Characterization of Mucins and Proteoglycans Synthesized by a Mucin- Secreting HT-29 Cell Subpopulation

Journal of Cell Science 108, 1275-1285 (1995) 1275 Printed in Great Britain © The Company of Biologists Limited 1995

Characterization of mucins and proteoglycans synthesized by a mucin- secreting HT-29 cell subpopulation

G. Huet1,*, I. Kim2, C. de Bolos3, J. M. Lo-Guidice1, O. Moreau1, B. Hemon1, C. Richet1, P. Delannoy2, F. X. Real3 and P. Degand1 1INSERM U377, Place de Verdun, 59045 Lille cedex, France 2UMR CNRS no. 111, UST Lille, 59655 Villeneuve d’Ascq cedex, France 3Institut Municipal d’Investigacio Mèdica, Universitat Autonoma de Barcelona, Spain *Author for correspondence

SUMMARY

HT-29 cells selected by adaptation to 10−5 M methotrexate A and the sugar analog GalNAc-α-O-benzyl on the (HT-29 MTX) are a homogeneous cell population synthesis and biochemical properties of mucins synthesized producing high amounts of mucin. Intracellular mucins by HT-29 MTX cells was examined. Brefeldin A induced and proteoglycans were isolated from these cells by ultra- the synthesis of more-sulfated mucins. GalNAc-α-O-benzyl centrifugation of cell lysates on a cesium bromide gradient treatment resulted in mucins with an increased content of and further separated by anion-exchange high perfomance T antigen and a 13-fold lower sialic acid content. We show liquid chromatography. The major mucin fraction isolated that GalNAc-α-O-benzyl was metabolized by the cells to was characterized by a high hydroxy amino acid content Galβ1-3GalNAc-α-O-benzyl, which, in turn, was a potent (40%), a Thr/Ser ratio of 1.52, a high sialic acid content, competitive inhibitor of the O-glycan α-2,3-sialyltrans- and a low sulfate content. When the same procedure was ferase. These results illustrate the suitability of HT-29 applied to undifferentiated HT-29 cells, a minor mucin MTX cells as a model to analyse mucin synthesis and sia- fraction was isolated which appeared less sialylated and lylation. more sulfated. The major proteoglycan species identiﬁed in HT-29 MTX cells showed less acidic behavior than the pro- Key words: mucin, proteoglycan, HT-29, aryl-N-acetyl-α- teoglycan isolated from HT-29 cells. The effect of brefeldin galactosaminide, brefeldin

INTRODUCTION clonal cell line (cl.16-E) has been derived from HT-29 cultures after treatment with sodium butyrate (Augeron and Laboisse, Mucins and proteoglycans are high molecular mass glycopro- 1984) and a homogeneous mucin-secreting population has teins whose biochemical functions depend on post-transla- been obtained by stepwise adaptation of HT-29 cells to 10−6 tional modifications. Alterations in these processes commonly or 10−5 M methotrexate (MTX) (Lesuffleur et al., 1990). The occur in transformed cells (Hardingham and Fosang, 1992; use of another antimetabolite, 5-fluorouracil (5-FU), has Devine and McKenzie, 1992) and may contribute to the neo- resulted in the isolation of a population containing both absorp- plastic phenotype. In human colorectal carcinomas, abnormal- tive and mucin-secreting cells (Lesuffleur et al., 1991). Anti- ities in the oligosaccharide structures carried on mucins bodies raised against cl.16-E mucins stain goblet cells in both (Kurosaka et al., 1983; Kim, 1992) and on proteoglycans normal colonic and normal gastric mucosa (Maoret et al., (Iozzo and Wight, 1982) have been described. 1989). Lesuffleur et al. (1990) have used rabbit antisera raised The analysis of the biosynthesis and composition of mucins against normal colonic mucosa and absorbed with stomach and proteoglycans, and the study of structure function rela- tissue to identify mucins of ‘colonic’ specificity, and rabbit tionships, would be greatly facilitated by the availability of cell antisera raised against normal gastric mucosa and absorbed cultures with a wide range of phenotypic properties. The with colonic tissue to identify mucins of ‘gastric’ specificity. parental HT-29 cell line fulfills this criterion. HT-29 cultures The low proportion of mucin-secreting cells in parental HT-29 are heterogeneous and in the postconfluent state consist of cultures expresses mucins of either gastric or colonic immuno- >95% undifferentiated cells and a small proportion of differ- logical specificity, whereas HT-29 MTX cells synthesize entiated mucin-secreting and absorptive cells (Pinto et al., mainly gastric-type mucins and HT-29 FU cells synthesize 1982; Augeron and Laboisse, 1984; Lesuffleur et al., 1990). colonic-type mucins (Lesuffleur et al., 1990, 1991). The char- Differentiated populations of either absorptive or mucin- acterization of these cell populations has been facilitated by the secreting phenotype can be obtained under various conditions isolation of cDNAs encoding human mucins (Gendler et al., of metabolic stress. A stably differentiated mucin-secreting 1990; Gum et al., 1990, 1992; Porchet et al., 1991; Guyonnet- 1276 G. Huet and others

