Inhibition of fibronectin-mediated adhesion of hamster fibroblasts to substratum: effects of tunicamycin and some cell surface modifying reagents

Abstract

Using baby hamster kidney (BHK) fibroblasts we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the ability of trypsinized cells to attach and spread onto fibronectin. Tunicamycin inhibited mannose incorporation into total acid-precipitable glycoproteins by at least 95% while glucosamine and leucine incorporation were less or hardly inhibited. Hydrolysis and analysis of [3H]glucosamine-labelled glycoproteins showed that radioactivity incorporated into cells exposed to tunicamycin was present predominantly as galactosamine, presumably present in O-glycosidically linked glycan chains whose assembly is insensitive to the drug. Treated cells exhibit reduced amounts of surface-associated fibronectin and adhere relatively poorly to plastic or collagen surfaces pre-coated with plasma or BHK cell-derived fibronectins at the minimum concentrations required to induce nearly quantitative attachment and spreading of untreated cells. Drug-treated cells do adhere and spread into a bipolar configuration on surfaces saturated with fibronectin. Cells treated with tunicamycin and then grown in the absence of the drug revert to a more normal behaviour, indicating that under certain conditions the effects of the drug are reversible. Fibronectin-mediated spreading of trypsinized BHK cells is also inhibited by pre-treatment of cells with several non-penetrating reagents reactive with cell surface amino groups, namely pyridoxal phosphate, trinitrobenzene sulphonate and fluorescein 5-isothiocyanate. Analysis of surface substitution indicates a strong correlation between the extent of amino group substitution and inability of treated cells to interact with a fibronectin lattice. While the extent of attachment under these conditions is normal, cells pretreated with a specific non-penetrating thiol reagent, p-chloromercuribenzenesulphonate fail to attach to fibronectin-coated culture dishes in a dose-dependent fashion, indicating that a biochemical distinction can be made between the processes of attachment and spreading. We conclude that both N-glycosidically linked carbohydrate moieties of BHK cell surface glycoproteins and primary amine groups present in surface proteins or lipid head groups play a role in interactions of cells with fibronectin, leading to the formation and maintenance of a stable well-spread morphology. Both N-linked glycans and surface sulphydryl groups appear to be required for an attachment process which precedes spreading.