Sialylation and Fucosylation of Epidermal Growth Factor Receptor Suppress Its Dimerization and Activation in Lung Cancer Cells

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

Ying-Chih Liua,b,1, Hsin-Yung Yena,b,1, Chien-Yu Chena,b, Chein-Hung Chenb, Ping-Fu Chenga,c, Yi-Hsiu Juand, Chung-Hsuan Chenb, Kay-Hooi Khooc, Chong-Jen Yud, Pan-Chyr Yangd, Tsui-Ling Hsub,2, and Chi-Huey Wongb,2

aInstitute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan; bGenomics Research Center, Academia Sinica, Taipei 115, Taiwan; cInstitute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan; and dDepartment of Internal Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan

Contributed by Chi-Huey Wong, May 19, 2011 (sent for review February 1, 2011)

Protein glycosylation is an important posttranslational process, domains II and IV maintains EGFR in a tethered conformation which regulates protein folding and functional expression. Studies to inhibit dimerization (8), and this conformation is opened have shown that abnormal glycosylation in tumor cells affects up by EGF binding to expose the dimerization interface for re- cancer progression and malignancy. In the current study, we have ceptor activation (9, 10). After autophosphorylation, dimeric identified sialylated proteins using an alkynyl sugar probe in two EGFR recruits and activates various downstream signaling pro- different lung cancer cell lines, CL1-0 and CL1-5 with distinct inva- teins to direct cell proliferation, migration, differentiation, and siveness derived from the same parental cell line. Among the iden- apoptosis (11). tified sialylated proteins, epidermal growth factor receptor (EGFR) Twelve N-linked glycosylation sites were reported to exist in was chosen to understand the effect of sialylation on its function. the extracellular region of EGFR (12). The major glycoforms We have determined the differences in glycan sequences of EGFR in of EGFR in an epidermoid carcinoma cell line, A431, have been both cells and observed higher sialylation and fucosylation of EGFR identified (13). Some previous studies have indicated that the in CL1-5 than in CL1-0. Further study suggested that overexpression glycans could participate in regulating certain functions of EGFR of sialyltransferases in CL1-5 and α1,3-fucosyltransferases (FUT4 or (14). For example, mutagenesis to remove the Asn 420 and 579- FUT6) in CL1-5 and A549 cells would suppress EGFR dimerization linked glycans of EGFR and Asn 418-linked glycans of ErbB3 and phosphorylation upon EGF treatment, as compared to the causes ligand-independent spontaneous dimerization (15–17). control and CL1-0 cells. Such modulating effects on EGFR dimeriza- Besides, overexpression of sialidase in A431 cells would enhance tion were further confirmed by sialidase or fucosidase treatment. EGFR activity, implying that sialylation of EGFR might inhibit Thus, increasing sialylation and fucosylation could attenuate EGFR- its activation (18). On the other hand, core fucosylation was re- mediated invasion of lung cancer cells. However, incorporation of ported to aid EGFR activation: knocking down fucosyltrans- the core fucose by α1,6-fucosylatransferase (FUT8) would promote ferase (FUT) 8 attenuates EGFR phosphorylation (19), and EGFR dimerization and phosphorylation. knocking out FUT8 reduces the EGF-binding ability of EGFR and down-regulates EGFR-mediated MAPK signaling (20). sialic acid ∣ glycoproteomics ∣ glycan sequencing ∣ click chemistry ∣ Further studies have indicated that overexpressing FUT8 could mass spectrometry increase the susceptibility of EGFR to specific tyrosine kinase inhibitors, whereas knockdown of FUT8 decreases the susceptibil- rotein glycosylation accounts for the vast majority of posttran- ity (21). Thus, delineating the roles of glycosylation on