The Structure and Function of Mouse Thrombomodulin

Vol. 263, No. 30, Issue of October 25, PP. 15815-15S22,1988 Printed in U.S.A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

The Structure and Function of Mouse Thrombomodulin PHORBOLMYRISTATE ACETATE STIMULATES DEGRADATION AND SYNTHESIS OF THROMBOMODULIN WITHOUT AFFECTING mRNA LEVELS IN HEMANGIOMA CELLS* (Received for publication, May 3, 1988)

William A. Dittman, Toshihiko Kumada,J. Evan Sadlert, and Philip W. Majeruss From the Division of the HematologylOncology, Departments of Internal Medicine and Biological Chemistry, Washington University School of Medicine and the $.Howard Hughes Medical Institute, St. Louis,Missouri 63110

Thrombomodulin is an endothelial membrane anti- inhibited by protein C but not by activated protein C (7). coagulant protein that is a cofactor for proteinC acti- Thrombomodulin is an important antithrombotic component vation. We have evaluated the expression of thrombo- of endothelium. We have shown that injection of anti-thrommodulin in cultured mouse hemangioma cells before bomodulin antibodies in mice decreases the expression of and after treatment with phorbol myristate acetate endothelial thrombomodulin cofactor activity andthereby C. We potentiates the toxicity of injected thrombin (8).Increases or (PMA), an agent that stimulates protein kinase alsoisolated a cDNA encoding 481 aminoacids of decreases in endothelial cell surface thrombomodulin might mouse thrombomodulin and the entire 3“untranslated alter the occurrence of vascular thrombosis; therefore, we portion of its mRNA. The deduced amino acid sequencehave studied the regulation of thrombomodulin expression in of mouse thrombomodulin is similar to those deter- cultured endothelial cells. Wehave used a mouse hemangioma mined for human and bovine thrombomodulin. An S1 nuclease protection assay was used to measure throm- cell line (9) for this purpose since itis stable, expresses bomodulin mRNA in hemangioma cells. The half-life thrombomodulin in large amounts, and retains properties of for thrombomodulin mRNA was 8.9 f 1.8 h (S.D.) in normal endothelium. We prepared mouse thrombomodulin cells treated withactinomycin D. Treatment withPMA cDNA in order to characterize thrombomodulin transcription had noeffectonthrombomodulin mRNA levels. in these cells. We also measured the rates of synthesis and Thrombomodulin turnover was evaluated by immuno- degradation of thrombomodulin and have studied the mechprecipitation of [36S]methionine-labeled thrombomod- anism by which phorbol myristate acetate (PMA)’ decreases ulin. The ts was 19.8 f 3.9 h (S.D.); PMA treatment cell surface thrombomodulin. decreased the tu to 10.9 f 1.1h (S.D.) while increasing EXPERIMENTALPROCEDURES the rateof synthesis toa maximum of 190%of control. Protein C cofactor activity on hemangioma cells was Materials-Radioisotopes were obtained from Amersham Corp., reduced 35 f 4% by treatment with PMA within 30 except [cY-~’P]~CTP whichwas from ICN Radiochemicals (Irvine, min. This decrease was associated with a parallel de- CA) and [32P]orthophosphatewhich was from Du Pont-New England cline in cell surface thrombomodulin antigen and withNuclear. Restriction endonucleases, calf intestine alkaline phosphaenhanced phosphorylation of thrombomodulin on tase, T4 polynucleotide kinase, single strand binding protein, and T7 serine residues. We conclude that thrombomodulin is DNA polymerase were from United States Biochemical Corporation phosphorylated in response to treatment of heman- (Cleveland, OH). Maloney murine leukemia virus reverse transcriptase, S1 nuclease, RNase H, and DNA and RNA molecular weight gioma cellswith PMA which leads to decreased protein markers were from Bethesda Research Laboratories, EcoRI methylase C cofactor activity andboth increased degradtion and and T4DNA ligase were from New England BioLabs (Beverly, MA), synthesis of thrombomodulin. oligo(dT)12-18,individual nucleotides, EcoRI linkers, and oligo(dT)

Thrombomodulin is an endothelial cell membrane protein that is a cofactor for thrombin-catalyzed activationof protein C (1,2). Activated protein C is an anticoagulant that inactivates coagulation factors Va and VIIIa (3-5). Thrombomodulin on the cell surface also removes thrombin from the circulation by binding it; thrombinbound to thrombomodulin stimulates endocytosis and degradation of the thrombinthrombomodulin complex in lysosomes (6). Endocytosis is

* This research was supported by Grants HLBI 14147 (Specialized Center for Research in Thrombosis), HLBI 16634 (to P. W. M.), Training Grant T32 HLBI 07088 from the National Institutes of Health, and by the Monsanto-Washington University Biomedical Research Grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s)reported in this paper has been submitted to the GenBankTM/EMBL Data Bank withaccessionnumber(s) 504060. §To whom reprint requests should be addressed.

cellulose were from Pharmacia LKB Biotechnology Inc. X packaging extracts and Bluescript plasmids were from Stratagene Cloning Systems (La Jolla, CA). Actinomycin D and PMA were from Sigma. Other chemical reagents were from Sigma or Fluka Chemical Corporation (Ronkonkoma, NY). Human thrombin (lo), human protein C (4), andpolyclonal anti-rat thrombomodulin IgG (8) were isolated as described previously. Modified Medium 199 (“199; M199 with basal medium Eagle’s vitamins and amino acids and Earle’s salts), “199 without phosphate or without methionine, and Dulbecco’s phosphate-buffered saline were from the Washington University Medical School Center for Basic Cancer Research. Tissue culture flasks, wells, and dishes were from Becton Dickinson Labware (Lincoln Park, NJ) or Corning Glass Works (Corning, NY). Preparation of Mouse Lung cDNA Library and Isolation of Mouse Thrombomodulin cDNA Clones-RNA was isolated from 10 g of fresh mouse lungs by repeated ethanol precipitation of a guanidine hydrochloride extract as described by MacDonald et al. (11).Polyadenylated RNA was selected on oligo(dT) cellulose (12). First strandcDNA was synthesized by Maloney murine leukemia virus reverse transcriptase treatment of oligo(dT) primed RNA, and the second strand was produced by Escherichia coli DNA polymerase I synthesis on RNase The abbreviations used are: PMA, 4@-phorboll2@-myristate1301acetate; “199, modified medium 199; SDS, sodium dodecyl sulfate; EGF, epidermal growth factor.