Duperat et al., 1994; Toribara et al., 1993; Bobek et al., 1993). in threonine-free Dulbecco’s minimum essential medium (Institut HT-29, HT-29 MTX and HT-29 FU cells show distinct Jacques Boy, Reims, France). After labeling, medium was collected patterns of MUC1-MUC5 mucin gene expression and HT-29 and cells were lysed in RIPA buffer (0.01 M Tris-HCl, pH 8.0, 0.01 MTX cells express mainly MUC1, MUC2, MUC3 and MUC5C M NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 0.5% genes (Lesuffleur et al., 1993). However, less is known sodium deoxycholate, 1% phenylmethylsufonyl fluoride, 0.001 M regarding the main carbohydrate structures carried on the sodium ethylene diaminotetra-acetate). In some experiments, the effect of GalNAcα-O-benzyl (Sigma, St products of these genes in HT-29 MTX cells (Dahiya et al., Louis, MO; 5 mM) or BFA (Sigma; 5 µg/ml) on mucin biosynthesis 1992). was examined. Cells were cultured for 20 days as described above, This paper describes a first step towards the biochemical and GalNAcα-O-benzyl or BFA were added for 24 hours, and cells were structural characterization of the mucins and proteoglycans processed. synthesized by HT-29 MTX cells in comparison with those produced by the parental cell population. To gain insight into Biochemical characterization mucin biosynthesis in HT-29 MTX cells, we have used two Ultracentrifugation compounds that could potentially interfere with glycosylation Twenty-one days after seeding, cells were rinsed twice with PBS, and alter the biochemical properties of mucins, brefeldin A scraped with a rubber policeman, and directly lysed by ultrasonica- (BFA) and benzyl-N-acetyl-α-galactosaminide (GalNAcα-O- tion in PBS. Cell extracts were collected after centrifugation for 5 benzyl). BFA is a fungal antibiotic that disrupts protein minutes at 1000 g at 4¡C. After adding cesium bromide (0.42 g/ml), transport from the rough endoplasmic reticulum (RER) to the cell lysates were ultracentrifuged (200,000 g for 72 hours) using a Beckman 70.1 Ti rotor (Houdret et al., 1986). Fractions of 1 ml were Golgi apparatus, inducing a redistribution of Golgi proteins to α collected, weighed to determine density, and analysed for absorbance the RER (Lippincott-Schwartz et al., 1989). Aryl-N-acetyl- - at 280 nm, orcinol reactivity, and dimethylmethylene blue (DMB) galactosaminides have been initially used as potential com- reactivity (De Jong et al., 1992). petitors of the glycosylation of GalNAc residues linked to the core protein and have been shown to inhibit UDP- HPLC chromatography Gal:GalNAcβ1-3-galactosyltransferase in vitro (Kuan et al., Anion-exchange high performance liquid chromatography (AE- 1989). However, it was observed that GalNAcα-O-benzyl HPLC) was carried out using a TSK DEAE 5PW (7.5 mm × 75 mm) treatment of HM7 colon cancer cells leads to an increased Spherogel column (Beckman). The system was equilibrated with expression of Tn antigen (GalNAc-Thr/Ser), but also of T 0.005 M sodium/potassium phosphate buffer, pH 6.0, at a flow rate antigen (Galβl-3GalNAc), together with a decrease in sialyl- of 0.8 ml/min. A total of 2 mg protein was injected. Elution was x a carried out using a NaCl gradient (0 to 1 M) in the same buffer. Le , sialyl-Le and sulfomucin epitopes (Huang et al., 1992). Eluates were collected in 0.8 ml fractions and analysed as described To explain the unexpected increase in T antigen activity, it has above. been proposed that the in situ synthesis of Galβ1-3GalNAcα- O-benzyl might lead to a more efficient inhibitor than Compositional analyses GalNAcα-O-benzyl itself; the benzyl disaccharide would For amino acid analysis, samples were hydrolysed in 5.6 M HCl for inhibit the elongation of T antigen by N-acetylglucosaminyl- 24 hours under a vacuum and processed using a 7300 Beckman amino transferases, sialyltransferases and/or fucosyltransferase. Here, acid analyser (Palo Alto, CA) equipped with a high performance we provide evidence that GalNAcα-O-benzyl induces a sodium column (4 mm × 120 mm) (Beckman). Sugar analysis was marked increase in T antigen and a marked decrease in sialic carried out by gas-liquid chromatography of trimethylsilyl derivatives acid content of HT-29 MTX mucins. Furthermore, the of methyl glycosides formed by methanolysis in 1.5 M HCl in methanol at 80¡C for 24 hours (Lamblin et al., 1984). To determine enzymatic mechanisms responsible for these changes in mucin sulfate content, samples were hydrolysed in 1 M HCl for 5 hours at synthesis are partially elucidated. 100¡C and sulfate was determined by AE-HPLC (Lo-Guidice et al., 1994). All compositional analyses were performed twice to examine the reproducibility of the results. MATERIALS AND METHODS Electrophoresis and western blotting Cell culture SDS-PAGE Parental HT-29 cells (referred to as HT-29) and HT-29 cells selected SDS-PAGE was performed with 2% to 5% gradient polyacrylamide by adaptation to 10−5 M methotrexate (MTX) were obtained from Dr gels (Laemmli, 1970). For autoradiogaphy, gels were fixed in 40% Thécla Lesuffleur (Unité INSERM U178, Villejuif, France). Cells ethanol, 10% glycerol, 10% acetic acid (by vol.), soaked in Amplify were grown in Dulbecco’s modified Eagle’s minimal essential (Amersham, UK) for 20 minutes, dried on Whatman paper, and medium (Eurobio, Paris, France), supplemented with 10% inactivated exposed to Cronex 4 NIF film (Dupont). (30 minutes, 56¡C) fetal bovine serum (Boehringer, Mannheim, Germany). Cells were seeded at 1.5×106 cells in 75 cm2 flasks Agarose gel electrophoresis (Corning Glassworks, Corning, NY) and cultured at 37¡C in a 10% Mucin glycopeptides were obtained by Pronase digestion for 48 hours CO2/90% air atmosphere. The medium was changed daily. Cultures at 37¡C in 0.01 M calcium acetate buffer, pH 7, at an enzyme/substrate were studied in the late post-confluent period (21 days after seeding), ratio of 1/40 (w/w). Fresh enzyme was added after 24 hours of when all cells display a mucin-secreting phenotype (Lesuffleur et al., digestion. Agarose gel electrophoresis was then performed in veronal 1990). Cell viability was determined by trypan blue dye exclusion. buffer at pH 8.2 and gels were stained with Schiff periodate (Marianne For metabolic labeling, cells were cultured as described above until et al., 1986). day 20 and were then labeled for 24 hours with [3H]glucosamine (1.48 MBq/ml) in low-glucose Dulbecco’s minimum essential H-16 Cellulose acetate electrophoresis medium (Gibco, Gaithesburg, MD), with [35S]sulfate (1.48 MBq/ml) After immersion of cellulose acetate plates in 0.05 M barium acetate, in Ham’s F-12 medium (Gibco), or with [3H]threonine (1.48 MBq/ml) migration was carried out for 20 minutes. The plates were then stained Biochemical characterization of HT-29 mucins 1277 in 0.02% DMB in 1% acetic acid for 10 minutes and destained in 10% samples were centrifuged at 3,000 g for 5 minutes and supernatants acetic acid (Wesslet, 1968). were directly processed for descending paper chromatography in ethyl acetate/ pyridine/ water (10/4/3, by vol.) (Delannoy et al., 1993). Western blotting After separation by 2-5% SDS-PAGE, proteins were transferred to a nitrocellulose membrane as described by Vaessen et al. (1981). RESULTS Membranes were incubated with 10 µg/ml peroxidase-labeled lectins (Sigma) in 10 mM Tris-glycine buffer containing 0.9% NaCl and 3% Comparative analysis of mucins and proteoglycans bovine serum albumin at 37¡C for 2 hours. After washing, membranes were incubated with 4-chloro-1-naphthol (Sigma). from HT-29 MTX and HT-29 cells The secretion of mucins and proteoglycans by HT-29 MTX Immunocytochemistry and HT-29 cells was first studied by metabolic labeling with Indirect immunofluorescence was performed on cryostat sections of [3H]glucosamine and with [35S]sulfate followed by analysis of cell layer rolls as reported by Lesuffleur et al. (1990), with minor culture medium by SDS-PAGE. After [3H]glucosamine modifications. Sections were air-dried and fixed with acetone for 10 labeling (Fig. 1A), a broad band was observed in the case of minutes. After washing with PBS, 5% normal horse serum was added. HT-29 MTX cells whereas HT-29 cells yielded a diffuse Sections were incubated with FITC-labeled soybean (SBA), peanut smear. This result is relevant to the high rate of mucins (PNA), and wheat germ (WGA) lectins for 60 minutes. After washing, produced by HT-29 MTX cells and to the diffuse high sections were mounted with glycerol. Alternatively, mouse mono- molecular mass band described by Dahiya et al. (1992). SDS- clonal antibodies (mAb), that detect a large panel of mucin-associated 35 carbohydrate epitopes were used. mAb Cu-1, detecting Tn (Takahashi PAGE analysis of culture media from [ S]sulfate-labeled HT- et al., 1988), was obtained from Dr S. I. Hakomori (The Biomem- 29 MTX and HT-29 cells showed diffuse smears with a brane Institute, Seattle, WA) and mAbs B72.3 and CC49, detecting mobility similar to that of [3H]glucosamine-labeled cells (Fig. sialyl-Tn (Nuti et al., 1982), were obtained from Dr Kenneth O. Lloyd 1B). Although [3H]glucosamine is predominantly incorporated (Sloan-Kettering Institute, New York, NY). To obtain better semi- into mucins and [35S]sulfate into glycosaminoglycans, it was quantitative data, several lectin and antibody dilutions were used in not possible to distinguish between mucins and proteoglycans each experiment. Reactions were developed with goat anti-mouse- using SDS-PAGE. TRlTC (Pierce, Rockford, IL) (5 µg/ml) and visualized using a Zeiss Mucins and proteoglycans synthesized by HT-29 MTX and Axioscop equipped with Plan-Neofluar lenses and scored as follows: × HT-29 cells were isolated by ultracentrifugation through a +++, when fluorescence signal was clearly seen at 100 magnifica- CsBr gradient. Fig. 2A shows the absorbance profiles obtained tion; ++, when it was seen at ×200 magnification; +, when signal was seen only at ×400 magnification. Percentage reactive cells was after the addition of orcinol, to detect carbohydrates; or DMB, expressed as an average of the whole section. Scoring was performed to detect glycosaminoglycans. AE-HPLC was then used to independently by two investigators (C. de B. and F.X.R.) and all separate mucins and proteoglycans from selected fractions of experiments were repeated twice independently. HT-29 MTX (1.42-1.51 g/ml) and HT-29 (1.39-1.53 g/ml) cells and the elution profiles are shown in Fig. 2B. In the HT- Sialyltransferase (ST) assays 29 MTX fraction, orcinol reactivity eluted as a major peak with Preparation of cell extracts a retention time of 24 minutes, whereas DMB reactivity eluted Cells were lysed at 0¡C with 10 mM sodium cacodylate buffer, pH later and showed a complex pattern of peaks at the 26-37 6.5, containing 1% Triton X-100, 20% glycerol, 0.5 mM dithiotreitol, 2 and 5 mM MnCl2 (1 ml per 170 cm flask). After a 10 minute incubation under continuous stirring, cell homogenates were centrifuged at 10,000 g for 15 minutes and the supernatants were used for enzymatic assays. Protein concentration was determined according to the method described by Peterson (1977) using bovine serum albumin (BSA) as standard.

