The Cartilage-specific Fibronectin Isoform Has a High Affinity Binding Site for the Small Proteoglycan Decorin*

Rina Gendelman, Nancy I. Burton-WursterDagger, James N. MacLeod, and George Lust

From the James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

Received for publication, November 19, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Binding of fibronectin to the small proteoglycan decorin plays an important role in cell differentiation and cell migration. The cartilage-specific (V+C)- fibronectin isoform, in which nucleotides that normally encode the protein segments V, III15, and I10 are spliced out, is one of the major splice variants present in cartilage matrices. Full-length and truncated cDNA constructs were used to express recombinant versions of fibronectin. Results demonstrated that the (V+C)- isoform has a higher affinity for decorin. Dissociation constants for decorin and fibronectin interaction were calculated to be 93 nM for the V+C+ isoform and 24 nM and 223 nM for (V+C)- fibronectin. Because heparin competed with decorin competitively, binding of decorin to fibronectin likely occurs at a heparin-binding region. We propose that alternative splicing of the V and C regions changes the global conformation of fibronectin in such a way that it opens an additional decorin-binding site. This conformational change is responsible for the higher affinity of the (V+C)- fibronectin isoform for decorin.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fibronectin (FN)1 is a major constituent of extracellular matrices in many tissues. The FN protein is folded into a series of globular homologous repeats of three distinct types (I, II, and III) composed of approximately 45, 60, and 90 amino acids, respectively (Fig. 1A) (1). It is secreted as a dimer with two interchain disulfides formed at the C terminus (2, 3). Although there are multiple isoforms of FN produced by different cell types, a single gene encodes all of these variants (4). The molecular heterogeneity of FN protein results primarily from alternative splicing of its pre-mRNA and different dimerization patterns (5). The (V+C)- isoform lacks protein segments V, III15, and I10 and forms primarily homodimers (6, 7).


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Fig. 1.   Schematic representation of fibronectin and mininectin constructs. A, full-length (V+C)- and V+C+ FN. The shaded areas indicate regions of alternative splicing. EDA and EDB are spliced in or out in their entirety. V region splice variants occur in plasma and other tissues. The (V+C)- FN isoform, however, is created when the V and C regions are removed entirely by alternative splicing, and a new splice junction joining III14 with I11 is created. Expression of this isoform is cartilage-specific. B, mininectin constructs. MNs are truncated FN constructs that originate at three different sites within the Hep II region and extend part of the way into segment I12. Thus, they all lack the major alpha 5beta 1 integrin site in III10, a GAG-binding site in III5, and a putative cryptic binding site for decorin near III10. They also lack interchain C-terminal disulfide bonds that are essential for dimerization and therefore are monomeric. The MN3 series, which delete V, III15, and I10, are truncated versions of the (V+C)- isoform and were derived from canine cartilage RNA. MN3-III12 includes the major heparin-binding site in III13. MN3-III14 lacks the major heparin-binding site in III13 but includes putative GAG and alpha 4beta 1 integrin sites in III14. The structure of type III repeats consists of seven linked beta -sheets, designated by convention as A-G from the N to the C terminus. The MN3-III14fg construct originates in the junction between beta -sheets E and F in III14.

Studies of FN in cartilage and other tissues have revealed that expression of (V+C)- FN is tightly linked to the cartilaginous phenotype (8). In articulator cartilage, the (V+C)- isoform represents 55-80% of total tissue FN. RNase protection assays determined that steady-state levels of canine (V+C)- FN mRNA range from 11% of total FN in the nucleus pulpous to 71% in the rib, with expression of this isoform rapidly lost if chondrocytes are cultured as monolayers and allowed to de-differentiate (8). These data suggest that the (V+C)- isoform plays an important role in cartilage matrix organization and perhaps even in regulating or maintaining the differentiated phenotype of chondrocytes.

