The COS cell line, derived from the African Green Monkey kidney CV-1 cells transformed with origin defective SV-40 mutant virus (Gluzman, 1981), can efficiently support the episomal replication of bacterial plasmids containing the SV-40 origin of replication and the ability to replicate and amplify transfected cDNA provides an effective means of obtaining high level transient expression of DNA directly introduced into the cells (Mellon et al., 1981). This cell line has frequently been used for transient expression of cloned [alpha]-l-fucosyltransferases cDNAs (Goelz et al., 1990; Lowe et al., 1991; Weston et al., 1992a,b; De Vries et al., 1995; Gersten et al., 1995; Clarke and Watkins 1996; Sajdel-Sulkowska et al., 1997), and the levels of enzyme activity in cell extracts and of fucosylated antigens expressed on the surface of untransfected cells were considered too low to be of significance. The cells are not, however, entirely lacking in endogenous fucosyltransferase activity and they also weakly express the fucosylated and sialylated, sialyl-Lex, antigen on their surfaces (Clarke and Watkins, 1996). In order to determine whether the endogenous activities could interfere with interpretation of results obtained with the transfected fucosyltransferase genes, and also whether they might reveal as yet unidentified fucosyltransferases, a more detailed examination was undertaken (Clarke and Watkins, 1997a). Fucosyltransferase tests showed the presence in extracts of the COS cells of an enzyme, provisionally identified as an [alpha]1,3- fucosyltransferase, that utilized the low molecular weight Type 2 disaccharide acceptor N-acetyllactosamine (Gal[beta]1-4GlcNAc), and a second enzyme, provisionally identified as an [alpha]1,6-fucosyltransferase, that utilized the glycoprotein, asialo-agalacto-fetuin, as acceptor substrate. Both enzymes had pH optima of 7.0 and were resistant to the action of N-ethylmaleimide (NEM). Incorporation of radioactive fucose into the glycoprotein, asialo-fetuin, was also observed: this activity was optimal at pH 5.5 and was less resistant to NEM than the other two activities. The difference in acceptor substrate specificity, together with the differences in properties, suggested the presence of a third fucosyltransferase although the linkage of the transferred sugar was not identified (Clarke and Watkins, 1997a).
We now report results of RT-PCR analyses of COS cell mRNA amplified with primers designed from the cDNA sequences of eight cloned human fucosyltransferase genes (FUT1-8), and demonstrate that five of these genes have homologs expressed in COS cell mRNA. Further characterization has shown that the fucosyltransferase activity previously detected in COS cells with asialo-fetuin as acceptor substrate is a manifestation of an [alpha]1,6-fucosyltransferase in which the properties measured are determined by the simultaneous action on the glycoprotein acceptor substrate of a weak endogenous [beta]-galactosidase.
RT-PCR analyses of fucosyltransferases expressed in COS cells
RT-PCR analyses were performed using specific primers for the human [alpha]1,3-, [alpha]1,6-, and [alpha]1,2-fucosyltransferase genes. FUT4 and FUT7 are so-called myeloid [alpha]1,3-fucosyltransferase genes which are known to be expressed in the human promyelocytic HL-60 cell line (Lowe et al., 1991; Natsuka et al., 1994). Amplification of the COS cell reverse transcribed mRNA with primers specific for FUT4 revealed low expression of a homolog for this gene of the same size (516 bp) as the amplified fragment from control HL-60 cells (Figure
Figure 1. RT-PCR analyses of fucosyltransferase genes expressed in COS cell RNA. Total RNA was prepared from COS cells, and from control HL-60, A431, K562, and HepG2 cells, and subjected to RT-PCR analyses as described in the Materials and methods section. Arrows indicate the amplified bands with the expected sizes. (a) Fragments amplified with primers for G6PD, FUT4 and FUT7 genes from COS and HL-60 mRNA. (b) Fragments amplified from COS and A431 mRNA with primers for FUT3/5 genes, and digestion products with Pfm1 and Nae1. (c) Fragments amplified from HepG2 and COS mRNA with primers for FUT6 gene, and digestion product with PvuII. (d) Fragments amplified from K562 and COS cell mRNA with primers for FUT1 gene, fragments amplified from A431 and COS cell mRNA with primers for FUT2 gene, and fragments amplified from K562 and COS cell mRNA with primers for FUT8 gene.
