2 Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
3 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusong-gu, Taejon 305-701, Korea
4 USDA Forest Service, Northeastern Research Station, Forestry Sciences Laboratory, 359 Main Road, Delaware, OH 43015, USA
5 Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
Received on January 9, 2003; revised on March 14, 2003; accepted on March 14, 2003
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Abstract |
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Key words: Baculovirus / Gypsy moth / Insect cells / Lymantria dispar nucleopolyhedrovirus / N-glycan
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Introduction |
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Earlier studies using dipteran insect cells (Butters and Hughes, 1981; Hsieh and Robbins, 1984
) and lepidopteran insect cells with the baculovirus system (Kuroda et al., 1990
) indicated that insect cells produce mainly high-mannose-type and paucimannosidic-type N-glycans. Similar results have been repeatedly published (Altmann et al., 1999
; März et al., 1995
). In addition,
(1,3)-fucosylation of Asn-linked GlcNAc is often observed in insect cellderived glycoproteins, and this modification represents a potential allergen to humans (Fotisch and Vieths, 2001
; Tretter et al., 1993
; Weber et al., 1987
). The inability of lepidopteran insect cells to synthesize sialylated complex-type N-glycans and the presence of
(1,3)-fucosylation have limited the utility of insect cells as host cells for production of pharmaceutical glycoproteins. The limitations of currently used insect cell lines may potentially be overcome by means of genetic manipulation to include the necessary processing enzymes (Ailor et al., 2000
; Aumiller and Jarvis, 2002
; Breitbach and Jarvis, 2001
; Hollister et al., 1998
, 2002
; Hollister and Jarvis, 2001
; Tomiya et al., 2003
), or by the use of an alternative insect cell line that may contain mammalian-like N-glycan processing capabilities.
As an attempt to survey insect cells potentially better suited for the production of pharmaceutical glycoproteins, a protein expression system based on a gypsy mothderived cell line and a virus, Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) (Yu et al., 1992), was tested. LdMNPV was used in the earlier study to express a bacterial ß-galactosidase in tissue culture cells derived from gypsy moth (Yu et al., 1992
). So far, however, the glycosylation pattern of glycoproteins expressed in gypsy moth cells has not been reported. LdMNPV will infect a number of cell lines derived from L. dispar, including the cell line Ld652Y, used in the present study. The Ld652Y cell line was chosen for the current study because it grows well in suspension in serum-free media at densities and growth rates similar to those of other popular insect cells, such as Sf9 and Tn-5B1-4. In this study, a recombinant human transferrin (hTf) was expressed as a model protein in Ld652Y cells using a recombinant LdMNPV. Structure of most (
90%) of the N-glycans released from the recombinant hTf was identified by a combination of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) and a 2-D mapping technique (Tomiya et al., 1988
).
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Results |
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No sialylated glycans were observed when a total mixture of the released glycans was analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The released N-glycans were then reductively aminated with 2-aminopyridine, and the resultant pyridylamino (PA) derivatives of glycans were first separated by reversed-phase high-performance liquid chromatography (HPLC) (Tomiya et al., 1988), from which 14 peaks were obtained (Figure 2). Each of these fractions was further separated by normal-phase HPLC using an amide-silica column (Figure 3). After the two chromatographic steps, 19 different PA-glycans were isolated. The eight major PA-glycans (7A, 8A, 9A, 10A, 11A, 12A, 13A, and 14A), accounting for 86.5% of the total glycans, were analyzed by MALDI-TOF MS for confirmation. MS spectra of these major glycans are shown in Figure 4. The elution positions of the major PA-glycans, together with those of several standard PA-glycans having related structures, are shown on a 2D map in Figure 5.
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The difference between the MS data of PA-glycans 11A and 14A ([M+Na]+=1361.02) was equivalent to one Fuc. The coordinates of PA-glycan 14A on a 2-D map (12.8 GU, 5.0GU) coincided with those of the following standard PA-glycan (110.1) within the experimental error (see Scheme 2). Furthermore, the coordinates of PA-glycan 14A on a 2D map shifted to those of PA-glycan 11A (and a standard PA-glycan [100.1]) after -L-fucosidase digestion, and the magnitude of the change in the coordinates (-3.4 GU/-0.4 GU) was consistent with the unit contribution value of Fuc, which is
(1,6)-linked to GlcNAc adjacent to Asn. These results suggest that PA-glycans 11A and 14A are unfucosylated and
(1,6)-fucosylated monoantennary glycans, respectively, as shown in Table I.
