the of biological chemistry val. no. 3, 25, pp. w printed in … · 2001-06-06 · 0-linked core 2...

THE JOURNAL OF BIOLOGICAL CHEMISTRY W 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Val. 266, No. 3, Issue of January 25, pp. 1772-1782,1991

Printed in U. S. A .

Increased UDP-GlcNAc:Gal@1-3GalNAc-R (GlcNAc to GalNAc) ,8-1,6- N-Acetylglucosaminyltransferase Activity in Metastatic Murine Tumor Cell Lines CONTROL OF POLYLACTOSAMINE SYNTHESIS*

(Received for publication, February 20, 1990)

Shida YousefiS, Elizabeth HigginsSP, Zhuang DaolingS, Annette Pollex-KriigerS, Ole Hindsgaulv, and James W. DennisSPII From the $Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, M5G 1x5, the §Department of Medical Genetics, University of Toronto, Ontario, and the TDepartment of Chemistry, Faculty of Science, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada

Malignant transformation of rodent cell lines by polyoma virus and by activated ras genes is associated with increased UDP-G1cNAc:Mana-R 8- 1,6-N-acetylglucosaminyltransferase V (GlcNAc-transferase V) activity and its product -GlcNAcB~Mana1-6Man/31- branched Asn-linked oligosaccharides. In this report, we have compared p1-6GlcNAc branching of core 0- and N-linked oligosaccharides in three experimental models of malignancy, namely (a) rat2 fibroblasts and their malignant T24H-ras-transfected counterpart; ( b ) benign SP1 mammary carcinoma cells and two metastatic sublines of SPl; and ( c ) the metastatic MDAY-D2 lymphoma cell line and its poorly metastatic glycosylation mutant KBL- 1. In addition to the previously reported increase in GlcNAc-transferase V activity, UDP-GlcNAc:GalB1-3GalNAca-R (GlcNAc to GalNAc) 8- 1,6-N-acetylglucosaminyltransferase (core 2 GlcNAc-transferase, EC 2.4.1.102) activity was found to be elevated by 70% in the malignant rat2 and SP1 cell lines while several other glycosyltransferase activities were not significantly different.

The action of core 2 GlcNAc-transferase followed by 01-4Gal-transferase provides an N-acetyllactosamine antenna that can be extended with polylactosamine (i.e. repeating GalBl-4GlcNAcPl-3) provided UDP- G1cNAc:GalD-R 81-3GlcNAc-transferase (GlcNAc- transferase (i)) activity is present. Polylactosamine content in microsomal membrane glycoproteins was quantitated by labeling the GlcNAc termini resulting from the action of Escherichia freundii endo-0-galactosidase with bovine galacto~yltransferase/UDP-[~H] Gal. Glycopeptidase F- sensitive and -insensitive frac- tions were measured to assess the N- and 0-linked components. In the SP1 tumor model, the metastatic sublines showed increased core 2 GlcNAc-transferase and GlcNAc-transferase V activities but no change in GlcNAc-transferase (i) activity, yet polylactosamine was increased in both 0- and N-linked oligosaccharides. In rat2 cells, down-regulation of GlcNAc-transferase (i) following transformation was associated with decreased polylactosamine even though core 2 GlcNAc-

* This work was supported by research grants from the National Cancer Institute of Canada (to J. W. D.) and from the Medical Research Council (to 0. H.) and by Grant MA6499 (to Carbohydrate Research Center). The costs of publication of this article were de- frayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

( 1 A research scholar of the National Cancer Institute of Canada.

transferase and GlcNAc-transferase V were elevated in the cells. Finally, a %fold decrease in GlcNAc-transferase V in KBL- 1, the glycosylation mutant of MDAY- D2 cells, resulted in complete loss of polylactosamine in N-linked but no change in 0-linked polylactosamine content. These results suggest that, provided GlcNAc- transferase (i) is not limiting, the B1-6-branching enzymes core 2 GlcNAc-transferase and GlcNAc-transferase V regulate the levels of polylactosamine in 0- and N-linked oligosaccharides, respectively. In addition, malignant transformation in the rat2 and SP1 tumor models is associated with increased 81-6GlcNAc branching of 0-linked as well as N-linked oligosaccharides.

Malignant transformation of both murine and human cells is commonly associated with expression of larger N-linked oligosaccharides (1-3). The increased size of N-linked carbo- hydrates in polyoma and Rous sarcoma virus-transformed BHK cells has been attributed to increased -GlcNAcpl- 6Manal-6Manp- branching of complex-type oligosaccharides as well as increased polylactosamine content (4-6). Consistent with these observations, UDP-G1cNAc:Manal-6Man-R p l - 6GlcNAc-transferase (GlcNAc-TV),’ the enzyme which ini- tiates the 61-6-linked antenna, was shown to be elevated in Polyoma virus-transformed BHK cells while GlcNAc-transferases I, 11, 111, and IV activities remained unchanged (7). Increased branching has also been observed in human breast carcinomas (8) and has been correlated with invasiveness by human uroepithelial cell lines (9). Transformation of rat:! fibroblasts with either T24H-rm, v-K-ras, or with the tyrosine kinase oncogene v-fps induces both increased GlcNAc-TV activity and invasive and metastatic potential (10). Finally, loss of GlcNAc-TV activity in the class 3 glycosylation mutants (i.e. KBL-1) of the highly metastatic tumor cell line MDAY-D2 was associated with loss of metastatic potential in mice (11).

GlcNAc-T(i), the enzyme required for polylactosamine synthesis, has been partially purified from Novikoff tumor cells and shown to preferentially substitute the Galpl-4GlcNAcpl- 2(Gal@l-4GlcNAc@l-6)Man portion of N-linked oligosaccharides (12). Therefore, the observation that increased GlcNAc-

The abbreviations used are: T, transferase; SA, sialic acid MES, 4-morpholineethanesulfonic acid; HPLC, high performance liquid chromatography; HEPES, 4-(2-hydroxyethyl)-l-piperazineethane- sulfonic acid; pNp, para-nitrophenyl.

1772

0-Linked Core 2 and Polylactosamine Synthesis in Cancer Cells 1773

T V in transformed fibroblasts is associated with increased polylactosamine content (6) suggests that, if GlcNAc-T(i) is not limiting, GlcNAc-TV may control the addition of polylactosamine. A similar situation may exist in the biosynthetic pathway for 0-linked oligosaccharides. The disaccharide core structure of 0-linked oligosaccharides is known to be a substrate for the branching enzyme core 2 GlcNAc-T which catalyzes the conversion of core 1 (i.e. Gal(31-3GalNAca) to core 2 ( ie. Gal~l-3(GlcNAc~l-6)GalNAca) (13, 14). The action of core 2 GlcNAc-T followed by 01-4Gal-T provides a lactosamine substrate for further extension by GlcNAc-T(i). Therefore, @1-6GlcNAc branching of core 1 may also control the polylactosamine content in 0-linked oligosaccharides. Al- though truncated structures such as T and Tn antigens (ie. GalB1-3GalNAc and GalNAc) are commonly associated with "mucin-type" glycoproteins in human carcinomas (15), transformation-related changes in the biosynthesis of the larger branched 0-linked oligosaccharides have not been examined.