ST assay using asialofetuin as acceptor Cell extracts (40 µl) were carried to a final volume of 120 µl with 0.1 M sodium cacodylate, pH 6.5, 1% Triton X-100, 0.1% BSA, 0.2 M galactose (an inhibitor of β-galactosidase), 1 mM 2,3-dehydro-2- deoxy-Neu5Ac (an inhibitor of sialidases), 52.9 µM CMP- [14C]Neu5Ac (0.58 GBq/mmol, 3.67 kBq/120 µl), containing 480 µg of asialofetuin (prepared by mild acid hydrolysis of fetuin) as exogenous substrate, and incubated at 37¡C for 1 hour. The reaction was stopped by adding 1 ml of ice-cold phosphotungstic acid (5% in 2 M HCl). The precipitate was collected on glass fiber filters, washed extensively with 5% trichloroacetic acid, distilled water and ethanol, and processed for scintillation counting (Vandamme et al., 1992).

ST assay using Galβ1-3GalNAcα-O-benzyl as acceptor Fig. 1. SDS-PAGE autoradiogram of culture medium from HT-29 Incubations were performed under the conditions described above and HT-29MTX cells labeled with [3H]glucosamine (A) or using 40 µl of cell extract or 1 mU of puriﬁed porcine liver Galβ1- [35S]sulfate (B). Lane 1, HT-29 cells; lane 2, HT-29 MTX cells. 3GalNAc α-2,3-ST (Boehringer, Mannheim, Germany) as enzyme Equal amounts of protein were loaded for each cell type. source and Galβ1-3GalNAcα-O-benzyl (Sigma) (1 mM ﬁnal con- Electrophoretic migration was compared with that of a 200 kDa centration). The reaction was stopped by adding 1 volume of ethanol; molecular mass marker. 1278 G. Huet and others

Fig. 2. (A) Ultracentrifugation proﬁles of lysates from HT-29 MTX and HT-29 cells. (B) HPLC proﬁles of ultracentrifugation fractions from HT-29 MTX and HT-29 cells. minute time periods. For HT-29 cells, a low orcinol reactivity Table 1. Amino acid composition of HT-29 MTX and was detected showing three small peaks at retention times of HT-29 HPLC fractions (%) 4, 25, and 31 minutes, whereas, a high DMB reactivity was HT-29 MTX HPLC fraction HT-29 HPLC fractions apparent at the 26-40 minute time points. Preparative HPLC chromatography was carried out for both cell types. Three and A B1 B2 A B two fractions were collected from HT-29 MTX (top Fig. 2B, Cys 0.73 1.96 2.47 0.51 0.97 fraction A, 21-25 minutes; B1, 26-29 minutes; and B2, 30-37 Asp 4.80 6.76 6.90 10.17 8.55 Thr 24.18 19.53 16.61 8.98 5.77 minutes) and HT-29 cells (bottom Fig. 2B, fraction A, 21-27 Ser 15.87 14.96 14.90 7.85 9.72 minutes; B, 28-37 minutes), respectively. These fractions were Glu 7.17 10.88 11.32 11.23 11.77 analysed for amino acid (Table 1), carbohydrate, and sulfate Pro 11.83 9.73 8.62 6.22 4.45 composition (Table 2). Gly 6.62 8.77 11.14 10.15 31.69 Ala 7.12 7.26 7.26 10.48 7.77 HT-29 MTX fraction A showed a high carbohydrate/protein Val 5.18 4.44 4.46 6.21 4.32 content (6.8 g sugar/g protein), high hydroxy amino acid Ile 2.40 2.21 2.37 3.72 2.22 content (40.05%) with a Thr/Ser ratio of 1.52, and a high Leu 3.11 3.13 3.66 6.48 4.23 proline content (11.83%), all typical features of mucins. Car- Tyr 0.08 1.24 1.47 2.45 bohydrate analysis revealed mainly GalNAc, Gal, GlcNAc and Phe 2.20 1.86 2.12 3.36 2.46 His 1.55 1.55 1.57 1.69 1.52 sialic acid. The sialic acid content was very high (1.3 molar Lys 2.59 2.96 2.35 5.14 4.58 ratio to GalNAc) whereas the sulfate content was very low (0.1 Arg 4.57 2.75 2.76 5.37 molar ratio to GalNAc). HT-29 MTX fractions B1 and B2 also showed very high hydroxy amino acid contents (34.49% and 31.51%, respectively) with lower Thr/Ser ratios (1.30 and 1.11, respectively), and lower Pro contents (9.73% and 8.62%, fractions A, B1 and B2. In contrast, fractions B1 and B2 had respectively). Other major amino acid changes consisted of a lower relative sialic acid contents (0.9 and 0.5, respectively) decrease in Arg and an increase in Cys, Glu/Gln, Gly and Tyr. and higher sulfate contents (1.2 and 0.6, respectively). These The relative content of neutral monosaccharides was similar in results suggest that fractions B1 and B2 correspond to more- Biochemical characterization of HT-29 mucins 1279

