Glycobiology Advance Access originally published online on December 22, 2004
Glycobiology 2005 15(5):541-547; doi:10.1093/glycob/cwi032
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Glycobiology vol. 15 no. 5 © Oxford University Press 2004; all rights reserved.
Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII
Mammalian Cell Technology, Novo Nordisk A/S, Novo Allé, 2880 Bagsværd, Denmark
1 To whom correspondence should be addressed; e-mail: bolt{at}novonordisk.com
Received on October 22, 2004; revised on December 15, 2004; accepted on December 15, 2004
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Abstract |
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N-glycosylation is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. In the present study, however, we demonstrate posttranslational N-glycosylation of recombinant human coagulation factor VII (FVII) in CHO-K1 and 293A cells. Human FVII has two N-glycosylation sites (N145 and N322). Pulse-chase labeled intracellular FVII migrated as two bands corresponding to FVII with one and two N-glycans, respectively. N-glycosidase treatment converted both of these band into a single band, which comigrated with mutated FVII without N-glycans. Immediately after pulse, most labeled intracellular FVII had one N-glycan, but during a 1-h chase, the vast majority was processed into FVII with two N-glycans, demonstrating posttranslational N-glycosylation of FVII. Pulse-chase analysis of N-glycosylation site knockout mutants demonstrated cotranslational glycosylation of N145 but primarily or exclusively posttranslational glycosylation of N322. The posttranslational N-glycosylation appeared to take place in the same time frame as the folding of nascent FVII into a secretion-competent conformation, indicating a link between the two processes. We propose that the cotranslational conformation(s) of FVII are unfavorable for glycosylation at N332, whereas a more favorable conformation is obtained during the posttranslational folding. This is the first documentation of posttranslational N-glycosylation of a non-modified protein in mammalian cells with an intact N-glycosylation machinery. Thus, the present study demonstrates that posttranslational N-glycosylation can be a part of the normal processing of glycoproteins.
Key words: factor VII / mammalian cells / posttranslational N-glycosylation / protein folding / pulse-chase
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Introduction |
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N-glycosylation is carried out by transfer of Glc3Man9 (GlcNAc)2 to the asparagine residue in the target sequence N-X-T/S. This reaction is catalyzed by the oligosaccharyltransferase (OST) complex (reviewed by Silberstein and Gilmore, 1996
; Yan and Lennarz, 1999). The OST complex is associated with the translocon complex in the endoplasmic reticulum (ER) membrane and add N-glycans to nascent protein as they grow from the translocation pore into the lumen of the ER (Glabe et al., 1980; Kelleher et al., 1992; Kiely et al., 1976; Nilsson and von Heijne, 1993). Thus N-glycosylation is normally a cotranslational process. Rare examples of posttranslational N-glycosylation taking place in mammalian cells with a deficient N-glycosylation machinery or in cells expressing truncated proteins have been reported (Duvet et al., 2002; Kolhekar et al., 1998; Ronnet and Lane, 1981). In the present study, we demonstrate posttranslational N-glycosylation of human coagulation factor VII (FVII) expressed in rodent (Chinese hamster ovary [CHO] K1) and human (human embryonal kidney [HEK] 293A) cells. To our knowledge, this is the first documentation of posttranslational N-glycosylation of an unmodified protein in mammalian cells with a normal N-glycosylation machinery.
FVII is a glycoprotein secreted by hepatocytes. FVII is synthesized as a 444-amino-acid precursor that undergoes several different posttranslational modifications before being secreted as a 406-amino-acid single-chain zymogen (Hagen et al., 1986; Jurlander et al., 2001; Kaufman, 1998). Secreted FVII consists of a N-terminal domain containing gamma-carboxy glutamic acids (Gla domain), two epidermal growth factor–like (EGF) domains modified with two O-glycans, a connecting region (CR) modified with one N-glycan, and a C-terminal serine protease domain modified with another N-glycan (Figure 1) (Bjoern et al., 1991; Jurlander et al., 2001; Thim et al., 1988). After secretion, FVII can be activated into disulfide-linked two-chain FVIIa by cleavage in the CR (Figure 1). Activated FVII (FVIIa) initiates the extrinsic coagulation pathway by binding to tissue factor on the surface of cells that have become exposed to circulating blood by injury (Rapaport and Rao, 1992). Recombinant human FVIIa can compensate for the lack of factor VIII (FVIII) or factor IX (FIX) and is therefore used for treatment of bleeding in hemophilia A or B patients that produce antibodies (inhibitors) against FVIII or FIX (reviewed by Astermark, 2003).
