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.
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Abstract |
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Key words: CHO cells/fucosyltransferase/secretion
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Introduction |
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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., 1990), FucT IV (Goelz et al., 1990
; Lowe et al., 1991
; Kumar et al., 1991
), FucT V (Weston et al., 1992a
), FucT VI (Weston et al., 1992b
; Koszdin and Bowen, 1992
), FucT VII (Sasaki et al., 1994
; Natsuka et al., 1994
), and FucT IX (Kaneko et al., 1999
). 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., 1997
). 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., 1996
).
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., 1992), 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., 1996
). 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, 1989). However, soluble forms have been demonstrated in such body fluids as milk, serum, amniotic fluid, seminal plasma, and saliva (Mollicone et al., 1990
; De Vries et al., 1997
and references therein). Production methods for FucT VII have been published, but involve either full-length enzyme (Natsuka et al., 1994
; Britten et al., 1998
) or truncated protein A fusion proteins (Sasaki et al., 1994
; Smithers et al., 1997
). 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., 1998 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., 1998
; Grabenhorst et al., 1998
). Interestingly, Sasaki et al. (1994)
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 4054) between the FucT VII catalytic domain (amino acids 39342) 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 163) with the catalytic domain of FucT VII (amino acids 39342). 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.
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Results |
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Discussion |
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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) 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., 1999
). Grabenhorst and Conradt (1999)
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., 1998
) 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) 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)
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, 1999).
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 3363 of FucT VI. Interestingly, this includes amino acids 4054 (of FucT VI), reported by Sasaki et al. (1994) to be necessary for enzymatic activity of protein A fused, truncated FucT VII. Out of a panel of type 1 and type 2 chaincontaining 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., 1997
), full-length FucT VII expressed in insect cells (Britten et al., 1998
), 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.
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Materials and methods |
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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 TrisHCl, 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 phosphatasetreated pNGV1 and ligated overnight, prior to amplification in the DH5 strain of Escherichia coli. Nucleotide sequencing was performed by the dideoxy chain termination method (Sanger et al., 1977
) 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
, 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 (Dulbeccos 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 (45 Ci/mol); 2.5 µmol TrisHCl 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., 1997). 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 min1.
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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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