Increasing the intra-Golgi pH of cultured LS174T goblet-differentiated cells mimics the decreased mucin sulfation and increased Thomsen-Friedenreich antigen (Galß1-3GalNac{alpha}-) expression seen in colon cancer

Barry J. Campbell1, Gillian E. Rowe, Keith Leiper and Jonathan M. Rhodes

Glycobiology Group, Gastroenterology Research Unit, Department of Medicine, University of Liverpool, Daulby Street, Liverpool, L69 3GA, UK

Received on October 16, 2000; revised on December 18, 2000; accepted on January 11, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Mucins in ulcerative colitis and colon cancer share common properties of reduced sulfation and increased oncofetal carbohydrate antigen expression. It has previously been shown that there is no simple correlation between these changes and the activity of the relevant glycosyl-, sialyl-, and sulfo-transferases. We examined mucin sulfation and expression of oncofetal Thomsen-Friedenreich (TF) antigen (galactosylß1-3N-acetylgalactosamine{alpha}-) in the goblet cell–differentiated human colon cancer cell line LS174T following treatment with bafilomycin A1, which raises intra-Golgi pH, or monensin, which disrupts medial-trans Golgi transport.

Cells were dual-labeled with sodium [35S]-sulfate and D-[6-3H(N)]-glucosamine hydrochloride, or labeled with L-[U-14C]-threonine alone. Mucin was purified using Sepharose CL-4B gel filtration. Mucin sulfo-Lewisa and TF antigen expression were assessed using the F2 anti-sulfo-Lewisa monoclonal antibody and peanut agglutinin binding respectively.

Bafilomycin (0.01 µM; 48 h) reduced total mucin sulfation, expressed relative to incorporation of glucosamine, to 0.50 ± 0.04 d.p.m. [35S]-sulfate per d.p.m. [3H]-glucosamine compared to control, 0.84 ± 0.05 (p < 0.001, n = 16). This was accompanied by 50.3 ± 8.0% increased expression of TF antigen (p < 0.01) and 50.1 ± 5.5% decreased expression of sulfo-Lewisa (p < 0.01). The reduced sulfate:glucosamine ratio was largely due to increased incorporation of glucosamine into newly synthesized mucin rather than reduction in total sulfate incorporation. In contrast, monensin only reduced total mucin glycosylation at concentrations > 0.1µM and had no significant effect on mucin sulfation or TF expression.

Intra-Golgi alkalinization affects mucin glycosylation, resulting in decreased mucin sulfation and increased expression of TF antigen, changes that mimic those seen in cancerous and premalignant human colonic epithelium.

Key words: mucin/sulfation/Thomsen-Friedenreich antigen/colon/Golgi


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Similar mucosal glycosylation abnormalities have been demonstrated in ulcerative colitis, Crohn’s disease, colon cancer, and metaplastic polyps (Rhodes et al., 1997Go). The described changes include reduced mucin sulfation (Filipe, 1979Go; Raouf et al., 1992Go; Corfield et al., 1992Go, 1996); increased expression of oncofetal carbohydrate antigens, such as Thomsen-Friedenreich (TF; galactosylß1-3N-acetylgalactosamine{alpha}-) (Campbell et al., 1995Go) and sialyl-Tn (sialyl{alpha}2-6N-acetylgalactosamine{alpha}-) (Ta et al., 1997Go; Karlén et al., 1998Go); and altered expression of ABO/Lewis blood groups (Kim et al., 1986Go). It is commonly assumed that most of these changes are secondary to disease, although we are considering the hypothesis that one or more of the changes could be a genetically determined factor in disease aetiology (Rhodes, 1996Go). The alteration of sulfation could itself explain some of the other changes because the TF antigen has been shown to be concealed by O-sulfate esters in the normal colon (Martinez-Menarguez et al., 1992Go), and a colonic mucin sulfotransferase, which undergoes progessively reduced expression from adenoma to cancer, has as its preferred acceptor the TF antigen (Kuhns et al., 1995Go).

The mechanisms that determine the glycosylation abnormalities in colon cancer and precancer are unclear. In a meticulous study, Brockhausen and colleagues showed that, although there are changes in expression of the relevant glycosyl-, sialyl-, and sulfo-transferases in colon cancer, these changes correlate relatively poorly with the changes in carbohydrate expression (Yang et al., 1994Go). They speculated that other explanations, including altered arrangement of transferases within the Golgi, might be responsible.

