Cell, Vol. 48, 185-191, January 30, 1997, Copyright 0 1987 by Cell Press

Recognition Site D irecting Vitamin K-Dependent y=Carboxylation Resides on the Propeptide of Factor IX Maria J. Jorgensen,’ Alan B. Cantor: Barbara C. Furie: Cheryl L. Brown: Charles B. Shoemaker,+ and Bruce Furie’ l Center for Hemostasis and Thrombosis Research Division of Hematology-Oncology Department of Medicine New England Medical Center and Tufts University School of Medicine Boston, Massachusetts 02111 tGenetics Institute 87 Cambridge Park Drive Cambridge, Massachusetts 02140

Summary

Posttranslational processing of vitamin K-dependent proteins includes ytarboxylation of specific glutamic acid residues to form y-carboxyglutamic acids. To de- termine whether carboxylation is directed by the pro- peptide sequence, homologous among the precur- sors of these proteins, alterations were made in the Factor IX propeptlde cDNA. The extent of y-carboxyl- ation of recombinant Factor IX was assessed using conformation-specific antibodies directed against the y-carboxyglutamlc acid-dependent, metal-stabilized structure. Deletion of the propeptide (residues -18 to -1) abolished carboxylatlon, but not secretion, of Fac-

tor IX. Substitution of alanine for phenylalanine -18 or glutamic acid for alanine -10 also impaired carboxyl- ation. These results indicate that the Factor IX propep- tide participates in defining a recognition site that designates an adjacent glutamic acid-rich domain for y-carboxylation. The association of the propeptide with the y-carboxylation recognition site provides the first demonstration of a specific function served by a propeptide in posttranslational protein processing.

Introduction

Factor IX is a plasma glycoprotein (M, 56,000) necessary for the efficient progression of blood coagulation. This serine protease zymogen is synthesized in the liver as a precursor molecule that undergoes extensive modifica- tion prior to secretion (for review, see Nemerson and Furie, 1980; Thompson, 1986). The posttranslational pro- cessing steps include glycosylation, cleavage of the pre- and propeptides, vitamin K-dependent y-carboxylation of the 12 most amino-terminal glutamic acid residues (DiSci- pio and Davie, 1979), and f3-hydroxylation of aspartic acid at residue 64 (McMullen et al., 1983). The y-carboxyglu- tamic acid (Gla) residues confer metal-binding properties upon Factor IX and the other vitamin K-dependent proteins, including prothrombin (Stenflo et al., 1974; Nelsestuen et al., 1974; Sperling et al., 1978). In the presence of metal ions, these proteins undergo two sequential confor- mational changes that are essential for the expression

of membrane-binding properties and coagulant activity (Borowski et al., 1986b; Liebman et al., 1985b). The sig- nals which designate the vitamin K-dependent proteins for y-carboxylation have not been defined.

Based upon the Factor IX cDNA nucleotide sequence (Choo et al., 1982; Kurachi and Davie, 1982; Jaye et al., 1983; Anson et al., 1984) the primary translation product (preproFactor IX) is predicted to include a 46 residue amino-terminal extension of the mature protein sequence. The amino-terminal hydrophobic portion of this prepro re- gion appears to be a prototypic signal peptide, required for translocation of the nascent polypeptide into the en- doplasmic reticulum. After cleavage of the signal peptide, the Factor IX precursor (proFactor IX) includes an 18 resi- due propeptide. This propeptide is normally removed prior to secretion by cleavage between arginine -1 and tyro- sine 1 to yield the mature protein sequence. The length of the propeptide, and therefore the location of the signal peptidase cleavage site, has recently been determined by analyses of three naturally occurring mutants: Factor IX Cambridge (Diuguid et al., 1986), Factor IX Oxford-3 (Bentley et al., 1986), and Factor IX San Dimas (Ware et al., 1986). Point mutations have resulted in substitution of serine for arginine -1 in Factor IX Cambridge and gluta- mine for arginine -4 in the other two mutants. As a conse- quence, these mutant Factor IX molecules circulate with the propeptide still attached. Amino-terminal sequence analyses have demonstrated the presence of residues -18 to -1 in these molecules, thereby defining the length

of the Factor IX propeptide. The DNA coding sequences have been determined for

most of the known vitamin K-dependent plasma proteins, including prothrombin (Degen et al., 1983; MacGillivray and Davie, 1984; Jorgensen et al., submitted), Factor X (Fung et al., 1984, 1985) Factor VII (Hagen et al., 1986) protein C (Long et al., 1984; Foster et al., 1985; Beckmann et al., 1985) and protein S (Dahlback et al., 1986; Lund- wall et al., 1986). These plasma proteins are known to have marked sequence homology (Furie et al., 1982) among both their serine protease domains (with the ex- ception of protein S) and their y-carboxyglutamic acid-rich domains. The vitamin K-dependent bone Gla protein (os- teocalcin) has no homology, in its mature form, with the vitamin K-dependent plasma proteins. The cDNA se- quence for bone Gla protein has recently been reported (Pan and Price, 1985; Celeste et al., 1986). Analysis of the predicted translation products indicates that all of these proteins, like Factor IX, are first synthesized as a precursor molecule, including both a hydrophobic signal sequence and a propeptide. Although the signal peptide sequences vary among these proteins, considerable sequence ho- mology is apparent among the propeptide regions (Figure 1). For example, phenylalanine -16, alanine -10, arginine -4, and arginine -1 are nearly invariant. Hydrophobic amino acids are conserved at residues -17, -7, and -6, while residue -2 is generally an arginine or lysine. In addition, the sequence of another vitamin K-dependent

Cell 166

-24 -23 -22 -7.1 -20 -19 -18 -17 -16 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -3 -4 -3 -7. -1 1

PAcTus Ix Len Len Ser Ala 01~ Cys Tht Val Phe Lea Asp Sir 01~ Asn Ala Asn Lyw 110 Lou As,, Ar; Pro Lys Ar, l)r

PEQllIEQWJIN Set Len Vd Eis Ser Qln Bis Val Phe Len Ala Pro 010 Qln Ala ArS Ser Lou Leo Qln Ar8 Vd Arl Ar‘ Ala

PACTDR X Len Lou Len Len Qly Qln Ser Lou Phs 11s Arg Arg 011 Qln Ala Asn Aso 11s La Ala Arfi Val Thr ArS Al.

