HEPARAN SULFATE PROMOTES INITIATION AND POLYMERIZATION OF FIBRONECTIN MATRIX THROUGH INTERACTION WITH THE 13TH TYPE III MODULE

Abstract

Fibronectin (FN), a critical component of the extracellular matrix, is assembled into fibrils through a cell-mediated process. Our previous work identified the glycosaminoglycan heparan sulfate (HS) as essential for the formation of insoluble FN fibrils. My work focuses on uncovering how HS is involved in FN matrix assembly. I investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis. Fibroblasts with decreased EXT1 expression exhibited a significant reduction in FN fibril initiation and polymerization, steps that are required before formation of the matrix. EXT1 knockdown cells had stunted polymerization of nascent FN fibrils which could be rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The dependence on HS chain length suggests that HS gathers FN molecules together to promote fibril assembly. To determine if HS acts through a direct interaction with FN to control assembly, the main HS binding site on FN was deleted in fibroblasts using the CRISPR-Cas9 system. These knockout cells assembled less FN matrix than wild type cells and little if any mutant FN matrix was assembled when purified knockout FN was provided to cells without endogenous FN. This reduced assembly of knockout FN shows that lack of binding between HS and FN caused the deficiency in assembly by knockout cells. Addition of heparin promoted the assembly of wild type FN, but it had no affect on the assembly of FN lacking the HS binding site. Furthermore, heparin binding stabilized the folded conformation of its binding domain and prevented it from self-associating under increasing temperature suggesting that stabilization by HS binding might regulate interactions between FN modules. This effect would be particularly important at fibril nucleation sites where our data show that knockout cells require both exogenous wild type FN and heparin in the culture medium to maximize fibril nucleation. We conclude that HS binds to FN to regulate the nucleation and extension of FN fibrils.