2 Pathology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan
3 Department of Biochemistry, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
4 Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
Received on December 2, 2002; revised on March 3, 2003; accepted on March 5, 2003
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Abstract |
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Key words:
benzyl--GalNAc
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cellcell adhesion
/
dysadherin
/
E-cadherin
/
O-glycosylation
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Introduction |
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Dysadherin is a molecule that we identified as a tumor-associated antigen in many cancer tissues (Ino et al., 2002; Shimamura et al., 2003
). The transfection of this molecule causes E-cadherin down-regulation and disturbs homotypic cell adhesion in human hepatocellular carcinoma cell line PLC/PRF/5 (Ino et al., 2002
). The amino acid sequence of dysadherin predicted from cDNA sequence indicated that it is a type 1 transmembrane protein with features typical of membrane-associated mucins, including serine-, threonine-, and proline-rich ectodomains. There was no tripeptide sequence, Asn-X-Ser/Thr, known as a potential N-glycosylation site in the predicted amino acid sequence of dysadherin. Thus it has been suggested that dysadherin expresses only O-glycans.
Some mucin-associated molecules have been reported to show anti-adhesive effects by steric hindrance owing to their size and/or by charge repulsion due to their negatively charged sialylated glycans. Episialin, also known as MUC1, is a glycoprotein shown in vitro to reduce E- cadherin-mediated cellcell adhesion by steric hindrance (Wesseling et al., 1996). Using full-length or shortened episialin transfectants, it has been clearly shown that length of the episialin molecule is mainly responsible for its anti-adhesive effect. Sialidase treatment only partially restored the aggregation capacity of the transfectants (Ligtenberg et al., 1992
; Wesseling et al., 1995
). Podocalyxin is another membrane-associated mucin that has an anti-adhesion function (Takeda et al., 2000
). In this case, a negative charge from sialic acid is primarily responsible for its anti-adhesive effect. Similarly, epiglycanin, which is highly expressed on the TA3/Ha mouse mammary tumor cell line, inhibits E-cadherin and integrin-mediated adhesion (Kemperman et al., 1994
).
In this article, we show direct evidence of O-glycosylation of dysadherin and propose the structures of O-glycans. Then we evaluate the importance of glycosylation in dysadherin function by examining biological and biochemical changes resulting from treatment of dysadherin overexpressing hepatocellular cells with benzyl--GalNAc, a modifier of O-glycosylation.
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Results |
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Down-regulation of dysadherin and up-regulation of E-cadherin after benzyl--GalNAc treatment
It has been shown that E-cadherin was markedly decreased at protein level but not at mRNA level in dysadherin transfectants. After benzyl--GalNAc treatment, expression of dysadherin was significantly reduced (Figure 2A). Western blot analysis showed a slight increase of apparent molecular weight of dysadherin. Furthermore, E-cadherin expression, both in Triton-X 100soluble fraction and Triton-X 100insoluble fraction, was up-regulated. On the other hand, benzyl-
-GalNAc treatment did not affect E-cadherin expression in mock transfectants. To assay for a dose-dependent effect of benzyl-
-GalNAc, cells were treated with different concentrations of the drug. Down-regulation of dysadherin and up-regulation of E-cadherin were dependent on the concentration of benzyl-
-GalNAc (Figure 2B). These results suggest that there is an inverse relation between expressions of dysadherin and E-cadherin. Moreover, down-regulation of dysadherin expression caused by benzyl-
-GalNAc treatment was also observed in other human cell lines (AsPC-1; pancreas carcinoma, MKN28; stomach carcinoma, TE6; small cell lung carcinoma) (data not shown). These observations indicate that O-glycosylation is important for stable expression of dysadherin.
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Sialidase treatment did not affect cell aggregation
To investigate whether the altered sialylation per se causes the decrease in cell aggregation, the mock and dysadherin transfectants were treated with sialidase for 30 min before cell aggregation assay. As shown in Figure 4, no significant effect on either cell was observed (P>0.2). Apparent molecular weight of membrane-associated dysadherin in western blot analysis of whole cell lysate was slightly increased as in the case of dysadherin-Ig (data not shown), suggesting that sialidase treatment was effective.