The primers used for the detection of FUT3 also amplify FUT5 but the products are distinguishable because the target DNA fragments have different sizes and are susceptible to cleavage by different restriction enzymes. Control human epidermoid carcinoma A431 cells which express FUT3 but not FUT5 (Yago et al., 1993) gave a single amplified fragment (447 bp) which was cleaved by Pflm1 but not by Nae 1 whereas COS cells gave a single 486 bp fragment which was cleaved by Nae1 but not by Pflm1 (Figure
Primers designed from the reported nucleotide sequence of the human [alpha]1,6-fucosyltransferase gene, FUT8 (Yanagidani et al., 1997), amplified a fragment of the expected size (600 bp) with control human erythroid K562 cell cDNA and an identical fragment was strongly expressed in the amplified COS cell product (Figure
Flow cytometry
Indirect immunofluorescence analysis of COS cells treated with undiluted monoclonal antibodies confirmed sialyl-Lex (with two monoclonal antibodies, CSLEX1 and KM93) and weak Lex expression on their cell surfaces (Clarke and Watkins 1997a). Ley activity was examined with two different monoclonal antibodies and one, H18A, gave weak but definite evidence of activity whereas the second AH6 gave a very low response. Similarly, the response with the monoclonal anti-Type 2 H reagent was very weak or doubtful, but weak A activity was definitely detected with a monoclonal anti-A reagent (Figure
Figure 2. Flow cytometric analyses of COS cells with monoclonal antibodies to Lex, sialyl-Lex, Ley and blood-group A and Type 2 H determinants. Flow cytometric analyses were carried out as described in the Materials and methods section with monoclonal antibodies to Lex, sialyl-Lex (positive with two different antibodies; only histogram with KM93 shown), Ley (two different antibodies H18A and AH6), blood-group A, and blood-group Type 2 H. The dotted lines represent the fluorescence given by the negative controls. Reactivity of the fucosyltransferase in COS cell extracts with a polyclonal antibody raised against human milk [alpha]-3/4-fucosyltransferase
An antiserum raised in a rabbit against a partially purified [alpha]1,3/4-fucosyl-transferase from human milk was previously shown to react with both the Lewis-gene associated [alpha]1,3/4-fucosyltransferase and the Lewis-gene independent [alpha]1,3-fucosyltransferase isolated from human milk, and also with the [alpha]1,3-fucosyltransferases from human plasma and liver (Watkins et al. 1993; Johnson et al., 1995). This antibody failed to react with the [alpha]1,3-fucosyltransferases in normal or leukemic myeloid cells, expressing FUT4 and FUT7 genes, and its behavior is therefore consistent with the interpretation that it recognizes the products of the chromosome 19 cluster of [alpha]1,3-fucosyltransferase genes (FUT3, FUT5, and FUT6; Weston et al.,1992b). In the present experiments the antibody failed to neutralize the activity of the enzyme in COS cells acting on N-acetyllactosamine at pH 7.0 or on asialo-fetuin at pH 5.5. The antibody also failed to inhibit the activity of the [alpha]1,3-fucosyltransferase in control HL-60 cells acting on N-acetyllactosamine at pH 7.0 but strongly inhibited the activity of the human plasma enzyme on asialo-fetuin or N-acetyllactosamine at pH 7.0 (Figure
Figure 3. Reactivity of fucosyltransferases in COS cell extracts with a polyclonal antibody to [alpha]1,3/4-fucosyltransferase. Tests for neutralization of fucosyltransferase activity by the antibody were carried out with COS cell and HL-60 cell extracts and human plasma as the enzyme sources. Mixtures of enzyme and antibody were prepared and assayed for fucosyltransferase activity as described in the Materials and methods section. Aliquots of the COS cell mixture were tested for [alpha]-fucosyltransferase activity at pH 5.5 with asialo-fetuin as substrate and at pH 7.0 with N-acetyllactosamine as substrate Aliquots of the human plasma-antibody mixture were tested for [alpha]-fucosyltransferase activity at pH 7.0 with N-acetyllactosamine and asialo-fetuin as substrates and the HL-60 cell-antibody mixture was tested for [alpha]-fucosyltransferase activity at pH 7.0 with N-acetyllactosamine as substrate. The columns represent the amount of [alpha]-fucosyltransferase activity remaining after treatment of the enzyme source with the antiserum. Characterization of the fucosyltransferase acting on asialo-fetuin
Activity with N-glycans. Evidence that the fucose was transferred to the N-linked oligosaccharides in asialo-fetuin was earlier shown by the release of the [14C]fucose labeled-chains on treatment with N-glycanase (Clarke and Watkins, 1997a), an enzyme preparation that hydrolyses the aspartyl-glycosylamine bond between asparagine and the proximal N-acetylglucosamine unit of the glycan chain (Hirani et al., 1987). In the present experiments the N-glycans shown in Figure
Figure 4. Structures of the N-glycan oligosaccharides tested for specificity with the COS cell fucosyltransferases.
Table I.
These results strongly suggested that in NA3, and also by analogy in asialofetuin, substitution was taking place to one of the core N-acetylglucosamine units despite the fact that neither of these compounds would be expected to function as substrates for an [alpha]-fucosyltransferase acting at these sites.
Figure 5. Action of glycosidases on the product of fucosyltransfer to NA3 N-glycan. The [14C]fucosylated NA3 product was treated with glycosidases and analyzed by descending paper chromatography as described in the Materials and methods section. (a) Untreated NA3 control run in solvent a (Rfuc = 0.2). (b) NA3 product treated with [alpha]1,3/4-fucosidase and run in solvent a (Rfuc = 0.2). (c) NA3 product treated with [alpha]1,6-fucosidase and run in solvent a (Rfuc = 1.0). (d) NA3 product treated with Endo-F3 and run in solvent b (Rfuc = 0.66).
Endoglycosidase F3 (Endo-F3) cleaves within the diacetylchitobiose core of N-linked oligosaccharide chains and would therefore release a radioactive disaccharide ([14C]Fuc-GlcNAc) if the fucose is attached to the innermost N-acetylglucosmine unit. Treatment of the NA3 product with Endo-F3, followed by paper chromatography, gave a labeled product which migrated close to fucose in solvent a (Rfuc = 0.93) but had an Rfuc = 0.66 in solvent b. The radioactive product was eluted from the paper and treated with [alpha]1,2-, [alpha]1,3/4- and the bovine [alpha]1,6-fucosidases. The products were run on TLC in solvent c. Hydrolysis occurred with the bovine [alpha]1,6-fucosidase and not with the [alpha]1,2- or [alpha]1,3/4-fucosidases. The untreated disaccharide product cochromatographed with an authentic sample of Fuc[alpha]1-6GlcNAc on TLC in solvent c (Rfuc = 0.66) and fucose was also released from this control disaccharide by the [alpha]1,6-fucosidase and not by the other fucosidases.