EGFR or Pslational processes to affect protein folding, stability, solubi- other proteins may provide previously undescribed insights into lity, and function. Abnormal glycosylation has been reported to disease biology and therapeutic strategy. associate with malignant transformation of tumor cells. In parti- Lung cancer cells are known to express various sialylated cular, the terminal position of glycans with sugars such as sialic or fucosylated glycan epitopes on the surface including sLex, acid and fucose is usually exhibited at a high level in tumor cells sLea, sTn, Ley, Globo H, polysialic acid, fucosyl GM1, GM2, (1). These two major terminal sugars may participate in impor- etc. (1). To uncover the differences of protein glycosylation tant cell–cell interactions and cell migrations in cancer metastasis and further link to protein functions, we labeled glycosylated pro- and angiogenesis (2). However, little evidence has been shown teins with alkyne-sugar probes, followed by Cu [I]-catalyzed al- to support the causative roles of sialylation and fucosylation in kyne-azide click chemistry (22) to identify the sialylated and malignant transformation. Because sialylation and fucosylation fucosylated proteins from lung cancer cell lines CL1-0 and occur globally, their exact roles in the growth and metastatic be- CL1-5 (23), two subpopulations that are derived from the same havior of tumors, especially at the molecular level, remain to be parental cell line with distinct invasion capabilities. Here, we investigated. showed that the more invasive cell line CL1-5 exhibited higher Among the membrane glycoproteins, epidermal growth factor sialylation and fucosylation levels and expressed more sialylated receptor (EGFR) with tyrosine kinase activity has been the target proteins. Among these identified sialylated proteins, we chose for drug discovery as its activity correlates with tumor progression. EGFR is overexpressed in various kinds of cancer cells and participates in the regulation of cancer invasion, metastasis, Author contributions: Chung-Hsuan Chen, K.-H.K., C.-J.Y., P.-C.Y., T.-L.H., and C.-H.W. designed research; Y.-C.L., H.-Y.Y., C.-Y.C., Chein-Hung Chen, P.-F.C., Y.-H.J., and T.-L.H. and angiogenesis (3). It belongs to the EGFR family and trans- performed research; Y.-C.L., H.-Y.Y., C.-Y.C., Chein-Hung Chen, P.-F.C., Y.-H.J., and T.-L.H. duces cell signaling through ligand (EGF)-induced receptor analyzed data; and Y.-C.L., T.-L.H., and C.-H.W. wrote the paper. dimerization (4), which then initiates its tyrosine kinase activity The authors declare no conflict of interest. to direct the malignant behaviors of cancers (5). The extracellular 1Y.-C.L. and H.-Y.Y. contributed equally to this work. domains of the EGFR family members are divided into four sub- 2To whom correspondence may be addressed. E-mail: [email protected] or domains, with domains I and III participating in ligand binding [email protected]. (6) and domains II and IV for dimerization (7). In the state with- This article contains supporting information online at www.pnas.org/lookup/suppl/ out ligand stimulation, an intramolecular interaction between doi:10.1073/pnas.1107385108/-/DCSupplemental.

11332–11337 ∣ PNAS ∣ July 12, 2011 ∣ vol. 108 ∣ no. 28 www.pnas.org/cgi/doi/10.1073/pnas.1107385108 Downloaded by guest on September 30, 2021 EGFR to further study the effect of sialylation and fucosylation on its functions, especially dimerization and phosphorylation. Results Identification of Sialyl Glycoproteins in CL1 Lung Cancer Cells. By em- ploying alkynyl sugars as probes for metabolically labeling and enriching cellular glycoproteins (22, 24), we compared the sialyl glycoproteins expressed on the lung cancer cells CL1-0 and CL1- 5. Fig. S1A shows the flowchart of the glycoproteomic approach. Fig. S1B