Production of soluble human {alpha}3-fucosyltransferase (FucT VII) by membrane targeting and in vivo proteolysis

Theodora de Vries1,2, Janet Storm2, Francien Rotteveel3, Geert Verdonk3, Marcel van Duin3, Dirk H. van den Eijnden2, David H. Joziasse2 and Hans Bunschoten3

2Department of Medical Chemistry, Vrije Universiteit Amsterdam, Van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands, and 3Research and Development Group, N.V. Organon, Oss, the Netherlands

Received on January 30, 2001; revised on April 23, 2001; accepted on April 23, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The rational design of fucosyltransferase (FucT VII) inhibitors as potential medication in the treatment of rheumatoid arthritis requires the three-dimensional structure of this member of the glycosyltransferase family. Structure determination by X-ray diffraction analysis needs purified, soluble enzyme protein. For this purpose we developed a novel method for the high-yield production of soluble FucT VII by in vivo proteolysis. To obtain a soluble form of FucT VII a mammalian expression construct was made encoding an N-terminal portion of FucT VI (amino acids 1–63) fused with the stem region and catalytic domain of FucT VII (amino acids 39–342). Chinese hamster ovary cells stably transfected with this construct produced FucT activity in the supernatant, which has the same catalytic properties as wild-type FucT VII. This soluble form of FucT VII can be obtained in high amounts (1 mg/L) and can be efficiently purified by GDP-hexanolamine affinity chromatography. In conclusion, it was demonstrated that the intrinsic properties of FucT VII could be transferred to secreted FucT VII constructs, which may open possibilities for production of soluble forms of other members of the glycosyltransferase family as well.

Key words: CHO cells/fucosyltransferase/secretion


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The {alpha}3-fucosyltransferases (FucTs) belong to a group of 100 or more membrane-bound glycosyltransferases, mostly located in the Golgi apparatus, that are collectively responsible for the synthesis of the sugar chains of soluble and cell-surface glycoconjugates (Kukuruzinska and Lennon, 1998Go and references therein). FucTs catalyze the reaction GDP-Fuc + Galß1->4GlcNAc-R {Rightarrow} GDP + Galß1->4[Fuc{alpha}1->3]GlcNAc-R.

The human FucT family consists of six members, of which the cDNAs have been cloned. Their protein products show a high degree of sequence similarity and have been named FucT III (Kukowska-Latallo et al., 1990Go), FucT IV (Goelz et al., 1990Go; Lowe et al., 1991Go; Kumar et al., 1991Go), FucT V (Weston et al., 1992aGo), FucT VI (Weston et al., 1992bGo; Koszdin and Bowen, 1992Go), FucT VII (Sasaki et al., 1994Go; Natsuka et al., 1994Go), and FucT IX (Kaneko et al., 1999Go). Some of these cloned FucTs have been related with enzyme activities detected in human tissues. Based on expression pattern and substrate specificity, FucT VII is the likely candidate responsible for the synthesis of functional selectin ligands on the cell surface of leukocytes (Austrup et al., 1997Go). These ligands, which are 3'-sialylated- and/or sulfated-LewisX structures, are essential in the recruitment of leukocytes to the site of an inflammation by mediating adhesion to the vascular endothelium via the endothelial receptors E- and P-selectin. Previously, the relevance of the FucT VII enzyme for migration of T cells to inflammatory sites has been confirmed by targeted mutation ("knock-out") of the FucT VII gene in mice (Maly et al., 1996Go).

Because inhibitors specific for FucT VII may have potential as anti-inflammatory drugs, for example in rheumatoid arthritis, where leukocytes extravasate from the circulation to the synovia of the joints leading to cartilage destruction (Cush et al., 1992Go), FucT VII is our target molecule for the design of inhibitors. Up to now, no inhibitors specific for any FucT have been described other than guanosine diphosphate (GDP) or derivatives thereof (Murray et al., 1996Go). For the rational design of inhibitors the 3D structure of FucT VII will be necessary. One way of obtaining a 3D structure is through X-ray analysis of protein crystals, in which case large quantities of soluble, pure enzyme have to be produced.