Oligosaccharide
% [14C]Fucose incorporated
pH 5.5
pH 7.0
A3
1
1
NA3
52
32
NGA3
43
61
NA2
39
-
NA2F
4
-
NGA2F
10
-
GlcNAc2Man3
0
-
Man3GlcNAc2
3
-
N-Acetyllactosamine
-
4
Treatment of NA3 and NGA3 products with [beta]-N-acetylhexosaminidase
Treatment of NGA3 product with jack bean [beta]-N-acetylhexosaminidase shifted its mobility in solvent a from Rfuc= 0.31 to Rfuc =0.44 indicating the release of N-acetylglucosamine residues. However, treatment of NA3 fucosylated product with the [beta]-hexosaminidase under the same conditions did not appreciably alter its mobility in this solvent, suggesting that if some galactose residues had been removed by an endogenous enzyme the number of N-acetylglucosamine residues exposed was insufficient for their removal to visibly change the mobility of the fucosylated product. Similarly, the faster movement on paper chromatography of the fucosylated NGA3 product than the NA3 product indicated that not all the terminal galactose units had been removed from the acceptor.
Glycosidase activities in COS cell extract
Despite the result obtained on treating the NA3 product with [beta]-N-acetylhexosaminidase, the characterization of the products obtained with the oligosaccharides suggested that NA3 and asialo-fetuin were being modified by endogenous glycosidases in the COS cell extract in such a way as to expose terminal nonreducing N-acetylglucosamine residues that allowed the compounds to function as acceptors for an [alpha]1,6-fucosyltransferase.
Tests for glycosidase activities in the COS cell extracts with p-nitrophenyl substrates revealed very weak [beta]-galactosidase activity and considerably stronger [beta]-N-acetylglucosaminidase and [beta]-N-acetylgalactosaminidase activities; all three enzymes had low pH optima (Figure
Figure 6. pH Activity curves for glycosidases in COS cell extracts measured with p-nitrophenyl substrates. Glycosidase activities of the COS cell extracts were measured by the release of nitrophenol from nitrophenyl glycosides as described in the Materials and methods section. Sodium cacodylate buffer was used for the range pH 3.5-7.0 and Tris/HCl buffer for the range pH 7.0-9.0. Results are expressed as nmol nitrophenol released/min/ml of cell extract.
Figure 7. Action of NEM on (a) fucosyltransferases acting on asialo-fetuin and asialo-agalacto-fetuin and (b) [beta]-galactosidases acting on nitrophenyl galactoside and lactose The enzymes were pretreated with different concentrations of NEM and then assayed for activity as described in the Materials and methods section.
Figure 8. Comparison of pH activity curves for transfer of fucose to asialo-fetuin and asialo-agalacto-fetuin with curve for hydrolysis of [14C]lactose by endogenous [beta]-galactosidase in COS cell extracts. (a) Transfer of fucose to asialo-fetuin compared with hydrolysis of [14C]lactose at different pH values and (b) pH activity curve for transfer of fucose to asialo-agalacto-fetuin under prolonged incubation conditions. Fucosyltransferase and [beta]-galactosidase assays were carried out as described in the Materials and methods section except that the incubation period for the transfer of fucose to asialo-agalacto-fetuin was continued for 18 h to match the incubation time used for measurement of the incorporation of fucose into asialo-fetuin.
The transfer of fucose to asialo-agalacto-fetuin showed a fairly sharp pH optimum at 7.0 when the reaction mixtures were incubated at 37°C for 5 h with very low activity at pH 5.5 (Clarke and Watkins, 1997a). However when the incubations were continued for 18 h, in order to match the time required for readily detectable incorporation of fucose into asialo-fetuin, the pH curve showed a much broader peak from pH 5.5-8.0 indicating that there was ample active [alpha]1,6-fucosyltransferase at pH 5.5 to account for the fucosylation of the asialo-fetuin (Figure
Effect of D-galactono-1,4-lactone on fucosyltransferase activity
The aldonolactones are known to function as inhibitors of a wide variety of glycosidases (Conchie and Levvy, 1957). To further test for the presence of a [beta]-galactosidase that was modifying substrates for the fucosyltransferases in COS cell extracts, assays were carried out in the presence of D-galactono-1,4-lactone with acceptors asialo-fetuin and asialo-agalacto-fetuin. The transfer of fucose to asialo-fetuin by the COS cell extract was almost completely abolished in the presence of D-galactono-lactone (Table II) whereas no inhibition occurred with asialo-agalacto-fetuin as substrate; demonstrating that the [alpha]1,6-fucosyltransferase activity itself was not inhibited by this reagent. The breakdown of [14C]lactose by the endogenous [beta]-galactosidase in the COS cell extract was 70% inhibited by a similar concentration of D-galactono-lactone.
Table II.