shows the patterns and labeling intensity of the protein extracts derived from these two cell lines labeling with alkynyl ManNAc [ManNAcyne, the precursor to CMP-alkynyl sialic acid (CMP-NeuAcyne)], and azido biotin probe. Consistently, more sialyl proteins were identified in CL1-5 cells (Fig. S1C and Table S1). Again, the merit of this labeling method was demonstrated by the results that >95% of the enriched peptides bore NXS/T sequon, and most of them were derived from membrane (72%) or secreted (13%) proteins. The MALDI-TOF MS profiles of permethylated N glycans from CL1-0 and CL1-5 also showed different sialylation levels between CL1-0 and CL1-5 cells and a higher level of fucosylation in CL1-5 cells (Fig. S1 D–F), although the branching level of N glycans in CL1-0 and CL1-5 were similar (biantennary: 56.1% vs. 60.3%; triantennary: 28.5% vs. 26.7%; Fig. 1. EGF-induced EGFR dimerization and tyrosine phosphorylation. tetraantennary: 15.4% vs. 13.0%). (A) Dimerization of EGFR on cell surface. (Lower) Quantitative results: first cal- culate EGFR dimer∶monomer ratio in each sample and then normalize with Sialylation of EGFR in CL1 Cells. Among these CL1-5 uniquely the ratio of CL1-0/EGFR without EGF treatment. (B and C) Tyrosine phosphor- expressed sialyl proteins (Table S1), EGFR was chosen for ylation of EGFR upon EGF stimulation at different doses (B) or for different further investigation for its important role in promoting tumor durations (C). (Right) The relative intensity that was compared with phos- growth and metastasis, and the link of EGFR N-linked glycosyla- pho-EGFR∶EGFR ratios and normalization with the ratio of CL1-0/EGFR with- tion to its function (14, 16, 17). To verify if EGFR in CL1-5 was out EGF treatment. (D) Dimerization of sialidase-treated soluble form EGFR. oversialylated, we next examined the expression and sialylation of EGFR in CL1-0 and CL1-5 cells. As shown in Fig. S2A, anti- Role of Fucosylation on EGFR Activation. Our results that sialylation EGFR antibody immunoprecipitated similar amounts of EGFR on EGFR could attenuate EGFR dimerization and phosphoryla- from very different quantities of CL1-0 and CL1-5 protein lysates tion prompted us to inspect the effect of terminal fucosylation, (8∶1), indicating that EGFR was up-regulated in CL1-5 cells. another kind of glycosylation associated with inflammation and CHEMISTRY Nevertheless, the sialylation level of EGFR as demonstrated in cancers, in CL1 cells. We performed quantitative-PCR to exam- ManNAcyne-treated cells was higher in CL1-5. To further con- ine the expression of FUT transcripts in CL1 cells. Comparing to firm the sialylation status, CL1-0/EGFR, an EGFR stable clone CL1-0, CL1-5 showed a higher expression of FUT2 (2.5 folds), of CL1-0 that expressed an equal level of EGFR as CL1-5 cells, FUT4 (2.4 folds), FUT5 (2.4 folds), FUT6 (3.7 folds), FUT8 and CL1-5 cells were subjected to lectin pull-down experiment by (22.5 folds), and FUT11 (4.3 folds) (Fig. S4A), suggesting that SNA (Sambucus nigra lectin, binds α2,6-linked sialic acid) and CL1-5 cells displayed higher α1,3- and α1,6-linked fucosylation MALII (Maackia amurensis lectin II, binds α2,3-linked sialic (core fucosylation). To understand the significance of α1,3-linked acid). Fig. S2B showed that SNA and MALII pulled down more fucosylation on EGFR, we established FUT4 and FUT6 stable EGFR from CL1-5 lysates. Accordingly, the relative percentages clones in lung adenocarcinoma A549 cells (A549-FUT4 and BIOCHEMISTRY of sialylated glycans in CL1-5 were also higher than in CL1-0 A549-FUT6) with Tet-off inducible protein expression system. (Figs. S2C and S1 E and F). These experiments confirmed that Equal expression of EGFR in/on stable clones was confirmed the EGFR in CL1-5 had higher sialylation than in CL1-0 cells. by