[CANCER RESEARCH 50, 6787-6792. November 1, 1990]

Identification and Characterization of Two Distinct a-(l-3)-L-Fucosyltransferase

Activities in Human Colon CarcinomaGeorge B. Stroup, Kalyan R. Annullila. Thomas F. Kline, and Madelyn M. Caltabiano1

Departments of Cell Sciences [G. B. S., T. F. K., M. M. C.J and Analytical Chemistry [K. R. A.], SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania19406

ABSTRACT

Two distinct a-(l-3)-fucosyltransferase activities have been ¡dentiCivilin the colon carcinoma cell lines HT-29 and COLO-205. While bothenzymatic activities exhibit similar aCCinitiesfor a synthetic a-(l-3)acceptor and GDP-fucose, they differ with respect to divalent cationrequirements, /V-ethylmaleimide inhibition, and glycoprotein substratespecificity. The COLO-205 a-(l-3) activity exhibits maximal enzymaticactivity in the presence of 20 miviMn2+ but retains less than 10% activityin the absence of divalent cations. In contrast, the optimal Mir'* concen

tration for the HT-29 enzyme is 1 HIM,although this activity is relativelyinsensitive to divalent cation stimulation. In addition, the HT-29 <*-(!-3)-fucosyltransferase activity is resistant to inhibition by 30 HIMN-ethylmaleimide and relatively inactive toward the glycoprotein substratefetuin as compared to its desialylated derivative, asialofetuin. The COLO-205 activity is inhibited approximately 90% by A'-ethylmaleimide and is

equally active with either glycoprotein acceptor.Although the «-(1-3)specific activities are similar in both cell lines,

A'-ethylmaleimide-sensitive «-(1-4)fucosyltransferase activity is 40-foldhigher in COLO-205 as compared to HT-29, suggesting that the COLO-205 fucosyltransferase activity may be an «-(1-3/4) enzyme, while theHT-29 activity appears to be an «-(1-3)specific form.

Further examination of a panel of cell lines, tumor biopsies, andxenografts, based on the effect of metal ions and /V-ethylmaleimide,indicated that both enzyme activities are similarly expressed in humancolon carcinoma tissue.

INTRODUCTION

Although the precise molecular mechanisms involved in thepathogenesis of solid tumors still remain largely undefined, thecell surface has long been suspected of being critically involvedin the neoplastic process (1). Alterations in cell surface antigensinclude deletions or modifications in the expression of normalglycoconjugate structures and the synthesis of new carbohydrateepitopes. These changes, resulting in the aberrant glycosylationof cell surface lipids and proteins, have been strongly implicatedin inappropriate cell-cell and cell-substrate interactions such astissue invasion, evasion of host defenses, and extravasation toand proliferation in secondary organs (2, 3).

Altered expression of the blood group antigens A, B, H,Lewis8, Lewis0, Lewis", and Lewis5 is a constantly recurringtheme in colorectal carcinoma (4-10). Of particular interest arethe Lewis" and Lewis' glycoconjugates which accumulate mark

edly in both preneoplastic and neoplastic tissues and are eitherabsent or minimally expressed in normal adult colon (9-13).The Lewis* structure is further associated with malignant pro

gression since elevated expression of this epitope is correlatedwith more advanced stages of the disease such as villous adenomas and adenocarcinomas as compared to benign tubularadenomas (12, 13).

Received 6/5/90; accepted 7/17/90.The costs oCpublication oCthis article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this Caci.

1To whom requests for reprints should be addressed, at Department of CellSciences, L-109, SmithKline Beecham Pharmaceuticals. P.O. Box 1539, King ofPrussia, PA 19406.

The biosynthesis of any blood group antigen proceedsthrough the sequential action of a number of glycosyltransfer-ases responsible for assembly of the oligosaccharide chains. Inthe case of the Lewis" and Lewis' determinants this pathway

results in terminal fucosylation of type 2 polylactosaminylbackbones. Since aberrant expression of cell surface glycoconjugates is thought to be a reflection of alterations in glycosyl-transferase activities (14-17), any study of blood group antigenexpression and regulation or the role of these oligosaccharidesin the neoplastic process must, by necessity, include an investigation of the enzymes involved in their biosynthesis. We havebegun to examine the terminal glycosyltransferases responsiblefor the production of the Lewis" and Lewis' structures in colon

carcinoma cells and in this study describe the expression andbiochemical characterization of two distinct human colon carcinoma a-(l,3)-fucosyltransferase activities.

