[CANCER RESEARCH 51. 3136-3142. June 15. 1991] Glycosyltransferase Changes upon Differentiation of CaCo-2 Human Colonie Adenocarcinoma Cells1

I. Brockhausen,2 P. A. Romero, and A. Herscovics

Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 ¡I.B.];and McGill Cancer Centre, McGill University, Montreal, Quebec, Canada H3G l Y6 [P. A. R., A. H.J

ABSTRACT primarily associated with /V-linked carbohydrate chains, a sig nificant proportion of glucosamine-labeled glycopeptides were The spontaneous differentiation of CaCo-2 human colonie adenocar- also found in alkali-labile O-linked oligosaccharides (2). The cinuma cells to enterocytes in culture is associated with a decrease in polylactosaminoglycans, particularly those attached to the lysosomal proportion of fucosylated polylactosaminoglycans known to membrane glycoprotein h-lamp-1 (Youakim et al., Cancer Res., 49:6889- carry oncodevelopmental antigens decreases significantly dur 6895, 1989). To elucidate the biosynthetic mechanisms leading to these ing differentiation (2). In particular, the lysosomal membrane alterations we have compared glycosyltransferase activities that are protein h-lamp-1 was shown to be affected by these glycosyla- involved in the synthesis of polylactosaminoglycans and of the ,V-and O- tion changes (3). glycan structures that provide the framework for the attachment of these To elucidate the mechanisms responsible for the decrease in chains. Glycosyltransferase activities in cell homogenates obtained from polylactosaminoglycans with differentiation, we compared the undifferentiated and differentiated CaCo-2 cells were assayed by high glycosyltransferase activities that are involved in the synthesis pressure liquid chromatography separation of enzyme products. The 0- of polylactosaminoglycan chains (Fig. 1) and of the backbone galactosidase activities and extremely high pyrophosphatase activities in structures of O- and TV-linked oligosaccharides in UD3 and D differentiated cells were effectively inhibited by 5 m\i 7-galactonolactone and 10 HIMAMP, respectively. CaCo-2 cells contain most of the enzymes CaCo-2 cells. The synthesis of core structures and of branches of A'-and O-glycans requires distinct sets of glycosyltransferases that are involved in /V-glycan branching jiY-acetylglucosamine (GlcNAc) transferases I to V| with the exception of GlcNAc transferase VI. (4, 5). However, most of the enzymes involved in the elongation The levels of GlcNAc transferase I activities were comparable in un of N- and O-glycans and in the addition of terminal antigens differentiated and differentiated cells, but GlcNAc transferase II to V are the same for both types of glycans. We found increased activities were significantly increased upon differentiation. The enzyme activities for both O- and A'-glycan branching enzymes and activities that are directly involved in the synthesis of linear polylacto decreased activity of the transferase responsible for the syn saminoglycans (Gal|84GlcNAc/33- repeating units), blood group i UDP- thesis of the O-glycan core 1 (Gal/33GalNAc-R) upon differ GlcNAc:Gal/9-R 03-GlcNAc transferase and UDP-Gal:GlcNAc 04-Gal entiation. Unexpectedly, the enzymes directly involved in transferase, were found at similar levels in undifferentiated and differ polylactosaminoglycan biosynthesis, Le, UDP-Gal:GlcNAc entiated CaCo-2 cells. Since GlcNAc transferase III activity is known /34-Gal transferase (/34-Gal-T) and blood group i UDP- to inhibit further branching and galactosylation, these results suggest that its increased activity in differentiated CaCo-2 cells may be partly GlcNAc:Gal,94GlcNAc-R 03-GlcNAc transferase (i 03-Gn-T), responsible for the decreased synthesis of fucosylated polylactosamino remain essentially unchanged with differentiation of CaCo-2 glycans. Differentiated cells showed a 2-fold increase in O-glycan core cells; blood group I UDP-GlcNAc: GlcNAc/33Gal/3-R (GlcNAc 2 UDP-GlcNAc:Gal#JGalNAca-R |GlcNAc to A'-acetylgalactosamine to Gal) ß6-GlcNActransferase, responsible for branching, was (GalNAc)l /?6-GlcNAc transferase activity. In contrast, O-glycan core 1 undetectable. UDP-Gal:GalNAca-R 03-Gal transferase activity was found decreased. Several enzymes that are found in homogenates from normal human colonie tissue are absent or barely detectable in CaCo-2 cells. MATERIALS AND METHODS These include blood group I UDP-GlcNAc:GlcNAc03Gal/3-R (Glc Materials. AG 1-X8 (100-200 mesh, Cl~ form) was purchased from NAc to Gal) 06-GlcNAc transferase, O-glycan core 3 UDP-Glc- Bio-Rad. Bovine serum albumin, Gal/3l-4GlcN Ac, AMP, Triton X- NAc:GalNAca-R /83GlcNAc transferase and O-glycan core 4 UDP- 100, 7-galactonolactone, Galß3GalNAca-Bn, GlcNAc/33 Gal/3-methyl, GlcNAc:GlcNAc/33GalNAc-R (GlcNAc to GalNAc) 06-GlcNAc and GalNAca-Bn were purchased from Sigma. Acetonitrile (190 UV transferase. cutoff) was from Fisher Scientific Co. or Caledon Laboratories. UDP- A'-[l-'4C]acetylglucosamine was synthesized as described previously (6) and diluted with UDP-GlcNAc from Sigma. UDP-[t/-14C]galactose was INTRODUCTION purchased from Amersham and diluted with UDP-Gal from Sigma. CaCo-2 human colonie adenocarcinoma cells differentiate Substrates and Standards. GlcNAc/31-3 GalNAc«-Bn, GlcNAc/31-6 spontaneously into cells with the properties of enterocytes upon (Gal/il-3) GalNAca-Bn, GlcNAc01-6(GlcNAc/31-3)GalNAca-Bn, and GlcNAc/31-6(GlcNAc/31-3Gal£il-3)GalNAc«-Bn were synthesized prolonged culturing (1). This differentiation is accompanied by by Dr. K. L. Matta, Buffalo, NY. GlcNAc/32Man«6 (GlcNAc/32 the acquisition of a brush border and of hydrolytic enzymes Man«3)Man/J-(CH2)8COOCH, |2,2mco) was donated by Dr. O. Hinds- typical of absorptive intestinal cells. CaCo-2 cells were previ gaul, Edmonton, Alberta, Canada. GlcNAc06 (GlcNAc/32) Mana- ously shown to contain polylactosaminoglycans, which could methyl was provided by Dr. R. Shah, Toronto, Ontario, Canada. Man«6 be labeled with fucose and glucosamine. Although these are 3The abbreviations used are: UD, undifferentiated CaCo-2 cells; Bn, benzyl; Received 12/10/90; accepted 4/4/91. D. differentiated CaCo-2 cells; Gal, G, D-galactose; GalNAc, GA, A/-acetyl-D- The costs of publication of this article were defrayed in part by the payment galactosamine: GlcNAc, Gn, A/-acetyl-D-glucosamine; HPLC, high pressure liquid of page charges. This article must therefore be hereby marked advertisement in chromatography; Man, M, D-mannose; meo, (CH2)gCOOCH3; MES, 2-(A/-mor- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. pholino)ethanesulfonate; nmr, nuclear magnetic resonance; T, transferase; ' This research was supported by a grant from the Canadian Cystic Fibrosis 10.2+FI, M«6(GnfJ2M«3)M/i4-Gníí4(F«6)Gn;|2,2+F|, Gn/32 M«6(Gn/32 Foundation to Harry Schachter (Hospital for Sick Children, Toronto), and by M«3) M/i4-Gn/i4(F«6) Gn; |2.24|. Gnf¡2M«6 (Gnß4[Gnß2]Mai) Mß4- grants from the Cancer Research Society and the Canadian Foundation for lleitis GníMGn; |26,24|, Gnfíó(Gnß2)Ma6 (Gnß4[Gnß2]Mai) Mß4-Gnß4Gn; and Colitis to A. H. and the Medical Research Council of Canada to I. B. |bis26.24|, Gnrfó (Gn/i2) M«6(Gnß4[Gnß2]M«3)[Gn/34] M/í4-GníJ4Gn; 2To whom requests for reprints should be addressed, at the Research Institute. |2,2mco|. Gnß2M«6(Gnp¡2M«3)M/3-mco: |bis2,2mco|, Gn/J2 M«6(Gn/J2 The Hospital for Sick Children. 555 University Avenue. Toronto, Ontario M5G M«3)[Gnfí4]Mff-mco; |2,24mco|, Gnf)2 Mnó (Gn/34 [Gnfi2) M«3)Mo-mco; 1X8, Canada. |bis2,24mco|, Gnß2M«6(Gnß4[Gnß2]M«3)[Gnf)4] M/J-mco. 3136

