J. Biochem. 102, 1261-1273 (1987)

Structure and Expression of cDNA for an Inhibitor of Blood Isolated from Human Placenta: A New Lipocortin-Like Protein

Akio IWASAKI,* Makoto SUDA ,* Hiroshi NAKAO,* Takao NAGOYA,* Yushi SAINO,* Koichi ARAI ,** Toshimi MIZOGUCHI,** Fumiyasu SATO,** Hideo YOSHIZAKI ,** Mitsuteru HIRATA,** Toshiyuki MIYATA,*** Yoshihiro SHIDARA ,**** Makoto MURATA,**** and Masahiro MAKI****

*Department of Cell Biology , Kowa Research Institute, Kowa Co.. Ltd., Yatabe, Tsukuba-gun, Ibaraki 305; **Tokyo Research Laboratories , Kowa Co., Ltd., Higashimurayama, Tokyo 189; ***Department of Biology , Faculty of Science, Kyushu University 33, Higashi-ku, Fukuoka , Fukuoka 812; and ****Department of Obstetrics and Gynecology, School of Medicine, Akita University, Akita, Akita 010

Received for publication, May 23, 1987

An inhibitor of blood coagulation, a new protein with an apparent molecular weight

of 34,000 and an isoelectric point of 4.9, was purified from human placental tissue

by EDTA extraction. Five cDNA clones were isolated from the human placental

ă gtll cDNA library using the mouse monoclonal antibody raised against the coagu

lation inhibitor as the probe. The longest insert consists of 1,566 nucleotides, and

contains 960 nucleotides entirely encoding the 320 amino acids of the inhibitor, and

a poly A tail. The deduced amino acid sequence was corroborated by chemical

analyses of the protein. The entire amino acid sequence shows homology to those

of lipocortin I, lipocortin II, and endonexin-related proteins. The cDNA for the

inhibitor was expressed in Escherichia coli under the regulation of the trc promotor

of the plasmid pKK233-2. The resulting recombinant protein manifested inhibitory

activities against both blood coagulation and phospholipase A2 activity, as did the

coagulation inhibitor isolated from human placenta.

The placenta is rich in tissue thromboplastin (1) able state. In the placenta, inhibition of platelet and inhibitor of (2), sug aggregation by prostacyclin-like activity (3), inhibi gesting that it is inclined towards a hyper-coagul tion of blood coagulation by thrombomodulin (4),

Abbreviations: APTT, activated partial thromboplastin time; PT, prothrombin time; IgG, immunoglobulin G;

SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FAB-MS, fast-atom-bombardment mass spectrometry; PTH, phenylthiohydantoin; IPTG, isopropyl-ƒÀ-D-thiogalactoside.

Vol. 102, No. 5, 1987 1261 1262 A. IWASAKI et al. and by tissue plasminogen activator (U.S.A); restriction endonucleases from Toyobo (5) have been reported to play important roles in Co., Ltd., Osaka, Bethesda Research Laboratories, the balance between blood coagulation and fibri Md. (U.S.A.), and New England BioLabs, Mass. nolysis. In particular, the regulation of the ex (U.S.A.); plasmid pUC118 and DNA ligation kits trinsic pathway of blood coagulation which is from Takara Shuzo Co., Kyoto; Exo-Mung dele triggered by the tissue thromboplastin-Factor VII tion system from Stratagene, Calif. (U.S.A.); complex in the presence of Cal+ (6) is important human placental cDNA library from Clontech in the placenta, though physiological control of Laboratories, Inc., Calif. (U.S.A.); Protoblot the Factor VII-thromboplastin complex is still immunoscreening and immunoblotting systems poorly understood. Recently, Maki et al. (7) and from Promega Corporation, Wis. (U.S.A.); M13 Reutelingsperger et al. (8) isolated inhibitors of sequencing kit, [ƒ¿-35S]dATPƒ¿S, and 1-stearoyl-2-[l- blood coagulation from the microsomal fraction of 13C]arachidonyl phosphatidylcholine from Amer- a human placenta and from a human umbilical sham Japan, Tokyo; (twice-crystallized cord, respectively. These coagulation inhibitors and treated with L-(1-tosylamido-2-phenyl)ethyl probably inhibit clot formation induced by throm chloromethylketone) from Worthington Biochem boplastin without inhibition of fibrin formation icals, Co., N.J. (U.S.A.); lysyl initiated by . (from Achromobacter lyticus M497-1) from Wako During the course of our studies on tissue Pure Chemicals, Co., Osaka; Ellman's reagent thromboplastin, several inhibitors of blood coagu from Pierce Chemical Company, Ill. (U.S.A.); lation were found in the EDTA extract of human Cosmosil 5C18 column from Nakarai Chemical placental tissue. We report here the molecular Co., Kyoto. cloning and the expression of cDNA for one of Purification of the Inhibitor of Blood Coagula these coagulation inhibitors. We also discuss the tion-All the purification procedures were carried amino acid sequence of the inhibitor as compared out at 4°C. Human full-term placentae were ob with those of lipocortins (9, 10) and endonexin tained at the Department of Obstetrics and Gyne (11, 12). cology, School of Medicine, Akita University, and were frozen within 30 min of delivery. Five frozen MATERIALS AND METHODS placentae (approximately 2,500 g) were thawed and cut into cubes, followed by washing with 0.15 M Materials-The materials were obtained from NaCl. The washed placental tissue was homoge the following commercial sources and were used nized in 2 liters of 50mM Tris-HCl (pH 7.4) with according to the procedures given by the respec a Waring blender and washed again with the same tive suppliers: Sephadex G-100, Blue-Sepharose buffer. The placental debris (wet weight 930 g) CL-6B, protein A-Sepharose CL-4B, CNBr-acti was rehomogenized in 1,800 ml of 50mM Tris-HCl

