Preparation of lipid-derivatized glycosaminoglycans to probe a regulatory function of the carbohydrate moieties of proteoglycans in cell-matrix interaction

We have shown previously that chondroitin sulfate, but not heparan sulfate/heparin, linked to either natural core proteins or serum albumin interferes with cell-to-substrate adhesion, provided that the external proteoglycans are topologically immobilized on plastic plates. In order to study the roles of glycosaminoglycan chains (GAGs) as recognition structure, a new assay system is now developed which involves the conversion of free GAGs to reactive lactone derivatives selectively modified at the reducing end. The modified GAGs can be coupled to the amino group of phosphatidylethanolamino (PE) for use as probes on either plastic plates or cell surfaces. Incubation of GAG-PE solutions in polystyrene plates results in a time- and dose-dependent increase of the density of the GAG chains noncovalently immobilized onto the plates. No immobilization is detected with any of the GAG-PE samples that have been treated with phospholipase D. A M(r) 30,000 chondroitin sulfate conjugate to PE (CS-PE), when immobilized onto a fibronectin-coated well for 2 h at an initial concentration of 0.06 microgram/100 microliters/well, inhibits the adhesion of baby hamster kidney (BHK) cells to the substratum by approximately 50%, whereas heparin-, heparan sulfate-, hyaluronic acid-, and dermatan sulfate-PE do not. The effect of CS-PE is abolished by treating the CS-PE-coated plates with chondroitinase ABC. A similar level of inhibition by CS-PE is found when the RGD-containing 120-kDa fragment of fibronectin is used in place of fibronectin. CS-PE in soluble form, once exposed to BHK cells in suspension, can be associated with the cell surfaces, thereby exerting some inhibitory effects on cell-to-substrate adhesion. On a per mol basis, however, the activity of cell-associated CS-PE is far lower than that of substrate-associated CS-PE. Together the results indicate that our GAG-PEs offer useful tools for probing regulatory function of the GAG moieties of proteoglycans and further support the hypothesis that the inhibitory regulation of cell-to-matrix adhesion by large chondroitin sulfate proteoglycans is caused by an interaction between the cell surface and the chondroitin sulfate chains topologically immobilized on extracellular matrices.