Electrophoretic characterization of species of fibronectin bearing sequences from the N-terminal heparin-binding domain in synovial fluid samples from patients with osteoarthritis and rheumatoid arthritis

Abstract

Fragments of fibronectin (FN) corresponding to the N-terminal heparin-binding domain have been observed to promote catabolic chondrocytic gene expression and chondrolysis. We therefore characterized FN species that include sequences from this domain in samples of arthritic synovial fluid using one-and two-dimensional (1D and 2D) Western blot analysis. We detected similar assortments of species, ranging from ~47 to greater than 200 kDa, in samples obtained from patients with osteoarthritis (n = 9) versus rheumatoid arthritis (n = 10). One of the predominant forms, with an apparent molecular weight of ~170 kDa, typically resolved in 2D electrophoresis into a cluster of subspecies. These exhibited reduced binding to gelatin in comparison with a more prevalent species of ~200+ kDa and were also recognized by a monoclonal antibody to the central cell-binding domain (CBD). When considered together with our previous analyses of synovial fluid FN species containing the alternatively spliced EIIIA segment, these observations indicate that the ~170-kDa species includes sequences from four FN domains that have previously, in isolation, been observed to promote catabolic responses by chondrocytes in vitro: the N-terminal heparin-binding domain, the gelatin-binding domain, the central CBD, and the EIIIA segment. The ~170-kDa N-terminal species of FN may therefore be both a participant in joint destructive processes and a biomarker with which to gauge activity of the arthritic process.

Figure 1

Structure of fibronectin (FN), including recognition sites for the monoclonal anti-FN antibodies used in this study. The structure of an intact FN subunit is shown, with the approximate binding sites for the three anti-FN monoclonal antibodies used in this study denoted by brackets at the top and binding specificities for various domains and structural motifs shown at the bottom. The primary FN sequence extends from the amino (N) terminus (NH₂, left) to the carboxy (C) terminus (COOH, right) and consists of repeating motifs designated type I, II, and III repeats. In addition to the 10th (counting rightward from the N terminus) type III repeat, cell surface integrin-binding motifs ('Cell') have been localized to the alternatively spliced EIIIA and V segments. The cysteine residues through which subunits are dimerized are depicted near the C terminus.

Figure 2

~170-kDa N-terminal species of fibronectin (FN) in samples of synovial fluid (SF) from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) bind to gelatin less avidly than do larger species bearing sequences from the N-terminal heparin-binding domain. Samples of SF from patients with OA (samples 1–3, 5, and 7–9 in the upper panels) or RA (samples 10–19 in the lower panels) were mixed with gelatin Sepharose beads, flow-through fractions were collected, and the beads were washed and boiled in reduced sample buffer to elute bound FN species. SF starting material ('S'), flow-through fractions ('F'), and bead eluates ('E') were then subjected to reduced 4–15% SDS–PAGE followed by Western blot analysis using mAb 1936 specific for an epitope in the N-terminal heparin-binding domain, followed by iodinated secondary antibodies. With the exception of RA SF samples 16 and 19, for which staining was restricted mainly to an ~200+-kDa band, the starting samples included two major species of FN, migrating at ~200+ and ~170 kDa, respectively. Whereas the ~200+-kDa band was stained more intensely than the ~170-kDa band in most samples, the flow-through fractions typically contained greater quantities of ~170- than ~200+-kDa species. Equivalent quantities of flow-through fractions and starting material were subjected to electrophoresis, whereas the volume of gelatin eluate was equivalent to four times the volume of starting material. OA samples 4 and 6 also exhibited lower '200+:170' ratios in flow-through fractions than in the starting fractions (not shown). The positions of molecular weight standards are denoted to the left of each panel, whereas the positions of the two predominant species of SF FN ('200+' and '170') are denoted by arrows to the left of the far left upper and lower panels only. The figure represents a composite derived from one autoradiagram, which was exposed overnight.