PBmBIT4 c Thr Pro Ala Pro Len Asp Ser Vd Phe Ser Ser Ser Qln Ar3 Ala Eis Qln Val Len At; 110 Ar; Lys Ar‘ Al,

PACTtIlt VII Trp Lys Pro Qly Pro Bi8 Ar; Val Pbs Val Thr Qln Qln Qlu Ala His Qly Vd Len Eis Arg Ar; Arl Ar, Aim

PmTBMS Vd Lem Pro Val Len Qln Ala Asn Phe Len Ser Arl Qln Bis Ala Ser Qln Val La., 110 Arl Arg Ar‘ ArS Al.

BIMBQLA PliUIBIN Sar Qly Ala Qlu Set Ser Lya Ala Phe Vd Set Lys Qln 011, Qly Ser Qln Val Val LJB Atg Pro Arg Ar‘ Tyr

Figure 1. Sequence Homology of the Propeptide Regions of the Vitamin K-Dependent Proteins

The predicted amino acid sequences are based upon the published cDNA sequences for human Factor IX (Kurachi and Davie, 1962; Anson et al., 1984; Jaye et al., 1963) human prothrombin (Degen et al., 1963) human Factor X (Fung et al., 1965) human Factor VII (Hagen et al., 1966) human protein C (Foster et al., 1965; Beckmann et al., 1965) bovine protein S (Dahlback et al., 1966) and human bone Gla protein (Celeste et al., 1966). The y-carboxyglutamic acid residues are contained within the domains immediately carboxy-terminal to the propeptide regions. The amino-termini of the mature, secreted proteins are aligned at residue 1. The propeptide of Factor IX extends from residue -16 to residue -1. The sites of signal peptide cleavage in the-other proteinshave not been defined:

bone protein, Gla matrix protein (Price and Williamson, 1985) reveals a domain in the mature protein homologous to this propeptide sequence. These observations have led to speculation that this particular propeptide structure participates in designating specific precursor polypep- tides for vitamin K-dependent ycarboxylation (Pan and Price, 1985; Celeste et al., 1986; Diuguid et al., 1986; Bentley et al., 1986; Dahlback et al., 1986; Beckmann et al., 1985).

Several laboratories have described mammalian ex- pression systems for recombinant Factor IX which, in the presence of vitamin K, generate ycarboxylated Factor IX (Anson et al., 1985; de la Salle et al., 1985; Busby et al., 1985; Kaufman et al., 1986). In our work (Kaufman et al., 1986) recombinant y-carboxylated Factor IX that was iso- lated by immunoaffinity chromatography using conforma- tion-specific antibodies (Liebman et al., 1985) had the NHp-terminal sequence of plasma Factor IX and had a y-carboxyglutamic acid content and a coagulant-specific activity about 65% that of plasma Factor IX. In the current study, we used a modification of this expression system to explore the effect of mutations in the propeptide on ycarboxylation. To facilitate analysis of y-carboxylation, we employed polyclonal conformation-specific antibodies directed against the metal-stabilized form of Factor IX. These antibodies do not bind to des-y-carboxy Factor IX, even in the presence of metal ions, because the y-car- boxyglutamic acid residues form the metal binding sites that stabilize the three-dimensional structure (Liebman et al., 1985b). Using immunoassays to measure levels of na- tive, metal-stabilized Factor IX antigen and total Factor IX antigen, we show that deletion of the propeptide or muta- tion of residue -16 or -10 eliminates normal y-carboxyl- ation. These results demonstrate that a recognition site directing vitamin K-dependent y-carboxylation is associ- ated with the propeptide of Factor IX.

Results

Expression of Factor IX cDNA in Chinese Hamster Ovary Cells The Factor IX expression plasmid, pMT2-IXAvt, contains the SV40 origin of replication, the adenovirus major late

promoter, the Factor IX coding region, the dihydrofolate reductase coding region, the SV40 early polyadenylation site, the adenovirus virus-associated genes, and the pBR322 sequences needed for propagation in E. coli. This plasmid is similar to p91023-IX, which has been pre- viously described (Kaufman et al., 1986). Factor IX was ex- pressed in Chinese hamster ovary cells by introducing pMT2-IX/wt and selecting for cells expressing the di- hydrofolate reductase-positive phenotype. In contrast with earlier work describing a similar system (Kaufman et al., 1986), Factor IX expression was not amplified by selecting for methotrexate resistance. Culture media containing secreted Factor IX were assayed by competition radioim- munoassay using anti-Factor IX:total antibodies, which bind to all Factor IX species regardless of the extent of y-carboxylation. The amount of total Factor IX antigen ex- pressed by these primary transfectants varied, with con- centrations between 0.09 and 1.1 ug/ml observed in six separate transfections (Figure 3, WT). The same samples were assayed for native Factor IX antigen using polyclonal conformation-specific antibodies, anti-Factor IX:Mg(II), which bind to specific determinants expressed on Factor IX in the presence of metal ions. These determinants are present only when Factor IX is sufficiently y-carboxylated to undergo its metal-induced conformational transitions, and their presence correlates closely with coagulant activ- ity. The concentration of native Factor IX antigen varied between 0.04 and 0.76 pglml in these samples. The con- centration of native Factor IX antigen was compared with that of total Factor IX antigen to determine the extent of y-carboxylation. By these criteria, 64% f 17% of the Factor IX molecules synthesized using the unmodified, wild-type coding sequence were sufficiently y-carboxyl- ated to undergo the metal-dependent conformational transitions.

