Article
J. Korean Soc. Appl. Biol. Chem. 52(1), 11-16 (2009)
Expression of Recombinant Cyclooxygenase 1 in BTI Tn 5B1-4 Cells Transformed with Human β1,4-galactosyltransferase and Galβ1,4-GlcNAc α2,6-sialyltransferase Trichoplusia ni
Kyung Hwa Chang , Ki Hyun Yoo , Jeon Hwang-Bo , Yeon Ju Seok , Jong Min Lee , Kyung Il Kim , Youn Hyung Lee , Jai Myung Yang , Nam In Baek , and In Sik Chung * 1
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Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University, Yongin 446-701, Republic of Korea 2 College of Life Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea 3 Department of Life Science, Sogang University, Seoul 121-742, Republic of Korea Received October 21, 2008; Accepted December 20, 2008
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Expression of the human cyclooxygenase 1 (COX-1) in BTI Tn 5B1-4 cells transformed with cDNAs encoding β1,4-galactosyltransferase (GalT) and Galβ1,4-GlcNAc α2,6sialyltransferase (ST6Gal) was examined. Southern blot analysis indicated that the glycosyltransferase genes were integrated into the Tn 5B1-4 cell genome. A lectin blot analysis also indicated that the recombinant COX-1s from the Tn 5B1-4/COX-1/GalT-ST cells contained the glycan residues of β1,4-linked galactose and α2,6-linked sialic acid. The specific peroxidase activity of the recombinant sialylated COX-1 from Tn 5B1-4/COX-1/GalT-ST cells was 23,930 units/mg, indicating an increase of approximately 17% compared with the non-sialylated control (20,500 units/mg) from the Tn 5B1-4/COX-1 cells. Key words: recombinant cyclooxygenase 1, Trichoplusia ni BTI Tn 5B1-4 cells, β1,4Trichoplusia ni
galactosyltransferase, Galβ1,4-GlcNAc α2,6-sialyltransferase
COXs are the key enzymes in the biosynthesis and function of prostaglandins. These enzymes have two types of activities: a cyclooxygenase activity, which converts arachidonate into PGG2, and a peroxidase activity which reduces prostaglandin G2 (PGG2) to form prostaglandin H2 (PGH2) [Miyamoto et al., 1976; Van der Ouderaa et al., 1977]. Two isoforms of COX are now known. The genes for COX-1 and COX-2 appear to be regulated differently. COX-1 is expressed constitutively, whereas the expression of COX-2 is inducible [Kujubu and Herschman, 1992]. Both isoforms of COX are the *Corresponding author Phone: +82-31-201-2436; Fax: +82-31-202-9885 E-mail:
[email protected]
Abbreviations: COX, cyclooxygenase; hGalT, human β1,4galactosyltransferase; hST6Gal, Galβ1,4-GlcNAc α2,6-sialyltransferase; PCR, polymerase chain reaction; RCA, Ricinus communis agglutinin; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate-ployacrylamide gel electrophoresis; SFM, serum free medium; SNA, Sambucus nigra agglutinin; SSC, saline-sodium citrate; ST, sialyltransferase; TBS, tris buffered saline; TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine doi:10.3839/jksabc.2009.002
therapeutic target of nonsteroidal anti-inflammatory drugs, which exhibit antipyretic, analgesic, and antiinflammatory effects in humans via inhibition of the prostaglandin biosynthesis [Smith et al., 1996]. COX-1 is an integral membrane glycoprotein concentrated in the endoplasmic reticulum and the nuclear envelope [Rollins and Smith, 1980; Reiger et al., 1993]. Approximately 8% of the mass of ovine COX-1 is carbohydrate, consisting of three Asn-linked, high mannose oligosaccharides [Van der Ouderaa et al., 1977]. Although high expression levels of COX-1 proteins are achieved in the baculovirus system, the yield of the active enzymes is low, apparently resulting from the inefficient N-glycosylation [Shimokawa and Smith, 1992]. It has also been reported that the glycosylation of COX-1 appeared to play an important role in obtaining proteins with full enzyme activity [Otto et al., 1993]. In our recent study, the expression of β-secretase in Trichoplusia ni BTI Tn 5B1-4 cells transformed with cDNAs encoding GalT and ST6Gal yielded sialylated βsecretase glycoproteins [Chang et al., 2005]. This finding indicates that the extension of the N-gylcan structure by the expression of the glycosyltransferases GalT and
