Analysis of the expression and enzymatic properties of [alpha]1->3fucosyltransferase from human lung carcinoma NCI-H69 and PC9 cells

Anne L. Sherwood and Eric H. Holmes1

Northwest Hospital, Pacific Northwest Cancer Foundation, Dept. of Cell Surface Biochemistry, 120 Northgate Plaza, Suite 218, Seattle, WA 98125, USA

Received on November 20, 1997; revised on January 11, 1999; accepted on January 14, 1999

An analysis of [alpha]1->3fucosyltransferase expression and enzyme properties has been conducted in human lung carcinoma NCI-H69 and PC9 cells. The results indicate that multiple forms of [alpha]1->3 fucosyltransferase are found in these cells. RT-PCR experiments using total RNA from NCI-H69 and PC9 cells amplified transcripts for three of these enzymes, FucT-IV, -VI, and -VII. Fucose transfer into glycolipid acceptors mediated by truncated chimeric and full length recombinant FucT-IV and -VI enzymes was examined. Both enzymes were found to be type 2 chain specific, but only FucT-VI efficiently transferred fucose to both neutral and sialylated acceptors. A truncated recombinant form of FucT-VI was capable of fucose transfer to the internal Glc residue of a variety of glycolipid acceptors. This property was not observed with the recombinant full length enzyme, suggesting the N-terminal portion of the protein, composed of the intracellular domain, transmembrane domain, and a part of the stem region, is involved in interactions with glycolipid acceptors. Using taurodeoxycholate as the detergent, the distribution of initial fucose transfer into nLc6 catalyzed by recombinant full length enzyme indicated 34% of the mono-fucosyl product was fucosylated at the III-GlcNAc and 66% at the V-GlcNAc for FucT-IV, and almost all of the FucT-VI mono-fucosyl product was III-GlcNAc fucosylated. Similar experiments with VI2NeuAcnLc6 as the acceptor resulted in predominantly III-GlcNAc monofucosylation, although detectable V-GlcNAc monofucosylation was obtained with FucT-VI. When the cationic detergent G-3634-A was used, substantially greater initial transfer into the V-GlcNAc of both neutral and sialylated acceptors with FucT-VI was observed. Using nonsialylated acceptors, total [alpha]1->3 fucosyltransferase activity in NCI-H69 cells was analyzed and found to be diminished 25-30% by exposure to 30 mM NEM, which can be attributed to FucT-VI inactivation. The remaining 70-75% of NEM-resistant activity is attributed to FucT-IV, an NEM-resistant enzyme form capable of fucosylating nonsialylated acceptors. These results suggest that multiple forms of [alpha]1->3fucosyltransferase are expressed in NCI-H69 and PC9 cells, which may account for the observed properties of enzyme derived from these cell lines.

Key words: fucosyltransferases/fucosylated glycoconjugates/NCI-H69 cells/N-ethylmaleimide (NEM) inactivation/RT-PCR

Introduction

[alpha]1->3Fucosylated glycoconjugates expressed on the surfaces of mammalian cells function as blood group and developmental antigens (Watkins, 1980; Hakomori, 1981, 1984, 1989; Feizi, 1985; Alhadeff, 1989; Larsen et al., 1990) and are involved in hematopoeitic cell differentiation, tumorigenesis and normal leukocyte trafficking (Macher et al., 1988; Fukuda, 1992; Koszdin and Bowen, 1992; Lasky, 1992). Sialylated/fucosylated structures have been shown to serve as ligands for E-,P- and L- selectins, which are adhesion molecules involved in leukocyte recruitment into lymphoid tissues and sites of inflammation extravasation (Lowe et al., 1990; Phillips et al., 1990; Walz et al., 1990; Polley et al., 1991; Tiemeyer et al., 1991; Imai et al., 1992; Koszdin and Bowen, 1992; Lasky, 1992; Rosen and Bertozzi, 1994; Stroud et al., 1996). It is crucial to understand not only the mechanisms whereby the biosynthesis of these fucosylated oligosaccharides is regulated in different cells, but the roles of these determinants in various biological processes. The specific types of fucosylated structures observed within a given cell type are a function of the acceptor specificities of [alpha]1->3fucosyltransferases (FucTs) expressed by that cell (Sadler, 1984; Kukowska-Latallo et al., 1990) and the of acceptor substrates which are present (Holmes, 1993).

