All post-translational modifications except propeptide cleavage are required for optimal secretion of coagulation factor VII [98/5]

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© 2007 Schattauer GmbH, Stuttgart

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

All post-translational modifications except propeptide cleavage are required for optimal secretion of coagulation factor VII Gert Bolt, Thomas D. Steenstrup, Claus Kristensen Mammalian Cell Technology, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark

Summary Human coagulation factor VII (FVII) has two N-glycosylation sites (N145 and N322) and two O-glycosylation sites (S52 and S60). In transiently transfected COS-7 cells, all combinations of N- and O-glycosylation knock-out mutations reduced the release of FVII to the medium.Pulse-chase analysis of CHO-K1 cell lines expressing recombinant FVII demonstrated that virtually all wild-type FVII synthesized was secreted from the cells, whereas both N- and O- glycosylation knock-out mutations induced partial intracellular degradation of the synthesized FVII. Likewise, two thirds of the FVII synthesized in vitamin K-depleted and warfarin-treated CHO cells was degraded intracellularly, demonstrating the importance of gamma-carboxylation for the secretion of FVII. The furin inhibitor decanoylKeywords Factor VII, protein processing, post-translational, glycosylation, gamma-carboxylation

Introduction Coagulation factor VII (FVII) is a glycoprotein secreted by hepatocytes. Activated FVII (FVIIa) initiates the extrinsic coagulation pathway by activating factor IX and factor X upon binding to tissue factor on the surface of cells that have become exposed to circulating blood by injury (1). When given in pharmacologic doses, sufficient amounts of FVIIa binds to activated platelets and activates factor X, thus by-passing the tenase complex and inducing thrombin burst. By this mechanism, recombinant human FVIIa produced in mammalian cells can compensate for the lack of factor VIII or IX and is therefore used for treatment of bleeding in haemophilia A or B patients that produce antibodies (inhibitors) against factor VIII or IX (2–4). FVII belongs to a group of vitamin K-dependent glycoproteins associated with the coagulation system. Besides FVII, this group consists of factor IX, factor X, protein C, protein S, pro-

R-V-K-R-chloromethylketone inhibited propeptide cleavage,but FVII with propeptide appeared to be secreted equally well as FVII without propeptide. Propeptide cleavage was not inhibited by vitamin K depletion and warfarin treatment, suggesting that for FVII, correct gamma-carboxylation is not required for optimal processing of the propeptide. In conclusion, all post-translational modifications of FVII except propeptide cleavage were important for complete secretion of the synthesized FVII and to avoid intracellular degradation. Thus, the extensive post-translational modification of FVII seems critical for the intracellular stability of the protein and is required for keeping the protein in the secretory pathway.

Thromb Haemost 2007; 98: 988–997

tein Z, and prothrombin. These proteins have similar domain organization (reviewed in [5]) and are synthesized as precursors with a N-terminal propeptide followed by the mature amino acid sequence. The propeptide contains a docking site for gammacarboxylase, which converts glutamic acids into gamma-carboxy glutamic acids (Gla) in the adjacent Gla domain. In FVII, the Gla domain is followed by two epidermal growth factor-like (EGF) domains, a connecting region (CR), and a C-terminal serine protease domain. Prior to secretion, the propeptide is cleaved from the protein and after secretion, the protein can be activated into a disulfide-linked heterodimer by cleavage in the CR. Human FVII contains two O-glycans in the EGF-1 domain (S52 and S60), a N-glycan in the CR (N145), and another N-glycan in the catalytic domain (N322) (6–8). Thus, FVII and other vitamin K-dependent glycoproteins associated with coagulation undergo extensive post-translational modification (reviewed in [5, 9, 10]). In pulse-chase assays, se-

Received May 7, 2007 Accepted after revision July 4, 2007

Correspondence to: Gert Bolt Novo Nordisk A/S Novo Nordisk Park, 2760 Måløv, Denmark Tel.: +45 30756654, Fax: +45 444 44008 E-mail: [email protected]

Prepublished online October 11, 2007 doi:10.1160/TH07–05–0332

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cretion of FVII (11, 12) and other vitamin K-dependent proteins (13–16) from producer cells was a relatively slow process, indicating that the post-translational processing of these proteins is a challenge for the cellular secretory machinery. In the present study, we therefore examined the role of the post-translational modifications for secretion of FVII.

