Matrilin-3 Forms Disulfide-linked Oligomers with Matrilin-1 in Bovine Epiphyseal Cartilage*

Jiann-Jiu WuDagger and David R. Eyre

From the Orthopaedic Research Laboratories, University of Washington, Seattle, Washington 98195

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.M guanidine HCl, 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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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; alpha 1(II), type II collagen alpha  chain; bands A and B, upper and lower halves of the reduced 60-kDa monomer.

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).

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).

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.

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).

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).

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.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants AR36794 and AR37318.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.

Dagger To whom correspondence should be addressed: Dept. of Orthopaedics, University of Washington, Box 356500, Seattle, WA 98195-6500. Tel.: 206-543-4700; Fax: 206-685-4700; E-mail: wujj{at}u.washington.edu.

1 The abbreviations used are: CMP, cartilage matrix protein, also known as matrilin-1; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; HPLC, high performance liquid chromatography; EGF, epidermal growth factor.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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