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Discussion |
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To assess the importance of O-glycans expressed on the cell surface dysadherin, dysadherin transfectant of PLC/PRF/5 cells was treated with benzyl--GalNAc, which is known to modify O-glycosylation. The anti-adhesive effect of dysadherin, which was shown by the previous observation that dysadherin transfectants were less aggregated than mock transfectants (Ino et al., 2002
), was significantly reduced after treatment. Benzyl-
-GalNAc treatment of dysadherin transfectants also resulted in a remarkably decreased expression of cellular dysadherin with a concomitant increase of E-cadherin expression. As expression of E-cadherin in mock transfectant was not affected by benzyl-
-GalNAc treatment, the theory that benzyl-
-GalNAc directly affects E-cadherin expression is excluded.
Considering these results and the previous observation that dysadherin transfection resulted in decreased protein expression of E-cadherin without affecting its expression on the mRNA level, it is likely that benzyl--GalNAc treatment primarily inhibits dysadherin expression and then induces E-cadherin expression by unknown mechanisms. Presently we do not know how O-glycans regulate expression of dysadherin. However, it will be important to consider the possible role of O-glycosylation in metabolic fate or stability of dysadherin as suggested in the cases of other glycoprotein receptors. For example, it has been shown that inhibition of O-glycosylation of very-low- density lipoprotein receptors results in a rapid cleavage from the cell and release of a large amino-terminal fragment into the culture medium (Magrane et al., 1999
). Similarly, in the absence of O-glycosylation, the decay-accelerating factor is proteolytically cleaved soon after reaching the cell surface with release of its large fragment into the culture medium, resulting in low expression of this molecule on the cell surface (Reddy et al., 1989
).
Benzyl--GalNAc was initially reported to selectively inhibit O-glycosylation through its ability to compete with GalNAc-O-Ser/Thr for the ß1,3-galactosyltransferase involved in the biosynthesis of O-glycan (Kuan et al., 1989
). However, in mucin-secreting HT-29 cells, it was shown that the major step inhibited by treatment with this sugar analog is sialylation rather than the transfer of Gal to GalNAc-
-O-Ser/Thr (Hennebicq-Reig et al., 1998
; Huet et al., 1998
, 1995
). In this article, we also indicate that sialylation of O-glycan attached to dysadherin produced in PLC/PRF/5 cells is almost completely inhibited by benzyl-
-GalNAc treatment. It is suggested that sialylation of O-glycans is involved in stable expression of dysadherin. However, the direct contribution of sialic acid to the anti-adhesive function of dysadherin expressed on the cell surface is not likely because sialidase digestion of dysadherin transfectant does not significantly change cellcell adhesion.
There are several mucins affecting cell adhesion of cancer cells. Epiglycanin, which is highly expressed on the TA3/Ha mouse mammary tumor cell line, inhibits E-cadherin and integrin-mediated adhesion (Kemperman et al., 1994). MUC1 mucin (episialin) is expressed in a wide variety of tumors, and its anti-adhesion ability and important role in tumor development have been shown in various cells. A normal mammary epithelial cell line and a melanoma cell line do not aggregate efficiently after transfection of cDNA encoding MUC1 (Ligtenberg et al., 1992
). MUC1 transfectants of human gastric cancer cells (Suwa et al., 1998
) and pancreatic cancer cells (Satoh et al., 2000
) exhibit enhanced in vitro invasion, increased motility, and decreased binding to extracellular matrix components. Adhesive properties of the transfected cells are abolished by benzyl-
-GalNAc treatment in both cases (Satoh et al., 2000
; Suwa et al., 1998
), suggesting an important role of O-glycans in MUC1 function. Decreased MUC1 expression in breast cancer cell lines induced by an antisense oligonucleotide induces E-cadherin-mediated cell adhesion (Kondo et al., 1998
). Sialomucin complex (SMC), a rat homolog of the human mucin MUC4, is a large, membrane-bound mucin complex originally isolated from highly metastatic ascites 13762 (Komatsu et al., 1997
). Overexpression of SMC induces morphology changes, cell detachment, and cellcell dissociation of transfected A375 human melanoma cells in culture. Intravenous injection of SMC-overexpressing cells also results in substantially greater lung metastasis than injection of SMC-repressed cells (Komatsu et al., 2000
). As previously demonstrated with MUC1 (Wesseling et al., 1996
), these mucins seem to sterically disrupt molecular interactions for cell adhesion. There is a relationship between expression of MUC1 and functional down-regulation of E-cadherin. However, there is no evidence showing that MUC1 regulates expression of E-cadherin molecules at protein level, and this fact contrasts with dysadherin which expression down-regulates E-cadherin expression (Ino et al., 2002
).