anti-EGFR immunoblotting and flow cytometric analysis (Fig. S4 B and C), and the increased fucosylation of EGFR in Role of Sialylation on EGFR Activation. To understand how different A549-FUT4/6 cells was detected by alkynyl fucose (Fucyne) label- EGFRs might act in CL1-0 and CL1-5 cells and whether the sia- ing (Fig. S4D). The results from lectin blotting confirmed that the lylation status could influence EGFR activation, we next analyzed increased fucosylation of EGFR in these cells was mainly α1,3- EGFR dimerization and phosphorylation upon EGF stimulation linked, because AAL (Aleuria aurantia lectin, recognizes α1,2, in both cells. Fig. 1A revealed that EGFR dimerization occurred α1,3/4, and α1,6-linked fucose), but not Lens culimaris aggluti- in CL1-0/EGFR without EGF treatment, and EGF induced less nin-A (recognizes α1,6-linked fucose) or Ulex europaeus agglu- EGFR dimers in CL1-5 than in CL1-0/EGFR cells. To evaluate tinin (recognizes α1,2-linked fucose), showed a stronger EGFR- the phosphorylation status of these two cells, cells were treated binding signal in lectin blotting (Fig. S4E). with EGF at various concentrations (Fig. 1B), or for different per- We next analyzed EGF-induced EGFR dimerization in A549- iods of time (Fig. 1C), and then examined for EGFR tyrosine FUT4/6 cells. Compared with mock control, A549-FUT4 and phosphorylation. Consistent with the dimerization results, CL1- A549-FUT6 cells showed less EGFR dimerization (Fig. 2A) 5 cells showed weaker EGFR tyrosine phosphorylation compared and lower EGFR tyrosine phosphorylation levels (Fig. 2B). After to CL1-0-EGFR. Because CL1-5 showed higher sialylation than treating the cells with doxycycline to turn off FUT4 and FUT6 CL1-0 and CL1-0-EGFR cells, we subsequently tested if EGF-in- expression, EGFR in these cells showed lower AAL binding duced EGFR dimerization would be intensified in CL1-5 cells (Fig. S5A), but increasing dimerization and tyrosine phosphory- after sialidase treatment. Accordingly, CL1-5 cells treated with lation levels (Fig. S5 B and C) upon EGF stimulation. These re- α2,3/6/8-linked sialidase showed stronger EGFR dimerization sults suggested that increasing α1,3-linked fucosylation on EGFR (Fig. S3A) and phosphorylation (Fig. S3B), suggesting that sialy- reduced receptor dimerization and activation. lation suppressed ligand-induced EGFR dimerization and the Because CL1-5 expressed a very high level of FUT8 mRNA downstream signaling. compared to CL1-0 (Fig. S4A), we then tested if overexpressing

Liu et al. PNAS ∣ July 12, 2011 ∣ vol. 108 ∣ no. 28 ∣ 11333 Downloaded by guest on September 30, 2021 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 Fig. 3. Summary of the glycoforms.(A) MALDI-TOF MS profiles of the per- N and normalization with the ratio of A549-Mock without EGF treatment. methylated glycans from EGFR immunoprecipitated from the lysates of (C) Dimerization of fucosidase-treated soluble form EGFR. CL1-0 (Upper) and CL1-5 (Lower) cells. The molecular compositions of the major ½M þ Naþ molecular ion signals detected were assigned as annotated. High mannose-type N glycans were labeled simply as M5–M9, representing or knocking down FUT8 could influence the behavior of EGFR. Man5–9ðGlcNAcÞ2. Monosaccharide symbols used were as follows: triangle, For this purpose, we established CL1-0-FUT8, A549-FUT8 Fuc; square, HexNAc; circle, Hex; diamond, NeuAc. Glucose oligomer contami- stable lines (FUT8 overexpression), and CL1-5-FUT8 knock- nants were labeled as X. (B) The glycoforms analyzed according to glycopep- down stable clones to examine EGF-induced EGFR dimerization tides profiles were detected from the EGFR samples derived from CL1-0 and and tyrosine phosphorylation (Fig. S6). Different from what we CL1-5 and site-specific sialic acid and fucose indexes (C) of the