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Gß4Gn I antigen Nuclear Magnetic Resonance. Samples were prepared by exchanging 106 G(54Gn03 G|M Gnß-H twice with 99.8% D,O (Aldrich) and twice with 99.96% D2O (Merck, Sharpe and Dohme). Samples were dissolved in 99.96% D;O with Sa-Gal-T acetone as the internal standard. Proton nmr spectra were recorded at the Toronto Carbohydrate Research Centre with a Bruker 500-MHz Gn spectrometer. The acetone signal was set at 2.225 ppm. 106 Gnß3Gß4Gnß-R Gn(33G(54Gnß3Gß4-Gnß-R High Pressure Liquid Chromatograph). HPLC separations were car ried out with an LKB or a Waters system as described (12, 13). k i S3-Gn-T Acetonitrile/water mixtures were used as the mobile phase for all columns at a flow rate of 1 ml/min. To separate enzyme products with ß4-Gal-T meo, hexyl, benzyl, and methyl groups, a reverse phase Cm column was Gnß3G(}4Gn(Jfl •*•Gß4Gnß3G(i4Gn0-R used. Free reducing sugars or methylglycosides were separated on a propylamine (NH;) column (Waters carbohydrate analysis column). i antigen Elution of compounds was monitored by measuring the absorbance at 195 nm and counting the radioactivity of collected fractions (12). Gß4Gnß-R Assays for GlcNAc Transfcrases (Table 1). The standard assay mix ture for GlcNAc transferases contained the following ingredients in a total volume of 40/il: 0.125 M GlcNAc; 0.125 M MES, pH 7; 0.125% Gnßfl Triton X-IOO; 12.5 mM MnCl:; 10 niM AMP; 5 mM -y-galactonolactone Fig. 1. Pathways of polylactosaminoglycan synthesis. The enzymes involved (only for substrates with terminal Gal at the nonreducing end as in the synthesis of the blood group i antigen attached to R (R = jV-linked or O- indicated in Table 1); 0.87 mM UDP-[l-'4C]acetylglucosamine (5486 linked oligosaccharides) are present in CaCo-2 cells. The fíó-GIcNActransferase dpm/nmol); substrate (as indicated in Table 1); and 10 >i\cell homog- activity synthesizing the branch of the blood group I antigen is absent from CaCo- enate (0.057 or 0.068 mg protein/ml for D or UD cells, respectively). 2 cells but is high in normal human colon. ffo-GlcNAc transferases that do not require MnCl: (GlcNAc trans ferase V. blood group I, core 2, and core 4 ffo-GlcNAc transferases) (Man«3) Manfi-hexyl was synthesized by Dr. H. Paulsen, Hamburg, were measured in the absence of exogenous MnCli to reduce any Germany. Man«6(['4C]GlcNAcff2 Man«3)Man/i-hexyl was prepared enzyme reactions requiring MnCI?. Mixtures were incubated for l h at from Man«6 (Man«3) Man/i-hexyl with purified rat liver GlcNAc 37°C.Reactions were stopped by the addition of 400 p\ 20 mM sodium transferase I and subsequent purification by AG 1-X8 and Bio-Gel P- tetraborate/1 mM EDTA, pH 9, and freezing. Mixtures were passed 4. It was shown that the purified enzyme attaches GlcNAc in fi2-linkage through Pasteur pipets filled with AG 1-X8. 100-200 mesh, Cl" form. only to the Man«3residue of the substrate4 |0.2+F|, |2,24|, |bis2,24|. After these were washed with 2.6 ml water, eluates were lyophilized, taken up in 200 ^1 water, and stored at -20°C. We used 100 p\ for |26,24|. and |bis26.24| were isolated as reducing oligosaccharides from glycoproteins (7). GIcNAc/ió [l4C-GlcNAcfi4] (GlcNAcf¡2) Mana- HPLC analysis. Results from assays lacking the acceptor were routinely methyl was prepared from GlcNAcfíó(GlcNAc/i2)Mann-methyl with subtracted in the calculations of activities. hen oviduct microsomal GlcNAc transferase VI (8). |bis2,2mco), Assays for Gal Transferases (Table 1). The assays for Gal transferases |2,24mco|, and |bis2,24mco| were isolated as described before (8). were carried out as described for the GlcNAc transferases, except that ('4C]GlcNAcff6 (GlcNAcf¡3)Gal/i-methyl was prepared by incubation the assay mixtures contained 1.86 mM UDP-[i/-'4C]galactose (3266 of GlcNAc/J3 Galrf-methyl with pig gastric mucosa microsomal blood dpm/nmol) instead of UDP-GlcNAc and 5 mM >-galactonolactone, group I (i6-GlcNAc transferase (9. 10) as follows: 3.2 ^mol GlcNAC|tf3 and the addition of 0.125 M GlcNAc was omitted. Gal/i-methyl were incubated with pig gastric mucosal microsomes (4.8 Assays for 0-Galactosidase. /i-Galactosidase was assayed according mg protein) for 2 h at 37°Cin a total volume of 1.32 ml containing to the method of Distler and Jourdian (14) in phosphate-citrate buffer 2.62 mM UDP-|'4ClGlcNAc (3287 dpm/nmol), 0.15% Triton X-100, (pH 4.3) or MES buffer (pH 5 to 8) with GaM-p-nitrophenyl as the 76 mM MES buffer (pH 7), 1.5 mM AMP, and 76 mM GlcNAc. To substrate and 30 min incubation time. stop the reaction. 8 ml 20 mM sodium tetraborate/1 mM EDTA, pH 9. Enzyme Kinetics. K\t and Vma>values were determined from 5 to 6 were added. The mixture was passed through a 20-ml column of AG different substrate concentrations by linear double reciprocal Line- 1-X8, 100-200 mesh, CI form. The column was washed with 52 ml weaver-Burk plots. water, and the eluate was lyophilized. Enzyme product was purified twice by HPLC, using a Waters carbohydrate (propylamine) column with acetonitrile/water = 85/15 at a 1 ml/min flow rate. The 500-MHz RESULTS proton nmr spectrum of the product showed an additional -V-acetyl signal at 2.033 ppm and an 8.5-Hz doublet at 4.538 ppm characteristic Glycosyltransferases that assemble and elongate the branches of/i6-linkedGlcNAc(9, 11). of A'-linked oligosaccharides and 0-glycan cores were assayed The structures of all substrates and standards were confirmed by nmr in UD and D CaCo-2 cells. The enzymes; structures of their spectroscopy. substrates and products; the HPLC conditions of product sep Preparations of Cell Homogenates. CaCo-2 cells were maintained in aration, including retention times; and enzyme activities are culture as described previously (3). Cells were harvested after 4 days in summarized in Table 1. The assays were complicated by high culture for rapidly growing UD cells, or after 25 days for late confluent pyrophosphatase activities observed especially in D cells. These D cells. Cells were washed three times in 0.99c NaCl solution followed activities were calculated from the total amount of free radio by centrifugation. Pellets were frozen and thawed in 0.9% NaCl solution active sugars arising from the breakdown of radioactive UDP- and centrifuged. The pellets were suspended in 0.25 M sucrose (1 ml/ 10" cells), homogenized with a Potter-Elvehjem homogenizer, and Gal or UDP-GlcNAc during the incubation. Without the ad stored at -70°C. Human colonie homogenates were prepared from dition of AMP, UD cells exhibited reasonable levels (<5%) of colonie tissue obtained from tissue resections of normal colonie mucosa breakdown; however, pyrophosphatases in D cells cleaved more adjacent to colon cancer: tissue was hand homogenized in 0.25 M than 80% of the nucleotide sugar donor. It was thus essential sucrose. to add 10 m.M AMP as a pyrophosphatase inhibitor to the Protein Determinations. Protein was determined by the Bio-Rad assay. Under these conditions, less than 29¿of the nucleotide method using bovine serum albumin as the standard. sugars in both cell populations were degraded during a 1-h 4G. Möller.M. Sarkar, F. Reck. H. Paulsen. and I. Brockhausen, manuscript incubation. in preparation. The absorbance scans of HPLC graphs indicated that the 3137