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) in the cells. The EGF1 is O-glycosylated at S52 and S60 of secreted FVII. The CR contains the cleavage site for activation of FVII after secretion. N-glycans are found at N145 and N322 of secreted FVII. |
Results |
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A N-glycan is posttranslationally added to nascent FVII
The posttranslational processing of FVII was studied by pulse-chase labeling of CHO-K1 cells expressing human FVII. FVII immunoprecipitated from cell lysates initially migrated as two bands of
54 kDa and 56 kDa (Figure 2A). Phosphorimager quantification of gels from four independent experiments indicated that immediately after a 5-min pulse, the 54-kDa form constituted an average of 55.8 ± 7.5 % of the labeled FVII. However, during chase, the intensity of the 54-kDa band decreased, whereas the intensity of the 56-kDa band increased, and 1 h after pulse only 5.6 ± 1.0 % of the labeled FVII migrated as the 54-kDa band (Figure 2A). This indicated that the 54-kDa FVII form was processed into the 56-kDa form. A slow but steady decrease in the amount of labeled intracellular FVII began 1–1.5 h after pulse (Figure 2A). From this time point, increasing amounts of labeled FVII was detected in the medium. The vast majority of labeled FVII secreted to the medium (93.9 ± 0.6 % measured 4 h after pulse) migrated as a band of 58 kDa. But a form migrating as a band of 53 kDa was also detected. (Figure 2B). The molecular weight of intracellular and secreted FVII cannot be compared directly, because a propeptide of 18 amino acids is believed to be cleaved from intracellular FVII immediately prior to secretion (Figure 1) (Kaufman, 1998).
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To examine the role of N-glycosylation in the generation of the species of intracellular FVII, we treated immunoprecipitated FVII with peptide: N-glycosidase F (PNGase F), which removes all N-glycans (Tarantino et al., 1985). PNGase F treatment converted both the 54-kDa and the 56-kDa band of intracellular FVII into a single band of 52 kDa (Figure 2A). This demonstrated that the difference in molecular weight between the 54-kDa and the 56-kDa FVII forms was caused by heterogenous N-glycosylation. Likewise PNGase F converted both the 53-kDa and the 58-kDa band of secreted FVII into a single band of 49 kDa (Figure 2B). Thus, the detection of two bands of secreted FVII could also be attributed to heterogenous N-glycosylation.
Human FVII has two potential N-glycosylation sites (Hagen et al., 1986) (Figure 1). The most obvious interpretation of the results is that the 54-kDa and 56-kDa forms represent intracellular FVII with one and two N-glycans, respectively, that a N-linked glycan was added to most 54-kDa FVII during chase, and that the vast majority of secreted FVII therefore had two N-glycans. Another less likely explanation was a leaky pulse-chase assay that allowed the translation and cotranslational N-glycosylation of labeled FVII after the end of the 5 min pulse. To exclude the latter possibility, we added 500 µM cycloheximide to the medium immediately after pulse. This treatment has been shown to stop translation instantly (Braakman et al., 1991), but the conversion of the 54-kDa FVII form into the 56-kDa form was not affected by the cycloheximide treatment (Figure 3A). Thus the apparent N-glycosylation of the 54-kDa form was indeed posttranslational. We also carried out a pulse-chase analysis of HEK293A cells expressing FVII. In lysates from these cells, we detected the same pattern of FVII bands as in CHO-K1 cells (Figure 3B). In conclusion, the processing of the 54-kDa form into the 56-kDa form appeared to be an intrinsic property of human FVII and not due to expression in the heterologous rodent CHO-K1 cells.