Generally, FucTs are membrane-bound and reside on the luminal side of the Golgi vesicles (Paulson and Colley, 1989Go). However, soluble forms have been demonstrated in such body fluids as milk, serum, amniotic fluid, seminal plasma, and saliva (Mollicone et al., 1990Go; De Vries et al., 1997Go and references therein). Production methods for FucT VII have been published, but involve either full-length enzyme (Natsuka et al., 1994Go; Britten et al., 1998Go) or truncated protein A fusion proteins (Sasaki et al., 1994Go; Smithers et al., 1997Go). Ideally, for successful crystallization both the transmembrane region and the protein A portion should be absent.

Previous reports have suggested that the expression level of FucT VII is low relative to other FucTs (Britten et al., 1998Go and references therein). In contrast, FucT VI has a relatively high expression level. Furthermore, expression of FucT VI in Chinese hamster ovary (CHO) or BHK-21 cells yields a truncated protein, which is efficiently secreted in the medium (Borsig et al., 1998Go; Grabenhorst et al., 1998Go). Interestingly, Sasaki et al. (1994)Go reported that enzymatic activity of a truncated form of FucT VII could only be achieved by incorporating a portion of the FucT VI sequence (amino acids 40–54) between the FucT VII catalytic domain (amino acids 39–342) and the protein A part. In the present report we utilized those properties of FucT VI to create a soluble form of FucT VII with high specific activity. For this purpose we fused the cytoplasmic, transmembrane, and stem (CTS) regions of FucT VI (amino acids 1–63) with the catalytic domain of FucT VII (amino acids 39–342). CHO cells stably transfected with this construct indeed produced a soluble FucT activity in the supernatant. The enzymatic properties of this enzyme were characterized and compared to the enzymatic properties of cell homogenates of CHO cells stably transfected with full-length, wild-type FucTVII cDNA. Indeed, the characteristics of our soluble enzyme were demonstrated to match those of wild-type FucT VII. This soluble form of FucT VII can be obtained in high amounts and is easy to purify. Furthermore, our procedure yields a concentrated enzyme solution that seems suitable for crystallization studies and might be more generally applicable for other members of the FucT family as well as other glycosyltransferases.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Expression and purification of soluble FucT VII
To construct a hybrid FucT VI–VII gene, first the coding sequences for both genes have to be cloned. The absence of intronic sequences allowed direct polymerase chain reaction (PCR) amplification of the FucT VI gene. Due to the existence of an intron in the FucT VII gene (Britten et al., 1998Go), reverse transcription (RT)-PCR was used to amplify the FucT VII cDNA from poly(A)+ messenger RNA from U937 cells. For PCR amplification of FucT VII addition of 15% glycerol was necessary to obtain a distinct DNA fragment of the expected size. An expression vector (pNGV1.FTVII.pepVI) was constructed as follows. First, a fragment coding for the FucT VII stem region and catalytic domain (amino acids 39–342) was obtained by digestion of pNGV1.FTVII. Subsequently, the sequence coding for the CTS regions (amino acids 1–63) of FucT VI was synthesized by PCR. This sequence coding for the N-terminal part of FucT VI was ligated upstream to the fragment coding for the C-terminus of FucT VII. The complete DNA and amino acid sequences are shown in Figure 1. Expression of this construct in CHO cells resulted in the secretion of a soluble, enzymatically active FucT. Culture of the CHO cell clone in a serum-free fermentor system consistently produced 1.6 U/L. Pooled supernatants were kept at 4°C for several months without loss of FucT VII activity. FucT VII could be purified to homogeneity in a one-step procedure, on GDP-hexanolamine-Sepharose, in almost quantitative yield. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) revealed that the purified protein consisted of two glycoforms with apparent molecular weights of 55,000 and 49,000, respectively (Figure 2). Treatment of FucT VII with PNGaseF yielded one single band of 39,000 (PNGaseF is visible as well as a band of 36,000). N-terminal sequencing revealed that the protein started at either amino acid Tyr33 or Val36 in a 1:1 ratio. The specific activity of soluble FucT VII was 1.6 U/mg using the protein assay by Bradford (1976)Go, with bovine serum albumin (BSA) as standard, and optimal reaction conditions with fetuin as acceptor substrate. We subjected this purified enzyme preparation to an extended characterization study to ensure that no properties of FucT VI were conferred with the introduction of the stem region of FucT VI and that the substrate preferences of FucT VII were not modified.



