Sekiguchi K, Hakomori S, Funahashi M, Matsumoto I, Seno N
J Biol Chem. 1983 Dec 10;258(23):14359-65.
Binding of intact plasma fibronectin and its proteolytic fragments to glycosaminoglycans immobilized on agarose beads was systematically compared at different ionic strengths. In low ionic strength buffer, intact fibronectin bound to heparin and high sulfated heparan sulfate, but not to low sulfated heparan sulfate, dermatan sulfate, chondroitin sulfates A and C, or hyaluronic acid. Fractionation of the thermolysin digest of fibronectin on the glycosaminoglycan-Sepharoses at low ionic strength revealed that three groups of fragments, i.e. Mr = 150,000-140,000, 24,000, and 16,000 (150K-140K, 24K, and 16K) fragments, were capable of binding to glycosaminoglycans with different specificities and affinities. The 150K-140K fragments exhibited the same specificities as intact fibronectin, binding only to heparin and high sulfated heparan sulfate. However, the 24K fragment bound not only to these two glycosaminoglycans but also to low sulfated heparan sulfate and other glycosaminoglycans as well. The 16K fragments were also capable of binding to most glycosaminoglycans with lower affinity than the 24K fragment. These results suggest that the binding sites in the 24K and 16K fragments are cryptic in the intact protein, but are exposed after limited proteolysis. The binding of fibronectin and its fragments to glycosaminoglycans is dependent on the ionic strength. At physiologic ionic strength, only heparin-Sepharose could bind intact fibronectin. Similarly, only heparin-Sepharose could bind the 150K-140K and 24K fragments, but not the 16K fragments, at the same ionic condition. Other glycosaminoglycan-Sepharoses did not retain significant amounts of any of the fibronectin fragments, suggesting that the affinity of plasma fibronectin and its fragments to heparan sulfate and other glycosaminoglycans, except heparin, is not strong enough to achieve stable mono- (or di-) valent binding under physiologic conditions.
在不同离子强度下，系统比较了完整血浆纤连蛋白及其蛋白水解片段与固定在琼脂糖珠上的糖胺聚糖的结合情况。在低离子强度缓冲液中，完整的纤连蛋白与肝素和高硫酸化硫酸乙酰肝素结合，但不与低硫酸化硫酸乙酰肝素、硫酸皮肤素、硫酸软骨素A和C或透明质酸结合。在低离子强度下，对纤连蛋白的嗜热菌蛋白酶消化产物在糖胺聚糖 - 琼脂糖凝胶上进行分级分离，结果显示三组片段，即分子量为150,000 - 140,000、24,000和16,000（150K - 140K、24K和16K）的片段，能够以不同的特异性和亲和力与糖胺聚糖结合。150K - 140K片段表现出与完整纤连蛋白相同的特异性，仅与肝素和高硫酸化硫酸乙酰肝素结合。然而，24K片段不仅与这两种糖胺聚糖结合，还与低硫酸化硫酸乙酰肝素和其他糖胺聚糖结合。16K片段也能够与大多数糖胺聚糖结合，但其亲和力低于24K片段。这些结果表明，24K和16K片段中的结合位点在完整蛋白质中是隐蔽的，但在有限的蛋白水解后会暴露出来。纤连蛋白及其片段与糖胺聚糖的结合取决于离子强度。在生理离子强度下，只有肝素 - 琼脂糖凝胶能结合完整的纤连蛋白。同样，在相同离子条件下，只有肝素 - 琼脂糖凝胶能结合150K - 140K和24K片段，而不能结合16K片段。其他糖胺聚糖 - 琼脂糖凝胶没有保留大量的任何纤连蛋白片段，这表明血浆纤连蛋白及其片段对硫酸乙酰肝素和除肝素外的其他糖胺聚糖的亲和力在生理条件下不足以实现稳定的单（或双）价结合。
