TY - JOUR
T1 - The hydrolysis of cell surface glycosphingolipids by endoglycoceramidase reduces epidermal growth factor receptor phosphorylation in A431 cells
AU - Ji, Li
AU - Ito, Makoto
AU - Zhang, Gu
AU - Yamagata, Tatsuya
N1 - Funding Information:
The authors are grateful to Dr S.Iwashita, of this institute, for his helpful suggestions on EGFR experiments, Dr J.Inokuchi, Seikagaku Co., Japan, for kindly providing the D-PDMP and Y.Ikegami, of this laboratory, for preparing EGCase and the activator. Thanks are also due to Dr S.P.Damle for his critical comments in preparing the manuscript. This work was supported in part by a Grant-in Aid for Scientific Research on Priority Areas (no. 05274107 to M.I., nos 02259103 and 05274106 to T.Y.) from the Ministry of Education, Science, and Culture of Japan.
PY - 1995/5
Y1 - 1995/5
N2 - 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.
AB - 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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U2 - 10.1093/glycob/5.3.343
DO - 10.1093/glycob/5.3.343
M3 - Article
C2 - 7655171
AN - SCOPUS:0029054205
VL - 5
SP - 343
EP - 350
JO - Glycobiology
JF - Glycobiology
SN - 0959-6658
IS - 3
ER -