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ST6Gal in the transformed BTI Tn 5B1-4 insect cells is feasible, and that the extended glycosylation possibly affects the activity of the recombinant COX-1. Therefore, we investigated the expression of the recombinant COX-1 in BTI Tn 5B1-4 cells transformed with cDNAs encoding the hGalT and hST6Gal. Trichoplusia ni
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Materials and Methods
Cell line, plasmids, and enzymes. BTI Tn 5B1-4 (Tn 5B1-4) cells were grown at 27οC in a T-25 culture flask (Nunc, Roskilde, Denmark) in Sf900II-SFM (Invitrogen, Carlsbad, CA) medium. The pIZT/V5-His plasmid (3.3 kb; Invitrogen) contained an OplE2 promoter, a V5 epitope tag, a polyhistidine region, and a zeocin resistant gene under the control of the EM7 promoter. The plasmid containing cDNA, which encodes human COX-1, was pIZT/BiP/His-COX-1. DH5α was used as the primary host for constructing and propagating the plasmids. cells were routinely grown in the Luria-Bertani medium [1% tryptone, 0.5% yeast extract, and 0.5% NaCl (pH 7.3)] containing 50 µg/ mL of ampicillin. DNA restriction enzymes from either Promega (Madison, WI) or Takara (Shiga, Japan) were used according to the manufacturer’s instructions. Construction of expression plasmids. The human COX-1 gene was amplified from pMT/BiP/His-COX-1 [Chang ., 2007] using PCR to construct the expression vector pIZT/BiP/His-COX-1. The sense and the antisense primers respectively were 5'-GGTACCATG AAGTTATGC-3' and 5'-AAGGGCCCTCTAGACTC-3'. The amplified His-COX-1 sequence was inserted into the pGEM-T vector to yield pGEM-T-His-COX-1. The pIZT/BiP/His-COX-1 was constructed by inserting I and I fragments of pGEM-T-His-COX-1 between the I and I sites of pIZT/BiP/V5-His. The cDNAs encoding the hGalT and hST6Gal were synthesized by reverse transcription-PCR using total RNA isolated from the human placenta cells [Kim ., 2003] and T. ni
E. coli
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subcloned into pIZT for constitutive expression in the insect cells. pIZT/GalT-ST was designed to express hGalT I (EC2.4.1.22) and hST6Gal I (EC 2.4.99.1) in Tn5B1-4 cells (Fig. 1). The proper orientation and reading frame of the gene inserted in all recombinant plasmids described above were confirmed by restriction enzyme mapping and DNA sequencing. Stable transformation and cell culture. Tn 5B1-4 cells were transfected with pIZT/BiP/His-COX-1, pIZT/ GalT-ST using the lipofectin method as described by Chang [2004]. For the control, Tn 5B1-4/COX-1 cells were transfected with pIZT/BiP/His-COX-1. The Tn 5B1-4/COX-1 cells and the Tn 5B1-4/COX-1/GalT-ST cells, Tn 5B1-4 cells co-transformed with cDNAs encoding pIZT/BiP/His-COX-1 and glycosyltransferases, GalT and ST6Gal, were isolated after 4 weeks of selection with zeocin at 400 µg/mL. Zeocin was maintained continuously at 200 µg/mL in the medium after the selection procedure. Tn 5B1-4/COX-1/GalT-ST cells were grown at 27οC in T-25 flasks with 3 mL of Sf900II-SFM medium containing 200 µg/mL zeocin. Tn 5B1-4/COX-1 