Proc. Nail. Acad. Sci. USA Vol. 87, pp. 3200-3204, April 1990 Cell Biology
Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues (liver regeneration/growth factor/hepatotrophic factor/hepatitis)
KOSUKE TASHIRO*, MITCHIO HAGIYAt, TSUTOMU NISHIZAWAt, TATSUYA SEKIt, MANABU SHIMONISHIt, SHIN SHIMIZUt, AND TOSHIKAZU NAKAMURA** *Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812, Japan; and tPharmaceutical Research Center, Toyobo Co., Ohtsu 520-02, Japan
Communicated by Gordon H. Sato, February 5, 1990
ABSTRACT The primary structure of rat hepatocyte growth factor (HGF) was elucidated by determining the base sequence of the complementary DNA (cDNA) of HGF. The cDNA for rat HGF was isolated by screening a liver cDNA library with oligonucleotides based on the partial N-terminal amino acid sequence of the P subunit of purified rat HGF. HGF is encoded in an mRNA of about 6 kilobases. Both a and 13 subunits of HGF are specified in a single open reading frame for a 728-amino acid protein with a calculated molecular weight of 82,904. The N-terminal part of HGF has a signal sequence and a prosequence with 30 and 25 amino acid residues, respectively. The mature heterodimer structure is derived proteolytically from this single pre-pro precursor polypeptide. The calculated molecular weights of the a and (3 subunits are 50,664 and 25,883, respectively, and each subunit has two potential N-lked glycosylation sites. The amino acid sequence of HGF is 38% identical with that of plasminogen. The a subunit of HGF contains four "Ikringle" structures, and the (3 subunit has 37% amino acid identity with the serine protease domain of plasmin. Northern blot analysis revealed that HGF mRNA was expressed in rat various tissues, including the liver, kidney, lung, and brain.
were shown to be markedly elevated in the blood plasma of rats with hepatitis induced by administration ofa hepatotoxin such as CC14 or D-galactosamine (18). In humans, similar findings were observed in the sera of patients after partial hepatectomy (19) and with fulminant hepatitis (33). HGF has been isolated as a homogeneous material from rat platelets and shown to be a heat-labile protein of -82 kDa, composed of a 69-kDa a subunit and a 34-kDa (3 subunit (20, 21). HGF at 1 ng/ml markedly stimulated DNA synthesis in adult rat hepatocytes and its effect was additive or synergistic with those of insulin and epidermal growth factor. HGF has no species specificity; for example, rat HGF also stimulated DNA synthesis in human, canine, and porcine hepatocytes in primary culture. In contrast, HGF did not stimulate DNA synthesis in established cell lines such as Swiss mouse 3T3 fibroblasts and BRL (buffalo rat liver) epithelial cells. Here we report the complete amino acid sequence of rat HGF obtained by cloning and sequence analysis of its cDNA.§ We have found that HGF mRNA is expressed in various rat tissues including the kidney, heart, lung, and brain, as well as in injured liver. MATERIALS AND METHODS Materials. Materials used for the purification of HGF were as described (20). The cDNA synthesis system and the multiprime labeling system were from Amersham. Gigapack Gold, AgtlO and M13 phage, and pBluescript were from Stratagene. Enzymes for DNA manipulation were obtained from Toyobo and New England Biolabs. Radioisotopic materials were from Amersham. Purification and Sequencing ofRat HGF. The purification of HGF from rat platelets was essentially as reported (20). Briefly, the procedures involved the following successive steps: stimulation of HGF release from rat platelets by thrombin, cation-exchange FPLC on a Mono S column, affinity chromatography on heparin-Sepharose CL-6B, and reverse-phase HPLC on a C4 column. Purification was monitored by the HGF activity, which was defined as the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture (8). The yields were about 60 tkg of pure HGF from the platelets of 3000 rats, corresponding to a recovery of 23%. The a and P subunits of HGF were separated by reverse-phase HPLC on a C4 column after reduction and carboxymethylation (22) of purified HGF. The subunits were separately subjected to N-terminal amino acid sequence analysis with an Applied Biosystems model 470A sequencer. Synthesis and Labeling of Oligonucleotides. Oligonucleotides corresponding to parts of the amino acid sequence of the
Liver regenerates actively after partial hepatectomy and liver injury. Early investigations (1-4) showed that hepatotrophic factors existed in the blood of partially hepatectomized animals. However, no one succeeded in purifying and characterizing such a humoral factor until recently, largely because no simple, reproducible, and sensitive assay for the factor has been available. Although many in vitro experimental methods using established liver and hepatoma cell lines have been employed in attempts to identify the hepatotrophic factor, these assays are unsuitable because these cell lines have lost almost all liver functions and normal growth properties. In contrast, adult rat hepatocytes in primary culture retain many normal liver functions and respond to various hormones, behaving more like the in vivo cells (5, 6). Moreover, they can proliferate in culture at low cell density when a mitogen such as epidermal growth factor is added to the culture medium (7-11). Primary cultured adult hepatocytes are thus the most suitable system for identification and purification of a growth factor for mature parenchymal hepatocytes. Applying this system, Nakamura et al. (12) were able to isolate a factor from the sera of partially hepatectomized rats that stimulated DNA synthesis in adult rat hepatocytes, which was named hepatocyte growth factor (HGF). Other investigators found a hepatotrophic factor in the sera of partially hepatectomized rats (13-15) and in rat platelets (16, 17). Recently, HGF levels
Abbreviation: HGF, hepatocyte growth factor. tTo whom reprint requests should be addressed. §The sequence reported in this paper has been deposited in the GenBank data base (accession no. M32987).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Proc. Natl. Acad. Sci. USA 87 (1990)
Biology: Tashiro et al.
N-terminal portion of the HGF /B subunit were synthesized on an Applied Biosystems DNA synthesizer (model 381A) with the procedures and reagents recommended by the manufacturer. Oligonucleotides were end-labeled with T4 polynucleotide kinase and [y-32P]ATP (23) and used for screening a cDNA library Extraction of Poly(A)+ RNA. Total RNA was extracted from rat tissues by the guanidinium chloride method (24). Poly(A)+ RNA was purified by chromatography on oligo(dT)-cellulose (23). Usually, 3 mg of total RNA was obtained from 1 g of liver, which yielded about 30 Ag of poly(A)+ RNA. Construction and Screening of cDNA Libraries. Poly(A)+ RNA was purified from rat liver 15 hr after administration of 20% CCL4 (1 ml/100 g of body weight) and used for the construction of a cDNA library. Double-stranded cDNA was synthesized with oligo(dT) as a primer, methylated by EcoRI methylase, and ligated with an EcoRI linker. After EcoRI digestion, the cDNAs were ligated into EcoRI-digested and phosphatase-treated AgtlO arms and packaged in vitro using Gigapack Gold. Two micrograms of poly(A)+ RNA yielded 2 x 106 phage transformants. Replica filters were prepared (25) and hybridized with the 32P-end-labeled 44-mer probe (Fig. 1) for 12 hr at 450C in 5x SSC (lx SSC is 0.15 M NaCl/0.015 M sodium citrate, pH 7.0) containing 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.1% SDS, and 100 ,g of denatured salmon testis DNA per ml. The filters were then washed at 50°C in 5x SSC/0.1% SDS, air-dried, and autoradiographed on Fuji x-ray film. The oligonucleotide-primed cDNA library was constructed as described above, except that the oligodeoxynucleotide 5'-AAATCCTCCATATTCTTGTC-3' was used as a primer. Recombinant phages were screened using the 32P-labeled 1.4-kilobase (kb) EcoRI fragment of cDNA clone RBC1 (multiprime labeling kit, Amersham) as a probe according to Maniatis et al. (23). Determination of Nucleotide Sequence. The DNAs of the isolated phage clones were extracted (23), digested with the appropriate restriction enzyme, and then electrophoresed in a 1% agarose gel. The digested fragments were extracted from the gel, subcloned into M13mpl8, M13mpl9, or pBluescript, and subjected to nucleotide sequence determination (26). Northern Hybridization. Poly(A)+ RNAs (2 ,ug) purified from various rat tissues were denatured with formaldehyde and electrophoresed in a 1% agarose/0.7 M formaldehyde gel (23). RNAs were transferred to a Zeta-Probe filter (Bio-Rad) and hybridized with the 32P-labeled 1.4-kb EcoRI fragment of RBC1 for 15 hr at 42°C in 50% (vol/vol) formamide/5 x SSC/10 mM sodium phosphate, pH 6.8/0.5% SDS containing 500 ,g of salmon sperm DNA per ml. The filter was then washed three times with 0.2x SSC/0.1% SDS for 30 min at 60°C, air-dried, and autoradiographed on Fuji x-ray film.