Effects of Propeptide Mutations on yCarboxylation of Factor IX Oligonucleotide-directed site-specific mutagenesis was used to modify the Factor IX propeptide coding sequence. The deletion mutant, FIX/d( - 18, - l), lacked the coding sequence for the entire 18 amino acid propeptide (Figure 2). The two substitution mutants, FIWFA-16 and FIXIAE-

Factor IX Propeptide Signals y-Carboxylation 187

PACMB IX PROPEPTIDE ALTERATIONS

-18 -10 -1 +1 .

rt Glu Cys . Thr Val Phe Lou Asp His Glu Asn Ala Aso Lyr 110 Len Am Arg Pro Arg Arp . br Asn . . . . . .

6(-18,-l) - . . - . . . .

AE-10 - . Cl- . - . . . . . . .

Figure 2. Factor IX Propeptide Alterations

Oligonucleotide-directed mutagenesis was used to modify the Factor IX coding sequence in the region encoding the propeptide. The wild-type se- quence, FIWwt, encodes the amino acid sequence illustrated. FIX/d(-18, -1) lacks the coding sequence for the 18 amino acid residues of the propep- tide. FDUFA-18 encodes Factor IX with substitution of alanine for phenylalanine at residue -18. FIWAE-10 encodes Factor IX with substitution of glutamic acid for alanine at residue -10. The signal peptidase cleavage site is shown between residue -19 and residue -18. The propeptide cleavage site is shown between residue -1 and the amino terminus of the mature protein, residue +l.

10, had changes leading to substitution of alanine for phenylalanine - 16 or glutamic acid for alanine - 10, respectively (Figure 2). Nucleotide sequencing of the region which was single-stranded during the mutagenesis procedure confirmed incorporation of only the desired changes.

Expression plasmids containing the modified Factor IX coding sequences were used to transfect Chinese ham- ster ovary cells in parallel with the unmodified pMT2- IX/v& Cell culture supernatants were analyzed by radioim- munoassay using the two antibody populations described above to determine levels of total and of native Factor IX antigen. The results are shown in Figure 3. Transfection of cells with pMT2-IX/d(-18,-l) led to secretion of total Factor IX at concentrations between 0.16 and 3.2 pg/ml, comparable to amounts observed when pMT2-IX/wt was used. Unlike the wild-type product, however, this Factor IX expressed undetectable levels of native Factor IX antigen, indicating the absence of functionally important y-car- boxyglutamic acid residues. These result8 indicate that deletion of the propeptide interferes with vitamin K-de- pendent y-carboxylation.

Because of the possibility that deletion of the entire propeptide might interfere with processing steps beyond y-carboxylation, two single amino acid substitutions in the propeptide were also made. The effects on carboxylation were evaluated when alanine was substituted for phenyl- alanine at residue -16, a position perfectly conserved among the vitamin K-dependent proteins, or when glu- tamic acid was substituted for alanine at residue -10, a well-conserved position. The products of pMT2-IX/FA-16 and pMT2-IX/AE- 10 transfections contained substantial levels of total Factor IX antigen but only minimal levels of native Factor IX antigen. Only 6.1% f 0.6% of the FIXIFA-16 total Factor IX antigen expressed native Factor IX antigen, and only 2.4% rt 0.5% of the FIX/AE-10 total Factor IX antigen expressed native Factor IX antigen. The significant reduction in the extent of carboxylation of Fac- tor IX observed when position -16 or position -10 is mu- tated indicates that the region including these residues plays a critical role in the y-carboxylation process.

Western Blot Analysis of Factor IX Propeptide Mutant Products In order to determine whether the products of the Factor IX propeptide coding sequence mutants were prOCesSed properly with regard to cleavage of the pre- and propep- tides, the total Factor IX secreted by the transfected cells was analyzed by Western blotting (Figure 4). The wild- type product WT (lanes 1 and 6), as well as the mutant products d(-18,-l) (lane 2), FA-16 (lane 8), and AE-10 (lane 5) migrated equivalently with plasma-derived Factor IX (lane8 4, 7, and 10). No product migrated equivalently with Factor IX Cambridge (lanes 3 and ll), which contains the additional 18 amino acid residues of the propeptide still attached. Most importantly, the d(-18,-l) product did not migrate more slowly than the wild-type product, indi- cating that the signal sequence is cleaved efficiently even when the propeptide is deleted.

Discussion

Factor IX and the other vitamin K-dependent proteins are a unique class of calcium-binding proteins that contain y-carboxyglutamic acid. This amino acid is synthesized posttranslationally by carboxylation of select glutamic acid residues in a reaction that requires vitamin K. A major unsolved question relating to vitamin K-dependent y-car- boxylation is the mechanism by which certain proteins and specific glutamic acid residues therein are desig- nated for carboxylation. The marked sequence homology of the propeptides, predicted from the cDNA sequences of vitamin K-dependent proteins, has led to speculation that this region serves to identify these proteins for post- translational y-carboxylation (Pan and Price, 1985).