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To further explore the possibility of posttranslational N-glycosylation, we established CHO-K1 cell lines stably expressing FVII proteins with mutated N-glycosylation sites. By site-directed mutagenesis, we constructed one double and two single N-glycan knockout mutants (Figure 4A). The FVII-N322Q mutant comigrated with the 54 kDa form of wild-type FVII (Figure 4B). The FVII-N145Q mutant migrated as two bands, one comigrating with the 54-kDa band and one migrating at 52 kDa. The latter band comigrated with the FVII-N145/322Q mutant (Figure 4B). This confirmed that the 52-, 54-, and 56-kDa bands are FVII with zero, one, and two N-linked glycans, respectively. Taken together, the data demonstrate that the processing of the 54-kDa FVII form into the 56-kDa form during chase represents posttranslational N-glycosylation of FVII with one N-glycan into FVII with two N-glycans.
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Glycosylation of N145 and N322 is co- and posttranslational, respectively
Immediately after a 5-min pulse, labeled FVII-N145Q mutant protein migrated either as the 52-kDa form without N-glycans or as the 54-kDa form with one N-glycan (Figure 4C). During chase, the 52-kDa form was processed into the 54-kDa form, and 1 h after pulse, only the 54-kDa form was detected (Figure 4C). During the same interval, the FVII-N322Q mutant solely migrated as the 54-kDa form with one N-linked glycan (Figure 4C). Thus it seems that posttranslational N-glycosylation of FVII takes place at N322 but not at N145, which is glycosylated cotranslationally. This is consistent with the detection of the FVII forms with one or two N-glycans but not the form without N-glycans during processing of wild-type FVII (Figure 2A).
The pulse-chase experiments were carried out with a 5-min pulse. This was the shortest pulse that gave sufficient labeling for subsequent glycosidase treatment. To better investigate the events taking place immediately after translation, we examined the processing of wild-type FVII after a pulse lasting only 30 s (Figure 5). In the lanes of lysates harvested immediately after this pulse, a smear that migrated faster than the band of FVII with one N-glycan was seen (Figure 5). Braakman et al. (1991) noticed a similar smear after short pulses and demonstrated that it represented a population of unfinished protein chains that were still under elongation when the cells were lysed. They also demonstrated that the completion of such chains is the main reason for the increase in band intensity seen in the first minutes after pulse (Figure 5). Immediately after the 30-s pulse, labeled FVII migrating as the 54-kDa band was detected, whereas the 56-kDa band could not be discriminated from the background (Figure 5). Thus the vast majority or all of the labeled FVII appeared to have only one N-linked glycan immediately after the short pulse. During the following 15 min, the proportion of FVII with two N-linked glycans gradually increased (Figure 5). Combined with the previous experiments, this strongly suggests that nascent FVII is cotranslationally glycosylated at N145 as the protein grows into the ER. In contrast, glycosylation at N322 predominantly or even exclusively seems to take place posttranslationally.
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Folding and posttranslational N-glycosylation takes place in the same time frame
To examine a possible connection between folding and N-glycosylation of the nascent FVII polypeptide chain, we followed the migration of pulse-chase labeled FVII in nonreduced sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). To prevent disulfide bond formation after cell lysis, the alkylating agent N-ethylmaleimide was present during lysis and immunoprecipitation. Immediately after pulse, nonreduced labeled FVII migrated as a smear believed to represent different folding forms (Figures 5 and 6). During chase, the smear gradually condensed into a single fast-migrating band (Figures 5 and 6). This shift most likely reflects the folding of the labeled FVII into the more compact conformation of correctly folded FVII. One hour after pulse, most of the labeled nonreduced FVII appeared to migrate as the condensed band. This band had slightly faster mobility than the band of nonreduced FVII secreted to the cell culture medium (Figure 6). The slower migration of secreted FVII probably reflects the addition of saccharide residues to the glycan chains in distal compartments of the secretory pathway. Thus FVII appears to obtain a secretion-competent conformation within 1 h after translation. In the same experiment, posttranslational N-glycosylation also took place within the first hour of pulse, as judged from the lanes with reduced FVII (Figure 6). This kinetics for posttranslational N-glycosylation is in agreement with the results of Figures 2A and 4B. In conclusion, posttranslational glycosylation of N322 and folding of FVII seem to take place within the same time frame.
