0 1987 by The American Society of Biological Chemists, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 262, No. 14, Issue of May 15, pp. 6729-6734,1957

Printed in U. S. A.

Expression of Completely y-Carboxylated Recombinant Human Prothrombin*

(Received for publication, September 9, 1986)

Maria J. JorgensenS, Alan B. Cantor$, Barbara C. Furie, and Bruce Furie From the Center for Hemostasis and Thrombosis Research, Division of Hemntolngy-Oncology, Department of Medicine, New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts 021 11

Human prothrombin cDNA has been expressed in mammalian cells to yield biologically active, fully y- carboxylated prothrombin, A 2.0-kilobase cDNA en- coding full-length prothrombin was isolated from a human fetal liver library using a cDNA fragment re- covered from a hg t l l human hepatoma expression li- brary. Prothrombin cDNA was cloned into a mamma- lian expression vector and transfected into Chinese hamster ovary cells. Selection for expression of dihy- drofolate reductase yielded cell lines secreting up to 0.55 pg/ml of prothrombin. Recombinant prothrombin synthesized in the presence of vitamin K was quanti- tatively recovered from tissue culture medium by af- finity chromatography using conformation-specific antibodies directed against the metal-stabilized, y-car- boxylated conformer. The purified material migrated as a single band on denaturing polyacrylamide gels with an electrophoretic mobility equivalent to that of plasma-derived human prothrombin. Automated Ed- man degradation of recombinant prothrombin re- vealed a single amino-terminal sequence identical to that of plasma-derived prothrombin. Recombinant and plasma-derived prothrombin interacted similarly with antibodies specific for total prothrombin, abnormal des-7-carboxyprothrombin, and two metal-stabilized conformers of prothrombin. Recombinant prothrombin exhibited a specific coagulant activity equivalent to that of plasma-derived prothrombin. The y-carboxy- glutamic acid analysis of recombinant prothrombin demonstrated 9.9 f 0.4 mol of y-carboxyglutamic acid/ mol of prothrombin. These results represent the first description of the expression of a recombinant vitamin K-dependent protein in which all of the expressed pro- tein is y-carboxylated.

Prothrombin is a plasma glycoprotein ( M , 72,000) which participates in the final stages of blood coagulation (1, 2). Prothrombin is synthesized in the liver and undergoes several post-translational modifications prior to secretion. These modifications include glycosylation, cleavage of the pre- and propeptides (3, 4), and vitamin K-dependent y-carboxylation of the 10 amino-terminal glutamic acid residues (5, 6). The

* This work was supported by Grants HL21543 and HL18834 from the National Institutes of Health and a grant from Seragen, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ This work was performed by M. J. J. in partial fulfillment of the requirements for the degree of Doctor of Philosophy from Tufts University.

Louis, MO 63110. 5 Present address: Washington University School of Medicine, St.

y-carboxyglutamic acid residues confer metal-binding prop- erties upon prothrombin and the other vitamin K-dependent proteins (7,8), allowing these proteins to bind to lipid surfaces (9, 10). In the presence of metal ions, prothrombin undergoes a conformational change (11-13) which is required for binding to phospholipid surfaces and for coagulant activity (14, 15).

Expression of recombinant prothrombin in mammalian cells would provide a system for identifying important func- tional domains. For example, structure-function relationships in prothrombin might be elucidated using site-directed mu- tagenesis. Such studies should yield information about the roles of specific y-carboxyglutamic acid residues, the location of the lipid binding sites, or the regions of the molecule which are needed to signal y-carboxylation. For these purposes an expression system is needed in which all of the secreted recombinant prothrombin is y-carboxylated. To date, expres- sion of completely processed recombinant vitamin K-depend- ent coagulation proteins has not been reported. Recombinant human Factor IX, a related vitamin K-dependent protein, has been expressed in several mammalian host cells (16-19). In each of these studies only a fraction of the secreted protein has undergone sufficient post-translational processing to ex- press biological activity. In the current study, human pro- thrombin cDNA has been cloned and expressed in Chinese hamster ovary cells. The expressed recombinant protein has been purified to homogeneity and shown to be equivalent to plasma-derived prothrombin in specific coagulant activity, expression of metal-stabilized antigenic determinants, and y- carboxyglutamic acid content.