Changes in mucin sulfation, which in the gastrointestinal tract is relatively specific to the colon, may have particularly important functional effects because the presence of O-sulfate esters imparts an increased negative charge and confers increased resistance against degradation by enteric bacterial enzymes (Carter et al., 1988Go; Tsai et al., 1992Go, 1995; Roberton et al., 1993Go; Campbell, 1999Go). Mucin sulfation occurs within the trans-Golgi network as a late step in glycoprotein synthesis (Nieuw Amerongen et al., 1998Go). Sulfate esters can be added to terminal or internal galactose (Gal) residues. It has been shown to exist on various sites within the core oligosaccharides that are O-linked to serine/threonine in the protein core (see Hounsell et al., 1996Go for review of the eight well-characterized oligosaccharide core structures), for example, on C-3 of Gal linked ß1-3 to N-acetylglucosamine (GlcNAc) in the core 2 sequence Galß1-3{GlcNAcß1-6}GalNAc{alpha}-, on C-6 of GlcNAc in core 2, and on C-4 of terminal Gal or terminal GalNAc linked ß1-4 to GlcNAc (Hooper et al., 1995Go; Karlsson et al., 1996Go, 1997; Lo-Guidice et al., 1997Go). Sulfated oligosaccharides have been described in the mucins of porcine small intestine (Karlsson et al., 1996Go), rat small and large intestine (Karlsson et al., 1997Go), and human respiratory mucosa (Lo-Guidice et al., 1997Go) but have yet to be fully characterized in human colon, although sulfated Lewisa, recognized using the F2 monoclonal antibody, is strongly expressed by goblet cells throughout the normal human colon (Veerman et al., 1997Go). Sulfation is controlled by Golgi-localized O-glycan sulfotransferases which catalyse the transfer of sulfate moieties from the donor adenosine 3'-phosphate-5'-phosphosulfate to mucin-type carbohydrates. In addition to the TF-antigen sulfotransferase described by Brockhausen’s group (Kuhns et al., 1995Go), two other sulfotransferases have been characterized from human respiratory mucosa that are responsible for the 3-O and 6-O sulfation of terminal galactose and N-acetylglucosamine of mucin carbohydrate chains (Lo-Guidice et al., 1995Go; Degroote et al., 1997Go). Recently, a 6-O-N-acetylglucosamine sulfotransferase that is highly restricted to intestinal tissue has been cloned (Lee et al., 1999Go). It is not known how many sulfotransferases are involved in the synthesis of sulfated mucus glycoproteins, and it is also unclear what mechanisms regulate their activity.

An increased sulfate content of mucins is seen in the bronchial mucosa from patients with cystic fibrosis (Roussel and Lamblin, 1996Go) and in cultured cystic fibrosis nasal epithelial cells even after several passages (Cheng et al., 1989Go). In addition to hypersulfation, recent studies from cystic fibrosis patients have reported increased sialylation of bronchial mucins (Davril et al., 1999Go). It has been suggested that the increased sulfation (Zhang et al., 1995Go) may be related to defective organelle acidification of the trans-Golgi as a result of mislocation of the cystic fibrosis transmembrane conductance regulator apical membrane chloride channel within cells. It is an attractive hypothesis that defective acidification might account for many of the observed cellular alterations involving abnormal posttranslational modifications, as a consequence of altered activity of pH-dependent glycosyltransferases and sulfotransferases in the trans-Golgi. However, there is both evidence for (Barasch et al., 1991Go) and against (Seksek et al., 1996Go) defective trans-Golgi acidification in cystic fibrosis.

We have speculated that mucin sulfation in the colon could be affected by alterations in trans-Golgi pH. In this study, using the human colon carcinoma cell line LS174T, a cell type that forms well-differentiated goblet cells expressing the secretory mucins MUC2, MUC5AC, and MUC6 (van Klinken et al., 1996Go), we investigated the synthesis and sulfation of mucins following treatment with (1) bafilomycin A1, a specific inhibitor of the Golgi vacuolar H+-ATPase (Bowman et al., 1988Go), and (2) monensin, a cationophore that perturbs medial-trans Golgi transport (Tartakoff, 1983Go).