Figure 3

Nonreduced analysis of species of osteoarthritis (OA) synovial fluid fibronectin (FN) that bear sequences from the N-terminal heparin-binding domain. (a) OA sample 6 was subjected to gelatin affinity isolation, and the starting material ('SM') and flow-through ('FT') fractions were submitted to 5% nonreduced SDS–PAGE followed by Western blot analysis in duplicate using monoclonal antibodies (mAbs) specific for the N-terminal heparin-binding domain ('anti-N-term') or the gelatin-binding domain (GBD) (mAb 1892, 'anti-GBD'). In the starting material and the flow-through fraction, the anti-N-terminal mAb recognized a fragment species with mobility expected of a reduced protein of ~140 kDa ('F'), in addition to dimeric ('D') and monomeric ('M') species. Although staining of all three species was less in the flow-through fraction than in the starting material, the reduction in staining of the dimeric and monomeric forms was substantially greater than for the fragment species. In contrast, the anti-GBD mAb produced staining of species with mobility expected of dimeric ('D') and monomeric ('M') FNs but did not stain a fragment species in the starting material or the flow-through fraction. The two pairs of lanes were derived from one autoradiogram, which was exposed overnight. Similar results, in which dimeric and monomeric species of FN were stained by anti-GBD mAb to the exclusion of the smaller fragment species, were obtained for OA samples 1, 4, and 9 (not shown). (b) Purified 30-kDa N-terminal heparin-binding ('30 K') and 45-kDa gelatin-binding ('45 K') fragments of human FN (2.5 μg each), as well as the 170-kDa-enriched fraction derived from OA synovial fluid sample 6 ('170 K') (5 μl) [15], were subjected to duplicate 4–15% nonreduced SDS–PAGE and Western blot analysis using mAbs to the N-terminal heparin-binding domain (left) or to the GBD (right). The anti-N-terminal mAb produced staining of the 30-kDa fragment and a species with migration expected of a reduced protein of ~140-kDa within the 170-kDa-enriched fraction, but failed to stain the 45-kDa fragment. In contrast, the anti-GBD mAb produced bright staining of the 45-kDa fragment, but failed to stain the 30-kDa fragment or material in the 170-kDa-enriched fraction. The 30-kDa fragment migrated faster than expected from the positions of migration of reduced molecular weight standards shown to the left of each panel, possibly reflecting the effect of maintenance of type I repeat intrachain disulfide bonds upon conformation under nonreducing conditions. Autoradiagram exposure times were 4 hours for the 30 K and 45 K lanes, and overnight for the 170 K lanes.

Figure 4

2D Western blot analysis of species of osteoarthritis (OA) synovial fluid fibronectin (FN) that contain sequences from the N-terminal heparin-binding domain (HBD). Samples of OA synovial fluid (5 μl) were subjected to isoelectric focusing in linear pH gradients followed by reduced 5% SDS–PAGE and Western blot transfer analysis, using anti-N-terminal-HBD mAb 1936 followed by iodinated secondary antibodies. Sample numbers are shown in the right upper corner of each panel. Except for sample nine, blots resulting from pH 4–7 first-dimension isoelectric focusing are presented. The pH 3–10 gradient used for sample nine (i) permitted detection of an ~130-kDa species which was also evident in the three other samples (OA samples 3, 5, and 8) that were submitted to pH 3–10 gradients (not shown). A portion of each synovial fluid sample (5 μl) was submitted to 1D electrophoresis in a lane at the left of each SDS–PAGE gel, and asterisks denote the approximate positions of migration of the ~200+- and ~170-kDa species in these lanes. At least part of the staining of material that migrated as a diffuse band at or near the dye-front in 1D lanes appeared to be nonspecific, since similar staining was present in 1D Western blot analysis of unconcentrated synovial fluid samples in the absence of primary mAbs (not shown). (a) Schematic diagram of the typical 2D migration of three predominant species of synovial fluid FN bearing sequences from the N-terminal HBD: (1) ~170-kDa (major cluster denoted by brackets facing upward): Eight of the nine OA samples contained between two and six ~170-kDa subspecies that migrated as a nearly horizontal array of spots in the cathodic half of the first dimension (pI ~6.0 to ~7.0). In sample number 2 (c), little or no such staining of a ~170-kDa species could be detected, and this correlated with an absence of staining of this species in the 1D lane. Additional ~170-kDa material that migrated much closer to the anode (pI ~4.3) was detected in samples 4 (arrowhead pointing to the right) and 9 (not visible in the pH 3–10 blot in panel i). A species possessing an apparent molecular weight slightly greater than 170-kDa (~180-kDa) was detected as a small spot beneath the cathodic aspect of the ~200+ kDa cluster in samples 1, 3, 4, 7, and 8 (diagonal arrows pointing upward and to the left). (2) ~185-kDa (denoted by small brackets facing downward): OA samples 1 and 3 (b,d), and 5 and 9 (f,g) (blots/exposures not shown) contained an additional fragment species, comprising between one and four faint spots. Similar to the ~170-kDa species, these forms migrated as a near-horizontal array of spots, but farther toward the anode and more slowly (Table 1). (3) ~200+ kDa (denoted by large brackets facing downward): This was detected in all OA samples tested, typically as a large and poorly defined cluster that migrated in the right upper quadrant of each blot. Additional material of ~200+ kDa that migrated farther toward the anode than the major 'cluster' is evident in samples 2, 4, 5 and 9 (c,e,f,i) (short arrows pointing toward the right) (see Table 1). Autoradiogram exposure times were 5 days for samples 1, 3, 5, and 8; 6 days for samples 2, 4, and 7; and 10 days for sample 9. A blot of OA sample 6 is not included in this figure, but can be seen in Figure 6.