Acceptor | d-Galactono-1,4-lactone | Incorporation of [14C]fucose (c.p.m.) | Inhibition (%) |
Asialo-fetuin | Absent | 1779 | - |
Present | 25 | 99 | |
Asialo-agalacto-fetuin | Absent | 6345 | - |
Present | 6338 | 0 |
Time course for the fucosyltransferase acting on asialo-fetuin and the [beta]-galactosidase acting on lactose
A comparison of the time course for the transfer of fucose to asialo-fetuin (Figure
Figure 9. Time curves for (a) the transfer of fucose to asialo-fetuin and asialo-agalacto-fetuin by the fucosyltransferases in COS cell extracts and (b) for the hydrolysis of [14C]lactose by the [beta]-galactosidase in COS cell extracts. The fucosyltransferase and [beta]-galactosidase assays were performed as described in the Materials and methods section except that incubations were carried out for various lengths of time between 0 and 50 h. Tests for blood group A and H glycosyltransferases in COS cell extracts
The presence of weak blood group A antigenic activity on the surface of COS cells implies the expression in the cells of both an [alpha]1,2-fucosyltransferase to synthesize the precursor H structures and an [alpha]1,3-N-acetylgalactosaminyl transferase to complete the A determinants (cf. Watkins, 1995). Domino et al. (1997) demonstrated the presence in COS cells of blood group A and B ([alpha]1,3-galactosyl) transferases that led to the cell surface expression of A and B structures in cells transfected with the mouse FUT1 ([alpha]1,2-fucosyltransferase) gene. Tests with UDP-[14C]GalNAc as donor substrate and 2[prime]-fucosyllactose as acceptor confirmed the presence of an [alpha]1,3-N-acetylgalactosaminyltransferase in the untreated COS cell extracts (Table III). However, tests with substances with terminal unsubstituted Gal-1-[beta]- residues, which should act as substrates for [alpha]1,2-fucosyltransferases, i.e., phenyl-[beta]-d-galactoside, Type 1 galactosyl[beta]1-3N-acetyl-(4-deoxy)-d-glucosamine-R and Type 2 galactosyl[beta]1-4N-acetyl-(3-deoxy)-d-glucosamine-R failed to reveal evidence of [alpha]-1,2-fucosyltransferase activity in the COS cell extracts, although the enzymes in A431 cells gave the expected results with these substrates for a cell line expressing [alpha]1,2-, [alpha]1,3/4-, and [alpha]1,3-fucosyltransferases (Table III). Boosting the phenyl [beta]-d-galactoside acceptor concentration 6-fold over that normally used did not give rise to significant incorporation of [14C]fucose into this substrate by the enzymes in the COS cell extract, thus failing to give any evidence of [alpha]1,2-fucosyltransferase activity.
The finding of weak sialyl-Lex expression on the surface of untreated COS cells (Clarke and Watkins, 1996) led us to question which fucosyltransferase genes were expressed in these cells and how they related to the cloned human fucosyltransferase genes. Our preliminary results suggested the presence of three different fucosyltransferases (Clarke and Watkins, 1997a). One of the activities detected with N-acetyllactosamine as a substrate was considered to be an [alpha]-1,3-fucosyltransferase by virtue of the fact that the product cochromatographed with an authentic sample of Lex (Gal[beta]1-4[Fuc[alpha]1-3]GlcNAc) and was susceptible to hydrolysis by an [alpha]1,3/4-fucosidase and not by [alpha]1,2- or [alpha]1,6-fucosidases. RT-PCR analysis has revealed expression of three homologs of human [alpha]1,3-fucosyltransferase genes (FUT4, 5, and 7) in COS cell mRNA. The very low activity with 3[prime]-sialyl-N-acetyllactosamine (Clarke and Watkins, 1997a) in the COS cell extract, suggests a FUT4-like enzyme (Lowe et al., 1991) rather than one resembling the products of the FUT5 gene which has a preference for sialylated Type 2 substrates (Weston et al., 1992b) or the product of the FUT7 gene which only transfers fucose to sialylated Type 2 substrates (Natsuka et al., 1994). This analogy is further supported by the failure of the COS cell [alpha]1,3-fucosyltransferase acting on N-acetyllactosamine to react with a polyclonal antibody directed towards the enzymes encoded by the FUT-3-5-6 gene-cluster.
Table III.