EXPERIMENTAL PROCEDURES

Screening of cDNA Libraries-A human hepatoma cDNA expres- sion library, prepared in the Xgtll vector of Young and Davis (20), was generously provided by J. R. de Wet (University of California, San Diego) (21). The library was screened for clones expressing prothrombin antigen using a chromogenic immunodetection system (21). Approximately 100,000 plaques were transferred to nitrocellu- lose filters and incubated overnight in Tris-buffered saline containing 3% bovine serum albumin, 0.02% sodium azide, and 0.5 pg/ml of immunoaffinity-purified rabbit anti-total prothrombin antibodies. After washing several times in Tris-buffered saline, the filters were incubated for 4 h in Tris-buffered saline containing 3% bovine serum albumin and goat anti-rabbit immunoglobulin conjugated with horse- radish peroxidase (Bio-Rad, 1:1,000 dilution). After further washing, positive plaques were detected upon addition of the substrate 1- chloro-2-naphthol (Bio-Rad). Seven positive clones were isolated by three rounds of rescreening, and phage DNA was purified from plate lysates (22). The cDNA inserts were excised with EcoRI and sub- cloned for analysis by restriction enzyme mapping. One 650-base pair insert was tentatively identified as the 3’-end of prothrombin cDNA based upon its restriction pattern. This identity was confirmed by nucleotide sequencing using the chemical cleavage method of Maxam and Gilbert (23).

To obtain the full-length coding sequence of prothrombin, a human fetal liver cDNA library in Charon 21A (24) was screened using the

6729

6730 Recombinant y-Carboxylated Prothrombin mel:lod of Benton and Davis (25). Restriction fragments of prothrom- bin cDNA, derived from the cDNA insert obtained from the human hepatoma expression library, were radiolabeled to a specific activity of approximately 10Rcpm/pg by random hexanucleotide priming using [32P]dCTP and the Klenow fragment of DNA polymerase (26). Hy- bridization for 16 h at 68 "C in 90 mM sodium citrate, pH 7.0, 900 mM sodium chloride, 5 X Denhardt's solution (27), 10 mM EDTA, and 0.5% sodium dodecyl sulfate was followed by extensive washing at 68 "C in 30 mM sodium citrate, pH 7.0, 300 mM sodium chloride, and 0.5% sodium dodecyl sulfate. Duplicate positives were plaque- purified and phage DNA was isolated from plate lysates (22). Desired restriction fragments of cDNA inserts were subcloned into appropri- ate M13 vectors (28) for restriction enzyme mapping and sequencing by the dideoxynucleotide chain termination method (29).

Construction of Prothrombin Expression Plasmid pMT2-PT-The full-length prothrombin coding sequence was reconstructed from two overlapping cDNA inserts, each digested at the single HindIII site, by cloning the appropriate fragments into M13mp18. A 2.0-kilobase EcoRI fragment encoding the complete prothrombin sequence was then isolated and inserted into the EcoRI site of the mammalian expression vector, pMT2. This plasmid, a slightly modified form of the expression vector p91023 (19, 30), was generously provided by R. Kaufman (Genetics Institute, Cambridge, MA). The resultant pro- thrombin expression plasmid, pMT2-PT, was shown by restriction mapping to contain the prothrombin coding region in the proper orientation with respect to the adenovirus major late promoter.

Cell Culture, DNA Transfection, and Cell Line Selection-The dihydrofolate reductase-deficient Chinese hamster ovary cell line, CHO DUKX-B11, was grown and maintained as described (31, 32). The cells were transfected with 20 pg of the prothrombin expression plasmid, pMT2-PT, by calcium phosphate coprecipitation (32). After transfection, cells were grown in a-modified Eagle's medium lacking nucleosides (GIBCO) containing 10% heat-inactivated fetal bovine serum, 5 pg/ml vitamin K1 (Aquamephyton, Merck Sharp and Dohme), and thymidine, adenosine, deoxyadenosine, penicillin, and streptomycin (10 pg/ml each). The cells were subcultured 2 days later into the same medium except that the nucleosides were omitted and dialyzed serum was used ("selective medium"). Transfected cells were fed every 3-4 days with selective medium until colonies were visible, about 10-12 days after subculturing. These initial transformants were pooled and grown in selective medium until confluent. At confluence, a 10-cm dish contained approximately 1.7 X lo' cells in 10 ml of medium. Large volumes of conditioned media were collected by re- feeding flasks of confluent cells every 3-4 days for several weeks. Conditioned media were stored at -20 "C until needed. For experi- ments requiring amplification of prothrombin expression levels, cells from the pool of initial transformants were selected for expression of high levels of dihydrofolate reductase by sequential subculturing in selective medium containing increasing concentrations of methotrex- ate (0.02, 0.2, and 0.5 pg/ml).