vated Sepharose 413, and pKK233-2 expression kit (pH 7.4) containing 50mM EDTA and then cen from Pharmacia (Sweden); DEAE-Toyopearl trifuged at 10,000 x g for 20 min. The EDTA 650M from Toyo Soda Mfg. Co., Ltd.. Tokyo; extract was fractionated between 30 and 80 Diaflo YM10 from Amicon Ireland, Ltd. (Ireland); saturation of ammonium sulfate and dialyzed over Ampholine PAGPLATE, pH 3.5-9.5 from LKB - night against an excess of 50mM Tris-HCl (pH (Sweden); citrated human plasma for normal co 7.4). The dialysate was applied to a DEAE- agulation control and activated Thrombofax re Toyopearl 650M column (5.5 x 18 cm) equilibrated agent from Ortho Diagnostic Systems Inc., N.J. with 50mM Tris-HCl (pH 7.4) and eluted with a (U.S.A.); thromboplastin and bovine thrombin linear gradient formed from 2 liters of the equi from Mochida Pharmaceutical Co., Ltd., Tokyo; libration buffer and 2 liters of the same buffer heparin from Shimizu Pharmaceutical Co., Ltd., containing 0.3 M NaCl. Activity to prolong the Shimizu; pristane (2,6,10,14-tetramethylpentade blood coagulation time in each fraction was mea cane), bovine serum albumin (fatty acid- and sured by the method described below. The active globulin-free), and phospholipase AL (from por fractions that eluted at 0.18M NaCl were pooled cine pancreas) from Sigma, Mo. (U.S.A.); silicic and dialyzed overnight against an excess of 20 acid (100 mesh) from Mallinkrodt Inc., Ky. mM potassium phosphate buffer (pH 7.0). The

J. Biochem. cDNA CLONING OF BLOOD COAGULATION INHIBITOR 1263

dialyzed active fraction was passed through a volume of 0.15 M NaCl. Measurement of APTT Blue-Sepharose CL-6B column (3 x 15 cm) to re was carried out in a mixture consisting of 50 pl move serum albumin and then concentrated by of a sample and 50 pl of activated Thrombofax, ultrafiltration using a Diaflo YM10 . The concen and the mixture was preincubated for 2 min. Sub trated protein was further purified by gel filtration sequent measurement was performed by the pro on a Sephadex G-100 column (4 .8 x 75 cm) equi cedure given by Ortho Diagnostic Systems Inc.

librated with 0.15M NaCl (data not shown) . For the measurement of thrombin time, 100µl of Analytical Methods-The molecular weight of a sample was combined with 100ƒÊl of human the coagulation inhibitor was estimated by sodium plasma and preincubated for 3 min. Coagulation dodecyl sulfate-polyacrylamide gel electrophoresis was started by the addition of 100ƒÊl of bovine

(SDS-PAGE) by the method of Weber and Osborn thrombin (2 units/ml). Recalcification time was

(13). The reference proteins used for molecular measured in terms of clot formation at 37°C in a

weight estimation were phosphorylase b (M .W. siliconized test tube containing 100 p1 of a sample

94,000), bovine serum albumin (67,000), ovalbumin and 100ƒÊl of human plasma. After a 3 min pre

(43,000), carbonic anhydrase (30,000), soybean incubation, 100 pl of 0.025M CaCl2 was added to

trypsin inhibitor (20,100), and ƒ¿-lactalbumin initiate coagulation.