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Table 1 Glycosyltransferase changes upon differentiation ofCaCo-2 cells: summary of enzymes assayed in CaCo-2 cells, structures of substrates and products, HPLC conditions, and enzyme activities"

(nmol/h/mg)% time SubstrateGn-TEnzyme (min)Col.c35 D12AN UD 1 0.5 mM M«6(M«3) M/3-hexProductMn6(Gntf2M«3) CuActivity 11.0 12.2 M/j-hexRetention*

Gn-T II" 0.25 mM 10.2+FI 14 NHj 74 12,2+FI 30 NHj 74 7.8 15.7

Gn-T III 1 mM |2.2mco| 25 C,. 16 |bis2,2mco| 38 C« 16 2.9 10.2 0.5 mM |26,24| 65 NHj 70 |bis26,24| 80 NH2 70 2.6 4.9

Gn-T IV 1 mM |2.2mco) 25 C,, 16 |2,24mco| 26 C,, 16 0.6 4.7

Gn-TV 0.2 mM 12,24) 17,28' C,, 0 |26,24| 11, 17' C,, 0 0.9 2.4

Gn-T VI 4 mM Gn/i6(Gn/J2) 12 M«-me Gn/J6([Gn/34]Gn/32) 46 C,, ND ND M«-me

Core 1 fiS- 2 mM GA«-Bn 31 CM 10 G/33GAtt-Bn 28 CM 10 38.3 21.4

Core 2 ffó-Gn-T7 2 mM Gfi3GAtt-Bn 26 CM 10 Gn 17 CM 10 8.9 20.1 \P6 G/33GA

Core 3 03-Gn-T 2 mM GA«-Bn 31 CM 10 Gn/J3GAn-Bn 25 CM 10 ND ND

Core 4 06-Gn-T 2 mM Gnf)3GA«-Bn 25 CM 10 Gn 16 CM 10 0.46 0.64 1/36 Gntf3GA«-Bn

Elongation /¿3-Gn-T^ Gn 32 1/36 4 mM G/33GA«-Bn Gn 30 CM 0.09 0.04

Gf)3GA«-Bn

i 03-Gn-Tf 4 mM G/34Gn 20 NH2 86 Gnf¡3G/í4Gn 64 NHj 86 0.81 0.62

I 06-Gn-T 2 mM Gnfi3G/J- 18 NH2 86 methyl Gn 64 NH2 ND ND 1/36 Gní¡3G/í-meth>l

2 mM Gn 16 NH2 84 G(i4Gn 43 NH2 84 50.3 58.8 •Enzyme(5) abbreviations are: Gn-T I, II, III, IV, V, VI; UDP-GlcNAc: Man-R GlcNAc transferases I, II, III, IV, V, VI (8, 12, 15, 22); core 1 /33-Gal-T, core 1 UDP-GaI:GalNAc-R /33-Gal-T (33, 34); core 2 06-Gn-T, core 2 UDP-GlcNAc:Gal/33 GalNAc-R /36-GlcNAc-T (35, 36, 37); core 3 03-Gn-T, core 3 UDP- GlcNAc:GalNAc-R 03-GlcNAc-T (6); core 4 /36-Gn-T, core 4 UDP-G!cNAc:GlcNAci33 GalNAc-R 06-GlcNAc-T (6); elongation /33-Gn-T, elongation UDP- GlcNAc:Ga!/J3 (R,-6) GalNAc-R2 /33-GlcNAc-T (16, 17); i 03-Gn-T, blood group i UDP-GlcNAc:Gal/34 GlcNAc-R 03-GlcNAc-T (19); ] 06-Gn-T, blood group I UDP-GlcNAc:GlcNAcfi3 Gal-R /%-GlcNAc-T (10); i)4-Gal-T, UDP-Gal:GlcNAc /34-Gal-T (26). Enzymes were assayed at least in duplicate determinations as described in "Materials and Methods." AN, acetonitrile; Bn, benzyl; hex, hexyl; G, Gal. D-galactose; GA, A'-acetyl-D-galactosamine; Gn, /V-acetylglucosamine; M, D- mannose; me, methyl; meo. (CH2)>COOCHj; ND, not detected; T, transferase. * Retention times may change according to the HPLC system and the age of the column. c Col., HPLC column; C|8, silica-bound C,t; NH3, silica-bound propylamine. ' Activity includes minor subsequent reactions by Gn-T III, IV, and V. ' Reducing oligosaccharides elute as two peaks on reverse phase columns. '5 mM Y-galactonolactone was added to the assay mixture. substrate Gal(33GalNAc«-Bn was degraded by the action of ß- D was 250% of the activity in UD cells. The optimal pH of ß- galactosidases in D cells to GalNAca-Bn. Degradation of galactosidase activity in both UD and D cells was pH 4.3 or Gal/33GalNAc«-Bn was barely detectable in UD cells. ß-Galac- below. This indicates that the /3-galactosidases probably origi tosidase activity measured with Gal/3-p-nitrophenyl substrate in nate from the lysosomes. However, the addition of 2.5 or 5 3138

