Cloning and Expression of a Cdna Coding for the Anticoagulant Hirudin
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Proc. Nati. Acad. Sci. USA Vol. 83, pp. 1084-1088, February 1986 Medical Sciences Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis (thrombosis/antithrombin/recombinant DNA/coagulation) R. P. HARVEY*t, E. DEGRYSE*, L. STEFANI*, F. SCHAMBER*, J.-P. CAZENAVEt, M. COURTNEY*§, P. TOLSTOSHEV*¶, AND J.-P. LECOCQ* *Transgtne, S.A., 11, rue de Molsheim, 67000 Strasbourg, France; and tCentre R6gional de Transfusion Sanguine, Strasbourg, France Communicated by J. Bracket, October 24, 1985 ABSTRACT Cloned cDNAs have been isolated that encode thrombin-induced disseminated intravascular coagulation in a variant of hirudin, a potent thrombin inhibitor that is rats (12). Endotoxin-induced disseminated intravascular co- secreted by the salivary glands of the medicinal leech, Hirudo agulation is prevented in newly weaned pigs (15). medicinals. This variant probably corresponds to a form that Purification of large quantities of hirudin from leeches for has been purified from leech heads but differs in amino acid further clinical testing or eventual clinical use is highly sequence from the hirudin purified from whole leeches. There impractical, but this problem can potentially be solved by are at least three hirudin transcripts detectable in leech RNAs recombinant DNA technology. In addition, cloning of the that are different in size, site of synthesis, inducibility by gene(s) for hirudin should help to resolve questions about starvation, and relationship to hirudin activity. The new different hirudin forms and possible precursor proteins. hirudin variant predicted by the cDNA and the heterodisperse Here we report the cloning ofa cDNA encoding one variant transcription products suggest a hirudin protein family. The of H. medicinalis hirudin and its expression in Escherichia hirudin cDNA was expressed in Esckerichia coli under the coli to yield a biologically active product. control of the bacteriophage X PL promoter. The recombinant product is biologically active, inhibiting the cleavage by throm- MATERIALS AND METHODS bin of fibrinogen and a synthetic tripeptide substrate. Leeches. Live Hirudo medicinalis were purchased from Leech hirudin is the most potent natural inhibitor of coagu- Ricarimpex (Audenge, France) and kept in aerated water lation known (1-4). A very stable noncovalent 1:1 complex containing 0.63 mM NaCl, 0.07 mM CaCl2, 0.05 mM MgSO4, is rapidly and specifically formed with a-thrombin, thereby and 0.05 mM KCl at ambient temperature. They were fed on abolishing its ability to cleave fibrinogen (4). To date there is citrate-treated rabbit blood from an inflated porcine bladder. no evidence that it can interact with other components ofthe Fed leeches were kept in a separate aquarium. human coagulation cascade (5, 6). Protein Extracts. Frozen leech segments were homoge- Hirudin is a polypeptide of65 amino acids that is stable to nized in phosphate-buffered saline (0.1 M Na3PO4, pH extremes ofpH and heat (4). It contains six cysteine residues 7.0/0.15 M NaCl) with a Polytron homogenizer (Brinkmann), grouped in the NH2-terminal half of the protein, an acidic and particulate material was sedimented. For bacterial ex- COOH-terminal half, and one sulfated tyrosine (7). A hirudin tracts, cells were disrupted by sonication in TGE buffer (25 form with isoleucine at the NH2 terminus was first purified mM Tris-HCl, pH 8/50 mM glucose/10 mM EDTA) and from leech heads (H. medicinalis) (4, 8) in which activity was cleared by centrifugation. found to be concentrated in the salivary glands. Subsequent- RNA Extraction. Powdered, frozen leech sections were ly, Bagdy et al. (9) adopted new purification schemes using added to lx NETS buffer (0.1 M NaCl/1 mM EDTA/10 mM whole leeches instead ofheads, yielding a form with Val-Val Tris HCl, pH 7.5/0.5% sodium dodecyl sulfate) containing as the first two NH2-terminal amino acids. The amino acid 50% phenol at 95°C and were homogenized immediately. sequence ofthe "whole body form" has been determined by After a cooling period, phases were separated by centrifu- independent groups (8, 10), and valine residues at positions gation. The phenol phase was reextracted with 2x NETS, 1 and 2 have been confirmed. Both forms had a specific and the aqueous phase was reextracted with phenol. Nucleic activity of around 8000-10,000 antithrombin units/mg. How- acid was precipitated from the pooled aqueous phases with 2 ever, more recently, Baskova et al. (11) described two vol of ethanol. After centrifugation, the pellet was dissolved distinct hirudins: a highly active form in heads with an Ile-1 in H20, and the solution was adjusted to 2.5 M LiCl and kept NH2 terminus and an inactive form in bodies (pseudohirudin) at 4°C overnight. To pellet precipitated RNA, the solution with a Val-Val NH2 terminus. The potency and specificity of was underlaid with 0.25 vol of 3 M LiCl and centrifuged