(14,400). The molecular weight of the inhibitor During the purification of the inhibitor, the

was also estimated from its gel chromatographic biological activity of each fraction was measured

behavior on a Sephadex G-100 column (1.9x98 by means of PT.

cm) equilibrated with 0.15M NaCl, according to Phospholipase A2 inhibition was assayed ac

the method of Andrews (14). The standard pro cording to the method described by Khanna et al.

teins used were bovine serum albumin (M.W. (16) with some modification. For each experi 67,000), ovalbumin (43,000), low-molecular-weight mental point, 45 pl of a sample was combined with

(34,000), chymotrypsinogen (25,000), 100 Id of 0.1M Tris-HCl (pH 8.0) containing 0.5

and myoglobin (17,800). The isoelectric point of mM CaC12 and with 50ƒÊl of porcine pancreatic

the coagulation inhibitor was determined by poly phospholipase A2 that contained 100ng of acrylamide gel isoelectric focusing according to the and 125ƒÊg of fatty acid-free bovine serum albumin.

method of Wrigley (15) using an Ampholine Samples were kept on ice for 1 h. The phospho

PAGPLATE, pH 3.5-9.5. The pI markers used lipase A2 assay was started by the addition of 5 pl

were amyloglucosidase (p13.50), soybean trypsin of the substrate solution that contained 2 nmol of inhibitor (4.55), ƒÀ-lactoglobulin A (5.20), bovine 1-stearoyl-2-[1-14C]arachidonyl phosphatidylcholine

carbonic anhydrase B (5.85), human carbonic an (0.025ƒÊCi/nmol). Reactions were terminated after hydrase B (6.55), horse myoglobin acidic band 5 min at 25°C by the addition of 3 ml of chloro

(6.85), horse myoglobin basic band (7.35), lentil form : methanol (2 : 1) and 1 ml of 0.01 N H2SO4. lectin acidic band (8.15), lentil lectin middle band Tubes were vortexed and centrifuged at 3,000 rpm

(8.45), lentil lectin basic band (8.65), and trypsino for 5 min. An aliquot (1ml) of the lower phase gen (9.30). was transferred into another tube and chloroform Assay of Biological Activity-Modified pro was removed in a stream of nitrogen. The resi thrombin time (PT), modified activated partial dual material was dissolved in 2 ml of the upper

thromboplastin time (APTT), and thrombin time phase of isopropanol:heptane : water : 0.1N were assayed by optical detection of fibrin forma H2S04 (1.5:1.5:1.3:0.2) and 200 mg of silicic tion at 37°C in a glass cuvette using a Coagtec acid was added and vortexed. After centrifugation, TE-600 (Erma Inc., Tokyo). For the measure 0.4 ml of the upper phase was subjected to 14C- ment of PT, 50 ill of a sample was mixed with 50 scintillation spectrometry using a Packard 1500

pl of 20mM Tris-HC1 (pH 7.4) containing 0.15M liquid scintillation analyzer. NaCl and 0.5%. human serum albumin and with Preparation of Monoclonal Antibody-Male 100 pi of thromboplastin (diluted to 0.1 mg/ml BALB/c mice were immunized by intraperitoneal with 20mM CaC12). After incubation for 3 min, injection of the purified coagulation inhibitor. coagulation was started by the addition of 200 pl Sensitized spleen cells were fused with murine PAI of human plasma which was diluted with an equal myeloma cells, which were derived from P3-X63-

Vol. 102, No. 5, 1987 1264 A. IWASAKI et al.

Ag8 (ATCC CRL1597), in the presence of poly with 4-vinylpyridine, followed by digestion with ethylene glycol 4,000. The culture media of lysyl endopeptidase. The resultant peptides were hybridomas were screened by means of an enzyme separated and purified by reversed-phase HPLC linked immunosorbent assay (ELISA). The posi on a Cosmosil 5C18 column with a linear gradient tive hybridoma cells were cloned and transplanted of acetonitrile (0-80%) in 0.1% trifluoroacetic to pristane-treated male BALB/c mice. Mono acid. The peptides contained in a few peaks were clonal antibody was purified from the murine further digested with trypsin and chromatographed ascitic fluid on a protein A Sepharose CL-4B under the same conditions. Some contaminating

column. Two mg of the purified antibody desig peptides in a few peptide peaks were removed by nated as A46 was coupled to 0.4 g of a CNBr- rechromatography on the same column with a activated Sepharose 4B according to the manu linear gradient of acetonitrile (0-50 %) in 10mM facturer's instructions. The resultant monoclonal triethylamine-acetic acid, pH 6.25. Amino acid antibody immobilized on Sepharose 4B was used analyses of the resultant peptides were done by the for immunoaffinity chromatography of the coagu use of a Waters Pico-tag amino acid system (25) lation inhibitor. after hydrolysis in HCl vapor at 110°C for 20 h. Screening of eDNA Library-A library of hu Amino acid sequences of the peptides were ana man placental cDNA inserted into the bacterio lyzed by automated Edman degradation in a Beck-