MATERIALS AND METHODS

Materials. Colon carcinoma cell lines were purchased from theAmerican Type Culture Collection (Rockville, MD), with the exceptionof HT-29 which was obtained from Dr. I. J. Fidler (M. D. AndersonHospital and Tumor Institute, Houston, TX). Dulbecco's modifiedessential medium, PBS,2 penicillin, streptomycin, and fetal bovine

serum were obtained from GIBCO Laboratories (Grand Island, NY).Triton X-100 was purchased from Boehringer Mannheim (Indianapolis, IN). GDP-[t/-'4C]fucose (270 mCi/mmol) was obtained from Amer-sham Corp. (Arlington Heights, IL) and AG 1-X8 anión exchangeresin, 200-400 mesh, Cl~ form, was obtained from Bio-Rad Laborato

ries (Richmond, CA). Protein assay reagents were obtained from Pierce(Rockford, IL). The «-(1-4) and «-(1-3) specific fucosyltransferaseacceptors, LNF1 and H-type 2-TAPE, respectively, were purchasedfrom BioCarb Chemicals (Lund, Sweden) (18). All additional reagentswere purchased from Sigma Chemical Co. (St. Louis, MO).

Cell Lines, Tumors, and Xenografts. Cell lines were propagated inDulbecco's modified essential medium supplemented with 5% fetal

bovine serum, penicillin (50 units/ml), and streptomycin (50 Mg/ml) ¡na humidified atmosphere of 95% air and 5% CO2 at 37°C.Cells were

grown to confluency in tissue culture trays (245 x 245 x 20 mm),medium was removed, and the cultures were washed with 10 ml ofPBS. Cultures were subsequently harvested in 10 ml of PBS using arubber cell scraper. The cell suspension was centrifuged at 1000 x gfor 10 min and the resulting pellet was stored at -20°Cuntil extraction.

Primary human colon tumors, collected from community hospitalarchives, were snap frozen in liquid nitrogen upon surgical excision andstored at —70°C.Xenografts from nude mice were obtained as describedpreviously, and stored in PBS at -70°C(19).

Enzyme Extracts. All procedures were carried out at 4°C.Samples

were thawed and suspended in 10 volumes of 25 HIMMES (pH 6.8).In the case of human tumors or xenografts from nude mice, the materialwas then minced with surgical scissors. Homogenization was carriedout by four successive 30-s pulses with a Polytron tissue homogenizer(Brinkmann Instruments, Westbury, NY). The homogenate was centri-

2The abbreviations used are: PBS, phosphate-buffered saline (137 mivi NaCl,2.7 HIMKCI, 1.5 mM KH;PO4, 8 mM NaHPO, 7H2O); LNF1, Fucal-2Gal/31-3GlcNAcííl-3Galfíl-4Glc;H-type 2-TAPE, Fuc«l-2Galffl-4GlcNAcfi-O-CH2-CH2-C6H4-NH-CO-CF3; MES, 2-(A'-morPholino)ethanesulfonicacid; NEM,N-ethylmaleimide; CHO, Chinese hamster ovary'.

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,,-(l-3)-L-FUCOSYLTRANSFERASES IN COLON CARCINOMA

fuged at 30,000 x g for 30 min. The resulting pellet was resuspendedin 2-4 ml of 25 m\i MES (pH 6.8), containing 2% Triton X-100 (w/v), 100 mM EDTA, and 1 mivi GMP, and solubilized with 20 strokesin a Dounce homogenizer. The solution was stirred constantly for 12to 18 h and then clarified by centrifugation at 100,000 x g for 30 min.The lipid layer was removed and the supernatant was desalted byexhaustive dialysis against 25 mM MES (pH 6.8) in membrane tubing(molecular weight cutoff, 14,000) Fucosyltransferase activity in thisextract was stable at 4°Cfor at least 3 months. Protein determinations

were routinely performed by using the Pierce bicinchoninic acid method(Pierce. Rockford, IL).