The protein structure of FN can be related to its known functions. In different tissues, FN has a role in cell adhesion and differentiation, wound healing, blood clotting, cell migration, and spreading (9, 10). The high affinity heparin-binding site (Hep II) is located within the C-terminal half of the molecule in domains III12-14 with a Kd close to 10 nM, although reported dissociation constants vary widely (11-14). Small proteoglycans (PG) have been reported to bind to plasma FN, presumably at the heparin-binding sites. The alpha 4beta 1 integrin facilitates cell binding to two sites within the V region of FN (15). More recently, Sharma et al. (16) demonstrated the presence of two additional alpha 4beta 1 integrin-binding sites as follows: one at the junction of III13-III14 containing the amino acid sequence IDAPS, and the other in the III14 segment (PRARI motif homologous to PHSRN in III9). Only the former site appears to be accessible to the integrin. Moreover, Moyano et al. (17) uncovered a site in the III5 segment for cooperative binding of chondroitin sulfate proteoglycan and alpha 4beta 1 integrin. The cooperative binding at III5 segment appears to be important for FN matrix assembly (17). Close proximity of heparin and alpha 4beta 1 integrin-binding sites suggests that coordinated interactions of alpha 4beta 1 integrin and chondroitin sulfate proteoglycans with FN may be an important theme in FN biology. Consistent with this model, FN fragments with mutations in the Hep II domain adjacent to a functional V region decreased pp125FAK protein levels downstream of integrin signaling and regulated apoptosis in fibroblasts. These effects were mediated via cell surface chondroitin sulfate (18).

Decorin is a member of a family of small leucine-rich PGs. Depending on the species, it can have chondroitin- or dermatan sulfate glycosaminoglycan (GAG) chains attached. Its role in cartilage matrix biology is unclear. Data substantiate that decorin controls the diameter of type I collagen fibrils (19), and it is assumed to have a similar role for type II collagen organization in cartilage. Decorin may modulate transforming growth factor-beta signaling pathways by binding and sequestering transforming growth factor-beta (20).

The cartilage-specific (V+C)- isoform is structurally different from the widely expressed V+C+ and V-C+ FN variants. In (V+C)- FN, the V, III15, and I10 segments adjacent to the Hep II site are spliced out. The omission of a large part (257 amino acids) near the heparin/PG/alpha 4beta 1 integrin-binding site may result in altered function. In this study, we demonstrate that (V+C)- FN retains its ability to bind both heparin and chondroitin sulfate PGs. Moreover, we show that the (V+C)- FN contains a high affinity decorin-binding site not found in V+C+ FN.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Materials-- 35SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> sodium salt was purchased from Amersham Biosciences. Rabbit anti-decorin and anti-biglycan antibodies were provided by Dr. Gabriella Cs-Szabo (Chicago, IL). The anti-fibromodulin antibody was provided by Dr. Peter Roughley (Montreal, Quebec, Canada). Mouse anti-decorin monoclonal antibody was obtained from Dr. Paul Scott (Edmonton, Alberta, Canada). Peroxidase-linked goat anti-rabbit and rabbit anti-mouse IgG were purchased from Cappel Biomedicals (West Chester, PA). Ham's F-12 medium, Gey's balanced salt solution, Hepes buffer, gentamicin, penicillin G/streptomycin sulfate (20 units/ml, 20 µg/ml), and L-glutamine were purchased from Invitrogen. alpha -Ketoglutarate, papain, N-acetylcysteine, EDTA-tetrasodium salt, were obtained from Sigma. Calcium chloride was purchased from Mallinckrodt (Phillipsburg, NJ). L-Ascorbic acid phosphate was purchased from Wako Pure Chemical Industries (Dallas, TX). ITSTM Premix (insulin, transferrin, selenious acid, bovine serum albumin, and linoleic acid) was obtained from BD Biosciences. Spodoptera frugiperda (Sf21) insect cells were obtained from Invitrogen. Murine monoclonal anti-rat FN antibody (IC3) and the pVL1392 baculovirus cloning vector (Invitrogen) containing cDNA encoding V+C+ FN were generous gifts from Dr. Jean Schwarzbauer (Princeton, NJ). EX-CELL 400 serum-free insect medium was purchased from JRH Biosciences (Lenexa, KS). Gelatin-Sepharose was purchased from Amersham Biosciences.