Materials and methods Plasmids and site-directed mutagenesis We have previously described the construction of pTS39, consisting of wild-type human FVII cDNA placed under the transcriptional control of the myeloproliferative sarcoma virus promoter in the pMPSVHE vector (12). Constructs encoding FVII with the O-glycosylation site knock-out mutations S52A, S60A, or S52/60A were generated by site-directed mutagenesis of pTS39 using the QuickChange kit (Stratagene) as recommended by the manufacturer. The S52A mutation was introduced with the 5’-GGGGACCAGTGTGCCGCAAGTCCATGCCAGAATGG-3’ forward primer (mutation in bold) and the complementary reverse primer, and the S60A mutation was introduced with the 5’-GCCAGAATGGGGGCGCCTGCAAGGACCAGCTCCA-3’ forward primer and the complementary reverse primer. The creation of similar constructs encoding FVII with the N-glycosylation knock-out mutations N145Q, N322Q, or N145/322Q were previously described (12). A construct encoding FVII devoid of both N- and O-glycosylation sites (FVII-N145/ 322Q-S52/60A) was constructed by introducing the S52/60A mutations in the N145/322Q encoding construct as described above. Cell culture Chinese hamster ovary (CHO)-K1 and African green monkey kidney CV-1 cells transformed with origin-defective SV40 (COS-7) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with non-essential amino acids, 10% fetal calf serum, 5 µg/ml vitamin K1, 100 units/ml penicillin and 100 µg/ml streptomycin. For transient transfection, 35-mm wells were seeded with 2.5 x 105 COS-7 cells each, and transfected 24 hours (h) later with 1 µg of the above FVII-coding plasmids using FuGENE 6 transfection reagent (Roche). Two days after transfection, the medium was collected and stored. The FVII content of medium from each well was determined by enzyme-linked immunosorbent assay (ELISA). We previously described the generation of CHO-K1-derived clones with stable expression of wild-type human FVII, FVII-N145Q, FVIIN322Q, or N145/322Q (12). Similar clones stably expressing FVII-S52A, FVII-S60A, FVII-S52/60A, or FVIIN145/322Q-S52/60A were established by co-transfection with the pMPSVHE-derived plasmids encoding each mutant and pSV2-neo (17) using FuGENE 6 transfection reagent. Transfected cells were selected with 600 µg/ml G418 and cloned by limiting dilution. The clones were screened for FVII production by testing cell culture supernatants for FVII by ELISA. For vitamin K depletion, a CHO-K1-derived clone producing wild-type FVII was cultured for more than a month in the absence of vitamin K.