In conclusion, this article reveals that dysadherin is a heavily O-glycosylated mucin-like molecule of which cellular expression is regulated depending on its glycosylation status. Aberrant O-glycosylation caused by benzyl-- GalNAc treatment inhibits stable expression of dysadherin and leads to up-regulation of E-cadherin by an unknown mechanism, resulting in increased cellcell adhesion. Therefore regulation of dysadherin expression might be a new strategy for cancer therapy, and the carbohydrate-directed approach is expected to aid its development.
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Materials and methods |
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To prepare stable dysadherin-Ig transfectant, pCDNA3 vector containing an extracellular domain of dysadherin fused with Fc fragment of human immunoglobulin G was transfected into PLC/PRE/5 cells using LipofectAMINE (Life Technologies). Stable transfectants were isolated by growing them in G418-containing medium.
Preparation of dysadherin-Ig
Dysadherin-Ig transfectants were cultured subconfluently, and after washing three times with phosphate buffered saline (PBS), the media were changed into Opti-MEM (Life Technologies). Cells were cultured for another 3 days, and dysadherin-Ig molecules secreted into the media were collected and purified using Protein-G Sepharose column (Life Technologies) according to the manufacturer's instructions. Its purity was checked using PAGE followed by silver staining.
Analysis of carbohydrate composition and O-glycans
Dysadherin-Ig was heated in 0.5 ml 4 N trifluoroacetic acid at 100°C for 3 h for analysis of neutral and amino sugars, or in 0.01 N HCl at 100°C for 20 min for analysis of sialic acid. The hydrolysates were freed from acid by repeated evaporation with water and analyzed by HPAEC-PAD using a Bio-LC system (Dionex, Sunnyvale, CA) equipped with a CarboPac PA-1 column (4x250 mm). PAD response was monitored by a Power Chrom system (AD Instruments, Tokyo) connected to a personal computer. An isocratic elution with 22 mM NaOH was used for analysis of neutral and amino sugars (Hardy et al., 1988), and an elution with 150 mM sodium acetate in 100 mM NaOH was used for analysis of sialic acid. The flow rate was 1 ml/min under both conditions.
O-glycans were liberated from dysadherin-Ig by ß-elimination (Iyer and Carlson, 1971) and purified as previously described (Kotani and Takasaki, 1997
). HPAEC-PAD of the O-glycans was carried out by elution with a linearly increasing concentration of 0400 mM sodium acetate in 100 mM NaOH from 030 min. The flow rate was 1 ml/min.
Benzyl--GalNAc treatment
Benzyl--GalNAc and benzyl-
-GlcNAc, negative control for benzyl-GalNAc (Sigma, St. Louis, MO), were dissolved in dimethylsulfoxide to 800 mM. Cells were seeded; the following day the mediums were changed to ones containing various concentrations of the drug and cultured for 72 h.