RESULTS Isolation of a cDNA Encoding Rat HGF. We isolated a and ,B subunits from 60 ,ug of pure HGF and determined 19 and 27 amino acid residues of the N-terminal portions, respectively (Fig. 1 a and b). These amino acid sequences had no homology to known growth factors (GenBank, August 1989). As a probe for the isolation of the cDNA encoding rat HGF, a 44-mer oligonucleotide was synthesized based on amino acid residues 1-15 of the / subunit (Fig. lc). It contained deoxyinosine at the positions where the nucleotide could not be specified because of codon degeneracy. We chose injured liver as a source of poly(A)+ RNA for the construction of the cDNA library, because we recently found that there was a large amount of HGF in the injured liver produced by the administration of CC14 into rats, and the HGF purified from liver was identical to platelet-derived HGF, judging by their chemical and biological properties, including the N-terminal amino acid sequence (unpublished
a)
3201
10
Pro-Leu-Val- Lys- lIe- Lys-Th r-Lys-Lys-VaI-Asn-Ser-AIa-Asp19
15
Glu-Cys-Ala-Asn-Arg b)
10
1
Val-Val-Asn-GIy- Ile-Pro-Th r-G In-Thr-Th r-Val-GIy-Trp-Met27
20
15
Val-Ser-Leu-Lys-Tyr-Arg-Asn-Lys-His-IIe-Cys-GIy-GIy C)
CAICAITTICCITAIGGITGIGTITGITGICAICCIACCTACCA
FIG. 1. (a and b) N-terminal amino acid sequences of the a and
,8 subunits, respectively, of purified rat HGF. (c) Sequence (3' to 5') of the 44-mer oligodeoxynucleotide probe synthesized on the basis of the N-terminal amino acid sequence of the 83 subunit. I, deoxyinosine.
data). Therefore, in order to isolate the cDNA encoding HGF, a cDNA library was constructed using poly(A)+ RNA extracted from rat liver 15 hr after administration of CCl4 This cDNA library was screened with the 32P-end-labeled 44-mer oligonucleotide. The screening of 2 x 106 recombinant phages yielded one positive clone, RBC1 (Fig. 2). The insert of RBC1 was 1.4 kb long and contained an open reading frame encoding 404 amino acid residues, followed by a 3' noncoding sequence. The 27-amino acid sequence of the N-terminal portion of the ( subunit was confirmed to be in this open reading frame. This 1.4-kb insert contained the full 233-amino acid sequence of the 3 subunit of rat HGF. The 1.4-kb insert coded for a further 171 amino acid residues in front of the ,/-subunit sequence, but no ATG start codon was observed in this reading frame. Moreover, Northern blot analysis of poly(A)+ RNA from the livers of CC14treated rats with the RBC1 cDNA insert as the probe revealed that the rat HGF mRNA was about 6.0 kb long (see Fig. 5). Therefore, clone RBC1 did not contain the whole sequence of rat HGF. To isolate the full-length cDNA for HGF, another cDNA library was constructed by the primerextension method, using a 20-mer oligonucleotide as a primer (noncoding strand, positions 1249-1268 in Fig. 3). The oligonucleotide-primed cDNA library was screened with the 1.4kb insert of RBC1 as a probe. Screening of 2 X 105 recombinant phages from the oligonucleotide-primed cDNA library and of 106 recombinant phages from the oligo(dT)-primed cDNA library yielded two (RAC1 and RAC2) and some (RBC3) positive clones, respectively (Fig. 2). The nucleotide sequence of the inserts of these E
B
6
EA
EA
0.'5 a
HBH
1
It
1 111
1.
1.5
I
(1.32 Kb) I
-
5.8 Kb
2.
(0.70Kb)
b
3.58 Kb
(A)n 3' RBC1
, ,RBC3 , RAC1
,
RAC2
FIG. 2. Schematic representation of rat HGF mRNA and cDNA. Restriction sites (B, Bgl II; E, EcoRI; A, Ava I; H, HindIII) are shown on the kilobase scale. The rat HGF mRNA is diagrammed below the scale. Noncoding sequences are represented by a line and the coding region is boxed. Black and hatched areas show the signal peptide and the prosequence, respectively. Open box indicates the coding regions for the a and /3 subunits. cDNA clones are represented below the mRNA; small black boxes indicate the location of the 20-mer primer on clones RBC1, RACi, and RAC2.
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clones revealed that the 1.4-kb insert of RACi contained the ATG initiation codon as well as 120 bases of 5' upstream sequence. The inserts of RBC3 encoded part of the Cterminal portion of the /3 subunit and contained a long 3' noncoding region (3.58 kb). Both RACi and RAC2 had an open reading frame coding for the N-terminal sequence of the a chain, which was determined by protein sequencing. Thus, the overlapping clones RACi, RBC1, and RBC3 covered the entire coding region and the 3' untranslated region. The complete cDNA and translated protein sequence of rat HGF are shown in Fig. 3. It contains two possible initiation codons at nucleotides 1-3 and 4-6 and a stop codon (TAA) at nucleotides 2185-2187. The first methionine was designated as residue 1 in the amino acid sequence.
Proc. Natl. Acad. Sci. USA 87 (1990)
Primary Structure of Rat HGF. Rat HGF cDNA encodes a polypeptide of 728 amino acids. The structure of pre-proHGF is represented in Fig. 4. Both the a and the P chain are encoded in a single open reading frame. Fifty-five amino acid
residues lie in front of the N terminus of the a subunit. The first 30 residues have hydrophobic side chains, characteristic of a signal peptide, and the next 25 residues appear to constitute a prosequence. The C terminus of the a subunit is followed directly by the N terminus of the /3 subunit. The sequence at the cleavage site between the a and p subunits is Arg-Val (residues 495 and 496), and this sequence may be recognized and cleaved by a trypsinlike protease. We condlude that the rat HGF precursor is synthesized as a prepro-protein and that mature HGF is formed by proteolytic GTTTAGTCCTAGATCTTTCCAGT
TAATCCACACAAATTAGCCATCCAATAAAGCGCTCGAACGACCGCTTGAACAGATTTTTCACCCGCATCCCTGCGACCATCACCTGCCGAATGCAGC 1 10 20 ATG ATG TGG GGG ACC AAA CTT CTG CCG Met Met Trp Gly Thr Lys Leu Leu Pro --ProGGA CAG AAG AAG AGA AGA AAT ACT IGiu Gly Gin Lys Lys Arg Arg Asn Thr
