This paper presents a new method to evaluate the biological significance of glycosphingolipids (GSLs) using a GSL-spedfic enzyme, endoglycoceramidase (EGCase), by which GSL-sugar chains are removed from the cell surface of living cells. In this report, the effects of EGCase on epidermal growth factor (EGF)-dependent tyrosine-specific EGF receptor (EGFR) phosphorylation of A431 cells are described. After treatment of A431 cells with EGCase II (20 mU/ml) in the presence of the activator for 12 h, all acidic GSLs tested were reduced to ∼70% of control, but no hydrolysis occurred on Galα1,4Galcβ1,4Glcβ1,1Cer (Gb3Cer) and GalNAcpβ1 Galα1,4Galβ1,4Glcβ1,1Cer (Gb4Cer). In plasma membrane fractions of A431 cells, the reduction of gangliosides by EGCase II was found to be much faster than that of intact cells and reached 54.8% reduction of total gangliosides after 2 h incubation with the enzyme. EGF-dependent phosphorylation of EGFR of A431 cells was inhibited by the reduction of cell surface GSLs by EGCase when either intact A431 cells or their plasma membrane fractions were used, while EGF binding to their receptors was not changed. Neither hydrolysis of cell surface GSLs nor reduction of EGFR phosphorylation occurred when A431 cells were incubated with the activator or EGCase alone. The exogenous addition of ceramides or sphingosines, and treatment of cell membranes with sphingomyelinase, had no effect on the EGFR phosphorylation of purified membrane fractions, while the inhibitory effect of EGCase II on EGFR phosphorylation was restored by the addition of GM3-sugar chains, but not by lactose or sialic acid. The data presented in this paper indicate that the removal of sugar chains of GSLs, but not the accumulation of ceramides, reduces EGFdependent EGFR phosphorylation of A431 cells, suggesting that endogenous GSLs, possibly GM3, may be one of the integral constituents for supporting (or stimulating) the EGFR phosphorylation of A431 cells in situ.
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