at hirudin have generated interest in its possible use as a clinical 15,000 x g for 10 min at 4°C. The pellet was dissolved in H20 reagent in treatment of thrombotic diseases, but a detailed and reprecipitated with ethanol. pharmacological assessment is prevented by the cost and Hirudin Activity. Antithrombin activity in leech or bacte- supply ofpurified material. In animal studies (12-14), hirudin rial extracts was measured in a clotting assay (4) using was shown to be pharmacodynamically inert apart from its citrated human platelet-poor plasma as a fibrinogen source or anticoagulant activity. It has extremely low toxicity (LD50 > in a colorimetric assay using the thrombin chromogenic 500,000 antithrombin units/kg in rats; ref. 12), appears to be substrate Tos-Gly-Pro-Arg-p-nitroanilide (Chromozym TH, nonantigenic, and is eliminated almost completely via the Boehringer Mannheim; Tos = tosyl) (7). Standard curves kidneys in a biologically active form (12). It is effective in preventing venous thrombosis, vascular shunt occlusion, and Abbreviation: kb, kilobases. tPresent address: Department of Biochemistry and Molecular Biol- ogy, Harvard University, 7 Divinity Avenue, Cambridge MA 02138. The publication costs of this article were defrayed in part by page charge §To whom all reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" Present address: Biotechnology Australia, Pty Ltd., 28 Barcoo in accordance with 18 U.S.C. §1734 solely to indicate this fact. Street, East Roseville, NSW 2069, Australia. 1084 Downloaded by guest on September 28, 2021 Medical Sciences: Harvey et al. Proc. Natl. Acad. Sci. USA 83 (1986) 1085 were established with standardized bovine thrombin (Roche, 1 21 Neuilly-sur-Seine, France) in the case of the clotting assay a GCA ATC TGC GTG TCT CAA GCA and with standardized hirudin (a gift from F. Markwardt) in b Ala Ile Cys Val Ser Gln Ala the case of the chromogenic assay. 22 66 cDNA Cloning. cDNA banks were constructed in pBR322 a ATT ACT TAC ACT GAT TGT ACA GM TCG GGT CM MT TTG TGC CTC 1 15 by standard procedures (16). Screening of banks with oligo- b Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu nucleotides was as described (16), with the stringency of 1 2 washes for the 48-mer probe being 0.3 M NaCl/0.03 M C Val Val --------------------------------------------------- sodium citrate/0.1% NaDodSO4 at 500C. Oligonucleotides 67 1ll were synthesized by the phosphotriester method on an a TGC GAG GGA AGC MT GTT TGC GGT AM GGC MT MG TGC ATA TTG 16 30 inorganic support (17). DNA sequence analysis ofclones was b Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys Ile Leu performed by the dideoxy chain-termination method (18) 24 after subcloning into an M13 vector. C ------------Gln------------------------ Thrombin-Sepharose. Thrombin (61 National Institutes of 112 156 a GGT TCT MT GGA MG GGC MC CM TGT GTC ACT GGC GAA GGT ACA Health units/mg) was bound to CNBr-activated Sepharose 31 45 beads (Pharmacia) by using the manufacturer's recommend- b Gly Ser Asn Gly Lys Gly Asn Gln Cys Val Thr Gly Glu Gly Thr ed protocol. For affinity selection ofbacterial hirudin, throm- 33 35 36 bin-Sepharose was added, in batch, to bacterial extracts so C ------- Asp --- Glu Lys ----------------------------------- that all activity was bound. The Sepharose was sedimented 157 201 a CCG MC CCT GAA AGC CAT MT MC GGC GAT TTC GAA GM ATT CCA by gravity, washed twice with excess 0.5 M NaCl, and then 46 60 eluted with 4 vol of 0.1 M 4-aminobenzamidine/25 mM HCl. b Pro Asn Pro Glu Ser His Asn Asn Gly Asp Phe Glu Glu Ile Pro All incubations and elutions were at ambient temperature for 47 49 53 10 min. Carrier bovine serum albumin (30 pg/ml) was added C --- Lys --- Gln ------------Asp --------------------------- eluted and 4-aminobenzamidine was removed 202 256 to the proteins, a GAA GAA TAT TTA CM TGAAMATGAAAGAATATCMTCATAGAGAATTTTGATTT by dialysis against 25 mM HCl and then H20. [35S]Cysteine- 61 65 labeled proteins were analyzed on 15% NaDodSO4/polyac- b Glu Glu Tyr Leu Gln rylamide gels (19) after reduction/denaturation in 2.3% 257 316 NaDodSO4/6.25% 2-mercaptoethanol at 100°C. a MAAACATTTCCATAGCTMGCTATTTACCMTAAATAMTTMTTTTTCCATTGMTCT 317 376 RESULTS a CMTCATATTTACTCTCMTCATATTCAGCTATTTACCAATMATAMTTMTTTTTCCA 377 Cloning ofHirudin cDNAs. Complementary DNAbanks were a TGA constructed from mRNA purified from the crudely dissected head region of starved leeches. Hirudin clones were identified FIG. 1. DNA sequence ofthe insert ofpTG717 (row a), the amino by screening with a long synthetic oligonucleotide (16), the acid sequence predicted by pTG717 in the reading frame that bears design of which was based upon the published amino acid homology to the published hirudin sequence (6, 9) (row b), and the sequence ofhirudin (8, 9) and on codon usage datafrom insects, residues in the published hirudin sequence that differ from the the closest evolutionary relatives of segmented worms for sequence in row b (row c) are shown.