Preparation of Protein Standards and Antibodies-Human pro- thrombin was purified from plasma by barium citrate adsorption, DEAE-cellulose chromatography, and affinity chromatography using dextran-Sepharose (33, 34). Human abnormal des-y-carboxypro- thrombin was purified from plasma by DEAE-Sephacel chromatog- raphy and affinity chromatography using anti-prothrombin-sepha- rose (35). The protein concentration of purified prothrombin was measured using A'./, at 280 nm of 14.4. Prothrombin and abnormal prothrombin were iodinated with NalZ5I using the lactoperoxidase method (36).

Rabbit anti-prothrombin. Ca(I1) antibodies were purified by im- munoaffinity chromatography on prothrombin-Sepharose in the pres- ence of Ca(I1) followed by elution with EDTA (35). Anti-prothrombin antibodies which bound to prothrombin-Sepharose in the presence of EDTA were eluted with 4 M guanidine hydrochloride and were termed anti-total prothrombin. Anti-abnormal prothrombin antibodies were prepared by sequential immunoaffinity chromatography using des-y- carboxyprothrombin-Sepharose as described (35). Anti-prothrombin. Mg(I1) and anti-prothrombin.Ca(I1)-specific antibodies were pre- pared by sequential immunoaffinity chromatography using prothrom- bin-Sepharose in the presence of either Mg(I1) or Ca(I1) followed by elution with EDTA (37).

Radioimmunomsays-The displacement of lZ5I-labeled prothrom- bin from anti-prothrombin antibodies was studied using a competi- tion radioimmunoassay. Anti-total prothrombin antibodies (1.1 X 1 O P M ) were added to a reaction mixture which included '251-labeled prothrombin (1.7 X 10-'" M) and varying concentrations of competi- tors. All components were diluted in Tris-buffered s-line containing

1 mM benzamidine, 0.1% bovine serum albumin, 3 mM EDTA, and carrier rabbit y-globulin. Anti-prothrombin. Ca(I1) antibodies (3.4 X

M), anti-prothromhin.Mg(I1) antibodies (1.0 X lo-' M), or anti- prothrombin. Ca(I1)-specific antibodies (1.3 X lo-' M) were added to the same reaction mixture except that 3 mM calcium chloride replaced EDTA. Anti-ahnormal prothrombin antibodies (4.0 X lo-' M) were added to a similar mixture containing '251-labeled abnormal pro- thrombin (2.8 X M ) and 3 mM EDTA. After overnight incubation at 4 "C, the bound '251-labeled prothrombin or abnormal prothrombin was precipitated by the addition of goat anti-rabbit immunoglobulin. The precipitate which formed was removed by centrifugation and was assayed for '''I in a Beckman Gamma 8000 spectrometer. For deter- mination of unknown antigen concentrations a standard curve was prepared using human plasma-derived prothrombin or abnormal pro- thrombin of known concentration.

Purification of Recombinant Prothrombin-Recombinant pro- thrombin was purified from conditioned medium by immunoaffinity chromatography using conformation-specific antibodies (38,39). The antibodies employed, anti-prothrombin . Ca(II), bind to the antigenic determinants expressed on y-carboxylated prothrombin in the pres- ence of metal ions. Conditioned medium containing 10 mM calcium chloride and 0.02% sodium azide was applied to a column of anti- prothrombin.Ca(I1)-Sepharose at 4 "C. After exhaustive washing with Tris-buffered saline containing 10 mM calcium chloride and 0.5 M sodium chloride, the calcium was eluted with 10 mM EDTA in Tris-buffered saline. The protein concentration of purified recombi- nant prothrombin was measured using Ala at 280 nm of 14.4. Elec- trophoresis was carried out on 10% polyacrylamide gels in the pres- ence of sodium dodecyl sulfate (40). Samples were incubated for 5 min at 100 "C in sample buffer containing 2-mercaptoethanol prior to loading on the gel. Gels were stained after electrophoresis with Coomassie Blue R-250.

Prothrombin Coagulation Assay-Prothrombin activity was deter- mined in a two-stage assay using prothrombin-deficient plasma (35). Human prothrombin of known concentration was used to prepare the standard curve.