cells, Tn5B1-4 cells transformed with only COX-1 cDNA, as the control were grown at 27οC in the T-25 flasks with 3 mL of Sf900II-SFM medium containing 200 µg /mL zeocin. Southern blot analysis. Genomic DNA was prepared from both Tn 5B1-4/COX-1 and Tn 5B1-4/COX-1/GalTST cells using a standard procedure [Sambrook ., 1989], and 20 µg aliquots were digested with either RI and I or RI and I. A probe was prepared from a 600-bp RI and a I fragment of pIZT/GalT-ST, including part of the coding region of human β1,4-galactosyltransferase, and also from a 680bp RI and a I fragment of pIZT/GalT-ST, including part of the coding region of human Galβ1,4GlcNAc α2,6-sialyltransferase. The digests and equivalents were resolved on a 0.8% agarose gel and transferred to the Hybond-N membranes following the manufacturer’s instructions (Amersham Biosciences, Uppsala, Sweden). Membranes were prehybridized for 2 h at 65οC in 6 × et al
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Fig. 1. A schematic representation of the plasmid expression. (A) pIZT/BiP/His-COX-1; (B) pIZT/GalT-ST. COX-1, human cyclooxygenase-1; GalT, β1,4-galactosyltransferase; ST6Gal, Galβ1,4-GlcNAc α2,6-sialyltransferase
Expression of Recombinant Cyclooxygenase 1 in
SSC (1× is 0.15 M NaCl/15 mM sodium citrate, pH 7)/5 × Denhardt’s solution/0.5% SDS with 0.1 mg/mL of denatured salmon sperm DNA and were hybridized for an additional 16 h in the same solution with a 32P-labeled probe (Prime-A-Gene Labeling System; Promega, Madison, WI). After hybridization, the membranes were washed in 2× SSC/0.1% SDS at 65οC for 10 min, in 1× SSC/0.1% SDS at 65οC for 10 min, and finally in 0.5× SSC/0.1% SDS at 65οC for 10 min, then sealed in a bag. Kodak Xray film was then exposed to the membranes. SDS-PAGE and Western blot analysis. Protein samples were separated by electrophoresis on the 8-10% polyacrylamide-SDS gels, and analyzed by silver staining [Sambrook et al., 1989] and Western blot. The electrophoresised proteins on the gel were transferred onto the nitrocellulose membrane (Amersham Biosciences), blocked with 3% bovine serum albumin or 3% skim milk, incubated with mouse anti-human COX-1 (1:1000 dilution in TBS, Cayman Chemical, Ann Arbor, MI), and probed with the alkaline phosphatase-conjugated goat anti-mouse IgG antibody (1:1000 dilution in TBS, Sigma). After washing the membranes BCIP/NBT solution (Amresco, Solon, OH) was added. The reaction was quenched with distilled water. Purification of recombinant cyclooxygenase 1. Tn 5B1-4/COX-1 and Tn 5B1-4/COX-1/GalT-ST cells (Tn 5B1-4 cells co-transformed with cDNAs encoding COX1 and glycosyltransferases, GalT and ST6) were cultured for 7 days in T-75 flasks with 10 mL of Sf900II-SFM medium containing 200 µg/mL of zeocin. All steps were performed at 4οC. These stably transformed Tn 5B1-4 cells were centrifuged at 3,000 rpm for 5 min to recover the medium, which was then dialyzed in Tris buffer (20 mM Tris-Cl, 500 mM NaCl, pH 7.9) for 48 h. Imidazole was then added to the dialyzed medium at a final concentration of 10 mM, followed by incubation with the Fast-flow Ni-NTA resin (Qiagen, Valencia, CA) in a chromatography column, according to the manufacturer’s instructions. Weakly bound proteins were washed with three volumes of Tris buffer containing 10 mM imidazole. Recombinant COX-1s from Tn 5B1-4/COX-1 and Tn 5B1-4/COX-1/GalT-ST cells were then eluted with the Tris buffer containing 200 mM imidazole. Fractions containing the recombinant proteins were pooled and dialyzed in the buffer containing 20 mM Tris-Cl (pH 6.0) to remove the imidazole.