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Discussion |
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N-glycans are normally transferred to proteins cotranslationally as growing polypeptide strands are translocated from ribosomes into the ER (Glabe et al., 1980
; Kiely et al., 1976). The transfer is carried out by the OST complex, which is attached to the translocon (Kelleher et al., 1992; Nilsson and von Heijne, 1993; Silberstein and Gilmore 1996). However, not all potential N-glycosylation sites are used during translocation (Gavel and von Heijne, 1990; Pohl et al., 1984), and in some instances, sites that were not cotranslationally glycosylated may be glycosylated posttranslationally. In the present study, we have demonstrated posttranslational N-glycosylation of human FVII expressed in rodent and human cell lines. We believe that this is the first documentation of posttranslational N-glycosylation of a nonmodified protein in mammalian cells with an intact N-glycosylation machinery.
Previously, posttranslational N-glycosylation in mammalian cells has been documented in cells with a disrupted N-glycosylation machinery or for modified proteins. Thus posttranslational N-glycosylation has been described in tunicamycin-treated cells (Ronnet and Lane, 1981), in mannosylphosphoryldolichol-deficient mutant cells (Duvet et al., 2002), and for truncated proteins (Kolhekar et al., 1998). Evidence for posttranslational glycosylation of calreticulin in the prompt glycosylation response to heat shock has been presented (Jethmalani et al., 1994). Furthermore, unoccupied N-glycosylation sites of denatured glycoproteins were glycosylated in vitro (Pless and Lennarz, 1977), and tripeptides with target sequences for N-glycosylation (N-X-T/S) exogenously added to cell cultures were taken up by cells, glycosylated, and secreted (Geetha-Habib et al., 1990). The detection of two glycoforms of intracellular murine tyrosinase by western blotting has been suggested to reflect posttranslational N-glycosylation (Olivares et al., 2003), but pulse-chase studies indicate that the two glycoforms represent the ER form with high-mannose N-glycan chains and the Golgi form with complex chains, respectively (Halaban et al., 1997; Svedine et al., 2004; Watabe et al., 2004). Among nonmammalian cells, an example of posttranslational N-glycosylation was recently demonstrated in Drosophila cells (Tanaka et al., 2002).
The present study raises questions regarding the molecular and subcellular requirements for posttranslanstional N-glycosylation. Evidently, only proteins that remain in the vicinity of the OST complex after translation can be posttranslationally N-glycosylated. The present finding that folding of FVII takes place in the same time frame as posttranslational N-glycosylation is in agreement with FVII being retained in the ER or being recycled to the ER from a more distal compartment. There is increasing evidence that the ER is divided into different functional and structural domains (reviewed by Baumann and Walz, 2001). Thus the extent and duration of a proteins access to the luminal side of ER membrane domains with translocon complexes after translation may be a determining factor for the ability of the protein to undergo posttranslational N-glycosylation.
Another obvious requirement for posttranslational N-glycosylation is that the protein possesses a N-glycosylation site that is not cotranslationally utilized. Several reasons for potential N-glycosylation sites not being utilized have been described. Both the X and the Y residue of the N-X-S/T-Y target sequence influence the utilization of a N-glycosylation site. Proline in the X or the Y position, for example, is highly unfavorable for glycosylation (Bause, 1983; Gavel and von Heijne, 1990; Shakin-Eshleman et al., 1996; Mellquist et al., 1998). However, the context of the glycosylation site at N322 of FVII (N-I-T-E) is not considered unfavorable for glycosylation.
The use of N-glycosylation sites may also be less efficient near the C-termini of glycoproteins. It has been suggested that N-glycosylation sites are less likely to be utilized if they have not passed the OST, before the nascent protein looses its ribosomal attachment (Gavel and von Heijne, 1990). The distance between the peptidyl transferase site of a ribosome attached to a translocation pore and the active site of the OST complex have been estimated to span 65 amino acids of a polypeptide strand (Glabe et al., 1980; Whitley et al., 1996). Kolhekar et al. (1998) noticed that wild-type peptidylglycine 
-amidating monooxygenase-3 (PAM-3) was N-glycosylated cotranslationally. PAM-3 has a single N-glycosylation site at N765, which is located 125 amino acids from the C-terminal. However, in truncated PAM-3 proteins with N765 located only 55 or 13 amino acids upstream of the C-termini, cotranslational N-glycosylation was almost or completely absent. Truncated PAM-3 proteins that were not cotranslationally glycosylated could be posttranslationally glycosylated (Kolhekar et al., 1998). In FVII, N322 is located 84 amino acids upstream of the C-terminus, and thus we cannot exclude the possibility that the proximity of the C-terminal plays a role for the inefficiency or absence of cotranslational glycosylation of N322.