A number of different forms of [alpha]1->3 FucTs in human tissues have been distinguished based upon acceptor substrate specificity and other biochemical properties (Holmes et al., 1985, 1995; Mollicone et al., 1990; Stroup et al., 1990; Kumar et al., 1991; Macher et al., 1991). Multiple human FucTs have been cloned and partially characterized. These include FucT-III, the Lewis enzyme (Kukowska-Latallo et al., 1990), FucT-IV, myeloid (Goelz et al., 1990; Kumar et al., 1991; Mollicone et al., 1994); FucT-V (Weston et al., 1992a); FucT-VI, plasma (Koszdin and Bowen, 1992; Weston et al., 1992b; Mollicone et al., 1994); and FucT-VII (Natsuka et al., 1994; Sasaki et al., 1994), also from cells of myeloid lineage. It was found that Fuc T-III, -V, and -VI are inactivated by NEM in a GDP-fucose protectable manner through reaction with a specific Cys residue present in these forms. In contrast, Fuc T-IV and -VII, which have Ser and Thr residues, respectively, in the analogous position, are not NEM sensitive (Holmes et al., 1995).

Previous results have indicated that [alpha]1->3fucosyltransferase activity in human lung NCI-H69 cells displays qualitatively different behavior compared to enzyme from either COLO 205 or HL-60 cells (Holmes and Macher, 1993). In this study, we report results of analyses of acceptor properties of recombinant forms of FucT-IV and -VI and present data to suggest that total FucT activity in NCI-H69 and PC9 cells is comprised of a combination of active enzyme forms, primarily FucT-IV and -VI.

Results

RT-PCR analysis of [alpha]1->3 fucosyltransferase expression in NCI-H69, PC9, and HL-60 cells

RT-PCR analysis was performed on total RNA extracted from NCI-H69, PC9, and HL-60 cells using primers specific for FucT-IV and -VII and those based upon consensus sequences of FucT-III, -V, and -VI (primers I and II). As shown in Figure 1 (lanes 7-9), a single 1 kb product was generated from NCI-H69, PC9, and HL-60 RNA with primers I and II. An identical product was amplified from an NCI-H69 cell cDNA library (results not shown). Based on partial sequencing of the PCR products directly, it was determined that this was FucT-VI. This was found to be a single product and not a mixture, since clear FucT-VI sequence was obtained in every case. Complete sequencing on both strands of the NCI-H69 PCR product and plasmid constructs made by inserting this PCR fragment into pZErO vector (pZErO-PCR), showed this coding sequence to be a variant of the FucT-VI gene reported by Koszdin and Bowen (1992) wherein three nucleotide differences were observed in the sequence at nctd 99, T->C; nctd 283, G->C; nctd 855, G->A. None of these changes resulted in an amino acid substitution. While it is possible that such changes can be introduced by PCR error, in this case it is highly unlikely since products from several different PCR reactions yielded the same sequence results.


Figure 1. RT-PCR analysis of total RNA isolated from NCI-H69, PC9 and HL-60 cells for the presence of FucT-IV,-VI, and -VII. Lanes 1-3, PCR products generated using primers homologous to FucT-VII; lanes 4 - 6, PCR products generated using primers homologous to FucT-IV; lanes 7- 9, PCR products generated using primers designed based upon the consensus sequence of FucT-III,-V, and -VI (primers I and II). Lanes 1, 4, and 7 show PCR performed on total HL-60 RNA. Lanes 2, 5, and 8 show PCR performed on total NCI-H69 RNA. Lanes 3, 6, and 9 show PCR performed on total PC9 RNA. Five microliters of each PCR mix was electrophoresed in a 0.8% agarose gel in 1× TBE buffer. Gel was stained with ethidium bromide.

The PCR cDNA encoded a slightly truncated enzyme missing the first 77 nucleotides at the N-terminus which was fully active and transferred fucose in [alpha]1->3-linkage to neutral and sialylated acceptors (Table II, see below). Primers I and II also effectively amplified a 1 kb product from a clone of FucT-III, pCDM8-FucT-III, demonstrating as expected that these primers were not restricted only to FucT-VI. However, these primers were not specific for FucT-IV and failed to amplify a PCR product from a full length FucT-IV construct (data not shown).

As expected, based upon earlier findings (Goelz et al., 1990; Lowe et al., 1990; Kumar et al., 1991; Yago et al., 1993; Natsuka et al., 1994; Sasaki et al., 1994; Clarke and Watkins, 1996), both FucT-IV and -VII were determined to be present in HL-60 cells since cDNAs of correct size and of the proper sequence were amplified from total HL-60 RNA using primers specific for these enzymes (Figure 1, lanes 1 and 4). Transcripts for FucT-VI were also found (Figure 1, lane 7). All three FucT transcripts were determined to be present in NCI-H69 and PC9 cells (Figure 1, lanes 2, 3, 5, 6, 8, and 9). Nucleotide sequencing also confirmed the identities of FucT-IV and -VII transcripts in NCI-H69 cells. The consistent and unambiguous sequences obtained from the RT-PCR products generated with multiple primer sets for FucT-IV suggests that no other transcripts which have significant homology for this enzyme form are expressed by these cell lines (results not shown).