The second activity detected with asialo-agalacto-fetuin was tentatively characterized as an [alpha]1,6-fucosyltransferase adding fucose on to the proximal N-acetylglucosamine residue in the N-linked chains (Clarke and Watkins, 1997a). The presence in the COS cell extracts of an enzyme with this specificity has now been confirmed by the enzymic fragmentation of the fucosylated product of the low molecular weight N-glycan, NGA3 (Figure
The third activity detected in the COS cell extracts was the transfer of fucose that took place with asialo-fetuin as acceptor. Optimal transfer occurred at pH 5.5 and the apparent properties of the enzyme differed from the [alpha]1,3-fucosyltransferase activity by virtue of its susceptibility to heat and from the [alpha]1,6-fucosyltransferase activity by its susceptibility to NEM. These differences led us to postulate the presence of a third fucosyltransferase in the COS cell extracts (Clarke and Watkins, 1997a). The fucose was not released from the fucosylated asialo-fetuin product by treatment with [alpha]1,2, [alpha]1,3, or [alpha]1,6-fucosidases. Fetuin has six carbohydrate moieties per molecule, three oligosaccharide chains O-linked to serine and threonine (Spiro and Bohyroo, 1974) and three oligosaccharides N-glycosidically linked to asparagine (Spiro, 1964). The release of the [14C]labeled oligosaccharides from the fucosylated asialo-fetuin product on treatment with N-glycanase had earlier demonstrated that the added fucose residues were attached to the N-linked chains (Clarke and Watkins, 1997a). Frequently, asialo-fetuin is used as a substrate for [alpha]1,2-fucosyltransferases acting on the terminal nonreducing galactose residues or for [alpha]1,3-fucosyltransferases acting on the subterminal N-acetylglucosamine residues of the complex triantennary N-linked oligosaccharide chains. However, the fact that the triantennary low-molecular-weight glycan, NA3 (Figure
COS cells have previously been used for the expression of cloned human [beta]-galactosidase genes (Oshima et al., 1988; Moreau et al., 1989) and small amounts of endogenous activity have been reported with methyl umbelliferyl galactoside as substrate. This enzyme was therefore a potential candidate for the breakdown of the asialo-fetuin and NA3. Glycosidase tests with p-nitrophenyl glycosides revealed a very weak [beta]-galactosidase and stronger [beta]-N-acetylglucosaminidase (Figure
The enzyme activities measured in the COS cell extracts do not appear to readily account for the antigens expressed on the cell surface. Although homologs of FUT5 and FUT7 were detected in the PCR products, and the [alpha]1,3 fucosyltransferases encoded by these genes have the capacity to complete the synthesis of sialyl-Lex (Weston et al., 1992a; Natsuka et al., 1994), the enzyme measured in the cell extracts resembles the FUT4 gene product which has only very low activity towards sialylated Type 2 acceptors (Lowe et al., 1991). Similarly, the synthesis of the Ley structure requires expression of an [alpha]1,2 fucosyltransferase and, although a homolog of FUT1 was detected in the PCR products, it has not been possible to find a measurable amount of [alpha]1,2 fucosyltransferase activity in the cell extracts. Domino et al. (1997) found that transfection of mouse FUT1 into COS-7 cells led to significant amounts of blood group A and B determinants through the presence of endogenous A ([alpha]1,3-N-acetylgalactosaminyl-) and B ([alpha]-1,3-galactosyl-) transferases that were adding sugars to the precursor H structures synthesized by the transfected gene product. We confirmed the presence of A transferase activity in the COS cell extracts and found weak expression of A antigenic activity on the surface of the untreated COS cells. Therefore, although it was not possible to detect any [alpha]1,2-fucosyltransferase activity, the expression of both Ley and blood group A antigens on the cell surface indicate that formation of H structures must have occurred at some stage to provide the requisite precursor substrates for the [alpha]1,3-fucosyltransferase and [alpha]1,3-N-acetylgalactosaminyltransferase, respectively, to complete the syntheses of the Ley and A determinant structures.
Discussion
Nucleotide sugar
Acceptor
Enzyme source (% [14C]sugar incorporated)
COS cells
A431 cells
UDP-[14C]GalNAc
Fuc[alpha](1-2)Gal[beta](1-4)Glc
16.5
59.0
GDP-[14C]fucose
Phenyl [beta]-d-Gal(1.6 mM)
0
9.5
Phenyl [beta]-d-Gal(10 mM)
1
-
Gal([beta]1-3)-4-deoxy GlcNAc-R
0
16.0
Gal([beta]1-4)-3-deoxy-GlcNAc-R
0
3.6
Gal([beta]1-4)GlcNAc-R
7.2
14.9
Gal([beta]1-3)GlcNAc-R
0
71.2
Materials
GDP-l-[14]fucose (290 mCi/mmol), UDP-N-acetyl-d-[14C]-galactosamine (60 mCi/mmol), and [14C]lactose (60 mCi/mmol) were purchased from Amersham, UK N-Acetyllactosamine (Gal[beta]1-4GlcNAc) and 2[prime]fucosyllactose (Fuc[alpha]1-2Gal[beta]1-4Glc) were gifts of Dr. A. S. R Donald (formerly of the MRC Clinical Research Centre, Harrow, UK). Compounds with hydrophobic spacer arms attached Gal[beta]1-4GlcNAc-R, Gal[beta]1-3GlcNAc-R, Gal[beta]1-4-(3-deoxy)GlcNAc-R and Gal[beta]1-3-(4-deoxy)-GlcNAc-R (where R = (CH2)8COOMe), were the gifts of Professor R.U.Lemieux, University of Alberta, Edmonton. Fuc[alpha]1-6-GlcNAc was purchased from Dextra Laboratories Ltd., Reading, UK. Dowex 1, Sephadex-G50, D-galactono-1,4-lactone, asialo-fetuin, and N-ethylmaleimide (NEM) were purchased from Sigma UK Asialo-agalacto-fetuin was prepared as described previously (Clarke and Watkins, 1997a).
The structures of the bi- and tri-antennary N-glycans used as low-molecular-weight enzyme substrates are shown in Figure
Jack bean [beta]-N-acetylhexosaminidase, recombinant Endoglycosidase F3 (EndoF3) from Flavobacterium meningosepticum and bovine kidney [alpha]1,6-fucosidase were purchased from Oxford Glycosciences, Oxford, UK. [alpha]1,3/4-fucosidase from Streptomyces spec. was obtained from Boehringer Mannheim, Lewis, UK, and [alpha]1,2-fucosidase from Xanthomonas maniatis from New England Biolabs (UK) Ltd., Hitchin, UK.
Cell lines and cell culture
All commercial growth media were supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. COS-7 cells were used for all the experiments involving COS cells described in this article.