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Presence of (1,3)-fucosylation in the minor glycans (each
1% of the total glycans) was also investigated by comparison with the known
(1,3)-fucosylated glycans. Figure 6 shows the elution positions of 11 minor PA-glycans and those of the known glycans having
(1,3)-fucosylated GlcNAc adjacent to Asn, except xylose-containing glycans from plants (Takahashi and Tomiya, 1998
; Takahashi et al., 1999
; Tomiya et al., 2003
). None of these minor PA-glycans coincided with any of the known PA-glycans containing Fuc
(1,3)GlcNAc linked to Asn, suggesting the absence of core
(1,3)-fucosylation.
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In summary, the most abundant glycans were Man13(±Fuc6)GlcNAc, representing 75.5% of the total N-glycans, followed by GlcNAcMan3(±Fuc
6)GlcNAc2, representing 7.4% of the total. There was only
6% of high-mannose-type glycans identified. Nearly half (49.8%) of the total N-glycans contained
(1,6)-fucosylation (12A, 13A, and 14A), but
(1,3)-fucosylation on the Asn-linked GlcNAc residue could not be detected within experimental error.
Effects of serum supplementation to the culture medium on the N-glycosylation of recombinant hTf
N-glycans of hTf from the Ld652Y cells grown in culture media containing 10% FBS were also analyzed exactly as described for the product from serum-free medium. The N-glycan profile on the ODS column showed little difference between these samples, except the total amount of fucosylated glycans (12A, 13A, and 14A) was slightly increased from 49.8% to 55.8% in serum.
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Discussion |
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It is worth noting that the cell death following viral infection with LdMNPV is more gradual than that observed using the AcMNPV virus in the previous studies (data not shown). Alternatively, Ld652Y cells may include higher levels of mannosidases, which convert high-mannose-type structures to paucimannosidic structures. Interestingly, 56% of the total N-glycans (7A, 10A, and 13A) do not have the Man1-3GlcNAc branch, and Man1GlcNAc2 (7A), which has only one Man residue, was as high as 7%. The high proportion of these three N-glycans indicates the existence of a highly active
(1,3)-mannosidase in Ld652Y cells.
Ld652Y cells also produced significant amounts of GlcNAc-terminated N-glycans (7.4% of the total) as did Tn-5B1-4 cells (Ailor et al., 2000). In Tn-5B1-4 cells, the N-glycans with GlcNAcß2Man
3 branch were 10% of the total and those with GlcNAcß2Man
6 branch, 4% of the total. However, in Ld652Y cells, GlcNAc was found only in the GlcNAcß2Man
6 branch. It may be that Tn-5B1-4 cells have a higher GlcNAc transferases I activity than Ld652Y, and Ld652Y cells have a higher GlcNAc transferase II activity. Alternatively, the Ld652Y cells may contain a higher level of N-acetylglucosaminidase than Tn-5B1-4 cells. An N-acetylglucosaminidase that specifically cleaves terminal GlcNAc residues from the GlcNAcß2Man
3 branch of N-glycans has been found in insect cells from Spodoptera frugiperda, Mamestra brassicae, Bombyx mori, and Trichoplusia ni (Altman et al., 1995a
; Kubelka et al., 1994
; Wagner et al., 1996
).
Galactosylated N-glycans are generally not found in glycoproteins derived from insect cells. Some exceptions are galactosylated N-glycans in interferon expressed in Estigmene acrea cells (Ogonah et al., 1996
) and in a mouse IgG produced by Tn-5B1-4 cells (Hsu et al., 1997
). However, no galactosylated N-glycan was found when human serum transferrin was expressed in Tn-5B1-4 cells (Ailor et al., 2000
). This is confirmed by the fact that no detectable change in reverse-phase HPLC was observed (data not shown) after the total mixture of PA-glycans was digested by ß-galactosidase.
N-glycans containing core (1,3)-fucosylation were found in honey bee venom phospholipase A2 and membrane glycoproteins from Sf-21, Mb-0503, Bm-N cells (Kubelka et al., 1994
). They were also found in a recombinant mouse IgG (Hsu et al., 1997
) expressed in Tn-5B1-4 cells. On the other hand, no core
(1,3)-fucosylation was found in the glycans of human interferon
expressed in Sf9 cells (Voss et al., 1993
), interferon
expressed in Sf9, and Estigmene acrea cells (Ogonah et al., 1996
). Core
(1,3)-fucosylation was not found in the third eight-cysteine domain of LTBP-1 (Rudd et al., 2000
) expressed in Sf9 cells, but it was present in the same protein expressed in Tn-5B1-4 cells (Rudd et al., 2000
).
(1,6)-Fucosylation of the Asn-linked GlcNAc is common in mammalian N-glycans (Kobata, 1992
) and considered innocuous. However,
(1,3)-fucosylation at the same GlcNAc is not found in mammalian glycoproteins and can cause an allergic reaction in humans. Furthermore, the presence of antibodies specific for such a Fuc residue in human blood can possibly affect in vivo activity and clearance of biopharmaceutical glycoproteins (Bardor et al., 2003
). To have such structures in a glycoprotein makes it less than ideal for therapeutic use (Prenner et al., 1992
; Wilson et al., 2001
).