Fucosyltransferase Assays. The standard «-(1-3) fucosyltransferasereaction mixture (70 pi) consisted of 40 ^1 of 25 mM MES buffer (pH6.8) containing 1.25 m\i MnCl2, 0.625% bovine serum albumin (w/v),and 0.25% Triton X-100 (w/v), 10 u\ of the acceptor H-type 2-TAPE(4.7 nmol) in H2O, 10 Mlof enzyme extract, and 10 ^1 of GDP-[I4C]-

fucose (110,000 dpm, 0.185 nmol) in H2O. The reaction was startedwith the addition of GDP-['4C]fucose or enzyme. Following a 30-minincubation at 37°Cfor 30 min the reaction was terminated by adding

20 fil of 0.2 M EDTA in 7.5% sodium tetraborate. The sample wasvortexed and added to an AG 1-X8 column with a bed volume not lessthan 0.5 ml. The column was subsequently washed with 2.9 ml of H:Oand the total eluate (3.0 ml) was added to 17 ml of Ready Solve HPscintillation fluid (Beckman, Fullerton, CA) and counted in a Beckmanscintillation counter (Beckman). Galactose ¡3(1-4)mannose (1.7 ^mol)and LNF1 (35 nmol) were used as substrates for the determination ofa-(l-2) and «-(1-4)fucosyltransferase activities, respectively. Controlassays were run in the absence of acceptor in order to correct forendogenous acceptor activity. One unit of enzyme activity is defined asthe amount of enzyme that will transfer 1 ptno\ of fucose/min underthe conditions of the assay.

Assays with the glycoprotein acceptors fetuin and asialofetuin (0.5mg/assay) were carried out as described above but were without bovineserum albumin and were terminated by the addition of 500 ni 20%perchloric acid and 2% phosphotungstic acid. Precipitated proteinswere collected on 0.45-Mm BA-85 nitrocellulose filters (Schleicher &Schuell, Keene, NH), washed with 2 ml of the acid solution, and countedin a Beckman scintillation counter using 10 ml of Ready Proteinscintillation fluid (Beckman).

RESULTS

Fucosyltransferase Activities in HT-29 and COLO-205 Extracts. As shown in Table 1, greater than 90% of the «-(1-3)-fucosyltransferase activity present in either HT-29 or COLO-205 human colon carcinoma extracts was membrane associated.Following solubilization of the membrane preparation withTriton X-100, 30-40% of this enzyme activity, with a 2- to 3-fold increase in specific activity, was present in the high-speedsupernatant (Table 1). By comparison, «(1-^-fucosyltransferase activity in the same preparations was 45 times lower in HT-29 than in COLO-205 (1.1 /junits/mg and 48.7 Munits/mg,respectively), while «-(l-2)-fucosyltransferase activity was min

imal in both cell extracts (2.1 ^units/mg and 4.7 /uunits/mg,respectively). To ensure that the specific «-(1-3)acceptor, H-type 2-TAPE, was not subject to exoglycosidase degradation,we examined its stability in our enzyme incubations. Whenanalyzed by thin-layer chromatography, the integrity of theacceptor was maintained in the reaction mixture during thecourse of the assay, assuring that only «-(l-3)-fucosyltransfer-ase activity was observed (data not shown). Furthermore, underthe assay conditions, fucosyltransferase activities were linearwith respect to time and enzyme concentration, and were unaffected by 0.2 to 1.0% Triton X-100. Since further isolationand purification of membrane bound fucosyltransferases proveddifficult, this detergent supernatant was used for biochemicalcharacterization of «-(1-3)enzyme activity.

