Purification and characterisation of recombinant rabbit plasminogen activator inhibitor-1 expressed in Saccharomyces cerevisiae

Purification and Characterisation

of Recombinant Rabbit Plasminogen

Activator Inhibitor-l

Expressed in Saccharomyces

K. J. Hofmann,

L. D. Schultz.

E. J. Mayer,

S. H. Socher,

cerevisiae

C. F. Reilly

SUMMARY. The rabbit plasminogen activator inhibitor-l (PAI-1) cDNA has been isolated from a rabbit cornea1 cell cDNA library. The cDNA encodes a 402-amino acid (AA) protein that shares an overall 66% AA sequence identity with the rat, mouse, bovine and human forms of PAI- and exhibits the greatest AA sequence identity (85%) with human PAI-1. Three potential N-linked glycosylation sites and the PI, Pl’ reactive centre of PAI- are conserved among all five species of animals. The cDNA encoding the proposed mature form of rabbit PAI- was cerevisiae as an intracellular, non-glycosylated protein. The purified, recombinant expressed in Saccharomyces rabbit PAI- (R-rPAI-1) has an apparent M, of 39 100 and exists primarily in a latent form which can be activated by guanidine HCI treatment. Activated R-rPAI-1 exhibits in vitro functional properties which are virtually indistinguishable from a recombinant, non-glycosylated form of human PAI- and from fully glycosylated, native human PAI-1.

Clot dissolution is initiated by the plasminogen activators (PAS), tissue-type plasminogen activator (t-PA) and urokinase (u-PA), which convert the inactive proenzyme plasminogen to the fibrindegrading protease, plasmin.’ Since strict control of t-PA and u-PA may be crucial for maintaining the balance between coagulation and fibrinolysis, the activities of these PAS are governed by a number of mechanisms including regulated expression and secretion, rapid hepatic clearance, specific interactions with fibrin, as well as inactivation by plasminogen activator inhibitor-l (PAI-l).‘PAI-1 is a member of the SERPIN family of protease inhibitors’ and is synthesised by several cell types including endothelial cells,’ monocytes/macrophages’ and vascular smooth muscle ce11s.“~7 I_Jpon activation. platelets release large amounts of PAI-I”,” which may accumulate locally at sites of vascular injury resulting in thrombus stabilisation. In plasma, PAIexists in active and latent forms or as a rapidly-formed, inactive complex with t-PA.2 Exposure to protein denaturants such as sodium dodexyl sulphate (SDS) or guanidine HCI can regenerate active PAIfrom latent PAI-I .“’ Elevated levels of plasma PAI-I activity and/or

K. J. Hofmann, L. D. Schultz, Department of Cellular and Molecular Biology, E. J. Mayer, S. H. Socher, C. F. Reilly. Pharmacology. Merck, Research Laboratories. West Point, Pennsylvania 19486. USA.

antigen have been detected in patients with coronary artery disease, ” deep venous thrombosis’” and mvocardial infarction.“,‘” These observations suggest that elevated levels of PAI- retard fibrinolysis which results in clinical thrombosis. However, increased levels of PAIhave also been observed following surgery” and in non-thrombotic disease states including non-insulin-dependent diabetes’” and systemic lupus erythematosus.‘7 Therefore, it is unclear whether the increased levels of PAI- are the cause or result of the disease. In vivo studies using human recombinant PAI(H-rPAI-1) have begun to define the precise roles for the protein in the fibrinolytic process. High levels of circulating H-rPAI-1 inhibit clot lysis in rabbits” and in rats’” and the administration of H-rPAI-1 in a canine model of occlusive thrombus formation shortened the time required to form occlusive thrombi.2” Blood clots enriched with H-rPAI-1 resist lysis in vivo, thus supporting the hypothesis of localised PAIhaving a thrombus-stabilising effect. ‘y.2’ To date, the studies in experimental animal models have utilised the human form of PAI-1. However, PAI- from various animal species may exhibit significant differences in t-PA and u-PA binding affinities, pharmacokinetic behaviour, and interactions with regulatory components such as vitronectin22 or fibrin.23 Therefore, more informative and controlled in vivo studies could be obtained if the PAI-I was derived from the animal species employed

264

Purification

and Characterisation

of Recombinant

Rabbit

PAI-

in the study. We have chosen the rabbit model to study fibrinolysis and consequently have cloned the rabbit PAIcDNA. In this study, we describe the cloning, expression, purification and in vitro characterisation of rabbit recombinant PAI(R-rPAI-1).