COS-7 cells, obtained from the European Collection of Animal Cell Cultures (PHLS Centre for Applied Microbiology, Porton Down, UK) and A431 cells supplied by Dr. W. Gullick (Imperial Cancer Research Fund Unit, Hammersmith Hospital, London) were grown in Dulbecco's Modified Eagle Medium (DMEM; Life Technologies Inc., UK). The cells were grown to confluence in a 5% CO2 atmosphere and the adherent cells were detached by treatment for 5 min with a solution of trypsin (0.5%) and EDTA (0.2%). The detached cells were washed with PBS and counted in a hemocytometer. HepG2 cells provided by Dr. Naveenan Navaratnam, Hammersmith Hospital, London, were grown to confluence in RPM1 1640 medium (Life Technologies Inc. Ltd., UK) and harvested, as described above. HL-60 cells and K562 cells, obtained from the European Collection of Animal Cell Cultures, were grown in free suspension in RPMI 1640 medium and were harvested by centrifugation at 800 × g for 5 min, washed with PBS, and counted.
RT-PCR analysis
RNA extraction. Total RNA was isolated according to the manufacturer's instructions from 107 cells using an RNeasy kit (Qiagen Ltd., Crawley, UK) Contaminating genomic DNA was removed by digestion with 20 U of RNase-free DNase (RQ1) (Promega Ltd., Southampton, UK) for 1 h at 37°C. The RNeasykit was used again to repurify the total RNA after DNase digestion.
Reverse transcription. A reverse transcription kit from Promega Ltd. was used according to the manufacturer's instructions to transcribe 1 µg of total RNA. The reaction was carried out for 1 h at 42°C in a total volume of 20 µl, containing 1 µg oligo(dT) primers to initiate first-strand cDNA synthesis and 25 U of AMV reverse transcriptase. The enzyme was inactivated by heating for 5 min at 99°C .PCR reactions. Reactions were carried out in a final volume of 25 µl containing the reverse transcribed product ( in 2 µl), 12.5 pmol of each sense and anti-sense primer (Life Technologies Ltd., U.K) and 12.5 µl of Taq PCR Master mix from Qiagen Ltd. The PCR reactions for FUT1 and FUT4 were performed in the presence of 5% dimethyl sulfoxide. For detection of FUT4, FUT7, FUT8, and G6PD cDNA the PCR protocol was 1 min at 94°C, 1 min at 60°C and 1 min at 72°C for 35 cycles. For detection of FUT3, FUT5, and FUT6 cDNA the PCR protocol was 1.5 min at 94°C and 3.5min at 72°C for 35 cycles. The PCR protocol for detection of FUT1 cDNA was 20 s at 94°C, 1 min at 65°C, and 1 min at 72°C for 30 cycles and for FUT2 cDNA was 1.5 min at 94°C and 3 min at 72°C for 30 cycles.PCR products were separated by electrophoresis on a 1.4% agarose gel and visualized with ethidium bromide. Amplification of G6PD cDNA was used as a positive control reaction to confirm successful first strand cDNA synthesis and reactions without reverse transcriptase were carried out to exclude the possibility of amplification of contaminating genomic DNA.
Primer sequences and product sizes. All PCR reactions were carried out with primer sequences previously published for human fucosyltransferase genes except those used for FUT8. For this gene the sense primer 5[prime]-CACTTGGTACGAGATAATGAC-3[prime] (nucleotides 82-103) and antisense primer 5[prime]-CACATGATGGAGCTGACAGCC-3[prime] (nucleotides 661-682) were designed from the human FUT8 sequence (Yanagidani et al., 1997) to amplify a 600 bp product.Published primers were used to amplify an 890 bp FUT1 product (Koda et al., 1997), a 1149 bp FUT2 product (Yu et al.,1995), a 516 bp FUT4 product (Sasaki et al., 1994), and a 497 bp FUT7 product (Sasaki et al., 1994). Published primer sequences were used to amplify a 342 bp G6PD transcript (Hochhaus et al., 1996) included as a positive control. A single set of primers (Weston et al., 1992a) were used to amplify FUT3 and 5. To distinguish the two products digestion was carried out with Nae1 and Pflm1. The FUT5 fragment (486 bp) is digested by Nae1 whereas the FUT3 fragment (447 bp) is digested by Pflm1. The primers used to amplify FUT6 gave a fragment of 404 bp, which can be digested by PvuII, and were designed not to amplify the closely similar FUT3 or FUT5 (Yago et al., 1993).
Indirect immunofluorescence
Antibody binding was assessed by indirect immunofluorescence as described previously (Clarke and Watkins, 1996). The following monoclonal antibodies were used undiluted as supplied: LeuM1 (anti-Lex) (Bettleheim, 1989) was purchased from Becton Dickinson (Mountain View, CA), CSLEX1 (anti-sialyl-Lex; Fukushima et al., 1984) was a gift from Dr. Paul Terasaki (UCLA Tissue Typing Laboratory, CA): KM93 (anti-sialyl-Lex; Shitara et al., 1987) was purchased from Serotec Ltd., UK; AH6 (anti-Ley; Nudelman et al., 1986) was supplied by Dr. S. Hakomori (Pacific Northwest Research Foundation, Seattle, WA) and HI8A (anti-Ley; Hirashima, 1990) was purchased from Serotec Ltd., UK; H11 (anti-H Type 2; Knowles et al., 1982) was supplied by Dr. G. L. Daniels, MRC Human Blood Group Unit, University College, London, and anti-blood-group-A was obtained from Seraclone, UK. Antibody bound to the cells was stained with R-phycoerythrin-conjugated goat F(ab[prime])2 anti-mouse immunoglobulins (Dako Ltd., UK) and analyzed by flow cytometry on a Becton Dickinson FACScan analyzer.