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Fig. 1. Nucleotide and amino acid sequences of FucT VII.pepVI. The transmembrane domain is underlined. The FucT VI fragment is indicated in boldface type. The proteolytic cleavage sites are indicated by arrows.

 


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Fig. 2. SDS–PAGE electrophoresis of recombinant soluble FucT VII. Lane Mw, molecular weight markers; lane +, FucT VII treated with PNGaseF; lane –, untreated FucT VII. For each lane 1 µg of pure FucT VII was applied.

 
Optimal reaction conditions
To determine the optimal pH for FucT VII, the activity of the soluble enzyme was measured in six different buffers in the standard activity assay. As is shown in Figure 3 the enzyme was active at a broad pH range. Only at low pH the FucT VII activity was decreased. In contrast, enzyme activity was not affected by higher pH values. Divalent cations were tested for their ability to activate the enzyme (Figure 4). The activity was stimulated by the presence of Mn2+ and Mg2+, and to a lesser extent by Ca2+ and Co2+. Cu2+, Fe2+, and Zn2+ had an inhibitory effect on the activity of FucT VII. Mn2+ enhanced the activity best and was tested at a concentration range of 0–45 mM. At 15 mM the enhancing effect was optimal. Because FucTs generally are assayed in the presence of 100 mM NaCl, the effect of increasing amounts of NaCl in the reaction mixture was tested. At 250 mM the activity was inhibited by 50% and at concentrations higher than 500 mM the enzyme activity was absent.



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Fig. 3. Effect of pH on the FucT VII activity. FucT VII activity was measured at different pH levels in 50 mM sodium acetate (filled diamonds), sodium cacodylate (open diamonds), 2-[N-morpholino]ethanesulfanic acid (filled triangles), 3-[N-morpholino]propanesulfonic acid (open squares), Tris (filled squares), and glycine (open triangles) buffer.

 


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Fig. 4. Effect of divalent cations on the FucT VII activity. FucT VII activity was measured in the presence of 15 mM of the indicated cation or 100 mM ethylenediaminetetraacetic acid.

 
Acceptor substrate specificity of soluble FucT VII
The acceptor substrate specificity of soluble FucT VII toward a variety of type 1 (Galß1->3GlcNAc-R) and type 2 (Galß1->4GlcNAc-R) chain-based oligosaccharide and glycoprotein substrates is shown in Table I. FucT VII preferentially acted on sialylated type 2 chain structures. FucT VII failed to act on any other substrate, only sialylated type 1 was used with poor efficiency. The apparent V and Km values for sialyl-N-acetyllactosamine (sialyl-lacNAc), fetuin, and GDP-fucose are listed in Table II. The affinity for glycoprotein substrates (fetuin) was 10 times higher than for oligosaccharide substrates (sialyl lacNAc), yet the maximum velocities were in the same range. The Km for GDP-fucose was in the same range (6–9 µM) as the Km from FucTs III–VI (De Vries et al., 1997Go). The acceptor specificity of soluble FucT VII was demonstrated to match the acceptor specificity of enzyme activity in cell lysates of CHO cells stably transfected with full length FucT VII cDNA (data not shown).


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Table I. Acceptor specificity of recombinant soluble FucT VII
 

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Table II. Kinetic parameters of recombinant soluble FucT VII
 
Inhibition by nucleotides
Measuring the activity in the presence of various nucleotide diphosphates (Figure 5, top), determined the specificity of FucT VII for the nucleotide portion of the donor sugar. FucT VII was only inhibited by GDP, indicating that there is a strict preference for guanosine. The number of phosphate groups seemed less critical because FucT VII was also inhibited by guanosine triphosphate and guanosine monophosphate, but to a lesser extent than by GDP (Figure 5, bottom).




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Fig. 5. Effect of nucleotides on the FucT VII activity. The inhibition by various nucleotides was measured by assaying the FucT VII activity in the presence of (top) ADP (filled squares), GDP (triangles), CDP (diamonds), UDP (open squares) or (bottom) GMP (squares), GDP (triangles), GTP (diamonds) at concentrations indicated in the standard reaction mixture.