15815

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The Structure and Function of Mouse Thrombomodulin

H-treated heteroduplexes (13). After methylation, addition of EcoRI linkers and size selection in low melting temperature agarose (3.04.5 kilobase pairs) double-stranded cDNA wasligated into Xgt.10 (14). The X g t l O library was screened (14) by colony hybridization with random hexadeoxyribonucleotideprimed 32P-labeledprobes (15) generated from a human thrombomodulin cDNA, HTMlO (16). Positive clones pMTM.lb1 and pMTM.2b2 were subcloned into Bluescript plasmids and were sequenced by the dideoxy chaintermination method (17) with [ Q - ~ ' S ] ~ A Tand P the T7 DNA polymerase system (181, using double-stranded plasmid as templates and synthetic oligonucleotide primers (16). Areas of compression on gradient, denaturing sequencing gels wereresolved with a combination of dITP and single strand binding protein, used according to the manufacturer's instructions. S l Nuclease and RNAAnalysis-A 187-base fragment including the 128 5' bases of mouse thrombomodulin cDNA and 59 bases from the plasmid was prepared by TaqI and Sac1 restriction endonuclease digestion of pMTM.2b2. The probe was labeled on the 5' end of the TaqI site with [y-32P]ATP and polynucleotide kinase and purified by electrophoresis for use as an S1 probe as described by Favaloro et al. (19). A probe generated by EcoRI and Hind111 digestion of a plasmid containing sequence for murine Pz-microglobulin was labeled at the EcoRI site as described above and was used as control for total amounts of RNA (20, 21). RNA was harvested from cells grown to confluence in 100-mm plates by the method of Chomczynski and Sacchi (23) and measured by absorbance at 260 nm. The mouse thrombomodulin probe was hybridized with RNA samples overnight at 55 "C, whereas the pp-microglobulinprobe was hybridized at 50 "C. Fifty or 100 units of S1 nuclease were incubated with the hybrids for 30 min a t 37 "C, and the reaction products were analyzed on a gel containing 6% acrylamide and 8.3 M urea. After autoradiography, radioactive bands were cut from the gel, and radioactivity was measured by scintillation counting. Northern blot analysis was performed as described by Lehrach et al. (22). Total RNA was electrophoresed in 1%agarose in the presence of 2.2 M formaldehyde, transferred to nitrocellulose, hybridized with 32P-labeled probes prepared from cDNAs as described above, and washed with 300 mM sodium chloride, 30 mM sodium citrate (pH 7.0), 0.1% sodium dodecyl sulfate (SDS) at 25 "C for 30 min, and then in 15 mM sodium chloride, 1.5 mM sodium citrate, 0.5% SDS at50 "Cfor 30 min, and autoradiographed. Metabolic Labeling-The rate of degradation of thrombomodulin was determined as follows. Hemangioma cells in 35-mm dishes (approximately 4.5 X lo5 cells/dish) were incubated for 48 h in methionine-free "199, 20% fetal calf serum, with [35S]methionineat 400 pCi/ld ml/dish. Cells were washed twice with Dulbecco's phosphatebuffered saline and incubated for the indicated times with complete medium containing PMA (200 ng/ml) or dimethyl sulfoxide (0.01%). PMA was dissolved in dimethyl sulfoxide at a concentrationof 2 mg/ ml and stored at -20 "C. Maximal effects on synthesis and degradation of thrombomodulin were found at PMA concentrations of 100 ng/ml. Thrombomodulin synthesis was measured after cells were incubated for 0-12 h with complete medium containing PMA (200 ng/ ml) or dimethyl sulfoxide (0.01%), washed, and then labeled for 4 h in methionine-free "199 containing [35S]methionineat 200 pCi/ 1.5 ml/dish and PMA or dimethyl sulfoxide. Thrombomodulin was labeled with [32P]orthophosphateat 2 mCi/ 2 ml/dish in 60-mm dishes (approximately 1.4 X lo6 cells/dish) for 2 or 18 h in phosphate-free "199 supplemented with 10% fetal calf serum and 10% dialyzed fetal calf serum. Labeled cells were treated with PMA (200 ng/ml) or dimethyl sulfoxide (0.01%) for 10 min a t 37 "Cand then the medium was removed.The cell layers were washed with cold 10 mM Tris-HC1 (pH 7.2) 150 mM sodium chloride, 1 mM EDTA, and then solubilized in cold 10 mM sodium phosphate (pH 7.2) 150 mM sodium chloride, 1%Triton X-100, 1%sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 23 pg/ml aprotinin (solubilization buffer) with 2.9 mM diisopropylfluorophosphateadded. After centrifugation at 30,000 X g for 30 min at 4 "C, the supernatant fraction of the cell extracts and themedium were used for immunoprecipitation. Trichloroacetic acid-precipitable radioactivity was measured in portions of the samples. Thrombomodulin Immunoprecipitation-Immunoprecipitation was performed by a modification of the method of Kessler (24). 35SLabeled cell extracts and medium were treated twice with 150 pl of Pansorbin (10% in solubilization buffer containing 1 mg ovalbumin/ ml (Calbiochem, Behring Diagnostics, La Jolla, CA)) followed by treatment with 40 pg of pre-immune IgG and 150 p1 of Pansorbin. The supernatantfractions were then incubated at 4 "Cfor 16 h with