CTG TTG CTG CAG CAT GTC CTG CTG CAC CTC CTC Leu Leu Leu Gin His Val Leu Leu His Leu Leu 50 CAT GAA TTC AAA AAG TCA GCA AAA ACT ACT CTT His Giu Phe Lys Lys Ser Ala Lys Thr Thr Leu 80 TGT GCC AAC AGG TGC ATC AGA AAC AAG GGC TTT Cys Ala Asn Arg Cys Ilie Arg Asn Lys Gly Phe 110 CCT TTC AAT AGT ATG TCA AGT GGA GTG AAA AAA Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys 140 ATT GGT AAA GGA GGC AGC TAT AAG GGG ACA GTA Ilie Gly Lvs Gly Gly Ser Tvr Lys Gly Thr Val 170 CAC AGC TTT TTG CCT TCG AGC TAT CGC GGT AAA His Ser Phe Leu Pro Ser Ser Tyr Ara Gly Lys 200 TTC ACA AGC AAT CCA GAG GTA CGC TAC GAA GTC Phe Thr Ser Asn Pro Glu Val Ara Tyr Glu Val 230 AGA GGT CCC ATG GAT CAC ACA GAA TCA GGC AAG Ara Gly Pro Met Asp His Thr Glu Ser Gly Lvs 260 TAT CCC GAC AAG GGC ITT GAT GAT AAT TAT TGC Tvr Pro Asp Lvs Gly Phe Asp Asp Asn Tvr Cvs 290 TGG GAG TAT TGT GCA ATT AAA ATG TGC GCT CAC Tro Glu Tvr Cvs Ala Ilie Lvs Met Cvs Ala His 320 GGA GAA GGT TAC AGG GGA ACC ACC AAT ACC ATT Gly Glu Gly Tvr Ara Gly Thr Thr Asn Thr Ilie 350 ACT CCC GAG AAC TIC AAA TGC AAG GAC CIT AGA Thr Pro Glu Asn Phe Lvs Cvs Lys Ase Leu Ara 380 CCA AAC AIC CGA GTl GGT TAC TGC ICT CAA All Pro Asn Ilie Ara Val Gly Tyr Cvs Ser Gin Ilie 410 AIG GGC AAC ITA ICC AAA ACA AGG ICT GGA CIC Met Gly Asn Leu Ser Lvs Thr Ara Ser Gly Leu 4 40 CCA GAC GCT AGC AAG TIG ACT AAG AAT TAC TGC Pro Asp Ala Ser Lys Leu Thr Lys Asn Tyr Cvs 470 CCI TGG GAT TAT TGC CCI ATT ICC CGT IGI GAA Pro Iry Ase Tyr Cvs Pro le Ser Ara Cvs§ Giu 500 ACA AAA CAA CIG CGA G TlGI AAT GGC All CCA Thr Lys Gin Leu Airg IVal Val Asn Gly Ilie Pro 530 GGG GGA ICA TIG AlA AAG GAA AGI TGG GTl CIT Gly Gly Ser Leu Ilie Lys Giu Ser Trp Val Leu 560 CIT GGA AIC CAT GAl GIC CAT GAG AGA GGC GAG Leu Gly Ilie His Asp Val His Giu Arg Gly Giu 590 GAl TIG GTl TIA CIG AAG CIT GCT CGC CCI GCA Asp Leu Val Leu Leu Lys Leu Ala Arg Pro Ala 620 GAA AAG ACT ACT TGC AGI All TAC GGC TGG GGC Giu Lys Thr Ihr Cys Ser Ilie Tyr Gly Trp Gly 650 All AIG GGG AAT GAG AAA TGC AGI CAG CAC CAT CAA GGC Ilie Met Gly Asn Giu Lys Cys Ser Gin His His Gin Gly 670 ~...680 ICA GGA CCI IGI GAG GGA GAlTT GGT GGC CCA CIC All Ser Gly Pro Cys Glu Gly Asp \ lr.Gy Gly Pro Leu Ilie 700 710 GGA IGI GCC AIC CCA AAT CGT CCI GGT All ITT GTl CGA Gly Cys Ala Ilie Pro Asn Arg Pro Gly Ilie Phe Val Arg
GTC Val 40 CTT Leu 70 AAA ACC AAA AAA GTG AAC TCT GCA GAT GAG Lys Thr Lys Lys Val Asn Ser Ala Asp Gin 100 GAT AAG TCG AGA AAA CGA TGC TAC TGG TAT Ser Asp Lys Arg Lys Arg Cys Tyr Trp Tyr 130 GAA AAC AAA GAC TAT ATT AGA AAT TGC ATC Giu Asn Lys Asp Tyr Ilie Arg Asn Cvs Ilie Ki 160 CAG CCT TGG AAT TCC ATG ATC CCC CAT GAA Gin Pro TrD Asn Ser Met Ilie Pro His Glu 190 CCT CGA GGG GAA GAA GGG GGA CCC TGG TGT Pro Ara Gly Glu Glu Gly Gly Pro Tro Cvs 220 GAA TGC ATG ACC TGC AAC GGT GAA AGC TAC Giu Cvs Met Thr Cvs Asn Gly Glu Ser Tyr K2 250 CCA CAC CGG CAC AAA TTC TTG CCG GAA AGA Pro his Ara His Lys Phe Leu Pro Glu Ara 280 TGG IGC TAC ACT CTT GAC CCT GAC ACC CCT Tre Cvs Tyr Thr Leu Asp Pro Aso Thr Pro 310 ATG GAA ACA ACT GAA TGT ATA AAA GGC CAA Met Giu Thr Thr Giu Cvs Ilie Lys Gly Gin 340 K3 GAT TCG CAG TAC CCC CAC AAG CAT GAC ATC Aisp Ser Gin Tyr Pro His Lys His Asp Ilie 370 GCT GAA ICA CCA IGG IGI ITT ACC ACT GAT Ala Glu Ser Pro Ire Cvs Phe Thr Thr Asp 400 GAT IGI TAT CGT GGC AAT GGG AAA AAC TAC Asp Cvs Tyr Ara Gly Asn Gly Lvs Asn Tyr 430 K4 GAl GTITA CAC CGI CAT AIC TIC TGG GAG Leu His His Ire GuAsp Ara le Phe Glu 460 CCI TGG TGC TAC ACA GGG AAT CCI CIC GTl Pro Ire Cvs Tyr Thr Gly Asn Pro Leu Val 490 TIG GAC CAT CCI GIA AlA ICC ~GCC AAA Leu Asp His Pro Val Ilie SerL sAla Lys 520 AGI TIG AAA TAC AGG AAT AAA CAC AIC IGI Ser Leu Lys Tyr Arg Asn Lys His Ilie Cys 550 AAC AAA GAC TIG AAA GAC TAT GAA GCT IGG Asn Lys Asp Leu Lys Asp Trp Giu Ala Irp 580 ICC CAG CTA GIC TAT GGA CCT GAA GGC ICA §_~ Gin Leu Val Tyr Gly Pro Giu Gly Ser 610 GAT TIA CCI AGI TAT GGC T.GTL ACA AIC CCI Leu Pro [~JThr Pro Ser Ilie Asp Tyr Gly
~GAA
ITA TIA CGA GIA GCT CAT CIG TAT Leu Leu Arg Val Ala His Leu Tyr IGI GCT GGG GCT GAA AAG All GGA Cys Ala Gly Ala Giu Lys Ilie Gly
CTG CTT CCT GTC ACC ATC Leu Leu Pro Val Thr Ilie - ( ACC AAG GAA GAC CCA TTA Thr Lys Giu Asp IPro Leu
CCA TTC ACT TGC AAG GCC Pro Phe Thr Cys Lys Ala
GGG TTT GGC CAT GAA TTT
30 CCC TAT GCA Pro Tyr Ala 60 GTG AAG ATT Val Lys Ile 90 TTT GTT TTT Phe Val Phe 120 GAC CTC TAT
Gly Phe Gly His Giu Phe Asp Leu Tyr
150 TCC ATC ACT AAG AGT GGC ATC AAG TGC Ser Ilie Thr Lys Ser Gy Ilie Lys Cvs 180 GAC CTA CAG GAA AAC TAC TGT CGA AAT Leu Gin Glu Asn Tyr Cvs Ara Asn Asp 210 TGT GAC ATT CCT CAG TGT TCA GAA GTT Cvs Asp Ilie Pro Gin Cvs Ser Giu Val 240 ACA TGT CAG CGC TGG GAT CAG CAG ACA Thr Cvs Gin Ara Tro Asp Gin Gin Thr 270 CGC AAT CCC GAT GGC AAG CCG AGG CCA Ara Asn Pro Asp Gly Lys Pro Ara Pro 300 AGT GCT GTG AAT GAG ACT GAT GTT CCC Ser Ala Val Asn Giu Thr Asp Val Pro 330 TGG AAT GGA ATT CCG TGT CAG CGT TGG Tre Asn Gly le Pro Cvs Gin Ara Tre
-120 -1 90
180 270 360 450 540 630
720 810 900
990
360
GAA AAT TAT
IGC CGC AAT CCG GAl GGG
Glu Asn Tyr Cvs Ara Asn Pro Asp Gly
390 CCC AAA IGI GAC GIG ICA AGI GGA CAA Pro Lvs Cvs Asp Val Ser Ser Gly Gin 420 ACA IGI ICC AIG TGG GAC AAG AAI AIG Thr Cvs Ser Met Ireso v s e 450 CGG AAC CCC GAT GAC GAC GCC CAT GGA Ara Asn Pro Asp Asp Asp Ala His Gly 480 GGA GAl ACT ACA CCI ACA All GIC AAT Thr Thr Ihr Pro Val Ilie Asn Gly Asp 510 ACA CAA ACA ACA GIA GGG TGG AIG GTl Thr Gin Thr Thr Val Gly Trp Met Val 540 ACT GCA CA'TGT ITT CCA GCT AGA Thr Ala Arg \Q~Cys Phe Pro Ala Arg 570 GAG AAA CGC AAA CAG AIC TIA AAC All Giu Lys Arg Lys Gin Ilie Leu Asn Ilie
AGG(
AIC CIG GAl AAC III GIC AGI ACA AlT Ilie Leu Asp Asn Phe Val Ser Thr Ilie 630 TAC ACT GGA TIG AIC AAC GCA GAl GGT Thr Leu Ala Ilie Asn Tyr Gly Asp Gly 660 AAG GIG ACT TIG AAT GAG ICT GAA TIA Lys Val Thr Leu Asn Giu Ser Giu Leu 690 IGI GAA CAA CAC AAA AIG AGA AIG GTl Cys Giu Gin His Lys Met Arg Met Val 720 GIA GCA TAT TAT GCA AAA IGG AlA CAC Val Ala Tyr Tyr Ala Lys Trp Ilie His
CIT GGT GIC All GTl CCI GGT CGT Leu Gly Val Ilie Val Pro Gly Arg 728 AAA GIA All TIG ACA TAC AAG TIG TAATAGCCATAGAAGAGGCCAGTGTATTTGAAGCATCCATGGATACAGGAAGATTTCCAAGACTTCAGGATTAAAATGTCACCTAAA
1080 1170 1260 1350 1440
1530 1620 1710
1800 1890 1980
2070 2160 2271
FIG. 3. Nucleotide sequence of rat HGF cDNA and the deduced amino acid sequence. Nucleotide numbers are at right and amino acid numbers are given above the sequence. Underlines indicate the kringle domains. The primer for the construction of the oligonucleotide-primed cDNA library is shown by the broken underline. The putative N-glycosylation sites are marked by wavy lines. The glutamine at position 535 and the tyrosine at 676 (circled) correspond to the residues of the active center in plasmin. The cysteine residues that form an interchain disulfide bond between the a and 83 subunits are boxed.