To address these questions, we have compared glycosyltransferase levels and N- and 0-linked polylactosamine content in immortalized rat2 fibroblasts and their T24H-ras- transformed counterpart (IO), and also in the spontaneous mammary carcinoma cell line SP1, and two metastatic sublines. SP1 tumor cells were previously shown to be tumorigenic in syngeneic CBA mice but nonmetastatic from a subcutaneous site of injection. SPlT24H-ras and A3a cells are metastatic sublines of SP1 generated either by transfection with activated T24 H-ras or by treating the cells with 12-0- tetradecanoylphorbol-13-acetate and Ca2+ ionophore, respectively (16, 17). Our results show that the activities of both the 0- and N-linked 01-6 branching enzymes (i.e. core 2 GlcNAc- T and GlcNAc-TV, respectively) are elevated in the T24H- ras-transformed rat2 cells and in metastatic SP1 mammary carcinoma cells compared to their nonmalignant counter- parts. We have also quantitated the polylactosamine content in N- and 0-linked oligosaccharides using a sensitive assay employing E. freundii endo-P-galactosidase and labeling of the resulting GlcNAc termini with UDP-[3H]Gal/bovine milk galactosyltransferase (18). The results suggest that the activities of core 2 GlcNAc-T and GlcNAc-TV, as well as the polylactosamine extension enzyme GlcNAc-T(i), can inde- pendently regulate polylactosamine content in 0- and N- linked oligosaccharides.

MATERIALS AND METHODS

Chemicals and Transferase Substrates

UDP-6-[:'H]N-acetylglucosamine (26.8 Ci/mmol, Du Pont-New England Nuclear), and UDP-6-[3H]galactose (20 Ci/mmol, Amer- sham, UK) were diluted with the respective unlabeled sugar-nucleo- tides purchased from Sigma. Galpl-3GalNAca-pNp was purchased from Toronto Research Chemicals (Toronto); GalNAca-phenyl from Genzyme. GlcNAc~l-2Manal-6Man~-O(CH2),COOCH3and Manal- 3(Manal-6)Manpl-O(CH,),COOCH3 were made as previously described (19).

The asialo-biantennary glycopeptide from pig fibrinogen (Sigma) was purified from a Pronase digest and used as a substrate for GlcNAc-T(i) (20). The protein was digested four times with 1% (w/ w) Pronase in 0.1 M Tris-HC1, pH 7.9, at 37 "C for 24 h, then size- fractionated on a G-25 Sepharose column developed in H,O. The G- 25-excluded material was separated on a DEAE-Sephacryl column run in 2 mM Tris-HC1, pH 7.0, with a NaCl gradient, 0-200 mM over 1 h. The fraction corresponding to sialylated glycopeptide was subject to mild acid hydrolysis in 2 M acetic acid at 100 "C for 30 min. The sample was desalted on a G-25 column and the neutral glycopeptide fraction was obtained as the void peak on a DEAE-Sephacryl column run. The material was analyzed by 500 mHz 'H NMR spectroscopy and found to have equal representation of unsubstituted nonreducing Gal termini. The spectra confirmed that the structure was Gal- terminating biantennary glycopeptide with a l -6 fucosylation in the

core (53). The phenolsulfuric acid assay for hexose sugars was used to determine the molar concentration of glycopeptide.

Cell Lines rat2 fibroblast cells and rat2 cells transfected with pSV2neoT24H-

ras have previously been characterized for expression of T24H-ras and for malignant growth in nude mice (10). SP1 is a nonmetastatic mammary adenocarcinoma which arose spontaneously in a CBA/J mouse (21). SPlT24H-rasl is a metastatic subline derived by transfection with pSV2neoT24H-ras (16). A3a is a metastatic subline of SP1 derived by treating SP1 with 12-O-tetradecanoylphorbol-13- acetate in uitro, subcutaneous injection of the cells into mice, and recovery of cells from metastatic nodules in the lung (17). The origin and analysis of oligosaccharides in the highly metastatic DBA/2 strain lymphoid tumor called MDAY-D2 have been previously described (22, 23). D36W25 and KBLl cell lines were selected as spontaneous lectin-resistant mutants of MDAY-D2 and both showed loss of metastatic potential (11). The class 1 mutant D36W25 is deficient in transport of UDP-galactose into the Golgi apparatus (23), while KBL1, a class 3 mutant, showed a 4-&fold decrease in pl- 6GlcNAc-transferase V using GlcNAc2Man3GlcNAc2-Asn as substrate (11).

Glycosyltransferase Assays Cells were washed in phosphate-buffered saline and lysed in 0.9%

NaC1, 1% Triton X-100 at 0 "C for all assays except core 2 GlcNAc- T where cells were lysed in 0.4%, Triton X-100, 0.9% NaC1. The lysates were adjusted to 8-12 mg/ml protein with lysis buffer and used as the source of transferases. The reactions contained 25 pl of cell lysate, 0.5 pCi of 3H-sugar-nucleotide donor (approximately 4400 cpm/nmol) in a total volume of 50 pl and were incubated for 1 or 2 h a t 37 "C followed by processing as indicated for each substrate. For all transfer assays, endogenous activity was measured in the absence of acceptor and subtracted from values determined in the presence of added acceptor.

Core 2 GlcNAc-T-The reaction contained 50 mM MES, pH 7.0, 1 mM UDP-['HH]GlcNAc, 0.1 M GlcNAc, 1 mM Galpl-3GalNAca-pNp as substrate. The reaction was diluted to 5 ml in H20, applied to a C18 Sep-Pak (Waters) in H20, washed with 20 ml of H,O, and the product was eluted with 5 ml of methanol.

pl-ZGlcNAc-TI-The reaction contained 50 mM MES, pH 7.0, 1 mM UDP-['HIGlcNAc, 0.1 M GlcNAc, 25 mM MnCI,, and 1 mM of Manal-3(Manal-6)Manpl-O(CH,)&OOCH3 as substrate. Product was isolated by C18 Sep-Pak and radioactivity measured.

/31-6GlcNAc-TV-The reactions contained 50 mM MES, pH 7.0,l mM UDP-('H)GlcNAc, 0.1 M GlcNAc, 10 mM NaeEDTA, 1 mM of G1cNAc~1-2Mancu1-6Manp1-0(CH2)&00CH3. The product was isolated, separated by C18 Sep-Pak, and radioactivity was measured.

pl-3GlcNAc-Tfi)"The reactions contained 50 mM MES, pH 7.0,l mM UDP-[3H]GlcNAc, 0.1 M GlcNAc, 25 mM MnC12, and 1 mM pig fibrinogen glycopeptide as acceptor (20). The reactions were passed over a Dowex AG-1x8 (Bio-Rad) column and the excluded material was concentrated, spotted onto paper, and washed in 80% ethanol by descending paper chromatography for 48 h. The product was eluted from the origin and radioactivity was measured. N-Acetyllactosamine at 20 mM was also used as substrate and the reactions were passed over Dowex AG-1x8 followed by separation of the product on a Glyco-Pak N HPLC column (Waters) developed in 82% acetonitrile run at 1 ml/min. When Galp1-3(GlcNAcpl-6)GalNAccu-pNp and Gal~l-3(Gal~l-4GlcNAc~l-6)GalNAca-pNp were used as substrates, the products were separated by C18 Sep-Pak.

pl-3Gal-T-The reactions contained 50 mM MES, pH 7.0, 1 mM UDP-[3H]Gal, 25 mM MnC12, and 1 mM GIcNAca-phenyl. Product was isolated by C18 Sep-Pak.

SI-4Gal-T-The reactions contained 50 mM MES, pH 7.0, 1 mM UDP-[3H]Gal, 25 mM MnCl,, and 20 mM GlcNAc as substrate. The reactions were passed over Dowex AG-1x8 in H20 and excluded radioactivity was counted.