Amino-terminal Sequence Analysis-Automated Edman degrada- tion was performed on an Applied Biosystems Model 470A gas-phase Protein Sequencer equipped with a Model 120 PTH Analyzer (41). Recombinant prothrombin was desalted by high pressure liquid chro- matography using an RP-300 Brownlee guard cartridge prior to analysis.

y-Carboxyglutamic Acid Analysis-Amino acid analyses were per- formed on a Beckman Model 119CL amino acid analyzer equipped with a Beckman Model 126 data system. The proteins were hydro- lyzed in 2 M potassium hydroxide for 22 h at 110 "C (42). The y- carboxyglutamic acid composition was quantitated after alkaline hy- drolysis by automated amino acid analysis using a ninhydrin detection system.

RESULTS

Cloning of Prothrombin cDNA-A human hepatoma cDNA expression library was screened using affinity-purified anti- prothrombin antibodies. One positive clone was shown by restriction enzyme mapping and nucleotide sequencing to contain an insert of 650 base pairs comprising the 3'-end of prothrombin cDNA (4). In order to obtain the full coding sequence for prothrombin, this fragment was used to screen a fetal liver cDNA library. Restriction enzyme analysis of the numerous positive clones which were isolated revealed that these inserts also were incomplete a t their 5"ends. A restric- tion fragment was prepared from the 5'-end of the insert which contained the most complete 5'-sequence, and this fragment was used to rescreen the library. Several clones were recovered which encoded the entire 5'-end including the ini- tiator codon. The full-length prothrombin coding sequence was reconstructed from two overlapping cDNA inserts.

Extensive restriction enzyme analysis and partial nucleo- tide sequencing of the prothrombin cDNA gave results iden- tical to those predicted from the previously reported partial sequence (4). The cDNA obtained is 2.0 kilobases in length and contains approximately 100-150 nucleotides of the 3'- untranslated region and 1 2 bases of 5"untranslated sequence.

Recombinant y-Carboxylated Prothrombin 6731

The nucleotide sequence of the 5’-end and the corresponding predicted amino acid sequence are shown in Fig. 1. The human prothrombin leader sequence begins with residue -43 and is homologous in its predicted amino-terminal sequence to the bovine prothrombin predicted sequence (3).

Expression of Recombinant Prothrombin in Chinese Ham- ster Ovary Cells-The prothrombin expression vector, pMT2- PT, contains the SV40 origin of replication, the adenovirus major late promoter, the prothrombin coding region, the di- hydrofolate reductase coding region, the SV40 early polyade- nylation site, the adenovirus virus-associated genes, and the pBR322 sequences needed for propagation in Escherichia coli. Details of the components of this vector have been described (19,30). Plasmid pMT2-PT was introduced into dihydrofolate reductase-deficient Chinese hamster ovary cells, and cells were selected for the dihydrofolate reductase-positive pheno- type by subculturing in selective medium. Culture media harvested when the cells reached confluency were assayed by competition radioimmunoassay using anti-total prothrombin antibodies. Total prothrombin antigen was found to be ex- pressed by these primary transfectants a t varying concentra- tions up to 0.55 pg/ml (100 ng/106 cells/24 h). The same samples were assayed using conformation-specific antibodies, anti-prothrombin. Ca(II), which bind to specific determinants expressed on prothrombin in the presence of metal ions. These determinants are present only when prothrombin is suffi- ciently y-carboxylated to undergo its metal-induced confor- mational transition, and their presence correlates closely with coagulant activity. Levels of native prothrombin measured using this assay were equivalent to the levels determined for total prothrombin antigen, suggesting that all of the pro- thrombin expressed was carboxylated and biologically active. Indeed, no (abnormal) des-y-carboxyprothrombin could be detected, using anti-abnormal prothrombin antibodies, to a limit of 0.03 pg/ml. When prothrombin expression levels were amplified by subculturing transfected cells in the presence of 0.5 pg/ml of methotrexate, 6.5-8.2 pg/ml(1.3-1.6 pg/106 cells/ 24 h) of total prothrombin antigen was secreted. At these levels of prothrombin expression, native prothrombin concen- trations were 4.2-4.4 pg/ml and abnormal prothrombin con- centrations were 0.8-1.1 pg/ml.