40 pg of anti-rat thrombomodulin IgG. The immune complexes were harvested with 30 pl of Pansorbin or 40 pl of protein A-Sepharose (25%, Sigma) and sequentially washed five times with solubilization buffer, two times with 20 mM Tris-HC1 (pH 7.4) 150 mM sodium chloride, 2 M urea, 100 mM glycine, and two times each with 50 mM Tris-HC1 (pH 8.0) 500 mM sodium chloride, and with water. The proteins were dissolved in 40 p1 of Laemmli's gel sample buffer (25) by incubation for 5 min in boiling water and electrophoresed through a 7.5% polyacrylamide SDS gel (25). Measurement of %-labeled thrombomodulin was performed by fluorography (26) with EN3HANCE solution (Du Pont-New England Nuclear) and by densitometry of autoradiograms using a softlaser scanning densitometer (Zeineh, Biomed Instruments, Chicago, IL). Immunoprecipitation of 32P-labeledthrombomodulin was performed as above except that all the solubilization and washing buffers contained 200 p~ sodium vanadate and 10 mM sodium fluoride to inhibit dephosphorylation reactions. Liquid scintillation countingwas performed in a Beckman LS 6800 using ScintiVerse I (Fisher). Assay of Thrombomodulin CofactorActivity-Assayof thrombomodulin cofactor activity was performed on monolayers of hemangioma cells (1-2 X lo4 cells/well) in 96-well tissue culture plates. The cells were washed three times with "199 and then treated with 40 pl of"199 containing PMA (100 ng/ml) or dimethyl sulfoxide (0.005%) for the indicated times at 37 "C. In some experiments, the medium was removedafter a 30 min incubation and complete medium or "199 was added back. After incubation, the treated cells were washed three times with 150 mM NaCl, 20 mM Tris-HC1 (pH 7.4) 1 mM CaClZ,5 Tg/ml bovine serum albumin, and then incubated in 40 pl of the same buffer with 4 units/ml thrombin, and 0.5 PM protein C for 15 or 30 min a t 37 "C. Activated protein C generated in 30 pl of the reaction mixture was determined by measuring the cleavage of the chromogenic substrate S-2238 (Kabi Vitrum, Stockholm, Sweden) in the presence of antithrombin I11 and hirudin as described previously (27). Control inct bations in the absence of cells were performed. Cell number was measured in an automated cell counter (Coulter Electronics, Hialeah, FL). Binding of Anti-rat Thrombomodulin IgG to Hemangioma CellsAffinity purified polyclonal anti-rat thrombomodulin IgG (8) was isolated on a human thrombomodulin Affigel-15 column as described previously for affinity-purified anti-human thrombomodulin IgG (16) and radiolabeled with carrier-free NalZ6Iusing Bolton-Hunter reagent (28) to a specific activity of 20-4500 cpm/ng protein. Labeled antibody was used a t 100 nM to measure hemangioma cell surface thrombomodulin as described previously (6, 7). Nonspecific binding was determined in the presence of 10 p~ unlabeled antibody. Protein concentrations were determined by a dye binding assay with bovine serum albumin as the standard(Bio-Rad). Phosphoamino Acid Analysis-Phosphoamino acid analysis was performed on thrombomodulin isolated from [32P]orthophosphatelabeled cells by immunoprecipitation followed by polyacrylmide gel electrophoresis. After autoradiography, the excised gel slices corresponding to the 32P-labeledthrombomodulin were hydrated in 2 ml of 50 mM ammonium bicarbonate, 100 pg/ml L-1-tosylamido-2-phenylethylchloromethyl ketone trypsin (Cooper Biomedical, Malvern, PA), 1.5 mM dithiothreitol. After incubation for 24 h a t 37 "C, the eluted tryptic peptides were dried in a Speed-Vac (Savant Instruments, Hicksville, NY) and hydrolyzed in6 N hydrochloric acid (Pierce Chemical Co.) for 1 h at 110 "C. Samples were dried and dissolved in 10 ml of water, and phosphoamino acids were isolated on Dowex AG 1-X8 as described by Cooper et al. (29). The eluted samples were electrophoresed for 1.5 h a t 700 V a t 4 "C on a 20 X 20cm thin layer cellulose plate (Eastman Kodak) at pH 3.5 in pyridine:acetic acidwater (1:10189). Phosphoamino acid standards were localized by ninhydrin, and radioactivity was detected by autoradiography with EN3HANCE spray and intensifying screens (Du Pont Cronex Lightning Plus, E.I. du Pont de Nemours & Co.) and X-Omat film (Eastman Kodak). RESULTS

Mouse lung was chosen as the source for RNA isolation because of its high thrombomodulin levels. A size-selected cDNA library was prepared in XgtlO and screened with a human thrombomodulin cDNA probe. Two clones were isolated from 2 X IO4 recombinant clones screened. The longer of these, pMTM.2b2,was sequenced on both strands and contains 3212 nucleotides (Fig. 1). An open reading frame of

15817


thrombomodulin and 86% of the nucleotide sequence including all of the 3”untranslated region by comparison with the human cDNA. The mouse and human proteins are similar with 68% identity of amino acids. The mouse cDNA is also

1443 nucleotides, encoding 481 amino acids, is followed by a 3”untranslated region of 1745 nucleotides and a poly(A) tail. Apotential polyadenylation signal (AATAAA)begins 23 bases 5’ to the poly(A) tail. The cDNA encodes 83% of mouse 1

CCGGTGCATCTCGGGCCCCTGCGCGGCTTCCAGTGGGTTACTGGCGATAACCACACCAGTTACAGCAGGTGGGCGCGGCCCAACGACCAGACGGCTCCAC P V H L G P L R G F Q W V T G D N H T S Y S R W A R P N D Q T A P L

100

101

TCTGCGGCCCTCTGTGCGTCACGGTCTCGACAGCAACTGAAGCTGCACCCGGCGAGCCGGCCTGGGAAGAGAAGCCATGCGAGACTGAGACCCAGGGTTT C G P L C V T V S T A T E A A P G E P A W E E K P C E T E T Q G F

200

201

CCTCTGTGAGTTTTACTTCACAGCTTCCTGCAGGCCTCTGACGGTGAATACTCGCGATCCTGAGGCTGCCCACATCTCTAGTACCTACAACACCCCGTTC L C E F Y F T A S C R P L T V N T R D P E A A H I S S T Y N T P F

300

301

GGGGTCAGTGGTGCGGACTTTCAAACGCTGCCGGTAGGCAGTTCCGCCGCGGTGGAGCCCCTTGGCTTGGAGCTGGTGTGCAGGGCCCCGCCCGGAACTT G V S G A D F Q T L P V G S S A A V E P L G L E L V C R A P P G T S

400

401

CAGAGGGACACTGGGCTTGGGAAGCGACAGGAGCCTGGAATTGCAGCGTGGAGATGGTGGCTGTGAGTACTTGTGCAATAGGAGCACGAATGAACCCAG E G H W A W E A T G A W N C S V E N G G C E Y L C N R S T N E P R