EGFR proteins observed that α1,3-linked fucosylation suppressed EGF-induced derived from CL1-0 and CL1-5. Types of glycans: Man, high mannose; Bi, biantennary; Tri, triantennary; Tetra, tetraantennary; Penta, penta-antennary; receptor dimerization and phosphorylation, overexpressing N A–F Hexa, hexa-antennary; F, fucose; S, sialic acid; N, acetylhexosamine. FUT8 in CL1-0 and A549 did not influence (Fig. S6 ), Sialic acid index ¼½ð%with one sialic acid × 1Þþð%with two sialic acids × 2Þþ whereas knocking down FUT8 in CL1-5 cells reduced (Fig. S6 G ð%with three sialic acids × 3Þþð%with four sialic acids × 4Þ∕100; Fucose and H) EGFR dimerization and phosphorylation. Our results index ¼½ð%with one fucose × 1Þþð%with two fucoses × 2Þþð%with three were consistent with the report that FUT8 knockout cells are fucoses × 3Þ∕100. less sensitive to EGF treatment, and this is possibly due to the reduction of EGF-binding affinity when EGFR bears no core every individual EGFR glycopeptide derived from CL1-0 and fucoses (20). CL1-5 cells was compositionally assigned and quantified in a site-specific manner. Site-Specific Glycoform Mapping and Glycan Sequencing of EGFR in Te n N-linked glycosylation sites on EGFR peptides were iden- CL1-0 and CL1-5 Cells. To profile the glycoforms of EGFR, the tified by Asn to Asp conversion after PNGase F treatment full-length EGFR was overexpressed and purified for analysis. (Table S2). Within these 10 sites, 3 of them (Asn positions The MALDI-TOF MS profiles (Fig. 3A) showed that EGFR from 328, 337, and 599) were identified to compose mostly high man- CL1-5, compared to CL1-0, displayed a higher level of sialylated nose-type N glycans (Man5 to Man9), 6 of them (Asn positions glycan structures (highlighted by red squares). The detailed gly- 32, 151, 389, 420, 504, and 579) were attached mainly with com- cosylation patterns on EGFR were subsequently analyzed by plex-type N glycans, and Asn 544 was ligated with both high man- liquid chromatography-MS/MS to reveal the glycoforms (Fig. 3B nose- and complex-type N glycans (Table 1 and Fig. S7). The and Figs. S7 and S8). Through matching with the calculated glycosylation analysis revealed that EGFR from CL1-0 displayed masses of both tryptic peptide fragments and glycans from more Man8 structure, and CL1-5 bore more bi- and triantennary Consortium for Functional Glycomics carbohydrate databases, glycans attached with at least one sialic acid and one fucose re- and the appearance of fragmented glycans in MS/MS spectra, sidue (Fig. 3B). The branching levels of complex-type N glycans

11334 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1107385108 Liu et al. Downloaded by guest on September 30, 2021 Table 1. Site-specific representative glycans of EGFR influence EGF-mediated CL1-5 cell invasion. CL1-5 cells were first confirmed for the low expression level of surface sialic acid 6 h after sialidase treatment (Fig. S4H). EGF-mediated CL1-5 cell invasion was then evaluated by matrigel-coated transwell assay within 6 h. The results in Fig. 4A showed a higher invasion ability of CL1-5 cells when exposed to EGF. Notably, treating CL1-5 cells with sialidase enhanced EGF-mediated cell invasion. Nevertheless, A549-FUT4 and A549-FUT6 cells showed weaker invasion ability than mock control cells, whereas doxycycline treated A549-FUT4/6 and mock cells showed similar invasion ability (Fig. 4B). Here, our results suggested that sialylation and fucosylation could suppress the metastatic ability of cancer cells possibly via affecting EGFR dimerization and activation. In summary, we have identified sialylated and fucosylated proteins by metabolically labeling the glycoproteins of lung cancer cells with ManNAcyne and Fucyne and compared the sialyl and fucosyl protein profiles. Our results revealed a regulatory