Biosynthesis of Prothrombin: Intracellular Localization of the Vitamin K-Dependent Carboxylase and the Sites of y-Carboxylation

By J. Andrew Bristol, Jennifer V. Ratcliffe, David A. Roth, Margaret A. Jacobs, Barbara C. Furie, and Bruce Furie

Prothrombin is a vitamin K-dependent blood coagulation protein that undergoes posttranslational y-carboxylation and propeptide cleavage during biosynthesis. The propep- tide contains the y-carboxylation recognition site that di- rects y-carboxylation. To identify the intracellular sites of carboxylation and propeptide cleavage, we monitored the synthesis of prothrombin in Chinese hamster ovary cells sta- bly transfected with the prothrombin cDNA by immunofluo- rescent staining. The vitamin K-dependent carboxylase was located in the endoplasmic reticulum and Golgi complex. Antibodies specific to prothrombin processing intermedi- ates were used for immunocytolocalization. Anti-des-y-car- boxyprothrombin antibodies stained only the endoplasmic reticulum whereas antiproprothrombin antibodies (specific

ROTHROMBIN, a plasma glycoprotein, is a component P of the blood coagulation process and is the zymogen of the serine protease thrombin.’ The synthesis of this protein requires vitamin K, and as such has been a prototype for the study of the calcium-binding proteins that contain y- carboxyglutamic acid. Prothrombin is synthesized in the liver, along with the other vitamin K-dependent blood clot- ting proteins. The prothrombin cDNA encodes a multido- main protein that includes a signal peptide, a propeptide, a domain rich in y-carboxyglutamic acid (Gla), a short aro- matic amino acid stack domain, two kringle domains, and the serine protease domain.’ The N-terminus includes 10 y- carboxyglutamic acid residues at residues 6, 7, 14, 16, 20, 21, 25, 26, 29, and 32 in human prothrombin. The x-ray crystal structure of the N-terminal third of bovine prothrom- bin has shown that many of these y-carboxyglutamic acid residues coordinate internal calcium ions, and thus stabilize the phospholipid binding structure of the Gla d0main.l

The vitamin K-dependent proteins contain an N-terminal extension during intracellular synthesis. The propeptide, de- fined as residues - 18 to - l in factor IX: contains the y- carboxylation recognition site.’ The propeptides of the ho- mologous vitamin K-dependent proteins share sequence similarity in that the carboxylation recognition site and the propeptide cleavage site are conserved! The propeptide is an amphipathic a helix, with the carboxylation recognition site located just N-terminal to the helix.7 This recognition element directs carboxylation’ and mediates the binding of the propeptide in the prozymogen to the vitamin K-depen- dent carboxylase.* The model that has been proposed for the biosynthesis of the vitamin K-dependent proteins, including prothrombin and factor IX, is that the uncarboxylated precur- sor or prozymogen is first carboxylated.’ The fully carboxyl- ated prozymogen is then processed proteolytically by a pro- peptidase, furin, to remove the propeptide before secretion of the mature protein into the plasma.” We have recently isolated and characterized fully carboxylated profactor M and found that, in contrast to factor IX, it does not bind to membrane surfaces in the presence of calcium ions?

The vitamin K-dependent carboxylase is a microsomal membrane protein that catalyzes the conversion of glutamic acid residues to y-carboxyglutamic acid residues. The cDNA

for the propeptide) and antiprothrombin:Mg(lI) antibodies (which bind the carboxylated forms of proprothrombin and prothrombin) stained both the endoplasmic reticulum and the Golgi complex. Antiprothrombin:Ca(lI)-specific antibod- ies (which bind only to the carboxylated form of prothrom- bin lacking the propeptide) stained only the Golgi complex and secretory vesicles, and colocalired with antimannosi- dase II and anti-p200 in the juxtanuclear Golgi complex. These resutts indicate that uncarboxylated proprothrombin undergoes complete y-carboxylation in the endoplasmic re- ticulum and that y-carboxylation precedes propeptide cleav- age during prothrombin biosynthesis. 0 1996 by The American Society of Hematology.

of the human” and the bovine’’ y-carboxylase encodes a single-chain polypeptide composed of 758 amino acids. The active site responsible for glutamate binding and catalysis as well as the complementary site for the propeptide carbox- ylation recognition site are located in the N-terminal one third of the p r~ te in . ’~ . ’~ Vitamin K epoxidase activity is also catalyzed by the carbo~ylase’~; a C-terminal region of this enzyme is required for this activity.I6 Furin is a membrane- associated, calcium-dependent serine endopeptidase homol- ogous to the bacterial subtilisins.” This enzyme catalyzes precursor processing at Arg-X-Lys/Arg-Argl, a motif in pro- prothrombin (Arg-Val-Arg-Arg) at residues -4 to - 1. Furin, synthesized as a zymogen and autoproteolytically acti- vated,” is concentrated in the trans Golgi network.”