MATERIALS Molecular

AND METHODS

Cloning of the Rabbit PAI-

cDNA

Poly (A)+ selected RNA was isolated from rabbit cornea1 (SIRC) cells (ATCC CCL No. 60) using the Fast TrackTM mRNA isolation kit (Invitrogen, Inc.). The cDNA was synthesised using the Gubler and cDNA was Hoffman protocol. *’ Double-stranded ligated with synthetic EcoRI linkers and subcloned into the lambda ZAP IITM vector (Stratagene, Inc.). The recombinant phage were screened by plaque hybridisation with a radiolabelled rabbit PAI- DNA fragment generated by polymerase chain reaction (PCR) using oligomer primers that were designed based on the human PAIsequence*” in regions most homologous to other SERPINS.26 The 5’ oligomer, 5’-GTATGATCAGCAACTTGCTTGGGAA-3’, and the 3’ oligomer, 5’-GGG’ITCCATCACTTGGCCCATGAAAAGG-3’, generated a 0.7-kbp DNA fragment consistent in size with the human PAIsequence, when used as primers in a PCR reaction. The template for the PCR reaction was the first-strand cDNA made from the SIRC cell mRNA. Hybridisation-positive clones were converted to the pBluescriptTM phagemid vector plus insert by the helper phage-mediated excision protocol (Stratagene, Inc.). Clones containing a 3-kbp insert were identified by digestion with EcoRI and the sequence of the cDNA insert was determined by dideoxy-DNA sequencing using SequenaseTM (United States Biochemical, Inc.). The sequence data was analysed with the IntelliGenetics Sequence Analysis Software Package version 5.37 (IntelliGenetics, Inc.) and the Sequence Analysis Software Package version 6.2 of the Genetics Computer Group at the University of Wisconsin.27 Construction

of the R-rPAI-1 Expression

Vector

Two DNA fragments which together encode R-rPAI1 were generated by PCR. The 5’ PCR primer, 5’-TCGGATCCACAAAATGTCATACCCTCACCAGCCCCAGG-3’, contains a Bum HI restriction site, a yeast-like 5’-untranslated leader sequence (underlined) *s and an NH;?-terminal Met codon adjacent to the Ser codon located at base 201 of the rabbit PAIcDNA (AA 24) (Fig. 1). This Ser residue is predicted to be at the mature NH*-terminus of rabbit PAI(Fig. 2). The 3’ PCR primer, 5’CGGAAAAGTCGACCTGCTTG-3’, corresponds to bases 563-582 of the rabbit PAIcDNA and contains a Sal1 site (underlined). To incorporate the

Sal1 site into the rabbit PAIDNA sequence, the Val codon at AA 147 was conservatively changed from GTG to GTC. Using the rabbit PAI- cDNA as PCR template, the 5’ portion of the rabbit PAIopen reading frame (ORF) was synthesised as a 440-bp Bum HI-Sal1 fragment. Following digestion with BumHI and Sull, the 440-bp PCR fragment was agarose-gel purified. The 3’ portion of the rabbit PAIORF was generated as a 775-bp Sull-Bglll fragment. The 5’ PCR primer, 5’-CAAGCAGGTCGACTTTTCCG-3’, is the complement to the 3’ PCR primer used to generate the 440-bp BumHI-Sal1 fragment described above. The 3’ PCR oligomer, 5’-GGAGATCTCAGAGTTCCATCACCTGGC3’, contains 20 bases of the rabbit PAIDNA sequence including the stop codon and a B&II restriction enzyme site. Following digestion with Sal1 and Bglll, the 775-bp PCR fragment was agarose-gel purified. The 440-bp BumHI-Sal1 and the 775-bp Sall-Bglll rabbit PCR fragments were cloned into the BumHI site of the yeast expression vector pCl/lADH2p-ADHlt-1 ,2’) to yield the plasmid pADH2PA1 (Fig. 3). Sequencing of the rabbit PAIORF within pADH2-PA1 ensured that no errors had occurred during PCR. Expression

of R-rPAI-1 in S. cerevisiae

The S. cerevisiue strain BJ1995 (MATa, leu2, trpl, prb l-1122,pep4-3, guf2) was a gift from Dr Elizabeth Jones, Carnegie-Mellon University. The BJ1995 cells were transformed”” with pADH2-PA1 and clonal isolates were grown at 30°C in YEHD complex medium”’ for 30 h following depletion of glucose from the medium as described.*” After harvesting the cells, the cell pellets were broken with glass beads and cell lysates analysed for the expression of R-rPAI-1 by an in vitro t-PA inhibition assay (see Determination of PAI- Activity in Yeast) and by immunoblot analysis using goat anti-human PAIIgG (American Diagnostica, Inc., no. 3956) as primary antibody and alkaline-phosphatase-conjugated rabbit ant-goat IgG (Organon-Teknika-Cappel, Inc., no. 8606-0082) as secondary antibody. The nitrocellulose filters were stained using an alkaline-phosphatase ImmunBlotTM assay kit (Bio-Rad, Inc.). Based on the results of these studies, a clonal isolate of BJ1995/pADH2-PA1 was selected to prepare a master seed which was used for all further cell growth and purification studies. The master seed was stored at -70°C in the presence of 17% (v/v) glycerol. Large-scale cultures were grown in multiple shake flasks (6-l total culture volume) containing YEHD complex medium for 46 h at 30°C. Cells were harvested by centrifugation and cell pellets (17Og wet weight) were stored at -70°C. Determination

of PAI-

The PAIactivity tially as described.“*

Activity in Yeast

in yeast was determined essenCell lysates (25 ~1) were diluted

Fibrinolysis

loo-, 200-, and 600-fold in TT buffer (5OmM Tris, pH 8.3, 0.01% Tween SO) and incubated with 2_5l.~l (50 W/ml) of single chain t-PA (sc-tPA) for 30 min at 25°C. The samples were frozen on dry ice, thawed, diluted with lml TT buffer, and tested for residual t-PA activity in the coupled amidolytic assay (see PAIActivity Assay). PAIactivity was determined by comparing t-PA activity in the presence of the cell lysates to the t-PA activity in the absence of the cell lysates. The protein concentrations of the cell lysates were determined by the Bio-Rad Protein Assay (Bio-Rad, Inc.).