Product Identification by HPLC and ' H NMR The products of core 2 GlcNAc-T, GlcNAc-TV, GlcNAc-T(i), and

Dl-3Gal-T were separated on Glyco-Pak N HPLC columns run iso- cratically in 82% acetonitrile. Their mobility was compared to standard compounds as indicated in the figure legends. A core 2 standard was prepared using an MDAY-D2 tumor cell lysate as the source of enzyme (i.e. approximately 10 nmol/mg/h) and Galpl-3GalNAca- pNp as substrate. The reaction was increased 30-fold (i.e. 1.5-ml final

1774 0-Linked Core 2 and Polylactosamine Synthesis in Cancer Cells

volume) over that used for analytic assays. The conversion of substrate into product was 20% during the 4-h incubation at 37 "C. The 300 nmol or approximately 2 mg of product was isolated by C18 Sep- Pak and Glyco-Pak N HPLC. The purity and identity of the core 2 product ( i e . Galpl-3(GlcNAcpl-6)GlcNAccu-pNp) and the tetrasaccharide Gal~l-3(Gal/31-4GlcNAc~l-6)GalNAccu-pNp were determined by 'H NMR. The tetrasaccharide was produced by incubating the core 2 trisaccharide with 2 mM UDP-Gal and 2 units of bovine 01-4Gal-T overnight.

The sample was dissolved in 99% DzO and lyophilized three times in order to sufficiently exchange the hydroxyl groups. The final D20- dried compound was transferred to a Wilmad 520-1 micro NMR tube in 100% Dz0. A trace of acetone as an internal standard (chemical shift set at 2.225 ppm of relative sodium 2,2-dimethyl-2-silapentane- sulfonic acid) was used. NMR experiments were carried out at 300 K on a Bruker AM 500 spectrometer as previously described (24). The Aspect 3000 computer was used for ID processing, and pVAX I1 data stations and Hare's software were used for processing the two-dimensional NMR data. Both two-dimensional Homonuclear-Hartmann- Hahn (HOHAHA) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) experiments used "reverse" mode con- figuration. In this case the decoupler channel is used as the transmit- ter and provides the exciting pulses. For the HOHAHA experiment, the MLEV-17 composite pulse sequence was used to obtain an efficient spinlock pulse. A 90" pulse of 27 ps generates a 9.2-kHz spinlock field. A mixing time of 85 ms was used to ensure magnetization coherence transfer throughout most of the scalar spin-subsystems of the sugars. In the ROESY experiment, a 10"-60"-140" composite pulse was used for the exciting pulse. The 90" pulse length was 70 ps which produced a relatively weak spinlock field of 3.6 kHz. The transmitting frequency was selected on the up-field edge of the spectrum outside of the region of interest in order to suppress HO- HAHA cross-peaks and false peaks. To accomplish this, the sweep width of the ROESY spectrum was increased to 7000 Hz. A mixing time of 400 ms was used. Both HOHAHA and ROESY spectra were recorded in the phase-sensitive mode with quadrature detection in F1 which was obtained with time-proportional phase incrementation. The number of scans per tl experiment was 32. Data sets consisted of 512 t l increments and every free induction decay contained 2048 data points. Before Fourier transformation, time domain data was zero filled to 2048 points in t l and the final matrix was 2K X 2K complex data points in both dimensions. Window functions of skewed phase-shifted sine bell (50, 0.7) in both dimensions were performed prior to processing. No data manipulations were applied to the data after Fourier transformation. The spectra were presented as contour plots in which HOHAHA peaks appeared as positive and ROESY as negative.

Microsomal Membranes Tumor cells were disrupted by nitrogen cavitation. Approximately

3 X 10' washed cells were suspended in 4 ml of 20 mM HEPES, pH 7.4, 0.5 mM MgC12, 0.13 M NaCI, and stirred while 5 ml of 0.5 M sucrose was added slowly. After adding 100 pl of Trasylol (Sigma) and 50 p1 of 200 mM phenylmethylsulfonyl fluoride, the cells were placed in a nitrogen cavitation bomb (Artisan metal products) and equilibrated with 600 p s i . of N2 for 20 min at 4 'C with constant stirring. Cell disruption occurred after dropwise release of the suspen- sion from the bomb. The lysate was made 1 mM with Na2EDTA, centrifuged at 1000 X g for 10 min, and the resulting supernatant was centrifuged at 20,000 X g for 20 min. Microsomal membrane vesicles in the supernatant were then pelleted at 120,000 X g for 1 h, resus- pended in 1 mM HEPES, pH 7.5, and stored at -20 "c. Protein concentrations were determined using the BCA reagent supplied by Pierce Chemical Co.

Quantitation of Endo-B-galactosidase-sensitive Polylactosamine Microsomal membranes (2 mg) in 1 mM HEPES, pH 7.0, were

heated to 65 "C for 15 min to inactivate 04-galactosidases. The samples were diluted to adjust the protein to 3.0 mg/ml with 0.15 M NaC1,lO mM HEPES, pH 7.0,0.5% Triton X-100, The samples were then galactosylated with bovine galactosyltransferase and unlabeled UDP-Gal to substitute terminal GlcNAc residues, particularly 0- linked GlcNAc residues (25). The reaction buffer A contained 100 mM galactose, 100 mM HEPES, pH 7.3, 0.15 M NaCI, 50 mM MnC12, 1.2 mM AMP, 1 mM UDP-Gal, 0.5% Triton X-100, 300 milliunits/ml of bovine milk Gal-T (Sigma), and 1.0 mg/ml of microsomal membrane protein. After 2 h at 37 "C, the samples were exhaustively

dialyzed into 0.15 M NaCI, 50 mM MnCl,, 25 mM sodium acetate, pH 5.0. The Gal-T and UDP-Gal concentrations as well as the time of incubation were optimized to saturate available GlcNAc residues in D36W25 tumor cells. This was assessed by subsequent galactosylat,ion with UDP-[3H]Gal/Gal-T. The samples were either incubated alone or with 5 milliunits of endo-P-galactosidase per 100 pg of protein for 16 h at 50 "C. The samples (25 pg of protein) were then diluted 1:l with buffer A but with the omission of nonradioactive UDP-Gal and the inclusion of 2 pCi of UDP-[3H]Gal (20 Ci/mmole, Amersham Corp.). After 2 h, the reaction was terminated by the addition of ice- cold 10% trichloroacetic acid and the pellet was washed four times with 10% trichloroacetic acid and counted in a liquid scintillation counter, For sodium dodecyl sulfate-polyacrylamide gel electrophoresis samples were precipitated and washed in ice-cold acetone.

Alternatively, the 0- and N-linked oligosaccharides were released separately. Following the [3H]galactose addition, the samples (25-30 pg of protein) were heated to 65 "C for 15 min, made 0.4% in sodium deoxycholate, and incubated either alone or with 1.5 units of glycopeptidase Flavobacterium meningosepticum (Boehringer) for 16 h at 37 "C. The samples were then trichloroacetic acid-precipitated, washed in 10% trichloroacetic acid and counted. For release of the labeled 0-linked oligosaccharides, the [3H]galactosylated samples were acetone-precipitated once, then reconstituted in either 0.05 M KOH either with or without NaBHl (7.5 mg/200 pl). Following an overnight incubation at 45 "C the samples were trichloroacetic acid- precipitated, washed in 10% trichloroacetic acid, and counted.