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bafilomycin A1 but not monensin caused marked reduction in mucin sulfation expressed relative to glucosamine incorporation
At 0.005 µM or higher concentration, bafilomycin significantly reduced total mucin sulfation as assessed by the ratio of d.p.m. [35S]-sulfate incorporated to d.p.m. [3H]-glucosamine incorporated; P < 0.001, ANOVA, n = 16 (Figure 1). The reduction in sulfation elicited by bafilomycin was reflected both in the cellular (0.85 ± 0.06, mean ± SD) and secreted (0.41 ± 0.03) mucin compared to control (cellular 1.25 ± 0.07 and secreted 0.73 ± 0.07, respectively). In contrast, monensin treatment showed no significant effect on total mucin sulfation. The reduction in the ratio of incorporation of sulfate to glucosamine that was induced by bafilomycin was mainly due to an increased incorporation of glucosamine (discussed later). Thus, at concentrations up to 1 µM, bafilomycin treatment had no significant effect on the incorporation of [35S]-sulfate into total mucin (cell plus medium mucin); for instance, at 0.01 µM bafilomycin: 538 ± 72 d.p.m. [35S]-sulfate incorporated into mucin/µg DNA compared to control (469 ± 37; n = 16).



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Fig. 1. Effect of bafilomycin (top panel) and monensin (bottom panel) on mucin sulfation. Seven-day overconfluent LS174T cells were treated for 48 h. Mucin sulfation was expressed as the ratio of d.p.m. [35S]-sulfate to d.p.m. D-[6-3H]-glucosamine incorporated into Sepharose CL-4B purified mucin (cellular [black bars], secreted [white bars], and total [shaded bars]). Increasing intra-Golgi pH with bafilomycin A1 significantly reduced sulfation of total mucin; ** P < 0.01 and *** P < 0.001, indicate significant differences from control; ANOVA, n = 16.

 
Bafilomycin but not monensin increased TF antigen and reduced sulfo-Lewisa expression by mucin
In parallel with the decreased ratio of sulfate to glucosamine incorporation, 0.01 µM bafilomycin significantly decreased (50.1 ± 5.5%) the expression of sulfo-Lewisa when expressed per 250 c.p.m. [14C]-threonine labeled total mucin (P < 0.01 ANOVA; n = 8; Figure 2), implying a reduced expression of sulfo-Lewisa per mucin glycosylation site. The decrease in sulfo-Lewisa expression by mucins was reflected both in the cellular and secreted components. In addition, a concomitant increase (50.3 ± 8.0%) in the expression of TF antigen/250 c.p.m. [14C]-threonine labeled total mucin was also seen with 0.01 µM bafilomycin treatment (P < 0.01 ANOVA; Figure 2). The increase in TF antigen expression by total mucin was principally due to increased expression by cellular mucin (40.3 ± 12.7%; P < 0.05). No change in Limax flavus agglutinin (LFA) reactivity was observed with bafilomycin treatment (n = 8). Monensin had no significant effect on either TF antigen or sulfo-Lewisa expression by LS174T mucins (data not shown).



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Fig. 2. Effect of bafilomycin A1 on expression of sulfo-Lewisa antigen (top panel) and Thomsen-Friedenreich (TF) antigen (bottom panel) by LS174T mucin. Seven-day overconfluent cells were treated for 48 h. Expression of sulfo-Lewisa and TF antigen were assessed by binding of the anti sulfo-Lewisa F2 monoclonal antibody and binding of PNA, respectively, per 250 c.p.m. L-[U-14C]-threonine labeled Sepharose CL-4B-purified mucin (cellular [black bars], secreted [white bars], and total [shaded bars]). * P < 0.05, ** P < 0.01 indicate significant differences from control; ANOVA, n = 16.

