HEPARIN-FIBRONECTIN INTERACTIONS IN THE DEVELOPMENT OF EXTRACELLULAR MATRIX INSOLUBILITY

Abstract

The extracellular matrix (ECM) is composed of a network of fibrous proteins and proteoglycans that together provide stability, support, and regulate cell behavior and organization. Fibronectin (FN) is one of these fibrous proteins, and cell-mediated interactions between FN dimers form fibrils that, over time, become insoluble. This insoluble FN matrix serves an important role as a foundational matrix for other ECM proteins to build upon. The insoluble nature of FN fibrils provides the ECM with its structure and rigidity, and is important for normal developmental processes. A dysregulation in the assembly or accumulation of these fibrils can lead to various developmental and progressive diseases. Understanding how FN fibrils become stable and thus insoluble can help in better understanding such complications. We have developed a procedure to isolate insoluble fibril fragments from a decellularized matrix using proteolysis, deoxycholate (DOC) detergent extraction, and gel electrophoresis. Analyses of these DOC-insoluble fragments using domain-specific antibodies and mass spectrometry have identified three regions that are resistant to proteolysis. Two of these regions contain known heparin/heparan sulfate (HS) binding sites in FN. To determine whether heparin-FN interactions facilitate fibril assembly, a HS-deficient CHO cell line was used for matrix assembly experiments. In the absence of heparin, these cells assembled a limited number of FN fibrils. In contrast, the addition of heparin significantly increased the amount of FN matrix. Time course experiments indicated that heparin acts at an early step in the assembly process, and is required to be present continuously for maximal matrix stimulation. Sulfated glycosaminoglycan (GAG) mimetics similarly increased FN assembly and demonstrated a dependence on GAG sulfation. The length of the heparin chains also played a role in assembly. Chains of sufficient length to bind to two FN molecules gave maximal stimulation of assembly, whereas shorter heparin had less of an effect. Heparin was also able to bind to multiple heparin-binding domain FN fragments, as revealed by size exclusion chromatography analyses. A model is proposed in which heparin/HS binding at the heparin-binding domains connects multiple FNs together and promotes conformational changes involved in forming insoluble protein interactions.