Cell aggregation assay
The assay used was a modification of a previously described method (Shimoyama et al., 1992). Cells were cultured on 100-mm dishes overnight in Dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum. Single-cell suspensions were prepared in accordance with an E-cadherin-saving procedure described previously (Takeichi, 1977
). Subconfluent cell layers were rinsed twice with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline (HCMF; 8.0 g/L NaCl, 0.4 g/L KCl, 0.12 g/L Na2HPO412H2O, 1.0 g/L glucose, 2.3 g/L HEPES in 0.005 M NaOH, pH 7.4) containing 5 mM CaCl2 and were incubated in HCMF containing 0.01% trypsin (Trypsin type IX; Sigma) and 5 mM CaCl2 at 37°C with gyratory shaking at 100 rpm for 15 min. Reaction was immediately stopped by adding trypsin inhibitor (Sigma). After washing twice in HCMF containing 1% bovine serum albumin (BSA) (HCMF/BSA), cells were resuspended (5x105 cells/ml) in HCMF/BSA containing 40 ng/ml DNAase (Sigma) and 1 mM MgCl2 by 10 passages through a 26-gauge needle. For removal of cell surface sialic acids, a cell suspension was incubated with 0.1 U/ml of neuraminidase from Streptococcus sp. (Seikagaku, Tokyo) in HCMF/BSA at 37°C for 30 min.
The cell suspension (0.5 ml/well) was seeded in a 24-well plate previously coated with 2% BSA in HCMF and allowed to aggregate for 30 min in the presence or absence of 5 mM CaCl2 at 37°C with gyratory shaking at 100 rpm. Aggregation was quantified by counting representative aliquots from each sample on a hematocytometer using phase-contrast optics. At least 500 cells were counted from each sample. Quantification of aggregation was estimated by the following formula: aggregation index=(N0-Nt)/N0, where Nt is the total number of particles at the incubation time t, and N0 is the total number of cells. Statistical analyses were performed by Mann- Whitney U test.
Cell extraction and western blot analysis
Triton X-100 soluble and insoluble lysates were isolated in accordance with the protocol described by Hinck et al. (1994) with a minor modification. The subconfluent cell layer was washed three times with PBS, followed by incubation at 4°C in 50 mM Tris buffer containing 50 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100, 300 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail tablet (Boehringer Mannheim, Mannheim, Germany) with gentle shaking at 800 rpm. Cell lysates were collected and centrifuged at 15,000 rpm for 30 min, and the supernatant was prepared for Triton Xsoluble faction. The remaining pellet was resuspended in SDS buffer (15 mM Tris, pH 7.3, 1% [w/v] sodium dodecyl sulfate, 5 mM ethylenediamine tetra-acetic acid). After boiling, the solution was used as Triton Xinsoluble fraction. The total protein concentration of each lysate was measured by DC protein assay kit (BioRad, Hercules, CA). An equal amount of protein was separated in ready-made 412% gradient SDSPAGE gel (Invitrogen, Carlsbad, CA) and electroblotted onto a polyvinylidene difluoride membrane (Immobilon, Millipore, Bedford, MA). Membranes were soaked in PBS containing 5% (w/v) nonfat dry milk followed by overnight incubation with primary antibody in PBS containing 2% (w/v) normal swine serum at 4°C. After washing three times with PBS containing 0.1% Tween20, the membrane was incubated with horseradish peroxidaseconjugated secondary antibodies (Amersham, Buckinghamshire, UK) in PBS containing 2% (w/v) normal swine serum at room temperature for 90 min. Proteins were visualized with enhanced chemiluminescent western blotting detection reagents (Amersham). NCC-M53 antibody is a mouse monoclonal antibody recognizing dysadherin (Shimamura et al., 2003
), and its hybridoma culture supernatant was used at 1:5 dilution. Purified HECD-1 antibody (a mouse monoclonal antibody against human E-cadherin) was from Takara Shuzo (Kyoto, Japan) and was used at 1:200 dilution.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: shirohas{at}ncc.go.jp
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Abbreviations |
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References |
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