12

27

QPQVAELAADFGVKV 213CnGUXCnGGIGOCAGR4C'l'GGCGGCAGXTTTca;GTGlipnGn;

42

VQASKDRNVVF F Q Q V 258 TltZCXCAGGTGGXC%u*:TAAGCX:cGc&UGiXGlTTIC

57

s P 303 n?ca

LAMLQLT Y G VA S V TATo;GGlGGcc 'ICGGTGCIGOJ3An;CTGC%~ACC

72

T A G E TQQQIQAAMRF 34B&'XGa;Gc4G4ApLy:CXCAGAltcnAGosGa;XTGpGATIC

87

KIDEQSTAPALRHLH 393 A%ATAGATCPGC4GAGCACGGCCCIXQXCXCGCC4CCIGCX

102

KELMGPWNKDEISTT 438 RpGGpGC?T:A~o;Gc(x:TGGFACFAGGRTGpGAx)pGT~AAa;

117

L V Q G D A I F V Q H D L K 483~GccA1c~Gx;cpGGa13ux:cr(;ppGcn;GrCc4GGGcTI13

265

ill3

1158

110:

:24a

1293

1339 :397 !257 3517 1577 1637 1697 1757 1817 1877 1937 1997 2057 2117 2177 2237 2297 2357 2417 2476 2536 2596 2655 2715 2775 2835 2895 2955

Fig.

F

132

MPHFFRLFRTTVKQV 528 ATGCCC Cpc TIC TICAG4TTG TIC 0;4PCCACGGTCFAGCAGGTG

147

D F S DVQRARFIINDW 573 ~llTTccG4TGl'G,cpG&X@Xi4XTICATCATCR4TGXTGG

162

VERHTKGMISDLLGE 618GTGGhGAG4C4CAcGApAGGcXtGATcpcCG.X'l-IGCJT@XGA4

177

GAMDQLTRLLLVNAL 663 ~G!XA'IGGXCJAClGAc4aC

192

PAI- I. The deduced AA sequence is shown above the nucleotide sequence. The proposed signal peptide cleavage site is indicated by the arrow after AA 23. Four potential N-linked glycosylation sites are indicated by bold-face type. An asterisk denotes the putative reactive centre. Within the 3’-untranslated region, the bracketed sequence represents the area of high homology between the human,*5 rat,4” mouse,“’ bovine? and rabbit PAI-I cDNAs and the underlined sequence is the presumed polyadenylation signal.

ClGClGClGGTGAATO(%CTC

YFNGQWKTPFSKSGT 70BTPII:TTC~CT*:C4GTGGAPGPCGCCCTICTCCRnG?CT~ACC

207

HHRLFHKSDGSTISV 753 ~CnCCQ)~~Cru)ffi?CGG4CCTC.AGCA~ATC?1STGrr

222

PMMAQTNKFNYTEFL 798 ~ATGATGCTT;C4GACCAPCR4GTTCFPI)TPCACTVIG~C~

237

TPDGHYYDILELPYH 252 843 XX Ccc G?X cGcC4C TPC TAC G4CATc TIGGPGCTGCCC IT\CCAT 267

LTNILSAQLI V P L S A 933 GlGccTCIC TCTGCIC'ICARCCFAC~

282 CIGAGTGX

CAGCICATC

S QWKGNMTRVTRLLV 978FGCCPATGG~GGCFACArrPCCPGAGn;ACC~Cr(;CrC;G~

297

LPKFSLESEVDLRAP .023Cr(;CCCPPG~TCCcn;GFGPGTG4Am(;GP13CTCPU;GCAC(X:

312

L E N L GMTDMFRPGQA 068 ~GpG~cx;GGGATGAcCG4CATG~Ao;oJ~ocx;GGo;

327

Purification

of R-rPAI-I

The yeast-expressed R-rPAI-1 was purified by the procedure described previously for H-rPAI-1.‘” Harvested cells (170g) were resuspended in 600ml of O.lM HEPES, pH 7.5, 10mM EDTA, 10 kg/ml pepstatin A, 0.13 kIU/ml aprotinin, 1OmM benzamidine, 2.5 PM trans-epoxysuccinyl-L-leucylamido-(4guanidino)butane, and 2mM phenylmethyl-sulphonyl fluoride, broken with a Microfluidizer 1lOY (Microfluidics, Corp.), and frozen at -80°C. After addition of Tween 80 (0.01%) to the thawed slurry, the solution was centrifuged at 13 680x g for 45 min at 4°C. The supernatant was diluted with 3 vol of 67mM Tris, pH 9.0, 40% glycerol, 0.01% Tween 80 and passed through a Q-Sepharose Fast Flow (Pharmacia, Inc.) anion-exchange column (33.5x2.6cm) equilibrated with 50mM Tris, pH 9.0, 30% glycerol,

266

Purification and Characterisation

of Recombinant

Rabbit

rabbit

MQMSPALACLALGWPLVSAGGSA

SYPH.

bovine

MRMSPVFACLALGLALIFGEGSA

t SYQP...

human

MQMSPALTCLVLGLALVFGEGSA

1 VHHP...

rat

MQMSSALTCLTLGLVLVFGKGFA

t SPLP...