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7-galactonolactone inhibited breakdown of Gal/í3GalNAcoligosaccharide is a specific substrate for GlcNAc-T III and showed a 2-fold increase of this enzyme in D cells (Table 1). |2,2mco| was used as the substrate to measure both GlcNAc-T III and GlcNAc-T IV activities in the same assay m (Fig. 4). GlcNAc-T III was increased more than 3-fold, and o> GlcNAc-T IV was increased more than 7-fold in D cells co (Table 1). 8 GlcNAc-T V assays were carried out in the absence of addi (O tional MnCl: to reduce the action of GlcNAc transferase III. o

O 20 40 60 ElutionTime(min) Fig. 3. Blood group I ¿6-GlcNActransferase assay by HPLC using GlcNAcrf.1 Galrf-melhyl as the substrate, and human colonie honiogenatc (A) and differen tiated CaCo-2 cells (B) as the enzyme source. Assays «erecarried out as described •IODO in "Materials and Methods" for GlcNAc-T. in the absence of MnCl2. Enzyme s product was separated on an amine column using acelonitrile/water = 86/14 as cS the mobile phase at I ml/min. The radioactive standard for the enzyme product, s (l4C]-GlcNAc/i6 (GlcNAc/J3) GaW-methyl eluted at 64 min (assays B). Human c S colonie homogenate shows good activity, but CaCo-2 cells lack the blood group •o I 06-GlcNAc transferase. 500 £

The enzymes assembling the repeating units of polylactosa- 100 minoglycans, i /i3-GlcNAc-T and 04-Gal-T, were found at a

0 20 40 comparable level in UD and D cells (0.81 and 0.62 nmol/h/mg for the i /i3-GlcNAc-T and 50.3 and 58.8 nmol/h/mg for the Elution Time (min) /i4-Gal-T) (Table 1). Enzyme kinetics for the /i4-Gal-T indicates Fig. 2. Core 2 (i6-GlcNAc transferase assay by HPLC usin Bn as the substrate and D cells as the enzyme source. Assays were carried out as similar A\, (6.3 mM for UD and 7.1 mM for D) and Vmax(167 described in "Materials and Methods" for GlcNAc-T. Enzyme product was nmol/h/mg for UD and 250 nmol/h/mg for D) values. separated on a reverse phase d8 column using acctonitrile/watcr = 10/90 as the The core 1 /¿3-Gal-Tthat synthesizes O-glycan core 1 (Gal/i3 mobile phase al I ml/min. There is no significant breakdown of the substrale to GalNAco-Bn. GalNAc-), using GalNAca-Bn as the substrate, showed a re- 3139

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cells, along with the other branching enzymes. It has been shown that the presence of the bisected GlcNAc has a profound effect on oligosaccharide conformation (23) and blocks further branching (24), lectin binding (25), and galactosylation (26). Bisected structures have also been found in differentiated HL •1000 60 cells associated with decreased high molecular weight oli gosaccharide chains (21). It seems possible, therefore, that the increased GlcNAc-T III activity observed in differentiated cells prevents subsequent reactions, including elongation to polylac tosaminoglycans. GlcNAc-T III may thus be an important