phage vector Agtll (17-19) was screened with a man 890D sequencer equipped with a Sequemat ProtoBlot immunoscreening system. As an anti P-6 autoconverter as previously described (26). body probe, a murine monoclonal antibody against Phenylthiohydantoin (PTH) derivatives were iden the inhibitor containing 10 jig/ml of IgG was used. tified by HPLC by the method of Glajch et al. Subcloning and Nucleotide Sequence Analysis (27). of cDNA Insert-The recombinant phage DNA Fast-atom-bombardment mass spectrometry was prepared according to the procedure described (FAB-MS) analysis on the peptide containing the by Perbal (20). The purified phage DNA was amino-terminal sequence of the inhibitor was car digested by EcoRI and subcloned into the EcoRI ried out using a JEOL JMS-D300 mass spectrom site of the plasmid vector pUC1I8. The cDNA eter. The carboxyl-terminal amino acid of the insert in the recombinant pUC118 plasmid was whole coagulation inhibitor was identified by the trimmed by several restriction and/or by hydrazinolytic procedure (28). Determination of exonuclease III and mung bean nuclease (21, 22). thiol groups in the inhibitor was achieved using Single-strand DNAs were prepared from the Ellman's reagent (29). trimmed recombinant plasmids according to the Expression of eDNA-The Ncol-HindIII frag

procedure provided by Takara Shuzo Co., Ltd., ment of the cloned cDNA was ligated into the Kyoto, using the helper phage M13KO7. DNA expression vector pKK233-2 (30) which was double sequencing of both strands was carried out by the digested by Ncol and HindIIl. For the produc dideoxynucleotide chain termination method (23) tion of the inhibitor of blood coagulation, the with some modification using a M13 sequencing resultant plasmid was introduced into Escherichia kit and [ƒ¿-35S]dATPƒ¿S. coli JM105 (22). The E. coli cells transformed by Amino Acid Composition and Sequence Anal the plasmid were cultured overnight at 37°C in 50 ysis-The entire amino acid composition of the ml of a medium containing 1.05% K2HPO4 , 0.45 coagulation inhibitor was analyzed on a Hitachi KH2PO2, 0.1% (NH4)2SO4, 0.05 % sodium citrate•

835 automatic analyzer according to the method 2H20, 0.02% MgSO4•E7H2O, 0.2% glucose , 0.5 of Spackman et al. (24). The protein was hydro casamino acids, 5.0ng/ml thiamine •HCl, and 50 lyzed in 5.7 M HCl in a tube sealed at low pressure ƒÊ g/ml ampicillin. To induce production of the at 110°C for 24, 48, and 72 h. To determine the recombinant protein, 1mM isopropyl-ƒÀ-D-thio content of tryptophan, the protein was hydrolyzed galactoside (IPTG) was added to the 3 h culture. in 3 M mercaptoethanesulfonic acid at 110°C for The E. coli cells were harvested and lysed by

24 h. freezing and thawing in 25mM Tris-HCl (pH 7 .4) The purified coagulation inhibitor was re containing 20mM EDTA. Subsequently, the cell duced with dithiothreitol and S-pyridylethylated extract was applied to a column in which the

d. Biochem. cDNA CLONING OF BLOOD COAGULATION INHIBITOR 1265

immobilized antibody A46 was packed , and the 1.5 kilobases. The common restriction sites PstI, column was washed with 0.1 M Tris-HCI (pH 7.4) SacI, SphI, and HindIII (Fig. 1) were observed in

containing 0.5 M NaCI. The purified recombinant all the inserts. The recombinant pUC118 plasmid

protein was eluted with 0.1 M acetate buffer (pH containing the longest insert was designated as 5.0) containing 0.5 M NaCI. pMKT7. The eDNA insert of pMKT7 was se

quenced according to the strategy shown in Fig. RESULTS I using the dideoxynucleotide chain termination method (23).

Purification of the Inhibitor of Blood Coagula- The nucleotide sequence of the longest eDNA

tion-Blood coagulation-inhibitory activity was insert consisting of 1,566 nucleotides and a poly

found in the EDTA extract of human placental A tail is shown in Fig. 2. The first ATG codon

tissue. Several active fractions were separated is preceded by a 5•L non-coding region of 135

from the EDTA extract on a DEAE-Toyopearl nucleotides that contains an in-frame stop codon

column (Fig. IS). The most active fractions, which TGA at residue -12. The coding region for the

eluted at 0.18 M NaCI, were pooled and further coagulation inhibitor includes 960 nucleotides for

purified as described in " MATERIALS AND 320 amino acids. The 3•L non-coding region con

METHODS." The purified coagulation inhibitor tains the polyadenylation signal, AATAAA, which

gave a single band on SDS-PAGE (Fig. 2S). The is located 22 nucleotides upstream of the poly A

overall yield of the inhibitor was approximately tract. Since the initiation codon for translation,