RESULTS

Glycosyltransferase Activity in rat2 Fibroblasts and rat2- T24H-ras Tumor Cells-The glycosyltransferase activities assayed in this study are listed in Fig. 1 along with a schematic of the residues added by each enzyme. The GlcNAc-TI and GlcNAc-TV are specific for N-linked oligosaccharide biosynthesis; p1-3Gal-T and core 2 GlcNAc-T for 0-linked; P1-4Gal- T and GlcNAc-T(i) appear to be utilized in the synthesis of both classes of oligosaccharide (26). In Table I, the activities of these enzymes are compared in rat2 and rat2-T24H-ras cells. The transferases were each assayed under saturating conditions for both acceptor and sugar-nucleotide. GlcNAc- TI, pl-4Ga1, and Dl-3Gal-T activities were the same in both cell lines. However, both pl-6 branching enzymes core 2 GlcNAc-T and GlcNAc-TV were significantly elevated in the transformed fibroblasts. The elevated core 2 GlcNAc-T activity in rat2-TZ4H-r~~ cells was associated with an increase in V,,, but the acceptor K, was unchanged (ie. 0.125 mM) (Fig. 2). Product formation was linear with increasing membrane lysate to 500 pg of protein per reaction (Fig. 2).

Product yield in the core 2 GlcNAc-T assay was not decreased by the addition of 10 mM Na2EDTA (data not shown),

Galpl-3 GalNAcol-Ser/Thr 0-linked

OlCNACpl-3 GAlPl-4 GlCNACP1-6/0

En7)mc Abbreviation

A) U U P - G ~ : ~ - G : I I N A ~ pl-3 palac,oryltranslerase

R) I J I X ~ G : I I - ~ . G I ~ N A ~ PI-4 g;Il,ciusyllransferasc

(') ~ : ~ p . ( j l ~ l i ~ ~ : ~ ~ l p ~ - . i G a l N A ~ 01.6 N-acetylglucosaminyltransferase Core 2 CilcNAc-T

v) UDP-CI~NA~:P-G~I 01-3 Nafefylgluco~aminyltransferase 01-3 GlcNAc Xi)

L) UDP.CICNAC:~ .M:~ pi-? N-acetylglucosaminyllransfe~~se I

I ) UDI'-GkNAc:o-M;tn p I-h N.acetylglucosaminyltransferase v

pl-3 Gal-T

01-4 Gal-?

GleNAc-TI

FlcNAc-TV

FIG. 1. Glycosyltransferases assayed in this study, their ab- breviated names, and a schematic of the residues added by each enzyme.


TABLE I Summary ofgbcosyltramferase activities in malignant and nonmalignant paired cell lines

Glycosyltransferase activities in rat2 and T24H-ras-transformed rat2 cell lines. The transferase reactions were prepared, and the products isolated as described under “Materials and Methods.” The specific activities were calculated from data generated at two protein concentrations per experiment and the average f S.D. is based on three experiments.

Enzymes/substrates Rata ratZ-T24-H-ras transformed cells Change in

nmoleslmglh %

/31-3Gal-T/

/31-4Gal-T/

Core 2 GlcNAc-T/

/31-3GlcNAc-T(i)/

GalNAccu-pNp 8.6 f 0.6 10.0 k 3.0 +16

GlcNAc 10.2 f 0.8 11.4 f 1.1 +12

Galj3l-3GalNAccu-pNp 0.43 f 0.06 0.73 f 0.05 + 70“

Asialoglycopeptide from fibrinogen 0.77 k 0.02 0.51 k 0.08 -34 Galj31-4GlcNAc 0.35 ? 0.03 0.11 f 0.01 -69”

Manal-G(Msncul-3)Man/3- 21.8 ? 1.6 19.0 f 0.4 -13 GlcNAc-TI/

O(CHdsCO&H, GlcNAc-TV/ GlcNAcj31-2Manorl-GManj3- 0.035 f 0.006 0.112 f 0.006 +320”

O(CH&COL% Denotes a significant difference compared to rat2 cells p < 0.05 using Student’s t test.

N-

P x

z 0

- 8 - 5 0 5 10

IUS (mM)

FIG. 2. Core 2 GlcNAc-T activity in rat2 fibroblasts (0). and T24H-rw-transfected rat2 cells (O), as a function of cell lysate concentration ( A ) ; and as a function of acceptor concentration (Le. Ga&31-3GalNAcc~-pNp) graphed as Line- weaver-Burk plot ( B ) . After a 2-h incubation at 37 “C, the product was isolated by C18 Sep-Pak and radioactivity measured in a j3- counter.

which is characteristic of the pl-6GlcNAc-transferases’ lack of divalent cation requirement. Core 2 GlcNAc-T product migrated as a single peak on a Glyco-Pak N HPLC column developed in 82% acetonitrile (Fig. 3). The elution times of the products by rat2 and rat2-T24-H-ras cells were identical to that of a standard of Gal~l-3(GlcNAc~l-6)GalNAc~~-pNp. The standard was prepared by a large scale enzyme reaction using MDAY-D2 tumor cell lysate as the source of core 2 GlcNAc-T activity. The product was isolated on C18 Sep-Pak and separated from substrate by HPLC and analyzed by ’H

S GlcNAc01-6’ GalOl-3GalNAc-pNp

::k” S Galpl-BGalNAc-pNp

120

90

60

30 ‘\

4 12 20 28 36 44

Time (min)

FIG. 3. HPLC analysis of glycosyltransferase products from reactions with rat2 cell (dotted line) and T24H-rw-transfected rat2 cells (solid line). The reaction products of core 2 GlcNAc-T ( A ) , GlcNAc-TV ( B ) , and @1-3Gal-T ( C ) were first separated on C18 Sep-Paks, then applied to a Glyco-Pak N HPLC column run in 82% acetonitrile under isocratic conditions. The elution time for each substrate ( S ) was detected by monitoring absorbance of the column elution at 200 nm.

NMR (Table 11). The proton assignments summarized in Table I1 for the substrate and product were based on analysis of homonuclear shift correlation spectroscopy (COSY) and HOHAHA two-dimensional spectra. The chemical shifts for the H1 and the H4 protons of the P1-3Gal as well as those of H1 and H6 of the Pl-GGlcNAc differ substantially from those previously reported for the corresponding di- and trisaccha-


TABLE I1 ' H NMR chemical shifts of structural-reporter groups for core I , core 2, and fll-4Gal-substituted core 2

Residue Reporter group Core 1" Core 2 P1-4Gal-substituted

core 2

6 ( p p d b JIHz) GalNAca-pNp H1 5.828 (3.6) 5.798 (3.7)

H2 H3 H4 H5 H6 H6' NAc

H1 4.550 (7.8) 4.542 (7.8) 4.543 (7.8) H2 H3

3.560 (9.9) 3.555 (10.0) 3.659 (3 .3 )

3.556 (9.9)

H4 3.656 (3.4)

3.934 (-0.8) 3.650 (3.4)

3.932 (-0.7) H5 NA'

3.92d (-0.6)

H6 3.691

NA 3.6gd

3.798 (7.9) H6' NA 3.752 (4.5, 11.8) 3.77d

3.80d

5.809 (3.6) 4.582 (10.5) 4.565 (10.3) 4.569 (10.5) 4.297 (3.1) 4.294 (3.1) 4.285 (3.1) 4.317 (-0.8) 4.304 (-0.8) 4.306 (-0.6) 4.024 (7.7) 3.794 (7.7)

4.183 (7.8) 4.000 (3.9)

3.744 (4.6, 12.0) 3.754 (7.8, 11.0) 2.015 2.006 2.007

4.179 4.016 (4.0)

3.754 (7.9, 11.2)

GalR1-3

GlcNAcBl-6

GalBl-4

H1 H2 H3 H4 H5 H6 H6' NAc

H1 H2 H3 H4 H5 H6 H6'

4.456 (7.8) 3.555 (10.3) 3.437 (9.0) 3.284 (9.7) 3.376 (9.9) 3.849 (2.1)

1.904 3.603 (6.2, 12.2)

4.474 (8.3) 3.61d 3.51d 3.67d 3.50d 3.93d 3.64d 1.901

4.386 (7.8) 3.535 (10.9) 3.660 (3.2) 3.916 (-0.6) 3.71d 3.77d 3.74d

Core 1 = Galpl-3GalNAccu-pNp; core 2 = Galp1-3(GlcNAc~1-6)GalNAca-pNp; /31-4Gal-substituted core 2 =

' Chemical shifts are in m m relative to an internal acetone standard at 2.225 ppm at 300 K acquired at 500 Gal/31-3(Gal~l-4GlcNAcpl-6)GalNAca-pNp.