Cell Biology: Tashiro et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
Arm
a-subunit
Val-
s S
N p -subunit
Leu
FIG. 4. Representation of rat pre-pro-HGF structure. The thick line and the wavy line represent the signal peptide and the prosequences, respectively. Sites of proteolytic cleavage are indicated by arrows.
cleavage. The a and /3 chains consist of 440 and 233 amino acids, respectively. The calculated molecular weight of the mature HGF after processing ofthe signal peptide and the prosequence is 76,529. The molecular weights of the a and a subunits are 50,664 and 25,883, respectively. These values are smaller than those determined by SDS/PAGE. Each subunit contains two AsnXaa-Thr/Ser sequences, potential sites for N-linked glycosylation (27); these are at residues 240-242 and 348-350 of the a subunit and at residues 74-76 and 161-163 of the /3 subunit. Thus, the differences between the molecular weights determined by SDS/PAGE and those calculated from the cDNAs may be due to the glycosylation of each subunit. HGF mRNA Expression in Rat Tissues. Recently, we examined the tissue distribution of HGF-like activity in rats and found that an HGF-like growth factor exists in various other tissues in addition to the liver (data not shown). We examined the expression of HGF mRNA in rat tissues by extracting poly(A)+ RNAs from the various tissues and analyzed them by Northern hybridization with the 1.4-kb EcoRP insert of RBC1 as a probe (Fig. 5). Rat HGF mRNA (-6 kb) was expressed in lung, kidney, and thymus as well as liver. Interestingly, we also detected HGF mRNA in samples from brain. The level of HGF mRNA in the liver increased markedly when CC14 was injected intraperitoneally into rats (Fig. 5). The HGF mRNA increased gradually in the liver from 5 hr after the administration of CCL4 and reached a maximum at 10-12 hr (data not shown).
DISCUSSION In this paper, we show the primary structure of rat HGF deduced from the nucleotide sequence of its cDNA. The deduced sequence contains the N-terminal sequences of both subunits of rat HGF, which were determined by sequence analysis of purified rat HGF. In the sequence of the HGF precursor, the a-subunit peptide is followed directly by the 1
2 3 4 5 6 7 8 9
3203
/3-subunit peptide. Moreover, the signal peptide and the prosequence immediately precede the N terminus of the a subunit. Therefore, HGF is synthesized as a pre-pro precursor. Presumably, the signal peptide and the prosequence are removed by processing, and the a and /3 subunits are formed by cleavage of the Arg-Val (positions 495 and 496) peptide bond by a trypsinlike protease. Recently, we determined the primary structure of human HGF by cloning and sequence analysis ofthe cDNA (28). The deduced amino acid sequence of human HGF is >90% identical with that of rat HGF (Fig. 6). There are some insertions and deletions in the amino acid sequence of the 83 subunit of human and rat. Therefore, the length of the / subunit of human and rat HGF is 234 and 233 amino acid residues, respectively. We also succeeded in expressing biologically active human HGF in COS-1 cells transfected with the plasmid carrying the entire protein-coding sequence of human HGF cDNA. Whereas HGF has no homology with various known growth factors, it has high homology with plasminogen (29). The amino acid sequence identity between rat pro-HGF and human plasminogen was calculated to be 38%. The a subunit of rat HGF has four "kringle" structures, like the A chain of plasminogen, which has five kringle domains. Kringle structures are also found in tissue plasminogen activator, urokinase, coagulation factor XII, and prothrombin (30). Although the kringle domain is thought to play a role in the interaction between macromolecules, its real functions are unknown at present. The / subunit of rat HGF shows a high degree of homology with various serine proteases, including the B chain of plasmin. However, the histidine and serine residues of the active sites of the proteases are replaced in the ,8 chain rat:
human: rat:
pro
MMWGTKLLPVLLLQHVLLHLLLLPVTIPYE GQKKRRNTLHEFKKSAKTT --V-----IA-IA--- -H------I---------a LTKE PLVKIKTKKVNSADECANRCIRNKGFPFTCKAFVFDKSRKRCYWY
human: -I-I +AL--------T--Q-----T--N-L-----------A--Q-L-F
50 49 100 99
PFNSMSSGVKKGFGHEFDLYENKDYIRNCIIGKGGSYKGTVSITKSGIKC 150 rat: human: -----------E----------------------R--------------- 149 rat:
human:
QPWNSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYE 200 199
rat: VCDIPQCSEVECMTCNGESYRGPMDHTESGKTCQRWDQQTPHRHKFLPER 250 human: -----------L--------I-----H------------ 249
rat: YPDKGFDDNYCRNPDGKPRPWCYTLDPDTPWEYCAIKMCAHSAVNETDVP 300 human: ----------------Q----------H-R-------T--DNT--D---- 299 rat: METTECIKGQGEGYRGTTNTIWNGIPCQRWDSQYPHKHDITPENFKCKDL 350 human: -------Q---------A---------------------M---------- 349
rat: RENYCRNPDGAESPWCFTTDPNIRVGYCSQIPKCDVSSGQDCYRGNGKNY 400 human: ----------S---------------------N--M-N------------ 399 rat: MGNLSKTRSGLTCSMWDKNMEDLHRHIFWEPDASKLTKNYCRNPDDDAHG 450 human: -----Q----------N-------------------NE------------ 449
28S-
m
W -HGF rat:
PWCYTGNPLVPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLI VVNGI 500
human: ---------I----------------------------------
18S-
----
499
rat: PTQTTVGWMVSLKYRNKHICGGSLIKESWVLTARQCFPARNKDLKDYEAW 550 human: --R-N----I--R-------------------------S- -------- 547 rat: LGIHDVHERGEEKRKQILNISQLVYGPEGSDLVLLKLARPAILDNFVSTI 600 human: -------G--------V--V--------------M------V--D--N-- 597 rat: DLPSYGCTIPEKTTCSIYGWGYTGLINADGLLRVAHLYIMGNEKCSQHHQ 650 human: ---N---------S--V----------Y---------------------R 647
FIG. 5. Northern hybridization analysis of poly(A)+ RNA (2 ,ug per lane) from various rat tissues. Samples from various rat tissues were electrophoresed in a 1% agarose/formaldehyde gel. RNAs were transferred to a Zeta-Probe filter and probed with the 32P-labeled 1.4-kb EcoRI insert of clone RBC1. Positions of 28S and 18S rRNA are shown at left. Lanes: 1, lung; 2, kidney; 3, pancreas; 4, brain; 5, submandibular gland; 6, thymus; 7, heart; 8, liver; 9, liver 15 hr after administration of CCl4.
rat:
GKVTLNESELCAGAEKIGSGPCEGDYGGPLICEQHKMRMVLGVIVPGRGC 700
human:
--------- I--------------------V-------------------
rat: AIPNRPGIFVRVAYYAKWIHKVILTYKL human: ---------------------I-----VPQS
697 728 728
FIG. 6. Alignment of amino acid sequences of rat and human HGF. Amino acid numbers are given at right. Identical amino acids are indicated by dashes in the sequence of human HGF.
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of rat HGF by glutamine and tyrosine, respectively. Similar replacements ofthe amino acids in the active site of the serine protease domain are found in protein Z (31) and haptoglobin (32), which have lost their proteolytic activities. On the other hand, neither plasminogen nor plasmin has HGF activity. Recently, other laboratories have also purified similar factors, hHGF (33) and HPTA (34), from human plasma. The molecular weights and subunit compositions of hHGF and HPT.A, estimated by SDS/PAGE, appear to be similar to that of HGF; all three factors are heat-labile proteins, and they bind to heparin and are eluted from it by '1 M NaCl. Also they reported the whole amino acid sequence of hHGF (35), which was deduced from the nucleotide sequence of cDNA derived from a placental cDNA library, and the N-terminal amino acid sequence of rabbit HPTA (36). There are a few differences between the amino acids sequence ofour human HGF and that of hHGF, but these differences are due to the existence of a few variants of human HGF (unpublished data). From the similarities of the biological and chemical properties and from the similarity of the amino acid sequences, we conclude that hHGF and HPTA are identical to HGF. Hepatotrophic factors for mature hepatocytes are known to increase in the sera of rats after partial hepatectomy (12, 14, 15). Epidermal growth factor and insulin have been shown to have growth-promoting activity for mature hepatocytes, but these factors did not increase in rat sera after partial hepatectomy. On the other hand, HGF increased in the sera of partially hepatectomized rats and hepatotoxin-treated rats (18). Recently, we found that the HGF that existed in normal rat liver markedly increased in liver injured by the administration of CC14 into rats, and the HGF activity in the injured liver paralleled the degree of liver injury (unpublished data). Northern blot analysis using the isolated HGF cDNA as probe confirmed that HGF mRNA was synthesized in the liver and that the amount of HGF mRNA dramatically increased in the injured liver after administration of CCl4 (Fig. 5). Recent work showed that HGF mRNA could be detected only in nonparenchymal cells in liver and that its level increased markedly after CCI treatment (37). From these observations, HGF appears to be a putative growth factor for mature hepatocytes, and hepatocyte growth during liver regeneration may be regulated by HGF in a paracrine fashion. In this paper, we showed that HGF mRNA is present not only in megakaryocytes and liver but also in various other tissues of the rat. It is possible that HGF produced in these other tissues also acts in liver regeneration via the blood circulation or that HGF plays an important role in the repair of these tissues themselves. The molecular cloning and expression of HGF should enable researchers to investigate the roles of HGF in the growth control of adult hepatocytes, to analyze and clone the HGF receptor, and to elucidate the molecular mechanism of liver regeneration in vivo. Moreover, HGF may be useful clinically for enhancing liver regeneration and for the diagnosis and therapy of patients with hepatitis. This work was supported by a Research Grant for Science and Cancer from the Ministry of Education, Science and Culture of Japan and by research grants from the Princess Takamatsu Cancer Research Fund, the Cell Science Research Foundation, and the Terumo Life Science Foundation. 1. Leong, G. F., Grisham, J. W., Hole, B. V. & Albright, M. L. (1964) Cancer Res. 24, 1496-1501. 2. Moolten, F. L. & Bucher, N. L. R. (1967) Science 158, 272274. 3. Fisher, B., Szuch, P., Levine, M. & Fischer, E. R. (1971) Science 171, 575-577. 4. Sakai, A. (1970) Nature (London) 228, 1186-1187.