To further understand the intracellular posttranslational processing that accompanies the synthesis of the vitamin K- dependent proteins, we have examined the biosynthesis of human prothrombin expressed in heterologous Chinese ham- ster ovary (CHO) cells. These cells synthesize biologically active prothrombin that is fully carboxylated when the cells are grown in the presence of vitamin K.” Using a panel of antibodies directed against various biosynthetic intermedi- ates of we have studied prothrombin bio- synthesis using immunofluorescence subcellular localization techniques. An antibody against the propeptide recognizes proprothrombin but not prothrombin; an antibody against

From the Center for Hemostasis and Thrombosis Research, the Division of Hematology-Oncology, New England Medical Center; and the Departments of Medicine and Biochemistry, Tufts University School of Medicine, Boston, MA.

Submitted March 21, 1996; accepted May 23, 1996. Supported by Grants No. HL38216, HL42443, and HU2574 from

the National Institutes of Health. Address reprint requests to Bruce Furie. MD, Division of Hema-

tology-Oncology, Tufts-New England Medical Center, 750 Washing- ton St, Boston, MA 02111.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 19% by The American Society of Hematology. 0006-4971/96/8807-0$3.00/0

Blood, Vol 88, No 7 (October 1). 1996: pp 2585-2593 2585

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2586 BRISTOL ET AL

abnormal (des-y-carboxy) prothrombin recognizes uncar- boxylated prothrombin and uncarboxylated proprothrom- bin.*’ The conformation-specific antibody antiprothrom- bin:Mg(II) recognizes fully carboxylated prothrombin regardless of the presence or absence of the propeptide.’ The conformation-specific antiprothrombin:Ca(II)-specific anti- bodies bind only to fully carboxylated prothrombin in which the propeptide was cleaved. We conclude that the y-carbox- ylase resides in both the endoplasmic reticulum and the Golgi complex, but carboxylation is completed in the endoplasmic reticulum. Complete y-carboxylation precedes propeptide cleavage during prothrombin biosynthesis.

EXPERIMENTAL METHODS

Antibodies. The preparation of the immunoaffinity-purified con- formation-specific antibodies, antiprothrombin:Mg(II) and antipro- thrombin:Ca(II)-specific, have been previously described.” Anti-ab- normal prothrombin antibodies were prepared in rabbits using purified des-y-carboxy (abnormal) prothrombin as previously de- scribed. **

Antipropeptide antibodies to the prothrombin propeptide were pre- pared by the identical methods used to prepare anti-factor IX pro- peptide antibodiesz4 A synthetic peptide, KGGHVFLAPQQARSL- LQRVRR, representing residues -18 to -1 of prothrombin with an additional N-terminal tripeptide KGG for purposes of chemical linking, was synthesized on an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, CA). After purifica- tion, the sequence of the peptide was verified by automated Edman degradation using an Applied Biosystems 470A Protein Sequencer. The peptide was coupled to bovine serum albumin (BSA) using glutaraldehyde. The proprothrombin propeptide (150 pL; S mmol/ L), dissolved in phosphate-buffered saline (PBS; SO mmoVL phos- phate, pH 7.41140 mmoVL NaC1) was mixed with 500 pL of 0.05 mmol/L BSA dissolved in PBS, and 350 pL of PBS was added. Glutaraldehyde ( 1 mL; 2.5 mmol/L; Sigma, St Louis, MO) was added dropwise to the peptide:BSA solution and stirred for 2 hours at room temperature. The final molar ratio of peptide:BSA:glutaral- dehyde was 30:l:lOO. The peptide conjugate was dialyzed against PBS and stored at -20°C. A New Zealand White rabbit was immu- nized subcutaneously with 500 pg of the peptide:BSA conjugate in Freund’s complete adjuvant ( I mL). The rabbit subsequently re- ceived monthly injections of 200 pg of the peptide:BSA conjugate in Freund’s incomplete adjuvant ( I mL). Antiserum was prepared 10 to 14 days after each immunization. Antiprothrombin propeptide antibodies were purified by immunoaffinity chromatography using the synthetic propeptide or BSA linked to cyanogen bromide-acti- vated Sepharose 4B (Sigma). Approximately 15 mL of antiserum was applied to a BSA-Sepharose column (7.5 mg BSA/mL Sepha- rose; 1 X 5 cm column) at 23°C to remove anti-BSA antibodies. Antibodies that failed to bind were applied to a propeptide-Sepharose column (6 mg of propeptide/mL of Sepharose 4B; I X 5 cm column). Bound antibody was eluted with 4 moVL guanidine HC1, and the guanidine eluate immediately dialyzed against Tris-buffered saline (TBS; 10 mmol/L Tris-HC1, pH 7.4/140 mmoVL NaCI) at 4°C.