_ _ mHz.

' NA, not assigned. Uncertainty in estimating shifts in the envelope.

ride alditols (Table 11) (27). This is not unexpected as the proximity of the pNp group would be expected to influence these chemical shifts. The substitution of core 1 with 01- 6GlcNAc results in relatively large changes in the H5 and H6 chemical shifts of GalNAc (ie. 0.15 and 0.22, respectively) as was previously reported for the alditols (27). The coupling constant for the H1 of GlcNAc, Jl,2 = 7.75 is similar to that previously reported for the core 2 alditol and is characteristic of the p linkage. ROESY experiments were performed on the core 2 product which had been further substituted by bovine pl-4Gal-T (i.e. Gal~1-3(Gal~1-4GlcNAc~l-6)GalNaca-pNp) (Fig. 4). Nuclear Overhauser enhancements were observed between the H1 proton of GlcNAc and the H5, H6a, and H6b of GalNAc. The nuclear Overhauser enhancement between H1 and H2 of GlcNAc was similar in magnitude to the nuclear Overhauser enhancement between H1 and the H6b of GalNAc, which is consistent with the interatomic distance of GlcNAc linked 1-6 to GalNAc. Therefore, substitution of GalPl-3GalNAca-pNp under the conditions used in the core 2 GlcNAc-T assay occurred exclusively at the 6 position of GalNAc.

The products of Dl-3Gal-T and GlcNAc-TV reactions were separated on a Glyco-Pak N HPLC column. In both instances, the product appeared to be a single major peak with elution times as expected for the respective products (Fig. 3). The amount of p1-3Gal-T product was similar for both rat2 and rat2-T24H-ras cells, whereas GlcNAc-TV product was ap-

proximately three times greater for the transformed rat2 cells consistent with the quantitation of product eluted directly from the C18 Sep-Paks (Table I).

GlcNAc-T(i) activity was decreased in rat2-T24H-ras compared to rat2 cells when either asialo-fibrinogen glycopeptide or N-acetyllactosamine was used as substrate (Table I). The addition of NA,EDTA completely inhibited the reaction with either substrate consistent with the known Mn2+ requirement of this enzyme (28) (Fig. 5). The product using N-acetyllactosamine as substrate, migrated on the Glyco-Pak N HPLC column as a single trisaccharide peak (Fig. 5).

Pig gastric mucosa has previously been shown to have a Dl- 3GlcNAc-T activity that utilizes Galfll-3GalNAca-pNp (as well as core 2) to yield GlcNAcpl-3Gal/31-3GalNAca-pNp (29). However, this enzyme appears to be absent in rat2 and SP1 cell lines since Gal~1-3(GlcNAc~l-6)GalNAca-pNp did not serve as an acceptor of GlcNAc (data not shown). In addition, Galpl-3GalNAca has previously been shown to be a poor substrate for human serum GlcNAc-T(i) (28). How- ever, Gal~l-3(Gal~l-4GlcNAc~l-6)GalNAc-pNp was a substrate for GlcNAc-T(i) and, at 0.5 mM, yielded 0.1 nmol/mg/ h of product using rat2 fibroblasts as the enzyme source.

Glycosyltransferase Activities in Spl Carcinoma Cells and Two Metastatic Sublines-Rat2 is an immortalized but non- tumorigenic cell line and, when transfected with T24H-ras, the cells become both tumorigenic and metastatic (10). In contrast, the SP1 mammary carcinoma cell line is tumorigenic


B

4 . 6 0 1.30 4 . 4 0 4.30 4 20 4 . 1 0 4 . 0 0 3 90 3 . 0 0 3.70 3.60 3.50 3.60 PPY

FIG. 4. 5OO-mHz NMR spectra of the tetrasaccharide Gal,91-3fGalj31-4GlcNAc~1-6)GalNAcax-pNp. A, Homonuclear- Hartmann-Hahn spectra; B, ROESY spectra; and C , one-dimensional ‘H NMR spectra. Different sugar units are indicated by superscripf primes and the abbreviations used are: G@3, BS-linked galactose; GP4, (%linked galactose; Gn@G, @6-linked N-acetylglucosamine. The dashed line moving up the diagonal in A traces the through-bond connections for the protons of GalNAc. Nuclear Overhauser enhancement signals between H1 of GlcNAc and H6a and H6b of GalNAc are marked in 8. Horizontal dashed lines in the lower part of A show the corresponding spin systems for G@4”’, GnPG”, and G@3’.

but nonmetastatic in mice and therefore serves as a model of tumor progression (11). In Table 111 the glycosyltransferase activities in SP1 were compared with those in two metastatic sublines. Of the six enzyme activities examined, only GlcNAc- TV and core 2 GlcNAc-T activities were altered. As with rat2 cells, the level of core 2 GlcNAc-T was elevated by 70%. The metastatic sublines of SP1 showed no change in GlcNAc-T(i) unlike the rat2 transfectant which showed a reduction in this enzyme activity. Specific activities of the various transferases differed in the rat2 and SP1 cell lines as might be expected for cells of different lineage and from different species. For example, the SP1 cell lines had higher core 2 GlcNAc-T but lower GlcNAc-T(i) compared to rat2 cell lines (Tables I and 111).

The use of GalNAca-phenyl as a substrate in a GlcNAc-T assay can yield the core 3 disaccharide (ie. GlcNAcO1- 3GalNAc) which is found in some mucin-producing tissues (30). However, GalNAca-phenyl was not an acceptor of GlcNAc (data not shown), suggesting that this is not a signif-

I GlcNAc Galfl-QGIcNAc

Time (min)

FIG. 5. HPLC separation of GlcNAc-T(i) product using N- acetyllactosamine as substrate. GlcNAc-T(i) reactions were prepared with rat2 cell lysates, either without (solid line) or with 10 mM Na2EDTA present (dotted line). After 2 h at 37 “ C , the reactions were passed over Dowex AG-1x8 Pasteur pipet columns followed by separation on a Glyco-Pak N HPLC column in 82% acetonitrile.

icant pathway in either the rat2 fibroblasts or SP1 cell lines. Core 3 has the potential to be extended with @1-4Gal, producing an N-acetyllactosamine sequence which could be extended further with polylactosamine (26). However, since GlcNAc was not added to GalNAca-phenyl, and both rat2 and SP1 cell lines show high levels of /31-3Gal-T activity, this would be expected to lead primarily to core 1 synthesis. Therefore, further extensions of the oligosaccharide with polylactosamine-based sequences would appear to be dependent upon expression of core 2 GlcNAc-T activity in these cells.

