Differences in hyaluronate binding to plasma and cell surface fibronectins. Requirement for aggregation.

Human plasma-derived fibronectin (pFbn) and chick embryo fibroblast cell surface fibronectin (cFbn) were compared in a series of in vitro assays for binding to glycosaminoglycans (GAG) from various sources. Substratum-attached material, adhesive material that remains attached to serum-coated tissue culture substrata following ethylene glycol bis(P-aminoethyl ether)-N,N,iV’JV”tetraacetic acid-mediated detachment of murine Balb/c 3T3 fibroblasts, was shown to be enriched, relative to whole cells, for highly N-sulfated heparan sulfate the only GAG in these preparations that binds to Sepharose columns derivatized with either pFbn o r cFbn. Adhesive material isolated from newly attaching and spreading fibroblasts contains 50% more of this heparan sulfate than material from long term substratum-attached cells. Both forms of fibronectin bound identically to preparations of 3H-heparin either under “univalent” binding conditions, in which fibronectin or heparin were immobilized onto inert matrices of Sepharose or gelatin-Sepharose, or under the aggregating conditions of Yamada et al. (Yamada, K. M., Kennedy, D. w., Kimata, K., and F’ratt, R. M. J. BioL Chem. 255,6055-6063), in which fibronectin: GAG aggregates are trapped on Millipore membranes. Neither form of fibronectin bound metabolically radiolabeled high molecular weight 3H-hyaluronate (HA) under the “univalent” binding conditions. cFbn, but not pFbn, displayed appreciable affinity for hyaluronate only as a consequence of aggregation of the fibronectin. HAso, a 50-sugar oligomer of HA, was as active in binding cFbn as high molecular weight HA, indicating that hyaluronate multivalency is not required for stable cFbn binding. These data are discussed with regard to the possible functional roles of cell surface heparan sulfate proteoglycans and hyaluronate binding to specific fibronectin classes in mediating physiologic adhesion and subsequent detachment of fibroblasts from the extracellular matrix.