Cartilage Culture and Labeling-- Canine articular cartilage was collected from knee, hip, and shoulder joints at necropsy from 2-year-old Labrador Retriever dogs, raised as part of a colony maintained at the James A. Baker Institute for Animal Health. Cartilage explants were washed with Gey's balanced salt solution and cultured for 48 h in Ham's F-12 complete medium supplemented with calcium chloride (485 µg/ml), Hepes buffer (25 mM), alpha -ketoglutarate (30 µg/ml), gentamicin (20 µg/ml), penicillin G/streptomycin sulfate (20 units/ml, 20 µg/ml), L-glutamine (0.3 mg/ml), ITS-CR+, L-ascorbic acid phosphate (0.05 mg/ml), 35SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, and [3H]leucine. Cartilage explants were metabolically labeled with 35SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> (39.4 µCi/ml) and [3H]leucine (14.6 µCi/ml). Explants were washed and extracted twice overnight, with agitation, at 4 °C using 4 M guanidine HCl in 0.05 M sodium acetate, pH 5.8, supplemented with 10% (v/v) protease inhibitor (PI) mixture (1.0 mM N-ethylmaleimide, 2.0 M EDTA, 30 mM benzamidine-HCl, 200 nM phenylmethylsulfonyl fluoride). Extraction buffer was added at 1 ml/100 mg wet weight of cartilage (21). Extracts were combined.

Purification of Small PGs from Cartilage-- Proteoglycans were separated from other proteins in the cartilage extract by DEAE chromatography. The cartilage extract was transferred by dialysis into DEAE column buffer (2 M urea, 0.05 M Tris-HCl, 10% PI, pH 8.0) and applied to a DEAE column at gravity flow (22). Proteins were eluted with the addition of 0.2 M NaCl to the DEAE column buffer and discarded. Proteoglycans were eluted with 4 M guanidine HCl, 0.1 M Na2SO4, 0.05 M sodium acetate, 0.1% Triton X-100, 10% PI, pH 6.1, and further purified by gel filtration chromatography on Sepharose CL-6B in order to separate the small PGs from aggrecan.

Decorin was separated from fibromodulin and biglycan by hydrophobic chromatography on octyl-Sepharose as described elsewhere (23), except that the material bound to the column was batch-eluted from the gel with increasing concentrations of guanidine HCl. Decorin was present in the flow-through and wash fractions. Samples were then re-purified by DEAE chromatography to remove any aggrecan spillover. Decorin was eluted with 0.3 M NaCl.

Gel Electrophoresis and Western Blot Analyses-- All fractions were analyzed by 5-15% SDS-PAGE in Tris/glycine buffer, pH 8.6 (7), and transferred to Hi-Bond N+ membranes (Amersham Biosciences) (22, 24). Blots were probed with rabbit polyclonal antibodies against decorin, fibromodulin, and biglycan, followed by peroxidase-linked goat anti-rabbit IgG. Peroxidase activity was detected by enhanced chemiluminescence (ECL Western blotting detection system, Amersham Biosciences). Dried blots were also exposed to a PhosphorImager plate to detect radioactive bands. The plate was read on a Fuji PhosphorImager and analyzed by MacBas software.

Expression and Purification of Recombinant FN and Recombinant MN Constructs-- Construction of recombinant baculovirus containing full-length rat V+C+ and (V+C)- FN cDNA has been described previously (25-27). Both isoforms were expressed following infection of Sf-21 insect cells according to the manufacturer's instructions. Fibronectin proteins were purified by sequential gelatin affinity and heparin affinity chromatography steps in a modification of the method proposed by Poulouin et al. (28). Fractions were analyzed by SDS-PAGE followed by silver staining to determine protein purity. Fibronectin concentration was quantified by a gelatin affinity enzyme-linked immunosorbent assay as described previously (29).