500

501

ATGCCTCTGCCCCAGAGACATGGACCTGCAGGCCGATGGACGTTCGTGTGCAAGACCTGTGGTTCAATCGTGCAACGAACTCTGCGAGCATTTTTGTGTC C L C P R D M D L Q A D G R S C A R P V V Q S C N E L C E H F C V

600

601

AGCAACGCTGAAGTGCCAGGCTCTTACTCCTGTATGTGTGAGACAGGCTACCAGTTGGCTGCAGACGGACACCGGTGTGAGGACGTGGATGACTGTAAGC S N A E V P G S Y S C M C E T G Y Q L A A D G H R C E D V D D C K Q

700

701

AGGGGCCCAATCCATGTCCCCAGCTCTGTGTTAACACCAAGGGCGGCTTCGAATGCTTCTGCTATGATGGCTATGAGTTGGTGGATGGAGAGTGCGTGGA

800

G

P

N

.

P

C

P

Q

L

C

V

N

T

K

G

G

F

E

C

F

C

Y

D

G

Y

E

L

V

D

G

E

C

V

E

900

801

GCTTCTGGATCCGTGTTTCGGATCTAACTGCGAGTTTCAGTGCCAGCCAGTGAGCCCCACCGACTACCGATGCATCTGCGCTCCAGGCTTCGCACCCAAG L L D P C F G S N C E F Q C Q P V S P T D Y R C I C A P G F A P K

901

CCGGATGAACCGCACAAGTGCGAAATGTTCTGCAATGACTTCGTGCCCAGCAGACTGTGACCCTAACTCTCCTACTGTTTGTGAATGCCCTGAAGGCT P D E P H K C E M F C N E T S C P A D C D P N S P T V C E C P E G F

000

1001

TCATCCTGGACGAGGGTTCCGTATGCACGGACATTGATGAGTGCAGTCAAGGCGAATGCTTCACCAGTGAATGTCGAAACTTCCCTGGCTCCTATGAGTG I L D E G S V C T D I D E C S Q G E C F T S E C R N F P G S Y E C

100

1101

TATCTGCGGGCCTGACACAGCCCTTGCTGGTCAGATTAGCAAAGACTGTGACCCCATCCCTGTTAGGGAAGACACCAAGGAAGAGGAGGGCTCTGGGGAG I C G P D T A L A G Q I S K D C D P I P V R E D T K E E E G S G E

200

1201

CCTCCAGTCAGCCCTACGCCAGGCTCTCCGACAGGTCCCCCTTCTGCAAGGCCAGTGCACTCTGGCGTGCTCATTGGCATTTCCATTGCCAGCCTGTCCC P P V S P T P G S P T G P P S A R P V H S G V L I G I S I A S L S L

300

1301

TGGTGGTGGCGCTTTTGGCGCTTCTCTGTCACCTGCGCAAGAAGCAGGGCGCTGCTCGTGCAGAGCTGGAGTACAAGTGCGCATCTTCCGCCAAGGAGGT V V A L L A L L C H L R K K Q G A A R A E L E Y K C A S S A K E V

1400

AGTGCTGCAGCACGTCAGGACTGATCGGACGCTGCAGAAGTTCTGAGGGATTTGCTCCAGAGACCCAGGTGGCCTTTGTCTTTCCGGGCTCTGTACCTCT

1500

*

1401

V

L

Q

H

V

R

T

D

R

T

*.

L

Q

K

.

* * .