Purification and Characterization of Recornbinant Pro- thrombin-Recombinant prothrombin was isolated from con- ditioned medium by immunoaffinity chromatography using conformation-specific antibodies (38, 39). The tissue culture supernatant was applied to a column of anti-prothrombin. Ca(I1)-Sepharose in the presence of Ca(I1). All prothrombin antigen was removed from the culture medium by this process; no prothrombin antigen was detected, using anti-total pro- thrombin antibodies, in the material that failed to bind to the column. The bound prothrombin was eluted with EDTA, and prothrombin was recovered quantitatively in this eluate.

Purified recombinant prothrombin migrated as a single major band on dodecyl sulfate gels in the presence of 2-

5‘ A G C TGA CAC ACT ATG GCG CAC GTC CGA OOC TTG GAG CTG CCT ...

3‘

Human Met Ala His Val Arg Gly Leu Gh Leu Pro ...

Bovine Met Ala Arg Val Arg Gly Pro Arg Leu Pro ... -43 -34

FIG. 1. Nucleotide sequence of the 5‘-end of human pro- thrombin cDNA. The nucleotide sequence of the 5’-end of the full- length prothrombin cDNA insert used to express recombinant pro- thrombin was determined by the dideoxy method. The deduced amino acid sequence is shown underneath with the corresponding sequence from bovine prothrombin (3). The amino acid residues encoded by DNA sequence not previously reported (4) are shown in italics.

mercaptoethanol (Fig. 2). Its electrophoretic mobility was identical to that of prothrombin derived from human plasma.

The coagulant activity of recombinant prothrombin was determined directly using prothrombin-deficient plasma. Pu- rified plasma prothrombin of known concentration was used to prepare a standard curve. Recombinant prothrombin was found to have 99 f 4% of the coagulant activity of plasma prothrombin (Table I).

The interactions of recombinant prothrombin with four distinct populations of anti-prothrombin antibodies were as- sessed by competition radioimmunoassay. Anti-total pro- thrombin antibodies bind to prothrombin regardless of its state of carboxylation or conformation. As expected, recom- binant prothrombin, plasma-derived prothrombin, and ab- normal des-y-carboxyprothrombin equally displaced ’2sI-la- beled prothrombin from these antibodies (Fig. 3A). Anti- abnormal prothrombin antibodies bind to antigenic determi- nants present only on des-y-carboxy forms of prothrombin (35,43). Recombinant prothrombin and plasma prothrombin did not displace ‘2sI-labeled abnormal prothrombin from these antibodies, demonstrating the absence of even trace quantities of des-y-carboxyprothrombin in the recombinant preparation (Fig. 3B) . Two antibody populations specific for different metal-dependent conformers of prothrombin have recently been described (37). Anti-prothrombin. Mg(I1) antibodies bind to antigenic determinants expressed on prothrombin only when it is sufficiently carboxylated to undergo the first conformational change which is induced by most divalent and trivalent metal ions. Anti-prothrombin. Ca(I1)-specific anti- bodies are directed against antigenic determinants exposed when prothrombin undergoes a second conformational change associated with expression of the lipid binding site. This change is supported only by Ca(II), not by most other metal ions. Recombinant prothrombin and plasma prothrombin equally displaced 9-labeled prothrombin from both of these

1 2

FIG. 2. Electrophoretic analysis of affinity-purified recom- binant prothrombin. Conditioned medium was applied to an anti- prothrombin.Ca(I1)-Sepharose column in the presence of 10 mM calcium chloride, and the bound recombinant prothrombin was eluted with 10 mM EDTA as described. The purified recombinant material and prothrombin purified from human plasma were analyzed on a 10% dodecyl sulfate-polyacrylamide gel in the presence of 2-mercap- toethanol. The gel was stained with Coomassie Blue R-250. L.une I , recombinant prothrombin; lane 2, plasma-derived prothrombin.

TABLE I Coagulant activity and y-carboxyglutamic acid content of

recombinunt Drothrombin Coagulant y-Carboxyglutnrnic

activity acid content 76 mOl

Plasma-derived prothrombin 100 10.0 Recombinant Drothrombin 99 f 4 9.9 f 0.4

6732 Recombinant y-Carboxylated Prothrombin

c A

0

* 20. .

Antl-PT: hlgl//l Antl-PT: cwlrl)-sp~clrlc

10.