-500 regulator of polylactosaminoglycan synthesis. Other factors may also be involved. As protein determinants are possible biosynthetic controlling factors (23, 27, 28), proteins may carry their individual carbohydrate structures. It is possible that •100 proteins undergo qualitative and quantitative changes upon differentiation, and these changes influence lactosaminoglycan O 20 40 O 20 40 60 synthesis. However, the decrease in polylactosaminoglycans Elution Time (min) Elution Time (min) with differentiation was clearly demonstrated for a specific Fig. 4. GlcNAc transferase III assay by HPLC using 1 m\i |2.2mco| as the glycoprotein, h-lamp-1 (3). This decrease was not due to a substrate, and UD (.-1)and D (B) cells as the enzyme source. Assays were carried out as described in "Materials and Methods" for GlcNAc-T. Enzyme product change in the amount of protein but to glycosylation changes. was separated on a reverse phase C|8 column and acetonitrile/water = 16/84 as Another possibility for the decrease in fucosylated polylacto the mobile phase at I ml/min. saminoglycans may be increased degradation of these chains in D, changes in the subcellular organization or intracellular trans port, or changes in the activity of glycosyltransferases that add auction in D cells (Table 1). Enzyme kinetics revealed similar A'Mfor the enzyme in UD and D (0.83 and 0.74 mivi respec terminal carbohydrate residues. Sialyltransferases have been tively), but a reduction in the Vmavfrom 50 nmol/h/mg in UD reported to increase upon differentiation of rat intestinal cells to 29.4 nmol/h/mg in D. In contrast, the core 2 06-GlcNAc-T (29), HL 60 cells (30), and human myeloblasts (31). If polylac activity that synthesizes core 2 (GlcNAc/36 [Gal/33] GalNAc-), tosaminoglycan chains became hypersialylated or increasingly using Gal03 GalNAov-Bn as the substrate, was increased in substituted with blood group antigens, extension would likely differentiated D cells (Table 1; Fig. 2). While the A"N1values be inhibited. However, for Chinese hamster ovary cells it has been suggested that the addition of terminal «3-Galand <*3- were similar for both UD and D cells (0.36 and 0.43 mM respectively), the Vmaxincreased from 9.6 nmol/h/mg in UD to sialic acid does not compete with polylactosaminoglycan syn 21.3 nmol/h/mg in D cells. thesis, probably because these reactions take place in different Only very low levels of the /13-GlcNAc-T that elongates core subcellular compartments (32). The subcellular distribution of 1 and core 2(16, 17) were found in CaCo-2 cells (Table 1). The terminal transferases in CaCo-2 cells is not known. activity that synthesizes GlcNAc06GalNAc-R could not be In previous work, it was concluded that the polylactosami demonstrated in either normal human colon or CaCo-2 cells. noglycans of CaCo-2 cells were highly branched, based upon the pattern of products obtained following endo-0-galactosidase digestion (2). It is possible that the mixture of products obtained DISCUSSION following endo-0-galactosidase digestion resulted from incom To test the possibility that the decreased proportion of fuco- plete degradation of linear polylactosaminoglycans due to ex sylated polylactosaminoglycans previously observed with differ tensive substitutions with fucose residues. No branching activity entiation of CaCo-2 cells (2, 3) is due to changes in the branch to form the blood group I antigen (10) was found in the present ing patterns of A'- and O-glycans, we have assayed the glycosyl- study, although there was considerable core 2 06-GlcNAc-T transferases that build and elongate the four common O-glycan activity. The 06-GlcNAc-T synthesizing the I antigen is present cores and the branches of /V-linked glycans (4, 18). Upon in normal human colonie tissue homogenate which consists of differentiation, we found a general increase in O- and /V-glycan a mixture of different cell populations. The activities synthesiz branching enzyme activities, while the enzymes directly in ing core 2, I antigen, and core 4 are probably due to the same volved in the synthesis of the repeating linear polylactosami- enzyme in mucin-secreting tissues (9). Apparently, this 06- noglycan units; i.e., the activities of 04-Gal-T and blood group GlcNAc-T in CaCo-2 cells is similar to the enzyme in human i /33-GlcNAc-T (19) were found to be unchanged. These obser leukocytes (13) but has a different specificity than the enzyme vations are rather unexpected since it has generally been as from pig gastric or human colonie mucosa inasmuch as it lacks sumed that the synthesis of polylactosaminoglycans is primarily the activities to synthesize core 4 and I antigen. regulated by the activities of these glycosyltransferases. Lee et The 03-Gal-T that actively synthesizes O-glycan core 1 al. (20) found a correlation between the blood group i 03- (Gal03 GalNAc«-)(18, 33, 34) in UD cells is reduced to about GlcNAc transferase activity and the increase in polylactosami- one-half upon differentiation in D cells. Enzyme kinetics sug noglycan chains during granulocytic differentiation of HL 60 gests that the same enzyme is expressed in both UD and D cells. However, HL 60 cells have also been reported to lose cells but that there is less enzyme protein in D cells. Core 2 06- their polylactosaminoglycan chains upon monocytoid differen GlcNAc-T [synthesizing core 2, GlcNAc/36 (Gal03) GalNAc«-] tiation (21). (35, 36, 37) is significantly increased in differentiated CaCo-2 The activity of GlcNAc-T III, the enzyme that introduces the cells, probably due to an increase in enzyme protein. The core bisecting GlcNAc linked to the 0-Man residue of A'-linked 2 06-GlcNAc-T has previously been found to be significantly oligosaccharides (22), was elevated in differentiated CaCo-2 elevated in the less mature granulocytes and blast cells from 3140