F

1501

CCTCTCCTCTCTCTCTCTCCAGCCTCCCAGCTGTGTTCTCTGGCAACTT~GCACCCTGGCTGGTATAATAACCAGAGAAGAGCCCATCCTCTCAGGA

1600

1601

CAAGCGAGGGTAGGGAGGACTTGAAGCAGGACAGCCCAGTTTCTTCCAAGTAGATACTGGACAACTGGGCGGAGGTGGCAAATACAGCGGAGATCCCAGA

1700

1701

GTACCCCAGTCCCTCACCTCACCTCCTAGTGCTGCTGATCTGTAGGCTTGAAGGCARACCTTGACCCCATGGGCTGGAGATGACCCAGATATTTATTTTT

1800

1801

TTTAAAGTATTTAGTATTTTCTTCCCTCCAGTTTTCTTCTGCTTGTAAGTCTCCAGCCCCCCACAGCTTTCTCAGTCCCTCCATTCCCCCCCTCCTGTCA

1900

1901

TTCTCCTCCCCAAACCTGATCATAACTTTGCCCTTACCGTTGTTTCCARACTCTTATGTGAAACAGARAAGACACTARAAGCAG~CGTTCTTTTT

2000

2001

CACTGGCTTTGGGTATTTAGTCAGAAATTTCAGGTAACCAAAGCAAAAGAATTTTAACAAAAGCTAAAATATTTCAGCTGAACACTMCTAGTCAATAGT

2100

2101

GCTGGMTGTCACAGAAAATAAACTTAAGGAAGTAGGGTTTTTTTTTTTTTTGAAATCTTTGTTTTTGAAAGGGTGAGCCTGGGTTTTATGATTGTTGCT

2200

2201

GTTGTTTTGAATGGGAATGACAAAAGAGGTCATTATTGTTAAGATTTTTATGCAGGCTCTACAGTGTTATTAATTTTTGACAGTGTTC~TGTGCAGA

2300

2301

GGATCCTTTGTCCAACCCTTTGACATGACAATAGGACATTGCTATCTTGAGACATACTGGGCCACATTCATAGCTTTCCAAGGATGTATGTGGTCCTGCC

2400

2401

TCAACATATCAGAGCCTGACAGATGGAAGCACCTTCCAGTAAAGCATGAGTTGTGTGCTTCGTGCCGAGCTGACTCTCAACTGTGCCTGCCCCTTGTAGT

2500

2501

CCCGAAATACAAGCAATGTGCTGCTGAGGGAAACATGGAAACTTGGGAATGGAGTCTGGGGGTGCCTAGATGGGGCTTTCTTTTMTGAGACTCTTGAAC

2600

2601

AATATCTCGTAATTCAGAGGGATCTTCTAGCCCTGGCCACTGGCCTGTACACAAGAATTGGGACCTCGCTTGGGATCTGGCTAGMTTGCAAAATCCTAG

2700

2701

CCCCCACCCCTGCCCCACCCCAGTGTGCCAGTTCATMGAATCTGCATTTTGACAACATCCACAGGGACATTGTCCAGTCATTTCAGGACAACTGGTCTT

2800

2801

AAGAGTTTCCAACCTTTGTAGAACATTTAAATGTCGGTTAATAATAAGTAGCAGGCCATGTTAAGGCCATTTATTATCAAGAAACTGAGGAATTTTCTCT

2900

2901

GCATAGCTTTGCTTTCTGGATACAATAAAATGAGAAGGTACACACCTCTAGATAGTGCCACACAGAGTCCAGAAGGGTTTTGTTTTAAGTAAGCTAGGAA

3000

3001

TGAGTTCATATGTTAGTGTAAGGAACAAATGTATTATATGTGTATCTTTTGTAAAGAAAGGTTTTTCTTTACGGTTTTGTAAGCTCAGCATATTTGTACA

3100

3101

TATTTATTTATTGGAGTTTCGCTAGAACACACAAGCAAAGCCTTTGCTTATGACGTCACATGTACAAAATAAATAGATGACAGTGTACTG~

3200

3201

AAAAAAAAAAAA

3212

FIG. 1. Nucleotide sequence of pMTM.2b2 and predicted amino acid sequence of mouse thrombomodulin. The EGF-like repeats similar to those found in humanand bovine thrombomodulin are underlined. The TTATTTAT motif described in the text is boldly underlined. Potential sites of cytoplasmic phosphorylation are starred.


15818

similar to a partial bovine cDNA (30) with 67% amino acid identity. The 3”untranslated region of the cDNAs, especially the extreme 3‘ ends, are alsohighly conserved. The entire 3’untranslated region is very AT-rich, and a specific AT-rich motif (TTATTTAT) (31) observed in clones for human and bovine thrombomodulin is present in themouse. We found a single mRNA of 3800 bases by Northern blot analysis of mouse lung RNA. We investigated the turnover of thrombomodulin mRNA by inhibiting transcription with actinomycin D and determining subsequent changes in mRNA levels by S1 nuclease analysis. The tH of thrombomodulin mRNA determined from five experiments was 8.9 f 1.8 h (S.D.) (Fig. 2). By 8 h, levels were 55 f 20% of control, andby 24 h levels were 22 k 9% of control. fi2-microglobulin mRNA was also measured as control for recovery of total mRNA in the reaction mixtures. Fig. 3 is a Northern blot analysis of mRNA before actinomycin D treatment and after 3 hdemonstrating relative stability of the message for thrombomodulin. The same blotwas hybridized with a c-myc probe (32) showing rapid disappearance of cmyc mRNA after actinomycinD treatment. Treatment of hemangioma cells with PMA had no effect on thelevels of thrombomodulin mRNA determined as a ratio of thrombomodulin mRNA to fi2-microglobulin mRNA. fi2microglobulin mRNA levels were also unchanged. One of four similar experiments with PMA treatment is shown inFig. 4. There was no difference in the results whetheror not serum was present during exposure to PMA. Thrombomodulin waslabeled with [35S]methionine in pulse-chase experimentsfollowed by immunoprecipitation of protein as demonstrated inFig. 5A. The proteinwas degraded with a tlAof 19.8 f 3.9 h (S.D.) in untreated cells. Treatment with PMA resulted in a decrease in tlhto 10.9 f 1.1 h (mean of four experimentsas shown inFig. 5B). The rateof synthesis of thrombomodulin was also measured as shown in Fig. 6. Synthesis increased 1.35 to times control whenmeasured over

Probe MTm cMyc Time(Hr) 0 3 0 3

c FIG. 3. Northern blot analysis of hemangioma mRNA. Total cellular RNA wasisolated as described in Fig.2, and 10 pgwas electrophoresed through 2.2 M formaldehyde, 1% agarose, and transferred to nitrocellulose. On the right, RNA isolated before and 3 h after exposure to 10pg actinomycin D/mlwashybridizedwitha probe toc-myc. The membranewas washed and reprobed with labeled mouse thrombomodulin (MTm)cDNA shown on the[eft. The migration of 28 S and 18 S ribosomal RNA subunits is indicated on the right.

Hours 0 I 4 8 PMA + + + +

I FIG. 4. S1 nuclease analysis of PMA-treated and control hemangioma cells. Cells were grown as described in Fig. 2. At the indicated times, medium was changed to M199 containing 20% fetal bovine serum and 100 ng PMA/ml, in 0.005% dimethyl sulfoxide or dimethyl sulfoxide alone. Cells were harvested, RNA isolated, and 10 pg of total RNA was analyzed by S1 nuclease protection as in Fig. 2. Control µglobulin (82M) levels are in the upper panel (205 bases), mouse thrombomodulin (MTm)in thelower (128 bases). This experiment is representativeof four experiments.

:h E

S l 0’

E

I

0

YWY

I

TWROUBOUOWLlN 5, P l O B L

10

20

30

TIME (hr) FIG. 2. S1 nuclease protection analysis of mRNA levels. Hemangioma cells were grown to confluence in P-100 tissue culture dishes. The mediumwas changed to M199, containing 20% fetal bovine serum and 10 pg actinomycin D/ml. At the times indicated, total RNA was extracted, and 10 pg was analyzed as described under “Experimental Procedures.” Upper right insert showscontrol µglobulin (j32M) protected fragments (upper panel)and the 128nucleotide-protected mouse thrombomodulin (MTm)fragment (lower panel). This experiment is representative of five individual experiments. The mouse thrombomodulin probe andexpected S1 nucleaseresistant product are shown in the lower left.