[COMPETITOR] ,ug/ml [COMPETITOR] #p/ml

FIG. 3. Binding of recombinant prothrombin to conformation-specific anti-prothrombin antibodies. The binding of various prothrombin species to antibody was evaluated by competition radioimmunoassay. Recombinant prothrombin (W), plasma-derived prothrombin (O), and abnormal prothrombin (A) were used to displace 1251-labeled prothrombin from: A, anti-total prothrombin antibodies (Anti-TPT); C , anti-prothrombin. Mg(I1) antibodies (Anti-PT:Mg(lZ)); and D, anti-prothrombin. Ca(I1)-specific antibodies (Anti-PT:Cu(ll)-spe- cific). In B, the same competitors were used to displace '251-labeled abnormal prothrombin from anti-abnormal prothrombin antibodies (Anti-APT).

antibodies (Fig. 3, C and D), demonstrating essential equiva- lence of recombinant prothrombin and plasma prothrombin by these criteria.

The amino-terminal sequence of recombinant prothrombin was established by automated Edman degradation. As shown in Table 11, the amino acid sequences of the first 16 amino acid residues of recombinant prothrombin and plasma-derived prothrombin are identical. Of particular interest is the fact

TABLE I1 Amino-terminal sequence of recombinant prothrombin

Residue Recombinant Plasma Prothrombin urothrombin

1 2 3 4 5 6 l 8 9

10 11 12 13 14 15 16

Ala Asn Thr Phe Leu -

Val Arg LY G ~ Y Asn Leu

-

- Arg

pmol 64 32 24 36 31

-

23 9

19 16 15 18

I -

-

Ala Asn Thr P he Leu - (Gla)b - (Gla) Val Arg LY s GlY Asn Leu - (Gla) '4% - (Gla)

that no secondary sequences, representing incompletely pro- cessed pre- or propeptide, were detected. Additionally, as the y-carboxyglutamic acid derivative is not released from the filter during the standard sequencing cycle, the undetectable levels of glutamic acid found at residues 6, 7, 14, and 16 are consistent with complete y-carboxylation of these residues.

The y-carboxyglutamic acid content of recombinant pro- thrombin was determined by amino acid analysis of the al- kaline hydrolysate. Purified recombinant prothrombin con- tained 9.9 f 0.4 mol of y-carboxyglutamic acid/mol of protein, using as the standard the value of 10 mol/mol for plasma- derived prothrombin (Table I).

DISCUSSION

Prothrombin, a vitamin K-dependent blood coagulation protein, is a model for the class of calcium-binding proteins that contain y-carboxyglutamic acid. Considerable informa- tion has accrued concerning the relationships of structure and function (l), but important insight would be gained into these relationships if recombinant prothrombin could be expressed and changes in the structure introduced by site-directed mu- tagenesis. Toward this objective, we have cloned the cDNA encoding full-length prothrombin, completing the known par- tial sequence at the 5'-end. These studies reveal that the human prothrombin nucleotide sequence predicts a prepro- prothrombin that has a 43-residue extension at the amino terminus when compared with plasma prothrombin. This prepropeptide has marked sequence homology with that pre- dicted from the bovine prothrombin cDNA (3).

Recombinant prothrombin was expressed in Chinese ham-

~

a -, not identified. * Gla, y-carboxyglutamic acid.

Recombinant y-Carboxylated Prothrombin 6733

ster ovary cells in a form that is fully active and completely y-carboxylated. The recombinant prothrombin antigen was quantitatively recovered from tissue culture medium by im- munoaffinity chromatography using conformation-specific antibodies (38, 39). These antibodies bind only to well y - carboxylated protein species that undergo a conformational change in the presence of metal ions. Purified recombinant prothrombin was identical to plasma-derived prothrombin upon dodecyl sulfate gel electrophoresis and amino-terminal amino acid sequence analysis, demonstrating that the recom- binant molecule undergoes normal proteolytic processing for removal of the propeptide in this expression system. The extent of y-carboxylation of recombinant prothrombin was assessed and compared with that of plasma-derived prothrom- bin. The prothrombin coagulant activity, impaired in the absence of even 1 or 2 y-carboxyglutamic acid residues (35), was similar to that of plasma-derived prothrombin. Recom- binant prothrombin and plasma-derived prothrombin bound equivalently to antibodies which recognize the metal-depend- ent conformational changes, including expression of the phos- pholipid binding site, which require full y-carboxylation (39, 44, 45). The y-carboxyglutamic acid content of recombinant prothrombin, quantitated by amino acid analysis of the al- kaline hydrolysate, was equivalent to that of plasma-derived prothrombin. In each of these analyses, recombinant pro- thrombin had properties identical with those of plasma pro- thrombin, confirming that the recombinant prothrombin was completely y-carboxylated and biologically active.