To test this hypothesis, we analyzed recombinant Factor IX mutants in which the propeptide sequence was deleted or altered while the sequence of the mature Factor IX poly- peptide, including the y-carboxyglutamic acid-containing region, remained unchanged. In these studies, we show that deletion of the propeptide, or mutation of residue -16 or residue -10 in the propeptide, abolishes or impairs y-carboxylation. The changes in propeptide structure had

Cell 188

1 3.2

f

m Netlve Antigen

123456 12345 123 123

WT d(-l&-l) FA-16 AE-10

Figure 3. Secretion of Recombinant Factor IX and Factor IX Propep- tide Mutants

Conditioned media from Chinese hamster ovary cells transfected with pMT2-IX/wt, pMT2-IXld(-18,-l), pMT2-IXIFA-16. and pMT2-IX/A&10 were analyzed by radioimmunoassay. The concentration of total Factor IX antigen was measured using anti-Factor IX:total antibodies (open bars). This antibody population binds equivalently to all Factor IX spe- cies regardless of the extent of y-carboxylation. The concentration of native Factor IX antigen was measured using anti-Factor IX:Mg(II) anti- bodies (solid bars). This antibody population binds only to well- carboxylated and fully carboxylated Factor IX molecules that are able to undergo the metal-dependent conformational changes and express the antigenic determinants characteristic of native Factor IX. Separate transfection experiments are numbered on the x-axis. Factor IX antigen concentration, either total or native, is shown on the y-axis in f@ml. Although the amount of total Factor IX antigen measured in each trans- fection is variable, the ratio of native antigen to total antigen is consis- tent among independent transfections of each plasmid.

no effect on the molecular size of secreted Factor IX molecules, regardless of the extent of carboxylation.

We employed conformation-specific antibodies to iden- tify Factor IX molecules that contain y-carboxyglutamic acid. Several observations support the concept that ex- pression of native Factor IX antigenic determinants on Factor IX demonstrates that the molecule is completely, or very nearly completely, y-carboxylated. In prothrombin, which undergoes similar conformational transitions in the presence of metal ions, the absence of only one or two y-carboxyglutamic acid residues is associated with com-

1234567891011

Figure 4. Western Blot Analysis of Recombinant Factor IX and Factor IX Propeptide Mutants

Factor IX species secreted by transfected CHO cells were immunoab- sorbed from conditioned media using anti-Factor IX:total antibodies coupled to Sepharose, run on a 10% SDS-polyacrylamide gel, and transferred to nitrocellulose. Factor IX antigen was detected using anti- Factor IX:total antibodies as primary antibody and goat anti-rabbit im- munoglobulin conjugated to horseradish peroxidase as secondary an- tibody. Lanes 1 and 6, wt. Lane 2,4(-18,-l). Lane 5, AE-IO. Lane 8, FA-16. Lanes 4, 7, and 10, plasma Factor IX. Lanes 3 and 11, Factor IX Cambridge.

plete loss of expression of native prothrombin antigen (Borowski et al., 1985, 1986a). Additionally, uncarbox- ylated Factor IX does not undergo a conformational transi- tion in the presence of metal ions and does not interact with anti-Factor IX:Mg(II) antibodies (Liebman et al., 1985b). Consequently, the ratio of the amount of native Factor IX antigen to the amount of total Factor IX antigen expressed on a given population of Factor IX molecules is an indication of the percentage of those molecules which are completely or nearly completely y-carboxylated.

Our results demonstrate that a recognition element lo- cated in the propeptide sequence is required for carboxyl- ation of Factor IX. This region, highly conserved among the vitamin K-dependent proteins, appears to serve as a y-carboxylation recognition site (-&RS) to direct carboxyl- ation of these proteins. Several observations suggest that it is the amino-terminal portion of the propeptide that com- prises the +33S. We have shown that mutation of residue -16 or -10 greatly impairs carboxylation of Factor IX, indi-

cating that these residues play essential roles in defining a functional T-CRS. Furthermore, analysis of sequence homologies among the various vitamin K-dependent pro- teins reveals that the amino-terminal portions of these regions are highly conserved (Figure 5). The lack of ho- mology, however, between the carboxy-terminal portions of the propeptide sequences and the corresponding seg- ment of the mature matrix Gla protein sequence favors a role for this region of the propeptide as a recognition site for propeptide cleavage rather than for y-carboxylation.

Our identification of the y-carboxylation recognition site provides insight into the pattern of carboxylation of glu- tamic acid residues within each vitamin K-dependent pro- tein. Although located outside the ycarboxyglutamic acid- containing domain, the r-CRS is adjacent to this domain. With the exception of matrix Gla protein, the vitamin K-dependent proteins contain y-carboxyglutamic acid

Factor IX Propeptide Signals yCarboxylation 189

Figure 5. Relationship of u-Carboxylation Recognition Site (VCRS) and Sites of v-Carboxylation in Vitamin K-Dependent Proteins The propeptide sequences containing the proposed y-CRS are shown aligned with the homologous internal y-CRS of mature bovine matrix Gla protein. The arrows indicate the amino-termini of the mature, secreted proteins. The y-carboxyglutamic acid residues, *, are located in domains adja- cent to the y-CRS. The role of the disulfide loop in these domains is unknown. The lengths of the lines representing the polypeptide backbone are proportional to the number of amino acid residues therein. Human Factor IX. hFIX; human prothrombin, h Pr; human bone Gla protein, hBGP; bovine matrix Gla protein, bMGl?

residues, instead of glutamic acid residues, within a re- stricted region carboxy-terminal to the y-C% (Figure 5). In matrix Gla protein, a glutamic acid in the region amino- terminal to the y-CRS is also designated for carboxylation by virtue of its proximity to this domain. Within this amino- terminal region, the three glutamic acid residues closest to the y-CR!3 remain uncarboxylated while only the one furthest away is carboxylated. These observations sug- gest that glutamic acid residues located in domains to ei- ther side of the y-CRS are designated for y-carboxylation, although carboxylation of those residues immediately ad- joining the y-CRS is hindered.