Table 2. Carbohydrate composition and sulfate content of HT-29 MTX and HT-29 HPLC fractions HT-29 MTX HPLC fractions HT-29 HPLC fractions A B1 B2 A B Mannose 0.1 0.2 0.5 ND Galactose 1.4 1.5 1.1 1 ND GalNAc = 1 = 1 = 1 = 1 ND GlcNAc 0.6 0.5 0.5 1.2 + Sialic acid 1.3 0.9 0.5 0.3 ND Sulfate 0.1 1.2 0.6 2.1 + Sulfate* 0.1 2.6 1.2 1.7 1.6 Fig. 3. Agarose gel electrophoresis of mucin glycopeptides and All results are expressed as molar ratios to GalNAc. staining with Schiff periodate. Fractions A from HT-29 MTX cells *The sulfate content was also expressed as molar ratio to GlcNAc. (lane 1) and from HT-29 cells (lane 2) were digested with Pronase ND, not detectable. for 48 hours at 37¡C and mucin glycopeptides were loaded on agarose gel slides. acidic mucins, mixed with proteoglycans, as predicted by the elution profile obtained after HPLC chromatography. HT-29 fraction A showed a relatively low carbohydrate/protein level (1.9 g sugar/g protein), a hydroxy amino acid content of 16.83% with a Thr/Ser ratio of 1.14, and a 6.22% Pro content. Carbohydrate analysis of this fraction revealed mainly mannose, Gal, GalNAc, GlcNAc and sialic acid. The sialic acid content was low (0.3 molar ratio to GalNAc) whereas the sulfate content was relatively higher (2.1 molar ratio to GalNAc). HT-29 fraction B had a hydroxy amino acid content of 15.49%, a Thr/Ser ratio of 0.59 and a high Gly content (31.69%). Carbohydrate analysis revealed only GlcNAc. Sulfate was also found in this fraction in a 1.6 molar ratio to GlcNAc. Therefore, the biochemical characteristics of HT-29 fraction B are those typical of a proteoglycan. Fig. 4. Proteoglycan analysis by cellulose acetate electrophoresis and The presence of mucins in fraction A from HT-29 MTX and DMB staining. Fraction B from HT-29 cells (A) and fraction B2 from HT-29 MTX cells (B) were compared with several standard HT-29 cells was further examined by Schiff periodate staining glycosaminoglycans. (A) Lane 1, chondroitin sulfate C; lane 2, HT- of Pronase digestion products fractionated by agarose gel elec- 29 fraction B; lane 3, chondroitin sulfate A; lane 4, dermatan sulfate; trophoresis: a fast migrating band which stained intensely was lane 5, heparan sulfate. (B) Lane 1, chondroitin sulfate A; lane 2, observed for HT-29 MTX cells whereas a weakly stained band HT-29 MTX fraction B2; lane 3, chondroitin sulfate C; line 4, was observed for HT-29 cells (Fig. 3). dermatan sulfate; lane 5, heparan sulfate. The electrophoretic behavior of proteoglycans from HT-29 MTX and HT-29 cells was analysed using cellulose acetate strips and coloration with DMB (Fig. 4). HT-29 MTX fraction GalNAcα-O-benzyl. SDS-PAGE analysis of culture media B2 contained a proteoglycan with a slower migration than the from BFA-treated and GalNAcα-O-benzyl-treated cultures heparan sulfate standard, whereas HT-29 fraction B contained revealed faint smears with mobility similar to that of the cor- a proteoglycan which migrated between the heparan sulfate responding cell lysates. This finding was in contrast to the and dermatan sulfate standards. Consequently, the main pro- discrete band observed in culture medium from control HT-29 teoglycan detected in HT-29 cells is more acidic than the HT- MTX cells. 29 MTX proteoglycan. Mucins from HT-29 MTX cells cultured for 24 hours in the presence of BFA or GalNAcα-O-benzyl were isolated by ultra- Effect of BFA and GalNAcα-O-benzyl on mucin centrifugation and AE-HPLC. Analysis of the mucin fractions biosynthesis by HT-29 MTX cells (Table 3) showed that BFA treatment led to a slight decrease The effects of BFA and GalNAcα-O-benzyl upon the biosyn- in the relative content of Gal and sialic acid and to a 4-fold thesis and secretion of mucins were first studied through con- increase in sulfate content. GalNAcα-O-benzyl treatment tinuous labeling with [3H]threonine. In the presence of BFA, induced a 13-fold decrease in the relative amount of sialic acid cell-associated and secreted radioactivity were 435.9% and in HT-29 MTX fraction A. The composition of B fractions and 42.1% of those of control cultures, respectively. In the the electrophoretic behavior of the HT-29 MTX proteoglycan presence of GalNAcα-O-benzyl, cell-associated and secreted were unchanged (data not shown). radioactivity were 428.9% and 49% of those of control To further study the effect of BFA and GalNAcα-O-benzyl cultures, respectively. Fig. 5 shows the electrophoretic pattern on HT-29 MTX mucins, their reactivity with a panel of mAbs of cell lysates and culture media after metabolic labeling with and lectins was examined by immunofluorescence on frozen [3H]threonine. A major band was observed in each cell lysate, sections of cell rolls (Fig. 6 and Table 4), or by western blotting the migration of which appeared slightly increased after (Fig. 7) on purified mucin fractions. BFA treatment did not treatment with BFA and slightly decreased after treatment with lead to major changes in the expression of mucin-associated 1280 G. Huet and others