 
Bafilomycin A1 causes increased mucin glycosylation
At concentrations up to 0.01 µM, bafilomycin treatment resulted in a significant increase in [3H]-glucosamine incorporation into total mucin (cell plus medium mucin)/µg DNA compared to the control (P < 0.05 ANOVA; n = 16; Figure 3). The increased mucin glycosylation was reflected by higher incorporation both into mucin within the cells (0.01 µM bafilomycin, 556 ± 33 d.p.m. [3H]-glucosamine/µg DNA compared with control, 234 ± 14) and into those mucins secreted to the medium (0.01 µM bafilomycin, 591 ± 19; control, 466 ± 13 d.p.m./µg DNA). Monensin treatment had little effect on mucin glycosylation; only concentrations that were antiproliferative for LS174T cells (0.1 µM and higher) significantly reduced the incorporation of [3H]-glucosamine/µg DNA into total mucin (P < 0.001, n = 16).



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Fig. 3. Effect of bafilomycin A1 (top panel) and monensin (bottom panel) on mucin glycosylation. Seven-day overconfluent cells were treated for 48 h. Synthesis of glycosylated total (cellular plus secreted) mucin was expressed as d.p.m. D-[6-3H]-glucosamine incorporated into Sepharose CL-4B purified total mucin/µg cellular DNA. * P < 0.05 and *** P < 0.001, indicate significant differences from control; ANOVA, n = 16. Bafilomycin caused a significant increase in glucosamine incorporation at concentrations that did not affect proliferation, whereas monensin only affected glucosamine incorporation at the highest concentration (0.1 µM), which significantly inhibits proliferation.

 
Bafilomycin A1 and monensin have little effect on mucin core protein synthesis
Bafilomycin at concentrations that reduced mucin sulfation had no effect on [14C]-threonine incorporation into total mucin (n = 8) (Figure 4). Although 0.05 µM bafilomycin significantly increased the [14C]-threonine/DNA ratio of total mucin compared to control (P < 0.01, ANOVA) this concentration may be somewhat antiproliferative for LS174T cells (Figure 5). Monensin had no effect on total mucin core synthesis (n = 8) (Figure 4).



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Fig. 4. Effect of bafilomycin A1 (top panel) and monensin (bottom panel) on mucin protein core synthesis. Seven-day overconfluent LS174T cells were treated for 48 h. Mucin protein core synthesis was expressed as c.p.m. L-[U-14C]-threonine incorporated into Sepharose CL-4B purified total mucin/µg cellular DNA. * P < 0.05 and ** P < 0.01 indicate significant differences from control; ANOVA, n = 8.

 


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Fig. 5. Effect of bafilomycin A1 (top panel) and monensin (bottom panel) on cell proliferation. Sixty percent confluent LS174T cells were treated for 48 h in the presence of [methyl 3H]-thymidine over the last 4 h of treatment. Results were expressed as c.p.m. incorporated per well. *** P < 0.001 and **** P < 0.0001, indicate significant differences from controls; ANOVA, n = 16. It should be noted that bafilomycin reduced sulfation and increased TF expression at a concentration (0.01 µM) an order of magnitude lower than that required to inhibit proliferation.

 
Effect of bafilomycin A1 and monensin on cell proliferation and cellular DNA content
No significant effect was seen on cell proliferation (thymidine incorporation/well) at 0.01 µM or lower concentrations of bafilomycin; levels that had significant effects on total mucin glycosylation and sulfation (n = 16) (Figure 5). At 0.1 µM and higher concentration, both bafilomycin and monensin significantly inhibited the incorporation of thymidine compared to control (P < 0.001 ANOVA, n = 16). However, bafilomycin treatment (0–1 µM) had no significant effect on cell monolayer DNA content (1 µM bafilomycin; 16.1 ± 1.6 µg DNA/well) compared to control (15.6 ± 1.2; n = 24), within a 48-h incubation period. Only at 10 µM bafilomycin was there any indication of reduced DNA content and cell monolayer damage within 48 h. Similarly, monensin treatment for 48 h only resulted in significant reduction in cellular DNA content at concentrations of 1 µM (23.2 ± 2.0 µg DNA/well; P = 0.045) and 10 µM (14.1 ± 2.8 µg DNA/well; P < 0.001) compared to control (28.8 ± 2.4), n = 24.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
These studies show that treatment of the goblet cell–differentiated colorectal cancer cell line LS174T with bafilomycin A1, a specific inhibitor of the vacuolar H+-ATPase responsible for acidification of intraorganelle vesicles (Bowman et al., 1988Go), results in the synthesis of undersulfated mucins, which overexpress the TF oncofetal carbohydrate antigen. It is interesting that, whereas bafilomycin-induced changes in sulfation are seen in both cellular and secreted mucins, the bafilomycin-induced increase in TF expression was seen only in cellular mucins, perhaps implying that these mucins, which are presumably less glycosylated and/or less sulfated, may be less easily secreted. The combination of increased TF expression of intracellular mucins and reduced mucin sulfation resembles the changes seen in colon cancer (Filipe, 1979Go; Campbell et al., 1995Go) and premalignant colonic disease (Raouf et al., 1992Go; Corfield et al., 1992Go, 1996; Campbell et al., 1995Go). The observed decrease in mucin sulfation seen in this study in response to bafilomycin results from a marked increase in incorporation of glucosamine into mucins that is not accompanied by a parallel increase in sulfate incorporation. These changes are not related to increased proliferation (with reduced sulfation still evident at 0.01 µM bafilomycin when proliferation was unaffected) and are similar to those seen in studies of mucus synthesis by cultured mucosal explants. Increased incorporation of N-acetylglucosamine into mucins has been reported in explants from Crohn’s disease (Ryder et al., 1995Go) and from hyperplastic and adenomatous polyps (Ryder et al., 1994aGo). Ulcerative colitis explants showed lower N-acetylglucosamine incorporation into mucins when expressed relative to explant protein content, but this probably reflects relative loss of epithelial cells in this condition.