Figure 5

2D Western blot analysis of species of rheumatoid arthritis (RA) synovial fluid fibronectin (FN) that contain sequences from the N-terminal heparin-binding domain. RA synovial fluid samples 10–19 were subjected to linear pH 4–7 first-dimension isoelectric focusing followed by reduced second-dimension 5% SDS–PAGE. After transfer, membranes were stained with mAb to the N-terminal heparin-binding domain followed by iodinated secondary antibodies. Sample numbers are shown in the right upper corner of each panel. Species of ~200+ kDa (large brackets facing down) that migrated at a position similar to corresponding species in OA samples (see Fig. 4) were evident in all 10 RA samples. An additional cluster of material of ~200+ kDa, denoted by short arrows pointing toward the right, was evident in samples 11–13 and 15–19 (see Table 1). This material was streaked upward in samples 12, 16, 17, and 18. Definitive staining of ~170-kDa species (large brackets facing up) was evident in samples 10–15, 17, and 18. Additional ~170-kDa material that migrated much closer to the anode (pI ~4.3) than the major cluster was evident in RA sample 17 (h) (arrowhead pointing toward the right). An additional species that possessed a molecular weight of approximately 180 kDa was detected as a spot beneath the cathodic aspect of the cluster of ~200+ kDa in samples 11–15 (diagonal arrows pointing upward and to the left) (see Table 1). An ~185-kDa species (small bracket facing down) is evident in samples 10, 11, 13–15, and 18. Autoradiograms were exposed overnight for sample 19, 2 days for sample 18, 4 days for samples 10, 13, and 16, 5 days for samples 11, 14, 15, and 17, and 6 days for sample 12. No definitive staining of ~170- or ~185-kDa species was observed in samples 16 or 19, even after exposure times as long as 10 days.

Figure 6

Sequences from the N-terminal heparin-binding domain and the 10th type III repeat reside together within common subspecies of ~170-kDa synovial fluid fibronectin (FN) fragment. Aliquots of osteoarthritis (OA) sample number 6 (5 μl) were subjected to isoelectric focusing in duplicate pH 4–7 first-dimension (1D) strips, each of which was then subjected to reduced 5% SDS–PAGE. A portion (5 μl) of the sample was also submitted to reduced 1D PAGE in a lane at the edge of each of the two second-dimension gels. After incubation with anti-N-terminal heparin-binding domain mAb followed by HRP-conjugated secondary antibodies, similar enhanced chemiluminescence (ECL) staining patterns were obtained for the resulting two membranes (a, c) after a film development time of 1 minute. Specifically, two major bands were evident in the 1D lane, representing ~200+ (upper arrow) and ~170-kDa (lower arrow) species. Additionally, three major 'spots' (denoted by three vertical arrows), consistent with ~170-kDa species (brackets facing upward), were evident as a nearly horizontal array in the cathodic half of each membrane, approximating the point of migration of the corresponding species within the 1D lane. A cluster of staining with migration approximating that of the ~200+ kDa band was also evident in each membrane (brackets facing downward). The membranes were stripped of antibodies for 30 min at 50°C in 6.25 mM Tris pH 6.7 containing 100 mM β-mercaptoethanol and 2% SDS, then washed in TBST and reblocked with blotto. One was stained with mAb A2C2 diluted in blotto (panel B), whereas the other was incubated in blotto alone (panel D). After incubation with HRP-conjugated secondary antibodies, both membranes were again subjected to ECL development and film exposure for 10 min. Staining was evident in the membrane that had been incubated sequentially with anti-CBD mAb followed by secondary antibodies (b), but not in the membrane exposed only to secondary antibodies (d). When the films shown in (a) and (b) were overlaid using membrane 'edge staining' as a guide, the three ~170-kDa spots present in (a) were found to occupy indistinguishable spatial positions as compared with the corresponding spots evident in (b). In comparison with the anti-N-terminal mAb, mAb A2C2 produced preferential staining of the ~200+ in comparison with the ~170-kDa species.