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ick, S. W., and Carver. J. P. Control of glycoprotein synthesis. Detection patients with chronic and acute myelogenous leukemia (13). and characterization of a novel branching enzyme from hen oviduct, UDP- Piller et al. (38) reported a dramatic increase in core 2 ß6- A/-acetylglucosamine:GlcNAcfM-6(GlcNAc/il-2)Man«-R (GlcNAc to Man) GlcNAc-T activity upon activation of lymphocytes by anti-CD3 fi-4-A/-acetylglucosaminyltransferasc VI. J. Biol. Chem.. 264: 11211-11221. antibody or interleukin 2. This enzyme appears to be develop- 9 1989. Brockhausen. I.. Malta. K. L.. Orr. J.. Schachter. H.. Koenderman. A., and mentally regulated and may be important in the differentiation van den Eijnden. D. H. Mucin synthesis. Conversion of R,-iil-3Gal-R2 to R,-/il-3(GlcNAc/il-6)Gal-R2 and of R,-fu-3GalNAc-R2 to R,-(il- process. CaCo-2 cells have only low levels of the /36-GlcNAc- 3(GlcNAc(il-6)GalNAc-R2 by a f)6-A'-acetylglucosaminyltransferase in pig T-synthesizing core 4 (GlcNAcf!6 [GlcNAc/S3] GalNAc-) (6) gastric mucosa. Eur. J. Biochem., 757:463-474, 1986. which in various mucin-secreting tissues, including human co- io. Piller. F., Cartron. J-P.. Maranduba, A.. Veyrières. A.. Leroy, Y., and Fournet, B. Biosynthesis of blood group I antigens. Identification of a UDP- Ion, accompanies core 2 ß6-GlcNAc-Tactivities at fairly high GlcNAc:GlcNAc/carbohydrates related to glycoproteins. Adv. Carbohyd. Chem. regulating enzyme specificity. Biochem., 41: 209-374. 1983. O-Glycan cores are usually made by the attachment of a 3- 12. Brockhausen, L, Carver. J. P.. and Schachter. H. Control of glycoprotein synthesis. The use of oligosaccharide substrates and HPLC to study the linked residue prior to introducing the 6-linked branch (18). sequential pathway for A'-acetylglucosaminyltransferases I. II. III. IV, V and Human glycoproteins have been reported to contain O-glycans VI in the biosynthesis of highly branched A-glycans by hen oviduct. Biochem. with the GlcNAc/tó GalNAc«- structure (39, 40). In normal Cell Biol.. 66: 1134-1151. I988b. 13. Brockhausen. L. Kuhns. W., Schachter, H., Matta. K. L.. Sutherland. D. R., human colon or in CaCo-2 cells we could not detect an enzyme and Baker. M. A. Biosynthesis of O-glycans in leukocytes from normal that can synthesize the GlcNAc/36 GalNAc-R linkage without donors and from patients with leukemia. Increase in O-glycan core 2 UDP- the /33-linked GlcNAc. It is possible that this linkage was made GlcNAc:Gal/i3GalNACrt-R (GlcNAc to GalNAc) fi(l-6-A'-acetylglucosami- nyltransferase in leukemic cells. Cancer Res., 51: 1257-1263. 1991. from core 2 (GlcNAc/36 [Gal/tt] GalNAc-R) by the action of ß- 14. Distler, J. J.. and Jourdian, G. W. The purification and properties of ß- galactosidase, because human cells studied in this report and galactosidase from bovine testis. J. Biol. Chem.. 248: 6772-6780, 1973. Pierce, M.. Arango. J.. Tahir. S. H., and Hindsgaul. O. Activity of UDP- previously (13) contained a high activity of a ß-galactosidase 15. GlcNAc:i»-mannoside fi(1.6)A'-acetylglucosaminyltransferase (Gn-T V) in capable of cleaving a 03-linked galactose. cultured cells using a synthetic trisaccharide acceptor. Biochem. Biophys. Normal human colonie mucins contain a high proportion of Res. Commun.. 146: 679-684. 1987. 16. Brockhausen. I.. Williams. D.. Malta, K. L„Orr. J„and Schachter, H. oligosaccharides with the core 3 (GlcNAc/33 GalNAc-R) struc Mucin synthesis. III. UDP-GlcNAc:Galí¿l-3 (GlcNAcjil-6) GalNAc-R ture (41, 42). The lack in CaCo-2 cells of the 03-GlcNAc (GlcNAc to Gal) fí.VA'-acetylglucosaminyltransferase, an enzyme in porcine gastric mucosa involved in the elongation of mucin-type oligosaccharides. transferase activity synthesizing core 3, an activity characteristic Can. J. Biochem. Cell Biol.. 61: 1322-1333. 1983. of normal colonie tissue (6), suggests that this enzyme is turned 17. Brockhausen. I., Orr. J.. and Schachter. H. Mucin synthesis. The action of pig gastric mucosa! UDP-GlcNAc:Galrfl-3 (R,) GalNAc-R2 (GlcNAc to off in colonie adenocarcinoma cells. However, these cells, al Gal) /IVA'-acetylglucosaminyltransferase on high molecular weight sub though capable of O-glycan synthesis, have properties analo strates. Can. J. Biochem. Ceil Biol.. 62: 1081-1090. 1984. gous to those of intestinal columnar cells and may therefore 18. Schachter. H., and Brockhausen, I. The biosynthesis of O-glycans. Symp. not contain the spectrum of enzymes found in colonie goblet Soc. Exp. Biol.. 43: 1-26. 1989. 