PAI-I -

..

signal peptides. The signal peptide Fig. 2 Comparison of PAIcleavage sites of the bovine,” human” and rat“” PAI-1s are indicated with arrows.

0.01% Tween 80. The column was eluted with a 0- to 0.5-M NaCl gradient, and fractions were analysed by 0.1% SDS-12.5% PAGE followed by immunoblotting as described above. R-rPAI-I-containing fractions were pooled (75 ml), diluted with an equal vol of 0.1 M sodium acetate, pH 5.5, 0.2M NaCl, 2% polyethylene glycol 6000 (PEG), 30% glycerol, 0.01% Tween 80 and the pH lowered to 5.5 with HCl. After loading onto a S-Sepharose Fast Flow (Pharmacia, Inc.) cation-exchange column (13.0x2.6cm) equilibrated in acetate buffer (50mM sodium acetate, pH 5.5, O.lM NaCl, 1% PEG, 30% glycerol, 0.01% Tween SO), the R-rPAI-1 was eluted with a 0.2- to 1.0-M NaCl gradient in acetate buffer. The R-rPAIl-containing fractions (75 ml) were identified as described above and combined with sufficient NaClfree acetate buffer to lower the concentration of NaCl to O.lM. The pooled R-rPAI-1 was loaded onto a second S-Sepharose Fast Flow column (3.1 x 1 .Ocm) to concentrate the R-rPAI-1 and eluted with Tris buffer (50mM Tris, pH 8.0, 0.5M NaCl, 30% glycerol, 0.01% Tween SO). This concentrated R-rPAI-1 solution (3ml) was applied to a Sephacryl S-100 (Pharmacia, Inc.) gel filtration column (80~1.6cm) equilibrated and eluted with Tris buffer. The final product was analysed using 0.1% SDS-12.5% PAGE and immunoblotting, silver and Coomassie Brilliant Blue R-250 staining. The concentration of R-rPAI-1 was determined by AA analysis on a Beckman 6300 AA analyser. A non-glycosylated form of H-rPAI-1 was expressed intracellularly from S. cerevisiae and purified as described.2” Native, human PAI-1, purified from dexamethasone-treated human fibrosarcoma cells (HT1080),“’ was obtained from American Diagnostica, Inc.

Activation

of PAI-

The purified recombinant PAI(rPAI-1) was in a latent, inactive form, that could be activated by treatment with guanidine HCl.“’ Latent rPAT-1 was incubated with 4.OM guanidine HCl for 5 min at 25°C in TNT buffer (50mM Tris, pH 7.4, O.lM NaCl, 0.01% Tween 80). The guanidine HCl was removed by gel filtration on a Pharmacia, Inc. NAPTM-5 column in TNT buffer at 4°C.

PAI-

Activity Assay

The inhibitory activity of PAIwas determined as previously described. 7.2’ In a microtitre plate, increasing amounts of activated PAI(0.125-g molar equivalents) or latent PAI(l-10000 molar equivalents) were added to a constant amount (0.2IU) of two chain t-PA (2c-t-PA) in 4Oul of TNT buffer. After 1 h at 37”C, 160 ~1 of TT buffer containing 0.625 yM glu-plasminogen, 162.5 kg/ml CNBr fibrinogen fragments, and 1.25mM S-2251 (Kabi Vitrum, Inc.) were added. Residual t-PA activity was determined by measuring the increase in A4,i5 after incubation for 1 h at 37°C. The linear portion of the inhibition curve was extrapolated to obtain the concentration of PAI- necessary for complete inhibition of the t-PA. One arbitrary unit (AU) is defined as the amount of PAIrequired to completely inhibit one IU of t-PA. Determination

of Rate Constants

The second order association rate constants were determined by mixing 2c-t-PA (50pM) or SC-t-PA (243pM) with excess activated R- or H-rPAI-1 in 80~1 TNT buffer at 37°C for t&60 min. The reaction was terminated by the addition of 12Ol.~l TT buffer

Fig. 3 Structure of the pADH2-PAI expression vector. The PCR-generated rabbit PAIORF contains a yeast-like 5’. untranslated leader and NH,-terminal Met fused to the rabbit PAI-I cDNA beginning at the Ser codon at base 201 (Fig. 1). The ADH2 promoter-driven vector contains the entire yeast 2-km DNA, leu2-d gene for selection of Leu+ prototrophs and pBR322 sequence from the yeast shuttle vector, pCI/l.“’ The rabbit PAIORF was cloned into the unique BamHI site located between the promoter and the ADH 1 terminator sequences. The E. co/i and S. cerwisiae origins of replication (Ori E and Ori Y, respectively) and the ampicillin resistance marker (AmpR) are indicated. The arrow denotes the direction of transcription.