 
Inhibition by NEM
Fucosyltransferases have been classified by their sensitivity to inhibition by N-ethylmaleimide (NEM), a cysteine modifying reagent (Mollicone et al., 1990Go). FucTs III, V, and VI are significantly inhibited by NEM (more than 98%), due to a free cysteine residue in the middle of the catalytic domain. FucT IV, which has a serine in the equivalent position, is resistant to NEM (Holmes et al., 1995Go). Recombinant soluble FucT VII appeared to be resistant to inhibition by NEM, which is consistent with the presence of a threonine residue at that position.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Structural studies on glycosyltransferases require purified, soluble enzyme protein. However, most glycosyltransferases are type II membrane proteins residing in the endoplasmic reticulum and the vesicles of the Golgi apparatus, where they glycosylate newly formed proteins in an ordered manner. Yet some of these enzymes appear in soluble forms in serum and in secretions such as milk, saliva, and other body fluids (Beyer et al., 1981Go; Paulson and Colley, 1989Go). It is assumed that they are a result of proteolytic cleavage in their stem region by specific proteases. Among glycosyltransferases, the soluble forms appear to be predominantly galactosyltransferases, sialyltransferases, and fucosyltransferases. However, when the stem regions of these enzymes are compared, it is not clear why those enzymes are cleaved and others are not. For instance, in bovine milk two forms of the ß1,4-GalT have been demonstrated by D'Agostaro et al. (1989)Go, starting at residues 79 and 106. A soluble form of Gal {alpha}2,6-sialyltransferase starting at residue 63 (Weinstein et al., 1987Go) was suggested to be released from rat liver in response to induced inflammation, as a result of cleavage by a cathepsin D–like protease within the acidic trans-Golgi compartment (Lammers and Jamieson, 1989Go). Among FucTs, the ones that are found in body fluids and secretions are the plasma type (FucT VI) and the Lewis type (FucT III) enzyme. Mitsakos and Hanisch (1989)Go demonstrated a soluble fucosyltransferase activity in amniotic fluid, matching the acceptor specificity of FucT VI. Sarnesto et al. (1992)Go purified a similar activity from serum, and Brinkman-Van der Linden et al. (1996)Go demonstrated that this activity originates from the FUT 6 gene and is probably released from the liver. The soluble FucT activity in milk probably originates from a mixture of two FucTs, FucT III and FucT VI, secreted from mammary gland epithelial cells (De Vries et al., 1997Go).

Soluble forms of glycosyltransferases have also been detected in vitro in cell culture supernatants: For instance, glycosyltransferases are secreted from cell lines transfected with cDNA coding for full-length enzymes. Borsig et al. (1998)Go demonstrated secretion of recombinant FucT VI from CHO cells. The same group reported the presence of FucT VI in the hepatoma cell line HepG2, which was partially released into the medium by proteolytic cleavage (Borsig et al., 1999Go). Grabenhorst and Conradt (1999)Go studied the targeting signals in the CTS region of several glycosyltransferases in BHK-21 cells. Apparently, there are at least three different signals contained in the CTS region mediating Golgi retention, targeting to specific functional areas, and susceptibility toward intracellular proteolysis. Previously, the same group (Grabenhorst et al., 1998Go) reported that when each of the five FucTs (III to VII) is expressed in BHK-21 cells, FucT III, IV, and VI were proteolytically cleaved and released into the medium in significant amounts, whereas FucT V and VII were found to be largely resistant toward proteolysis. The function of these soluble versions of glycosyltransferases remains unknown. It is questionable whether they can exert a functional role, because the proper nucleotide donor sugars and acceptor substrates are not necessarily present in the same surroundings.

Although at present much effort is being put into resolving the 3D structure of FucT VII by X-ray analysis on protein crystals, studies on large-scale production of soluble FucT VII are limited. So far, all recent studies involve protein A chimeric molecules. Shinoda et al. (1997)Go expressed FucT VII as a soluble protein A chimeric form in a human cell line (Namalwa KJM-1), producing 0.6 mg/L. Smithers et al. (1997)Go replaced the cytoplasmic and transmembrane domains of FucT VII by a single protein A domain and used the baculovirus system to express the protein to a level of 2 mg/L. To obtain free, or "nonchimeric" FucT VII molecules, it might be possible to release protein A by proteolysis, when a cleavage site is added between the two fusion partners. However, contaminating proteins will have to be removed.