ELISA and clot assay EGF domain-dependent FVII ELISA was carried out with standard ELISA reagents using the monoclonal antibody (Mab) FVII-4F9 (Novo Nordisk) as capture antibody and horse-radish peroxidase (HRP)-labelled FVII-4F7 Mab (Novo Nordisk) as detection antibody. Both these two antibodies recognize epitopes in the EGF domains of FVII. For Gla domain-dependent FVII ELISA, wells in microtiter plates were coated with polyclonal rabbit anti-FVII antibody (Novo Nordisk). Samples, detection antibody and HRP-labelled conjugate were all applied in dilution buffer consisting of 20 mM Hepes, 100 mM NaCl, 20 mM CaCl2, 0.2% bovine serum albumin (BSA), 0.02% Tween 80, pH 7.4. Bound FVII was detected with the Mab FVII-3F1 (Novo Nordisk), which recognizes an epitope in the FVII Gla domain, followed by HRP-labelled goat anti-mouse IgG (DAKO) and TMB PLUS Ready-to-use substrate (Kem-En-Tec). All washes were carried out with dilution buffer without BSA. ELISA data was analysed by one-way analysis of variance (ANOVA) with Tukey’s post test using the PRISM software (GraphPad). The clotting activity of wild-type and mutant FVII in medium from FVII-producing CHO-K1 cells was measured by Prothrombin time (PT) assay on an ACL 9000 coagulometer (Instrumentation Laboratory). Cell culture supernant (40 µl) diluted in saline was mixed with 40 µl FVII-deficient plasma, and 80 µl PT reagent with recombinant rabbit tissue factor (all reagents for PT assay were from Instrumentation Laboratory). Pulse-chase Normal cell culture medium was replaced with methionine- and cysteine-deficient DMEM (Sigma), and the cells were starved for 30 minutes (min) followed by a 5 min pulse with 100 µCi/ml [35S]methionine and [35S]cysteine (Pro-mix, Amersham Biosciences). After pulse, the deficient medium was replaced with normal medium, and the cells were chased for various time intervals. In some experiments, various chemicals were added at different times during the pulse-chase and then maintained in the medium until harvest of cells and medium. Dithiothreitol (DTT) (Sigma, 5 mM) was added 5 min before pulse, whereas lactacystin (Calbiochem, 50 µM), chloroquine (Sigma, 100 µM), or decanoyl-R-V-K-R-chloromethylketone (Alexis Biochemicals, concentrations indicated in the text) was added at the start of starvation. For pulse-chase of vitamin K-depleted cells, 1 µg/ml warfarin (Sigma) was present in the media from seeding until harvest of the cells. In all experiments, cells were washed in Hank’s solution and lysed in cold lysis buffer consisting of 50mM TrisHCl pH 7.5, 150 mM NaCl, 10 mM MgCl2, protease inhibitor cocktail (Complete, Roche) and Igepal CA630. For experiments on the folding of FVII, 20 mM N-ethylmaleimide was added to the lysis buffer. Pulse-chase assays on the kinetics of FVII secretion was carried out 2–4 times for each FVII variant. Radioimmuno precipitation assay (RIPA) and barium citrate precipitation For RIPA, 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 La-boratories) in lysis buffer was added, and rotation was continued for another hour. The sepharose beads were pelleted, and the pre-

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cleared supernatants were rotated for 1 h at 4°C with polyclonal goat antibodies against FVII (Novo Nordisk). Protein G Sepharose was added as described above and rotated with the lysate for another hour. The beads were then pelleted and washed five times in lysis buffer. For experiments on the folding of FVII, 20 mM N-ethylmaleimide was added to the lysis buffer. The washed and pelleted beads (approximately 50 µl) were resuspended in 50 µl of 2 X SDS-PAGE sample buffer (NuPAGE, Invitrogen) and boiled for 5 min. The beads were pelleted, and the labelled proteins in the supernatants were separated by SDSPAGE 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 above protease inhibitor cocktail were added to the supernatants, which were then incubated for 1 h at 37°C with peptide: N-glycosidase F (PNGase F) (New England Biolabs). The glycosidase reaction systems were mixed with SDS-PAGE sample buffer and analysed 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). In some experiments, FVII in media from pulse-chased cells were separated into the non-barium citrate precipitable fraction and the barium citrate precipitate prior to RIPA. Barium citrate precipitation was performed by a slightly modified version of the method described by Berkner et al. (18). Briefly, medium was initially incubated 10 min on ice with 22.5 µM sodium citrate. Then 45 µM BaCl2 was added and the medium was incubated 1 h on ice. The medium was then centrifuged for 5 min at 10,000 x g, and the non-barium citrate precipitable supernatant stored. The pellet was resuspended in 100 µM BaCl2 and 100 µM NaCl, incubated 1 h on ice and centrifuged as above. The supernatant was discarded, and the barium citrate precipitable pellet was resuspended in PBS with 150 µM sodium citrate and 0.1% BSA. FVII in the non-barium citrate precipitable supernatant and the resuspended barium citrate precipitate was detected by RIPA as described above.

Results Both N- and O-glycans are required for optimal secretion of FVII To estimate the influence of N- and O-glycans on the secretion of FVII, we mutated the expression construct pTS39 encoding wild-type human FVII and established similar constructs encoding FVII with glycosylation site knock-out mutations. Thus, constructs encoding FVII without N-glycans (FVII-N145/ 322Q), without O-glycans (FVII-S52/60A), or without both Nand O-glycans (FVII-N145/322Q-S52/60A) were generated. The efficiency of these mutations in eliminating the glycosylation sites have previously been demonstrated (12, 19). The amount of FVII in medium from COS-7 cells transiently transfected with these constructs was measured by ELISA. According to EGF domain-dependent FVII ELISA, knock-out of either the N- or the O-glycosylation sites reduced secretion approximately five-fold compared to wild-type FVII (Fig. 1A). Knock-out of both the N- and the O-glycosylation sites reduced secretion more than 30-fold (Fig. 1A). In the EGF domain-dependent FVII

