and their
molar ratio was about 1:1. Finally, matrilins-1 and -3 coexisted in the
same apparent 1:1 molar ratio throughout the fractionation range of the
high molecular weight polymers (Fig. 6). We interpret from these
observations that matrilins-1 and -3 chains are co-assembled into a
disulfide-linked heterotetramer of composition,
((matrilin-1)2(matrilin-3))2. These oligomeric
molecules are able to form disulfide-bonded polymers of higher
molecular weight.
The above conclusions are consistent with the observed CMP property of
forming oligomers and a filamentous network (23, 24). The exact
polymeric form of the protein in vivo is still uncertain.
The CMP-A1 domain and/or the EGF domain appear to be necessary for
filament assembly by CMP (matrilin-1) (24). It is notable that similar
domains also exist in matrilin-3 in addition to the carboxyl-terminal
coiled-coil domain.
From the cDNA sequence, matrilin-3 contains four characteristic
seven-residue repeats, (abcdefg)4, in which positions a and d are hydrophobic residues that form the core of the coiled-coil (25,
26). Only 10 of the 28 residues forming the heptad repeat domain of
human matrilin-3 are conserved in the corresponding sequence of human
matrilin-1. It has been demonstrated that substitution of a single
residue 487 arginine by glutamine in the heptad-repeat domain of
matrilin-1 can alter the helix assembly from a trimer to a tetramer
(27). Mutations in the hydrophobic core of the coiled-coil domain of
the transcription factor, GCN4, also affect this protein's preference
for forming two-, three-, or four-stranded structures (28). It remains
to be demonstrated which amino acid residues in the heptad repeats of
matrilin-1 and -3 are responsible for determining the specificity of
helix assembly into trimer or tetramer and why matrilin-3 chains
preferentially oligomerize with matrilin-1 as the results would
suggest.
In summary, we report observations at the protein level on the nature
of a novel matrix protein (matrilin-3) in cartilage. The protein is a
relative of CMP (matrilin-1), with which it appears to co-exist
exclusively in the form of heteromeric molecules which appear to be
characteristic of skeletal growth cartilage. These findings imply that
matrilin-3 may have a special role in endochondral bone formation and
skeletal development.
We are grateful to Dr. Lawrence Rosenberg for
a gift of matrix proteins from fetal bovine cartilage, and we thank
Aydin Ghajar for preparing the figures and Kae Ellingsen for preparing
the manuscript.