Bacterial expression constructs for truncated canine FN cDNA fragments termed mininectins (MNs) were produced using the pTrcHis-TOPO system (Invitrogen) according to the manufacturer's instructions (Fig. 1B). Total RNA was purified from articular cartilage as described earlier (6). The cDNA fragments of interest were amplified by RT-PCR using the primer pairs listed in Table I. The three MN3 constructs represent the (V+C)- isoform (Fig. 1B). The amplified cDNA fragments were ligated into the pTrcHis-TOPO vector containing a 6-histidine tag at the N terminus and transfected into TOP-10 bacterial cells. Mininectins were purified on ProBond nickel resin (Invitrogen) according to the manufacturer's protocol. Protein purity was assessed by SDS-PAGE followed by a silver stain. Recombinant proteins (1 nmol/column for full-length FNs and 7 nmol/column for MNs) were immobilized in duplicate on the CNBr-activated Sepharose-4B. Similarly, purified rat plasma FN was immobilized and used as a positive control, and BSA was immobilized and used as a negative control.


                              
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Table I
Primer names and sequences for PCR amplification of mininectin constructs
Primers 6a and 6s have been described previously (6). The sequence of the primer III-14 is located in the linker region between III13 and III14. The sequence of the primer III-14fg is located at the junction between beta  sheets E and F in III14. Primer 6a was used as an antisense primer for all constructs. Complementary DNA derived from cartilage RNA was used for amplification of MN3 constructs.

Binding of Small PGs to FNs-- Purified small PGs in 30 NTE (30 mM NaCl, 20 mM Tris-HCl, 2 mM EDTA, pH 7.3) buffer were applied to the columns with immobilized FN and to the column with immobilized BSA and allowed to bind for 15 min. The optimal binding time was determined empirically. Columns were washed with 30 NTE buffer until no radioactivity above background was detected. Columns were then eluted with 1 M NaCl in NTE buffer to determine total PGs bound. Fractions were analyzed for radioactivity by scintillation counting in order to quantitate bound PGs as a percentage of the total. Bound and unbound (free) fractions were resolved on 5-15% SDS-PAGE, stained with toluidine blue dye, dried, and exposed to a PhosphorImager plate. The plate was read by a Fuji PhosphorImager and analyzed with MacBas software.

Competition between Heparin and Small PGs for Binding to FNs-- Small PGs were mixed with heparin (0.2-25 µg) to test for competitive binding to FN. Samples containing heparin were applied to the columns with immobilized FN and analyzed as described above. Binding of decorin was compared with maximum binding in the absence of heparin, and the data were fitted to a curve for competitive inhibition using GraphPad PRISM3 software (GraphPad Software, San Diego, CA).

Determination of Dissociation Constants for Decorin-FN Interactions-- A range of concentrations of purified decorin in a standard volume of 1 ml were applied to columns containing either immobilized FN or BSA, followed by elution with 1 M NaCl. The molar concentration of free decorin was plotted as a function of the molar concentration of bound decorin, and curve fitting was performed using the Kaleidagraph software to Equations 1 and 2,


Y=X×B<SUB><UP>max,1</UP></SUB><UP>/</UP>(X+K<SUB>d,1</SUB>) (Eq. 1)

<UP>+</UP>X×B<SUB><UP>max,2</UP></SUB><UP>/</UP>(X+K<SUB><UP>d,2</UP></SUB>) (<UP>two binding sites</UP>)

Y=X×B<SUB><UP>max</UP></SUB><UP>/</UP>(K<SUB>d</SUB>+X) (<UP>one binding site</UP>) (Eq. 2)
where Y is the concentration of bound decorin, X is the concentration of free decorin; Bmax is the concentration of maximum sites occupied on FN, and Kd is the dissociation constant.