Analysis of fucosyltransferase activity in expressed enzyme forms

COS-7 cells which had been transfected with pcDNA3-FucT-VI, pcDNA3-FucT-VI(-) (reverse orientation), pCDM8 FucT-VII (positive control), and untransfected COS-7 cells were assayed for [alpha]1->3fucosyltransferase activity. Figure 2 shows that a single reaction product from transfer of [14C]fucose to the acceptor IV3NeuAcnLc4 was detected in cells transfected with both the positive control and pcDNA3-FucT-VI, but not in untransfected COS-7 cells or cells transfected with pcDNA3-FucT-VI(-).


Figure 2. Assay of COS-7 cells for [alpha]1->3FucT activity on IV3NeuAcnLc4 substrate following transfection with various constructs. Lane 1, Untransfected COS-7 cells; lane 2, transfection with pCDM8-FucT-VII plasmid; lane 3, transfection with pCDNA3-FucT-VI (-) (reverse orientation); lane 4, transfection with pCDNA3-FucT-VI. Arrow shows where reaction product migrates on a TLC plate chromatographed with CHCl3:CH3OH:ddH2O, 60:40:9 containing 0.02% CaCl2.

Protein-A fusion proteins secreted from COS-7 cells transfected with various pPROTA FucT plasmids were purified from spent medium on IgG-agarose beads. The constructs, which all contained FucT inserts truncated at the N-terminus, were pPROTA FucT-IV (amino acids 58-405), pPROTA FucT-VI, (amino acids 27-359, both orientations, cloned from NCI-H69 cells) and pPROTA FucT-VII (amino acids 40-342). Table I shows that, when assayed in the presence of LacNAc-R1 acceptor, high FucT activity was isolated from the medium of COS-7 cells transfected with pPROTA FucT-IV and pPROTA FucT-VI, but not pPROTA FucT-VI(-) or pPROTA FucT-VII. In the presence of sialyl-LacNAc-R1 acceptor, moderate activity was detected with both FucT-VI and -VII chimeric enzymes. The activity of chimeric FucT-VI in the presence of a panel of oligosaccharide acceptors is shown in Table II. These results indicate that the chimeric FucT-VI is type 2 chain specific and efficiently transfers fucose to both neutral and sialylated acceptors. These results are identical to those obtained with isolated plasma FucT-VI enzyme (Koszdin and Bowen, 1992; Weston et al., 1992b).

Table I. Activity of pPROTA-expressed [alpha]1->3 fucosyltransferases
Plasmid Presence of acceptor pmol/h
pPROTA FucT-IV Gal[beta]1->4GlcNAc[beta]->R1a
(LacNAc-R1)
262
pPROTA FucT-VI   210
pPROTA FucT-VI (-)b   1.2
pPROTA FucT-VII   3.9
pPROTA FucT-VI NeuAc[alpha]2->3Gal[beta]1->4GlcNAc[beta]->R1
(3[prime] sialyl LacNAc-R1)
83
pPROTA FucT-VII   67
aR1 = O-(CH2)8-COOCH3.
bFucT-VI sequence inserted in reverse orientation.

Table II. Oligosaccharide acceptor specificity of pPROTA-expressed FucT-VI
Acceptor pmol/h
Gal[beta]1->4 GlcNAc[beta]->R1a 87
NeuAc[alpha]2->3 Gal[beta]1->4 GlcNAc[beta]->R1 83
Gal[beta]1->3GlcNAc[beta]->R1 0
(3-SO4)Gal[beta]1->4GlcNAc[beta]->R1 79
Fuc[alpha]1->2Gal[beta]1->4GlcNAc [beta]->R1 104
aR1 = O-(CH2)8- COOCH3.

Table III. Fucose transfer to lacto- and neolacto-series acceptors by recombinant full length FucT-IV and FucT-VI
Acceptor FucT-IV (pmol/h/ml) FucT-VI (pmol/h/ml)
TDOC G-3634-A TDOC G-3634-A
Lc4 NDa ND 290b ND
nLc4 300 30 12,300 280
IV3NeuAcnLc4 70 30 13,700 420
nLc6 490 100 7100 320
VI3NeuAcnLc6 40 60 3800 190
aNone detected.
bTransfer to internal Glc residue.