Fig. 5. SDS-PAGE autoradiogram of HT-29 MTX cell lysates (A) and culture media (B) after labeling with [3H]threonine. Lane 1, control HT-29 MTX cells; lane 2, HT-29 MTX cells cultured in the presence of GalNAcα- O-benzyl for 24 hours; lane 3, HT-29 MTX cells cultured in the presence of BFA for 24 hours. Equal amounts of protein/sample were loaded. carbohydrate epitopes. GalNAcα-O-benzyl treatment resulted decrease in the transfer of [14C]sialic acid was detected in in an increase in the expression of Tn and T antigens as GalNAcα-O-benzyl-treated cells (Fig. 8A). detected with SBA and mAb Cu-l (Tn antigen) or PNA (Fig. GalNAcα-O-benzyl cannot be used as a substrate by sialyl- 6 and Table 4). No changes in the reactivity with WGA were transferases (Van den Eijden and Joziasse, 1993). On the other observed (not shown). Western blotting particularly showed a hand, Galβ1-3GalNAcα-O-benzyl is a speciﬁc substrate for great increase in the T antigen reactivity of mucins obtained Galβ1-3GalNAcα-2,3-sialyltransferase (α-2,3-ST(O), EC after GalNAcα-O-benzyl treatment (Fig. 7). 2.4.99.4) and would therefore be able to compete for sialyla- To determine the mechanism(s) by which GalNAcα-O- tion of asialofetuin in an in vitro assay. benzyl treatment induced such a decrease in sialic acid content The ability of Galβ1-3GalNAcα-O-benzyl and GalNAcα- and the concomitant increase in T antigen in HT-29 MTX O-benzyl to inhibit the ST activity using asialofetuin as a mucins, ST assays were performed. First, ST activity was substrate was examined. As shown in Fig. 8B, Galβ1- measured using asialofetuin as an in vitro substrate: a 30% 3GalNAcα-O-benzyl was an effective inhibitor at a concentration of 100 µM, whereas GalNAcα-O-benzyl was not Table 3. Carbohydrate composition and sulfate content of inhibitory. HT-29 MTX HPLC fraction A obtained after treatment These results ﬁtted well the hypothesis proposed by Huang with brefeldin A or GalNAcα-O-benzyl et al. (1992), that GalNAcα-O-benzyl could be converted in β α HT-29 MTX HT-29 MTX vivo into Gal 1-3GalNAc -O-benzyl and compete for the HT-29 MTX fraction A fraction A elongation of T antigen in HM 7 colon cancer cells. fraction A of obtained after obtained after We then investigated the in situ synthesis of Galβ1- control HT-29 treatment with treatment with α α α 3GalNAc -O-benzyl in GalNAc -O-benzyl-treated HT-29 MTX cells brefeldin A GalNAc -O-benzyl MTX cells. When the in vitro α-2,3-ST(O) assay was Galactose 1.4 1.1 1.4 performed with homogenates from GalNAcα-O-benzyl-treated GalNAc = 1 = 1 = 1 cells in the absence of the exogenous acceptor Galβ1- GlcNAc 0.6 0.6 0.5 α Sialic acid 1.3 1.1 0.1 3GalNAc -O-benzyl, a sialylated product that was absent Sulfate 0.1 0.4 0.2 from control homogenates was detected. This sialylated product comigrated with NeuAcα2-3Galβl-3GalNAcα-O- All results are expressed as molar ratios to GalNAc. benzyl obtained by incubation of the Galβ1-3GalNAcα-O-

Table 4. Immunoﬂuorescence on cryostat sections of HT-29 MTX cells untreated, or treated with brefeldin A or GalNAcα-O-benzyl % of labeled cells Control HT-29 HT-29 MTX cells HT-29 MTX cells MTX cells treated with treated with Speciﬁcity (%) brefeldin A (%) GalNAcα-O-benzyl (%) Lectins SBA, 20 µg/ml Tn <10 − 100 PNA, 20 µg/ml T 40-50(+)* 30 70 Antibodies Cu-1 Tn 70 (++) 90 (+++) 100 (+++) B72.3 Sialyl-Tn 70 (++) 80 (+++) 30 (++) CC.49 Sialyl-Tn 80 (++) 60 (+++) 60 (++)

*See Materials and Methods for explanation of scoring method. Biochemical characterization of HT-29 mucins 1281

Fig. 6. Immunocytochemistry of cryostat sections of HT-29 MTX cell rolls using lectins and antibodies recognizing different glycan epitopes. Control untreated cells (a,d,g,j); BFA-treated cells (b,e,h,k); GalNAcα-O-benzyl-treated cells (c,f,i,l). SBA(a,b,c); PNA (d,e,f); mAb Cu-1 (g,h,i); mAb B72.3 (j,k,l). Bar, 100 µm. benzyl substrate with puriﬁed porcine liver α-2,3-ST(O) (Fig. ST(O) were used as enzyme source, NeuAcα2-3Galβ1- 9A). Furthermore, when denatured extracts from GalNAcα-O- 3GalNAcα-O-benzyl was also detected (Fig. 9B and C). Taken benzyl-treated HT-29 MTX cells were used as substrates and together, these results indicate that GalNAcα-O-benzyl is HT-29 MTX control extracts or puriﬁed porcine liver α-2,3- converted to Galβ1-3GalNAcα-O-benzyl, which in turn

1282 G. Huet and others kDa kDa

Fig. 7. Immunoblot with peroxidase-labeled Helix pomatia lectin (A) and PNA lectin (B). Lane 1, untreated HT-29 MTX cells; lane 2, BFA-treated HT-29 MTX cells; lane 3, GalNAcα-O-benzyl-HT-29- treated MTX cells. inhibits the α-2,3-ST(O) activity, resulting in the observed increase in T antigen and decrease in sialic acid content of mucins from HT-29 MTX cells.

