Interaction of Cartilage Collagens with Heparin

Abstract

Type XI collagen (1α2α3α) extracted from bovine articular cartilage by pepsin digestion binds strongly to heparin immobilized on agarose. The collagens from cartilage will bind to heparin-agarose in 0.1 M NaCl/2 M urea/0.01 M Tris-HC1 and can be eluted with a linear gradient of NaCl. Type XI begins to elute at NaCl concentrations higher than 0.28 M and is totally eluted at 0.40 M NaCl. The peak of elution occurs at 0.37 M NaCl. The other collagens of cartilage bind more weakly and are fully eluted when the NaCl concentration reaches 0.25 M. Collagen types I, III, and V are also bound to the column at low salt concentrations but are fully eluted before type XI begins to elute. All of the type XI preparation binds to heparin-agarose, suggesting that there is at least one binding site per molecule. Denatured 1 α and 2α chains bind strongly, suggesting that at least one binding site exists on both of these chains. These data confirm that the interaction between polyanions and type XI collagen is stronger than that with other known collagens and provide a method for purification of type XI without any type II contamination.

Cartilage collagens include several minor collagen types in addition to type II (Burgeson and Hollister, 1979; Reese and Mayne, 1981). Type XI, the collagen composed of the a chains designated lα, 2α, and 3α, binds to the high density proteoglycans of cartilage in vitro, apparently in a non-specific manner (Smith et al., 1985). This binding is stable in increasing concentrations of NaCl until the collagen fibrils begin to dissolve. Heparin and dextran sulfate are extremely potent inhibitors of the interaction.

The ability of heparin and dextran sulfate to inhibit the binding of fibrillar collagen to cartilage proteoglycans suggested that binding might also occur between type XI collagen and these highly sulfated polyanions. If so, this would provide a method to purify the cartilage collagens and to assess the relative strength of the interactions between the polyanion and the various collagen types in cartilage.

In this paper, we demonstrate the binding of soluble collagens to heparin immobilized on Sepharose 4B and examine the concentration of NaCl required to disrupt this binding.