Fucose transfer catalyzed by the chimeric FucT-VI enzyme to a variety of glycolipid acceptors was tested. The results are shown in Figure 3A. Interestingly, the chimeric FucT-VI enzyme was capable of transferring fucose to LacCer, Lc3, and Lc4 in addition to type 2 chain acceptors, in contrast to what was observed above with short chain oligosaccharide acceptors. The position of fucose substitution in the Lc3 and Lc4 products was analyzed after hydrolysis with either jack bean [beta]-N-acetylhexosaminidase, bovine testes [beta]-galactosidase, or both using the conditions described under Materials and methods. The results shown in Figure 3B indicate that [beta]-N-acetylhexosaminidase cleaved the Lc3 product to yield a product which retained the labeled fucose and comigrated with the LacCer product. Treatment of the Lc4 product with [beta]-galactosidase resulted in a faster migrating labeled product which also comigrated with the LacCer product after subsequent [beta]-N-acetylhexosaminidase treatment. Thus, the chimeric FucT-VI transfers fucose onto Glc of the internal Gal[beta]1->4Glc structure of glycolipids. This is in contrast to results obtained with full length recombinant FucT-VI (Table III). Little or no fucose transfer to the internal Glc of Lc4 was observed with the full length enzyme, in close agreement with previous findings indicating that full length FucT-VI transfers only to GlcNAc residues in Gal[beta]1->4GlcNAc linkage (Weston et al., 1992B).


Figure 3. Thin layer chromatography of reaction products from transfer of [14C]-fucose into glycolipid acceptors catalyzed by beaded chimeric FucT-VI (pPROTA-FucT-VI). (A) Fucose incorporation into the following glycolipid acceptors: lane 1, LacCer; lane 2, Lc3; lane 3, Lc4; lane 4, nLc4; lane 5, IV3NeuAcnLc4. (B) Enzymatic hydrolysis of products of fucose incorporation into glycolipid acceptors catalyzed by pPROTA- FucT-VI. Product from: lane 1, Lc3; lane 2, Lc3 following cleavage with jack bean [beta]-N-acetylhexosaminidase; lane 3, Lc4; lane 4, Lc4 following hydrolysis with bovine [beta]-galactosidase; lane 5, Lc4 following treatment with both [beta]-galactosidase and [beta]-N-acetyl-hexosaminidase; lane 6, nLc4; lane 7, nLc4 following hydrolysis with [beta]-galactosidase; lane 8, nLc4 following treatment with both [beta]-N-acetylhexosaminidase and [beta]-galactosidase. The solvent system used was CHCl3:CH3OH:H2O, 60:40:9 containing 0.02% CaCl2.

The distribution of fucose residues present in monofucosyl derivatives of nLc6 and VI3NeuAcnLc6 catalyzed by recombinant full length FucT-IV and FucT-VI was studied. In assays catalyzed by FucT-IV using TDOC as the detergent, 34% of monofucosyl derivative of nLc6 was present as III-GlcNAc product and 66% was present as V-GlcNAc product. In assays utilizing VI3NeuAcnLc6 as the acceptor for FucT-IV, only the III-GlcNAc product was detected. In equivalent assays catalyzed by FucT-VI, 98% of monofucosyl derivative of nLc6 was present as the III-GlcNAc product and 2% as the V-GlcNAc product when TDOC was used. In the presence of TDOC the monofucosyl derivative of VI3NeuAcnLc6 was also predominantly the III-GlcNAc product (92%). In contrast, in the presence of the cationic detergent G-3634-A, substantial amounts of the V-GlcNAc monofucosyl product was obtained with FucT-VI using both acceptors. In these experiments 41% and 80% of the monofucosyl products with nLc6 and VI3NeuAcnLc6, respectively, were fucosylated on the V-GlcNAc (results not shown).

Kinetics of chimeric FucT-VI activity

The effect of GDP-fucose concentration on transfer of fucose in [alpha]1->3 linkage to LacNAc by the FucT-VI was tested. The apparent Km for GDP-fucose was calculated to be 10 ± 2 µM. Similar analyses of the Km for the oligosaccharide acceptor LacNAc-R1 was determined to be 50 µM for this enzyme.

Effect of NEM on NCI-H69 cell [alpha]1->3fucosyltransferase activity

The effect of NEM treatment on detergent solubilized enzyme from NCI-H69 and COLO 205, and full length FucT-VII expressed in COS-7 cells is shown in Figure 4. The positive control COLO 205 enzyme (primarily FucT-III) was efficiently inactivated by increasing concentrations of NEM regardless of the nature of the acceptor substrate used. NEM treatment of FucT-VII expressed in COS-7 extracts was completely insensitive to inactivation even at 30 mM final NEM concentration. This is consistent with the expression of a Thr residue at position 127, the position analogous to that occupied by a catalytically essential Cys residue in FucT-III, -V, and -VI (Holmes et al., 1995). NEM treatment of solubilized enzyme from NCI-H69 cells, when assayed using the neutral acceptor nLc4, resulted in only partial inactivation (~25-30%) of total NCI-H69 FucT enzyme activity (Figure 4C). This indicates that neutral acceptor fucosylation activity is most probably due to the action of both the NEM sensitive FucT-VI and NEM insensitive FucT-IV enzyme forms. Assays of NEM treated solubilized NCI-H69 cell enzyme using IV3NeuAcnLc4 indicated that only about 15% of the activity was sensitive. This suggests that both FucT-VI and -VII are responsible for fucosylation of sialylated acceptors in NCI-H69 cells with FucT-VII likely the predominant enzyme catalyzing this reaction.