DISCUSSION

The availability of mucin-producing HT-29 MTX cells and the parental HT-29 cells from which the former population was isolated has allowed the analytical characterization of mucins Fig. 8. Effect of GalNAcα-O-benzyl and Galβ1-3GalNAcα-O- synthesized by these two cell types. To interpret the findings, benzyl on the transfer of Neu5Ac on asialofetuin. (A) Homogenates one must bear in mind that the HT-29 MTX line represents a of HT-29 MTX cells cultured with (᭿) or without (ᮀ) GalNAcα-O- homogeneous population of cells producing mucins of gastric benzyl were incubated with asialofetuin and the transfer of Neu5Ac type, whereas HT-29 cultures contain >95% undifferentiated was comparatively measured. Results are expressed as nmol of [14C]Neu5Ac transferred/mg of protein (mean values of two separate cells and a low proportion of cells producing mucins of gastric experiments). (B) Homogenates of HT-29 MTX cells were incubated as well as colonic immunological specificity. with asialofetuin in the presence of GalNAcα-O-benzyl (᭿) or HT-29 MTX mucins barely enter 2% to 5% gradient poly- Galβ1-3GalNAcα-O-benzyl (ᮀ) at different concentrations. Results acrylamide gels, in accordance with their formation of a are expressed as the percentage of remaining activity. secreted mucous layer (Lesuffleur et al., 1990). The major mucin fraction isolated by AE-HPLC is characterized by a high hydroxy amino acid content and a Thr/Ser of 1.52. Prior studies proline content; (2) higher GlcNAc/GalNAc ratio; (3) lower have shown that MUC1, MUC2, MUC3 and MUC5AC are sialic acid content; and (4) higher sulfate content. The distinct expressed in HT-29 MTX at the mRNA level, and the amino compositional features of HT-29 MTX and HT-29 mucins may acid composition of the major mucin fraction isolated is in reflect differences in expression of mucin genes (Lesuffleur et agreement with the amino acid sequences of the tandem repeats al., 1993) or be related to the altered compartmentalization of of these genes (Gendler et al., 1990; Gum et al., 1990, 1992; resident Golgi proteins in HT-29 MTX cells (Egea et al., 1993). Porchet et al., 1991; Guyonnet Duperat et al., 1994). The car- Besides, the HT-29 cell line likely contains mucin-secreting bohydrate composition of this fraction revealed a high GalNAc cells of different specificity, as shown using rabbit antisera and sialic acid content, and a low GlcNAc and sulfate content. detecting gastric or colonic mucins (Lesuffleur et al., 1990). These characteristics are similar to those described for the Current efforts aim at characterizing the structure of carbohy- cl.16-E mucin-secreting clone isolated from HT-29 cells after drate chains of HT-29 MTX and HT-29 mucins in an attempt treatment with sodium butyrate (Augeron and Laboisse, 1984). to analyse the relationship between oligosaccharide chains and The analysis of mucin-containing fractions from HT-29 cells the apomucin carrying them. revealed a lower amount of mucins, related to the small pro- The study of high molecular mass glycoproteins from these portion of mucin-producing cells in this population (less than two cell populations has also shown differences in the structure 0.5%) (Augeron and Laboisse, 1984; Lesuffleur et al., 1990, of proteoglycans, although this analysis is hampered by the 1991), as well as less glycosylated mucins. Furthermore, the high amount of mucins synthesized by HT-29 MTX. Indeed, a composition of HT-29 mucins was different from that of HT- certain level of mixing of these two types of molecules is 29 MTX mucins in having: (1) lower hydroxy amino acid and almost inevitable. In any case, the proteoglycans of HT-29 Biochemical characterization of HT-29 mucins 1283

proteoglycan analysis suggest that the degree of sulfatation of these molecules may be modulated by the state of differentiation of HT-29 cells. To examine mucin glycosylation in greater detail, we have ) 3 analysed the effect of BFA and GalNAcα-O-benzyl on the − structure of mucins synthesized by HT-29 MTX cells. BFA, 10

× which blocks transport from the RER to the Golgi apparatus and induces the redistribution of resident Golgi proteins to the RER (Lippincott-Schwartz et al., 1989), induced an increase in

c.p.m. ( c.p.m. sulfate content and an increase in the electrophoretic mobility of mucins in SDS-PAGE, events that are possibly related. Indeed, the high carbohydrate content of mucins may prevent effective binding of SDS to the protein core and the intrinsic negative charge of mucin molecules could inﬂuence their migration, as described for the MUC1 gene product (Hilkens and Buijs, 1988). The mechanisms by which these changes occur are not known, but our ﬁndings suggest that compartmentalization of sulfotransferases is affected by brefeldin A. GalNAcα-O-benzyl induced an increased expression of T antigen and a marked decrease in the sialylation of HT-29