Anticarboxylase antibodies directed against bovine carboxylase residues 86-99 and residues 661-675 were prepared as de- scribed.I2.l3 Antimannosidase I1 antibodiesz5 were the gift of Drs Marilyn G. Farquhar (University of California, San Diego) and Kelley Moremen (University of Georgia, Athens). A monoclonal antibody (MoAb), AD7, directed against the p200 protein associ- ated with the Golgi complex,” was the gift of Dr Brian Burke (Harvard Medical School, Boston, MA). Polyclonal anti-BiP (HSP78) antibodies were prepared in rabbits using a bovine liver

carboxylase preparation in which BiP was the major component.’ The IgG fraction of this antiserum was purified by affinity chroma- tography using protein A-Sepharose (Sigma).

Expression and pur$cation of proprothrombin/R-2D,R-IE. Mutant cDNA for proprothrombin/R-2D.R-lE was constructed using the site overlap extension method of the polymerase chain reaction (PCR).27 After gel purification and restriction digestion, the muta- genic DNA fragment was substituted for the wild-type fragment in pMT2-PT, a mammalian expression vector containing the cDNA for human prothrombin’“ and the gene for dihydrofolate reductase. The entire region generated by the PCR was sequenced before transfec- tion. A clonal population of dihydrofolate reductase-deficient CHO cells, CHO Dukx-B11/4,** was further selected for its ability to fully carboxylate prothrombin. Plasmid DNA was transfected into the cells using lipofectin (GIBCO-BRL Life Technologies, Gaithers- burg, MD). Clones expressing prothrombin were detected by a filter hybridization assay using polyclonal antiprothrombin antibodies. CHO cells producing the highest levels of the mutant proprothrombin were introduced in 2,000 mL of medium into Nunc cell factories and allowed to grow for 7 days. The collected medium was concen- trated in an Amicon Hollow Fiber Concentrator (model RA2000; Beverly, MA) and applied to an immunoaffinity column of poly- clonal antiprothrombin antibodies. The column was washed with borate-buffered saline ( I mol/L NaCI, 0. I m o m boric acid, pH 8.0) containing 2 mmom benzamidine and 0.1 % Tween 80. The column was washed with TBS (20 mmol/L Tris, 150 mmoUL NaC1, pH 7.5) before elution. The protein was eluted with 4 mol/L GuHCI, dialyzed three times against TBS containing I mmol/L benzamidine, and concentrated using Amicon Centriprep 30 concentrators.

A CHO cell line expressing prothrombin was grown to near confluence on glass coverslips (Corning Glass, Coming, NY) in selective media (AdT deficient) containing 10 pg/ mL of vitamin K, . The coverslips were transferred to ceramic boats submerged in TBS and the cells were fixed at 25°C by incubating the coverslips for 20 minutes in 3.7% formaldehyde (Fisher, Pitts- burgh, PA) diluted into TBS containing either 3 mmol/L CaClz or 3 mmoVL EDTA. After washing the coverslips in TBS, cells were permeabilized by incubating the coverslips at 25°C for 30 minutes in 0.5% Triton X-100 (Sigma) in TBS. After washing with TBS. coverslips were stored at 4°C in TBS/O.l% BSA/O.OS% NaN,. Anti- body ( I O pL at 5 to 100 pg/mL in 0.2% ovalbumin/TBS/O.OS%NaN,) was applied to the cells on the coverslips, and the coverslips incu- bated for 45 minutes at 37°C. After washing in TBS, the cells were stained with rhodamine-labeled goat-antirabbit IgG (Pierce, Rock- ford, IL). After washing in TBS, the coverslips were mounted on glass slides with Airvol (Air Products, Allentown, PA). ImmunoHu- orescence microscopy was performed on a Zeiss Axioscope fluores- cence microscope at 6 3 0 ~ magnification (Zeiss, Thornwood, NY). Micrographs were prepared using Kodak T-400 black and white film (Eastman Kodak, Rochester, NY).