Fibrinolysis

X7

containing 0.83 FM glu-plasminogen, 83 kg/ml desbase 132, and a 1654-bp 3’ untranslated sequence AA-fibrinogen, and 1.67mM S-2251. Control experiterminating in a poly (A) tail. There is a single ments in which PAI-I was added after substrate and consensus poly (A) addition signal. AATAAA, fibrin mimetic addition confirmed that no additional located 16bp 5 to the start of the poly (A) tail. t-PA was inhibited. Residual t-PA activity was Comparison of the rabbit PAIcDNA sequence determined by the increase in A4o5 after a 30 min with the PAI-I cDNA sequences of the previously incubation at 37°C. The second order association rate characterised bovine,3” human,2s mouse” and rat’” constants were determined from the slope of a plot of forms reveal an overall 73%, 71%. 66%) and 65% log[PA][PAI],,/[PA],,[PAI] vs time for t&20 min using sequence similarity, respectively. the standard second order rate equation: t kabSOC= 2.303 (~~([~~l,,-[~~~l,,)~~~g([~~I[~~~l,J[~~lAnalysis of the Deduced AA Sequence ,,[PAI]) where [PA],, and [PAI],, are the initial concentration of t-PA and PAI-I, and [PA] and [PAI] The rabbit PAI-I cDNA encodes a 402-AA protein are the concentration of t-PA and PAI- at time t.“4 containing four potential N-linked glycosylation acceptor sites. one Cys residue and a conserved Rs6’). M370 reactive centre (Fig. I). The mature NHL-terMiscellaneous mini (following cleavage of the hydrophobic signal The specific activity of 2c-t-PA (American Diagpeptide) of the bovine, human and rat PAI-I have nostica, Inc., Lot 1269425, 74.5 IU/pmole) was been identified by AA sequencing of purified native determined by site-specific titration using 4protein.“‘b42 Based on a comparison with these PAI-I methylumbelliferyl-p-guanidinobenzoate3’ and analspecies, we have predicted that the rabbit PAI-I signal peptide will be cleaved similarly, resulting in a ysis with software written by Graphpad, San Diego, 379-AA mature protein having an NHz-terminal Ser CA, USA. The specific activity of SC-t-PA (GenenLot L9114A, 51.4 IU/pmole) was residue (Fig. 2). There is a high degree of AA tech, Inc., sequence homology between the previously characdetermined by titration with known amounts of Hand R-rPAI-1, which had been previously titrated terised PAI-Is (402-AA form) and rabbit PAI-1. against 2c-t-PA. Protein concentrations of 2c-t-PA Rabbit PAI-I shares the greatest AA sequence and SC-t-PA were determined by AA analysis. Amino identity with human PAI(85% identity, 91% similarity), followed by bovine PAI-I (83% identity, terminal sequencing of R-rPAI-1 was determined on an Applied Biosystems Model 470A sequenator 92% similarity), rat PAI-I (78% identity. 87% similarity) and mouse PAI-I (75% identity, 86% simifollowing deacylation of the NHZ-terminus with larity). trifluoroacetic acid (TFA).‘” ‘251-t-PA (18000 cpm/ng) was prepared by the method of Husain et al .37 Expression in S. cerevisiae

RESULTS Isolation and Sequence Analysis of the Rabbit PAIcDNA The 0.7-kbp PCR-generated rabbit PAIfragment which corresponds to bases 637-1337 of the rabbit PAIcDNA (Fig. I), hybridised to a 3-kbp transcript when used as a probe on a northern blot of SIRC cell mRNA (results not shown). Approximately 100000 recombinant clones from the lambda ZAP BrM cDNA library made from the SIRC mRNA were screened in duplicate using the PCR-generated rabbit PAI-I probe. Eight positive plaques were identified through three rounds of plaque purification and screening. Following the conversion to insert-conphagemids, the sizes of the taining pBluescriptTM insert DNAs were determined by restriction endonuclease analysis. Four clones were shown to contain the largest inserts of approximately 3-kbp (the fulllength size based on the mRNA), and the complete DNA sequence was determined (Fig. 1). The 2991-bp cDNA consists of a 131-bp 5’ untranslated sequence, a 1206-bp ORF beginning at the first in-frame ATG at

Based upon our previous success in expressing high levels of active H-rPAI-1 internally in S. cerevisi~c,~’ we pursued the same strategy for the expression of active R-rPAI-1. The cDNA was restructured free of the 23-AA putative signal peptide encoding segment, and an ATG start codon was placed adjacent to the TCA codon for the Ser residue predicted to be at the mature NH2-terminus of rabbit PAI-I (Fig. 2). A pADH2-PA1 expression vector containing the yeast ADH2 promoter fused to the coding region of the proposed mature form of rabbit PAI-I (Fig. 3) was used to transform the protease-deficient yeast host strain BJ1995. Transformants were grown in complex medium containing glucose as the sole carbon source. The ADH2 promoter is derepressed when the glucose in the culture media becomes depleted. Immunoblot analysis of cell lysates prepared from BJ1995/pADH2-PAI cells revealed a protein with an apparent M, of 40000 (data not shown). The cell lysates also contained PAI-I activity as judged by measuring inhibition of t-PA activity in vitro. The BJ1995/pADH2-PA1 isolate that produced the highest level of R-rPAI-1 was grown in shake flasks (6-l total volume) to obtain a sufficient quantity of R-rPAI-1 for purification and characterisation.