) MTX mucins. These changes were accompanied by a decrease 3

− in the electrophoretic mobility of mucins by SDS-PAGE,

10 possibly due to the synthesis of less-acidic mucins. Indeed the × extensively sialylated form of MUC1 has a higher mobility in SDS-PAGE than the incompletely sialylated premature form (Hilkens and Buijs, 1988). GalNAcα-O-benzyl treatment of c.p.m. ( the HM7 high-mucin variant of LS174T colon cancer cells also led to an increase in T antigen reactivity. This result, now reproduced in HT-29 MTX cells, was unexpected as GalNAcα-O-benzyl behaves as an in vivo competitive inhibitor of the galactosyltransferase responsible for the synthesis of T-antigen (Huang et al., 1992). One of the hypotheses proposed by these authors was the in situ genera- Fig. 9. Detection of Galβ1-3GalNAcα-O-benzyl in the homogenates tion of the disaccharide Galβ1-3GalNAcα-O-benzyl, which of HT-29 MTX cells treated with 5 mM of GalNAcα-O-benzyl. would behave as an efficient inhibitor of the elongation of T (A) Homogenates of cells cultured with (᭿) or without (ᮀ) antigen. This hypothesis would be in agreement with the GalNAcα-O-benzyl were incubated in standard conditions of α-2,3- decreased sialylation of HT-29 MTX mucins observed in our ST(O) assay without exogenous acceptor, and sialylated products study. Galβ1-3GalNAcα-O-benzyl is a potential substrate for were detected as described in Materials and Methods. 1, CMP- α-2,3-ST(O) but also for Galβ1-3GalNAc-R β-1,6-N-acetyl- Neu5Ac; 2, Neu5Ac; 3, the arrowed bar indicates the position of α β α glucosaminyltransferase (EC 2.4.1.102) and sulfotransferases. Neu5Ac 2-3Gal 1-3GalNAc -O-benzyl obtained by the sialylation Thus changes in the incorporation of GlcNAc and sulfate might of Galβ1-3GalNAcα-O-benzyl with porcine liver α-2,3-ST(O). (B,C) Homogenates of cells cultured with GalNAcα-O-benzyl were also be expected. Our findings indicate that incorporation of denatured by heating and used as substrate source in α-2,3-ST(O) GlcNAc and sulfate was unaffected, whereas sialic acid incor- assay. Homogenates from control cells (B) or porcine liver α-2,3- poration into mucin was almost completely inhibited. In fact, ST(O) (C) were used as enzyme sources. Sialylated products were using asialofetuin as substrate, we have demonstrated that the detected as described in Materials and Methods. ST activity in GalNAcα-O-benzyl-treated homogenates is decreased and that Galβ1-3GalNAcα-O-benzyl is a powerful competitive inhibitor of the ST activity of HT-29 MTX homogenates. Among the different ST, only the Galβ1- MTX and HT-29 cells differ with regard to their elec- 3GalNAc α-2,3-ST can use Galβ1-3GalNAcα-O-benzyl as trophoretic behavior: the less-acidic nature of HT-29 MTX substrate. These observations suggest that α-2,3-ST(O) is the proteoglycans might result from a larger amount of unsulfated predominant ST expressed in HT-29 MTX cells, a finding that heparan chains. Such structures were shown to coexist with would be in agreement with the fact that NeuAcα2-3Galβ1- sulfated heparan chains on the same protein core of the heparan 3GalNAc-O is a major carbohydrate component of the mucins sulfate proteoglycan (HSPG) from the colon carcinoma cell synthesized by HT-29 MTX cells (unpublished observations) line WiDr (Iozzo, 1989), a cell line that has been reported by as well as of the mucins from HT-29-derived cl. 16E (Capon the ATCC as possibly being the same as HT-29. The studies et al., 1992). The marked inhibition of sialic acid incorpora- of high Mr HSPG produced by different cell types have tion into mucins may be related to the striking inhibition of suggested that a common precursor protein core could undergo mucin secretion in GalNAcα-O-benzyl-treated cells. cell-specific processing, thus generating different forms of pro- As mentioned above, GalNAcα-O-benzyl treatment did not teoglycans (Murdoch et al., 1992). The data from mucin and affect incorporation of GlcNAc or sulfate into mucins, sug- 1284 G. Huet and others gesting that the subcellular compartment in which the enzymes cDNAs derived from a novel human intestinal mucin gene. Biochem. responsible for this transfer are located is different from the Biophys. Res. Commun. 171, 407-415. compartment where α-2,3-ST(O) is present. This possibility Gum, J. R., Hicks, J. W., Toribara, N. N., Rothe, E. M., Lagace, R. E. and Kim, Y. S. (1992). The human MUC2 intestinal mucin has cysteine-rich would be in agreement with the concept that O-glycosylation subdomains located both upstream and downstream of its central repetitive proceeds in a stepwise manner according to the subcompart- region. J. Biol. Chem. 267, 21375-21383. mentalization of the enzymes that participate in it (Egea et al., Guyonnet Duperat, V., Audié, J. P., Debailleul, V., Laine, A., Buisine, M. 1993). In fact, sialylation and sulfation of the rat mammary P., Zouitina-Galiègue, S., Pigny, P., Degand, P., Aubert, J. P. and Porchet, N. (1995). Characterization of the human mucin gene MUC5AC: a sialomucin have been reported to occur in separate compart- consensus cysteine-rich domain for 11p15 mucin genes. Biochem. J. (in ments (Carraway and Hull, 1989). GalNAcα-O-benzyl may be press). of use in dissecting these steps of mucin maturation. Further Hardingham, T. E. and Fosang, A. J. (1992). Proteoglycans: many forms and studies may help to determine the influence of the compart- many functions. FASEB J. 6, 861-870. mentalization of ST and sulfotransferases upon the post-trans- Hilkens, J. and Buijs, F. (1988). Biosynthesis of MAM-6, an epithelial sialomucin. J. Biol. Chem. 263, 4215-4222. lational processing of these molecules. Houdret, N., Perini, J. M., Galabert, C., Scharfman, A., Humbert, P., Taken together our findings indicate that sialylation and sul- Lamblin, G. and Roussel, P. (1986). The high lipid content of respiratory fatation of mucins and proteoglycans are important post-trans- mucins in cystic fibrosis is related to infection. Biochim. Biophys. Acta 880, lational steps that may be modulated by the state of differen- 54-61. Huang, J., Byrd, J. C., Yoor, W. H. and Kim, Y. S. (1992). Effect of benzyl- tiation of HT-29-derived cell populations. Furthermore, α α α -GalNAc, an inhibitor of mucin glycosylation, on cancer-associated aryl-N-acetyl- -galactosaminides can impair the action of - antigens, in human colon cancer cells. Oncol. Res. 4, 507-515. 2,3-ST(O) in the O-glycosylation process of glycoproteins and Iozzo, R. V. and Wight, T. (1982). Isolation and characterization of this effect may be useful in investigating the activity of this proteoglycans synthesized by human colon and colon carcinoma. J. Biol. enzyme and the biological effects of its products. Chem. 257, 11135-11144. Iozzo, R. (1989). Presence of unsulfated heparan chains on the heparan sulfate proteoglycan of human colon carcinoma cells. J. Biol. Chem. 264, 2690- This work was supported in part by grant no. 2209 from the Asso- 2699. ciation pour la Recherche sur le Cancer and grant 93/1228 from the Kim, Y. S. (1992). Altered glycosylation of mucin glycoproteins in colonic Fondo de Investigaciones Sanitarias (Madrid, Spain). The authors neoplasia. J. Cell. Biochem. Suppl. 16G, 91-96. thank Drs S. I. Hakomori and K. O. Lloyd for providing antibodies, Kuan, S. F., Byrd, J. C., Basbaum, C. and Kim, Y. (1989). Inhibition of and Mrs E. Delaleau, M. T. Maillard and F. Roussez for typing the mucin synthesis by aryl-N-acetyl-α-D-galactosaminides in human colon manuscript. cancer cells. J. Biol. Chem. 264, 19271-19277. Kurosaka, A., Nakajima, H., Funakoshi, I., Matsuyama, M., Nogayo, T. and Yamashina, I. (1983). Structures of the major oligosaccharides from a human rectal adenocarcinoma glycoprotein. J. Biol. Chem. 258, 11594- REFERENCES 11598. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of Augeron, C. and Laboisse, C. L. (1984). Emergence of permanently the head of bacteriophage T4. Nature 227, 680-681. differentiated cell clones in a human colonic cancer cell line in culture after Lamblin, G., Boersma, A., Klein, A., Roussel, P., Van Halbeek, H. and treatment with sodium butyrate. Cancer Res. 44, 3961-3969. Vliegenthart, J. F. G. (1984). Primary structure determination of five Bobek, L. A., Tsai, H., Biesbrock, A. R and Levine, M. J. (1993). Molecular sialylated oligosaccharides derivated from bronchial mucus glycoproteins of cloning, sequence, and specificity of expression the gene encoding the low patients suffering from cystic fibrosis. J. Biol. Chem. 259, 9051-9058. molecular weight human salivary mucin (MUC7). J. Biol. Chem. 268, Lesuffleur, T., Barbat, A., Dussaulx, E. and Zweibaum, A. (1990), Growth 20563-20569. adaptation to methotrexate of HT-29 human colon carcinoma cells is Capon, C., Laboisse, C. L., Wieruszeski, J. M., Maoret, J. J., Augeron, C. associated with their ability to differentiate into columnar absorptive and and Fournet, B. (1992). Oligosaccharide structures of mucins secreted by mucus-secreting cells. Cancer Res. 50, 6334-6343. the human colonic cancer cell line Cl. 16E. J. Biol. Chem. 267, 19248-19257. Lesuffleur, T., Kornowski, A., Luccioni, C., Muleris, M., Barbat, A., Carraway, K. L. and Hull, S. R. (1989). O-glycosylation pathway for mucin- Beaumatin, J., Dussaulx, E., Dutrillaux, B. and Zweibaum, A. (1991). type glycoproteins. BioEssays 10, 117-121. Adaptation to 5-fluorouracil of the heterogeneous human colon tumor cell Dahiya, R., Lesuffleur, T., Kwak, K. S., Byrd, J. C., Barbat, A., Zweibaum, line HT-29 results in the selection of cells committed to diffentiation. Int. J. A. and Kim, Y. S. (1992). Expression and characterization of mucins Cancer 49, 721-730. associated with the resistance to methotrexate of human colonic Lesuffleur, T., Porchet, N., Aubert, J. P., Swallow, D., Gum, J. R., Kim, Y. adenocarcinoma cell line HT-29. Cancer Res. 52, 4655-4662. S., Real, F. X. and Zweibaum, A. (1993). Differential expression of the De Jong, J. G. N., Wevers, R. A. and Sambeek, R. L. V. (1992). Measuring human mucin genes MUC1 to MUC5 in relation to growth and urinary glycosaminoglycans in the presence of protein: an improved differentiation of different mucus-secreting HT-29 cell subpopulations. J. screening procedure for mucopolysaccharidoses based on Cell Sci. 106, 771-783. dimethylmethylene blue. Clin. Chem. 38, 803-807. Lippincott-Schwartz, J., Yvan, L. C., Bonifacino, J. S. and Klausner, R. D. Delannoy, P., Pelczar, H., Vandamme, V. and Verbert, A. (1993). (1989). Rapid redistribution of Golgi proteins into the ER in cells treated with Sialyltransferase activity in FR3T3 cells transformed with ras oncogene: brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56, 801- decreased CMP-NeuAc: Galβ1-3GalNAc-α-2,3-sialyltransferase. 813. Glycoconjugate J. 10, 91-98. Lo-Guidice, J. M., Wieruszeski, J. M., Lemoine, J., Verbert, A., Roussel, P. Devine, P. L. and Mckenzie, I. F. C. (1992). Mucins: structure, function, and and Lamblin, G. (1994). Sialylation and sulfatation of the carbohydrate associations with malignancy. BioEssays 14, 619-625. chains in respiratory mucins from a patient with cystic fibrosis. J. Biol. Chem. Egea, G., Franci, C., Gambus, G., Lesuffleur, T., Zweibaum, A. and Real, 269, 18794-18813. F. X. (1993). Cis-Golgi resident proteins and O-glycans abnormally Maoret, J., Font, J., Augeron, C., Codogno, P., Bauvy, C., Aubery, M. and compartmentalized in the RER of colon cancer cells. J. Cell Sci. 105, 819- Laboisse, C. L. (1989). A mucus-secreting human colonic cancer cell line. 830. Biochem. J. 258, 793-799. Gendler, S. J., Lancaster, C. A., Taylor-Papadimitriou, J., Duhig, T., Peat, Marianne, T., Périni, J. M., Houvenaghel, M. C., Tramu, G., Lamblin, G. N., Burchell, J., Pemberton, L., Lalari, E. and Wilson, D. (1990). and Roussel, P. (1986). Action of trifluoromethanesulfonic acid on highly Molecular cloning and expression of the human tumour-associated glycosylated regions of human bronchial mucins. Carbohydr. Res. 151, 7-19. polymorphic epithelial mucin, PEM. Biochem. J. 265, 15286-15293. Murdoch, A. D., Dodge, G. R., Cohen, I., Tuan, R. S. and Iozzo, R. V. Gum, J. R., Hicks, J. W., Swallow, D. M., Lagace, R. L., Byrd, J. C., (1992). Primary structure of the human heparan sulfate proteoglycan from Lamport, D. T. A., Siddiki, B. and Kim, Y. S. (1990). Molecular cloning of basement membrane (HSPG2/perlecan). J. Biol. Chem. 267, 8544-8557. Biochemical characterization of HT-29 mucins 1285