4h in the presence of PMA. Prior treatment with PMA increased synthesis to 1.9 times control at 2 h and 1.8 times control a t 6 h. Since PMA increases both the synthesisand degradation of thrombomodulin, there should be little net effect on cellular thrombomodulin levels. Thrombomodulin activity was assayed on intact cells by measuring thrombin-stimulated ratesof protein C activation after exposure to PMAas shown in Fig. 7. There was a rapid drop incofactor activity reaching65.1 f 3.7% of control levels within 30 min. In another experimentat times between 5 and 120 min (not shown), surface activity reaches a nadir a t 30 min. Thrombomodulinactivity reappeared after several hours. The same pattern of thrombomodulin cofactor activity decline and return to control values was observed in separate experiments where PMA was continuously present for 12 h (data not shown). Since the turnover of thrombomodulin is slow relative to the rapid changes incofactor activity, we evaluated the possibility that PMA induced endocytosis of thrombomodulin with loss of cell surface thrombomodulin. We measured cell

The S t r u c t u r e and Function of Mouse Thrombomodulin

15819

A

TIME(h1 PMA

MTm

0 6 6 12 12 24 24 - + - + - + -

-. TIME (hr 1

FIG. 6. Rate of thrombomodulin synthesis in hemangioma cells treated with PMA withvarious times. Monolayers of hemangiomacells were pretreated for 0-12 hwith PMA (0)or dimethyl sulfoxide (O), washed, andthen radiolabeledwith [35S] methionine for anadditional 4 h. Radiolabeled thrombomodulin content in cell extracts was determined as described in Fig. 5, and the change in radioactivity was expressed as the percentage of the pretreatment control k S.D. of two to six determinations, except for those of dimethylsulfoxide-treatedvalues a t 6 and 12hwhich represent individual dishes.

4ot FIG.5. Disappearance of “S-labeled thrombomodulin after incubation of hemangioma cells with PMA. Monolayers of hemangioma cells were radiolabeled for 48 h with [3sS]methionine and then chased for the indicated time in completemedium without ( X ) or with PMA (A,4, 0, and V) or dimethyl sulfoxide (O,O, A, and V). Radiolabeled thrombomodulin was isolated from cell extracts by immunoprecipitation followed by SDS, 7.5% polyacrylamide gel electrophoresis in 2-mercaptoethanol and fluorography ( A ) .Densitimetry was performed on the autofluorograms. Data derived from each of the four experiments is plotted inB with each experiment represented by a different symbol. MTm, mouse thrombomodulin.

surface thrombomodulin by determining the binding of Iz5Ianti-thrombomodulin IgG to hemangioma cells. These measurements proved difficult since 75% of the binding of this antibody to hemangioma cells was “nonspecific,” i.e. not displaced by unlabeled IgG. However, PMA did reduce specific binding of antibody to hemangioma cells from 0.10 pg/106 cells (total IgG bound 0.40 pg/106 cells, nonspecific IgG bound 0.30 pg/106 cells) to 0.06 pg/106 cells (total IgG bound 0.28 pg/106 cells, nonspecific IgG bound 0.22 pg/106 cells). In two other experiments, PMAreduced binding similarly. The PMA-induced decrease in surface antigen and protein C cofactor activity was associated with increased phosphorylation of thrombomodulin as shown in Fig. 8. PMA-treated cells showed a 38 f 14% increase in 32Pincorporated into thrombomodulin. Phosphoamino acid analysis of the labeled thrombomodulin showed increased incorporation into serine as shown in Fig. 9. DISCUSSION

Analysis of the cDNAs for murine,bovine, and human thrombomodulin shows similarity between the three species

TIME (hr) FIG. 7. Protein C activation on hemangioma cells after addition of PMA. Monolayers of hemangioma cells were treated with serum-free “199 containing PMA ( 0 )or dimethyl sulfoxide (0) for 30 min. The medium was changed and at the indicated times, thrombomodulin cofactor activity was assayed for 30 min. Thrombomodulin cofactor activity is expressed as the percentage of the pretreatment control &S.D. of three to five determinations from two experiments. Control values were 7.2 f 1.4 pmol of protein Caformed/ ml/104 cells/30 min (eight experiments).

and certainhighly conserved regionswhich may be important for thrombomodulin function (Fig. 10). Mouse thrombomodulin, like humanand bovine, contains a putativeaminoterminal ligand-binding domain, a region of epidermal growth factor (EGF)-like repeats, a serine/threonine-rich region, a membrane-spanning domain, and a cytoplasmic tail. EGFlike repeats may preserve the ability of intracellular receptors to effectively release ligand, avoid degradation, and recycle to the cell surface as has been suggested for the low density lipoprotein receptor (33,34),where alteration of these repeats by mutagenesis interfered with low density lipoprotein receptor recycling (35). EGF domains also have been reported to contain sequences that direct /3-hydroxylation of asparagine or aspartate residues (36). The consensus sequence described by Stenflo et al. (36) (CX(N/”)XXXX(,/~)XCXC) isfound in EFG repeats 3 and 6. Although not demonstrated for murine thrombomodulin, acid hydrolysates of bovine thrombomodulin have been shown to contain 8-hydroxyaspartic acid (37). Human (16),bovine (30), and murine thrombomodulin all have the consensussequence in EGF repeats3 and 6 implying that they contain the amino acid modification. Mouse throm-


15820

PMA

MTm

-

+

-

FIG.8. Autoradiogram of 32P-labeled thrombomodulin immunoprecipitated from hemangioma cells. Monolayers of hemangioma cells were radiolabeled for 18 h with [32P]orthophosphate and then treated with PMA or dimethyl sulfoxide for 10 min. 32PThrombomodulin was immunoprecipitated from cell extracts and analyzed by SDS/7.5% polyacrylamide gel electrophoresis in 2-mercaptoethanol and autoradiography. The proteins that migrate below thrombomodulin arecontaminants which are also observed in control precipitates with protein A prior to the addition of antibodies. MTm, mouse thrombomodulin.