Proc. Natl. Acad. Sci. USA 87 (1990) 5. Ichihara, A., Nakamura, T. & Tanaka, K. (1982) Mol. Cell. Biochem. 43, 145-160. 6. Guillouzo, A. & Guguen-Guillouzo, A. (1986) in Isolated and Cultured Hepatocytes (Libbey, London), pp. 1-397. 7. Tomita, Y., Nakamura, T. & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371. 8. Nakamura, T., Tomita, Y. & Ichihara, A. (1983) J. Biochem.
(Tokyo) 94, 1029-1035. 9. Michalopoulos, G., Cianciulli, H. D., Novotony, A. R., Kligerman, A. D., Strom, S. C. & Jirtle, R. L. (1982) Cancer Res. 42, 4673-4682. 10. McGowan, J. A., Strain, A. J. & Bucher, N. L. R. (1981) J. Cell. Physiol. 108, 353-363. 11. Hasegawa, K., Watanabe, K. & Koga, K. (1982) Biochem. Biophys. Res. Commun. 104, 259-265. 12. Nakamura, T., Nawa, K. & Ichihara, A. (1984) Biochem. Biophys. Res. Commun. 122, 1450-1459. 13. Michalopoulos, G., Houck, K. A., Dolan, M. L. & Luetteke, N. C. (1984) Cancer Res. 44, 4414-4419. 14. Thaler, F. J. & Michalopoulos, G. K. (1985) Cancer Res. 45, 2545-2549. 15. Diaz-Gil, J. J., Escartin, P., Garcia-Canero, R., Trilla, C., Veloso, J. J., Sanchez, G., Moreno-Caparros, A., Enrique De Salamanca, C., Lozano, R., Gavilanes, J. G. & Garcia-Segura, J. M. (1986) Biochem. J. 235, 49-55. 16. Russell, W. E., McGowan, J. A. & Bucher, N. L. R. (1984) J. Cell. Physiol. 119, 183-192. 17. Paul, D. & Piasecki, A. (1984) Exp. Cell Res. 154, 95-100. 18. Ando, T., Tomita, Y., Takai, S., Komi, N., Nakamura, T. & Ichihara, A. (1990) Hepatology, in press. 19. Selden, C., Jhostone, R., Darby, H., Gupta, C. & Hodgson, J. J. F. (1986) Biochem. Biophys. Res. Commun. 139, 361-366. 20. Nakamura, T., Teramoto, H. & Ichihara, A. (1986) Proc. Natl. Acad. Sci. USA 83, 6489-6493. 21. Nakamura, T., Nawa, K., Ichihara, A., Kaise, N. & Nishino, T. (1987) FEBS Lett. 224, 311-316. 22. Ling, N., Ying, S. Y., Ueno, N., Esch, F., Denorog, L. & Guillemin, R. (1985) Proc. Natl. Acad. Sci. USA 82,7212-7221. 23. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold
Spring Harbor, NY).
24. Cox, R. A. (1968) Methods Enzymol. 12, 120-129. 25. Benton, H. C. & Davis, R. W. (1977) Science 196, 180-182. 26. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 27. Pless, D. D. & Lernnarz, W. J. (1977) Proc. Natl. Acad. Sci. USA 74, 134-138. 28. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K. & Shimizu, S. (1989) Nature (London) 342, 440-443. 29. Sotrup-Jensen, L., Claeys, H., Zajdel, M., Peterson, T. E. & Magnussen, S. (1978) Progress in Chemical Fibrolysis and Thrombolysis, eds. Davidson, J. F., Roman, R. H., Samana, M. M. & Desroyers, P. C. (Raven, New York), Vol. 3, pp. 191-209. 30. Furie, B. & Furie, B. C. (1988) Cell 53, 505-518. 31. Hojurp, P., Jensen, M. S. & Peterson, T. E. (1985) FEBS Lett. 184, 333-338. 32. Kurosky, A., Barnett, D. R., Lee, T. H., Touchstone, B., Hay, R. E., Arnott, M. S., Bowman, B. F. & Fitch, W. M. (1980) Proc. Natl. Acad. Sci. USA 77, 3388-3392. 33. Gohda, E., Tsubouchi, H., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Miyazaki, H., Hashimoto, S. & Daikuhara, Y. (1988) J. Clin. Invest. 81, 414-419. 34. Zarnegar, R. & Michalopoulos, G. (1989) Cancer Res. 49, 3314-3320. 35. Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi, K., Okigaki, M., Arakaki, N., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Gohda, E., Daikuhara, Y. & Kitamura, N. (1989) Biochem. Biophys. Res. Commun. 163, 967973. 36. Zarnegar, R., Muga, S., Enghild, J. & Michalopoulos, G. (1989) Biochem. Biophys. Res. Commun. 163, 1370-1376. 37. Kinoshita, T., Tashiro, K. & Nakamura, T. (1989) Biochem. Biophys. Res. Commun. 165, 1229-1234.
Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues (liver regeneration/growth factor/hepatotrophic factor/hepatitis)
KOSUKE TASHIRO*, MITCHIO HAGIYAt, TSUTOMU NISHIZAWAt, TATSUYA SEKIt, MANABU SHIMONISHIt, SHIN SHIMIZUt, AND TOSHIKAZU NAKAMURA** *Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812, Japan; and tPharmaceutical Research Center, Toyobo Co., Ohtsu 520-02, Japan
Communicated by Gordon H. Sato, February 5, 1990
ABSTRACT The primary structure of rat hepatocyte growth factor (HGF) was elucidated by determining the base sequence of the complementary DNA (cDNA) of HGF. The cDNA for rat HGF was isolated by screening a liver cDNA library with oligonucleotides based on the partial N-terminal amino acid sequence of the P subunit of purified rat HGF. HGF is encoded in an mRNA of about 6 kilobases. Both a and 13 subunits of HGF are specified in a single open reading frame for a 728-amino acid protein with a calculated molecular weight of 82,904. The N-terminal part of HGF has a signal sequence and a prosequence with 30 and 25 amino acid residues, respectively. The mature heterodimer structure is derived proteolytically from this single pre-pro precursor polypeptide. The calculated molecular weights of the a and (3 subunits are 50,664 and 25,883, respectively, and each subunit has two potential N-lked glycosylation sites. The amino acid sequence of HGF is 38% identical with that of plasminogen. The a subunit of HGF contains four "Ikringle" structures, and the (3 subunit has 37% amino acid identity with the serine protease domain of plasmin. Northern blot analysis revealed that HGF mRNA was expressed in rat various tissues, including the liver, kidney, lung, and brain.