20 mg from a full-term placenta. The molecular ATG, is located in the NcoI recognition site, it is

weight of the inhibitor was estimated as 34,000 by possible to cleave off the coding region for the SDS-PAGE under both reducing and nonreducing inhibitor by Ncol digestion.

conditions (Fig. 2S) and by gel filtration on a Amino Acid Composition and Sequence Anal Sephadex G-100 column (data not shown). The ysis of the Inhibitor of Blood Coagulation-The isoelectric point of the inhibitor was found to be amino acid composition deduced from the eDNA

around 4.9. sequence was almost identical to the entire amino

Preparation of Monoclonal Antibodies-Six acid composition of the coagulation inhibitor

hybridoma cell lines producing monoclonal anti- (Table I). bodies against the coagulation inhibitor were To further confirm the predicted amino acid cloned. Among these 6 monoclonal antibodies, sequence, the coagulation inhibitor was digested

a combination of the monoclonal antibodies A39, by proteolytic enzymes and purified by reversed-

A46, and A180 was found to be sufficiently sen- phase HPLC according to the procedure shown in sitive to detect 30 pg of the inhibitor using a Fig. 3S. The results of sequence analyses of some

Protoblot immunoblotting system. Therefore, the of the peptides are given in Table IS and are sum- cultured media of these 3 hybridoma cell lines were mixed and used as the antibody probes for screening of the cDNA library. Cloning and Nucleotide Sequence Analysis of eDNA-The cDNA library constructed in the phage dgtll from 34-week-old human placental tissue was subjected to immunoscreening (17-19). Out of 3x106 recombinant phage plaques, 5 clones reacting with the monoclonal antibody probe were isolated. All cDNA inserts of the positive phage clones were introduced into the plasmid pUC118 Fig. 1. Restriction map and sequencing strategy for at the EcoRI site. To confirm the identity of the the cDNA inserts encoding the inhibitor of blood positive cDNA inserts, these 5 subclones were coagulation. The arrows indicate the direction and examined by means of digestion with several extent of the sequencing. The open boxes indicate restriction endonucleases. The sizes of the cDNA the non-coding regions and the hatched box indicates inserts, estimated by EcoRl digestion, were around the coding region for the polypeptide of the inhibitor.

Vol. 102, No. 5, 1987 1266 A. IWASAKI et al.

Fig. 2. Nucleotide sequence of cDNA for the inhibitor of blood coagulation. The open reading frame is trans lated to amino acids. Nucleotides are numbered in the 5•L to 3•L direction, and numbers are indicated on the left

of the sequence. Nucleotide residue 1 corresponds to the A of the initiating methionine codon. The amino acids are numbered sequentially from the amino-terminal alanine of the coagulation inhibitor. Numbers of amino

acids are shown below the sequence. The putative polyadenylation signal is underlined.

J. Biochem. cDNA CLONING OF BLOOD COAGULATION INHIBITOR 1267

TABLE I. Amino acid composition of the inhibitor sulfhydryl groups using Ellman's reagent (29) indi of blood coagulation . cated that the cysteine residue did not form a

disulfide bond in most of the inhibitor molecules.

Expression of cDNA-To obtain further proof

that we had indeed cloned the cDNA for the

inhibitor of blood coagulation, the coding sequence

of the cDNA was expressed in E. coll. A piece

consisting of 1,309 base pairs of the Ncol-HindIII

fragment including the coding region for the in

hibitor was introduced into the expression vector

pKK233-2 (30). The resultant plasmid, designated as pMKTXI, was expected to produce the full-

length protein for the inhibitor under the regula

tion of the trc promotor (30). The crude extract

of E. coli JM105 (22) transformed by pMKTXI

was examined by means of Western blot analysis

using the monoclonal antibody A46. The recom

binant protein was detected as a single immuno

reactive protein band showing identical mobility

to the coagulation inhibitor (Fig. 5S). The iso

electric point of the recombinant protein, which

was purified by the immunoaffinity chromatog

raphy, was estimated as 5.0 and is higher than the

pI4.9 of the coagulation inhibitor. The amino terminal sequence of the recombinant protein was

determined to be as follows: Ala-Gln-Val-Leu-

Arg-Gly-Thr-Val-Thr-Asp- (Table IIS). The initial

methionine was supposed to have been processed a Average values obtained from 24 , 48, and 72 h hydro since no methionine could be detected at the first lyses with 5.7M HCl. b Extrapolated to zero time. step of Edman degradation. C Taken from 72 h hydrolysis . d Obtained from 24 h Prolongation of blood coagulation by the in hydrolysis with 3 M mercaptoethanesulfonic acid. hibitor and that by the recombinant protein are e S-Pyridylethylcysteine . compared in Table II. Both the inhibitor and

the recombinant protein prolonged PT, APTT,

marized together with the amino acid compositions and recalcification time to the same extent, whereas

of the peptides in Fig. 3. they did not affect thrombin time at the concen

The amino-terminal residues of the whole tration of 125ƒÊg/ml of the protein.