For double-label experiments, rabbit antiprothrombin:Ca(II)-spe- cific antibodies (100 pg/mL) and AD7 (a murine antikp200 MoAb; ascites fluid I : 100 dilution) were mixed in dilution buffer (2% BSA, TBS, pH 7.4, 0.02% NaN,), applied to coverslips together, and incubated as described above. After washing with PIPES buffer ( IS mmol/L PIPES, pH 7.4, 140 mmol/L NaC1, 1 mmol/L CaCI,), the coverslips were incubated with a mixture of rhodamine-conjugated goat-antimouse IgG (Pierce; diluted 1 :250 into dilution buffer) and fluorescein-conjugated donkey-antirabbit IgG (Pierce; diluted 1 : LOO in dilution buffer). In control experiments, either rabbit antipro- thrombin:Ca(II) antibodies, AD7, or both were omitted to evaluate the specificity of staining with the second antibody. Samples were washed again in PIPES buffer and photographed at 630X magnifica- tion using a Zeiss Axioscope fluorescence microscope. Filter sets were used for excitation and visualization of fluorescein and rhoda-

Immunojluorescencr.

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BIOSYNTHESIS OF PROTHROMBIN 2587

A 6 1 2 3 1 2 3 4 5 6

87 + c 87

,_ -

140 140+

48 + c 48

Fig 1. Purification and characterization of proprothrombin. (A) SDS gel electrophoresis of prothrombin and proprothrombin forms. Lane 1, des-y-carboxy-proPT/R-2D,R-l E; lane 2, proPTIR-2D.R-lE; lane 3, plasma-derived prothrombin. Proteins were visualized with Coomassie blue. (B) Western blot of proprothrombin and prothrombin. Lane 1, plasma-derived prothrombin; lane 2, proPT/R-2D.R-1E; lane 3, des-y-carboxy- proPT/R-2D.R-1E; lane 4, plasma-derived prothrombin; lane 5, proPT/R-ZD.R-lE; lane 6, des-y-carboxy-proPT/R-2D,R-lE. Lanes 1 through 3 were developed with antiprothr0mbin:totaI antibodies. Lanes 4 through 6 were developed with anti-proPT antibodies. The blots depicted in lanes 1 through 3 and lanes 4 through 6 were performed separately with their respective antibodies.

mine signals. The sample was excited at 450 to 490 nm and emission monitored with a 5 15- to 565-nm barrier filter to observe the fluoro- scein signal. The rhodamine signal was visualized with excitation at 546 nm using a barrier filter at 590 nm.

RESULTS

PuriJication and characterization of antibodies directed against the prothrombin propeptide. We generated a poly- clonal antibody directed against the prothrombin propeptide

A

to examine the presence or absence of the propeptide in various intracellular prothrombin biosynthetic intermediates. This antibody was prepared in a rabbit using a synthetic peptide based on the sequence of proprothrombin (prom) (residues -18 to -1) that was coupled to BSA as a carrier protein. The antiprothrombin propeptide antibodies were pu- rified from rabbit antiserum by sequential immunoaffinity chromatography. Antibody that failed to bind to BSA-Sepha- rose was applied to a propeptide-Sepharose column. The

110 ~

C Fig 2. Interaction of anh-pro-

thrombin antibodies with pro- 'lo

thrombin, proPT/R-2D.R-lE, and des ycarboxyprothrombin. (A) 90

AntiproPT antibodies directed against the propeptide. (B) Anti- abnormal prothrombin antibod- E 'O

ies. (C) Antiprothrombin:MgllI) antibodies. ID) Antiprothrom- 5o

bin:Ca(ll)-specific antibodies. (MI, Prothrombin; (AI, proPT/R-tD,R- 1E; (01, des y-carboxyprothrom- 3o

bin; ( 1, des-ycarboxy-proPT/

p

Comptiia (nH) RD-2,RE-1.

B 110 , I

80

Ea

a 0

k. I

D

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2588 BRISTOL ET AL

? Fig 3. Localization of precursor prothrombin spe

cies and the vitamin K-dependent y-carboxylase. CHO cells expressing prothrombin were stained with antibodies, as indicated. (A) Anti-abnormal pro- thrombin; (6) anti-prom; IC) antibovine carboxylase; (D) anti-BiP. The arrowheads identify the juxta- nuclear Golgi complex.

antibody that bound was eluted with 4 mol/L guanidine HCI and is referred to as anti-prow antibody.