As shown in Fig. 1, divalent cations exhibited differentialeffects on the HT-29 and COLO-205 n-(l-3)-fucosyltransferaseactivities. The optimal concentrations of Mn2+ were 1 and 20mM for the HT-29 and COLO-205 activities, respectively. Inaddition, HT-29 retained 46% of maximal Mn:+ stimulated

enzyme activity in the absence of metal ions, whereas theCOLO-205 dropped to less than 10% of maximum activityunder the same conditions. Mg2+ (10 mM) caused a slightlyhigher activity in the HT-29 preparation compared to Mn2+(optimal concentration, 1 mM). In contrast, 50 mM Mg2+ wasrequired in COLO-205 to obtain an activity equivalent to 20mM Mn:+. In both HT-29 and COLO-205 preparations theeffect of Ca:+ was very similar to that of Mg:+ (data not shown).

Effect of Substrate Concentration on Fucosyltransferase Activities. As shown in Table 2, both the HT-29 and COLO-205 «-(l-3)-fucosyltransferase activities exhibited equal affinity forthe substrate, GDP-fucose (apparent A',,,= 2 to 3 n\\), and there

was no significant difference with respect to inhibition by GDPor GMP (data not shown). However, the COLO-205 «-(1-3)activity exhibited a higher affinity for the accepting substrate,H-type 2-TAPE (apparent A",,,= 62 ¿¿M),as compared to theHT-29 enzyme (apparent A',,, = 132 ^M). The difference inaffinity for H-type 2-TAPE was independent of Mn:+ concen

tration (data not shown).Effect of NEM on Fucosyltransferase Activities. To further

discriminate between the HT-29 and COLO-205 «-(l-3)-fucos-yltransferase activities, we examined the effect of NEM, shownpreviously to inhibit the «-(l-3)-fucosyltransferase activities inhuman milk and the LECH CHO cell mutant (20, 21). Asdemonstrated in Fig. 2, the «-(l-3)-fucosyltransferase activityin COLO-205 was inhibited approximately 90% following a30-min preincubation with 30 mM NEM. In contrast, the HT-29 «-(l-3)-fucosyltransferase activity was essentially insensitiveto the same treatment. The Mn2+ dependence of the residualNEM-resistant activity (10%) present in the COLO-205 extractwas further examined (Fig. 3, top right) and was found to exhibit

Table 1 i>-(l-3)-Fucosyltramferase activity in enzyme preparations from ¡IT-2Vand COLO-205

PreparationHT-29Homogenate30,000

x gsupernatant"100,000x gsupernatant'1COLO-205Homogenate.10,000

x gsupernatant"100.000x g supernatant*Volume

(ml)16.515.03.814.312.03.5Activity(/junits)142211547026671101169Protein(mg)100.338.212.6118.064.621.1Specificactivity

(fiunits/mg)14.23.037.322.61.755.4%ofyield100833100444Enhancement(fold)1.00.22.61.0O.I2.5

" Contains cytosolic proteins.'Contains detergent-solubilized proteins.

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,,-<l-3)-L-Fl'COSYl.TRANSFERASES IN COLON CARCINOMA

120rHT-29

Cation (mM)

120

COLO-2052 t

Cation (mM)Fig. 1. Divalent cation requirement of «-(l-3)-fucosyltransferase activity from

HT-29 and COLO-205. Enzyme activity was measured as described in "Materialsand Methods" at the indicated concentration of Mn2+ (O) or Mg2* (G). Points,

means of triplicate samples: han, SD.

Table 2 Affinity ofCDP-fucose and H-lype2-TAPEfor the a-(l-3)-fucosyltransferases from COLO-205 and HT-29

Concentrations of substrate were varied about the A'mwhile the other substrate

was held constant. Velocity data were fit directly to the Michaelis Mentenequation by an iterative method (32). The method for solving the nonlinearequations is based upon the marquardt algorithm (33). Values are the means ±SE of replicate experiments. Number of experiments are given in parentheses.