PMA

-

+ -Inorganic Phosphate

Phosphoserine

c

Phosphothreonine

-Phosphotyrosine

FIG.9. Phosphoamino acids of thrombomodulin immunoprecipitated from “P-labeled hemangioma cells. Monolayers of hemangioma cells labeled with [32P]orthophosphatefor 18 h were treated with PMA or dimethyl sulfoxide. The experiment was performed as describedin the legend to Fig. 8. The phosphorylated thrombomodulin hydrolyzed andphosphoamino acids were separated byhigh voltage electrophoresis as describedunder “Experimental Procedures.” The radioactive phosphoamino acids comigrated precisely with authentic phosphoserine internal standards, although the standards migrated slightly differently in the two lanes.

bomodulin has a 39-amino acid serine-threonine-rich domain compared to 34 for human and 42 for bovine, with 7 hydroxy amino acids in mouse and bovine thrombomodulin compared to 8 for human; presumably this is a region for 0-linked glycosylation of thrombomodulin by analogy to the low density lipoprotein receptor (38). The most highly conserved

region of thrombomodulin is the putative membrane-spanning domain with 20 of 23 amino acids identical in all three species; the nonidentical amino acids are conservative substitutions. The first 8 residues of the cytoplasmic tail of thrombomodulin and the potential sites for threonine and tyrosine phosphorylation are identical in all three species; serines are present but in different locations. In theregions where only mouse and human thrombomodulin can be compared, two regions have near identity; 18 of 20 amino acids are identical starting at human amino acid 207, and 22of 23 starting a t human amino acid 102. The similarity suggests an essential function for these regions. The conservation of the AT-rich motif in the 3”untranslated region of mRNA from all three species suggests a role for these sequences. Indeed, this motif is associated with short mRNA half-life for many cytokines (39, 40) and destabilization of hybrid mRNA with the 5’ sequence of globin and 3‘untranslated region with this motif has been demonstrated (39). We found, however, that despite the presence of the conserved AT motifs the half-life of thrombomodulin is 8.9 h (Figs. 2 and 3),too long to allow for rapid decreases of thrombomodulin activity due to changes in transcription. PMA had no effect on thrombomodulin mRNA levels (Fig. 4). The genomic sequence for human thrombomodulin (41, 42) does not contain a9-base pair 5’ motif common to PMAinducible genes (43), consistent with the observed lack of increase of thrombomodulin mRNA levels in response to PMA. PMA appears to stimulate endocytosis of thrombomodulin and also increases receptor degradation as indicated by the shortened survival of thrombomodulin (Fig. 5). Theincrease in thrombomodulin synthesis that follows treatment with PMA may compensate for the increased degradation (Fig. 6) and thereby maintain cellular thrombomodulin levels. The changes in protein degradation in response to PMA cannot explain the observed rapid decrease in protein C activation (Fig. 7). The half-life of the protein remains toolong to allow a rapid alteration in activity. Thrombomodulin is subject to endocytosis that is blocked by protein C (6,7) butstimulated in response to PMA as indicated by decreased binding of affinity-purified antibodies directed against thrombomodulin. Endocytosis of other receptors, including thetransferrin receptor (44), the tumornecrosis factor receptor (45), and the epidermal growth factor receptor (46, 47), increases in response to PMA. In addition, the transferrin receptor is phosphorylated on serine 24 of the cytoplasmic tail in response to PMA (48,49), although studies involving mutagenesis of this serine suggest that the phosphorylation is not essential for endocytosis in some cell lines (50-52). Whether this is true also for thrombomodulin will require alteration of the cytoplasmic phosphate acceptor. We have been unable to phosphorylate either human or murine thrombomodulin in uitro with protein kinase C, and therefore, the phosphorylation of thrombomodulin after PMA is probably not a direct effect of protein kinase C. The relatively slow turnover of thrombomodulin mRNA and protein implies that thrombomodulin levels in mouse hemangioma cells will not fall quickly in response to external stimuli. Presumably, thrombomodulin functions only under conditions where thrombin is generated, andthere is no obvious reason to propose a mechanism to decrease thrombomodulin levels quickly. Although thrombomodulin protein levels do not fall quickly, protein C-activatingcofactor activity does rapidly decline in response to PMA. Cofactor activity has been shown to fall on the surface of endothelial cells exposed to tumor necrosis factor (53), interleukin 1 (54), and


15821

1 MTm HTm BTm

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... .................... .........................................................................

MLGVLVLGAL ALAGLGFPAPAEPQPGGSQCVEHDCFALYPGPATFLNASQICDGLRGHLMTVRSSVAADVISLLLNGDGGVGRFUUWIGLQLPPGCG l . . . . . .

101 MTm HTm BTm

PaT;ai?J;

200

.................................................................................................... 201

MTm HTm BTm

.......................

MTm HTm BTm

30 AE PD LH

-EGFRXP

NAI PGAP EGFRll

E RCEDVDD RCEDVDD RCEDVDD

0 D

KQ IL AQ

H G

NQTSY EGFRX3

MTm HTm BTm

E E RGTPEDY EGFRXC

EGFRX5 MTm HTm BTm TRANSMEMBRANE

FIG. 10. Alignment of mouse (MTm),human (HTm),and bovine (BTm)thrombomodulin amino acid sequences. Identical amino acids are enclosed in boxes. The epidermal growth factor-like repeats (EGFR) are underlined as is the transmembrane domain.

endotoxin (55). The mechanism of decline in response to these cytokines has not been determined, but thelong survival for thrombomodulin implies that these cytokines, like PMA, may induce the endocytosis of thrombomodulin. Acknowledgments-We thank Dr. John C. Hoak for providing hemangioma cells, Drs. Peter Lobe1 and Tim Ley for advice on cloning and S1 analysis, Victoria Masakowski for providing µglobulin and c-myc probes, Lisa Westfield for synthetic oligonucleotide primers, Roger Inhorn and Dr. Tom Connolly for helpful discussions, and Lois Isenberg for typing this manuscript.