Expression and characterization of proPT/R-ZD, R-1 E, Proprothrombin is efficiently processed in heterologous CHO cells2o and no proprothrombin can be detected after secretion. To obtain proprothrombin, CHO cells were trans- fected with a mutated form of prothrombin cDNA. These cells expressed the mutant form of prothrombin, proPT/R- 2D,R-IE in which the arginine at -2 was mutated to an aspartic acid and the arginine at - 1 that is adjacent to the propeptide cleavage site was mutated to a glutamic acid. Cells were grown in the presence of vitamin K to obtain proprothrombin that contained y-carboxyglutamic acid; cells were grown in the presence of warfarin, a vitamin K antago- nist, to obtain proprothrombin that was deficient in y-car- boxyglutamic acid. This mutant proprothrombin is similar to the naturally occurring mutant, factor IX Cambridge: in which factor IX circulates with an 18-residue propeptide N- terminal extension. ProPTR-2D,R- 1 E, expressed in CHO cells in the presence of vitamin K or in the presence of warfarin, migrated as single bands after sodium dodecyl sul- fate (SDS) gel electrophoresis (Fig 1A). Direct y-carboxy- glutamic acid analysis of the alkaline hydrolysate showed that proPT/R-2D,R-IE expressed in CHO cells in the pres- ence of vitamin K contained 8.2 2 0.4 mol of y-carboxyglu- tamic acid per mol of protein; given the presence of minor contaminants, the prothrombin precursor is likely fully car- boxylated. In contrast, proPTR-2D,R- 1 E expressed in CHO cells in the presence of warfarin had no detectable y- carboxyglutamic acid. Upon Western blot analysis, both the carboxylated and the acarboxy form of prothrombinR-2D.R-

IE as well as prothrombin stained with antiprothrombin:total antibodies (Fig IB). However, the carboxylated and the acar- boxy form of proPTR-2D,R-lE stained with anti-prow an- tibodies but prothrombin, lacking the propeptide, did not (Fig I E). These results indicate that purified proprothrombin, in both its carboxylated and its uncarboxylated form, con- tains the propeptide and is reactive with anti-prow antibod- ies, in contrast to prothrombin.

Although we have previously established the specificity of antibodies for various synthetic prothrombin intermediatesZ3 and paral- lel studies with factor IX antibodies allowed us to anticipate certain results for prothrombin~2J~2Y the absence of prior studies on proprothrombin necessitated evaluation of the in- teraction of antipropeptide antibodies, antiabnonnal pro- thrombin antibodies, antiprothrombin:Mg(II) antibodies, and antiprothrombin:Ca(II)-specific antibodies with prothrom- bin, proPTR-2D.R- 1 E, des-y-carboxyprothrombin, and des-y-carboxyproPTR-2D,R-IE. The interaction of each an- tibody population was studied using a competition radioim- munoassay. Using 'ZSI-labeled proPTR-2D,R- 1 E, the inter- action of "'I-labeled proPTR-2D,R- 1E with anti-prow antibodies was inhibited with proPTR-2D,R-lE and des-y-carboxyproPTR-2D,R- 1 E but not prothrombin or des-y-carboxyprothrombin (Fig 2A). Using "'I-labeled des- y-carboxyprothrombin, the interaction of '2sI-labeled des-y- carboxyprothrombin with antiabnormal prothrombin anti- bodies was inhibited with des-y-carboxyprothrombin and, to a lesser extent, by des-y-carboxyproPTR-2D,R- 1 E, but not prothrombin (Fig 2B). ProPTR-2D.R-I E exhibited mini- mal reactivity with this antibody. The interaction of I2'I-

SpeciJcity of the antiprothrombin antibodies.

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BIOSYNTHESIS OF PROTHROMBIN 2589

Fig 4. Localization of fully carboxylated pro- thrombin species. CHO cells expressing prothrombin were stained with antibodies, as indicated. (A) Anti- prothrombin:Mg(ll) antibodies; IB) antiprothrom- bin:Mg(ll) antibodies, cells were fixed in the presence of EDTA; (C) antiprothrombin:Calll)-specific antibod- ies; (D) antiprothrombin:Ca(ll)-specific antibodies; cells were fixed in the presence of EDTA. The arrow- heads identify the juxtanuclear Golgi complex.

labeled prothrombin with antiprothrombin:Mg(II) antibodies was inhibited with proPT/R-2D,R-IE and prothrombin but not des-y-carboxyprothrombin or des-y-carboxyproPTIR- 2D,R- 1 E (Fig 2C). Using '2sI-labeled prothrombin, the interaction of '*'I-labeled prothrombin with antiprothrom- bin:Ca(lI)-specific antibodies was inhibited with prothrom-

bin but proPT/R-2D,R- 1 E, des-y-carboxyproPT/R-2D,R- 1 E and des-y-carboxyprothrombin were unreactive with this an- tibody (Fig 2D). These results indicate that anti-prow anti- bodies can be used to identify species that contain the pro- peptide, regardless of the state of carboxylation; that antiabnormal prothrombin antibodies can be used to identify

Fig 5. Intracellular localiza- tion of the juxtanuclear Golgi complex. Immunofluorescent staining of mannosidase II in CHO cells expressing prothrom- bin. Mannosidase II, a marker of the juxtanuclear Golgi complex, was stained with rabbk anti- mannosidase II antibodies.