CelllineCOLO-205

COLO-205HT-29HT-29[Mn2*](mM)200.1Apparent

A'm(^M)GDP-fucose

H-type2-TAPE2.25

±0.10 (2) 61.5 ±2.3(3)2.01 ±0.09 (2) 44.4 ±3.6(1)2.98 ±0.18 (2) 131.7 ±6.2(3)1.80 ±0.09 (2) 117.3 ±12.2(2)

a Mn2+ profile similar to that observed in HT-29 cells in either

the absence or presence of NEM (Fig. 3, bottom). Specifically,about one-half of the COLO-205 NEM-resistant activity isretained in the absence of divalent cations and the Mn2+ peak

shifts from 20 to 1 HIM(Fig. 3, top right). This is in contrast tothe COLO-205 «-(1-3)activity, in the absence of NEM, whichexhibited a Mn2+ optimum of 20 HIMand has minimal activity

in the absence of divalent cations (Fig. 3, top left). We furtherfound that «-(l-4)-fucosyltransferase activity, using the acceptor LNF1 (21), in both HT-29 and COLO-205 extracts wassensitive to NEM inhibition (data not shown).

Fucosyltransferase Activities with Glycoprotein Acceptors.Additional evidence for differences between the HT-29 andCOLO-205 «-(l-3)-fucosyltransferase activities was obtainedwith the glycoprotein substrates fetuin and asialofetuin. Table

3 demonstrates, first, that the COLO-205 «-(l-3)-fucosyltrans-ferase, compared to HT-29, is much more active with theseglycoprotein acceptors. Second, the HT-29 «(1-3)activity, inthe presence or absence of NEM, fucosylates asialofetuin morereadily than native fetuin. In contrast, the COLO-205 «-(1-3)activity, in the absence of NEM, is equally able to fucosylatefetuin and its desialylated derivative. However, preincubationof the COLO-205 extract with NEM results in nearly completeinhibition of this activity, similar to the result obtained withthe synthetic acceptor H-type 2-TAPE (Fig. 2). In addition, theresidual NEM-resistant «-(1-3)activity in the COLO-205 extract was greater with asialofetuin than with fetuin, similar tothe results obtained with the HT-29 «-(1-3)preparation.

Comparative Analysis of a-(l-3)-FucosyItransferase Activitiesin Human Colon Adenocarcinoma Specimens. Having identifieddistinct biochemical differences between the «-(l-3)-fucosyl-transferase activities in HT-29 and COLO-205 we further investigated the expression of these enzymes in a series of humancolon carcinoma cell lines, xenografts, and surgical biopsyspecimens. The «-(1-3) enzyme activities were prepared and

ECLO

E3EX(O

120

100

80604020

0•r^COLO-205COLO-205 HT-29HT-29-

NEM , Hi M . HIM + NEM

Fig. 2. Inhibition of fucosvltransferase activities by NEM. Extracts wereincubated prior to assay for 30 min at room temperature in the presence orabsence of 30 imi NEM. and were subsequently assayed for <*-(1-3)-fucosyltrans-ferase activity as described in "Materials and Methods." Columns, mean of

triplicate reactions; bars, SD.

EO.U

xCO

1XB100

80

60

40

20

0120

100

80

60

40

20COLO-205

-NEM,

,0HT-29

- NEMTCOLO-205*

NEM|1

20 0 120~T~rHT-29

+ NEMT

20 20

Mn2+(mM)Fig. 3. Effect of NEM on Mn2* activation in COLO-205 and HT-29. Extracts

were incubated prior to assay for 30 min at room temperature in the presence orabsence of 30 mM NEM and were assayed for tv-(I-3)-fucosyltransferase activityas described in "Materials and Methods" at the indicated Mn2+ concentration.COLO-205 plus NEM describes the effect of Mn2* on the residual enzyme activity

(column 2 of Fig. 2). Columns, means of triplicate samples: bars. SD.

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a-(l-3)-L-FUCOSYLTRANSFERASES IN COLON CARCINOMA

Table 3 Effect of NEM on fucosylation of glycoproteins by COLO-205 and HT-29 enzyme preparations

Enzyme extracts were incubated for 30 min prior to assay in the presence or absence of 30 min NEM. Specific activity values are from the means of triplicatesamples.