me- chanism of sialylation and fucosylation on EGF-mediated EGFR behavior: Increasing sialylation and α1,3-linked fucosylation would suppress EGFR dimerization and phosphorylation, which could in turn affect the metastatic ability of cancer cells (Fig. 4C). On the other hand, the core fucosylation would promote EGFR dimerization and phosphorylation. Discussion Aberrant glycosylation has been discovered in many kinds of cancer cells, but the exact functions of altered glycans on glyco- on EGFR were similar in CL1-0 and CL1-5 cells (biantennary: proteins or cellular tumorigenesis are still unclear. Many reports 82.8% vs. 82.3%; triantennary 11.8% vs. 15.4%; tetraantennary: suggest that structural changes in cell surface carbohydrates may 2.7% vs. 2.0%; pentaantennary: 2.4% vs 0.3%; hexaantennary: promote tumor transformation. For example, the α1,6-linked 0.4% vs. 0). fucosylation of complex-type glycans may be an important feature We next examined the level of sialylation and fucosylation on of tumor progression related to increased metastasis (26), over- N C each glycosite with sialic acid and fucose indexes (Fig. 3 ). expression of N-acetylglucosaminyltransferase V can enhance the Consistent with the result that EGFR in CL1-5 carried more sialic invasion ability of glioma cells (27), and overexpression of FUT4 acids and fucoses, CL1-5 was identified to have more sialylated would promote the proliferation of human epidermoid carcino- CHEMISTRY and fucosylated glycans than CL1-0, especially at positions 389, ma A431 cell by controlling the cell cycle machinery via MAPK 420, and 544. Considering that CL1-0 and CL1-5 expressed much higher FUT8 than other FUTs(Fig. S4A), the fucose residue was preferentially assigned to the core position in each glycan structure. Detailed percentages of individual forms of glycopeptides were shown in Fig. S8.

In Vitro Dimerization of Sialidase- and Fucosidase-Modified EGFR. To

further test whether sialylation and fucosylation have a role in BIOCHEMISTRY EGFR dimerization, the FLAG-tagged soluble EGFR (sEGFR) was overexpressed in 293F cells and treated with glycosidases (α2,3/6/8/9-sialidase and α1,3/4-fucosidase). The digestion of sEGFR with sialidase or fucosidase was confirmed by quantifying the remaining sialic acids attached on sialidase-treated sEGFR (Fig. S4F) or AAL lectin blotting (Fig. S4G), respectively. Ac- cording to the analysis, the sialic acids on sEGFR were removed completely by sialidase treatment (from approximately 6 to approximately 0.2 sialic acids per sEGFR molecule). We then tested the in vitro dimerization of sialidase-treated sEGFR. As shown in Fig. 1D, more dimers were formed in sialidase-treated sEGFR compared to the untreated control, whereas treating with heat- inactivated sialidase did not influence sEGFR dimerization. Also, in accordance with the observations from cell-based experiments, the fucosidase-treated sEGFR yielded higher dimerization upon EGF ligation (Fig. 2C). All these results confirmed that sialylation and fucosylation could suppress EGFR dimerization. Together with the previous reports that Lex and sLex epitopes Fig. 4. Sialylation and fucosylation suppress EGFR activation to modulate were detected in EGFR of A431 cells (13, 25), our results indi- EGF-mediated invasion in lung cancer cells.(A) EGF-mediated invasion of cated that Lex and sLex could affect EGFR dimerization. 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 calcu- Role of Sialylation and Fucosylation in EGF-Mediated Cell Invasion. To lated from five power fields for each sample for A and B.*,p < 0.05; **, p < understand the significance of sialylation and fucosylation on 0.001 by Student’s t test. (C) Sialylation and fucosylation suppress EGFR EGFR in tumor metastasis, we tested if sialidase treatment could activation.

