Sialylation and fucosylation of epidermal growth factor receptor suppress its dimerization and activation in lung cancer cells

Abstract

Protein glycosylation is an important posttranslational process, which regulates protein folding and functional expression. Studies have shown that abnormal glycosylation in tumor cells affects cancer progression and malignancy. In the current study, we have identified sialylated proteins using an alkynyl sugar probe in two different lung cancer cell lines, CL1-0 and CL1-5 with distinct invasiveness derived from the same parental cell line. Among the identified sialylated proteins, epidermal growth factor receptor (EGFR) was chosen to understand the effect of sialylation on its function. We have determined the differences in glycan sequences of EGFR in both cells and observed higher sialylation and fucosylation of EGFR in CL1-5 than in CL1-0. Further study suggested that overexpression of sialyltransferases in CL1-5 and α1,3-fucosyltransferases (FUT4 or FUT6) in CL1-5 and A549 cells would suppress EGFR dimerization and phosphorylation upon EGF treatment, as compared to the control and CL1-0 cells. Such modulating effects on EGFR dimerization were further confirmed by sialidase or fucosidase treatment. Thus, increasing sialylation and fucosylation could attenuate EGFR-mediated invasion of lung cancer cells. However, incorporation of the core fucose by α1,6-fucosylatransferase (FUT8) would promote EGFR dimerization and phosphorylation.

Fig. 1.

EGF-induced EGFR dimerization and tyrosine phosphorylation. (A) Dimerization of EGFR on cell surface. (Lower) Quantitative results: first calculate EGFR dimer∶monomer ratio in each sample and then normalize with the ratio of CL1-0/EGFR without EGF treatment. (B and C) Tyrosine phosphorylation of EGFR upon EGF stimulation at different doses (B) or for different durations (C). (Right) The relative intensity that was compared with phospho-EGFR∶EGFR ratios and normalization with the ratio of CL1-0/EGFR without EGF treatment. (D) Dimerization of sialidase-treated soluble form EGFR.

Fig. 2.

EGF-induced EGFR dimerization and tyrosine phosphorylation in A549-Mock, -FUT4, and -FUT6 cells. (A) Dimerization of cell surface EGFR. (Lower) The quantitative results calculated by EGFR dimer∶monomer ratio in each sample normalized with the ratio of A549-Mock treated with 20 ng/mL EGF. (B) EGF-induced EGFR tyrosine phosphorylation. The relative intensity indicated below was calculated as phospho-EGFR∶EGFR ratios and normalization with the ratio of A549-Mock without EGF treatment. (C) Dimerization of fucosidase-treated soluble form EGFR.

Fig. 3.

Summary of the glycoforms. (A) MALDI-TOF MS profiles of the permethylated N glycans from EGFR immunoprecipitated from the lysates of CL1-0 (Upper) and CL1-5 (Lower) cells. The molecular compositions of the major [M + Na]⁺ molecular ion signals detected were assigned as annotated. High mannose-type N glycans were labeled simply as M5–M9, representing Man_5–9(GlcNAc)₂. Monosaccharide symbols used were as follows: triangle, Fuc; square, HexNAc; circle, Hex; diamond, NeuAc. Glucose oligomer contaminants were labeled as X. (B) The glycoforms analyzed according to glycopeptides profiles were detected from the EGFR samples derived from CL1-0 and CL1-5 and site-specific sialic acid and fucose indexes (C) of the EGFR proteins derived from CL1-0 and CL1-5. Types of glycans: Man, high mannose; Bi, biantennary; Tri, triantennary; Tetra, tetraantennary; Penta, penta-antennary; Hexa, hexa-antennary; F, fucose; S, sialic acid; N, N acetylhexosamine. Sialic acid index = [(%with one sialic acid × 1) + (%with two sialic acids × 2) + (%with three sialic acids × 3) + (%with four sialic acids × 4)]/100; Fucose index = [(%with one fucose × 1) + (%with two fucoses × 2) + (%with three fucoses × 3)]/100.

Fig. 4.

Sialylation and fucosylation suppress EGFR activation to modulate EGF-mediated invasion in lung cancer cells. (A) EGF-mediated invasion of CL1-5 cells with or without sialidase treatment. (B) EGF-mediated invasion of A549-mock, A549-FUT4, and A549-FUT6 cells with or without doxycycline treatment. The average cell number under 100× magnifications was calculated from five power fields for each sample for A and B. *, p < 0.05; **, p < 0.001 by Student’s t test. (C) Sialylation and fucosylation suppress EGFR activation.