COLO-205AcceptorFetuinAsialofetuinFetuinAsialofetuinSpecific

activityNEM(/iunits/mg)43.0341.21+

0.50+1.67Fetuin/asialofetuin

ratio1.040.30HT-29Specificactivity

(nunits/mg)0.543.840.232.73Fetuin/asialofetuinratio0.140.09

characterized with respect to NEM inhibition and Mn2+ acti

vation. As shown in Table 4, most samples exhibited predominantly one form of the a-(l-3)-fucosyltransferase activity, similar to either HT-29 or COLO-205. In general, enzyme extractswhich showed greater than 40% inhibition by NEM were maximally activated by 20 miviMn2+, consistent with the propertiesobserved with the COLO-205 «-(1-3)activity. Likewise, mostsamples with less than 30% inhibition by NEM were maximallyactivated by 1 mM Mn2+ and therefore resembled the HT-29

enzyme activity.

DISCUSSION

Many alterations in blood group antigen expression havebeen documented in colorectal carcinoma. In particular, bloodgroup structures such as the Lewis* and Lewis* determinants

are consistently expressed at elevated levels in colon cancer aswell as other malignancies (1, 22). In an attempt to begin tounderstand the association between antigen expression andneoplastic development we have examined, in human coloncarcinoma cells, the biochemical characteristics of «-(1-3)-fucosyltransferase activity, one of a series of glycosyltransferaseessentially involved in the formation of the Lewis" and Lewis*

structures.

Table 4 Comparative NEM inhibition and A/n2* activation of'a-(l-S)-

fucosyltransferases from human colon carcinoma cells, tumors, and xenograftsNEM inhibition was assessed by preincubation of enzyme extracts for 30 min

in the presence of 30 mM NEM. Mn2* activation was determined by altering theMn2* concentration as indicated. Values represent the means of duplicate exper

iments except where indicated. Triplicate samples were run within each experiment.

SampleCell

lineCOLO-205XLS174T"LS174TSW-403COLO-201*SW-948SW-1116SW-480SW-1417"COLO-320HSRCACO-2DLD-1xHT-29°SW-620SKCO-1HT-29Tumors'SKF-045SKF-028SKF-047SKF-050SKF-048%

ofNEM inhibition878181614328282120191615119988567654421Mn2+

activation (% of maximumcpm)-Mn2*887112149404048494256544861484201012401

mMMn2*2825252650100100100100100100100100100100100193630327220mMMn2*100too1001001005570649680707557686660100100100too100fl

Xenograft.* Single experiment.

The results of the present study have shown that at least twoa-(l-3)-fucosyItransferase activities are present in human coloncarcinoma cells. Using the HT-29 and COLO-205 colon carcinoma cell lines as sources of enzyme activity, several lines ofevidence suggest that the a-(l-3)-fucosyltransferase activitieswithin each of these cell lines are distinct with respect to severalbiochemical characteristics. First, the «-(l-3)-fucosyltransfer-ase activities in HT-29 and COLO-205 differ with respect todivalent cations requirements. COLO-205 is activated maximally by a higher concentration of Mn2+ than HT-29 (20 mM

versus 1 mM, respectively) and retains less than 10% of maximalactivity in the absence of Mn2+, compared to about 50% activityfor HT-29. In HT-29, Ca2+ or Mg2+ (10 mM) causes a 25%higher activation of enzyme activity compared with Mn2*. This

property was not observed in the COLO-205 enzyme preparation. The differential effects of Mn2+ on the a-(l-3) activities

therefore provide a useful operational method for distinguishingthe two enzyme activities.

Second, although both enzyme preparations displayed similaraffinity for the substrates GDP-fucose and H-type 2-TAPE,they differed with respect to glycoprotein substrate specificity,a criterion used previously to discriminate two distinct c*-(l-3)-fucosyltransferase enzymes in CHO cell glycosylation mutants(20). Whereas the HT-29 a-(l-3)-fucosyltransferase activitywas relatively inactive toward fetuin compared to asialofetuin,the COLO-205 extract was equally active with either substrate.The differences in glycoprotein fucosylation between the HT-29 and COLO-205 enzyme preparations is presumably due onlyto a-(l-3)-fucosyltransferase activity, since both enzyme extracts exhibit minimal a-(l-2) activity and both fetuin andasialofetuin possess few a-(l-4)-fucosyltransferase sites [lessthan 3% of terminal residues have Gal/31-3GlcNac structures(23)]. These data suggest that sialic acid residues on nativefetuin carbohydrates block the action of the HT-29 enzyme butdo not interfere with COLO-205 activity.