Furthermore, the conformation of the nascent protein may make N-glycosylation sites unaccessible for the OST complex. Like N-glycosylation, folding of glycoproteins begins cotranslationally as the nascent protein grows from the translocon pore into the ER (Bergman and Kuehl, 1979; Chen et al., 1995). Thus, the two processes are likely to influence one another. In several studies, N-glycosylation sites, which were only partially utilized under normal conditions, became fully used when disulfide bond formation was abolished by adding dithiothreitol to the cell cultures (see, for instance, Allen et al., 1995). In another study, optimal utilization of N-glycosylation sites required downstream polypeptide sequences (Dubuisson et al., 2000). Also, nonutilized N-glycosylation sites can be glycosylated in vitro after denaturation of the protein (Pless and Lennarz, 1977). In the present study, the posttranslational glycosylation of N322 took place in the same time frame as folding of the protein. Thus it is tempting to speculate that the cotranslational conformation(s) of FVII are unfavorable for glycosylation of N322, whereas during posttranslational folding, the protein obtains a conformation that makes N322 accessible for the OST complex.
A recent database search identified several proteins with glycans attached to N-glycosylation sites within the last 10 amino acids of the C-terminal (Ben-Dor et al., 2004). Based on the notion that cotranslational glycosylation of such sites is unlikely, Ben-Dor et al. (2004) suggested that these sites are posttranslationally glycosylated. The present study demonstrates that posttranslational N-glycosylation can indeed be a part of the normal processing of glycoproteins. Furthermore, the present study suggests that posttranslational N-glycosylation can take place not only because the glycosylation site is located close to the C-terminal but also due to a requirement for posttranslational folding of the nascent protein. This raises questions about how many glycoproteins that undergo posttranslational N-glycosylation as part of their normal processing. The scarcity of reports on this phenomenon may suggest that posttranslational N-glycosylation is a rare event. On the other hand, many examples of posttranslational N-glycosylation may yet await discovery, because it often requires analysis of samples from pulse-chased cells harvested within a relatively narrow time frame. Thus the general importance of posttranslational N-glycosylation in the processing of glycoproteins remains to be determined.
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Materials and methods |
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Plasmids and site-directed mutagenesis
Full-length human FVII cDNA originating from the
HVII565 clone generated by Hagen et al. (1986) (accession number M13232
[GenBank]
) was inserted into the BamH I/EcoR I sites of pcDNA3.1+ (Invitrogen, Carslbad, CA) to create the pTS8 plasmid. Constructs encoding FVII with N-glycosylation site knockout mutations were generated by site-directed mutagenesis of pTS8 using the QuickChange kit (Stratagene, La Jolla, CA) as recommended by the manufacturer. The N145Q mutation was introduced with the 5'-TTCTAGAAAAAAGACAAGCCAGCAAACCCCAAGG-3' forward primer (mutation in boldface) and the complementary reverse primer, and the N322Q mutation was introduced with the 5'-GTGGGAGACTCCCCACAAATCACGGAGTACATG-3' forward primer and the complementary reverse primer. Wild-type FVII cDNA was subcloned from pTS8 into the Hind III/EcoR I sites of pMPSVHE (Artelt et al., 1988) to create the pTS39 plasmid. Likewise, mutated FVII cDNA encoding the single or double N-glycosylation site knockout mutations were inserted into the Mlu I/EcoR I sites of pMPSVHE. Thereby, the FVII genes were placed under the transcriptional control of the myeloproliferative sarcoma virus promotor. The inserted FVII genes were verified by DNA sequencing. The structure of the encoded wild-type and mutant FVII proteins is shown in Figure 4A.