Binding of Decorin GAGs and Core Protein to FNs-- Equivalent amounts of decorin were either digested with chondroitinase ABC to produce free core protein or digested with papain to produce free GAGs. The free GAG components and the free decorin core protein components were independently applied to the FN columns as above. Bound and free fractions were analyzed by scintillation counting for the presence of radioactivity. The percentages of bound core protein and GAGs were calculated and plotted.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

(V+C)- FN Binds Significantly More PGs Than Does V+C+ FN-- Analysis of small PG fractions bound to recombinant (V+C)- FN, recombinant V+C+ FN, and plasma FN revealed that the cartilage-specific isoform was able to bind small PGs despite the absence of protein segments V, III15, and I10 near the Hep II-binding domain. Fig. 2A shows an SDS-PAGE separation of both bound and free fractions of small PGs. In fact, (V+C)- FN bound significantly more PGs than equivalent amounts of V+C+ or plasma FN (p < 0.0004 and p < 0.0001, respectively) (Fig. 2B).


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Fig. 2.   Binding of small proteoglycans to fibronectins. Radioactively labeled small PGs were allowed to bind to equivalent amounts of immobilized recombinant FN isoforms [r(V+C)- and rV+C+], purified plasma FN, and BSA. Columns were washed with binding buffer followed by elution with 1 M NaCl. A, representative SDS-PAGE comparing the bound (B) and free (F) PGs from eluted and flow-through column fractions. B, flow-through, washes, and eluted PGs were analyzed by scintillation counting, and the percentage of bound PG was calculated. Bound and free fractions were analyzed in a total of 32 independent binding experiments with three different FN preparations. Standard deviations and the p values were calculated using Student's t test ((V+C)- compared to V+C+ - p < 0.0004, (V+C)- compared to pFN - p < 0.0001).

Further analysis indicated that all three small PGs, decorin, biglycan, and fibromodulin, bound to FN, and all three exhibited increased binding to the cartilage-specific FN isoform. Decorin and biglycan were identified by sensitivity to chondroitinase ABC and by reactivity with anti-decorin and anti-biglycan antibodies, respectively. Decorin and fibromodulin (67-78 and 84-90 kDa, respectively) were not resolved by SDS-PAGE. Therefore, to determine whether fibromodulin bound to FNs, small PGs were first digested with chondroitinase ABC to remove chondroitin/dermatan sulfate GAG chains. This reduced decorin and biglycan to their core proteins. The small PG with a molecular mass of 84-90 kDa that remained after this chondroitinase ABC digestion was identified by immunoblot analysis as the keratan sulfate containing small, leucine-rich PG, fibromodulin.

Cartilage-specific (V+C)- FN Contains Two Decorin-binding Sites-- The above results indicated that the (V+C)- FN isoform bound more small PGs than the V+C+ isoform. To assess if this reflected an increased binding affinity, we determined dissociation constants (Kd) for the binding of purified decorin. Decorin was separated from fibromodulin and biglycan by octyl-Sepharose chromatography, as described under "Experimental Procedures," and applied to the immobilized full-length FNs at a range of concentrations. Results were plotted, and Kd values were calculated (Fig. 3). The binding profile indicates two decorin-binding sites for (V+C)- FN with dissociation constants of 23 and 224 nM. Only one decorin-binding site is suggested for V+C+ FN with a dissociation constant of 93 nM.