Figure 4. Effect of NEM treatment on total [alpha]1->3 FucT enzyme activity. Detergent solubilized [alpha]1->3 FucT enzymes from a variety of sources were treated with varying concentrations of NEM in the presence of anionic TDOC detergent and either neutral (nLc4) or sialylated (IV3NeuAcnLc4) acceptor as described in Materials and methods. (A) COLO 205 cells (solid squares) and pCDM8- FucT-VII transfected COS-7 cells (solid circles) using IV3NeuAcnLc4 acceptor; (B) COLO 205 cells (solid squares) and NCI-H69 cells (solid circles) using IV3NeuAcnLc4 acceptor; (C) COLO 205 cells (solid squares) and NCI-H69 cells (solid circles) using neutral nLc4 acceptor. Control activity in the absence of NEM was 384 pmol/h for the COLO 205 enzyme and 285 pmol/h for the NCI-H69 enzyme. Control activity in the absence of NEM was 27 pmol/h for Fuc T-VII.

Discussion

Multiple, distinct [alpha]1->3 FucT's have been detected in a variety of human tissues and cells, five of which have been cloned, FucT-III, -IV, -V, -VI, and -VII (Goelz et al., 1990; Kukowska-Latallo et al., 1990; Lowe et al., 1991; Koszdin and Bowen, 1992; Weston et al., 1992a,b; Natsuka et al., 1994; Sasaki et al., 1994). These have been distinguished by extensive acceptor specificity studies and biochemical analyses (Goelz et al., 1990; Kukowska-Latallo et al., 1990; Lowe et al., 1990; Kumar et al., 1991; Koszdin and Bowen, 1992; Weston et al., 1992a,b; Mollicone et al., 1994; Natsuka et al., 1994; Sasaki et al., 1994; deVries et al., 1995).

Characterization of the various forms has been complicated by the fact that transcripts for multiple FucTs often have been detected in a given tissue or cell line (Stroup et al., 1990; Yago et al., 1993; Sasaki et al., 1994; deVries et al., 1995). The results presented indicate transcripts for FucT-IV, -VI, and -VII are present in NCI-H69 and PC9 cells. The acceptor properties of truncated, chimeric, and recombinant full length FucT-IV and -VI have been examined in this study. Analysis of reaction products obtained with recombinant FucT-IV enzymes reveal a close similarity with those obtained in earlier studies with the enzyme in HL-60 cells (Holmes and Macher, 1993).

An analysis of the acceptor substrate properties of recombinant FucT enzymes has been conducted. The results indicate that recombinant full length FucT-IV expressed in crude homogenates of COS-7 cells is capable of initial transfer of fucose to the V-GlcNAc of nLc6 (66% of total monofucosyl product with TDOC as detergent). Further, initial transfer of fucose to nLc6 catalyzed by FucT-VI indicates almost exclusive transfer to the III-GlcNAc when TDOC is used as the detergent, whereas in the presence of G-3634-A, 41% of the total monofucosyl product is the V-GlcNAc product. These results suggest that specificity of transfer to alternate GlcNAc residues in an extended chain may be modulated in part by factors influencing the interactions of the enzyme with acceptor.

Enzyme assays using the truncated, chimeric FucT-VI enzyme from NCI-H69 cells demonstrate that in addition to GlcNAc residues of type 2 acceptors, this form of the enzyme was capable of transferring fucose to the internal Glc residue of glycolipids. Thus, it appeared to be specific for acceptors containing either Gal[beta]1->4GlcNAc- or Gal[beta]1->4Glc- structures. This is rather unusual behavior for FucT-VI (Weston et al., 1992B). Previous results have indicated that only FucT-III in its native form is capable of catalyzing fucose transfer to internal Glc residues of glycolipids (Holmes, 1993). The results indicated that, in contrast, recombinant full length FucT-VI did not catalyze significant fucose transfer to the internal Glc residue of Lc4. Thus, the difference in transfer specificity to internal Glc residues of glycolipids between full length and chimeric FucT-VI reflects properties of the transmembrane domain missing from the chimeric enzyme. This, coupled with the observed modulation of transfer to specific GlcNAc residues of long chain acceptors depending on the nature of the lipidic environment, suggests more N-terminal protein segments may play a role in acceptor substrate interaction.