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2590 BRISTOL ET AL

Fig 6. Colocalization of fully carboxylated prothrombin and p200 to the Golgi complex. Affinity-purified rabbit antiprothrombin:Ca(ll)- specific polyclonal antibodies and AD7, a murine anti-p200 MoAb, were used in double-label immunofluorescence experiments as de- tailed in Experimental Methods. (A) carboxylated prothrombin lack- ing the propeptide; IBI p200.

des-y-carboxylated prothrombin species, regardless of the presence or absence of the propeptide; that antiprothrom- bin:Mg(II) antibodies recognize fully carboxylated pro- thrombin, regardless of the presence or absence of propep- tide; that antiprothrombin:Ca(II) antibodies recognize fully carboxylated prothrombin only after the propeptide has been cleaved.

Immunojluorescent staining of CHO cells expressing pro-

thrombin. Antiprothrombin:total antibodies bind to pro- thrombin regardless of the carboxylation state or the pres- ence of the propeptide. Antiprothrombin:total antibodies stain organelles in these cells, including the meshlike endo- plasmic reticulum and the perinuclear Golgi complex (data not shown). CHO cells transfected with the expression vector lacking prothrombin cDNA were not reactive with antipro- thrombin:total antibodies. Similarly, an irrelevant antibody, anti-factor IX:t~tal?~ failed to stain CHO cells expressing prothrombin nor did the fluorescent antibody alone interact with cells (data not shown). Furthermore, if CHO cells ex- pressing prothrombin were not permeabilized before immu- nofluorescent staining with antiprothr0mbin:totaI antibodies, no staining was evident (data not shown). Thus, these results indicate that these antibodies specifically bind to intracellular forms of prothrombin.

Figure 3A shows the staining with anti-abnormal prothrombin anti- bodies of cells synthesizing prothrombin. These antibodies react with uncarboxylated or poorly carboxylated prothrom- bin species regardless of the presence or absence of the propeptide. The endoplasmic reticulum is stained specifi- cally, whereas no staining of the Golgi complex can be ob- served. These results indicate that at the expression levels characteristic of these stably transfected CHO cells, y-car- boxylation is an efficient process and appears to be com- pleted in the endoplasmic reticulum.

Using antibovine carboxylase antibodies to stain the en- dogenous y-carboxylase of CHO cells, the endoplasmic re- ticulum and the Golgi complex represent the major sites of carboxylase (Fig 3C). An endoplasmic reticulum marker, BiP (HSWS), stained with anti-BiP antibodies (Fig 3D). These results indicate that the carboxylase resides in both the endoplasmic reticulum and the Golgi complex. However, carboxylation appears complete before transit of prothrom- bin from the endoplasmic reticulum to the Golgi complex because no des-y-carboxyprothrombin is observed in the Golgi complex.

Localization of propeptide-containing forms of prothrom- bin. Anti-prom antibodies reacted with proprothrombin regardless of the degree of y-carboxylation, but did not bind to prothrombin or des-y-carboxyprothrombin. As shown in Fig 3B, these antibodies stain both the endoplasmic reticu- lum and a component of the Golgi complex. The Golgi stain- ing in CHO cells is perinuclear, but the particular region of the Golgi cannot be determined from these light micro- graphs. Because these antibodies do not bind to the free propeptide (J.A.B., unpublished results, December 1990), interpretation of these micrographs is not complicated by the localization of cleaved propeptide.

Localization of fully carboxylated forms of prothrombin. Antiprothrombin:Mg(II) antibodies bind to the fully carbox- ylated form of prothrombin regardless of the presence or absence of propeptide. These antibodies bind prothrombin only in the presence of metal ions. As shown in Fig 4A, antiprothrombin:Mg(II) antibodies are reactive with antigen in the endoplasmic reticulum and in the Golgi complex. When cells were fixed in the presence of EDTA, this binding was abolished (Fig 4B). When CHO cells were cultured in

Localization of acarboxy forms of prothrombin.