Quantitation of Polylactosamine in Microsomal Mem- branes-Increased core 2 GlcNAc-T and GlcNAc-TV activity in transformed or metastatic cells as observed in the rat2 and SPI cell lines may be associated with increased levels of polylactosamine expression in both 0- and N-linked oligosaccharides. To examine this possibility, a method for measuring polylactosamine content in the microsomal membrane of cells was developed based upon labeling the GlcNAc termini produced by the action of endo-@-galactosidase with bovine milk Gal-T/UDP-[”]Gal (Fig. 6). This is an adaptation of a method previously used to measure polylactosamine on the surface of erythrocytes (18). E. freundii endo-@-galactosidase cleaves polylactosamine sequences including those substituted with SA or a1-2/3/4fucose (31). Although it cannot be excluded that some endo-p-galactosidase-resistant polylactosamine sequences may be present in the cells, the material labeled by this procedure is referred to as polylactosamine. The microsomal membranes were solubilized in 0.5% Triton X-100 and pretreated with unlabeled UDP-Gal and Gal-T to saturate the 0-linked and other terminal GlcNAc residues (25). After exhaustive dialysis to remove UDP-Gal, the samples were digested with endo-@-galactosidase then treated with UDP-[”HIGal and Gal-T. The protein was precipitated and the radioactivity measured in a @-counter. Acetone and trichloroacetic acid precipitated the same amount of radioactivity, suggesting that glycolipids had not been labeled. To distinguish N- and 0-linked polylactosamine, the portion of radioactivity that became soluble in ice-cold 10% trichloroacetic acid after glycopeptidase F treatment was determined.

To test the method and optimize the conditions for quantitation of polylactosamine, MDAY-D2 and D36W25 cell lines were used. The D36W25 cell line is a glycosylation mutant of MDAY-D2 which is deficient in UDP-Gal transport into the Golgi and consequently it produces complex-type oligosaccharides terminating in GlcNAc with no polylactosamine. The wild-type MDAY-D2 cell line has previously been shown to have polylactosamine in complex-type N-linked oligosaccha-


TABLE IIr Summary of glycosyltransferase activities in malignant and nonmalignant paired cell lines

Glycosyltransferase activities in SPl tumor under cells and metastatic sublines, SPlT24H-ras and A3a. The reactions were prepared and the products isolated as described under “Materials and Methods.” The specific activities were calculated from data generated at two protein concentrations per experiment and the average f S.D. is based on three experiments. The 76 change is an average of the two metastatic lines.

Enzymes/substrates SP1 SPlTP4-H-ras A3a Change in metastatic cells

nmoles/mg/h % p1-3Gal-T/

p1-4Gal-T/

Core 2 GlcNAc-T/

pl-3GlcNAc-T(i)/

GalNAca-pNp 11.9 f 0.25 11.7 f 2.7 10.4 f 2.5 -7

GlcNAc 46.1 f 1.0 51.2 f 2.2 49.7 k 0.6 +9

Gal@l-3GalNAcotl-pNp 3.2 f 0.47 5.0 f 0.02 5.9 f 0.71 +70“

asialoglycopeptide from fibrinogen 0.27 k 0.07 0.28 f 0.05 0.26 f 0.03 0

Mancvl-G(Manal-3)Manp- 6.7 f 0.41 5.6 f 0.23 6.0 f 1.05 -14 GkNAc-TI/

O(CHz)aCOKHa GlcNAc-TV/

GlcNAcpl-2Manal-6ManP- <0.01 0.091 0.120 + 1050” O(CHz)aCOKH3

Denotes a significant difference compared to SP1 cells p < 0.05 using Student’s t test.

8~-GaLpL-4Glc&bc~l-3Gal . . . O / a Gal-T + 1 ODP-Gal

B~-Galpl-4GlclUcpl-3Gal . . . O / A

GlclUca-0

Galpl-4GlclUca-0

I I dialyze. endo p- galactosidase

GlclUcp1-3Cal. o/A

Gal-T + ODP-[%]Gal

[%I]Galpl-4GlclUcpl-3Gal. . . O / A 1 TCA or [%I]~a~-glycoprotein

acetone

/ I \ p-c-t. SDS-PAGE. glycopeptidase I

FIG. 6. Scheme for the quantitation of polylactosamine in microsomal membranes prepared from tumor cells as described under “Materials and Methods.” GlcNAccv . . . 0 designates terminal GlcNAc residues which, in the first step of the procedure, are substituted with unlabeled Gal. Although, the unsubstituted GlcNAcs are shown as 0-linked, terminal GlcNAc residues associated with both 0- and N-linked oligosaccharides are galactosylated. The

then labeled with Gal-T and UDP-[3H]Gal. GlcNAc termini produced by the action of endo-p-galactosidase are

rides by ‘H NMR and fast atom bombardment spectroscopy (22). In Fig. 7, detergent-solubilized membranes were treated with or without cold galactosylation and endo-&galactosidase and then subjected to 3H-galactosylation. Prior galactosylation of D36W25 samples with unlabeled UDP-Gal precluded labeling with UDP-[3H]Gal/Gal-T as expected. Endo-P-galactosidase did not enhance labeling in either case, also as expected. The amount of unsubstituted GlcNAc in D36W25 membranes was determined to be 62.4 & 5.8 pmol/mg (Fig. 7 A ) . Galactosylation of MDAY-D2 membrane lysates with unlabeled UDP-Gal also saturated the existing unsubstituted GlcNAc residues. In MDAY-D2 membranes, the polylactosamine content could be detected, and was quantitatively similar either with or without prior cold galactosylation (Fig. 7B). However, in subsequent experiments, membrane preparations were routinely galactosylated with unlabeled UDP-

Gal, prior to quantitation of polylactosamine. The addition of [3H]Gal to endo-j3-galactosidase-sensitive polylactosamine sequences was determined over a range of membrane concentrations to assure that the UDP-[3H]Gal and Gal-T were not limiting and that cold UDP-Gal had been completely removed by the dialysis step. The net amount of [3H]Gal added to polylactosamine in MDAY-D2 microsomal membranes was 20.7 k 1.3 pmol/mg while in D36W25 cells there was essen- tially none (Fig. 8).

Galactosylated samples were also separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the labeled glycoproteins were visualized by autoradiography. MDAY-D2 membranes appeared to have two major glycoproteins species with endo-P-galactosidase-sensitive polylactosamine sequences approximately 125 kDa and 90 kDa in size (Fig. 9). Based on earlier studies, where the major leukoagglutinin-reactive glycoproteins in MDAY-D2 cells were purified and characterized (32), they are likely to be P2A (structurally similar to leukosialin/CD43) (33), and lysosomal-associated membrane glycoprotein (LAMP- l), respectively. Murine LAMP-I has 20 potential N-linked glycosylation sites, many of which have leukoagglutinin-reactive N-linked oligosaccharides with polylactosamine sequences (34, 35). The P2A glycoprotein in MDAY-D2 cells has a large number of 0-linked oligosaccharides (32). However, the polylactosamine content of this glycoprotein has not been determined.

Polylactosamine in N- and 0-linked Oligosaccharides of MDAY-D2, rat2, SPl, and Their Sublines-The KBL-1 glycosylation mutant of MDAY-D2 was selected for leukoagglutinin resistance and subsequently shown to be deficient in GlcNAc-TV activity as well as metastatic potential (11). Approximately equal proportions of the labeled polylactosamine in MDAY-D2 membranes was released by glycopeptidase F and by alkaline borohydride (Table IV). The total polylactosamine content in the KBL-1 glycosylation mutant was approximately half that in MDAY-D2 cells. The labeled polylactosamine in KBL-1 cells was insensitive to glycopeptidase F but sensitive to alkaline borohydride reduction suggesting that it was exclusively associated with 0-linked oligosaccharides (Table IV). Therefore, KBL-1 cells have an unaltered content of 0-linked polylactosamine while the N- linked sequences were lost. In KBL-1 cells, the GlcNAc-Tfi)


A) D36W25

0

endoa ” - - 0

- + . + - 0

+ + n

0 0

6 0 n 0

1’5 3il sb Protein bg)

FIG. 7. Galactosylation of unsubstituted GlcNAc in the membranes prepared from D36W25 mutant cells (A) and MDAY-D2 wild type cells ( B ) . Cold Gal refers to pretreatment of the membrane glycoproteins with unlabeled UDP-Gal and Gal-T, endo@ refers to samples treated with endo-@-galactosidase prior to labeling with UDP-[“HIGal and Gal-T.

