Domain structure of hamster plasma fibronectin. Isolation and characterization of four functionally distinct domains and their unequal distribution between two subunit polypeptides.

The domain structure of plasma fibronectin was studied by limited proteolysis with trypsin and thermolysin. Intact hamster plasma fibronectin is a disulfide-bonded dimer of two nonidentical subunit polypeptides with M, = 230,000 (a subunit) and 210,000 (B subunit). Mild trypsin treatment selectively released a small fragment with M, = 32,000 (designated as 32K fragment, others are designated in the same way) from both subunits, converting a and p subunits into 200K and l80K fragments, respectively. In contrast, thermolysin cleaved intact fibronectin into four fragments, 150K-l40K, 40K, 24K, and 21K, which represent distinct constitutive domains of intact fibronectin. Tryptic 200K and 180K fragments gave on thermolysin digestion 150K-l40K, 40K, and 21K fragments but not 24K fragment. On the contrary, thermolysin digestion of the 32K tryptic fragment generated only 24K fragment, indicating that both fragments are derived from the same domain. Of the four distinct thermolysin fragments, only 24K and 21K fragments bound to fibrin, whereas 150K-140K (which binds to heparin and promotes cell spreading) and 40K (which binds to gelatin) fragments failed to bind to fibrin. Of three major tryptic fragments, 200K, 180K, and 32K, only 200K and 32K fragments were capable of binding to fibrin. The failure of the 180K fragment to bind to fibrin is due to the absence of the 21K fibrinbinding fragment which was only generated from the 200K fragment by thermolysin treatment. In contrast, 150K-140K and 40K fragments were produced from both 200K and l8OK fragments. These results indicate that plasma fibronectin is composed of four structurally and functionally distinct domains, among which one of the fibrin-binding domains represented by 21K thermolysin fragment (“21K” domain) is only present in a subunit while the other three domains, represented by 150K-l40K, 40K, and 24K thermolysin fragments, are equally present in both subunits. The susceptible glutamyl residue(s) for Factor XIIIacatalyzed transamidation seemed to be located near the NH2 or COOH terminus of the 24K domain. Half-cystine was enriched in the 40K, 24K, and 21K domains but was scarce in the 150K-140K domain. Intact fibronectin was estimated to contain four carbohydrate units per subunit; three units were attached to the 40K domain and one unit was attached to the 150K-140K domain.