Identification and distribution of a novel, collagen-binding protein in the developing subepicardium and endomysium

A protein doublet (M(r) = 68,000) that copurifies with chicken cardiac collagen types I and III is purified and characterized in the present study. Peptide mapping and amino terminus sequencing for both 68-kDa polypeptides show they have similar structures. This is supported by amino terminus sequencing of a 39-kDa proteolytic fragment of each polypeptide. The 68-kDa polypeptides appear at pI 6.7-6.8 in two-dimensional gels. Under nonreducing, electrophoretic conditions, the doublet appears as a large multimer or aggregate. Amino acid sequencing of the protein shows that its amino terminus contains a heptapeptide (VCLXXGK) that appears in the heparin/fibrin-binding domain of fibronectin and the collagen-binding domain of laminin. Cardiac myocytes synthesize and secrete the protein in vitro onto cell surfaces and onto the substratum. Indirect immunofluorescence shows the protein first appears in the chicken subepicardium at approximately 10 days following fertilization. As collagen accumulates in the subepicardium and the volume of the subepicardial space increases, the 68-kDa protein is found predominantly at the interface between myocardial cells and the connective tissue and between epicardial cells and the connective tissue. In adult hearts, the protein is also present at lower concentrations in endomysial connective tissue. The 68-kDa protein is also present in the skeletal muscle endomysium of embryonic chickens. Electron microscopic immunocytochemistry shows the 68-kDa protein is located at the surface of subepicardial collagen fibers. In addition, a direct interaction between the 68-kDa protein and collagen are indicated by: 1) equilibrium gel filtration of the 68-kDa protein in the presence of gelatin, 2) gelatin affinity chromatography of the 68-kDa protein, and 3) comigration of type I collagen and the 68-kDa protein during gel filtration under reducing conditions. The 68-kDa protein exhibits no collagenase activity under native conditions or in zymograms. Together, the data indicate that the 68-kDa protein is a novel collagen-associated protein appearing in late epicardial development.