Cell culture
CHO-K1 and HEK293A cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with nonessential amino acids, 10% fetal calf serum, 5 µg/ml vitamin K1, 100 U/ml penicillin, and 100 µg/ml streptomycin. Transfection with the pMPSVHE-derived linearized plasmids was carried out with Lipofectamine (Invitrogen). Clones stably expressing the FVII proteins were selected with 450 µg/ml G418. Resistant clones were screened by testing cell culture supernatants for FVII by enzyme-linked immunosorbent assay.
Pulse-chase
Normal cell culture medium was replaced with methionine and cysteine deficient DMEM (Sigma, St. Louis, MO), and the cells were starved for 30 min. Then, 100 µCi/ml [35S]methionine and [35S]cysteine (Pro-mix, Amersham Biosciences, Little Chalfont, U.K.) was added, and the cells were pulsed for 30 s or 5 min. After pulse, the deficient medium was replaced with normal medium, and the cells were chased for various time intervals. In some experiments, 500 µM cycloheximide (Sigma) was added immediately after pulse and maintained in the medium until harvest of cells and medium. In all experiments, cells were harvested in cold lysis buffer consisting of 10 mM Tris–HCl, pH 7.8, 150 mM NaCl, 600 mM KCl, and 5 mM ethylenediamine tetra-acetic acid, protease inhibitor cocktail (Complete, Roche, Indianapolis, IN) and 2% Triton X-100. For experiments on the folding of FVII, 20 mM N-ethylmaleimide was added to the lysis buffer.
Radioimmunoprecipitation assay and glycosidase treatmen
Radioimmunoprecipitation assay was carried out by a modified version of the method described by Sheshberadaran et al. (1983). Lysates and media of pulse-chased cells were precleared by rotation for 1 h at 4°C with normal goat serum (Sigma). A slurry of Protein G conjugated Sepharose 4B (Zymed Laboratories, San Francisco, CA) in lysis buffer was added and rotation was continued for another hour. The Sepharose beads were pelleted, and the precleared supernatants were rotated for 1 h at 4°C with polyclonal goat antibodies against FVII (Novo Nordisk, Denmark). Protein G Sepharose was added as described and rotated with the lysate for another hour. The beads were then pelleted and washed three times in lysis buffer and two times in washing buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl).
For experiments on the folding of FVII, 20 mM N-ethylmaleimide was added to all buffers. The washed and pelleted beads ( 50 µl) were resuspended in 50 µl of 2x SDS–PAGE sample buffer (NuPAGE, Invitrogen) and boiled for 5 min. The beads were pelleted and the labeled proteins in the supernatants were separated by SDS–PAGE and visualized by autoradiography. For glycosidase treatment of FVII, however, the beads were resuspended and boiled for 10 min in 1% SDS, 2% 2-mercaptoethanol. Sodium phosphate at a final concentration of 50 mM, pH 7.5, 1% NP40, and the protease inhibitor cocktail were added to the supernatants, which were then divided in two and incubated for 1 h at 37°C with or without PNGase F (New England Biolabs, Beverly, MA). The glycosidase reactions were mixed with SDS–PAGE sample buffer and analyzed by SDS–PAGE and autoradiography. Quantification of the signals was carried out with a FLA-3000 phosphorimager and the Image Gauge 4.0 software (both from Fujifilm).
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Acknowledgements |
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We thank Else Jost Jensen and Gedske Thygesen for excellent technical assistance. We thank Drs. Esper Boel and Randal J. Kaufman for valuable comments and suggestions during the work and for critical reading of the manuscript. The pMPSVHE vector was kindly donated by Dr. Hansjörg Hauser.
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Abbreviations |
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CHO, Chinese hamster ovary; CR, connecting region; DMEM, Dulbecco’s modified Eagle medium; EGF, epidermal growth factor; ER, endoplasmic reticulum; Gla, gamma-carboxy glutamic acid; HEK, human embryonal kidney; OST, oligosaccharyltransferase; PAM-3, peptidylglycine
-midating monooxygenase-3; PNGase F, peptide: N-glycosidase F; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis |
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