Protein (re)

FIG. 8. The net incorporation of [‘HIGal into endo+galactosidase sensitive polylactosamine sequences in MDAY-D2 and mutant D36W25 cells. The data was calculated from the experiment shown in Fig. 6 by subtracting the -endo-@-galactosidase from the +endo-P-galactosidase curves.

GT + - - + - + + - + Reel + + - - + + - - + endo@ + + + + - - - ”

130 K. 75 K

50 K.

39 K

27 K,

MDAY-D2 D38w25

MDAY-D2 and D36W25 were treated as indicated in the FIG. 9. Microsomal membrane p ro t e ins (25 pg) f r o m

legend and separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis to visualize the glycoproteins labeled by UDP-[3H]Gal and Gal-T. The gel was stained with Coomassie Blue and embedded with Enhance (NEN), dried, and exposed to X-ray film for 2 weeks. The lanes are designated endo@ for treatment of the samples with endo-@-galactosidase, pre Gal for pretreatment with unlabeled UDP-Gal and Gal-T, and GT designates Gal-T in the final labeling step with UDP-[’HIGal.

TABLE IV Endo-P-galactosidase-sensitive polylactosarnine

Microsomal membrane glycoproteins were treated as outlined in Fig. 5 and as described under “Materials and Methods” for quantitation of endo-@-galactosidase-sensitive polylactosamine. The N- linked polylactosamine is the portion of 3H-galactosylated oligosaccharide that was sensitive to glycosidase F, the 0-linked is the glycopeptidase F-insensitive portion, and the numbers in parentheses are the fraction of glycopeptidase F-insensitive material that could be released by alkalfne-borohydride treatment.

Cell lines Total N-linked 0-linked

pmoleslw MDAY-D2 20.7 f 1.3 10.2 10.5 (94%) D36W25 0.0 0.0 0.0 (NDY KBLl 9.6 f 1.4 CO.1 9.6 (67%) rat2 24.5 & 3.6 <0.1 24.3 (ND) rat2-T24H-ras 8.0 f 0.8 co.1 8.0 (ND) SP1 0.33 f 0.3 CO.1 -0.3 (73%) SPl-TP4H-rasl 3.04 & 0.1 0.44 2.6 (87%) A3a 2.77 f 0.6 0.23 2.5 (ND)

., ND, not done.

TABLE V Glycosyltransferase activities in MDAY-D2 and the class 3

mutant KBL-1 Transferase reactions were prepared and processed as described

under “Materials and Methods.” The results are a representative experiment and the mean of duplicate determinations.

Enzvme/substrate MDAY-D2 KBL-1 nmoleslmglh

Core 2 GlcNAc-T/

@1-3GlcNAc-T(i)/ Gal@1-3GalNAccu-pNp 7.5 12.5

asialoglycopeptide 0.88 1.14

GlcNAcj31-2Mancul-GMan& 0.158 0.047 GlcNAc-TV/

O(CHqbCOXH2 ~~

and core 2 GlcNAc-T activities were slightly higher than in MDAY-D2 cells while GlcNAc-TV was decreased 3-fold using the synthetic substrate for GlcNAc-TV (Table V). Previous studies using GlcNAc2Man3GlcNAc2-Asn as a substrate for GlcNAc-TV showed 3-4-fold higher specific activities in these cell lines and a 5-fold loss of activity in the mutant (11). Taken together, the present results suggest that loss of GlcNAc-TV activity in KBL-1 cells precludes the addition of polylactosamine in N-linked oligosaccharides.

Although both core 2 GlcNAc-T and GlcNAc-TV are ele-


vated in rat2-T24H-ras cells, GlcNAc-T(i) activity was re- duced approximately %fold compared to the rat2 cell line. The consequences of these changes appear to be a net decrease (Le. 3-fold) in polylactosamine in the transformed cells (Table IV). In both cell lines the polylactosamine was glycopeptidase F-insensitive and therefore appeared to be located in 0-linked oligosaccharides. Although the Pl-6 branching enzymes were both increased following transformation in rat2 cells, the decrease in GlcNAc-T(i) activity appeared to be the dominant factor resulting in a decrease in polylactosamine content.

The tumorigenic but nonmetastatic SP1 carcinoma cells had negligible polylactosamine (Fig. lo), but the two metastatic sublines had significant amounts of polylactosamine, approximately 85% of which was located in 0-linked carbo- hydrates and 15% in N-linked (Table IV). The metastatic sublines showed no change in GlcNAc-T(i) activity suggesting that the increase in polylactosamine was due to increased PI- 6 branching of both N- and 0-linked oligosaccharides. This is the reverse of the MDAY-DI/KBL-l system where loss of GlcNAc-TV in the mutant cells with no change in GlcNAc- T(i) resulted in the loss of polylactosamine exclusively in N- linked oligosaccharides.

In Fig. 11, the @l-GGlcNAc-branched N- and 0-linked oligosaccharides are shown as preferential substrates for polylactosamine addition. Our results suggest that, if GlcNAc- T(i) activity is not limiting, transformation- or progression- related increases in Pl-6 branching of both core 0- and N - linked oligosaccharides can enhance polylactosamine content and presumably terminal blood group and embryonic antigens which are commonly associated with type 2 chains.

DISCUSSION

In this study, we have compared the activities of several glycosyltransferases in three models of malignancy to determine whether increased P1-6GlcNAc branching occurs in the core of 0-linked as well as N-linked oligosaccharides following transformation. rat2 fibroblasts are not tumorigenic when injected into rats or athymic nude mice but when transformed with activated ras (i.e.v-K-ras or T24H-ras), the cells acquire both tumorigenic and metastatic properties (10, 11). In contrast, SP1 cells are transformed and produce tumors when injected into mice, however, the tumor cells do not metastasize (11). Transfection of SP1 cells with activated T24H-ras ( e g ,

30 1 ,,i /

0

/'

I

O Ib ;o &I 4b i o Protein (pg)

FIG. 10. Galactosylation of endo-8-galactosidase-treated microsomal membranes prepared from SPl, 0; A3a, 0; and SPlT24H-ras, H, cell lines. The membrane preparations were pretreated with unlabeled UDP-Gal/Gal-T, followed by endo-P-galactosidase and UDP-['HH]Gal/Gal-T as described under "Materials and Methods."

FIG. 11. Complex-type N- and 0-linked oligosaccharide

both pathways. The second panel is GlcNAc branching which influ- biosynthesis scheme. The top punel shows common early steps in

punels. The pl-B-linked branched oligosaccharides, both N- and 0- ences the type of extensions and termini as shown in the lower two

linked are preferred substrates for extension with polylactosamine which are subject to further substitutions to produce Lewis and blood group antigens. The example shown is sialylated poly Le'. The Pl-6- linked GlcNAcs are shown as stippled squares and the branching reactions as dotted lines, these pathways being more utilized in malignant or metastatic cells. The swainsonine (SW)-induced diver- sion of the N-linked pathway is shown as a dashed line. Note swainsonine is a potent inhibitor of metastasis (51, 52).