The choice of glycoamidase A from sweet almond is important in the current work. It is known that glycoamidase A, but not glycoamidase F (from Flavobacter), can release N-glycans containing the Fuc(1,3)-GlcNAc-Asn moiety (Altmann et al. 1995b
; Fan and Lee, 1997
; Takahashi and Tomiya, 1992
; Tretter et al., 1991
). Using the glycoamidase A, we previously found that N-glycans in a recombinant hTf expressed in Tn-5B1-4 cells contained as much as 6% of difucosylated trimannosyl core structure (010.1 F) (Ailor et al., 2000
), which would have escaped detection if we had used the Flavobacter enzyme (see Scheme 3).
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Insect cells can grow in serum-containing, serum-free, or even protein-free growth medium. Our results show that inclusion of FBS did not affect N-glycosylation patterns in Ld652Y cells except for a small increase in (1,6)-fucosylated glycans. Elimination of serum from the culture medium will further contribute to lowering the production costs of glycoproteins.
Mammalian cells express sialylated complex-type N-glycans on glycoproteins, but Ld652Y cells, like most other insect cell lines, lack the capacity to produce complex-type N-glycans containing Gal and sialic acid residues. Other modifications leading to complex-type structures (including tri- and tetraantennary structures), are also not present in Ld652Y cells. To produce more "humanized" glycoproteins in insect cells, such deficiencies must be overcome. These include elimination of core (1,3)-fucosylation, enhancement of GlcNAc transferases I and II, and ß(1,4)-galactosyltransferase activity, introduction of the sialylation module (Altmann et al., 1999
; Hollister et al., 2002
; Lawrence et al., 2000
; Laurence et al., 2001
) and suppression of a certain specific ß-N-acetylglucosaminidase activity (Altman et al., 1995a
; Watanabe et al., 2002
). More "mammalian-like" N-glycans are being produced in insect cells by the introduction of genes of processing-related enzymes (Ailor et al., 2000
; Aumiller and Jarvis, 2002
; Breitbach and Jarvis, 2001
; Hollister et al. 1998
, 2002
; Hollister and Jarvis, 2001
; Tomiya et al., 2003
). Gypsy mothderived cell lines may become a preferable target for genetic engineering if the lack of
(1-3)-fucosylation can be profitably utilized.
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Materials and methods |
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Generation of a recombinant hTf-expressing virus strain of LdMNPV
Polyh-htf-pMT/V5-His was constructed to contain the hTf gene (Yang et al., 1984) with accompanied sequence at the 3' end to generate a V5 tag as well as six histidine residues under the control of the LdMNPV polyhedrin promoter (Bischoff and Slavicek, 1996). The following sequence was added to the 3' end of the hTf gene to generate the V5 epitope and the six his tag: CTC GAG TCT AGA GGG CCC TTC GAA GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG CGT ACC GGT CAT CAT CAC CAT CAC CAT TGA.
To generate a transplacement vector for construction of a recombinant virus, cosmid clone P313 (Riegel et al., 1994) was digested with BamHI and HindIII, and the 9294-bp fragment from 128,400137,694 (Kuzio et al., 1999
) was cloned into the BamHI and HindIII sites of pBS sk+ to generate pNP-EGT-9.3. The pNP-EGT-9.3 was digested with BsteII and blunt ended with Klenow. The polyh-htf-pMT/V5-His was digested with SpeI and PmeI, and the SpeI/PmeI fragment was isolated and the ends were filled with Klenow. The SpeI/PmeI fragment from clone polyh-htf-pMT/V5-His was ligated into the BsteII digested pNP-EGT-9.3 to generate the transplacement vector polyh-htf-his6EGT-. This transplacement vector contains the gene and lacks most of the EGT gene (from 120,641121,613). Viral strain 122bEGT-LacZ+ was used to generate a recombinant LdMNPV strain expressing the hTf gene (Slavicek et al., 2001
). This viral strain had the LacZ gene in place of the EGT gene. Viral isolate 122bEGT-LacZ+ genomic DNA and the transplacement vector polyh-htf-his6EGT- were cotransfected into Ld652Y cells as described previously (Bischoff and Slavicek, 1996
). Budded virus from the transfection was plaque-purified, and several clear plaques were isolated and plaque-purified again. The purified viral isolates were propagated in Ld652Y cells; genomic DNA was isolated and analyzed by restriction endonuclease digestion (data not shown).