It remains unclear whether the y-CRS, although re- quired for y-carboxylation, is sufficient to direct carboxyl- ation of adjacent domains. Additional structural features, such as the disulfide loop within the y-carboxyglutamic acid-rich regions of all these proteins, may be required for substrate specificity of the carboxylase. We are currently constructing cDNAs of chimeric proteins that include the y-CRS-containing propeptide of a vitamin K-dependent protein attached to a normally uncarboxylated protein. Analysis of the recombinant protein should reveal whether the y-CRS alone is sufficient to direct y-carboxylation in unrelated glutamic acid-containing regions placed adja- cent to it.

The association of the targeting structure for y-carbox- ylation, the y-CRS, with the propeptide of Factor IX, is the first identification of a specific function for a propeptide in processing and/or sorting of a secretory protein in eu- karyotic cells. In analogy with the function of signal se- quences in protein secretion, the propeptides of the vita- min K-dependent proteins serve a transient purpose in identifying these proteins for a specific posttranslational processing step. The expression of hierarchical signals for progressive steps in posttranslational processing might represent a general pattern in protein synthesis and suggests a role for transient propeptides on other proteins that undergo specific modifications prior to secretion.

Experimental Procedures

Construction of Factor IX Expression Plasmid Preparation of a 2.5 kb Pstl fragment containing the human Factor IX coding sequence has been described (Kaufman et al., 1986). This frag- ment was inserted into the Pstl site of the mammalian expression vec- tor. pMT2. which was provided by R. Kaufman. This plasmid is nearly

identical to the expression vector, p91023, previously used to express the Factor IX coding sequence in Chinese hamster ovary cells (Kaut man et al., 1966). The resultant Factor IX expression plasmid, pMT2- IX/v&, was shown by restriction mapping to contain the Factor IX cod- ing region in the proper orientation with respect lo the adenovirus major late promoter.

Mutagenesis Alterations were made in the Factor IX coding sequence correspond- ing lo the desired amino acid changes indicated in Figure 2. Synthetic oligonucleotides were utilized for site-directed mutagenesis (Oostra et al., 1983) of a heteroduplex form of the plasmid pMT2Wwt. This tem- plate, containing a 600 bp single-stranded gap at the B’end of the Fac- tor IX coding sequence, was prepared as follows. An aliquot (5 pg) of pMT2-IXIwt was cut with Bglll and EcoRV lo remove the 5’ end of the Factor IX coding sequence and was gel-purified (Maniatis et al., 1982). Another aliquot (5 pg) was linearized with Clal at a site several kilo- bases from the Factor IX coding sequence and was treated with calf intestinal phosphatase. These preparations were mixed in a final vol- ume of 40 ~1 and denatured by addition of 10 ul of 1 N NaOH. After 10 min at room temperature, the mixture was neutralized by addition of 450 pl of a solution containing 0.1 M Tris (pH 7.5), and 0.02 M HCI. The DNA was allowed to reanneal by incubating the mixture overnight at 68% and cooling gradually to room temperature over several hours. The resulting mixture of reannealed DNA included the desired het- eroduplex molecule, containing a single-stranded region lo which the mutagenic primer could anneal. Oligonucleotides for mutagenesis were synthesized on an Applied Biosystems 3806 DNA Synthesizer and were gel-purified prior lo use. For preparation of the mutants FIWFA-16 and FWAE-10, the mutagenic oligonucleotides incorpo- rated two base changes each and had the sequences B’-GTACAG- TTGCTCTTGATC and 5’TGAAAACGAGAACAAAAITC! respectively. For the deletion mutant, d(-18.-l). the 47 base mutagenic oligonu- cleotide, 5’-GATATCTACTCAGTGCTGAATGlTATAATTCAGGTA- AAlTGGAAGAG, was composed of blocks of 23 and 24 bases com- plementary lo the regions to be joined in the desired mutant. Afler 5’ phosphorylation using T4 polynucleotide kinase in a standard reaction mixture (Maniatis et al., 1982), 10 ~I(50 pmol) of the kinasing reaction was mixed with 30 PI (0.1 pmol) of the heteroduplex mixture. and 5 VI of 0.5 M NaCl was added. The annealing reaction was carried out by heating to 68°C for 5 min and cooling slowly to WC. The primer was extended and joined to the existing partial second strand during over- night incubation at 15% after addition of 5 ~1 containing 20 mM MgCI?, 40 mM dithiothreitol, 2 mM ATP, 1 mM each dATP, dTTP. dCTP, and dGTP, 5 U Klenow fragment of DNA polymerase I (Pharmacia) and 0.5 U T4 DNA ligase (Bethesda Research Labs). The reaction mixture was heated to 68% for 5 min, and 5 ~1 was used to transform E. coli strain TGl. For the point mutations. plasmids containing the desired mutation were identified by colony hybridization (Maniatis et al., 1982) using 32P-labeled mutagenic oligonucleotides. Melting from wild-type molecules occurred during washing at 44-52% and from mutant clones at about 10°C higher. Colony hybridization to identify the dele- tion mutant was performed using a 32P-labeled synthetic 16-mer, 5’-GAATTATAACATTCAG, which spanned the newly joined se- quence. The DNA obtained from a positive colony was used to retrans-

Cell 190

form E. coli, colony hybridization was repeated, and a single positive colony was selected for use as a source of plasmid DNA. Sequence analysis (Sanger et al., 1977) of the 5’ portion of each mutant Factor IX coding sequence verified that the desired mutation, but no other al- teration, had been incorporated.