coagulation inhibitor and the peptide K20T11 were In our assay system, heparin, a well-known

not reactive to phenylisothiocyanate, indicating anticoagulant, inhibited blood coagulation as fol

that their amino termini may be blocked. The lows. In the measurement of PT, 0.25 unit/ml (1

amino-terminal residue was identified as acetyl unit corresponds to approximately 10ƒÊg) of hepa

alanine, based on the FAB-MS analysis of the rin was equivalent to 2.5 Fig/ml of the inhibitor. peptide K20T11, which contains the amino-termi Complete inhibition (more than 360 s) of blood nal sequence (Fig. 4S). The carboxyl-terminal coagulation in terms of APTT and thrombin time aspartic acid residue was identified by hydrazine was caused by the addition of 1.25 units/ml and analysis. From the above results, the molecular 0.0375 unit/ml of heparin, respectively. The pro weight of the coagulation inhibitor isolated from longing effect of 0.033 unit/ml of heparin on the human placenta is estimated as 35,731. The unique recalcification time was equivalent to that of 0.33ƒÊ cysteine residue is located in the carboxyl-terminal g/ml of the inhibitor. region of the inhibitor. Quantitative analysis for In addition, the inhibitory activity of the

Vol. 102, No. 5, 1987 1268 A. IWASAKI et al.

Fig. 3. Amino acid sequence of the inhibitor of blood coagulation. The solid bars indicate peptides whose amino acid compositions were determined as described in " MATERIALS AND METHODS": K, peptides obtained by digestion with lysyl endopeptidase; K-T, peptides obtained by digestion with lysyl endopeptidase followed by trypsin digestion. Horizontal arrows below the residues indicate that the sequences were determined by a Beckman 890D sequencer.

recombinant protein toward phospholipase A2 was as the human placental protein inhibited blood of the same order of magnitude as that of the coagulation (Table II) in spite of the difference in coagulation inhibitor. Approximately 1ƒÊM of the amino-terminal acetylation. each protein was required for 50% inhibition of The predicted amino acid sequence was com-

phospholipase A2 activity. pared with those of known proteins using a com puter program for genetic information analysis, SDC-GENETYX Ver. 4.0 (revised on October 1, DISCUSSION 1986) (Software Development Co., Ltd., Tokyo). We have isolated cDNA encoding the 319-amino The coagulation inhibitor shows a marked homol acid sequence of a new human placental protein ogy with lipocortins (9, 10), potent inhibitors of which exerts an inhibitory effect upon blood phospholipase A2 (Fig. 4). More than 40%, ho coagulation. When the cDNA was expressed in mology was found between the coagulation in E. coli, the resultant recombinant protein as well hibitor and lipocortins after the 8th valine of the

J. Biochem. cDNA CLONING OF BLOOD COAGULATION INHIBITOR 1269 inhibitor, and this is only slightly lower than the mologous sequences found in lipocortin (31) are 50% homology between lipocortin I and lipocortin also found in the inhibitor. The fourth repeated II (10). The four repetitions of the internal ho- sequence of the inhibitor shares nearly 60%. ho mology with the fourth repeated sequence of lipo TABLE II. Comparison of the activity of the coagu cortin II. If these repeating regions participate in lation inhibitors isolated from human placenta and the some functional activity of the lipocortins, the recombinant E. coli cells. hIBC , inhibitor of blood inhibitor would be expected to inhibit phospho coagulation isolated from human placenta; rIBC, inhibitor of blood coagulation isolated from recombi lipase A2 activity. Indeed, the coagulation in nant E. coli cells; R.Ca, recalcification time. hibitor did inhibit phospholipase A2 activity. The unique cysteine residue located near the carboxyl terminus of the inhibitor is conserved in lipocortins. It would be worthwhile to perform further studies to clarify the role of the unique cysteine residue in the function of these proteins. In contrast to lipocortins, no potential attachment sites for carbohydrate, such as Asn-X-Ser, Asn-X- Thr, or Asn-X-Cys, are found in the inhibitor. No significant differences between the inhibitor and lipocortins seem to exist except for the amino terminal sequences. Elucidation of the biological role of the amino-terminal stretch remains to be done. Recently, Geisow et al. reported that there exists a conserved amino acid sequence of 17

Fig. 4. Homology of amino acid sequence among the inhibitor of blood coagulation, lipocortin I, lipocortin II, and endonexin. The amino acid residues of the proteins, which are identical to those of the coagulation inhibitor, are shaded. Boxed regions correspond to the two consensus sequences of lipocortins and endonexin (12). The four repeated regions in the sequence are aligned with the sequence of lipocortin in the manner described by Munn and Mues (31). Peptide fragments of endonexin are shared in the most homologous part of the inhibitor. IBC, the inhibitor of blood coagulation; Lipo I, lipocortin I; Lipo II, lipocortin 11; Endo, endonexin.