REFERENCES 1. Esmon, C. T., and Owen,W.G. (1981) Proc.Natl.Acad.Sci. U. S. A . 78,2249-2252 2. Esmon, C. T. (1987) Science 235, 1348-1352 3. Kisiel, W., Canfield, W. M., Ericsson, E. H., and Davie, E. W. (1977) Biochemistry 16,5824-5831 4. Suzuki, K., Stenflo, J., Dahlback, B., and Teodorsson, B. (1983) J. Biol. Chem. 258, 1914-1920 5. Fulcher, C. A., Gardiner, J. E., Griffin, J. H., and Zimmerman, T. S. (1984) Blood 6 3 , 486-489 6. Maruyama, I., and Majerus, P. W. (1985) J. Biol. Chem. 260, 15432-15438 7. Maruyama, I., and Majerus, P. W. (1987) Blood 69, 1481-1484 8. Kumada, T., Dittman, W. A., and Majerus, P. W. (1988) Blood 7 1,728-733 9. Fry, G.L., Czervionke, R.L., Hoak, J. C., Smith, J. B., and Haycraft, D. L. (1980) Blood 55,271-275 10. Miletich, J. P., Broze, G. J., and Majerus, P. W. (1980) Anal. Biochem. 105,304-310 11. MacDonald, R. J., Swift, G. H., Przybyla, A. E., and Chirgwin, J. M. (1987) Methods Enzyrnol. 152,219-227 12. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A . 6 9 , 1408-1412 13. D’Alessio, J. M., Noon, M.C., Ley, H. L., and Gerard, G. F. (1987) Focus 9 , 1-4 14. Huynh, T. V., Young, R. A., and Davis, R.W. (1985) in DNA Cloning: A Practical Approach (Glover, D. M., ed) pp. 49-78,

IRL, Oxford 15. Feinberg, A. P., and Volgelstein, B. (1983) A d . Biochem. 1 3 2 , 6-13 16. Wen, D., Dittman, W. A., Ye, R. D., Deaven, L. L., Majerus, P.

W., and Sadler, J. E. (1987) Bioc,kmistry 26,4350-4357 17, Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc.Natl. Acad. Sci. U. S. A . 74,5463-5467 18, Tabor, S., and Richardson, C.C. (1987) Proc.Natl.Acad.Sci.

U. S. A . 84,4767-4771 19. Favaloro, J., Treisman, R., and Kamen, R. (1980) Methods Enzymol. 65, 718-749 20, Kelly, K., Cochran, B. H., Stiles, C. D., and Leder, P. (1983) Cell 35,603-610 21, Parnes, J. R., and Seidman, J. G. (1982) Cell 29, 661-669 22, Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H. (1977) Biochemistry 16,4743-4751 23. Chomczynski, P., and Sacchi, N. (1987) Anal.Biochem. 162, 156-159 24. Kessler, S. W. (1981) Methods Enzymol. 7 3 , 442-459 25. Laemmli, U. K. (1970) Nature 227,680-685 26. Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochern. 46, 83-88 27. Salem, H. Maruyama, H., I., Ishii, H., and Majerus, P. W. (1984) J. Biol. Chem. 259,12246-12251 28. Bolton, A. E., and Hunter, W. M. (1973) Biochem. J. 133, 529539 29. Cooper, J. A., Sefton, B.M., and Hunter, T. (1983) Methods Enzymol. 99,387-402 30. Jackman, R. W., Beeler, D. L., VanDeWater, L., and Rosenberg, R. D. (1986) Proc. Natl. Acad. Sci. U. S. A . 83, 8834-8838 31. Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S., and Cerami, A. (1986) Proc. Natl. Acad. Sci. U. S. A . 83, 1670-1674 32. Harris, L. J., Remmers, E. F., Brodeur, P., Riblet, R., D’Eustachio, P., and Marcu, K. B. (1983) Nucleic Acids Res. 11,83038315 33. Brown, M. S., and Goldstein, J. L. (1986) Science 232, 34-47 34. Goldstein, J. L., Anderson, R. G . W., and Brown, M. S. (1979) Nature 279,679-685 35. Davis, C. G., Goldstein, J. L., Sudhof, T. C., Anderson, R. G. W., Russell, D. W., and Brown, M. S. (1987) Nature 326,760-765 36. Stenflo, J., Lundwall, A., and Dahlback, B. (1987) Proc. Natl. Acad. Sci. U. S. A . 84.368-372 37. Stenflo, J., Ohlin, A., Owen, W. G., and Schneider, W. J. (1988) J. Biol. Chem. 263, 21-24 38. Russell, D. W., Schneider, W. J., Yamamoto, T., Luskey, K. L., Brown, M. S., and Goldstein, J. L. (1984) Cell 37,577-585 39. Shaw, G., and Kamen, R. (1986) Cell 4 6 , 659-667

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The Structure and Function of Mouse Thrombomoddin

40. Treisman, R. (1985) Cell 42,889-902 41. Jackman, R. W., Beeler, D. L., Fritze, L., Soff, G., and Rosenberg, R. D. (1987) Proc. Natl. Acud. Sei. U. S. A. 84,6425-6429 42. Shirai, T.,Shiojiri, S., Ito, H., Yamamoto, S., Kusumoto, H., Deyashiki, Y., Maruyama, I., and Suzuki, K. (1988)J. Biochem. 103,281-285 43. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R. J., Rahmsdorf, H. J., Jonat, C., Herrlich, P., and Karin, M. (1987) Cell 49,729-739 44. Klausner, R. D.,Harford, J., and van Renswoude, J. (1984) Proc. Nut!. A d . Sci. U. S. A. 81, 3005-3009 53. 45. Aggarwal, B. B., and Eessalu, T.E. (1987) J . B i d . Chern. 262, 16450-16455 54. 46. Lee, L. S., and Weinstein, I. B. (1978) Science 202, 313-315 47. Salomon, D. S. (1981) J. Biol. Chem. 256,7958-7966 48. May, W.S., Jacobs, S., and Cuatrecasas, P. (1984) Proc. Natl.

Acud. Sci. U. S. A. 81,2016-2020 49. Davis, R. J., Johnson, G . L., Kelleher, D. J., Anderson, J. K., Mole, J. E., and Czech, M. P. (1986)J. Biol. Chem. 261,90349041 50. Zerial, M., Suomalaninen, M., Zanetti-Schneider, M., Schneider, C., and Garoff, H. (1987) EMBO J. 6,2661-2667 51. Rothenberger, S., Iacopetta, B. J., and Kuhn, L. C. (1987) Cell 49,423-431 52. Davis, R. J., and Meisner, H. (1987) J. Bid. Chern. 262, 1604116047 Nawroth, P. P., and Stern, D. M. (1986) J. Exp. Med. 163, 740745 Stern, and M. D. Nawroth, P. P., Handley, D. A., Esmon, C. T., (1986) Proc. Nutl. Acad. Sci. U. S. A. 83, 3460-3464 55. Moore, K. L., Andreoli, S. P., Esmon, N. L., Esmon, C. T.,and Bang, N. U. (1987) J. Clin. Znuest. 79, 124-130