It seems likely that the changes in mucin sulfation and TF expression seen here occur as a direct result of the effect of bafilomycin on intra-Golgi pH (Llopis et al., 1998Go). Although bafilomycin treatment has been shown not to alter Golgi morphology, the maintenance of a low pH is thought to be essential for protein trafficking through the trans-Golgi (Yilla et al., 1993Go; Bonay et al., 1996Go). The pH of various intracellular compartments, including the Golgi apparatus, is regulated by means of a vacuolar H+-ATPase that acts as an electrogenic pump (Stevens and Forgacs, 1997Go). The vacuolar H+-ATPase is assumed to maintain a normal pH between 5.9 and 6.7 in the trans-Golgi compartments (Stevens and Forgacs, 1997Go; Llopis et al., 1998Go), although other ATPases may also be involved (Yoshimori et al., 1991Go; Demaurex et al., 1998Go). Continued proton transport requires dissipation of the membrane potential, which occurs primarily through the action of a chloride channel (Glickman et al., 1983Go). In the present study bafilomycin is unlikely to have altered the availability of the sulfate donor adenosine 3'-phosphate-5'-phosphosulfate (PAPS) to the trans-Golgi sulfation processes; first, because PAPS is known to be stable at alkaline pH (Robbins and Lipmann, 1957Go) and hence would be unaffected by intravesicular alkalinization; second, because the binding of PAPS by a membrane-localized PAPS translocase (Mandon et al., 1994Go; Ozeran et al., 1996aGo), followed by its subsequent transport to the Golgi lumen, although a pH-dependent process (Vargas, 1990Go), is thought to be controlled on the cytoplasmic side of vesicles (Ozeran et al., 1996bGo).

In contrast, monensin, at concentrations that did not affect proliferation, did not affect the ratio of sulfate to glucosamine incorporation. Vesicular swelling induced by monensin is known to disrupt translocation of glycosyltransferases throughout the Golgi (Tartakoff, 1983Go; Mollenhauer et al., 1990Go). This could account for the reduction in mucin glycosylation that was observed with higher concentrations of monensin in this study. Both bafilomycin and monensin at 0.1 µM and above strongly inhibit DNA synthesis and therefore any affects on synthesis of glycans seen at these concentrations are probably of little biological importance.

It should be noted that colonic tissue and cultured colonic cells produce an insoluble form of MUC2 (Herrmann et al., 1999Go). It is unknown to what extent this would have been solubilized by the sonication used in the mucin extraction, but analysis of similarly prepared LS174T mucins in our laboratory has confirmed the presence of MUC2, MUC5AC, and MUC6 (using antibodies LUM2-3 EU, LUM5-1 EU, and LUM6-3 EU; data not shown). The relative expressions of sulfate and TF antigen by these secreted mucins is not known.

