Electrophoretic Characterization of Species of Fibronectin
Electrophoretic Characterization of Species of Fibronectin
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.
Fibronectins (FNs), a family of multifunctional adhesion proteins that differ from one another through alternative splicing of a pre-mRNA derived from a single gene, are found as soluble dimeric molecules in the blood and as insoluble multimers within the extracellular matrix of tissues, where they are concentrated in basement membranes and bloodvessel walls. They bind to cell-surface integrin receptors and participate in a variety of cellular processes, including adhesion, migration, transformation, and apoptosis, as well as wound healing, fibrosis, and hemostasis. FN is deposited in cartilage from osteoarthritis (OA), and fragmented forms of FN have been detected in synovial fluid (SF) and articular cartilage from patients with OA and patients with rheumatoid arthritis (RA). On the basis of such findings, plasma-derived FN (pFN) and specific purified pFN fragments have been tested for their capacity to regulate the function of chondrocytes in vitro. Whereas intact, soluble pFN has been observed to exert little or no effect, several purified, proteolytically derived pFN fragments have proved to be active. Additionally, mixtures of fragments derived from OA cartilage have been observed to promote chondrolysis in vitro.
Although fragments corresponding to the 29-kDa (also referred to as 30-kDa) amino-terminal (N-terminal) heparin-binding domain (HBD) have been studied most extensively, species derived from sites spanning most of the FN molecule have been observed totrigger catabolic gene expression in chondrocytes. For example, purified fragments of pFN corresponding to the 120- to 140-kDa central cell-binding domain (CBD), the 50-kDa gelatin-binding domain (GBD), and the 40-kDa C-terminal HBD have each been observed to trigger release of proteoglycans from cartilage slices in vitro, as has a recombinant version of the alternatively spliced EIIIA segment (Fig. 1). In addition, the 29-kDa N-terminal HBD has been observed to trigger gene expression for stromelysin, inducible nitric oxide synthetase, hyaluronan receptor proteins, and other biologically active molecules in cultured chondrocytes. Chondrolysis triggered by FN fragments occurs in association with local release of catabolic cytokines, including tumor necrosis factor ±, interleukin-1², and interleukin-1±. Furthermore, intra-articular injection of either N-terminal or central CBD fragments into rabbit joints triggers loss of cartilage proteoglycan, whereas injection of intact, dimeric pFN does not.
(Enlarge Image)
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.
Our goal in this study was to characterize and compare the assortments of N-terminal SF FN species in samples from OA versus RA patients with respect to their domain structures and ligand-binding properties. We have found that, among the two predominant species of SF FN that bear sequences from the N-terminal HBD in patients with OA or RA, the smaller, ~170-kDa species binds less readily to gelatin and to a monoclonal antibody (mAb) specific for the GBD than does the larger, ~200+-kDa species. Furthermore, 2D electrophoretic analysis reveals the ~170-kDa species to be comprised of distinct subspecies, most of which extend sufficiently toward the carboxy terminus (C terminus) to include the 10th type III repeat within the central CBD. In addition to prominent ~200+- and ~170-kDa species, several additional forms of FN that bear sequences from the N-terminal HBD were detected in OA and RA samples. Each of the soluble species identified in this study, in addition to its possible roles in the promotion of arthritic joint injury, is a candidate as a biomarker for the arthritic disease process.
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.
Fibronectins (FNs), a family of multifunctional adhesion proteins that differ from one another through alternative splicing of a pre-mRNA derived from a single gene, are found as soluble dimeric molecules in the blood and as insoluble multimers within the extracellular matrix of tissues, where they are concentrated in basement membranes and bloodvessel walls. They bind to cell-surface integrin receptors and participate in a variety of cellular processes, including adhesion, migration, transformation, and apoptosis, as well as wound healing, fibrosis, and hemostasis. FN is deposited in cartilage from osteoarthritis (OA), and fragmented forms of FN have been detected in synovial fluid (SF) and articular cartilage from patients with OA and patients with rheumatoid arthritis (RA). On the basis of such findings, plasma-derived FN (pFN) and specific purified pFN fragments have been tested for their capacity to regulate the function of chondrocytes in vitro. Whereas intact, soluble pFN has been observed to exert little or no effect, several purified, proteolytically derived pFN fragments have proved to be active. Additionally, mixtures of fragments derived from OA cartilage have been observed to promote chondrolysis in vitro.
Although fragments corresponding to the 29-kDa (also referred to as 30-kDa) amino-terminal (N-terminal) heparin-binding domain (HBD) have been studied most extensively, species derived from sites spanning most of the FN molecule have been observed totrigger catabolic gene expression in chondrocytes. For example, purified fragments of pFN corresponding to the 120- to 140-kDa central cell-binding domain (CBD), the 50-kDa gelatin-binding domain (GBD), and the 40-kDa C-terminal HBD have each been observed to trigger release of proteoglycans from cartilage slices in vitro, as has a recombinant version of the alternatively spliced EIIIA segment (Fig. 1). In addition, the 29-kDa N-terminal HBD has been observed to trigger gene expression for stromelysin, inducible nitric oxide synthetase, hyaluronan receptor proteins, and other biologically active molecules in cultured chondrocytes. Chondrolysis triggered by FN fragments occurs in association with local release of catabolic cytokines, including tumor necrosis factor ±, interleukin-1², and interleukin-1±. Furthermore, intra-articular injection of either N-terminal or central CBD fragments into rabbit joints triggers loss of cartilage proteoglycan, whereas injection of intact, dimeric pFN does not.
(Enlarge Image)
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.
Our goal in this study was to characterize and compare the assortments of N-terminal SF FN species in samples from OA versus RA patients with respect to their domain structures and ligand-binding properties. We have found that, among the two predominant species of SF FN that bear sequences from the N-terminal HBD in patients with OA or RA, the smaller, ~170-kDa species binds less readily to gelatin and to a monoclonal antibody (mAb) specific for the GBD than does the larger, ~200+-kDa species. Furthermore, 2D electrophoretic analysis reveals the ~170-kDa species to be comprised of distinct subspecies, most of which extend sufficiently toward the carboxy terminus (C terminus) to include the 10th type III repeat within the central CBD. In addition to prominent ~200+- and ~170-kDa species, several additional forms of FN that bear sequences from the N-terminal HBD were detected in OA and RA samples. Each of the soluble species identified in this study, in addition to its possible roles in the promotion of arthritic joint injury, is a candidate as a biomarker for the arthritic disease process.