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Fig. 3.   Binding of decorin to fibronectin isoforms. Decorin was applied to immobilized V+C+ (A) and (V+C)- (B) FNs at concentrations ranging from 43.6 to 320 nM. Decorin was allowed to bind, and concentrations of bound and free decorin were calculated as described under "Experimental Procedures." Molar concentration of bound decorin was plotted as a function of molar concentration of free decorin using the Kaleidagraph software. Curve fitting was performed with equations for one and two binding sites, and the best fit was chosen (R2 = 0.906, one site, for V+C+ and R2 = 0.958, two sites, for (V+C)-). Data represent the mean of at least two independent experiments. A high affinity site (Kd = 24 nM) was present in (V+C)- FN.

GAG Chains of Decorin and Not the Core Protein Are Responsible for Binding to FN-- There are conflicting reports with regard to what part of the PG molecule is responsible for interactions with FN. Barkalow and Schwarzbauer (30) showed that in 75 mM NaCl, binding of PGs is mainly due to GAG chains. Schmidt et al. (31) have demonstrated that in 150 mM NaCl, 94% of interactions are due to the core protein moiety of decorin. Our results in 30 mM NaCl indicate that binding of decorin to all three FN isoforms is due mainly to the GAG chains and not to the core protein. Binding of GAGs alone accounted for 70% of total binding by intact decorin, whereas core protein accounted for less than 5% (Fig. 4).


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Fig. 4.   Interactions of (V+C)- and V+C+ recombinant fibronectins with intact decorin, decorin core protein, and free glycosaminoglycans. Equivalent amounts of decorin were either digested with chondroitinase ABC to produce free core protein or digested with papain to produce free GAGs. Intact decorin, free GAGs, and core protein were independently allowed to bind to the FN columns. Percent bound PG, core protein, or GAGs were calculated as a relative fraction of total amount (in terms of radioactive counts/min) applied to the columns. Data show that binding of decorin to FN was mediated almost entirely by GAGs.

Binding of Decorin to MNs Requires Both III13 and III14 Repeats of the Hep II Domain-- Three FN constructs, termed MNs (Fig. 1B), were produced in bacteria in order to elucidate the domain responsible for binding of decorin to FNs. MN3-III12 contained segments III12 through I12 for the (V+C)- isoform. MN3-III14 contained segments III14 through I12, and MN3-III14fg contained the C-terminal part of III14 (beta -sheets F and G) through I12. All constructs lacked the disulfide dimerization domain near the C terminus. MN3-III12 was able to bind decorin, but further truncation to eliminate the III12-13 domains reduced decorin binding to background levels (Fig. 5).


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Fig. 5.   Binding of decorin to the recombinant mininectins. Mininectin constructs were immobilized on CNBr-activated Sepharose-4B at a concentration of 8 nmol/0.5 ml gel. The negative control was recombinant beta -galactosidase derived from transformation of TOP-10 Escherichia coli with the pTrcHis-TOPO®/lacZ control vector supplied by Invitrogen. Decorin was allowed to bind to the mininectins and control columns. After subtraction of nonspecific binding, the percentage of bound decorin was calculated.

Heparin Inhibits Binding of Small PGs to FNs-- Heparin has a higher affinity for FN than do small PGs (30). There are at least three known heparin-binding sites identified on FN (32). The reported high affinity site is located in repeats III13-III14 (Hep II) (Fig. 1A). Another site is located at the N terminus of the molecule. A third site or sites is within the first six type III repeats C-terminal to the gelatin-binding site. To determine whether the small PGs interact with FNs at or near a heparin-binding site, a competition binding experiment was conducted. Fig. 6, A and B, shows that all FN isoforms bound heparin and that binding of small PGs to FN was almost completely abolished in the presence of excess heparin. Increasing concentrations of unlabeled heparin inhibited the binding of decorin to FN in a dose-dependent manner. Interestingly, half-maximal inhibition of binding of equimolar amounts of decorin required three to four times as much heparin for (V+C)- FN than was required for V+C+ FN (Fig. 6C). The data fit theoretical curves for competitive inhibition with R2 values of 0.97. 