were shown to be markedly elevated in the blood plasma of rats with hepatitis induced by administration ofa hepatotoxin such as CC14 or D-galactosamine (18). In humans, similar findings were observed in the sera of patients after partial hepatectomy (19) and with fulminant hepatitis (33). HGF has been isolated as a homogeneous material from rat platelets and shown to be a heat-labile protein of -82 kDa, composed of a 69-kDa a subunit and a 34-kDa (3 subunit (20, 21). HGF at 1 ng/ml markedly stimulated DNA synthesis in adult rat hepatocytes and its effect was additive or synergistic with those of insulin and epidermal growth factor. HGF has no species specificity; for example, rat HGF also stimulated DNA synthesis in human, canine, and porcine hepatocytes in primary culture. In contrast, HGF did not stimulate DNA synthesis in established cell lines such as Swiss mouse 3T3 fibroblasts and BRL (buffalo rat liver) epithelial cells. Here we report the complete amino acid sequence of rat HGF obtained by cloning and sequence analysis of its cDNA.§ We have found that HGF mRNA is expressed in various rat tissues including the kidney, heart, lung, and brain, as well as in injured liver. MATERIALS AND METHODS Materials. Materials used for the purification of HGF were as described (20). The cDNA synthesis system and the multiprime labeling system were from Amersham. Gigapack Gold, AgtlO and M13 phage, and pBluescript were from Stratagene. Enzymes for DNA manipulation were obtained from Toyobo and New England Biolabs. Radioisotopic materials were from Amersham. Purification and Sequencing ofRat HGF. The purification of HGF from rat platelets was essentially as reported (20). Briefly, the procedures involved the following successive steps: stimulation of HGF release from rat platelets by thrombin, cation-exchange FPLC on a Mono S column, affinity chromatography on heparin-Sepharose CL-6B, and reverse-phase HPLC on a C4 column. Purification was monitored by the HGF activity, which was defined as the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture (8). The yields were about 60 tkg of pure HGF from the platelets of 3000 rats, corresponding to a recovery of 23%. The a and P subunits of HGF were separated by reverse-phase HPLC on a C4 column after reduction and carboxymethylation (22) of purified HGF. The subunits were separately subjected to N-terminal amino acid sequence analysis with an Applied Biosystems model 470A sequencer. Synthesis and Labeling of Oligonucleotides. Oligonucleotides corresponding to parts of the amino acid sequence of the
Liver regenerates actively after partial hepatectomy and liver injury. Early investigations (1-4) showed that hepatotrophic factors existed in the blood of partially hepatectomized animals. However, no one succeeded in purifying and characterizing such a humoral factor until recently, largely because no simple, reproducible, and sensitive assay for the factor has been available. Although many in vitro experimental methods using established liver and hepatoma cell lines have been employed in attempts to identify the hepatotrophic factor, these assays are unsuitable because these cell lines have lost almost all liver functions and normal growth properties. In contrast, adult rat hepatocytes in primary culture retain many normal liver functions and respond to various hormones, behaving more like the in vivo cells (5, 6). Moreover, they can proliferate in culture at low cell density when a mitogen such as epidermal growth factor is added to the culture medium (7-11). Primary cultured adult hepatocytes are thus the most suitable system for identification and purification of a growth factor for mature parenchymal hepatocytes. Applying this system, Nakamura et al. (12) were able to isolate a factor from the sera of partially hepatectomized rats that stimulated DNA synthesis in adult rat hepatocytes, which was named hepatocyte growth factor (HGF). Other investigators found a hepatotrophic factor in the sera of partially hepatectomized rats (13-15) and in rat platelets (16, 17). Recently, HGF levels
Abbreviation: HGF, hepatocyte growth factor. tTo whom reprint requests should be addressed. §The sequence reported in this paper has been deposited in the GenBank data base (accession no. M32987).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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N-terminal portion of the HGF /B subunit were synthesized on an Applied Biosystems DNA synthesizer (model 381A) with the procedures and reagents recommended by the manufacturer. Oligonucleotides were end-labeled with T4 polynucleotide kinase and [y-32P]ATP (23) and used for screening a cDNA library Extraction of Poly(A)+ RNA. Total RNA was extracted from rat tissues by the guanidinium chloride method (24). Poly(A)+ RNA was purified by chromatography on oligo(dT)-cellulose (23). Usually, 3 mg of total RNA was obtained from 1 g of liver, which yielded about 30 Ag of poly(A)+ RNA. Construction and Screening of cDNA Libraries. Poly(A)+ RNA was purified from rat liver 15 hr after administration of 20% CCL4 (1 ml/100 g of body weight) and used for the construction of a cDNA library. Double-stranded cDNA was synthesized with oligo(dT) as a primer, methylated by EcoRI methylase, and ligated with an EcoRI linker. After EcoRI digestion, the cDNAs were ligated into EcoRI-digested and phosphatase-treated AgtlO arms and packaged in vitro using Gigapack Gold. Two micrograms of poly(A)+ RNA yielded 2 x 106 phage transformants. Replica filters were prepared (25) and hybridized with the 32P-end-labeled 44-mer probe (Fig. 1) for 12 hr at 450C in 5x SSC (lx SSC is 0.15 M NaCl/0.015 M sodium citrate, pH 7.0) containing 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.1% SDS, and 100 ,g of denatured salmon testis DNA per ml. The filters were then washed at 50°C in 5x SSC/0.1% SDS, air-dried, and autoradiographed on Fuji x-ray film. The oligonucleotide-primed cDNA library was constructed as described above, except that the oligodeoxynucleotide 5'-AAATCCTCCATATTCTTGTC-3' was used as a primer. Recombinant phages were screened using the 32P-labeled 1.4-kilobase (kb) EcoRI fragment of cDNA clone RBC1 (multiprime labeling kit, Amersham) as a probe according to Maniatis et al. (23). Determination of Nucleotide Sequence. The DNAs of the isolated phage clones were extracted (23), digested with the appropriate restriction enzyme, and then electrophoresed in a 1% agarose gel. The digested fragments were extracted from the gel, subcloned into M13mpl8, M13mpl9, or pBluescript, and subjected to nucleotide sequence determination (26). Northern Hybridization. Poly(A)+ RNAs (2 ,ug) purified from various rat tissues were denatured with formaldehyde and electrophoresed in a 1% agarose/0.7 M formaldehyde gel (23). RNAs were transferred to a Zeta-Probe filter (Bio-Rad) and hybridized with the 32P-labeled 1.4-kb EcoRI fragment of RBC1 for 15 hr at 42°C in 50% (vol/vol) formamide/5 x SSC/10 mM sodium phosphate, pH 6.8/0.5% SDS containing 500 ,g of salmon sperm DNA per ml. The filter was then washed three times with 0.2x SSC/0.1% SDS for 30 min at 60°C, air-dried, and autoradiographed on Fuji x-ray film.
RESULTS Isolation of a cDNA Encoding Rat HGF. We isolated a and ,B subunits from 60 ,ug of pure HGF and determined 19 and 27 amino acid residues of the N-terminal portions, respectively (Fig. 1 a and b). These amino acid sequences had no homology to known growth factors (GenBank, August 1989). As a probe for the isolation of the cDNA encoding rat HGF, a 44-mer oligonucleotide was synthesized based on amino acid residues 1-15 of the / subunit (Fig. lc). It contained deoxyinosine at the positions where the nucleotide could not be specified because of codon degeneracy. We chose injured liver as a source of poly(A)+ RNA for the construction of the cDNA library, because we recently found that there was a large amount of HGF in the injured liver produced by the administration of CC14 into rats, and the HGF purified from liver was identical to platelet-derived HGF, judging by their chemical and biological properties, including the N-terminal amino acid sequence (unpublished
a)
3201
10
Pro-Leu-Val- Lys- lIe- Lys-Th r-Lys-Lys-VaI-Asn-Ser-AIa-Asp19
15
Glu-Cys-Ala-Asn-Arg b)
10
1
Val-Val-Asn-GIy- Ile-Pro-Th r-G In-Thr-Th r-Val-GIy-Trp-Met27
20
15
Val-Ser-Leu-Lys-Tyr-Arg-Asn-Lys-His-IIe-Cys-GIy-GIy C)
CAICAITTICCITAIGGITGIGTITGITGICAICCIACCTACCA
FIG. 1. (a and b) N-terminal amino acid sequences of the a and
,8 subunits, respectively, of purified rat HGF. (c) Sequence (3' to 5') of the 44-mer oligodeoxynucleotide probe synthesized on the basis of the N-terminal amino acid sequence of the 83 subunit. I, deoxyinosine.
data). Therefore, in order to isolate the cDNA encoding HGF, a cDNA library was constructed using poly(A)+ RNA extracted from rat liver 15 hr after administration of CCl4 This cDNA library was screened with the 32P-end-labeled 44-mer oligonucleotide. The screening of 2 x 106 recombinant phages yielded one positive clone, RBC1 (Fig. 2). The insert of RBC1 was 1.4 kb long and contained an open reading frame encoding 404 amino acid residues, followed by a 3' noncoding sequence. The 27-amino acid sequence of the N-terminal portion of the ( subunit was confirmed to be in this open reading frame. This 1.4-kb insert contained the full 233-amino acid sequence of the 3 subunit of rat HGF. The 1.4-kb insert coded for a further 171 amino acid residues in front of the ,/-subunit sequence, but no ATG start codon was observed in this reading frame. Moreover, Northern blot analysis of poly(A)+ RNA from the livers of CC14treated rats with the RBC1 cDNA insert as the probe revealed that the rat HGF mRNA was about 6.0 kb long (see Fig. 5). Therefore, clone RBC1 did not contain the whole sequence of rat HGF. To isolate the full-length cDNA for HGF, another cDNA library was constructed by the primerextension method, using a 20-mer oligonucleotide as a primer (noncoding strand, positions 1249-1268 in Fig. 3). The oligonucleotide-primed cDNA library was screened with the 1.4kb insert of RBC1 as a probe. Screening of 2 X 105 recombinant phages from the oligonucleotide-primed cDNA library and of 106 recombinant phages from the oligo(dT)-primed cDNA library yielded two (RAC1 and RAC2) and some (RBC3) positive clones, respectively (Fig. 2). The nucleotide sequence of the inserts of these E
B
6
EA
EA
0.'5 a
HBH
1
It
1 111
1.