Fucosyltransferase assays
Freshly harvested COS cells (0.4 × 108 cells) were suspended in 0.75 ml PBS and lysed by addition of 0.25 ml 1% Triton X-100 for 60 min at 4°C. Standard reaction mixtures for [alpha]1,2-, [alpha]1,3-, and [alpha]1,6-fucosyltransferase assays contained in a final volume of 80 µl: 0.48 µM GDP-[14C]fucose (24,000 c.p.m.), 12.5 mM MnCl2 , 63 mM sodium cacodylate buffer pH 7.0, 3 mM ATP, 0.06% (v/v) Triton X-100, 20 µl of cell extract, and either 3 mM low molecular weight oligosaccharide with free reducing group, 800 µM low-molecular-weight acceptor with hydrophobic spacer ({CH2}8COOMe) arm attached, or 400 µg of glycoprotein acceptor. The reaction mixtures containing the low molecular weight disaccharide acceptors or asialo-fetuin were incubated for 18 h. Assays for [alpha]1,6-fucosyltransferase with asialo-agalacto-fetuin as substrate were incubated for 5 h except in the experiment in which the incubation time was increased to 18 h in order to match the time used for the transfer of fucose to asialo-fetuin. For measurement of the acceptor specificity of the N-glycan oligosaccharides at pH 5.5, the reaction mixtures contained in a volume of 15 µl: 5.6 µM GDP-l-[14C]fucose (40,000 c.p.m.), 2 mM MnCl2, 63 mM sodium cacodylate buffer pH 5.5, 3.3 mM ATP, 0.06 % Triton X-100, 5 µl of COS cell extract (containing extract of 0.4 × 106 cells), and 666 µM di- or triantennary oligosaccharides. The composition of the mixtures for the reactions carried out with the N-glycans at pH 7.0 was essentially the same as that for the other low molecular acceptors except that the total volume was 15 µl, and the volumes of the other components were reduced accordingly. The reaction mixtures were incubated for 3.5 h at 37°C.
The incubation mixtures containing the neutral and sialylated low-molecular-weight oligosaccharides were separated on mini-columns of Dowex-1 resin (formate form) and those containing glycoprotein acceptors were separated on columns of Sephadex G-50 (Clarke and Watkins, 1997b). The mixtures containing compounds with hydrophobic groups were separated on Sep-Pak cartridges (Palcic et al., 1988). The eluates containing the radioactive products were mixed with water-miscible scintillant and counted in a Beckman scintillation counter.
Blood-group-A transferase activity
A transferase ([alpha]1,3-N-acetyl-d-galactosaminyltransferase) was measured with 2-fucosyllactose as substrate. The reaction mixture contained in a final volume of 100 µl: UDP-[14C]GalNAc (6.5 µM, 65,000 c.p.m..); 2[prime]-fucosyllactose, 2.5 mM; MnCl2, 20 mM; ATP, 5 mM; sodium cacodylate buffer pH 6.0, 50 mM; and 20 µl COS cell extract. Mixtures were incubated for 16 h and separated on columns of Dowex-1 as described previously (Navaratnam et al., 1990)
Antibody neutralization tests
Aliquots (76 µl) of a 1:2 dilution of a polyclonal rabbit anti-[alpha]1,3/4-fucosyltransferase serum directed towards partially purified [alpha]1,3/4-fucosyltransferase from human milk (Watkins et al., 1993; Johnson et al., 1995) were mixed with 76 µl aliquots of COS and HL-60 cell extracts and human plasma from a blood group Oh donor (Bombay) who lacked [alpha]1,2-fucosyltransferase activity. The cell extracts from HL-60 cells were prepared in the same way as the COS cell extracts from approximately the same number of cells. The mixtures were each left 4 h at 4°C and duplicate 20 µl aliquots were then assayed for fucosyltransferase activity by standard procedures. Asialo-fetuin and N-acetyllactosamine were the substrates for the COS cell and human plasma enzymes and N-acetyllactosamine the substrate for the HL-60 cell enzyme. The assays were performed at pH 7.0 except for the assay of the COS cell extract with asialo-fetuin, which was carried out at pH 5.5.
Glycosidase treatment of products of fucosyltransfer to N-glycans
The products formed with NA3 and NGA3 oligosaccharides were separated from unreacted GDP-[14C]fucose on Dowex-1x8 mini-columns. The eluate was concentrated on a Savant Speedivac Plus SC110A apparatus and aliquots containing ~7000 c.p.m. (20-40 µM) were then treated with either 36 mU of [alpha]1,6-fucosidase in 100 mM sodium citrate buffer pH 6.0, or 25 µU of [alpha]1,3/4-fucosidase in 100 mM sodium citrate buffer pH 6.0, or 5 mU Endo-F3 in 1% Triton X-100/200 mM sodium acetate buffer pH 4.5, or 1 U [beta]-N-acetylhexosaminidase in 100 mM sodium citrate/phosphate buffer pH 5.0. The mixtures were incubated at 37°C for 24 h and the products were examined by descending chromatography on Whatman no. 40 paper in pyridine/ethyl acetate/acetic acid /water (5:5:1:3 by vol, solvent a). The paper was cut into 2 cm strips and counted in a Beckman scintillation counter. The mobility of the compounds was measured relative to fucose (Rfuc).