SP1T24H-rasl), or selection of metastatic sublines using drugs coupled with in vivo passage of the tumor cells to isolate metastatic nodules (eg. A3a), is accompanied by increased activity of GlcNAc-TV and core 2 GlcNAc-T. These results suggest that increased branching of both 0- and N-linked oligosaccharides is associated with the acquisition o f invasive and metastatic potential rather than simply transformation. The increased level of core 2 GlcNAc-T was not associated with a change in acceptor K , but an increase in V,,,. This suggests that core 2 GlcNAc-T enzyme levels may be up- regulated following transformation or progression, but confir- mation of this will require cDNA or antibody probes to measure mRNA or enzyme levels. Consistent with our enzyme data, the 0-linked oligosaccharides of human chorionic gonadotropin in normal pregnancy urine were found to be mostly core 1, while human chorionic gonadotropin from choriocarcinoma patient urine was enriched in core 2-based oligosaccharides (36).

Core 2 GlcNAc-T also appears to be developmentally regulated in human lymphoid cells. The enzyme activity is low in resting peripheral T-cells which produce sialylated core 1 structures, whereas T-cells that have been stimulated to pro-

()-Linked Core 2 and Polylactosamine Synthesis in Cancer Cells 1781

liferate via the T-cell receptor with an anti-CD3 antibody show elevated core 2 GlcNAc-T activity and expression of the core 2 structures (37). However, concanavalin A-stimulated T-cells did not show elevated core 2 GlcNAc-T activity, suggesting that induction of the enzyme is not a simple consequence of T-cell proliferation. A concomitant decrease in a2-6SA-T activity was also observed in activated T-lym- phocytes. It is possible that the proportion of core 1 and 2 may also be affected by competition between a2-6SA-T and core 2 GlcNAc-T for a common substrate. This would be expected to occur only if the enzymes were present in the same Golgi stacks.

The 0-linked oligosaccharides found in most nonmucin- producing cells appear to be based on the core 1 and core 2 structures (26). In cells producing core 1-based structures, the oligosaccharides are generally completed by terminal sialyla- tion to produce SAa2-3Ga181-3(SAa2-6)GalNAc (Fig. 10). In cells with core 2 GlcNAc-T activity producing core 2-based structures, the sialylated structure commonly found is SAa2- 3Gal~l-3(SAa2-3Gal(31-4GlcNAc~l-6)GalNAc. However, the action of core 2 GlcNAc-T provides a lactosamine substrate for GlcNAc-T(i) and therefore the opportunity to extend 0- linked oligosaccharides with polylactosamine and terminal antigen sequences. Consequently, initiation of the Dl-6 branch by core 2 GlcNAc-T appears to be an important regulatory step in the synthesis of larger 0-linked structures containing polylactosamine and associated antigenic sequences.

The synthesis of polylactosamine requires GlcNAc-T(i), an enzyme which is also subject to oncodevelopmental regulation (38). In rat2-T24H-ras cells both 0- and N-linked (31-6 branching transferases were increased, however a decrease in GlcNAc-T(i) activity was associated with a loss of polylactosamine. In contrast to rat2 cells, transformation in the human colon appears to be associated with increased GlcNAc-T(i) activity, while it is absent in normal adult colon. Increased GlcNAc-T(i) activity in colon tumors appears to be a con- troling factor in expression of Lewis antigens (38). Polylac- tosamine content in oligosaccharides of mouse uteri has been shown to be subject to hormonal (estrogen and progesterone) regulation (39). Based on our observations in this study, hormonal regulation may be at the level of (31-6 branching of the core, extension, or both.

Several lines of evidence from earlier studies suggest that polylactosamine is preferentially added to the Dl-6-branched complex-type N-linked oligosaccharides. First, the loss of GlcNAc-TV activity and decreased Dl-6 branching in the BW5147-PHAR2.1 lymphoma mutant was associated with a loss of polylactosamine sequences in N-linked structures (40). Conversely, increase Dl-6 branching of N-linked oligosaccharides in Rous sarcoma-transformed BHK cells has been associated with increased polylactosamine content (6). Similar observations were made in the present study; that is the coordinate loss of GlcNAc-TV activity and N-linked polylactosamine in the ciass 3 glycosylation mutant of MDAY-D2 and the increase in both parameters in metastatic SP1 cell lines. In both instances GlcNAc-T(i) activity was not altered. In vitro, the preferred substrate for partially purified GlcNAc- T(i) has been shown to be the Dl-6-linked lactosamine substrate on the Manal-6 side of the trimannosyl core (12). Our results are consistent with the idea that most of the polylactosamine is added to a single antenna per N-linked oligosaccharide in MDAY-D2 cells. Only the N-linked oligosaccharides in D36W25 mutant cells are subject to galactosylation by Gal-T since the 0-linked are simply GalNAc-' and glyco-

' S. Yousefi, E. Higgins, Z. Daoling, A. Pollex-Kriiger, D. Hinds- gaul, and J. W. Dennis, unpublished observation.

lipids are largely glycosylceramide (41). Therefore, the number of GlcNAc termini labeled on D36W25 membranes provides an estimate of total antenna which was three to four times that labeled on MDAY-D2 cells following endo-P-galactosidase treatment. Therefore, based on previous studies showing that the majority of the complex-type N-linked oligosaccharide in MDAY-D2 cells containedpolylactosamine (231, this is consistent with a single polylactosamine chain per tetra-antennary oligosaccharide.

Several observations suggest that expression of polylactosamine or extended type 2 chains in glycoprotein is an important marker of malignancy. Studies on colon carcinoma and polyps adenoma have shown that extended type 2 Lewis antigens (Le. poly Le" and LeY) are more cancer-specific than their short chain versions (42,43). A major and diagnostically significant proportion of the Le" and LeY antigens in the serum of colon carcinoma patients was found on glycoproteins rather than glycolipids (44). In addition, the detection of the type 2-based Lewis antigens in colon carcinomas or the serum of patients has been correlated with more advanced and metastatic disease (44).

Polylactosarnine sequences have also been found in cells that are motile such as embryonic cell lines and lymphoid cells (45-47). Polylactosamine sequences are present in embryonic fibronectin but not in the adult form, and these sequences have been shown to reduce fibronectin binding to collagen (49). Similarly, binding between purified LAMP-1 glycoprotein and collagen was enhanced by removal of polylactosamine sequences in LAMP-1 (41). These observations suggest that polylactosamine may reduce cell-substratum adhesion and facilitate tumor cell invasion by enhancing cell motility on extracellular matrix (48). Polylactosamine and terminal sequences associated with these structures may serve as ligands for mammalian lectins and play a role in the homing of blood-borne tumor cells to specific secondary sites. In this regard, a mammalian lectin from calf heart has recently been shown to have specificity for polylactosamine (48).

The Dl-6GlcNAc-branched Asn-linked oligosaccharides in malignant cells appear to be required for efficient tumor cell metastasis but are not necessary for solid tumor growth (11). These conclusions are based in part on studies using the three tumor models employed in this study (50). Branching and extension of 0-linked oligosaccharides may also affect metastasis or tumor growth. However, genetic mutation and specific inhibitors of 0-linked branching are not readily available to test this possibility. In conclusion, our results show that transformation and tumor progression in two experimental models is associated with increased core 2 GlcNAc-T as well as GlcNAc-TV activity. The regulated expression of these Dl- 6GlcNAc branching enzymes appears to be a means of con- trolling polylactosamine addition (Fig. lo), which in turn may influence the structure and function of specific glycoproteins required for metastatic dissemination of tumor cells.

Acknowledgments-We would like to thank Frances H o p e a n d Lynda Woodcock for their secretarial assistance, M. Harris-Brandts for technical assistance, Dr. A. Grey at the Carbohydrate Research Center, University of Toronto, for assistance with 'H NMR, and Dr. H. Schachter for helpful suggestions and advice.

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