Lectin blot analysis of recombinant cyclooxygenase 1.
Equivalent amounts of the purified recombinant COX-1s were used for the lectin blotting assays [Jarvis and Finn, 1996]. The membranes were cut into strips, blocked, and probed with the mouse anti-human COX-1 monoclonal antibody or the biotinylated (Vector Laboratories,
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Burlingame, CA) lectins. The lectins used in this study were RCA, which binds the β-linked galactose, and SNA, which binds terminal α2,6-linked sialic acid. Bound lectins or antibodies were detected by secondary reactions with the alkaline phosphatase-conjugated goat antimouse IgG antibody, alkaline phosphatase-conjugated avidin (Vector Laboratories, Burlingame, CA). The membranes were washed, and the BCIP/NBT solution (Amresco) was added. For lectin blotting assays using RCA, the purified COX-1 was pretreated with 2000 U/ mL of Arthobacter urefaciens neuraminidase (Calbiochem, Madison, WI) for 6 h at 37οC as described previously [Laemmli, 1970]. After the incubation, the pretreated COX-1 was analyzed by SDS-PAGE and lectin blotting. Activity assay of cyclooxygenase 1. Protein concentrations were measured by the Bradford method using an RC-DC protein assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard. In vitro activities of the recombinant purified COX-1s from Tn 5B1-4/COX-1 and Tn 5B1-4/COX-1/GalT-ST cells were determined by the peroxidase assay [Raz and Needleman, 1990]. Peroxidase activity was estimated as the ability to oxidize TMPD in a substrate-dependent manner. Samples were added to the 96-well plates, and the reaction was initiated by the addition of assay buffer (100 mM Tris-Cl, 1 µM bovine hemin chloridem, and 170 µM TMPD) containing 100 µM arachidonic acid. After 10 min reaction at room temperature (25οC), the absorbance at 590 nm was measured. The peroxidase activity of the recombinant COX-1s was determined by comparison with the commercial ovine COX-1 (Cayman Chemical). Results and Discussion
Analysis of insertion of glycosyltransferase cDNAs in the genome of transformed Tn 5B1-4 cells. We
attempted to extend the glycosylation of the recombinant COX-1 expressed in the Trichoplusia ni BTI Tn 5B1-4 cell line by introducing the human glycosyltransferase genes hGalT and hST6Gal into the cell line. Stably transformed Tn 5B1-4 cells transfected with only COX-1 cDNA were obtained as a control and designated as Tn 5B1-4/COX-1 cells. Stably transformed Tn 5B1-4 cells co-transfected with cDNAs encoding COX-1 and glycosyltransferases were also obtained and designated as Tn 5B1-4/COX-1/GalT-ST cells. Southern blot analysis was used to investigate the insertion of glycosyltransferases genes into the stably transformed Tn5B1-4 cells (Fig. 2). Twenty micrograms of the genomic DNAs extracted from Tn 5B1-4/COX-1 and Tn 5B1-4/COX-1/ GalT-ST cells were digested with the restriction enzymes,
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Fig. 3. SDS-PAGE of the purified His-tagged COX-1 from the cellular fraction of stably transformed Tn 5B1-4 cell cultures. Before and after affinity purification Fig. 2. Southern blot analysis of genomic DNAs from Tn 5B1-4 cells. Genomic DNAs (20 µg) extracted from
Tn 5B1-4COX-1 and Tn 5B1-4COX-1/GalT-ST cells were digested with restriction enzymes. All digests were resolved by agarose gel electrophoresis as follows: Lane 1 for (A) and (B), Tn 5B1-4COX-1 (Tn 5B1-4 cells transformed with only COX-1 cDNA) cells; lane 2 for (A) and (B), Tn 5B1-4COX-1/GalT-ST (Tn 5B1-4 cells transformed with cDNAs coding COX-1 and glycosyltransferases) cells. The probe for (A) was a 600bp fragment of human β1,4-galactosyltransferase cDNA. The probe for (B) was a 680-bp fragment of human Galβ1,4-GlcNAc α2,6-sialyltransferas cDNA. Lanes 3, 4, and 5 for A and B represent equivalent DNAs of 1, 10 and 20 copies.