Finally, experiments with NEM clearly resolved the activitiesin COLO-205 and HT-29 preparations by showing a NEM-sensitive «-(l-3)-fucosyltransferase in COLO-205 (90% inhibition) and an NEM-resistant form in HT-29 (10% inhibition).

Since the a-(l-3)-fucosyltransferase activity in COLO-205cells was not completely inhibited by NEM, this characteristicwas used to determine if one or more a-(l-3)-fucosyltransferasewas present in this cell line. In fact the residual NEM-resistanta-(l-3)-fucosyltransferase activity in COLO-205 enzyme extracts exhibited a cation profile similar to that of HT-29 withmaximum activity at 1 mM Mn2+ and greater than 40% activity

in the absence of a divalent cation. This COLO-205 NEM-resistant activity also resembled that of HT-29 with respect toglycoprotein substrate specificity, exhibiting higher activitywith asialofetuin than with fetuin. Together, these data suggestthat COLO-205 possesses at least two distinct «-(l-3)-fucos-

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t>-(l-3)-L-FUCOSYLTRANSFERASES IN COLON CARCINOMA

yltransferase activities, one of which is similar to the activityobserved in HT-29 cells.

Both HT-29 and COLO-205 also express an «-(1-4)-fucos-yltransferase activity, as determined by using the specific acceptor LNF1. The amount of «-(1-4) activity in COLO-205 wassimilar to that of a-(l-3) and was at least 40-fold greater thanin HT-29, although both activities were inhibited by more than50% with NEM. This observation, in addition to the presentlydescribed biochemical differences, suggests that the HT-29 andCOLO-205 enzyme activities are the products of two distinctgenes. It is probable that the predominant COLO-205 activityhas, as has been observed for several other fucosyltransferases(21, 24), an «-(1-3/4) specificity, while the HT-29 enzymeappears to be a specific a-(l-3) form. Definitive proof willrequire characterization of enzymes purified to homogeneity.

«-(l-3)-Fucosyltransferase activities similar to those presentin COLO-205 and HT-29 have been previously reported inseveral mammalian sources, including COLO-205 cells (25),human milk (21), ovarian carcinoma (26), CHO mutants (20,27). Previous studies with COLO-205 demonstrated a membrane-bound enzyme with high a-( 1-3)-fucosyltransferase activity toward both siah lateti and desialylated glycolipid acceptors(25), consistent with our findings. Further competitive substrateinhibition studies with COLO-205 revealed that this enzymewas similar to a soluble «-(l-3/4)-fucosyltransferase identifiedin human milk (21 ). The present work confirms and strengthensthis comparison by demonstrating the NEM sensitivity of «-(l-3)-fucosyltransferase from COLO-205 and showing its profile of activity with divalent cations. Specifically, both theCOLO-205 and milk enzyme activities require divalent cations,are stimulated optimally by Mn2+, as compared to Mg2+ orCa2+, and are inhibited by NEM (21, 24). The similarity of

these two fucosyltransferases is further supported by the demonstration of NEM-sensitive «-(l-4)-fucosyltransferase activityof equal amounts to «-(1-3)activity in COLO-205.

Several other previously reported «-(l-3)-fucosyltransferasesalso appear to share characteristics with the COLO-205 «-(1-3) activity, suggesting that all of these enzymes may be membersof the same a-( 1-3/4) isozyme class. In particular, sensitivityto NEM was shown to be a characteristic not only of an «-(1-3)-fucosyltransferase in human ovarian carcinoma but also the

LECH CHO cell mutant (20, 27). The latter enzyme, a membrane-bound form, shares many characteristics with the «-(1-3) activity in COLO-205, such as optimal activation with 20mM Mn2+, less than 20% activity in the absence of a metal ion,

and the ability to fucosylate fetuin and the desialylated derivative with equal activity. However, the LECH and COLO-205enzymes differ by an order of magnitude with respect to affinityfor GDP-fucose (apparent Km= 20 and 2 /¿M,respectively). Anadditional cv-(l-3)-fucosyltransferase from human amnioticfluid has also been shown to fucosylate sialylated and desialylated acceptors (glycolipids as well as glycoproteins) equallywell (28), results similar to those of our study with COLO-205.