1.5
I
(1.32 Kb) I
-
5.8 Kb
2.
(0.70Kb)
b
3.58 Kb
(A)n 3' RBC1
, ,RBC3 , RAC1
,
RAC2
FIG. 2. Schematic representation of rat HGF mRNA and cDNA. Restriction sites (B, Bgl II; E, EcoRI; A, Ava I; H, HindIII) are shown on the kilobase scale. The rat HGF mRNA is diagrammed below the scale. Noncoding sequences are represented by a line and the coding region is boxed. Black and hatched areas show the signal peptide and the prosequence, respectively. Open box indicates the coding regions for the a and /3 subunits. cDNA clones are represented below the mRNA; small black boxes indicate the location of the 20-mer primer on clones RBC1, RACi, and RAC2.
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clones revealed that the 1.4-kb insert of RACi contained the ATG initiation codon as well as 120 bases of 5' upstream sequence. The inserts of RBC3 encoded part of the Cterminal portion of the /3 subunit and contained a long 3' noncoding region (3.58 kb). Both RACi and RAC2 had an open reading frame coding for the N-terminal sequence of the a chain, which was determined by protein sequencing. Thus, the overlapping clones RACi, RBC1, and RBC3 covered the entire coding region and the 3' untranslated region. The complete cDNA and translated protein sequence of rat HGF are shown in Fig. 3. It contains two possible initiation codons at nucleotides 1-3 and 4-6 and a stop codon (TAA) at nucleotides 2185-2187. The first methionine was designated as residue 1 in the amino acid sequence.
Proc. Natl. Acad. Sci. USA 87 (1990)
Primary Structure of Rat HGF. Rat HGF cDNA encodes a polypeptide of 728 amino acids. The structure of pre-proHGF is represented in Fig. 4. Both the a and the P chain are encoded in a single open reading frame. Fifty-five amino acid
residues lie in front of the N terminus of the a subunit. The first 30 residues have hydrophobic side chains, characteristic of a signal peptide, and the next 25 residues appear to constitute a prosequence. The C terminus of the a subunit is followed directly by the N terminus of the /3 subunit. The sequence at the cleavage site between the a and p subunits is Arg-Val (residues 495 and 496), and this sequence may be recognized and cleaved by a trypsinlike protease. We condlude that the rat HGF precursor is synthesized as a prepro-protein and that mature HGF is formed by proteolytic GTTTAGTCCTAGATCTTTCCAGT
TAATCCACACAAATTAGCCATCCAATAAAGCGCTCGAACGACCGCTTGAACAGATTTTTCACCCGCATCCCTGCGACCATCACCTGCCGAATGCAGC 1 10 20 ATG ATG TGG GGG ACC AAA CTT CTG CCG Met Met Trp Gly Thr Lys Leu Leu Pro --ProGGA CAG AAG AAG AGA AGA AAT ACT IGiu Gly Gin Lys Lys Arg Arg Asn Thr
CTG TTG CTG CAG CAT GTC CTG CTG CAC CTC CTC Leu Leu Leu Gin His Val Leu Leu His Leu Leu 50 CAT GAA TTC AAA AAG TCA GCA AAA ACT ACT CTT His Giu Phe Lys Lys Ser Ala Lys Thr Thr Leu 80 TGT GCC AAC AGG TGC ATC AGA AAC AAG GGC TTT Cys Ala Asn Arg Cys Ilie Arg Asn Lys Gly Phe 110 CCT TTC AAT AGT ATG TCA AGT GGA GTG AAA AAA Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys 140 ATT GGT AAA GGA GGC AGC TAT AAG GGG ACA GTA Ilie Gly Lvs Gly Gly Ser Tvr Lys Gly Thr Val 170 CAC AGC TTT TTG CCT TCG AGC TAT CGC GGT AAA His Ser Phe Leu Pro Ser Ser Tyr Ara Gly Lys 200 TTC ACA AGC AAT CCA GAG GTA CGC TAC GAA GTC Phe Thr Ser Asn Pro Glu Val Ara Tyr Glu Val 230 AGA GGT CCC ATG GAT CAC ACA GAA TCA GGC AAG Ara Gly Pro Met Asp His Thr Glu Ser Gly Lvs 260 TAT CCC GAC AAG GGC ITT GAT GAT AAT TAT TGC Tvr Pro Asp Lvs Gly Phe Asp Asp Asn Tvr Cvs 290 TGG GAG TAT TGT GCA ATT AAA ATG TGC GCT CAC Tro Glu Tvr Cvs Ala Ilie Lvs Met Cvs Ala His 320 GGA GAA GGT TAC AGG GGA ACC ACC AAT ACC ATT Gly Glu Gly Tvr Ara Gly Thr Thr Asn Thr Ilie 350 ACT CCC GAG AAC TIC AAA TGC AAG GAC CIT AGA Thr Pro Glu Asn Phe Lvs Cvs Lys Ase Leu Ara 380 CCA AAC AIC CGA GTl GGT TAC TGC ICT CAA All Pro Asn Ilie Ara Val Gly Tyr Cvs Ser Gin Ilie 410 AIG GGC AAC ITA ICC AAA ACA AGG ICT GGA CIC Met Gly Asn Leu Ser Lvs Thr Ara Ser Gly Leu 4 40 CCA GAC GCT AGC AAG TIG ACT AAG AAT TAC TGC Pro Asp Ala Ser Lys Leu Thr Lys Asn Tyr Cvs 470 CCI TGG GAT TAT TGC CCI ATT ICC CGT IGI GAA Pro Iry Ase Tyr Cvs Pro le Ser Ara Cvs§ Giu 500 ACA AAA CAA CIG CGA G TlGI AAT GGC All CCA Thr Lys Gin Leu Airg IVal Val Asn Gly Ilie Pro 530 GGG GGA ICA TIG AlA AAG GAA AGI TGG GTl CIT Gly Gly Ser Leu Ilie Lys Giu Ser Trp Val Leu 560 CIT GGA AIC CAT GAl GIC CAT GAG AGA GGC GAG Leu Gly Ilie His Asp Val His Giu Arg Gly Giu 590 GAl TIG GTl TIA CIG AAG CIT GCT CGC CCI GCA Asp Leu Val Leu Leu Lys Leu Ala Arg Pro Ala 620 GAA AAG ACT ACT TGC AGI All TAC GGC TGG GGC Giu Lys Thr Ihr Cys Ser Ilie Tyr Gly Trp Gly 650 All AIG GGG AAT GAG AAA TGC AGI CAG CAC CAT CAA GGC Ilie Met Gly Asn Giu Lys Cys Ser Gin His His Gin Gly 670 ~...680 ICA GGA CCI IGI GAG GGA GAlTT GGT GGC CCA CIC All Ser Gly Pro Cys Glu Gly Asp \ lr.Gy Gly Pro Leu Ilie 700 710 GGA IGI GCC AIC CCA AAT CGT CCI GGT All ITT GTl CGA Gly Cys Ala Ilie Pro Asn Arg Pro Gly Ilie Phe Val Arg
GTC Val 40 CTT Leu 70 AAA ACC AAA AAA GTG AAC TCT GCA GAT GAG Lys Thr Lys Lys Val Asn Ser Ala Asp Gin 100 GAT AAG TCG AGA AAA CGA TGC TAC TGG TAT Ser Asp Lys Arg Lys Arg Cys Tyr Trp Tyr 130 GAA AAC AAA GAC TAT ATT AGA AAT TGC ATC Giu Asn Lys Asp Tyr Ilie Arg Asn Cvs Ilie Ki 160 CAG CCT TGG AAT TCC ATG ATC CCC CAT GAA Gin Pro TrD Asn Ser Met Ilie Pro His Glu 190 CCT CGA GGG GAA GAA GGG GGA CCC TGG TGT Pro Ara Gly Glu Glu Gly Gly Pro Tro Cvs 220 GAA TGC ATG ACC TGC AAC GGT GAA AGC TAC Giu Cvs Met Thr Cvs Asn Gly Glu Ser Tyr K2 250 CCA CAC CGG CAC AAA TTC TTG CCG GAA AGA Pro his Ara His Lys Phe Leu Pro Glu Ara 280 TGG IGC TAC ACT CTT GAC CCT GAC ACC CCT Tre Cvs Tyr Thr Leu Asp Pro Aso Thr Pro 310 ATG GAA ACA ACT GAA TGT ATA AAA GGC CAA Met Giu Thr Thr Giu Cvs Ilie Lys Gly Gin 340 K3 GAT TCG CAG TAC CCC CAC AAG CAT GAC ATC Aisp Ser Gin Tyr Pro His Lys His Asp Ilie 370 GCT GAA ICA CCA IGG IGI ITT ACC ACT GAT Ala Glu Ser Pro Ire Cvs Phe Thr Thr Asp 400 GAT IGI TAT CGT GGC AAT GGG AAA AAC TAC Asp Cvs Tyr Ara Gly Asn Gly Lvs Asn Tyr 430 K4 GAl GTITA CAC CGI CAT AIC TIC TGG GAG Leu His His Ire GuAsp Ara le Phe Glu 460 CCI TGG TGC TAC ACA GGG AAT CCI CIC GTl Pro Ire Cvs Tyr Thr Gly Asn Pro Leu Val 490 TIG GAC CAT CCI GIA AlA ICC ~GCC AAA Leu Asp His Pro Val Ilie SerL sAla Lys 520 AGI TIG AAA TAC AGG AAT AAA CAC AIC IGI Ser Leu Lys Tyr Arg Asn Lys His Ilie Cys 550 AAC AAA GAC TIG AAA GAC TAT GAA GCT IGG Asn Lys Asp Leu Lys Asp Trp Giu Ala Irp 580 ICC CAG CTA GIC TAT GGA CCT GAA GGC ICA §_~ Gin Leu Val Tyr Gly Pro Giu Gly Ser 610 GAT TIA CCI AGI TAT GGC T.GTL ACA AIC CCI Leu Pro [~JThr Pro Ser Ilie Asp Tyr Gly