Increased expression of the TF blood group antigen, Galß1-3GalNAc{alpha}-, is probably the most common described glycosylation abnormality in malignant and hyperplastic epithelia (Samuel et al., 1990Go; Campbell et al., 1995Go; Shamsuddin et al., 1995Go). The TF disaccharide behaves as an oncofetal antigen and is expressed in neonatal colon and meconium, but is absent from normal adult epithelium (Hounsell et al., 1985Go). In the normal colon, TF is likely to be a cryptic antigen, with sialylation commonly assumed to be the mechanism for its concealment (Lance and Lev, 1991Go) although other studies have suggested fucosylation (Okada et al., 1994Go) or sulfation (Martinez-Menarguez et al., 1992Go) as the mechanism. Evidence from our own previous studies, that TF antigen can be revealed on normal colonic mucins by mild acid hydrolysis (Campbell et al., 1995Go) would suggest concealment by sialylation or fucosylation as the mechanism, or concealment by O-sulfate esters, which are also labile under these conditions. Brockhausen’s group have shown TF to be a major site for ester sulfation in the colonic epithelium (Kuhns et al., 1995Go). The increased glucosamine incorporation in response to bafilomycin could be a reflection of either increased mucin synthesis or increased mucin glycosylation; moreover, the glucosamine could be converted to GlcNAc, GalNAc, or sialic acid, each of which could be expected to have differing effects on TF expression. The reciprocal relationship between sulfation and TF expression also raises the interesting possibility that altered Golgi localisation of sulfotransferase(s) could account both for the increased TF antigen expression (Campbell et al., 1995Go) and decreased mucin sulfation (Filipe, 1979Go; Raouf et al., 1992Go; Corfield et al., 1992Go, 1996) seen in ulcerative colitis and colon cancer.

There is increasing evidence that changes in carbohydrate expression may play a key role in the invasive and metastatic behaviour of tumour cells (Irimura, 1994Go). Our own group has shown that increased TF antigen expression on hyperplastic and malignant epithelial cell-surface glycoconjugates may have functional significance by allowing interaction with TF-binding lectins of dietary origin (Yu et al., 1993Go; Ryder et al., 1994aGo,b, 1998). Anti-TF antibodies, which are present in all human sera, have also been shown to stimulate colorectal cancer cell proliferation (Yu et al., 1997Go).

This study shows that bafilomycin-induced increase in trans-Golgi pH results simultaneously in a decreased ratio of sulfate:glucosamine incorporation into mucins and increased expression of the TF antigen, thus mimicking the changes seen in colon cancer and precancer. Intra-Golgi pH has been little studied in cancer but there is evidence of defective Golgi acidification in MCF-7 breast cancer cells (Schindler et al., 1996Go). Further studies are indicated to assess intra-Golgi pH in epithelial cancers and precancerous states to see whether this is a widespread phenomenon that might explain many of the cancer-associated changes in glycosylation.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Reagents
Monensin and bafilomycin A1 were purchased from the Alexis Corporation (Nottingham, UK). The anti sulfo-Lewisa monoclonal antibody F2 was a kind gift from Dr Jan Bolscher (A.C.T.A., Vrije Universitet, Amsterdam, Netherlands) (Veerman et al., 1997Go). Peroxidase-labeled peanut agglutinin (PNA) was purchased from Sigma Chemical Co. (Poole, U.K.), and peroxidase-labeled LFA was purchased from EY Laboratories (San Mateo, CA). The TF antigen–expressing Antarctic fish (Trematomus borchgrevinki) antifreeze glycopeptides (1–5; BD-87-1-I) were a kind gift from Professor Arthur L. DeVries (University of Illinois, Urbana, IL). The biotinylated neoglycoconjugates sulfo-Lewisa (HSO3-3Galß1-3[Fuc{alpha}1-4]GlcNAc-) and sulfo-TF antigen (HSO3-3Galß1-3GalNAc{alpha}-) were purchased from Syntesome (Moscow).

Cell culture
The human colon adenocarcinoma cell line LS174T was obtained from the European Collection of Animal Cell Cultures (Porton Down, UK). Cells, at an initial density of 3 x 104 cells/ml in 24 well plates, were grown as monolayers in Dulbecco’s modified Eagle’s medium supplemented with 4 mM glutamine, 15% v/v fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin, in 5% CO2 /95% air at 37°C.