ELISA, both the capture and the detection antibodies are monoclonals recognizing epitopes in the EGF domains. Since the O-glycosylation sites of FVII are located in the EGF-1 domain, we speculated that the recognition of FVII without O-glycans by the EGF domain-dependent ELISA might be reduced. Therefore, we developed a Gla domain-dependent FVII ELISA with a polyclonal capture antibody and a monoclonal detection antibody, which recognizes an epitope in the Gla domain. This ELISA detected various FVII molecules containing the Gla domain including a truncated FVII molecule that did not contain the FVII domains C-terminal to the Gla domain (data not shown). Thus, the Gla domain-dependent FVII ELISA was not dependent on the glycosylation of FVII. This ELISA, however, gave almost identical results to those of the EGF domain-dependent ELISA (Fig. 1B). Thus, the reduced amounts of FVII glycosylation site knock-out mutants compared to wild-type FVII measured in the medium of transfected cells does reflect a quantitative influence of both N- and O-glycans on the secretion of FVII. In order to confirm and further analyse these findings, we examined the kinetics of FVII secretion by pulse-chase analysis. CHO-K1 cell lines stably expressing the four different FVII molecules were metabolically labelled with [35S]methionine and [35S]cysteine for 5 min and chased for different time intervals. FVII in cell lysates and media harvested from 0 to 8 h after pulse was immunoprecipitated and subjected to SDS-PAGE. The intensity of the resulting bands of labelled FVII was measured by phosphorimager quantification. Immediately after the 5 min pulse, wild-type FVII immunoprecipitated from lysates migrated as two bands. During chase, the fastest migrating of these two bands was processed into the slower migrating band (Fig. 2A). We have previously demonstrated that this phenomenon represents post-translational glycosylation of N322 (12). According to phosphorimager quantification, the intensity of the bands of intracellular FVII peaked 15–30 min after pulse (Fig. 2A). This increase in band intensity probably represents the completion of unfinished protein chains that were still under elongation at the end of pulse as previously described by Braakman et al. (20). Release of the labelled FVII to the medium was a protracted process. A slow but steady decrease in the amount of labelled intracellular FVII began 1–1.5 h after pulse, and from the same time-point, increasing amounts of labelled FVII was detected in the medium. Almost all labelled intracellular FVII measured shortly after pulse had been released to the medium 8 h later (Fig. 2A). Thus, intracellular degradation of wild-type FVII appears negligible. FVII without N-glycans (FVII-N145/322Q) appeared to be secreted faster than wild-type FVII. During the first 2 h after pulse both the level of intracellular labelled FVII-N145/322Q decreased and the level of labelled FVII-N145/322Q in the medium increased considerable faster than for wild-type FVII. However, only approximately 65% of all labelled intracellular FVIIN145/322Q measured early after pulse was released, indicating that the remaining labelled FVII-N145/322Q was intracellularly degraded (Fig. 2B). In contrast, the absence of O-glycans (FVII-S52/60A) seemed to delay the secretion of FVII. During the first 3 h after pulse, only 30% of the labelled FVII-S52/60A was released (Fig

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Figure 1: Secretion of double N- and/or O-glycosylation site knock-out mutants. Wild-type and glycosylation site knock-out mutant FVII proteins were transiently expressed in COS-7 cells. Two days post-transfection, the content of FVII in the medium was measured by EGF-domain dependent ELISA (A) and Gla domain-dependent ELISA (B). Results are shown as average ± standard deviation of three independent transfections. According to oneway ANOVA with Tukey’s post test, the yields of all three glycosylation site knock-out mutants measured with EGF-domain dependent ELISA were significantly different from the yield of wild-type FVII (P≤0.0001). Furthermore, the yields of the three mutants were significantly different from each other (P≤0.05). Identical results were obtained by statistical analysis of the yields measured with the Gla domain-dependent ELISA, except that the difference between the N145/322Q and the S52/60A mutants was not significant.