Previous results have shown that certain FucT enzymes (FucT -III,-V, and -VI) are inactivated by NEM, while others (FucT-IV and -VII) are insensitive toward inactivation by sulfhydryl group modifying reagents (Holmes et al., 1985, 1995; Mollicone et al., 1990; Stroup et al., 1990). Subsequent work indicated that NEM inactivation is due to modification of a GDP-fucose protected site on the enzyme (Holmes et al., 1995). Results presented in this paper indicate that 70-75% of the FucT activity with neutral acceptors present in NCI-H69 cells is resistant to NEM. In view of the enzyme expression in these cells, this is most probably attributable to FucT-IV. The remaining 25-30% of NEM sensitive neutral acceptor FucT activity is attributed to FucT-VI. Analysis of the effect of NEM treatment on transfer to sialylated acceptors suggests that most of this activity is catalyzed by the NEM insensitive FucT-VII enzyme, since FucT-IV transfers fucose to sialylated acceptors only poorly. Taken together, the results suggest that lung carcinoma NCI-H69 and PC9 cells express a combination of FucT enzyme forms, FucT-IV, -VI, and -VII, resulting in the observed enzymatic properties of the enzyme from these cell lines.

Interestingly, it has previously been found that lung cancer patients whose tumors showed strong expression of FucT-IV and/or -VII have a poorer prognosis than patients expressing neither, due presumably to participation in biosynthesis of sialyl Lewis x, the potential ligand for E-selectin, whose expression correlates with higher metastatic potential of cancer cells (Ogawa et al., 1996). The evidence appears to implicate both FucT-VII and -IV in ligand biosynthesis (Natsuka et al., 1994; Sasaki et al., 1994; Ogawa et al., 1996), given that polyfucosylated ligands for E-selectin are present coupled with the finding that FucT-IV fucosylates GlcNAc residues closest to sialyl substitutions of sialylated acceptors poorly if at all (Foster et al., 1991; de Vries et al., 1995) and FucT-VII is capable of fucosylating only that GlcNAc residue in close proximity to a terminal sialyl substitution (Stroud and Holmes, 1997). Our findings of the presence of FucT-IV and -VII transcripts in the transformed NCI-H69 and PC9 lung cell lines are consistent with the results of Ogawa et al.(1996) that FucT-VII expression and sLex could be a marker of invasive lung cancer.

The results presented in this study and others (Stroup et al., 1990; Yago et al., 1993; Sasaki et al., 1994; Clarke and Watkins, 1996; Ogawa et al., 1996) indicate multiple enzyme forms are present in cells derived from a number of different tissues. Given the multiplicity of enzymes and enzymatic properties which can potentially be expressed by cells, it is reasonable to conclude that the differing enzyme forms are responsible for slightly differing physiological functions. We are beginning to learn important information about specific functions of these enzyme forms. Given the importance of the reaction products of these enzymes to cellular properties and functions, a much greater understanding of this is needed.

Materials and methods

Materials

Human promyelocytic leukemia HL-60 cells, human small cell lung carcinoma NCI-H69 cells, human colonic adenocarcinoma COLO 205 cells, and the simian COS-7 cell line were obtained from the American Type Cell Collection (Rockville, MD). Human lung carcinoma PC9 cells were obtained from M.Adachi, Immunoresearch Laboratory, Takasaki, Japan. N-Acetyllactosamine (LacNAc), Na-taurodeoxycholate (TDOC), rabbit IgG-agarose beads, and DEAE-Dextran were obtained from Sigma (St. Louis, MO). Plasmids pcDNA3 and pZErO-1 were from Invitrogen (San Diego, CA), and pPROTA, pPROTA-Fuc T-IV, and pCDM8-Fuc T-VII were received from Dr. Bruce A.Macher (San Francisco State University). 8-Methoxycarbonyloctyl glycosides (see Table II for chemical composition) were received from Dr. Monica Palcic (University of Alberta, Canada). GDP-[14C] fucose (283 mCi /mmol) and [35S] dATP were obtained from Dupont NEN (Boston, MA). Jack bean [beta]-N-acetylhexosaminidase and bovine testes [beta]-galactosidase were obtained from Oxford GlycoSystems, Bedford, MA. DNA sequencing was done using the Sequenase Version 2.0 DNA sequencing kit or the Sequenase PCR product sequencing kit obtained from United States Biochemical Corp.(Cleveland, OH). PCR primers were obtained from Oligos, Etc. Inc. (Wilsonville, OR) or were made on a Beckman Oligo 1000 Synthesizer. All other reagents were of the highest quality commercially available.

Cell culture

HL-60, NCI-H69, and PC9 cells were grown in flasks in RPMI 1640 media supplemented with 20, 10, and 10% fetal calf serum, respectively. The cells were harvested and passed 1:3 every 5-6 days. COLO 205 and COS-7 cells were grown in tissue culture plates in RPMI 1640 and Dulbecco's modified Eagle's medium (DME), respectively, supplemented with 10% fetal calf serum. These were passed 1:4 every 5-6 days.