To determine the linkage specificity of the disaccharides released from NA3 and NGA3 by Endo F3 the products were separated from the remainder of the reaction products by descending chromatography on Whatman N[ogr]40 paper in ethyl acetate/pyridine/water (10:4:3 by vol., solvent b). The [14C]fucose labeled disaccharides were eluted from paper, the eluate was concentrated, and aliquots were treated for 24 h at 37°C with either 30 mU [alpha]1,6-fucosidase, 4 U of [alpha]-1,2-fucosidase, or 15 µU of [alpha]1,3/4-fucosidase. The products were separated by thin layer chromatography on a HPTLC silica gel 60 plate (Merck, UK) in chloroform/methanol/acetic acid /water (25:15:2:4 by vol., solvent c). The plate was sprayed with Enhance (Dupont) and exposed to Kodak X-Omat AR5 film at -70°C. [14C]Labeled fucose was run as control. The unlabeled Fucosyl[alpha]1-6-N-acetylglucosamine and its hydrolysis products were detected by spraying the TLC plate with orcinol ferric chloride (Sigma, UK), followed by heating for 5 min at 110°C.
Glycosidase activities in COS cell extracts
[beta]-Galactosidase activity with p-nitrophenyl [beta]-d-galactoside as substrate was measured by mixing, in a total volume of 300 µl, 3.3 mM substrate, 20 µl of COS cell extract, and 120 mM buffer (sodium cacodylate pH 3.5-7.0; Tris/HCl pH 7.0-9.0. The mixtures was incubated for 1 h at 37°C, and the reaction was then stopped by the addition of 2 ml of 0.2 M Na2CO2. The tubes were spun briefly at 10,000 × g and the absorbance of the liberated p-nitrophenol in the supernatant was measured at 420 nm. [beta]-Hexosaminidase activities were measured with p-nitrophenyl N-acetyl-[beta]-d-glucosaminide and p-nitrophenyl N-acetyl-[beta]-[delta]-galactosaminidase substrates under the same conditions as for [beta]-galactosidase activity.
[beta]-Galactosidase activity with (d-glucose-1-14C]lactose as substrate was measured by incubating 0.34 nmol of substrate (46,000 c.p.m.) with 15 µl COS cell extract and 30 mM sodium cacodylate buffer pH 5.5 for 22 h at 37°C. The products were separated by descending paper chromatography for 16 h on Whatman no. 40 paper in solvent b and the strips were cut up and counted in a scintillation counter as described above. For the pH curve sodium cacodylate buffer was used for pH values 4.0-7.0 and Tris/HCL for pH values 7.0-8.5. The mobility of the products was measured relative to lactose (Rlac) and fucose (Rfuc).
Treatment with NEM
NEM was added to the fucosyltransferase assay mixtures with either asialo-fetuin or asialo-agalacto-fetuin as substrates, or to [beta]-galactosidase assays with either [14C]lactose or p-nitrophenyl [beta]-d-galactosidase as substrates, immediately before incubation to give final NEM concentrations ranging from 0.01 mM to 10 mM. Assays were then carried out by the standard procedures.
Inhibition tests with d-galactono-1,4-lactone
To test for inhibition of [beta]-galactosidase activity with [14C]lactose as substrate, or inhibition of fucosyltransferase activity with either asialo-fetuin or asialo-agalacto-fetuin as substrates, enzyme activities were measured by the standard procedures except that d-galactono-1-4-lactone was added to the reaction mixtures at a final concentration of 0.5%.
We thank Drs. S. Hakomori and P. Terasaki for the gifts of monoclonal antibodies, Professor R. Lemieux and the late Dr. A. S. R. Donald for the provision of substrates and Drs. W. Gullick and N. Navaratnam for gifts of cell lines. We are grateful to Dr. Nadia Le Marer for advice on PCR procedures. This work was supported by a grant from the Wellcome Trust.
Lex, Gal[beta]1-4[Fuc[alpha]1-3]GlcNAc; sialyl-Lex, NeuAc[alpha]2-3Gal[beta]1-4[Fuc[alpha]1-3]GlcNAc; 2[prime]-fucosyllactose, Fuc[alpha]1-2Gal[beta]1-4Glc; Ley, Fuc[alpha]1-2Gal[beta]1-4[Fuc[alpha]1-3]GlcNAc; [alpha]1,3-fucosyltransferase, GDP-l-fucose: N-acetyl-[beta]-d-glucosaminide 3-[alpha]-l-fucosyltransferase; [alpha]1,2-fucosyltransferase, GDP-l-fucose: [beta]-d-galactoside 2-[alpha]-l-fucosyltransferase; [alpha]1,6-fucosyltransferase, GDP-fucose: N-acetyl-[beta]-d-glucosaminide: [alpha]1,6-fucosyltransferase; blood-group A transferase, UDP-N-acetylgalactosamine: [beta]-d-galactoside [alpha]1,3-N-acetylgalactosaminyltransferase; RT-PCR, reverse transcriptase-polymerase chain reaction; FUT1 and FUT2 , human [alpha]1,2-fucosyltransferase genes; FUT-3-7, human [alpha]1,3-fucosyltransferase genes; FUT8 , human [alpha]1,6-fucosyltransferase gene; kb, kilobase; bp. base pair; DMSO, dimethylsulfoxide; NEM, N-ethylmaleimide; FACS, fluorescence-activated cell sorter.
1To whom correspondence should be addressed