Vol. 102, No. 5, 1987 1270 A. IWASAKI et al. residues among Ca2+-binding proteins such as endonexin, calelectrin, and protein II (11). The REFERENCES homology between endonexin and lipocortin was discussed by Kretsinger and Creutz (32) and by 1. Williams, W.J. (1966) J. Biol. Chem. 241, 1840- Weber and Johnsson (33). Geisow and Walker 1846 indicated that there is an additional homology 2. Kawano, T., Morimoto, K., & Uemura, Y. (1970) between these proteins at another region (12). J. Biochem. 67, 333-342 The coagulation inhibitor shares homologous se 3. Myatt, L. & Elder, M.G. (1977) Nature 268, 159- 160 quences with endonexin (Fig. 4), as well as calelec 4. Freyssinet, J.M., Brami, B., Gauchy, J., & Cazenave, trin and protein II (comparisons are not shown). J.P. (1986) Thromb. Haemost. 55, 112-118 The homology between the inhibitor and endonexin 5. Kobayashi, T., Terao, T., Oike, J., & Kawashima, seems to be very high. Saris et al. (34) have Y. (1981) Sanfujinka Chiryo (in Japanese) 43, 476 classified Ca2+/phospholipid-binding proteins into 6. Hubbard, A.R. & Jennings, C.A. (1986) Thromb. 4 groups. Lipocortin I, lipocortin II, and endo Res. 42, 489-498 nexin have each been classified into a different 7. Maki, M., Murata, M., & Shidara, Y. (1984) Eur. group. On the basis of physicochemical prop J. Obstet. Gynecol. Reprod. Biol. 17, 149-154 erties such as isoelectric point (pI4.9), relative 8. Reutelingsperger, C.P.M., Hornstra, G., & Hemker, molecular weight (34,000), and homology of amino C. (1985) Eur. J. Biochem. 151, 625-629 acid sequence, our coagulation inhibitor can be 9. Wallner, B.P., Mattaliano, R.J., Hession, C., Cate, assumed to belong to the endonexin group. R.L., Tizard, R., Sinclair, L.K., Foeller, C., Chow, The homology between the inhibitor and E.P., Browning, J.L., Ramachandran, K.L., & Pepinsky R.B. (1986) Nature 320, 77-81 endonexin suggests that the inhibitor might bind 10. Huang K.-S., Wallner, B.P., Mattaliano, R.J., Ca2+ and phospholipid. This assumption has Tizard, R., Burne, C., Frey, A., Hession, C., been confirmed by our preliminary study, in which McGrey, P., Sinclair, L.K., Chow, E.P., Browning, the inhibitory effects of Ca 2+ and phospholipid J.L., Ramachandran, K.L., Tang, J., Smart, J.E., upon the interaction between the coagulation in & Pepinsky, R.B. (1986) Cell 46, 191-199 hibitor and the monoclonal antibody against the 11. Geisow, M.J., Fritsche, U., Hexham, J.M., Dash, inhibitor were examined (data not shown). On B., & Jonson, T. (1986) Nature 320, 636-638 addition of the inhibitor, PT, APTT, and recal 12. Geisow, M.J. & Walker, J.H. (1986) TIBS 11, 420- cification time were prolonged, whereas thrombin 423 time was not affected. The results described above 13. Weber, K. & Osborn, M. (1969) J. Biol. Chem. lead us to consider that the inhibitor may prevent 244,4406-4412 14. Andrews, S.P. (1964) Biochem. J. 91, 222-233 blood coagulation at steps such as the formation 15. Wrigley, C.W. (1971) Methods Enzymol. 22, 559- and the action of Factor VII-thromboplastin com 564 plex and at the level of the complete prothrom 16. Khanna, N.C., Hee-Chong, M., Severson, D.L., binase, in which Ca 2+ and phospholipid are re Tokuda, M., Chong, S.M., & Waisman, D.M. quired. To test these hypotheses, it is essential (1986) Biochem. Biophys. Res. Commun. 139, 455- to perform further structural and functional studies 460 on the binding sites for Ca2+ and phospholipid in 17. Huynh, T.V., Young, R.A., & Davis, R.W. (1985) our coagulation inhibitor. in DNA Cloning: A Practical Approach (Glover, D.M., ed.) Vol. 1, pp. 49-78, IRL, Oxford We are grateful to Professor S. Iwanaga of Kyushu 18. Young, R.A. & Davis, R.W. (1983) Proc. Natl. University for kind guidance during the structural Acad. Sci. U.S. 80, 1194-1198 analysis studies and for critical comments on the manu 19. Young, R.A. & Davis, R.W. (1983) Science 222, script. We thank Mrs. C. Sueyoshi and Miss S. Kaji 778-782 yama of Kyushu University for amino acid sequence 20. Perbal, B. (1984) in A Practical Guide to Molecular analyses. We also thank Dr. H. Tani, director of Cloning pp. 175-187, A Willey-Interscience Publica Kowa Research Institute for his support and encourage tion, New York ment during this work. 21. Henikoff, S. (1984) Gene 28, 351-359 22. Yanisch-Perron, C., Viera, J., & Messing, J. (1985) Gene 33, 103-119