and the digests were examined by Southern blot analysis. Two probes, which were prepared from a 600-bp R I and I fragment, and from a 680-bp R I and I fragment of pIZT/GalT-ST did not hybridize with the genomic DNA from the Tn 5B1-4/COX-1 cells (lane 1 in Fig. 2), but did hybridize with the genomic DNA from the Tn 5B1-4/COX-1/GalT-ST cells (lane 2 in Fig. 2), indicating that the glycosyltransferase genes were integrated into the Tn 5B1-4 cell genome. Eco
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Purification of recombinant COX-1 and lectin blot analysis of recombinant COX-1. The recombinant
COX-1 proteins tested were rapidly purified from Tn 5B1-4/COX-1 and Tn 5B1-4/COX-1/GalT-ST cells by Ni2+-affinity purification, and the recovery rates with Ni2+affinity purification were 64.9 and 59.1%, respectively. The purified COX-1 proteins were obtained without visibly contaminated proteins on silver nitrate-stained SDS-PAGE gel (Fig. 3). The lectin blot analysis revealed that RCA, which is specific to the β-linked galactose, bound to COX-1 produced by Tn 5B1-4/COX-1 and Tn
of the cellular fraction of Tn 5B1-4 cells transformed with only COX-1 cDNA are shown in lanes 1 and 2, respectively. Before and after affinity purification of the cellular fraction of Tn 5B1-4 cells transformed with cDNAs encoding COX-1 and glycosyltransferases are shown in lanes 3 and 4, respectively. Arrow indicates the recombinant COX-1 protein. M, molecular weight marker
5B1-4/COX-1/GalT-ST cells (Fig. 4), indicating the presence of the galactosylated glycan in COX-1 produced by the Tn 5B1-4/COX-1 cells. Hsu . [1997] reported the galactosylation of the recombinant IgG produced by the Tn 5B1-4 cell line. Tn 5B1-4 cells were also observed to contain approximately 10% of the GalT activity detected in Chinese hamster ovary cells [Abdul-Rahman B. ., 2002]. SNA, which specifically recognizes the terminal sialic acid, bound to COX-1 produced by the Tn 5B1-4/COX-1/GalT-ST cells (Fig. 4). However, the recombinant COX-1 produced by Tn 5B1-4/COX-1 cells did not acquire any detectable sialic acid, indicating that Tn 5B1-4/COX-1/GalT-ST cells produced the recombinant glycoproteins containing both β1,4-linked galactose and α2,6-linked sialic acid. Native COX-1, an integral membrane protein, has an observed molecular mass of 72 kDa and is glycosylated at three of four consensus N-glycosylation sites including Asn 68, Asn 144, and Asn 410 [Otto ., 1993]. In addition, the oligosaccharide structure of the sheep seminal vesicle COX-1 showed occupation by two acetylglucosamines and nine mannoses (Man9GlcNAc2) [Mutsaers ., 1985]. In the present study, the molecular weights of the recombinant COX-1s in the cellular fractions of Tn 5B1-4/COX-1 and Tn 5B1-4/ COX-1/GalT-ST cells were found to be approximately 74 kDa (Fig. 3). A potential ER-targeting sequence, (P/ S)TEL, is conserved at the COOH terminus of COX-1 et al
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Table 1. Peroxidase activity of purified COX-1 proteins from Tn 5B1-4COX-1 and Tn 5B1-4COX-1/GalT-ST cells Protein Specific Activity content activity (units) (mg) (units/mg) COX-1 0.09 1,845 20,500 COX-1/GalT-ST 0.072 1,723 23,930 Data represent the average of duplicate runs. a
Fig. 4. Lectin blot analysis of purified COX-1 from Tn 5B1-4COX-1 and Tn 5B1-4COX-1/GalT-ST cells.