In contrast to the COLO-205-like enzymes, a second mutantfrom CHO cells, LEC12, has been shown to have an «-(1-3)-fucosyltransferase activity that is insensitive to NEM, andwhich has a much lower activity with native glycoproteins,including fetuin, than with their desialylated derivatives (20).These findings are identical to the present investigations withHT-29 «-(l-3)-fucosyltransferase activity. In addition, neitherthe LEC12 nor HT-29 enzymes exhibit an absolute requirementfor metal ion, demonstrating further the similarities betweenthese two enzymatic activities. Other sources, including mouse

embryonal carcinoma cells and human lung carcinoma cells,have also identified «-(l-3)-fucosyltransferases that are unaffected by NEM (29, 30). In addition, the «-(1-3)activity frommouse embryonal carcinoma cells, like that in HT-29 cells, wasunable to fucosylate sialylated glycoproteins (29). Together,this group of enzymes may represent a second class of specifica-( 1-3)-fucosyltransferase isozymes.

Because of the occurrence of two distinct «-(l-3)-fucosyl-transferase activities within two different colon carcinoma celllines, namely HT-29 and COLO-205, both shown previouslyto be highly tumorigenic in nude mice (31), we examined anadditional panel of colon carcinoma cell lines, tumor biopsies,and xenografts. In general, both of these activities were equallyrepresented within the total population of samples examined.Specimens which showed less than 30% inhibition in the presence of NEM also had maximum activities at 1 mM Mn2+ andwere therefore similar to the HT-29 «-(1-3) activity. The remaining cell lines were highly sensitive to NEM and wereactivated maximally by 20 mM Mn2+, similar to the activityfound in the COLO-205 preparation. There was no apparentcorrelation between expression of either enzymatic activity inthe established cell lines and previously reported tumorigenic-ity, metastatic potential, or production of tumor-associatedmarkers (31).

Although the majority of human biopsy specimens reflecteda COLO-205-like activity, at least one sample behaved like HT-29 in that it retained activity in the absence of metal ions andwas only marginally inhibited by NEM treatment. The enzymatic activities from human xenografts also did not differ fromthe cell lines from which they were derived with respect toNEM sensitivity or optimal Mn2+ concentration. Together,

these data demonstrate that within human colon carcinomaneither the COLO-205- nor the HT-29-like «-(l-3)-fucosyl-transferase activity predominates. In fact, the two forms of theenzyme appear to be capable of being coexpressed within thesame biological sample, as demonstrated by COLO-205.

The results of the present investigation provide strong, although indirect, evidence for the existence of two distinct «-(1-3)-fucosyltransferase enzymes in human colon carcinoma cells.Confirmation of these conclusions will require isolation ofsufficient quantities of enzyme for unequivocal structural determination, although further investigation into the regulation ofaltered carbohydrate expression, specifically the Lewis" andLewis' epitopes, or the role of these antigens in the malignancy

of colon carcinoma, will undoubtedly require consideration ofboth enzymatic activities.

ACKNOWLEDGMENTS

We would like to thank Drs. Robert Clark, Walter DeWolf, FredDrake, and Juan Luengo for their thoughtful suggestions and excellentassistance, and Drs. Anthony M inassale and Herbert Spellman ofMetropolitan Hospital, Parkview Division, Philadelphia, PA, and theirstaff, for their help in tissue acquisition.

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1990;50:6787-6792. Cancer Res   George B. Stroup, Kalyan R. Anumula, Thomas F. Kline, et al.   3)-l-Fucosyltransferase Activities in Human Colon Carcinoma

−-(1αIdentification and Characterization of Two Distinct

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