268

Purification

and Characterisation

of Recombinant

Rabbit

PAI-

97K

w

66K

>

that R-rPAI-1 contains a blocked NH2-terminus. Following treatment of R-rPAI-1 with TFA,“6 a procedure which deacylates acylated Ser and Thr residues, the sequence Ser-Tyr-Pro-X-Gln-Pro-GlnVal-Ala-Glu was obtained. The sequence corresponds to the one beginning with AA 24 in the AA sequence predicted from the cDNA (Fig. 1).

45K

w

Inhibitory

31Kw

,

Fig. 4 Analysis of purified R-rPAI-1 by SDS-PAGE. Lane 1: R-rPAI-1, Coomassie staining. Lane 2: R-rPAI-1, immunoblotting. Lane 3: H-rPAI-1, silver staining. Lane 4: R-rPAI-1, silver staining. Lane 5: HT1080 PAI-1, silver staining. Protein molecular weight markers in kilodaltons are indicated.

Purification

and Characterisation

Activity of R-rPAI-1

The inhibitory activity of R-rPAI-1 was determined by adding increasing amounts of the inhibitor to a constant amount of t-PA and subsequently measuring the residual t-PA activity in a coupled amidolytic assay. A representative experiment depicting the inhibition of 2c-t-PA with non-activated and activated R-rPAI-1 is shown in Figure 5. Non-activated R-rPAI-l:t-PA molar ratios 31OOO:l were required for any inhibition to occur. Similar results were obtained with non-activated R-rPAI-1 and SC-t-PA. However, following activation of R-rPAI-1 with guanidine HCI, substantial inhibition was observed at a R-rPAI-l:t-PA molar ratio of 1:l and inhibition was essentially complete at a molar ratio of 4: 1. Extrapolation of the inhibition curve from linear plots to zero t-PA activity in this and other experiments (n=7)

of R-rPAI-1

The yeast-expressed R-rPAI-1 was purified by successive chromatographic steps on Q-Sepharose Fast Flow, S-Sepharose Fast Flow, and Sephacryl S-100. Since the lysate rapidly lost PAI- activity after preparation, the purification was monitored by immunoblotting using a primary antibody directed against human PAI-1. Amino acid analysis indicated that 82mg of purified R-rPAI-1 was obtained from 170g of cell paste. Purified R-rPAI-1 was examined by SDS-PAGE followed by Coomassie (Fig. 4, lane 1) and silver staining (Fig. 4, lane 4). The protein is >99% pure as judged by stained gels and AA analysis and has an apparent M, of 39 100. The M, obtained by SDS-PAGE is comparable with the theoretical molecular weight of 43 000 for non-glycosylated, mature rabbit PAI- based on the predicted AA sequence (Fig. 1). R-rPAI-1 migrated slightly faster than H-rPAI-1 (Fig. 4, lane 3), a non-glycosylated form of human PAI-1, and is clearly distinguishable from HT-1080-PAI- (Fig. 4, lane 5), a fully glycosylated form of human PAI-1. Since non-glycosylated H-rPAI-1 has a theoretical molecular weight of 42900, the slightly faster mobility of R-rPAI-1 could be due to the differences in AA composition. Analysis of R-rPAI-1 by immunoblotting (Fig. 4, lane 2) revealed a single band with a M, of 40000. Thus, R-rPAI-1 is immunologically related to human PAI-1. The initial NHz-terminal sequence analysis of R-rPAI-1 generated negative results which suggested

,LL, _^ 0

“2

1

10

100

1000

10000

PAIL1 (Molar Eqwalents)

Fig. 5 Inhibition

of 2c-t-PA by activated and latent R-rPAI-1. 2c-t-PA (0.2IU) was incubated with increasing concentrations of activated and latent R-rPAI-1 for 1 h at 37°C. Immediately afterwards CNBr fibrinogen fragments, plasminogen, and the chromogenic substrate, S-2251 were added as described (Materials and Methods) and incubated for 1 h at 37°C. The increase in A405 was measured and the percent of active 2c-t-PA remaining at each concentration was calculated from a standard curve of t-PA alone. Activity of R-rPAI-1 was calculated by extrapolating the linear portion of the curve from linear plots to the x-axis and from this data computing the number of molar equivalents of PAI- that are required to inhibit the t-PA present. 0, latent R-rPAI-1; 0, activated R-rPAI-1.

Fibrinolysis

_

123

Table Association rate constants with SC-t-PA and k-t-PA

of rabbit

and human

269

rPAI-I

k.,,wc (x10’ Mu Is-‘) SC-t-PA R-rPAI-1 H-rPAI-

I

0.75+0.04 0.71+0.03

2c-t-PA (n=3) (n=4)

3.3fJ+o.34 3.40+0.55

(n=3) (n=4)

97 K 66 K 45 K

31 K

employed, >75% inhibition occurred by 20 min. The data from this and other experiments involving R-rPAI-1 and 2c-t-PA or SC-t-PA were replotted as indicated in the inset and the second order rate association constants were determined (Table). The k,,,,, for R-rPAI-1 with 2c-t-PA and SC-t-PA equalled 3.36+0.34x lO’M_’ s-’ and 0.7SkO.04x lO’M_’ s-‘, respectively. These values are nearly identical to those obtained for H-rPAI-1 with 2c-t-PA and SC-t-PA (Table).