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Fig. 6.   Competition between heparin and small proteoglycans for binding to fibronectin. Small PGs were mixed with heparin and allowed to bind to FN. Proteoglycans alone were used as a control. Bound and free fractions were analyzed by scintillation counting and resolved on 5-15% SDS-PAGE. A, percent bound small PGs in the presence or absence of excess heparin. B, bound PG fractions in the absence (-Hep) or presence (+Hep) of heparin resolved by SDS-PAGE after elution from the column with 1 M NaCl. C, percent bound decorin in the presence of increasing concentrations of heparin (0.2-15 µg/ml) relative to maximum binding to V+C+ FN and (V+C)- FN in the absence of heparin. Curves were fit to an equation for one-site competitive inhibition with R2 values of 0.976 and 0.967, respectively. Half-maximum inhibition occurred at 30 nM for V+C+ and 93 nM for (V+C)-.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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In this report, we demonstrate that the cartilage-specific (V+C)- FN isoform interacts with leucine-rich small PGs. Interactions of decorin, biglycan, and fibromodulin with FN isoforms were studied in a solid-phase binding assay at low ionic strength (30 mM NaCl). Binding of small PGs to cartilage-specific (V+C)- FN was quantitatively different from binding to the other FN isoforms examined. (V+C)- FN consistently bound up to 50% more small PGs than did the V+C+ isoform. The dissociation constants for binding of decorin PG to recombinant (V+C)- FN suggest the presence of two binding sites: a high affinity site (Kd = 24 nM) and a low affinity site (Kd = 223 nM). Only one binding site was detected on the V+C+ FN with a Kd of 93 nM.

It was determined that decorin GAG chains were responsible for the binding to FN isoforms. Our data are in general agreement with Barkalow and Schwarzbauer (30), who found interactions of V+C+ and V-C+ FNs with chondroitin sulfate GAGs at low ionic strength (75 mM), which were eliminated at physiological salt concentrations (150 mM). Schmidt et al. (31) observed binding of plasma FN to decorin in 150 mM NaCl. They showed that decorin core protein, but not the GAG chains, was responsible for binding. Only very low levels of binding between decorin core protein and FNs was seen in the current study. A potential reason for why our results differ from that of Schmidt et al. (31) is that by working at 150 mM NaCl, their experimental conditions likely excluded binding to the GAG chain of decorin. One might question whether interactions between FN and chondroitin/dermatan sulfate GAGs are relevant in vivo because, in contrast to heparin, they only occur at less than the assumed physiological salt concentration. However, considerable evidence exists that chondroitin sulfate PGs, often cooperatively with alpha 4beta 1 integrins, contribute to cell adhesion to FN (discussed in Refs. 14, 30, 33).

The major heparin-binding site is thought to be located within repeats III12-III14. This domain also supports cell adhesion and spreading through five peptides composed of basic residues that, in vitro, have been shown independently to bind heparin and/or chondroitin sulfate (33-36). Repeat III14 contains three of the five basic peptides, one immediately N-terminal to the V region which may be inaccessible in the native molecule (37, 38). X-ray crystallography studies by Sharma et al. (16) confirmed the prediction that a cluster of six basic residues from one or more of these peptides come together to generate a positively charged "cradle" on one side of the molecule that forms the major heparin-binding site in III13. Structural analysis revealed a similar cluster of five basic residues in the putative additional heparin-binding site in III14. Based on this information, we hypothesized that altered conformation of the (V+C)- FN isoform, created by deletion of V, III15, and I10 through alternative RNA splicing, enhances exposure of a heparin-binding site in III14, making it more accessible to decorin and contributing to the increase in binding affinity between decorin and (V+C)- FN. To test this hypothesis, MN constructs were created. The largest construct, MN3-III12, contains an intact heparin-2 binding domain. The next construct, MN3-III14, is further truncated to remove domains III12 and III13 but retains an intact III14 domain. The smallest construct, MN3-III14fg, retains only the f and g beta -sheets of the III14 domain. The hypothesis predicts that MN3-III14 will bind decorin. If truncation of MN3-III14 disrupts the integrity of the binding site, then MN3-III14fg will fail to bind decorin. The data indicate, however, that only the MN containing domains III12-14 was able to bind decorin. The other two constructs did not bind decorin above background. Heparin inhibited binding of decorin to both FNs and MNs. These results suggest that binding of decorin to the (V+C)- and V+C+ FN isoforms requires the heparin-binding domain in III13, but the heparin-binding site in III14 does not bind decorin independent of the site in III13. Barkalow and Schwarzbauer (11) showed that both III13 and III14 domains bind to heparin, but binding is greater if both are present. They also showed that chondroitin sulfate GAGs bind at identical or overlapping sites in both III13 and III14 albeit with lower affinity (30). Our results are in partial agreement with this but do not support an independent GAG-binding site in III14. Interestingly, recent results reported by Sachchidanand et al. (39) do not support binding of heparin to III14.