J. Biochem. cDNA CLONING OF BLOOD COAGULATION INHIBITOR 1271

23. Sanger, F., Nicklen, S., & Coulson, A.R. (1977) Biophys. 118, 241-252 Proc. Natl. Acad. Sci. U.S. 74, 5463-5467 29. Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 24. Spackman, D.H., Stein, W.H., & Moore, S. (1958) 70-77 Anal. Chem. 30, 1190-1206 30. Amann, E. & Brosius, J. (1985) Gene 40, 183-190 25. Bidlingmeyer, B.A., Cohen, S.A., & Tarvin, T.L. 31. Munn, T.Z. & Mues, G.I. (1986) Nature 322, 314- (1984) J. Chromatogr. 336, 93-104 315 26. Miyata, T., Usui, K., & Iwanaga, S. (1984) J. 32. Kretsinger, R.H. & Creutz, C.E. (1986) Nature Biochem. 95, 1793-1801 320, 573 27. Glajch, J.L., Gluckman, J.C., Charikofsky, J.G., 33. Weber, K. & Johnsson, N. (1986) FEBS Lett. 203, Minor, J.M., & Kirkland, J.J. (1985) J. Chromatogr. 95-98 318,23-39 34. Saris, C.J.M., Tack, B.F., Kristensen, T., Glenny, 28. Braun, V. & Schroeder, W.A. (1967) Arch. Biochem. J.R., & Hunter, T. (1986) Cell 46, 201-212

Vol. 102, No. 5, 1987 1272 A. IWASAKI et al.

TABLE IIS . Parllal amino sequence of Supplemental Materials the recombinant protein produced by transformed _??_ JM105. Approximate quantities of PTH derivatives are shown.

TABLE IS. Complete and porn al amino acid sequences of lysyl endopeptldase peptlides and t ryplic peptides derived from the inbibitor of blood coaguation. Approximate quantities of PTH derivatives are shown.

a not quanlitated

a not quantitated.b not identified.c _??_ -pyridylethylcysteine.

TABLE IS. Continued.

J. Biochem. cDNA CLONING OF BLOOD COAGULATION INHIBITOR 1273

LEGENDS TO SUPPLEMENTAL FIGURES Fig. 55. Western blot analysis of the recombinant protein

extracted from transformed _??_ JM105 cells. Reduced Fig. 1S. Separation of the inhibitor of blood coagulation by DEAE-Toyopearl chromatography. The ammonium sulfate SDS-PAGE was performed on 10% gel. After electrophoresis,

fraction of EDTA extract of human placenta was dialyzed proteins were transferred electrophoretically to nitrocellulose filter. The coagulation inhibitor was against 50 MM Tris-HCl (pH 7.4) and applied to a DEAE- detected by using a Protoblot immunblotting system with Toyopearl 650M column. Linear NaCl gradient elution was monoclonal antibody A46 (10ƒÊg/ml). Ce11 extract (75 R g performed as described in "MATERIALS AND METHODS". The fraction volume was 25 ml and the flow rate was about 150 ml protein) of JM105 transformed by pMKTX1 induced (lane 1) or uninduced (lane 2) with IPTG and by pEK233-2 induced (lane per h: The protein elution profile was monitored at 280 em. 3) or uninduced (lane 4) with IPTG were compared with 0.3 Prolongation of clotting time by each fraction was measured by means of PT. Shaded active fractions eluted at 0.18 M fig of the coagulation inhibitor isolated from human placenta (lane 5). NaCl were pooled for further purification.

Fig. 2S SDS-PAGE of the inhibitor of blood coagulation.

SDS-PAGE (1µ g sample) was carried out on 12.5 z gel in the

presence (lane 1) or absence (lane 2) of 2-mercaptoethanol.

The gel was stained with Coomassie brilliant blue R-250.

The molecular weight of the coagulation inhibitor was

estimated as described in "MATERIALS AND METHODS".

Fig. 3S. Procedure for the elucidation of the structure.

IBC, inhibitor of blood coagulation.

Fig. 4S. FAB-MS of peptide K20TII.

Vol. 102, No. 5, 1987