Cell Culture, DNA Pansfection, and Cell Line Selection The dihydrofolate reductase-deficient Chinese hamster ovary cell line, CHO DlJKXBll, was grown and maintained as described (Chasin and Urlaub, 1980). Ten monoclonal cell lines were established by cloning these cells at limiting dilution. Using procedures described below, these ten lines were transfected with pMT2-IX/wt, selected for expres- sion of dihydrofolate reductase, grown to confluency, and the media as- sayed to determine the extent of y-carboxylation of secreted Factor IX. The CHO cell line which secreted Factor IX having the greatest ratio of native to total Factor IX antigen (7:lO) was used for all further experi- ments. Transfection of CHO cells with expression plasmids was ac- complished by electroporation (Potter et al., 1984). Cells (5 x 106) were suspended in 1 ml of phosphate-buffered saline at O°C in a disposable plastic cuvette (Sarstedt) fitted with aluminum foil electrodes on two sides. DNA (20 ug) was added, the mixture was incubated at OOC for 30 min, and a brief pulse of 4000 V was applied to the cuvette. The transfected cells were plated in a 10 cm culture dish containing 10 ml of a-modified Eagle’s medium lacking nucleosides (Gibco) with addi- tion of 10% heat-inactivated fetal bovine serum, 5 jrglml vitamin Kr (Aquamephyton, Merck Sharp and Dohme), and 10 ug/ml each of thymidine, adenosine, deoxyadenosine, penicillin, and streptomycin. The cells were subcultured two days later into the same medium, except that the nucleosides were omitted and dialyzed serq was used (selective medium). Transfected cells were fed every 3-4 days with selective medium until colonies were visible, about IO-12 days after subculturing. These initial transformants were pooled and grown in selective medium until confluent. At confluence, a 10 cm dish con- tained approximately 1.7 x lo7 cells in 10 ml of medium. Conditioned media were centrifuged to remove cellular debris and stored at -20°C.

Pmparatlon of Protein Standards and Antlbodles Human Factor IX (Liebman et al., 1985a) and Factor IX Cambridge (Diuguid et al., 1986) were purified from plasma by immunoaffinity chromatography. These preparations migrated as single bands upon dodecyl sulfate-gel electrophoresis. The protein concentration of puri- fied Factor IX species was measured using AIS at 280 nm of 13.3. Plasma-derived Factor IX was iodinated with Nar2sl using lactoperoxi- dase (Morrison, 1980). The rz51-labeled Factor IX was repurified using anti-Factor IX:Ca(ll)-Sepharose (Liebman et al., 1985a).

Rabbit anti-Factor IX:Mg(II) antibodies were purified by im- munoaffinity chromatography on Factor IX-Sepharose in the presence of Mg(ll) and were eluted with EDTA (Diuguid et al., 1988; Liebman et al., 1985b). Anti-Factor IX antibodies that bound to Factor IX- Sepharose in the presence of EDTA were eluted with 4 M guanidine hydrochloride and are termed anti-Factor IX:total antibodies.

Radlolmmunoassays The displacement of 1251-labeled Factor IX from anti-Factor IX antibod- ies was studied using a competition radioimmunoassay. Anti-Factor IX: total antibodies (1.2 x 10mg M) were added to a reaction mixture which included 1251-labeled Factor IX (1 x IO-r0 M) and varying concentra- tions of competitors. All components were diluted in Tris-buffered sa- line (50 mM Tris/O.lti M NaCl [pH 7.51) containing 1 mM benzamidine, 0.1% bovine serum albumin, 3 mM EDTA, and carrier rabbit gam- maglobulin. Anti-Factor IX:Mg(II) antibodies (1.3 x lO-9 M) were added to the same reaction mixture except that 1 mM CaCIz replaced EDTA. After overnight incubation at 4OC, the bound 1251-labeled Factor IX was precipitated by the addition of goat anti-rabbit immunoglobulin. The precipitate that formed was removed by centrifugation and was as- sayed for rzsl in a Beckman Gamma 8000 spectrometer. For determi- nation of unknown antigen concentrations, a standard curve was pre- pared using human plasma-derived Factor IX of known concentration. The contents of the tissue culture media, including fetal bovine serum, did not interfere with antibody recognition of human Factor IX. Mea- surements of both total and native Factor IX antigen concentrations were reproducible within *5% using these assays.

Western Blot Analysis Anti-Factor IX:total antibodies were coupled to cyanogen bromide- activated Sepharose to give 2 mg antibodies/ml packed beads. Ali- quots (0.5 ml) of conditioned media were immunoadsorbed by addition of 50 pl of a 1 :l slurry of the antibody-agarose conjugate in phosphate- buffered saline containing 10 mM benzamidine and 0.2% NaNs. After overnight incubation with continuous mixing, the beads were washed twice with Tris-buffered saline. Adsorbed Factor IX was eluted by heat- ing for 10 min at 95OC in the presence of 2% SDS, run on a 10% SDS- polyacrylamide gel (Laemmli, 1970) and transferred to nitrocellulose paper flowbin et al., 1979). The blot was incubated for 30 min at room temperature in Tris-buffered saline containing 3% gelatin and then overnight in the same solution containing also 2 pglml anti-Factor IX:total antibodies, 10% donor calf serum, and 0.02% NaNs. After washing three times over 30 min at room temperature in Tris-buffered saline, the blot was incubated for 3 hr in 1% gelatin containing goat anti-rabbit immunoglobulin coupled to horseradish peroxidase (Bio- Rad, 1:2000) and then washed again three times. The blot was devel- oped by immersion in a color-development reagent prepared by mixing 20 ml of methanol containing 60 mg of 4-chloro-1-naphthol (Bio-Rad) with 100 ml of Tris-buffered saline containing 60 WI of 30% HzOz.