In this study we used the targeting signals in the CTS region of FucT VI for the large-scale production of soluble, recombinant FucT VII. By genetically engineering the CTS region of FucT VI to the catalytic domain of FucT VII, the new chimeric molecule is released into the culture medium. Interestingly, release appeared to be increased when CHO cells were grown under stress circumstances, such as medium depleted from glucose or (fetal calf) serum or at fluctuating temperatures (data not shown). N-terminal sequencing demonstrated that cleavage occurred at the C-terminus of the transmembrane domain (Tyr33 or Val36). This site was identical to the cleavage site found for recombinant, full-length FucT VI, expressed in BHK-21 cells (Grabenhorst and Conradt, 1999Go).

The influence of the CTS region of FucT VI on the catalytic activity of FucT VII was tested because the resulting soluble FucT VII molecule still contains amino acids 33–63 of FucT VI. Interestingly, this includes amino acids 40–54 (of FucT VI), reported by Sasaki et al. (1994)Go to be necessary for enzymatic activity of protein A fused, truncated FucT VII. Out of a panel of type 1 and type 2 chain–containing structures, our chimeric FucT VII enzyme appeared to strictly prefer sialylated type 2 structures as substrate. This is similar to the acceptor specificity of the protein A fused, truncated FucT VII (Shinoda et al., 1997Go), full-length FucT VII expressed in insect cells (Britten et al., 1998Go), and full-length FucT VII expressed in CHO cells (this study, data not shown). In conclusion, our results demonstrate that the chimeric enzyme has catalytic properties very similar to wild-type FucT VII. A FucT VI acceptor specificity pattern (action on nonsialylated lacNAc) was not introduced by the addition of the FucT VI stem region.

We present here a novel methodology for the high-yield production of soluble FucT VII by in vivo proteolysis. We developed this technique by making use of the relatively higher expression levels of FucT VI and the secretion signals that are contained in the CTS region of FucT VI, thereby adding only 30 amino acids to the FucT VII catalytic domain. The advantage is that no additional proteins or fusion partners (protein A) are required for secretion and solubility, an essential condition for protein crystallography. We anticipate that this production method will be applicable not only for other FucTs but also for nearly all glycosyltransferases.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Human placental DNA was obtained from Clontech. The mammalian expression vector pNGV1 was constructed as described (EMBL/Genbank, acc. no. X99274). N-terminal sequencing (by Edman degradation) was performed by Eurosequence (Groningen). Unlabeled GDP-fucose was purchased from Boehringer Mannheim. GDP-[14C]Fuc (250 Ci/mol) was purchased from New England Nuclear Corp. (Boston, MA) and diluted with the unlabeled nucleotide sugar to obtain a specific radioactivity of 5 Ci/mol. Calf fetuin was obtained from Gibco. The 8-methoxycarbonyloctyl glycoside acceptors were kindly provided by Dr. Ole Hindsgaul (University of Alberta, Edmonton, Alberta). LacNAc was purchased from Sigma and 3'-sialyl-lacNAc from Oxford GlycoSystems. All other chemicals were obtained from commercial sources and were of the highest purity available.

Oligonucleotides
Oligonucleotides 2026, CCAGAATTCGCCACCATGGATCCCCTGGGCCCG, and 2027, CGCGAATTCCATGCCGGCCTCTCAGGTGAA, were used for amplification of FucT VI. Oligonucleotides 2617, CCAGAATTCGCCACCATGAATAATGCTGGGCACGG, and 2660, CACCCAGCCCTTCCACCCACAC, were used for amplification of FucT VII. Oligonucleotides 2934, CTCGGTACCCTCGAATTCGCACCATGGATCC, and 2935, CTCGGTACCCAGGGGGATGGAGTGGGCGG, were used for amplification of the FucT VI N-terminal fragment.