DISCUSSION Fig. 6 R-rPAI-I forms SDS-stable, high molecular weight complexes with t-PA. “‘I-t-PA (1.25 ng) was incubated with buffer only (lane l), latent R-rPAI-1 (3.9Sng. lane 2) or activated R-rPAI-1 (3.95ng. lane 3) in 40~1 of TNT buffer for I h at 37°C. Samples were denatured and examined by 0. I “h, SDS-12.S’X PAGE and autoradiographv.

indicates that 3.67+0.05 moles of activated R-rPAI-1 were required to inhibit one mole of 2c-t-PA. Since PAI- inhibits t-PA by forming 1:l molar complexes, these data indicate that the PAIis approximately 27% active following guanidine HCI treatment. Nearly identical results were obtained when activated R-rPAI-1 was titrated against SC-t-PA (complete inhibition at R-rPAI-l:t-PA molar ratio of 4.08:1, n=4). Thus, R-rPAI-1 isolated from the yeast cell lysates is in a latent inactive form (<0.05% active) that can be activated by treatment with guanidine HCl. To explore the mechanism underlying the t-PA inhibitory effects of R-rPAI-1, ‘*“I-t-PA was incubated with activated and latent inhibitor and the mixtures analysed by SDS-PAGE and autoradiography (Fig. 6). ‘*“I-t-PA only (lane 1) or after exposure to latent R-rPAI-1 (lane 2) migrated at its predicted M, of 68000. However, following incubation with activated R-rPAI-3, the ‘2sI-t-PA migrated at a M, of 114000 (lane 3). These data suggest that activated, but not latent, R-rPAI-1 inhibits t-PA by formation of 1: 1 molar, SDS-stable complexes.

Determination

of Rate Constants

An experiment depicting the time dependency for the inhibition of 2c-t-PA by R-rPAI-1 is shown in Figure 7. At the concentrations of R-rPAI-1 and 2c-t-PA

We have Isolated and characterised a cDNA encoding the rabbit PAIprotein. There is considerable DNA homology between the protein-coding regions of rabbit PAI- and the PAI- from four other animal species (rat, bovine, human and mouse) that have been cloned to date (data not shown). Previous comparisons of the mouse and human PAI- cDNA5” and the rat and human PAI- cDNAs,~” identified an unexpected span of approximately 100 bases within

,

Fig. 7 Time dependency for R-rPAI-1 inhibition of 2c-t-PA. Zc-t-PA (SOpM) was incubated with R-rPAI-1 (289pM) for increasing times before the reaction was stopped by the addition of des-AA-fibrinogen, plasminogen, and the chromogenic substrate. S-2251 as described (Materials and Methods). Results were plotted as the percentage of t-PA activity remaining versus time. Inset. Second order rate constants were calculated from the slope of the plot as described in Materials and Methods.

270

Purification

and Characterisation

of Recombinant

Rabbit

PAI-

GagGaCagAGtGGTtTCaAAttTtTcCA-a----Tt-aGaag c-gagtggg-aAgGGGgCTgtgtg[ACCTA-CAGGACAGAACtT TCcCCAATTACaGGGTGA--ctcacagC-gcacTGGTGACTCAc TTCAATGTGTCAtTTCCGGCTGCTGTgtGtGAGCaG-tGGACa CGTG]g-ggggcgg---ggggG-gGgaTGAaAGagaCaGccAGC TCggg-tCAAccACCT... Fig. 8 DNA sequence conservation within the PAI- 3’. untranslated region. A best-fit. sequence alignment among the 3’-untranslated regions of the human,” bovine.” rat,“” mouse”’ and rabbit PAI-I cDNAs revealed this area of high sequence similarity. The bold-face, capital letters represent bases that are conserved between all five species. Small-case letters are bases most often present at the site and the dashes are sites of little homology. The I lO-hp tract (designated by brackets) exhibits 75% homology between the PAI-1s of the five animal species and corresponds to the rabbit PAI- cDNA beginning at base 2493 (Fig. 1).

the 3’-untranslated region which exhibits >90% homology between the two species analysed. A sequence alignment of the 3’-untranslated regions of the rat, mouse, bovine, human and rabbit PAIcDNAs revealed that this tract is highly conserved among all five species (Fig. 8). The conserved region corresponds to bases 2429-2653 of the rabbit PAIcDNA. Within this region is a 1 lO-bp tract (beginning at base 2493) that exhibits 75% homology among all five species. This high degree of sequence similarity strongly implies that this region may be important for the regulation of PAIgene expression. A comparison of the predicted AA sequences of the rat, mouse, bovine, human and rabbit PAIreveals that the rabbit PAI- shares the greatest AA sequence identity (85%) with human PAIand that an overall 66% sequence identity exists among the PAI- AA sequences of all five species of animals. As seen in Figure 9, there are regions of PAIAA sequence that are 100% conserved among all five species of animals. Most notable are the three N-linked glycosylation sites located at AA 232, 288 and 352 and the conserved Pl, Pl’ reactive centre, R3hc1,Mx,,,. The greatest sequence divergence occurs within the first 37-AA of the PAIproteins. As region may previously suggested,40 this NHz-terminal be relatively nonessential to the t-PA inhibitory function of PAI-1. R-rPAI-1 was expressed in S. cerevisiae as an intracellular, non-glycosylated protein using the pCl/ I-ADH2p-ADHlt-1 expression vector. This system was employed since the intracellular expression of H-rPAI-1 in S. cerevisiae resulted in the accumulation of ample quantities of non-glycosylated inhibitor which exhibited functional in vivo properties that were very similar to the native, glycosylated human PAI- .*’ Earlier attempts to secrete H-rPAI-1 from