Acknowledgments

The authors are grateful to Dr. Richard Goodman for valuable advice and encouragement during these studies, to Dr. Randy Kaufman for providing the expression vector, pMT2, to Ron Kriz for DNA sequenc- ing, and to Gary Lampman for helpful discussions. This work was sup- ported by grants (HL21543 and HL18834) from the National Institutes of Health and a grant from Seragen, Inc. The Applied Biosystems 3808 DNA Synthesizer was purchased with funds provided in part by the Biomedical Research Support Grant Program, NIH (BRSG SO% RR05598-20). This work was performed by M. J. J. in partial fulfillment of the requirements for the degree of Doctor of Philosophy from Tufts University.

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.

Received November 3, 1988.

References

Anson, D. S., Austen, D. E. G., Brownlee, G. G. (1985). Expression of active human clotting factor IX from recombinant DNA clones in mam- malian cells. Nature 375, 683-885.

Anson, D. S., Choo, K. H., Rees, D. J. G., Giannelli, F.. Gould, K., Hud- dleston, J. A., and Brownlee, G. G. (1984). Gene structure of human anti- haemophilic factor IX. EMBO J. 3, 1053-1064.

Beckmann, R. J., Schmidt, R. J., Santerre, Ft. F., Plutzky, J., Crabtree, G. R., and Long, G. L. (1985). The structure and evolution of a 461 amino acid human protein C precursor and its messenger RNA, based upon the DNA sequence of cloned human liver cDNAs. Nucl. Acids Res. 13, 5233-5247.

Bentley, A. K., Rees, D. J. G., Rizza, C., and Brownlee, G. G. (1988). Defective propeptide processing of blood clotting Factor IX caused by mutation of arginine to glutamine at position -4. Cell 45, 343-348.

Borowski, M., Furie, 8. C., Goldsmith, G. H., and Furie, B. (1985). Metal and phospholipid binding properties of partially carboxylated human prothrombin variants. J. Biol. Chem. 260, 9258-9284. Borowski, M.. Furie, B. C.. and Furie, B. (1986a). Distribution of y-car- boxyglutamic acid residues in partially carboxylated human prothrom- bins. J. Biol. Chem. 267, 1624-1628.

Borowski, M., Furie, 8. C., Bauminger, S., and Furie, B. (1986b). Pro- thrombin requires two sequential metal-dependent conformational transitions to bind phospholipid. J. Biol. Chem. 267, 14989-14975.

Busby, S., Kumar, A., Joseph, M., Halfpap, L., Insley, M., Berkner, K., Kurachi. K., and Woodbury, R. (1985). Expression of active human fac- tor IX in transfected cells. Nature 318, 271-273.

Factor IX Propeptide Signals yCarboxylation 191

Celeste, A. J., Rosen, V., Buecker, J. L., Kriz, R., Wang, E. A., and Woz- ney, J. M. (1986). Isolation of the human gene for bone gla protein utilizing mouse and rat cDNA clones. EMBO J. 5, 1885-1890.

Chasin, G., and Urlaub, L. A. (1980). Isolation of Chinese hamster ovary cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. USA 77, 4216-4220.

Choo, K. H., Gould, K. G., Flees, D. J. G., and Brownlee, G. G. (1982). Molecular cloning of the gene for human anti-haemophilic Factor IX. Nature 299, 178-180.

Dahlback, B., Lundwall, A., and Stenflo, J. (1986). Primary structure of bovine vitamin K-dependent protein S. Proc. Natl. Acad. Sci. USA 83, 4199-4203.

Degen, S. J., MacGillivray, Ft. T., and Davie, E. W. (1983). Characteriza- tion of the cDNA and gene coding for human prothrombin. Biochemis- try 22, 2087-2097.

de la Salle, H., Altenburger, W., Elkaim, Ft., Dott, K., Dieterle, A., Dril- lien, R., Cazenave, J.-P, Tolstoshev, R, and Lecocq, J.-P. (1985). Active r-carboxylated human factor IX expressed using recombinant DNA techniques. Nature 316, 268-270.

DiScipio. R. G., and Davie, E. W. (1979). Characterization of protein S, a gammacarboxyglutamic acid containing protein from bovine and hu- man plasma. Biochemistry 78, 899-904.

Diuguid, D. L., Rabiet, M.-J., Furie, B. C., Liebman, H. A., and Furie, B. (1986). Molecular basis of hemophilia B: a defective enzyme due to an unprocessed propeptide is caused by a point mutation in the factor IX precursor. Proc. Natl. Acad. Sci. USA 83. 5803-5807.

Foster, D. C., Yoshitake, S., and Davie, E. W. (1985). The nucleotide se- quence of the gene for human protein C. Proc. Natl. Acad. Sci. USA 62, 4673-4677.

Fung, M. R., Campbell, R. M., and MacGillivray, R. T (1984). Blood coagulation factor X mRNA encodes a single polypeptide chain con- taining a prepro leader sequence. Nucl. Acids Res. 72, 4461-4492.