Nuti, M., Teramoto, Y. A., Mariani-Constantini, R., Horand Hand, P., Toribara, N. W., Robertson, A. M., Ho, S. B., Kuo, W. L., Gum, E., Hicks, Colcher, D. and Schlom, J. (1982). A monoclonal antibody (B72. 3) defines J. W., Gum, J. R., Byrd, J. C., Siddiki, B. and Kim, Y. S. (1993). Human patterns of distribution of a novel tumor-associated antigen in human gastric mucin. J. Biol. Chem. 268, 5879-5885. mammary carcinoma cell populations. Int. J. Cancer 29, 539-545. Vaessen, R. T. M. J., Kreike, J. and Groot, G. S. P. (1981). Protein transfer to Peterson, G. L. (1977). Modified procedure for protein assay. Anal. Biochem. nitrocellulose filters. FEBS Lett. 124, 193-196. 83, 346-356. Vandamme, V., Cazlaris, H., Le Marer, N., Laudet, V., Lagrou, C., Pinto, M., Appay, M. D., Simon-Assmann, P., Chevalier, G., Drocopoli, N., Verbert, A. and Delannoy, P. (1992). Comparison of sialyl- and β1-3- Fogh, J. and Zweibaum, A. (1982). Enterocytic differentiation of cultured galactosyltransferase activity in NIH3T3 cells transformed with ras human colon cancer cells by replacement of glucose by galactose in the oncogene: increase β-galactoside-2,6-sialyltransferase. Biochimie 74, 89- medium. Biol. Cell 44, 193-196. 100. Porchet, N., Nguyen, V. C., Dufossé, J., Audié, J. P., Guyonnet-Duperat, V., Van den Eijden, D. H. and Joziasse, D. H. (1993). Enzymes associated with Gross, M. S., Denis C., Degand, P., Bernheim, A. and Aubert, J. P. glycosylation. Curr. Opin. Struct. Biol. 3, 711-721. (1991). Molecular cloning and chromosomal localization of a novel human Wesslet, E. (1968). Analytical and preparative separation of acidic tracheobronchial mucin cDNA containing tandemly repeated sequences of glycosamino-glycan by electrophoresis in baryum acetate. Anal. Biochem. 48 base pairs. Biochem. Biophys. Res. Commun. 175, 414-422. 26, 439-444. Takahashi, H. K., Metoki, R. and Hakomori, S. I. (1988). Immunoglobulin G3 monoclonal antibody directed to Tn antigen (tumor associated α-N- acetyl-galactosaminyl epitope) that does not cross-react with blood group A antigen. Cancer Res. 48, 4361-4367. (Received 27 July 1994 - Accepted 15 November 1994)