19. van den Eijnden, D. H.. Koenderman, A. H. L., and Schiphorst. W. E. C. cells that synthesize mucins. M. Biosynthesis of blood group i-active polylactosaminoglycans. Partial purification and properties of an UDP-GlcNAc:A'-acetyllactosaminide /il,3A'-acetylglucosaminyltransferase from Novikoff tumor cell ascites fluid. ACKNOWLEDGMENTS J. Biol. Chem.. 26.5:12461-12471, 1988. 20. Lee, N., Wang. W-C., and Fukuda, M. Granulocytic differentiation of HL- The authors thank Ji-Mao Yang for technical assistance. 60 cells is associated with increase of poly-A'-acetyllactosamine in Asn-linked oligosaccharides attached to human lysosomal membrane glycoproteins. J. Biol. Chem.. 265: 20476-20487. 1990. 21. Mizoguchi. A.. Takasaki. S., Maeda, S., and Kobata. A. Changes in aspara- REFERENCES gine-linked sugar chains of human promyelocytic leukemic cells (HL-60) during monocytoid differentiation and myeloid differentiation. Decrease of 1. Pinto, M., Robine-Leon. S.. Appay. M. D.. Kedinger. M.. Triadou, N., high-molecular-weight oligosaccharides in acidic fraction. J. Biol. Chem., Dussaulx, E.. Lacrois, B.. Simon-Assmann, P.. Haffen, K.. Fogh. J.. and 259: 11949-11957. 1984. Zweibaum. A. Enterocyte-like differentiation and polarization of the human 22. Narasimhan, S. Control of glycoprotein synthesis. UDP-GlcNAc: colon carcinoma cell line CaCo-2 in culture. Biol. Cell. 47: 323-330. 1983. glycopcptide fi4-,Y-acetylglucosaminyltransferase III. an enzyme in hen ovi 2. Youakim, A., and Herscovics. H. Differentiation-associated decrease in the duct which adds GlcNAc in (il-4 linkage to the /i-linked mannose of the proportion of fucosylated polylactosaminoglycans of CaCo-2 human colonie trimannosyl core of A'-glycosyl oligosaccharides. J. Biol. Chem.. 257: 10235- adenocarcinoma cells. Biochem. J., 247: 299-306. 1987. 10242, 1982. 3. Youakim, A., Romero. P. A.. Yee, K.. Carlsson, S. R., Fukuda, M., and 23. Carver, J. P., and Cumming, D. A. Site-directed processing of AMinked Herscovics. A. Decrease in polylactosaminoglycans associated with lysosomal oligosaccharides: the role of three-dimensional structure. Pure Appi. Chem.. membrane glycoproteins during differentiation of CaCo-2 human colonie 59:1465-1476. 1987. adenocarcinoma cells. Cancer Res., 49: 6889-6895. 1989. 24. Schachter. H. Biosynthetic controls that determine the branching and micro- 4. Beyer. T. A.. Sadler. J. E.. Rearick. J. I., Paulson. J. C. and Hill. R. L. heterogeneity of protein-bound oligosaccharides. Biochem. Cell Biol.. 64: Glycosyltransferases and their use in assessing oligosaccharide structure- 163-181. 1986. function relationships. Adv. Enzymol. Relat. Areas Mol. Biol., 52: 23-175, 25. Narasimhan. S.. Freed. J. C.. and Schachter. H. The effect of a bisecting N- 1981. acetylglucosaminyl group on the binding of biantennary. complex oligosac 5. Schachter. H.. Brockhausen. !.. and Hull, E. High-performance liquid chro- charides to concanavalin A Phaseolus vulgaris erythroagglutinin (E-PHA) matography assays for A'-acetylglucosaminyltransferases involved in A'-and and Ricinus communis agglutinin (RCA-120) immobilized on agarose. Car- O-glycan synthesis. Methods Enzymol.. 179: 351-397. 1989. bohydr. Res.. 149: 65-83. 1986. 6. Brockhausen. I., Malta, K. L.. Orr. J., and Schachter, H. Mucin Synthesis. 26. Paquet. M. R.. Narasimhan. S.. Schachter. H., and Moscarello. M. A. Branch UDP-GlcNAc:GalNAc-R jlVA'-acetylglucosaminyltransferase and UDP- specificity of purified rat liver Golgi UDP-galactose:A'-acetylglucosamine ß- GlcNAc:GIcNAcfil-3 GalNAc-R (GlcNAc to GalNAc) ¿6-/V-acetylglucosa- 1.4-galactosyltransferase. J. Biol. Chem., 259:4716-4721, 1984. minyltransferase from pig and rat colon mucosa. Biochemistry. 24: 1866- 27. Brockhausen. !.. Möller.G., Merz. G., Aderniann. K., and Paulsen, H. 1874, 1985. Control of mucin synthesis: the peptide portion of synthetic O-glycopeptide 7. Brockhausen. I., Grey. A. A.. Pang. H.. Schachter. H., and Carver. J. P. N- substrates influences the activity of O-glycan core 1 uridine 5'-diphospho- galactose:A'-acetylgalactosaminidca-R j<3-galactosyltransferase. Biochemis Acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-,V-acetylglucosamine linkage. Purification try. 29: 10206-10212. 1990. and structural determination of oligosaccharides with mannose and A'-ace- 28. Lee, S-O., Connolly. J. M.. Ramirez-Solo. D.. and Porelz, R. D. The tylglucosamine at the non-reducing termini. Glycoconjugate J.. 5: 419-448. polypeptide of immunoglobulin G influences its galactosylation in viro. J. I988a. Biol. Chem., 265: 5833-5839. 1990. 8. Brockhausen, I.. Hull. E.. Hindsgaul. O.. Schachter. H., Shah, R. N., Michn- 29. Taatjes. D. J.. and Roth. J. Alteration in sialyltransferase and sialic acid 3141