Liu et al. PNAS ∣ July 12, 2011 ∣ vol. 108 ∣ no. 28 ∣ 11335 Downloaded by guest on September 30, 2021 and PI3K/AKT signaling pathway (28). Thus it is proposed that to only EGFR glycosylation in this complex situation. Neverthe- specific inhibitors targeting the glycosyltransferases associated less, our results clearly showed that reducing the terminal sialyla- with numerous cancers may help defend against cancers. How- tion and α1,3-fucosylation on EGFR enhanced its dimerization ever, some other reports mention that glycosylation may have and activation, and according to our data, the sialic acid and fu- negative effects on protein functions or cellular behavior. For cose on Asn 420 glycans may be most effective to inhibit EGFR instance, α2,6-sialylated glycans may inhibit glioma malignancy dimerization and phosphorylation. because decreased α2,6-sialylation on glycoproteins (29, 30) or This study raises a question that whether inhibiting sialyl- on gangliosides (31) enhances glioma formation and invasion; in- or fucosyltransferases is a practical way to counteract cancer, hibition of N-linked glycosylation by tunicamycin can induce because EGFR, and possible other sialyl/fucosyl receptors, are functional E-cadherin-mediated cell–cell adhesion (32); removal widely involved in the malignancy of many types of tumors. If sia- of sialic acids on cell surface by sialidase enhances connexin-43 lylation and fucosylation suppress receptor activation, inhibiting gap junction functions (33). In virus infection, it has been ob- sialylation and fucosylation may create more aggressive cancer served that removal of glycans on influenza hemagglutinin en- cells. Conversely, increasing sialylation or fucosylation in aggres- hances binding to cell surface receptors (34). Previous studies sive cancers may help counteract cancer progression; therefore, based on site-directed mutagenesis of EGFR also reveal that the enhancement of sialyl- or fucosyltransferase activity may be a the glycans at positions 420 and 579 of EGFR pose an inhibitory unique therapeutic approach to tumors. Further attempts to ex- effect on EGF-independent dimerization (14, 17, 35). amine how glycoforms alter protein conformation and protein– In our study, we found that EGFR in CL1-5 exhibited higher protein interaction, the events that closely link with signaling sialylation and fucosylation levels and resulted in lower dimeriza- and cellular behaviors, will provide more evidence to understand tion and tyrosine phosphorylation than in CL1-0 during EGF the multifaceted roles of protein glycosylation. stimulation. In addition, removal of sialic acids from EGFR by sialidase increased dimer formation of EGFR upon EGF treat- Materials and Methods ment on cell and in vitro, and pretreating EGFR with fucosidase EGFR Dimerization Assay. Purified FLAG-tagged sEGFR (1 μg), with or without also resulted in a similar dimerization enhancement in vitro. glycosidase treatment, was incubated with 100 μg∕mL human EGF in 100 mM NaCl∕20 mM Hepes buffer (pH 7.4) for 2 h at room temperature (RT). Moreover, induction of FUT4 and FUT6 overexpression in lung 3 Tet Cross-linker BS (Bis[sulfosuccinimidyl] suberate, Thermo Scientific; 0.25 mM) cancer A549 cells with -off system decreased EGFR dimeriza- was added into the protein mixtures and allowed to react for 1 h at RT. The tion and activation, and this FUT4/6-mediated suppression was samples were analyzed by SDS-PAGE and immunoblotting with Peroxidase- reverted when treating the cells with doxycycline to turn off conjugated anti-FLAG (M2) MAb (Sigma-Adrich). For analyzing the EGFR di- FUT4/6 overexpression (Fig. S5 and Fig. 4B). These results de- merization on cell surface, cells were first starved in serum-free medium over- monstrated that sialylation and terminal α1,3-fucosylation could night, followed by stimulation with EGF (0–50 ng∕mL) for 5 min. Cross-link inhibit EGFR activation in lung cancer cells, and these modifica- reaction was conducted by the addition of 1 mM BS3 and the incubation at RT tions could actually affect EGFR-mediated signaling and cellular for 20 min, and terminated by adding 50 mM Tris-HCl (pH 7.4). The samples behavior. Previous studies have reported the fractionation and were analyzed by SDS-PAGE and Western blot with anti-EGFR Ab (Cell identification of EGFR glycans in A431 cells and indicated that Signaling). the blood group A antigen, as exemplified by α-linked terminal N