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BIOSYNTHESIS OF PROTHROMBIN 2591

Fig 7. Schematic summary of the biosynthesis of prothrombin. Prothrombin is synthesized in the en- doplasmic reticulum lERl as a precursor protein (acarboxyproPT),including a propeptide N-terminal extension that contains the y-carboxylation recogni- tion site. This precursor protein is not carboxylated. Although the carboxylase (01 resides in both the en- doplasmic reticulum and the Golgi complex, carbox- ylation is complete before the protein leaves the endoplasmic reticulum. Fully carboxylated propro- thrombin (ProPT) is present in both the endoplasmic reticulum and in the Golgi complex. The propeptide is cleaved by furin, a protease of the trans Golgi net- work. Prothrombin, fully carboxylated and lacking the propeptide, appears only in the juxtanuclear Golgi complex and in secretory granules (SV) that represent the final step in the constitutive biosynthe- sis of prothrombin. (01. Carboxylase; (A), Bip; (31, p200; (01, mannosidase II; (11. furin; PM, plasma membrane. Propeptide (rectangular appendage), re- active with anti-proPT antibodies; uncarboxylated glutamic acids (I), define antigen reactive with anti- abnormal prothrombin; y-carboxyglutamic acid IYI, define antigen reactive with antiprothrombin:Mg(lll antibodies in the presence or absence of propeptide and define antigen reactive with antiprothrom- bin:Ca(lll-specific antibodies only in the absence of propeptide.

w

ProPT

CIS

h

sv

BLOOD

medium containing warfarin to inhibit y-carboxylation, they failed to interact with antiprothrombin:Mg(II), even in the presence of calcium ions (data not shown). These results confirm that the interaction of these antibodies with pro- thrombin species requires y-carboxylation and that carboxyl- ated prothrombin and carboxylated proprothrombin reside in both the endoplasmic reticulum and the Golgi complex.

Localization of fully carhmylatecl forms of protltrombin that lack the propeptide. Antiprothrombin:Ca(lI)-specific antibodies bind to the fully carboxylated form of prothrom- bin that lacks the propeptide. These antibodies bind pro- thrombin only in the presence of Ca(l1) ions and not other divalent metal ions. As shown in Fig 4C, antiprothrom- bin:Ca(ll)-specific antibodies are reactive with antigen in small granules within the cytoplasm as well as the Golgi complex. In the presence of EDTA, this binding was abol- ished (Fig 4D).

The juxtanuclear Golgi complex was localized using poly- clonal antibodies to mannosidase I1 (Fig 5 ) ; mannosidase I1 is concentrated in the medial Golgi compartment,'" but this compartment and the trans Golgi network cannot be distin- guished by immunofluorescence techniques.

To simultaneously localize prothrombin and the Golgi complex, we performed a double-label experiment to visual- ize carboxylated prothrombin, using antiprothrombin:Ca(II)- specific antibodies, and p200, a Golgi complex marker that colocalizes with mannosidase 11, using the MoAb AD7.l' p200 and carboxylated, propeptide-free prothrombin colocal- ized to the juxtanuclear Golgi complex and small cyto- plasmic granules (Fig 6). In control experiments, omission

of AD7 resulted in the absence of a rhodamine signal, omis- sion of antiprothrombin:Ca(II)-specific antibodies resulted in the absence of a fluorescein signal, and omission of both AD7 and antiprothrombin:Ca(II)-specific antibodies resulted in the absence of both rhodamine and fluorescein signals. Thus, mature prothrombin is located in the juxtanuclear Golgi complex and in small secretory granules.

DISCUSSION

The biosynthesis of the vitamin K-dependent proteins includes the posttranslational synthesis of y-carboxyglu- tamic acid. In this study we have identified the site of y- carboxylation and the site of propeptide cleavage by tracking immunochemically distinct forms of prothrombin through subcellular organelles. Using specific antibodies, we have been able to identify four chemically distinct prothrombin species: (1 ) uncarboxylated proprothrombin; (2) carboxyl- ated proprothrombin; (3) uncarboxylated prothrombin; and (4) carboxylated prothrombin, the mature and biologically active species that circulates in blood. We and others have previously shown that the vitamin K-dependent proteins lack biologic activity if the propeptide is not cleaved4,q,3' or if carboxylation is not complete."." Such forms lack the ability to bind to membrane surfaces in the presence of cal- cium.