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. GLYCOSYLTRANSFERASES IN CaCo-2 CELLS

expression accompanies cell differentiation in rat intestine. Eur. J. Cell Biol.. submaxillary glands of an A'-acetylglucosaminyltransferase which acts on «:289-298, 1988. mucin substrates. J. Biol. Chem., 255: 11247-11252, 1980. 30. Momoi. T., Shinmoto. M., Kasuya, J., Senoo. H.. and Suzuki, Y. Activation 36. Williams. D., Longmore, G., Matta, K. L., and Schachter, H. Mucin synthe of CMP-A'-acetylneuraminic acid:lactosylceramide sialyltransferase during sis. II. Substrate specificity and product identification studies on canine the differentiation of HL-60 cells induced by 12-O-tetradecanoylphorbol-13- submaxillary gland UDP-GlcNAc:Galifl-3GalNAc(GlcNAc-»GalNAc) (36- acetate. J. Biol. Chem., 261: 16270-16273. 1986. /V-acetylglucosaminyltransferase. J. Biol. Chem.. 255: 11253-11261, 1980. 31. Kanani. A., Sutherland. R. D., Fibach. E.. Malta, K. L.. Hindenburg, A.. 37. Wingert. W. E., and Cheng, P-W. Mucin biosynthesis: characterization of rabbit small intestinal UDP-A'-acetylglucosamine:galactose /J-3-A'-acetylglu- Brockhausen, I.. Kuhns. W.. Taub. R. N., van den Eijnden. D. H., and Baker. cosaminide (A'-acetylglucosamine to A'-acetylgalactosamine) /i-o-A'-acetylglu- M. A. Human leukemic myeloblasts and myeloblastoid cells contain the enzyme cytidine 5'-monophosphate-A'-acetylneuraminic acid: Galrfl- cosaminyltransferase. Biochemistry. 23: 690-697, 1984. 3GalNAc,<(2-3)-sialyltransferase. Cancer Res., 50: 5003-5007. 1990. 38. Piller, F.. Piller, V., Fox. R. I., and Fukuda, M. Human T-lymphocyte activation is associated with changes in O-glycan biosynthesis. J. Biol. Chem., 32. Smith. D. F.. Larsen. R. D.. Mattox, S., Lowe. J. B., and Cummings. R. D. Transfer and expression of a murine UDP-Gal:)i-r>-Gal-

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I. Brockhausen, P. A. Romero and A. Herscovics

Cancer Res 1991;51:3136-3142.

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