DNA constructs
U937 cells were washed in phosphate buffered saline (PBS) and the cell pellet was stored at –70°C. Poly(A)+ RNA was prepared from 107 cells using the "quick prep mRNA isolation kit" from Pharmacia. cDNA synthesis was performed on 100 ng of poly(A)+ RNA with 1 µg random hexanucleotides and 100 U Superscript reverse transcriptase (BRL). cDNA corresponding to 10 ng of mRNA was used for PCR. PCR reactions were performed in a final volume of 100 µl containing 250 ng of pooled human placental DNA (FucT VI) or 10 µl of the supernatant of the cDNA synthesis (FucT VII); 40 pmol of each primer; 100 µM dNTPs (Pharmacia); 100 mM Tris–HCl, pH 8.8; 25 mM KCl; 1.5 mM MgCl2; 5 µg/ml gelatin; 15% glycerol (FucT VII); and 0.5 µl Taq polymerase (5 U/µl, Perkin Elmer). The reaction mixture was overlaid with mineral oil and subjected to 30 cycles of amplification in a 480 Perkin Elmer thermocycler using the following conditions: 1 min 94°C, 1 min 60°C, 1 min 72°C. The PCR products were digested with 20 U EcoRI for 3 h and analyzed on a 1% agarose gel. Agarose containing the DNA was cut into pieces, and DNA was retrieved using spin columns (Millipore). Two hundred nanograms of FucT fragment was mixed with 100 ng EcoRI digested, alkaline phosphatase–treated pNGV1 and ligated overnight, prior to amplification in the DH5{alpha} strain of Escherichia coli. Nucleotide sequencing was performed by the dideoxy chain termination method (Sanger et al., 1977Go) to check for PCR-induced mutations. The T7 sequencing kit (Pharmacia), and several sequence-specific synthetic oligonucleotide primers were used. The FucT VI and VII constructs (pNGV1.FTVI, pNGV1.FTVII) appeared to have a sequence identical to the published sequences. These two constructs were used to prepare the soluble form of FucT VII as follows: PNGV1.FTVII was digested with KpnI and BamHI, and a FucT VII fragment of 992 bp was isolated. A second fragment from pNGV1.FTVI was synthesized by PCR using oligonucleotides 2934 and 2935 as described above. 2935 introduced a KpnI site at the 3' site of the fragment. The PCR product was digested with EcoRI and KpnI and isolated. EcoRI/BamHI-digested pNGV1 (7197 bp) was ligated with the 992-bp KpnI/BamHI FucT VII fragment and the 202-bp EcoRI/KpnI FucT VI PCR fragment. After amplification in DH5{alpha}, the clones carrying plasmid DNA of correct size and orientation were sequenced and the correct one (pNGV1.FTVII.pepVI) was used for transfection in CHO cells.

Transfection of CHO cells and functional FucT expression
Tissue culture dishes containing 5 x 105 CHO cells were cotransfected as follows. Ten micrograms pNGV1.FTVII.pepVI and 2 µg pGEM.MTIIA DNA (possibility for Cd selection) were dissolved in 0.5 ml transfection buffer (192 mM HEPES, 55 mM glucose, 1.37 M NaCl, 50 mM KCl, 70 mM Na2PO4, pH 7.05) and carefully mixed with 31 µl 2 M CaCl2. After precipitation the DNA was spread over CHO cells and incubated for 15 min. 10 ml M505 (Dulbecco’s modified Eagle medium/F12 [Gibco] with L-glutamine, antibiotics, and mercaptoethanol) containing 10% fetal calf serum (FCS) ,was added and the dishes were incubated for 4 h. Glycerol shock was performed by incubating the dishes with 15% glycerol in PBS for 90 s at 37°C. Finally, after washing two times with PBS, the dishes were incubated for 24 h in M505 containing 10% FCS. Subsequently, selection was performed by adding 0.8 mg/ml G418. After selection, neopools were tested for FucT activity and for the expression of sLex by fluorescence-assisted cell sorting analysis. The neopool showing the highest FucT activity was used for single cell cloning.

