Construction and characterization of a fusion protein with epidermal growth factor and the cell-binding domain of fibronectin.

An efficient expression system was constructed for C-EGF, a fusion protein made of a fragment of the cell-binding domain of human fibronectin (FN) bound with epidermal growth factor (EGF). C-EGF was produced in Escherichia coli HB101 cells carrying the recombinant plasmid pCE102 as inclusion bodies, which were solubilized and refolded after purification. C-EGF had both cell-adhesive and EGF activities, so it might be more effective than EGF in therapeutic applications. This fusion system would be useful for the construction of a recombinant drug delivery system for cells that have fibronectin receptors (integrins).

Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.

Effects of Fe2+, Fe3+ and desferrioxamine on paraquat accumulation by rabbit kidney cortex slices.

Effects of Fe2+, Fe3+ and desferrioxamine (DFO) on the accumulation of paraquat (PQ) into rabbit kidney cortical slices were studied. Fe3+ showed dose-dependent inhibition of PQ accumulation. Fe2+ had a more powerful stimulatory effect on the lipid peroxidation of cortical slices than Fe3+, but it did not reduce PQ accumulation. The addition of DFO made the inhibitory effect of Fe3+ on PQ accumulation more pronounced. These results suggest that Fe3+ is able to inhibit the accumulation of PQ independent of its peroxidative action, and that Fe(3+)-desferrioxamine (ferrioxamine) may serve as an antidote for the toxicity of PQ.

Role of type III homology repeats in cell adhesive function within the cell-binding domain of fibronectin.

Recombinant fibronectin (FN) fragments and their mutant proteins were produced to elucidate the role of type III homology repeats in cell adhesive activity within the cell-binding domain of FN. Cell adhesive activity of the 11.5-kDa fragment, the cell attachment site of the cell-binding domain, was less than 0.1% that of native FN despite the presence of the Arg-Gly-Asp-Ser sequence. The activity increased as type III homology repeats were added to the N terminus of the 11.5-kDa fragment, and a 52-kDa fragment with four additional type III repeats had almost the same activity of native FN. Deletion of Arg-Gly-Asp from the fully active fragments completely abolished the cell adhesive activity. Deletion of one or two repeats from the 52-kDa fragment affected the extent of the cell adhesive activity, the degree of the effect being inversely correlated with the distance of the deletion from the type III repeat containing Arg-Gly-Asp-Ser. Rearrangement of type III repeats caused much loss of activity. These results suggest that the number and kinds of type III repeats and their correct alignment rather than the putative synergistic site decide the extent of the specific cell adhesive activity.

Inhibition of lung metastasis by synthetic and recombinant fragments of human fibronectin with functional domains.

We have investigated the antimetastatic effect of synthetic or recombinant peptides containing the functional domains of fibronectin on experimental and spontaneous lung metastases of murine tumor cells. CS1 peptide which is present within type III homology connecting segment (IIICS) as well as C-274 (cell-binding domain) were able to inhibit experimental lung metastasis when co-injected intravenously (iv) with B16-BL6 melanoma cells, while H-271 (heparin-binding domain) could not. In the spontaneous metastasis model, multiple iv administrations of CS1 or C-274 after surgical excision of primary tumors caused a significant reduction of metastatic colonies in the lung. Both CS1 and C-274 significantly inhibited cell adhesion and migration to fibronectin-coated substrates when added freely in solution. CS1 peptide also inhibited the cell adhesion and migration to laminin-coated substrates, but C-274 did not. H-271 did not have any inhibitory effect on cell adhesion or migration to either of the substrates. Similarly, CS1 inhibited tumor invasion to both Matrigel/fibronectin- and Matrigel/laminin-coated filters, whereas C-274 inhibited the invasion to only Matrigel/fibronectin-coated filter. These results indicate that CS1 peptide of fibronectin, lacking the Arg-Gly-Asp-containing domain, actively inhibits tumor metastases in spontaneous and experimental metastasis models. The use of such a peptide might offer a promising therapeutic approach for combatting or preventing cancer metastasis.

Modulation of haptotactic migration of metastatic melanoma cells by the interaction between heparin and heparin-binding domain of fibronectin.

We utilized recombinant fibronectin polypeptides with cell-binding domain and heparin-binding domains (referred to as C-274 and H-271, respectively) and their fusion polypeptide (CH-271) to examine the role of sulfated polysaccharide heparin and/or the functional domains of fibronectin in modulating tumor cell behavior. Both C-274 and CH-271 polypeptides with cell-binding domains promoted the adhesion and migration of B16-BL6 melanoma cells, whereas H-271 did not. Heparin bound to the immobilized polypeptides with heparin-binding domain (H-271, CH-271, and a mixture of C-274 and H-271 or fibronectin) but did not affect the tumor cell adhesion to the substrates. At the same time, heparin or two monoclonal antibodies against the heparin-binding domain were able to inhibit the haptotactic migration to CH-271 or fibronectin, though not to C-274 or a mixture of C-274 and H-271. This suggests that although heparin did not affect tumor cell adhesion to the cell-binding domain near the heparin-binding domain in CH-271 or fibronectin, it did lead to a modulation of cell motility. It seems likely that the regulatory mechanism may depend on interaction between heparin-like molecules on the cell surface and the heparin-binding domain in fibronectin, rather than on simple steric hindrance or on the masking of the cell-binding domain caused by the binding of heparin to heparin-binding domain.