The effect of bafilomycin A1 and monensin on synthesis and sulfation of mucin
Seven- to eight-day overconfluent LS174T cell monolayers were treated with monensin or bafilomycin A1 (0–10 µM) for 48 h in medium containing 20 µCi/ml sodium [35S]-sulfate (1050–1600 Ci/mmol; NEN, Brussels, Belgium) and 2 µCi/ml D-[6-3H(N)]-glucosamine hydrochloride (20–45 Ci/mmol; NEN). 100% ethanol vehicle alone was used as control at a final concentration in the culture medium of 1% v/v. To assess mucin protein core synthesis, steady-state labeling of cells was performed in medium containing 0.1 µCi/ml [U-14C]-L-threonine (131 Ci/mmol; ICN Biomedical Ltd; Oxford, UK) alone. Following incubation, the medium was harvested and pooled with two 0.5 ml cell washes with serum-free medium containing a cocktail of protease inhibitors (2 µM aprotinin, 10 µM leupeptin, 5 mM benzamidine hydrochloride, and 0.01% w/v thimerosal). Cells were scraped and harvested using three washes of 0.5 ml serum-free medium (as above)and solubilized by ultrasonication (as per Campbell et al., 1995Go); supernatants collected following centrifugation at 14,000 x g for 20 min, 4°C. Two-hundred-microliter aliquots of either cell supernatants or medium samples were each subjected to gel filtration on Sepharose CL-4B mini-columns (5 x 1.5 cm) eluted with 0.01 M Tris–HCl pH 8 containing 0.01% w/v thimerosal (Finnie et al., 1995Go). Void volume fractions containing high molecular weight mucins were collected and 375-µl aliquots of each fraction were subjected to liquid scintillation counting. The degree of mucin sulfation was assessed by examining the ratio of d.p.m. [35S]-sulfate per d.p.m. [3H]-glucosamine incorporated, and total mucin sulfation was also expressed per µg DNA. Glycosylation of mucin was expressed as d.p.m. [3H]-glucosamine incorporated/µg cellular DNA, and mucin protein core synthesis was expressed as d.p.m. [14C]-threonine/µg cellular DNA. The contribution of contaminating proteoglycans was assessed by incubation of Sepharose CL-4B-purified [3H]-glucosamine-labeled mucin with protease-free chondroitin lyase ABC (EC 4.2.2.4), collagenase D (EC 4.24.3; both from Roche Diagnostics Ltd, Lewes, UK) and sheep testis hyaluronidase (EC 3.2.1.35; Sigma), conditions as per Campbell et al. (1995)Go. These incubations were followed by repeat gel filtration and liquid scintillation counting compared with untreated controls. Only very small losses of radioactivity from the mucin preparation were seen after incubation with hyaluronidase (3.7 ± 1.3%; n = 6) and collagenase (1.9 ± 2.6%; n = 6). Glycolipid contamination was assessed by extracting Sepharose CL-4B-purified [3H]-glucosamine-labeled mucin with two volumes chloroform:methanol (2:1) (Finnie et al., 1995Go). This reduced subsequent recovery of mucin by 2.7 ± 3.5% (n = 6), showing minimal contamination by glycolipids.

Cellular DNA measurements were performed using indirect fluorimetry (Ladarca and Paigen, 1980Go) with 200-µl samples of cell ultrasonicate added to 1.8 ml of 0.1 µg/ml bis-benzamide solution. DNA was quantified using a fluorescence spectrophotometer (excitation {lambda} 363 nm; emission {lambda} 460 nm) against a calibration curve of 0–100 µg/ml type 1 calf thymus DNA (Sigma). Coefficient of variation within assay was 5.1% (n = 6) and between assay was 3.7% (n = 3).