Cell culture procedures
To obtain large quantities of soluble FucT VII, transfected CHO cells were cultured in 250 ml spinner flasks in M505 containing 5% FCS in the presence of microcarriers (Cultisphere S, 2.5 g/L). Culture medium was replaced by medium with 1% FCS after 2 days, and by serum-free medium supplemented with insulin and transferrin after 5 days. After 7 days FucT containing supernatant was harvested by leaving the cells to settle, decanting the supernatant, and replacing with fresh medium. This way, FucT VII containing supernatant could be harvested for several months and was stored at 4°C until use. Larger production was obtained in a 3.6-L continuous flow fermentor system, which was kept at essentially the same culture conditions as above.

Activity assay of soluble FucT VII
The standard reaction mixture contained in 50 µl: 5 nmol GDP-[14C]Fuc (4–5 Ci/mol); 2.5 µmol Tris–HCl pH 8.0; 0.75 µmol MnCl2; 0.2 µmol ATP; 0.5 mg fetuin (110 nmol acceptor sites calculated from galactose content of the protein); and an amount of enzyme preparation that did not convert more than 10% of the substrate during the assay period. Reactions were incubated for 1 h at 37°C. Incorporation of fucose was determined as described (De Vries et al., 1997Go). Values were corrected for incorporation into endogenous acceptors. Three replicates were used per data point. One unit of activity was defined as the amount of enzyme catalyzing the transfer of 1 µmol of fucose min–1.

Purification of soluble FucT VII
One liter of CHO culture supernatant was stirred with 1 ml GDP-hexanolamine-Sepharose beads (6 µmol/ml) overnight at 4°C. After adsorption of the FucT to the beads, the beads were allowed to settle, supernatant was decanted, and the enzyme containing beads were collected. The resin was washed with 25 mM cacodylate buffer, pH 6.8; containing 2 M NaCl; 25 % glycerol; and 0.05 % Na-azide. At this stage, the enzyme-on-beads can be stored in 25 mM cacodylate, pH 6.8; 25 % glycerol; 0.05 % Na-azide at 4°C for many months without loss of activity. Elution was performed by head-over-head rotation for 2 h, at RT, in 10 volumes of 25 mM cacodylate, pH 6.8; 0.2 M NaCl; 10 mM GDP; 1 mM MnCl2; 15 % glycerol; and 0.05 % Na-azide. The supernatant was collected by centrifugation (5', 1500 r.p.m.) and stored at 4°C, or concentrated on centricon cartridges (Amicon) to a desired protein concentration.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Antoon van Doornmalen, Han Kok, and Wim Koot for cell and fermentor cultures, and Dr. Ole Hindsgaul for providing us with the 8-methoxycarbonyloctyl glycoside acceptors. This research was supported by the Technology Foundation STW, applied science division of NWO, and the technology program of the Ministry of Economic Affairs (Grant #349-4211).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
BSA, bovine serum albumin; CHO, Chinese hamster ovary; CTS, cytoplasmic, transmembrane, and stem; FCS, fetal calf serum; FucT, fucosyltransferase; GalT, galactosyltransferase; GDP, guanosine diphosphate; lacNAc, N-acetyllactosamine; NEM, N-ethylmaleimide; PBS, phosphate buffered saline; PCR, polymerase chain reaction; RT, reverse transcription; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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Borsig, L., Katopodis, A.G., Bowen, B.R., and Berger, E.G. (1998) Trafficking and localization studies of recombinant {alpha}1, 3-fucosyltransferase VI stably expressed in CHO cells. Glycobiology, 8, 259–268.[Abstract/Free Full Text]

Borsig, L., Imbach, T., Hochli, M., and Berger, E.G. (1999) {alpha}1, 3 fucosyltransferase VI is expressed in HepG2 cells and codistributed with ß1, 4 galactosyltransferase I in the Golgi apparatus and monensin-induced swollen vesicles. Glycobiology, 9, 1273–1280.[Abstract/Free Full Text]

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Brinkman-Van der Linden, E.C.M., Mollicone, R., Oriol, R., Larson, G., Van den Eijnden, D.H., and Van Dijk, W. (1996) A missense mutation in the FUT6 gene results in total absence of {alpha}3-fucosylation of human {alpha}1-acid glycoprotein. J. Biol. Chem., 271, 14492–14495.[Abstract/Free Full Text]

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