Detection of EGFR Tyrosine Phosphorylation. Cells were starved in serum-free acetylgalactosamine, can be highly expressed in EGFR (36, 37), 0–50 ∕ α medium overnight and stimulated with EGF ( ng mL) for 5 min or with whereas terminal sialylation and 1,3-fucosylation are limited in 10 ng∕mL EGF for 0–10 min. The cells were washed with cold PBS immedi- A431 cells. Thus, based on our finding, it is interesting to know if ately and harvested with lysis buffer (1% NP-40, 25 mM Tris, 150 mM NaCl, A431 cell has enhanced EGFR dimerization and signaling. It is 3 mM KCl, pH 7.4, 1 × EDTA-free protease inhibitor cocktail from Roche) with worthy of mentioning that the study also suggests a negative role 1× phosphatase inhibitor (Roche). The EGFR in cell lysates was immunopre- of blood group type A antigen/terminal N acetylgalactosamine cipitated with EGFR antibody (Cell Signaling) on protein G agarose (Thermo in EGFR affinity, tyrosine kinase activation, and receptor turn- Scientific). The immunoprecipitated samples were resolved SDS-PAGE and over (37). analyzed for the phosphotyrosine signals by Western blot with antiphospho- One of the major locations that showed elevated sialylation tyrosine antibody (4G10, Millipore). and fucosylation of EGFR in CL1-5 was Asn position 420 C N Glycosidase Treatment. For sialidase treatment on sEGFR, 1 μg sEGFR was (Fig. 3 ), the -glycosylation site that has been shown to prevent mixed with 28.6 mU∕μL sialidase (α2,3/6/8/9-linked, Prozyme) at 37 °C for EGFR spontaneous dimerization (17). This implies that sialyla- overnight in 5 μL as instructed by the vendor. For sialidase treatment of tion and fucosylation on Asn 420 can be critical in suppressing CL1-5 cells, 106 cells were incubated with 0.04 U∕mL sialidase (α2,3/6/8-linked, EGFR dimerization and activation. Regarding the core fucosyla- Roche) in 3 mL serum-free RPMI medium 1640 at 37 °C for 30 min. The reaction, although CL1-5 expressed a much higher level of FUT8 than tion efficiency was further detected by sialic acid quantitation kit (Sigma- CL1-0, overexpressing FUT8 in CL1-0 did not affect EGFR Aldrich). For cleaving fucoses on sEGFR, 1 μg sEGFR was mixed with 200 μU dimerization and phosphorylation, indicating that the core fuco- fucosidase (α1,3/4-fucosidase, from Calbiochem) in 10 μl of sodium phosphate sylation could be fulfilled in the cells with even a limited amount buffer (pH 5.0) at 37 °C overnight. of FUT8 expression. On the other hand, consistent with the results reported previously, knocking down FUT8 expression in EGF-Mediated Invasion Assay. The 24-well transwell plates with inserts [8-μm-pore polycarbonate membrane (Corning)] were set up for invasion CL1-5 cells showed a slight reduction of EGFR activation, which assay. First, the membranes of the inserts were coated with 50 μg Matrigel could be contributed by a reduced affinity to EGF (20). (BD Bioscience) in 100 μL RPMI medium 1640 (Invitrogen). Cells were resus- Our study provides a clear correlation between sialylation/ pended in RPMI medium 1640 at the density of 106 cells∕mL and seeded into fucosylation and EGFR dimerization/activation, but the conclu- the inserts (100 μL∕insert) of transwell plates. The attractant human EGF sion is contradictory to the malignant phenotypes of CL1-5 (20 ng∕mL, Millipore) was diluted in RPMI medium 1640 and dispensed into because CL1-5 cells display more sialylation/fucosylation and the lower chambers. Following the incubation for 6 h at 37 °C, the inserts were are still more invasive than CL1-0 (23). This is understandable removed and the numbers of cells that migrated to the reverse side of the because CL1-5 cells are selected based on the invasion ability in- membrane were quantified by microscopy after nuclear staining with DAPI. stead of glycosylation patterns of the cells. This sorting system results in two distinct cell populations that express very different ACKNOWLEDGMENTS. We thank Professor Mien-Chie Hung (MD Anderson Cancer Center, Houston, TX) for the EGFR construct, and Eric Huang, sets of proteins, especially those for cancer progression; for Bo-Hua Chen, and Po-Kai Chuang for technical support. The research was example, CL1-5 cells express more MMP-9 (23). Thus it is inap- supported by the National Science Council and Genomics Research Center, propriate to directly link the invasive ability of CL1-5 and CL1-0 Academia Sinica, Taiwan.

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