Isolation of cDNA encoding a major NCI-H69 fucosyltransferase

Total RNA was extracted from 1 × 107 NCI-H69 cells using the RNAzol B method (Tel-Test, Inc., Friendswood, TX). Isolated RNA in 10 mM Tris buffer, pH 7.5 was amplified by RT-PCR in a Coy thermocycler by 35 cycles of 95°C for 20 sec, 55°C for 20 sec, and 72°C for 1 min in the presence of 1.75 µg total RNA and 200 pM primer, using a Gene Amp PCR kit (Perkin-Elmer, Branchburg, NJ). Primers used in this step were based upon the consensus sequence of FT-III, -V, and -VI corresponding to regions of the protein in the stem and C-terminal. Thus, PCR would be expected to generate an amplified product of about 1000 base pairs coding for a protein encompassing the stem region to the C-terminal of the NCI-H69 cell enzyme. The more N-terminal regions of the protein were ignored in primer design at this stage due to the considerable sequence heterogeneity among known forms in this region. The primers used were: forward: 5[prime] GCGgaattcGGCTGTGTGTTTCTTCTCCTACC 3[prime] (Primer I) and reverse: 5[prime] GCTgaattcCTCTCAGGTGAACCAAGCCG 3[prime] (Primer II). These primers contained EcoRI sites (lower case above) within the ends and an additional G residue (bold/underlined) in the forward primer sequence to allow the PCR product to be kept in frame when cloned into the pPROTA expression vector. The PCR product resulting from these primers was briefly treated with mung bean nuclease and blunt-end ligated into the EcoRV site of pZErO-1 vector for sequencing. The DNA sequence was determined by the dideoxynucleotide chain termination method (Sanger et al., 1977) off this plasmid and directly off the PCR product using 10 pM primer and (5 µg alkaline denatured plasmid or 5 µl ExoI and shrimp alkaline phosphatase treated PCR product).

RT-PCR analysis of fucosyltransferases present in NCI-H69, PC9, and HL-60 cells

The same primers, described above, were also used in an RT-PCR analysis of total RNA extracted from 1 × 107 HL-60 and PC9 cells. Additionally, PCR analysis of total RNA derived from the NCI-H69, PC9, and HL-60 cell lines was conducted to determine the presence or absence of transcripts for FucT-IV and -VII. Primers for amplification of FucT-IV cDNA, used in earlier studies by Yago et al.(1993) were: forward, 5[prime] GGTGCCCGAAATTGGGCTCCTGCACAC 3[prime], and reverse: 5[prime] CCAGAAGGAGGTGATGTGGACAGCCTA 3[prime]. A 319 bp fragment spanning nctds 807-1125 was generated with these primers. Primers for amplification of FucT-VII cDNA, used in earlier studies by Natsuka et al.(1994), were: forward, 5[prime] CTCGGACATCTTTGTGCCCTATG 3[prime], and reverse, 5[prime] CGCCAGAATTTCTCCGTAATGTAG 3[prime]. A 288 bp fragment, within the coding region of FucT-VII, was generated with these primers. Primers for the amplification of full length FucT-VII (minus the transmembrane domain) used in later fusion protein studies were: forward: 5[prime] CTgaattcGACCCCGGCACCCCAGCCC 3[prime], and reverse: 5[prime] TCCgaattcCAGCGGATCTCAGGCCTG 3[prime]. EcoRI sites are lower case and the additional G residue (inserted for in-frame expression of this Fuc T-VII PCR product when inserted into pPROTA) is bold/underlined in the forward primer. The template for this PCR product was pCDM8 FucT-VII. The nucleotide sequences of all PCR products obtained were confirmed by sequencing using the procedures described above.

Construction of fucosyltransferase expression vectors and expression of RT-PCR cDNA in COS-7 cells

The FucT-PCR insert was excised from PCR-pZErO-1 with EcoRI and cloned into the EcoRI site of pcDNA3 and pPROTA. Correct orientation was established by ApaI digestion and ApaI/Hind III digestion, respectively. Constructs were transiently transfected into COS-7 cells by the DEAE Dextran method (Ausubel et al., 1993). Cells which had been transfected with pcDNA3-PCR constructs were harvested after 4-5 days. Secreted fusion proteins were purified from the conditioned medium of cells transfected with pPROTA-PCR after 4-7 days on IgG agarose beads as described previously (Holmes et al., 1995).

[alpha]1->3 Fucosyltransferase assays

Unless otherwise specified, [alpha]1->3 fucosyltransferase activity was assayed in reaction mixtures containing 2.5 µmol HEPES buffer, pH 7.2, 20 µg of acceptor glycolipid, 100 µg TDOC or G-3634-A, 1 µmol MnCl2, 15 nmol GDP-[14C]fucose (15,000 c.p.m./nmol), and 300-400 µg protein from sonicated crude cell homogenates in a total volume of 0.1 ml as described previously (Holmes and Macher, 1993). Analysis of fucose transfer into neutral and sialylated long chain glycolipid acceptors containing multiple GlcNAc residues was conducted as described previously (Holmes and Macher, 1993).

