Characterization of fibronectin interactions with glycosaminoglycans and identification of active proteolytic fragments.

TLDR

Fibronectin is a major cell‑surface glycoprotein known to interact with glycosaminoglycans. The authors developed a nitrocellulose filter‑binding assay to quantify fibronectin–glycosaminoglycan interactions at physiological conditions and used heparin affinity chromatography to isolate fibronectin heparin‑binding domains. Fibronectin binds hyaluronic acid and heparin with high, saturable, reversible affinity at distinct sites (KD ≈10⁻⁷–10⁻⁸ M), a binding that is not blocked by EDTA or other glycosaminoglycans and only modestly reduced by ionic strength, while chondroitin sulfate and glycopeptides bind minimally; heparin‑binding proteolytic fragments of 160 kDa and 50 kDa were isolated. The study discusses how these high‑affinity sites may mediate glycosaminoglycan binding to cell surfaces or extracellular matrix organization.

Abstract

Fibronectin is a major cell-surface glycoprotein which has been reported to interact with glycosaminoglycans. A nitrocellulose filter-binding assay was developed to quantitate these interactions at physiological pH and ionic strength. Fibronectin isolated from chick embryo fibroblasts binds both hyaluronic acid and heparin; heparan sulfate is bound less efficiently, and chondroitin sulfate and glycopeptides are bound minimally. The binding of hyaluronic acid and heparin to fibronectin is saturable and reversible and occurs at separate binding sites. The binding of both molecules to fibronectin is not blocked by EDTA or by other glycosaminoglycans, and is only moderately inhibited by elevated ionic strength. Scatchard analyses revealed nonlinear, high affinity binding to fibronectin with a KD of approximately 10(-7) to 10(-8) M for these glycosaminoglycans. The affinity for heparin was utilized for the isolation of heparin-binding domains of fibronectin on heparin-agarose affinity columns. Heparin-binding proteolytic fragments with apparent molecular weights of 160,000 and 50,000 were isolated following hydrolysis of fibronectin by chymotrypsin or pronase, respectively. The possible involvement of such high affinity binding sites of fibronectin in the binding of glycosaminoglycans to the cell surface or in the organization of extracellular matrices is discussed.