The thorough y-carboxylation of recombinant human pro- thrombin observed here is in contrast to that observed in the expression of recombinant human Factor IX, another vitamin K-dependent coagulation protein (16-19). The Factor IX gene has been introduced into a variety of cell types, including rat (16) and human (17) hepatoma cells, mouse fibroblasts (16, 17), baby hamster kidney cells (18), and Chinese hamster ovary cells (19). Although the levels of Factor IX produced in these studies varied widely, the fraction of total Factor IX antigen which was y-carboxylated was never more than 50- 70%. These figures represent upper limits since most of these analyses were based upon indirect measurements of extent of y-carboxylation as deduced from the ratio of antigen bound and unbound to barium salts and/or from measurement of Factor IX coagulant activity, using conditioned medium or the fraction recovered after adsorption to barium salts. In our earlier report (19) the active fraction of Factor IX was purified to homogeneity and extensively characterized. In that study, however, the active material represented only 1-2% of the total Factor IX antigen (40-100 pg/ml) produced in a highly amplified Chinese hamster ovary cell line. In a more recent study (46), approximately 60-70% of the Factor IX expressed by unamplified Chinese hamster ovary cell lines was suffi- ciently carboxylated to undergo its conformational transition in the presence of metal ions. In the current study, recombi- nant prothrombin synthesized at levels of up to 0.55 pg/ml in Chinese hamster ovary cells is completely y-carboxylated. When prothrombin expression levels are amplified 10-15- fold, however, only approximately 60% of the secreted pro- thrombin is sufficiently carboxylated to bind to the confor- mation-specific antibodies. Chinese hamster ovary cells are thus capable of more efficient y-carboxylation of both pro- thrombin and Factor IX when expressed at low levels than when expression is amplified. It appears that the y-carboxy- lation system has a limited capacity for the amount of sub- strate which can be efficiently processed over a given period of time; when the rate of substrate synthesis exceeds this limit, the extent of y-carboxylation is reduced.

Two differences are apparent in the relative efficiencies of y-carboxylation of prothrombin and Factor IX by Chinese hamster ovary cells. First, a greater percentage of prothrom- bin molecules are well-carboxylated when compared with Factor IX expressed at comparable levels. Second, the abso- lute level of well-carboxylated, native prothrombin antigen expressed by the amplified cells is approximately 5-fold higher than the levels of well-carboxylated Factor IX antigen ob- served even at 10-fold higher expression levels (19). Pro- thrombin may have some fundamental structural character- istics which make it a better substrate than Factor IX for the y-carboxylation system. The propeptides of all the vitamin K-dependent proteins demonstrate extensive homology with one another, and this region has been proposed to be impor- tant in targeting these proteins for y-carboxylation. Indeed, a recent study from this laboratory (46) showed that an intact Factor IX propeptide is required for y-carboxylation of Factor IX by Chinese hamster ovary cells. The prothrombin propep- tide might have certain features which make proprothrombin a better substrate for y-carboxylation than proFactor IX. TO assess whether differences in the propeptides are responsible for the observed differences in y-carboxylation of recombi- nant prothrombin and Factor IX, a chimeric protein which includes the signal sequence and propeptide of prothrombin attached to the Factor IX zymogen might be constructed using recombinant DNA techniques. Such a construction might allow more efficient post-translational processing and y-carboxylation of the recombinant Factor IX expressed.

The development of a system for the expression of fully carboxylated, biologically active human prothrombin now al- lows the complete examination of the role of the propeptide in post-translational processing, particularly as it relates to y-carboxylation and intracellular trafficking of proteins dur- ing protein synthesis. In addition, it will allow the systematic introduction of point mutations into the y-carboxyglutamic acid-rich region, the phospholipid binding site, the Factor V binding site, and the Factor Xa binding site of prothrombin, extending structure-function relationships previously based upon mutant prothrombins (eg. prothrombin Barcelona (48)) and variant prothrombins (39, 49).

Acknowledgments-We thank Drs. J. R. de Wet, J. J. Toole, and R. J. Kaufman for providing the human hepatoma cDNA expression library, the fetal liver cDNA library, and the expression vector pMT2, respectively. We are especially grateful to Drs. Richard Goodman and Charles Shoemaker for helpful advice and discussions and to Cheryl Brown and Karen Kotkow for selection of methotrexate-resistant cell lines.

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