Fung, M. R.. Hay, C. W., and MacGillivray, R. T. A. (1985). Characteriza- tion of an almost full-length cDNA coding for human factor X. Proc. Natl. Acad. Sci. USA 82, 3591-3595.

Furie, B., Bing, D. H., Feldmann, R. J., Robison, D J., Burnier, J. l?, and Furie, B. C. (1982). Computer-generated models of blood coagula- tion Factor Xa, Factor IXa, and thrombin based upon structural homol- ogy with other serine proteases. J. Biol. Chem. 257, 3875-3882.

Hagen, F S., Gray, C. L., G’Hara, P., Grant, F. J., Saari, G. C., Wood- bury, R. G., Hart, C. E., Insley, M., Kisiel, W., Kurachi, K., and Davie, E. W. (1986). Characterization of the cDNA coding for human factor VII. Proc. Natl. Acad. Sci. USA 83, 2412-2416.

Jaye, M., de la Sake, H., Schamber, F., Balland, A., Kohli, V., Findeli, A., Tolstoshev, P., and Lecocq, J. R (1983). Isolation of a human anti- haemophilic factor IX cDNA clone using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bo- vine factor IX. Nucl. Acids Res. 71, 2325-2335.

Kaufman, Ft. J., Wasley, L. C., Furie, B. C., Furie, B., and Shoemaker, C. B. (1986). Expression, purification and characterization of recom- binant ycarboxylated Factor IX synthesized in Chinese hamster ovary cells. J. Biol. Chem. 261, 9622-9628.

Kurachi, K., and Davie, E. W. (1982). Isolation and characterization of a cDNA coding for human factor IX. Proc. Natl. Acad. Sci. USA 79, 6461-6464.

Laemmli, U. K. (1970). Cleavage of structural proteins during the as- sembly of the head of bacteriophage T4. Nature 227, 680-685.

Liebman, H. A., Limentani, S. A., Furie, 8. C., and Furie, 8. (1985a). lmmunoaffinity purification of factor IX (Christmas factor) by using conformation-specific antibodies directed against the factor IX-metal complex. Proc. Natl. Acad. Sci. USA 82, 3879-3883.

Liebman, H. A., Eklund, D. M., Furie, B. C., and Furie, B. (1985b). Calcium-stabilized conformational determinants of Factor IX. Thromb. Haemost. 54, 226.

Long. G. L., Belagaje, R. M., and MacGillivray, R. T. A. (1964). Cloning and sequencing of liver cDNA coding for bovine protein C. Proc. Natl. Acad. Sci. USA 81. 5653-5656.

Lundwall, A., Dackowski, W., Cohen, E., Shaffer, M., Mahr, A., Dahl- back, B., Stenflo, J., and Wydro, R. (1986). Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation. Proc. Natl. Acad. Sci. USA 83, 67166720.

MacGillivray, R. T. A., and Davie, E. W. (1984). Characterization of bo- vine prothrombin mRNA and its translation product. Biochemistry 23, 1626-1634.

Maniatis, T, Fritsch, E. F., and Sambrook. J. (1982). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Har- bor Laboratory).

McMullen, B. A., Fujikawa, K., and Kisiel, W. (1983). Occurrence of f%hydroxyaspartic acid in the vitamin K-dependent blood coagulation zymogens. Biochem. Biophys. Res. Comm. 155, 8-14.

Morrison, M. (1980). Lactoperoxidase-catalyzed iodination as a tool for investigation of proteins. Meth. Enzymol. 70, 214-220.

Nelsestuen, G. L., Zytkovicz, T. H., and Howard, J. 8. (1974). The mode of action of vitamin K. Identification of y-carboxyglutamic acid as a component of prothrombin. J. Biol. Chem. 249, 6347-6350.

Nemerson, Y.. and Furie, 8. (1980). Zymogens and cofactors of blood coagulation. CRC Crit. Rev. Biochem. 9, 45-85.

Cc&a, 8. A., Harvey, R., Ely, B. K., Markham, A. F., and Smith, A. E. (1983). Transforming activity of polyoma virus middle-T antigen probed by site-directed mutagenesis. Nature 304, 456-459.

Pan, L. C., and Price, P. A. (1985). The propeptide of rat bone gamma- carboxyglutamic acid protein shares homology with other vitamin K-dependent protein precursors. Proc. Natl. Acad. Sci. USA 82, 6109-6113.

Potter, H., Weir, L., and Leder, P. (1984). Enhancer-dependent expres- sion of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc. Natl. Acad. Sci. USA 81, 7161-7165.

Price, f? A., and Williamson, M. K. (1985). Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein. J. Biol. Chem. 260, 14971-14975.

Sanger, F.. Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 54634467.

Sperling, R., Furie, B. C., Blumenstein, M., Keyt, B., and Furie, 8. (1978). Metal binding properties of y-carboxyglutamic acid. J. Biol. Chem. 253, 3898-3906.

Stenflo, J., Fernlund, f?, Egan, W., and Roepstorff, l? (1974). Vitamin K dependent modifications of glutamic acid residues in prothrombin. Proc. Natl. Acad. Sci. USA 77, 2730-2733.

Thompson, A. R. (1986). Structure, function and molecular defects of Factor IX. Blood 67, 565-572.

Towbin, H., Staehelin, T.. and Gordon, J. (1979). Electrophoretic trans- fer of proteins from polyacrylamide gels to nitrocellulose sheets: proce- dure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350- 4354.

Ware, J., Liebman, H. A., Kasper, C., Graham, J., Furie, 8. C., Furie, B., and Stafford, D. (1986). Genetic characterization of a hemophilia B variant: factor IX San Dimas. Blood 68 (suppl.). 343a.