Investigation of expression of 3'sulfo-Lewisa, TF antigen, and sialylation
Slot-blot analysis was performed on 250 c.p.m. aliquots of [14C]-threonine-labeled Sepharose CL-4B-purified mucins. Purified mucins were vacuum-blotted to nitrocellulose membrane, stained with 0.2% w/v Ponceau S in 1% v/v acetic acid, and then blocked for 1 h at room temperature with PBS pH 7.4 blocking buffer (containing 0.1% v/v Tween 20 and 1% w/v bovine serum albumin). Expression of TF antigen on mucins was measured using 1 mg/ml peroxidase-labeled PNA, at 1:3000 dilution in PBS blocking buffer for 2 h. Sialylation of mucins was assessed using 1 mg/ml peroxidase-labeled LFA at 1:500 dilution. Expression of sulfo-Lewisa carbohydrate epitope on mucins was measured using the monoclonal antibody F2 (Veerman et al., 1997Go) at 1:8 dilution in blocking buffer followed by application of a peroxidase-labelled rabbit anti-mouse immunoglobulins secondary antibody 1:2000 (Dako, Ely, UK). Between each incubation step, membranes were extensively washed in PBS–0.05% v/v Tween 20. Bound PNA or F2 antibodies were detected by ECL chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK), bound LFA was detected using chloronaphthol (3 mg/ml methanol) in PBS containing 0.1% v/v H2O2. A linear binding relationship for peroxidase-labeled PNA was seen with the TF antigen–expressing antifreeze glycopeptides, at concentrations from 6.25–100 µg/ml (data not shown). Specificity of F2 binding was assessed by enzyme-linked immunosorbent assay, as previously described (Veerman et al., 1997Go). Briefly, triplicate samples of oligosaccharide polyacrylamide-biotin conjugates were serially diluted in 0.1 M NaHCO3, pH 9.6, and incubated for 16 h at 4°C. After washing with PBS–Tween, mAb F2 (1:8) was added and incubated for 1 h at 37°C. Bound antibody was probed with peroxidase-conjugated rabbit anti-mouse immunoglobulin (1:600), using o-phenylene-diamine (0.4 mg/ml) and 0.012% v/v H2O2 as substrates. OD was measured at 490nm. The F2 antibody bound to biotinylated 3'sulfo-Lewisa but did not bind 3'sulfo-TF antigen (see Figure 6).



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Fig. 6. Monoclonal antibody F2 assessed by enzyme-linked immunoabsorbent assay against the neoglycoconjugates, 3'sulfo-Lewisa and 3'sulfo-TF. Neoglycoconjugates were coated onto polystyrene wells of microtiter plates by overnight incubation at 4°C. MAb F2 (1:8) in PBS containing 0.1% v/v Tween 20 and 1% w/v bovine serum albumin was added. Bound antibody was probed with anti-mouse immunoglobulins conjugated to peroxidase, using o-phenyl-diamine and H2O2 as substrate.

 
Effect of bafilomycin A1 and monensin on cell proliferation
Sixty percent confluent LS174T cell monolayers were incubated for 48 h in the presence of either 0–10 µM monensin or bafilomycin A1. 0.8 µCi/ml [methyl 3H]-thymidine (82 Ci/mmol; Amersham Pharmacia Biotech) was added to each well 4 h before the end of the treatment period. Cell precipitation and solubilization with 5% w/v trichloroacetic acid and 0.2 M sodium hydroxide, respectively, were performed as previously described (Yu et al., 1993Go). Solubilized cell precipitates were ß-counted, and results were expressed as cpm [methyl 3H]-thymidine incorporated per well.

Statistical analysis
Data are presented as mean ± SE. Sample groups were analysed using one-way analysis of variance followed by selected pair-wise comparisons of treatment means using Bonferroni’s modified t-test (Arcus Pro-Stat II; Medical Computing; Aughton, UK). Differences were considered significant when P < 0.05.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
BJC was supported by research awards obtained from the Nuffield Foundation (SCI/180/95/320/G), the Royal Society (RSRG 16731), and the University of Liverpool Research Development Fund (1978-ADPH5C). KL was the Leslie Parrott fellow of the National Association for Colitis and Crohn’s Disease (M/97/1). Further support was provided by an European Union Concerted Action Award (BMH4-CT98–3222), a Medical Research Council Co-operative Grant (GR990432), and a grant from the Biotechnology and Biological Sciences Research Council (D08258).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Gal, galactose; GalNAc, N-acetyl-galactosamine; GlcNAc, N-acetylglucosamine; PAPS, 3'-phosphate-5'-phosphosulfate; LFA, Limax flavus agglutinin; PBS, phosphate buffered saline; PNA, peanut agglutinin; TF, Thomsen-Friedenreich.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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