yeast had given lower yields than intracellular expression.4” In addition, H-rPAI-1 and native PAIpossess similar pharmacokinetic behaviour in the non-glycosylated, recomrabbits. 44 Therefore, binant and fully-glycosylated, native forms of human PAI- appear to be interchangeable in in vitro and in vivo studies. Since the human and rabbit PAI-1s share a 85% identity at the AA level, we predict that, like H-rPAI-1, the R-rPAI-1 would be functionally indistinguishable from its native form. The yeast-expressed R-rPAI-1 was purified to >99% homogeneity and exists primarily in a latent, inactive form. Treatment with guanidine HCI resulted in a >lOO-fold activation of R-rPAI-1, which is comparable with the activation of latent H-rPAIl*” and native human PAI-1.4’.4” Activated R-rPAI-1 exhibits efficient inhibition of both 2c-t-PA and SC-t-PA and forms SDS-stable complexes with t-PA as expected for a member of the SERPIN class of protease inhibitors. The second-order association rate constants of R-rPAI-1 and H-rPAI-1 with 2c-t-PA and SC-t-PA are nearly identical to the values obtained with H-rPAI-1 and those enzymes and are comparable to the rate constant observed for purified, native. human PAI- .2’),47Together, these data suggest that there are no obvious functional differences between the human and rabbit forms of PAI-1. In vivo animal studies may expose variations in the pharmacological properties of the human versus rabbit inhibitor and these studies are in progress.

t

1

MqMSpalaCLaLGl-Lvfg-Gsas-Ip-sh-AhlAtdFGV

41

kVFQqVvqASKDRNWFSPYGVaSVLAMLQ1TTaGeTrqQ

81

IQ-AMgFkidekgtApAlrhL-KELMGpWNKdEISTaDAI

12i

FVQrDLeLVqGFMPhFFrLFrttVKQVDFSeVeP.ARFIiN

161

DWVerETKC;MIsdLLgkGAvdqLTRLvLVNALYFnGQWKt

201

PF-essTH-RLFHKSDGSTiSVPMMAQt

24;

GhyYDilELPYhg-TLSMfIAAPyEK-VpLSAlTnILdAe

281

LIsqWKgNMTRlpP.LLvLPKSLEtEvDLRgPLEnLGMtD -

321

mFrptqADFtslSDQEqL-VaqALQISVkIEVNESGTvASS -

361

STai-vSA&APeEiimDRpFLF

401

EP

NKFNYTEFtTPD -

VVRRNPTgTiLFmGQvM

Fig. 9 Amino acid conservation among PAI- proteins. This consensus sequence was derived by an AA alignment between the 402.AA PAI-Is from human.” bovine,” rat,“” mouse’” and rabbit. The hold-face, capital letters, the small-case letters and the dashes represent conserved AA, AA present most often at that position, and positions of little homology, respectively. The arrow denotes the proposed signal peptide cleavage site. Conserved N-glycosylation sites are underlined and the reactive site is indicated by an asterisk.

Fibrinolysis

ACKNOWLEDGEMENTS WC would like to thank M. Neeper for advice on cDNA cloning, M. Sardana for performing AA sequence analysis, R. Ellis for critical reading of the manuscript and K. Short and P. Burke for synthesis of oligonucleotides.

LIST OF ABBREVIATIONS PA,: plasminogen activators t-PA: tissue-type plasminogen actlvatol u-PA: urokinase PAI-I: plasminogen activator inhibitor-l SERPIN: serine protease inhibitor SDS: sodium dodecyl sulphatc I-I-#AI-l: human recombinant PAI-I R-rPAI-I: rabbit recombinant PAI-I PCR: polymerase chain reaction khp: kilobase pair AA: amine acid(s) ORF: open reading frame bp: base pair(s) S. : Sacchuromyces IU: international unit sc-t-PA: single-chain t-PA klU: IOOO-IU PAGE: polyacrylamidc gel elcctrophoresis PEG: polyethylene glycol 6000 rPAI-I: recombinant PAI-I Zc-t-PA: two-chain t-PA Au: arbitrary unit TFA: trifluoroacetic acid

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Received: 21 October 1991 Accepted after revision: 31 December 1991 Offprint orders to: K. J. Hofmann, Department of Cellular and Molecular Biology, Merck, Research Labs, West Point, Pennsylvania 19486, USA. Tel: (215) 661-6611, Fax: (215) 661-7320.

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