A)

B)

2C) compared to 50% for wild-type FVII (Fig. 2A). In this period, the decrease in intracellular FVII-S52/60A was equalled by the increase in secreted FVII-S52/60A, but after this time-point, considerable amounts of intracellular FVII-S52/60A appeared to be degraded instead of being secreted. Approximately 50% of all initially labelled intracellular FVII -S52/60A was released to the medium (Fig. 2C). For FVII with neither N- nor O-glycans (FVII-N145/ 322Q-S52/60A), the decrease in labelled intracellular protein was much faster than the corresponding increase in secreted labelled FVII-N145/322Q-S52/60A almost from the beginning of the chase period (Fig. 2D). Thus, intracellular degradation of FVII-N145/322Q-S52/60A appeared even more pronounced than for FVII missing only the O-glycans, and only approximately 30% of all initially labelled intracellular FVII-N145/ 322Q-S52/60A was released to the medium (Fig. 2D). The pulse-chase experiments confirmed the finding from the transient expression system that both N- and O-glycans of FVII are required for optimal secretion and that the combined absence of both N- and O-glycans has an additive detrimental effect on secretion. A considerable fraction of the synthesized FVII lacking N- and/or O-glycans was intracellularly degraded suggesting

that both the N- and the O-glycans play a role for correct folding of FVII. Each individual N- and O-glycan of FVII favors secretion To examine the role of each N-glycan for secretion of FVII, we also transiently transfected COS-7 cells with constructs encoding mutated FVII lacking each of the two N-glycosylation sites (FVII-N145Q and FVII-322Q). The absence of either N-glycosylation site reduced secretion but not to the same extent as knock-out of both sites (Fig. 3A). Likewise, we also examined the effect of removing each O-glycosylation site with constructs encoding FVII-S52A and FVII-S60A. Each of these mutations reduced secretion by approximately 50%, while secretion of FVII without any O-glycans was reduced by a factor 5 (Fig. 3B). Thus, each N-glycan and each O-glycan contributes to the secretion of FVII in an additive manner. Gamma-carboxylation is required for efficient secretion of FVII Plasma-derived or recombinant FVII is gamma-carboxylated on nine or ten of the ten glutamic acid residues of the Gla domain

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B)

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D) Figure 2: Kinetics of secretion of N- and/or O-glycosylation site knock-out mutants. CHO-K1 cell lines were pulsed by metabolic labelling and chased for the indicated intervals. FVII was immunoprecipitated from lysates and media and analysed by SDS-PAGE. The resulting bands from wild-type FVII (A), FVII-N145/322Q (B), FVII-S52/60A (C), and FVII-N145/322Q-S52/60A (D) are shown to the left. For each FVII protein, the intensity of the bands of labelled FVII from lysates and media was measured by phosphorimager quantification and plotted in the chart shown to the right.

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Figure 3: Secretion of single or double Nor O-glycosylation site knock-out mutants. Wild-type FVII and N-glycosylation site (A) or O-glycosylation site (B) knock-out mutants were transiently expressed in COS-7 cells. Two days post-transfection, the content of FVII in the medium was measured by ELISA. Results are shown as average ± standard deviation of four independent transfections. According to one-way ANOVA with Tukey’s post test, the yields of all three N-glycosylation siteknock out mutants and all three O-glycosylation site knock-out mutants were significantly different from the yield of wild-type FVII (P≤0.0001). Furthermore, the yields of the three N-glycosylation mutants were significantly different from each other (P≤0.0001). The yields of the S52/A and S60A single O-glycosylation site knock-out mutants were significantly different from the yield of the S52/60A double O-glycosylation site knock-out mutant (P≤0.0001), but the difference between the two single O-glycosylation site knock-out mutants was not significant.

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(7). We investigated the importance of gamma-carboxylation for secretion of FVII by pulse-chase analysis of a FVII producing CHO-K1 cell line maintained under conditions that inhibit gamma-carboxylation. Gamma-carboxylase requires reduced vitamin K as a co-factor. The carboxylating reaction oxidizes vitamin K, but vitamin K epoxide reductase, which is inhibited by warfarin, can recycle oxidized vitamin K back to the reduced form required by the gamma-carboxylase (reviewed in [21]). Therefore, the cells were vitamin K-depleted by culturing without vitamin K for more than a month, and the pulse-chase assay was carried out in the presence of 1 µg/ml warfarin. To examine the effect of these conditions on gamma-carboxylation of FVII in our cells, medium harvested 8 h after pulse was analysed by barium citrate precipitation followed by immunoprecipitation of FVII. Only proteins with several gamma-carboxylated glutamic acids are precipitated with barium citrate (18, 22). Barium citrate precipitable FVII was not detected in the medium of vitamin K-depleted and warfarin-treated cells, whereas the vast majority of labelled FVII in medium of the same cell line grown with vitamin K and without warfarin was precipitated by barium citrate (Fig. 4). In agreement with these findings, FVII activity could not be detected by prothrombin time (PT) assay in the medium