Although an additional heparin-binding site in III14 may not explain the increased binding affinity to decorin, nevertheless, the protein segment immediately adjacent to the Hep II domain appears to be important for the function of this region. Santas et al. (40) showed that the amino acids adjacent to III14 influence cell spreading on FN and FN fibrillogenesis. Ten amino acids from the N terminus of the V region were sufficient to make fibrillogenesis and cell spreading responsive to interactions of cell surface heparin sulfate proteoglycans with the Hep II domain. Replacing these 10 amino acids with 6 amino acids from the III15 domain abolished this dependence. The altered binding capacity and affinity for small proteoglycans observed with (V+C)- FN further support the concept that native alternative splicing patterns, which position different amino acids immediately C-terminal to III14, alter functional properties of the Hep II region.

(V+C)- FN represents from 50 to 80% of total FN in adult canine and equine articular cartilage, respectively (8). During postnatal development, steady-state (V+C)- FN mRNA in cartilage increases slightly, then roughly doubles to adult levels during puberty (10 months of age in dogs (8)). By contrast, the splicing pattern that generates ED-A+ FN is turned off as mesenchymal cell precursors differentiate into chondrocytes. Interestingly, cell migration (41) and FN-induced cell cycle progression appear to be enhanced by ED-A+ FN (42). The onset of decorin expression is also correlated with differentiation of mesenchymal stem cells into chondrocytes occurring along with increases in aggrecan synthesis and just prior to the appearance of type II collagen (43). The migration of cells on FN is inhibited by decorin (44, 45). It has been suggested that the interaction of decorin with extra cellular matrix components such as FN and collagens supports formation of a pericellular matrix that stabilizes cellular differentiation (46). The higher affinity of (V+C)- FN for decorin appears to support this model, enhancing the interaction of extracellular matrix components that may help to stabilize the differentiated phenotype of chondrocytes.

    ACKNOWLEDGEMENTS

We thank Drs. Paul Scott (Edmonton, Alberta, Canada), Peter Roughley (Montreal, Quebec, Canada), and Gabriella Cs-Szabo (Chicago, IL) for generous gifts of anti-proteoglycan antibodies. We thank Dr. Jean Schwarzbauer (Princeton, NJ) for gifts of V+C+ FN constructs from which (V+C)- FN constructs were derived. We are grateful to Dr. Robert Oswald (Cornell University) for helpful discussions and advice in analyzing the binding data. We acknowledge the valuable technical contributions of Michael Jackson and Alma Jo Williams and the secretarial assistance of Dorothy Scorelle.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant AR44340 and the Arthritis Foundation.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. Tel.: 607-256-5651; Fax: 607-256-5608; E-mail: niw1@cornell.edu.

Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M211799200

    ABBREVIATIONS

The abbreviations used are: FN, fibronectin; PG, proteoglycans; GAG, glycosaminoglycan; BSA, bovine serum albumin; PI, protease inhibitor; MN, mininectin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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