Cell culture and virus infection
Ld652Y cells established from L. dispar and LdMNPV were used as a host and virus system. The cells were maintained as described previously (Bischoff and Slavicek, 1996). A suspension culture of Ld652Y cells was maintained at 27°C and rotated at 120 rpm in 250-ml shaker flasks containing 30 ml Excell 420 media. For the recombinant hTf production, 10 100-ml shake flasks, containing serum-free Excell 420 media with or without 10% FBS, were seeded with 7 x 107 Ld652Y cells. The cells were infected with LdMNPV-hTf at 1.0 tissue culture infectious dose (TCID50) unit per cell. TCID50 was determined as described previously (Slavicek et al., 2001
). The cells were incubated at 27°C and rotated at 120 rpm. Five days postinfection, the culture media containing hTf were harvested after removal of cells by centrifugation.
Purification of recombinant hTf from culture medium
The culture supernatant containing hTf (1 L) was concentrated by ultrafiltration, and proteins were precipitated by adding ammonium sulfate (50% final saturation). After removing the precipitate by centrifugation at 8000 rpm for 15 min at 4°C, proteins including hTf were precipitated by adding ammonium sulfate to the supernatant (80% final saturation) and collected by centrifugation. The precipitate was dissolved in buffer A (10 mM TrisHCl, pH 7.0), and dialyzed against buffer A at 4°C. The hTf-containing sample solution was then applied to a column (1.6 x 6 cm) of anti-hTf-IgG immobilized Sepharose 4 Fast Flow (Tomiya et al., 2003
) equilibrated with buffer A containing 0.5 M NaCl. After unbound proteins were washed off with the same buffer, hTf was eluted with 0.1 M glycineHCl, pH 2.7, containing 0.5 M NaCl. The eluate was immediately neutralized with 0.5 M TrisHCl, pH 8.3, dialyzed against water, lyophilized, and used for carbohydrate analyses. The purity of the recombinant hTf preparation was analyzed by SDSPAGE (10% acrylamide) under nonreducing condition, and proteins were visualized by Coomassie brilliant blue R-250 staining.
Purification and derivatization of N-glycans from recombinant hTf
N-glycans were prepared from the purified recombinant hTf as described previously (Ailor et al., 2000). Briefly, a trypsin-chymotrypsin (each 1%, w/w, of the substrate protein) digest of hTf (5 mg) was treated with glycoamidase A (0.4 mU) in 100 mM sodium citrate-phosphate, pH 5, at 37°C overnight, and the mixture was passed through a Dowex 50 x 2 (H+) column (1 ml). The purified glycans in the effluent thus obtained were lyophilized and derivatized by reductive amination with 2-aminopyridine and sodium cyanoborohydride (Nakagawa et al., 1995
; Yamamoto et al., 1989
), and the PA-derivatized glycans were purified by gel filtration on a Sephadex G-15 column (1.0 x 40 cm) using 10 mM NH4HCO3 as eluant.
Isolation and characterization of PA-glycans by two different HPLC steps
The LC-10A HPLC system (Shimadzu USA) was used to analyze the PA-glycans. The PA-glycan mixture was separated and characterized by 2D sugar mapping technique as described previously (Takahashi et al., 1995; Tomiya et al., 1988
). PA-derivatized glycans were monitored by fluorescence (
ex=300 nm,
em=360 nm). The HPLC conditions for analytical chromatography with two columns were the same as described previously (Tomiya et al., 1988
). The PA-glycans were successively separated on a reverse-phase column, Shim-pack CLC-ODS (6 x 150 nm), and a normal phase column, Amide-80 (4.6 x 250 mm). The elution time normalized as the elution position of GU, based on elution positions of PA- isomalto-oligosaccharides (DP 420). The elution times (in min) of the glycans of interest in ODS and amide-80 column chromatography were converted to GU and plotted on the x-axis (ODS column) and y-axis (amide-80 column). The resultant 2D map for all PA-glycans was compared with those of known PA-glycans and confirmed by cochromatography with reference glycans. The reference glycans were obtained from human immunoglobulin G, ribonuclease B, and recombinant hTf expressed in T. ni cells using the same procedure (Tomiya et al., 2003
).
MALDI-TOF MS analysis
The Kompact SEQ MALDI-TOF mass spectrometer (Kratos Analytical, Manchester, UK) was used to analyze PA-glycans in the linear positive-ion mode using 20 mg/ml of 2,5-dihydroxybenzoic acid as reported previously (Papac et al., 1998). The matrix was dissolved in a 1:1 (v/v) mixture of ethanol:10 mM sodium chloride. To crystallize the sample PA-glycans, 0.5 µl of the matrix was placed on a sample plate to which 0.5 µl of the sample (usually 10 pmol) followed by 0.5 µl of matrix were added, and air-dried. A mixture of PA-isomalto-oligosaccharides (DP=420) was used for calibration.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: yclee{at}jhu.edu
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Abbreviations |
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References |
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