from vitamin K-depleted and warfarin-treated FVII-producing cells (data not shown). Thus, vitamin K depletion and warfarin treatment efficiently blocked gamma-carboxylation. Under these conditions, the level of labelled intracellular FVII decreased relatively fast, whereas the amount of labelled FVII in the medium increased at a much slower rate, and only approximately one third of the total labelled intracellular FVII was secreted after 8 h (Fig. 5). Thus, most of the synthesized FVII appeared to be intracellularly degraded, when gamma-carboxylation was prevented. To confirm that intracellular degradation actually accounted for the difference between the total amount of labelled FVII measured early after pulse and the sum of labelled FVII recovered in lysate and medium late after pulse, we tried to inhibit intracellular degradation with the proteasome inhibitor lactacystin (23) and the lysosome inhibitor chloroquine (24). When present in the medium during chase, chloroquine and, to a lesser degree, also lactacystin increased the amount of labelled intracellular FVII in vitamin K-depleted and warfarin-treated cells examined 8 h after pulse (Fig. 6). Both inhibitors also appeared to inhibit FVII secretion (Fig. 6), which must be taken into account when evaluating their influence on intracellular degradation. According to phosphorimager analysis of the gel

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Figure 4: Gamma-carboxylation of FVII analyzed by barium citrate precipitation. CHO-K1 cells producing wild-type FVII were metabolically labelled and chased for 8 h. Medium (T) was fractionated into the non-barium citrate precipitable fraction (N) and the barium citrate precipitate (B). FVII was immunoprecipitated from all three samples and analyzed by SDS-PAGE. Vitamin K-deprived cells incubated with warfarin (- vit. K) and cells grown under standard conditions with vitamin K and without warfarin (+ vit. K) were compared.

shown in Figure 6, lactacystin and chloroquine increased the sum of labelled FVII in lysate and medium harvested 8 h after chase by approximately 9% and 42%, respectively. This is in good agreement with the pulse-chase analysis of vitamin K-depleted cells assayed without inhibitors of intracellular degradation shown in Figure 5, which suggests that approximately 62% of the total amount of labelled FVII measured early after pulse had disappeared 8 h after pulse. In conclusion, a substantial fraction of the FVII synthesized in the absence of gamma-carboxylation was indeed degraded intracellularly, and lysosomal degradation appeared to be the major pathway for this degradation. Propeptide cleavage is not required for FVII secretion Propeptide cleavage of FVII takes place at four consecutive arginine residues immediately before the N-terminal of the Gla domain (6, 25). This polybasic motif conforms to the substrate specificity of furin, a subtilisin-like serine endoprotease (26). To investigate the importance of propeptide cleavage for the secretion