We demonstrate by direct immunofluorescence studies that the carboxylase resides in the endoplasmic reticulum and the Golgi complex. Previously, Carlisle and Suttie" had identified the endoplasmic reticulum as the site of carboxyl- ase activity in studies based on the preparation and assay of

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2592 BRISTOL ET AL

carboxylase in disrupted bovine liver cells. Wallin et aPS extended these studies by isolating preparations of both en- doplasmic reticulum and the Golgi complex; both contained carboxylase activity. Because there remains uncertainty about the purity of these subcellular fractions, we used anti- carboxylase antibodies in situ to perform immunocytochemi- cal localization. These antibodies, including anti-carboxyl- ase 86-99 and anti-carboxylase 661-675, were prepared against peptides based on the sequence of bovine carboxyl-

Both reagents showed identical staining patterns of ase. 12.13

both the endoplasmic reticulum and the Golgi complex. Thus, the vitamin K-dependent carboxylase is resident in both the endoplasmic reticulum and the Golgi complex. However, the apparent absence, within experimental limits, of uncarboxylated proprothrombin in the Golgi complex sug- gests that prothrombin is efficiently carboxylated in the en- doplasmic reticulum before transit to the Golgi complex. The presence of carboxylase in the Golgi complex may be caused by membrane fusion and recycling of the carboxylase back to the endoplasmic reticulum, although the carboxylase lacks any of the known recognition elements that localize membrane proteins in the endoplasmic reticulum. ','*

As previously described for factor IX? a synthetic pro- peptide based on the sequence of prothrombin was prepared and, after conjugation to albumin, was used as immunogen to generate rabbit antiprothrombin propeptide antibodies. After immunoaffinity purification, these antibodies were not reac- tive with prothrombin, but were reactive with the mutant proprothrombin, proPT/R-2D,R- lE, that we used as a model for the propeptide-containing prothrombin species. In con- trast to the expression of recombinant factor IX CHO cells, where about 7% of the factor IX generated is profactor IX,9 CHO cells expressing prothrombin cleave the propeptide efficiently and no proprothrombin could be detected. Thus, we mutated residues -2 and - 1 so that the propeptide cleav- age site was altered and the propeptide remained attached to prothrombin. ProFT/R-2D,R- 1E was nearly fully carbox- ylated and yielded a single band upon analysis by SDS gel electrophoresis. It was reactive with anti-prom antibodies, in contradistinction to prothrombin. Thus, the identification of proprothrombin within the cell by anti-prom antibodies is based on the expression of antigenic determinants in wild- type proprothrombin and proprothrombin with residues - 1 and -2 altered.

This in situ analysis illustrates a pathway for the biosyn- thesis of the vitamin K-dependent proteins (Fig 7). Pro- thrombin is synthesized in the endoplasmic reticulum as a precursor protein, including a propeptide N-terminal exten- sion that contains the y-carboxylation recognition site.5 This precursor protein, proprothrombin, does not contain y-car- boxyglutamic acid. This is consistent with carboxylation as a posttranslational process rather than a cotranslational pro- cess. Proprothrombin undergoes subsequent y-carboxyla- tion. Although the carboxylase resides in both the endoplas- mic reticulum and the Golgi complex, carboxylation is essentially complete before the protein leaves the endoplas- mic reticulum. Thus, fully carboxylated proprothrombin ex- ists within the cell, and is present in both the endoplasmic reticulum and in the Golgi complex. This result confirms

our original hypothesis that the carboxylation recognition site on the propeptide directs y-carboxylation,5 and that pro- peptide cleavage must follow y-carboxylation. Fully carbox- ylated proprothrombin is further processed in the Golgi com- plex, with cleavage of the propeptide. Proprothrombin contains the furin concensus sequence. Because furin is known to reside in the trans Golgi network, and by immuno- fluorescence microscopy colocalizes to the Golgi complex with mannosidase II,I9 our observation of the conversion of proprothrombin to prothrombin in a Golgi compartment is consistent with the function and location of this enzyme." Prothrombin, fully carboxylated and lacking the propeptide, appears only in the Golgi complex and in secretory vesicles for constitutive secretion of this protein into the blood where it circulates as a plasma protein.

ACKNOWLEDGMENT

We thank Kerry Gowell and Drs Karen Kotkow and Rita Blanch- ard for providing some of the antiprothrombin antibodies, Dr Brian Burke for anti-p200 antibody, and Drs Marilyn Farquhar and Kelley Moremen for anti-mannosidase I1 antibodies. We are grateful to Dr Stuart Komfeld and Gary Thomas for helpful discussions.

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1996 88: 2585-2593  

JA Bristol, JV Ratcliffe, DA Roth, MA Jacobs, BC Furie and B Furie K-dependent carboxylase and the sites of gamma-carboxylationBiosynthesis of prothrombin: intracellular localization of the vitamin 

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