Recombinant COX-1 from Tn 5B1-4COX-1 (lane 1), and Tn 5B1-4COX-1/GalT-ST (lane 2) cells were transferred to an Immobilon filter. The filter was then cut into strips containing COX-1 from each source, and the strips were probed with anti-V5 (Ab) (A), RCA (B) or SNA (C). Bound lectins and antibodies were detected by secondary reactions with alkaline phosphatase-conjugated goat antimouse IgG antibody and alkaline phosphatase-conjugated avidin.
[Otto ., 1993]. This sequence may be a variation of the KDEL retention sequence observed in many soluble proteins that are retained in the ER lumen. Thus, the recombinant COX-1 proteins (post-translationally modified in the Golgi) could be retained in the ER via retrograde transport. Recombinant COX-1 from the cellular fractions of Tn 5B1-4/COX-1 cells could be a glycoprotein, consisting of a high mannose-type oligosaccharide. On the other hand, the recombinant COX-1 from the cellular fractions of the Tn 5B1-4/COX-1/GalT-ST cells might be a glycoprotein, consisting of a complex type of oligosaccharide, because the -linked glycan structure of the recombinant COX-1 could be further modified in the Golgi by the action of the glycosyltransferases GalT and ST. However, why the apparent molecular weights of these two types (high mannose-type and complex-type) of the recombinant COX-1 show similar values is not known. et al
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Peroxidase activity of recombinant COX-1 from Tn 5B1-4/COX-1and Tn 5B1-4/COX-1/GalT-ST cells.
The peroxidase activities of the purified recombinant proteins were also analyzed. The purified COX-1 from Tn 5B1-4/COX-1 cells had a specific peroxidase activity of 20,500 U/mg, whereas that of the purified recombinant COX-1 from Tn 5B1-4/COX-1/GalT-ST cells was 23,930 U/mg, indicating a small incremental change compared with the control (20,500 U/mg) (Table 1). This indicates that extension of the -linked glycan structure by expression of the glycosyltransferases GalT and ST in Tn 5B1-4 cells did not noticeably improve the activity of the recombinant cyclooxygenase 1, in contrary to our previous findings on the recombinant β-secretase [Chang ., 2005] that the sialylation of the N
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recombinant β-secretase improved the activity by 77%. Extension of the -linked glycan structure to yield a sialylated form of the recombinant COX-1 might not be essential for maintaining a catalytically active conformation of the enzyme, because natural COX-1 is a glycoprotein with a high level of mannose oligosaccharides. The native COX-1 enzyme, once in the active conformation state, does not appear to require attached carbohydrate for peroxidase activity [Otto ., 1993]. In the case of the recombinant β-secretase, silaylation is important for the biological activity, because the native form of β-secretase is a sialylated protein [Charlwood ., 2001]. In summary, our method can be a useful tool for producing recombinant glycoproteins with extended -linked glycans containing a penultimate galactose and a terminal sialic acid. However, the effect of the extension of the linked glycan structure on the activity of the recombinant proteins depended on the nature of the target glycoproteins. N
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Acknowledgment. This work was supported by grants
from the Biogreen 21 project (20070401034026), the Korea Science and Engineering Foundation (KOSEF) (R01-2006-000-10635-0), and KOSEF through the Plant Metabolism Research Center, Kyung Hee University.
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