of FVII, the pulse-chase assay was carried out in the presence of the furin inhibitor decanoyl-R-V-K-R-chloromethylketone. To better compare the migration of intracellular and secreted FVII, N-glycans were removed by treatment of the immunoprecipitated FVII with PNGase F prior to SDS-PAGE. In the absence of furin inhibitor (0 µM), intracellular FVII migrated more slowly than FVII secreted to the medium, demonstrating cleavage of the propeptide shortly before secretion (Fig. 7A). The furin inhibitor did not influence the migration of intracellular FVII (Fig. 7A). The migration of secreted FVII, however, reacted in a dose-dependent manner on addition of the furin inhibitor. With increasing concentrations of furin inhibitor, the migration of secreted FVII shifted towards a more slowly migrating form that comigrated with intracellular FVII (Fig. 7A). Thus, decanoylR-V-K-R-chloromethylketone inhibited propeptide cleavage of FVII. According to phosphorimager quantification, treatment with 0.2 or 1 µM of the inhibitor did not influence the level of labelled FVII in the medium, whereas 5 µM, 25 µM or 125 µM of inhibitor reduced the level of FVII in the medium by 30–50%. Nevertheless, there was no indication that FVII with propeptide was secreted less efficiently than FVII without propeptide. The reduced secretion of FVII treated with 5–125 µM of inhibitor most likely represents a general effect on the secretory pathway of the cells. Thus, propeptide cleavage did not appear to play a role for secretion of FVII. Above, we demonstrated that gamma-carboxylation is vital for correct processing and secretion of FVII. Therefore, we also examined whether gamma-carboxylation is required for propeptide cleavage. FVII in medium from vitamin K-depleted and warfarin-treated cells migrated faster than FVII in lysates of the same cells (Fig. 7B). However, when 25 µM decanoyl-R-VK-R-chloromethylketone was added to the medium, secreted FVII co-migrated with intracellular FVII (Fig. 7B). Thus, only FVII without the propeptide was detected in the medium from vitamin K-deprived cells, as was also seen in cells grown under standard conditions. As, we were unable to precipitate the FVII secreted from vitamin K-depleted and warfarin-treated cells

Figure 5: Role of gamma-carboxylation for secretion of FVII. Vitamin K-deprived FVII producing CHO-K1 cells incubated with warfarin were metabolically labelled and chased for the indicated intervals. FVII was immunoprecipitated from lysates and media and analyzed by SDS-PAGE. The resulting bands are shown to the left. The intensity of the bands of labelled FVII from lysates and media was measured by phosphorimager quantification and plotted in the chart shown to the right.

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with barium citrate (Fig. 4), it appears that gamma-carboxylation does not influence propeptide cleavage.

Discussion Secretion of nascent FVII was a slow protracted process, since most FVII molecules remained intracellular for several hours after their synthesis before being released from the cell. Nevertheless, intracellular FVII appeared stable, since intracellular degradation was negligible. In previously reported pulse-chase experiments, secretion of FVII (11, 12) and other vitamin K-dependent proteins (13–16) was also slow. The present study demonstrates that each post-translational modification of FVII except propeptide cleavage is essential for optimal secretion of FVII and for avoiding intracellular degradation of FVII. This strongly indicates that in the cell, the bare amino acid chain of FVII and similar vitamin K-dependent proteins is prone to malfolding and that extensive post-translational modification of the protein is required to balance this property and keep the protein in the secretory pathway until released from the cell. In the present study, FVII secreted by vitamin K-deprived cells was not gamma-carboxylated and did not exhibit measurable activity. A similar result was obtained with vitamin K-deprived FVII producing cells by Berkner et al. (27). In another study, Clarke and Sridhara (28) examined FVII secreted from cells that had been grown without vitamin K for 3–6 passages and found that the specific activity decreased from approximately 50% to 15% of normal activity during this period. Both Berkner et al. (27) and Clarke and Sridhara (28) as well as ourselves (data not shown) also found that the medium from FVII producing cells contained less FVII antigen when grown in the absence of vitamin K. In agreement with these findings in vitro, reduced levels of both the amount and the specific activity of circulating FVII has been measured in warfarin-treated patients (29). The present pulse-chase analysis of vitamin K-deprived FVII-producing cells demonstrates that the intracellular stability of human FVII is highly dependent on gamma-carboxylation of the Gla domain, and that the reduced secretion of FVII from these cells is due to intracellular degradation of most of the synthesized FVII. Among the other vitamin K-dependent proteins associated with coagulation, an important role of gamma-carboxylation for secretion has also been reported in pulse-chase experiments with human protein C, protein S, protein Z, and prothrombin (13, 16, 30–32). Notably, the secretion of human prothrombin was much less susceptible to vitamin K deprivation than that of protein C (33). In contrast, gamma-carboxylation did not appear to be important for secretion of human factor IX (34, 35) or factor X (36). Interestingly, these findings in vitro correlate well with results on warfarin treatment in vivo. In the blood of patients starting oral warfarin treatment, the levels of FVII and protein C antigen decreased much faster than the levels of prothrombin, factor IX and factor X antigen (37). The gamma-carboxyglutamic residues in the Gla domain of FVII and other vitamin K-dependent proteins associated with coagulation bind Ca2+, and this binding induces conformational shifts that are important for the activity of the protein (38–42). However, these conformational changes can only take place after removal of the propeptide (43). Thus, propeptide cleavage is also