Enzyme activities in supernatants from cells transfected with various pPROTA constructs were determined in reaction mixtures composed of 1 µmol HEPES buffer, pH 7.2, 6 nmol of GDP-[14C] fucose (15,000 c.p.m./nmol), 0.4 µmol of LacNAc or 0.072 µmol of 8-methoxycarbonyloctyl glycoside acceptor, 2 µmol of NaCl, 0.125 µmol of MnCl2, 10 µg of bovine serum albumin, 0.01 µmol of ATP, and chimeric enzyme bound to IgG agarose beads in a total volume of 0.02 ml as described previously (Holmes et al., 1995). Transfer to LacNAc was determined after passage of the reaction mixture over a Dowex-1 column using the conditions described previously (de Vries et al., 1995; Holmes et al., 1995). Transfer to oligosaccharides containing hydrophobic substitutions at the reducing end was determined after passage of the reaction mixture over a C18 reversed phase column (Bond-elut, Analytichem International) using the conditions described previously (de Vries et al., 1995).

Treatment of native and chimeric [alpha]1->3 fucosyltransferase enzymes with NEM

The effect of NEM on enzyme activity was determined using aliquots of detergent solubilized enzyme from COLO 205 or NCI-H69 cells or full length pCDM8-FucT-VII in transfected COS-7 cell extracts incubated in 50 µM HEPES buffer, pH 7.2, 25% glycerol, 1.0 mM DTE, 0.2% Triton X-100R. Thirty microliters of enzyme and 10 µl of ddH20 containing increasing amounts of NEM up to a final concentration of 30 mM were incubated at room temperature for 30 min. The reaction was stopped by the addition of 1.5 µmol DTE and the activity remaining was then determined.

Hydrolytic studies of FT-VI glycolipid products

Bands containing [14C]-labeled products from fucosylated glycolipid acceptors, which had been chromatographed and identified on autoradiographs (Figure 3A) were scraped from Whatman silica gel HPTLC plates, extracted with isopropanol:hexane:ddH2O (55:25:20) and dried down under an N2 stream. Approximately 5000 c.p.m. of each product was resuspended in a 0.1 ml reaction mix containing 100 µg TDOC, 100 mM Na citrate buffer, pH 4.0, 0.1 unit of [beta]-galactosidase and/or 0.1 unit of [beta]-N-acetylhexosaminidase and incubated at 37°C overnight. Reaction mixes were run over C18 reversed phase columns to remove salt and products were eluted with MeOH. Hydrolyzed glycolipid products were dried down under an N2 stream, dissolved in chloroform:MeOH (2:1) and separated by TLC followed by autoradiography as described earlier.

Acknowledgments

We thank Dr. Monica Palcic (University of Alberta, Canada) for the 8-methoxycarbonyloctyl glycosides for fucosyltransferase characterization, Dr. Stuart Swiedler (Glycomed, Inc., Alameda, CA) for the NCI-H69 cDNA library and the pCDM8-FucT-III clone and Dr. John Lowe (University of Michigan Medical School, Ann Arbor, MI) for the full length pcDNAI FucT-IV clone. We also thank Dr. Bruce Macher and Ms. Susan Shetterly (San Francisco State University, CA) for use of their DNA sequencing facility. This work was supported by Research Grant CA70740 from the National Cancer Institute, NIH.

Abbreviations

FucT, fucosyltransferase; NEM, N-ethylmaleimide; TDOC, taurodeoxycholate; LacNAc, N-acetyllactosamine; kb, kilobase; RT-PCR, reverse transcription polymerase chain reaction. Glycolipids are designated in the text according to the recommendations of the IUPAC Nomenclature Committee, but the suffix OseCer is omitted (IUPAC-IUB Commission on Biochemical Nomenclature, 1977). sLex (IV3NeuAcIII3FucnLcOse4Cer), NeuAc[alpha]2->3 Gal[beta]1->4 [Fuc[alpha]1->3] GlcNAc[beta]1->3 Gal[beta]1->4 Glc[beta]1->1Cer; sLea (IV3NeuAcIII4FucLcOse4Cer), NeuNAc[alpha]2->3Gal[beta]1->3[Fuc[alpha]1->4]GlcNAc[beta]1->3 Gal[beta]1->4Glc[beta]1->1Cer; nLcOse6Cer, Gal[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4GlcNAc[beta]1->3 Gal[beta]1->4Glc[beta]1->1Cer; nLcOse4Cer, Gal[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc[beta]1->1Cer; LcOse4Cer, Gal[beta]1->3GlcNAc[beta]1->3Gal[beta]1->4 Glc[beta]1->1Cer; LcOse3Cer, GlcNAc[beta]1->3Gal[beta]1->4Glc[beta]1->1Cer; and LacCer, Gal[beta]1-4Glc[beta]1->1Cer; Cer, ceramide.

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