Figure 6: Proteosomal and lysosomal degradation of FVII in the absence of correct gamma-carboxylation. Vitamin K-deprived and warfarin-treated FVII-producing CHO-K1 cells were pulsed by metabolic labelling and chased for 8 h with the proteasome inhibitor lactacystin (L), with the lysosome inhibitor chloroquine (C), or without inhibitor (-). FVII was immunoprecipitated from lysates and media and analyzed by SDS-PAGE.

A)

B) Figure 7: Role of propeptide cleavage for secretion of FVII. A) CHO-K1 cells incubated with the indicated concentrations of the furin inhibitor decanoyl-R-V-K-R-chloromethylketone were pulsed by metabolic labelling and chased for 4 h. FVII was immunoprecipitated from lysates and media, incubated with PNGase F, and analyzed by SDSPAGE. B) Vitamin K-deprived and warfarin-treated FVII-producing CHO-K1 cells were pulsed by metabolic labelling and chased with (+) or without (-) 25 mM decanoyl-R-V-K-R-chloromethylketone. Lysate and media were harvested 2 h and 8 h after pulse, respectively. FVII was immunoprecipitated, incubated with PNGase F, and analyzed by SDS-PAGE.

a prerequisite for optimal activity. In the present study, only FVII without propeptide appeared to be secreted from cells grown under standard conditions. In contrast, only FVII with propeptide was detected in the cells, which indicates that propeptide cleavage takes place immediately prior to secretion. Propeptide cleavage was inhibited in a dose-dependent manner by decanoylR-V-K-R-chloromethylketone, which inhibits furin and related endoproteases targeting multibasic cleavage sites (26). Furin resides primarily in the trans-Golgi network (26), a location that corresponds well with the above finding that propeptide cleavage occurs shortly before secretion. Furin has been shown to cleave the propeptides of factor IX and protein C (35, 44), and the present data suggest that furin is also important for propeptide cleavage of FVII. Secretion of FVII was, however, independent of propeptide cleavage.

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There are indications that propeptide cleavage of factor IX and factor X is inhibited by insufficient gamma-carboxylation (34–36). In the present study, however, propeptide cleavage of FVII in vitamin K-deprived cells appeared to be complete. Thus the propeptide cleavage of FVII seems to be independent of gamma-carboxylation. The O-glycans of FVII are unusual, as they consist of a di- or trisaccharide (xylose-glucose or xylose-xylose-glucose) on S52 and a single fucose residue on S60 (8, 45). Apparantly, such O-glycans are restricted to EGF-like domains and have only been detected on relatively few proteins including some of the vitamin K-dependent proteins and Notch receptors (reviewed in [46, 47]). In the present study, these short O-linked glycans were at least as important for the intracellular stability and secretion of FVII as the much larger N-glycans. Furthermore, the PT activity of the secreted FVII O-glycosylation knock-out mutants was reduced compared to wild-type FVII, whereas elimination of the N-glycosylation sites did not reduce the PT activity (data not shown). Previous studies have also shown that the O-glycans are required for full clotting activity of FVII (8, 19, 48). Taken to-

gether, these findings strongly suggest that the O-glycans of FVII are required for correct folding of FVII. The folding of secretory proteins takes place in the endoplasmic reticulum (ER), but O-glycosylation is generally believed to take place in the more distal Golgi cisternae and trans-Golgi network. However, two recent studies (49, 50) demonstrate that O-fucosylation of Notch EGF-like domains takes place in ER and suggests that O-fucosylation is involved in the ER quality control. The subcellular localization of O-glucosylation remains to be determined, but the present finding that the S52 and the S60 O-glycan of FVII contributed equally to secretion of FVII may indicate that both O-glycans on FVII play a significant role for quality control in the secretory pathway of FVII-producing cells. Acknowledgements We thank Berit Gerlach and Else Jost Jensen for excellent technical assistance. EGF domain-dependent ELISA was skilfully performed by Annemette Petersen and Michael Wilken. The pMPSVHE vector was kindly donated by Dr. Hansjörg Hauser.

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