~GAA
ITA TIA CGA GIA GCT CAT CIG TAT Leu Leu Arg Val Ala His Leu Tyr IGI GCT GGG GCT GAA AAG All GGA Cys Ala Gly Ala Giu Lys Ilie Gly
CTG CTT CCT GTC ACC ATC Leu Leu Pro Val Thr Ilie - ( ACC AAG GAA GAC CCA TTA Thr Lys Giu Asp IPro Leu
CCA TTC ACT TGC AAG GCC Pro Phe Thr Cys Lys Ala
GGG TTT GGC CAT GAA TTT
30 CCC TAT GCA Pro Tyr Ala 60 GTG AAG ATT Val Lys Ile 90 TTT GTT TTT Phe Val Phe 120 GAC CTC TAT
Gly Phe Gly His Giu Phe Asp Leu Tyr
150 TCC ATC ACT AAG AGT GGC ATC AAG TGC Ser Ilie Thr Lys Ser Gy Ilie Lys Cvs 180 GAC CTA CAG GAA AAC TAC TGT CGA AAT Leu Gin Glu Asn Tyr Cvs Ara Asn Asp 210 TGT GAC ATT CCT CAG TGT TCA GAA GTT Cvs Asp Ilie Pro Gin Cvs Ser Giu Val 240 ACA TGT CAG CGC TGG GAT CAG CAG ACA Thr Cvs Gin Ara Tro Asp Gin Gin Thr 270 CGC AAT CCC GAT GGC AAG CCG AGG CCA Ara Asn Pro Asp Gly Lys Pro Ara Pro 300 AGT GCT GTG AAT GAG ACT GAT GTT CCC Ser Ala Val Asn Giu Thr Asp Val Pro 330 TGG AAT GGA ATT CCG TGT CAG CGT TGG Tre Asn Gly le Pro Cvs Gin Ara Tre
-120 -1 90
180 270 360 450 540 630
720 810 900
990
360
GAA AAT TAT
IGC CGC AAT CCG GAl GGG
Glu Asn Tyr Cvs Ara Asn Pro Asp Gly
390 CCC AAA IGI GAC GIG ICA AGI GGA CAA Pro Lvs Cvs Asp Val Ser Ser Gly Gin 420 ACA IGI ICC AIG TGG GAC AAG AAI AIG Thr Cvs Ser Met Ireso v s e 450 CGG AAC CCC GAT GAC GAC GCC CAT GGA Ara Asn Pro Asp Asp Asp Ala His Gly 480 GGA GAl ACT ACA CCI ACA All GIC AAT Thr Thr Ihr Pro Val Ilie Asn Gly Asp 510 ACA CAA ACA ACA GIA GGG TGG AIG GTl Thr Gin Thr Thr Val Gly Trp Met Val 540 ACT GCA CA'TGT ITT CCA GCT AGA Thr Ala Arg \Q~Cys Phe Pro Ala Arg 570 GAG AAA CGC AAA CAG AIC TIA AAC All Giu Lys Arg Lys Gin Ilie Leu Asn Ilie
AGG(
AIC CIG GAl AAC III GIC AGI ACA AlT Ilie Leu Asp Asn Phe Val Ser Thr Ilie 630 TAC ACT GGA TIG AIC AAC GCA GAl GGT Thr Leu Ala Ilie Asn Tyr Gly Asp Gly 660 AAG GIG ACT TIG AAT GAG ICT GAA TIA Lys Val Thr Leu Asn Giu Ser Giu Leu 690 IGI GAA CAA CAC AAA AIG AGA AIG GTl Cys Giu Gin His Lys Met Arg Met Val 720 GIA GCA TAT TAT GCA AAA IGG AlA CAC Val Ala Tyr Tyr Ala Lys Trp Ilie His
CIT GGT GIC All GTl CCI GGT CGT Leu Gly Val Ilie Val Pro Gly Arg 728 AAA GIA All TIG ACA TAC AAG TIG TAATAGCCATAGAAGAGGCCAGTGTATTTGAAGCATCCATGGATACAGGAAGATTTCCAAGACTTCAGGATTAAAATGTCACCTAAA
1080 1170 1260 1350 1440
1530 1620 1710
1800 1890 1980
2070 2160 2271
FIG. 3. Nucleotide sequence of rat HGF cDNA and the deduced amino acid sequence. Nucleotide numbers are at right and amino acid numbers are given above the sequence. Underlines indicate the kringle domains. The primer for the construction of the oligonucleotide-primed cDNA library is shown by the broken underline. The putative N-glycosylation sites are marked by wavy lines. The glutamine at position 535 and the tyrosine at 676 (circled) correspond to the residues of the active center in plasmin. The cysteine residues that form an interchain disulfide bond between the a and 83 subunits are boxed.
Cell Biology: Tashiro et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
Arm
a-subunit
Val-
s S
N p -subunit
Leu
FIG. 4. Representation of rat pre-pro-HGF structure. The thick line and the wavy line represent the signal peptide and the prosequences, respectively. Sites of proteolytic cleavage are indicated by arrows.
cleavage. The a and /3 chains consist of 440 and 233 amino acids, respectively. The calculated molecular weight of the mature HGF after processing ofthe signal peptide and the prosequence is 76,529. The molecular weights of the a and a subunits are 50,664 and 25,883, respectively. These values are smaller than those determined by SDS/PAGE. Each subunit contains two AsnXaa-Thr/Ser sequences, potential sites for N-linked glycosylation (27); these are at residues 240-242 and 348-350 of the a subunit and at residues 74-76 and 161-163 of the /3 subunit. Thus, the differences between the molecular weights determined by SDS/PAGE and those calculated from the cDNAs may be due to the glycosylation of each subunit. HGF mRNA Expression in Rat Tissues. Recently, we examined the tissue distribution of HGF-like activity in rats and found that an HGF-like growth factor exists in various other tissues in addition to the liver (data not shown). We examined the expression of HGF mRNA in rat tissues by extracting poly(A)+ RNAs from the various tissues and analyzed them by Northern hybridization with the 1.4-kb EcoRP insert of RBC1 as a probe (Fig. 5). Rat HGF mRNA (-6 kb) was expressed in lung, kidney, and thymus as well as liver. Interestingly, we also detected HGF mRNA in samples from brain. The level of HGF mRNA in the liver increased markedly when CC14 was injected intraperitoneally into rats (Fig. 5). The HGF mRNA increased gradually in the liver from 5 hr after the administration of CCL4 and reached a maximum at 10-12 hr (data not shown).
DISCUSSION In this paper, we show the primary structure of rat HGF deduced from the nucleotide sequence of its cDNA. The deduced sequence contains the N-terminal sequences of both subunits of rat HGF, which were determined by sequence analysis of purified rat HGF. In the sequence of the HGF precursor, the a-subunit peptide is followed directly by the 1
2 3 4 5 6 7 8 9
3203
/3-subunit peptide. Moreover, the signal peptide and the prosequence immediately precede the N terminus of the a subunit. Therefore, HGF is synthesized as a pre-pro precursor. Presumably, the signal peptide and the prosequence are removed by processing, and the a and /3 subunits are formed by cleavage of the Arg-Val (positions 495 and 496) peptide bond by a trypsinlike protease. Recently, we determined the primary structure of human HGF by cloning and sequence analysis ofthe cDNA (28). The deduced amino acid sequence of human HGF is >90% identical with that of rat HGF (Fig. 6). There are some insertions and deletions in the amino acid sequence of the 83 subunit of human and rat. Therefore, the length of the / subunit of human and rat HGF is 234 and 233 amino acid residues, respectively. We also succeeded in expressing biologically active human HGF in COS-1 cells transfected with the plasmid carrying the entire protein-coding sequence of human HGF cDNA. Whereas HGF has no homology with various known growth factors, it has high homology with plasminogen (29). The amino acid sequence identity between rat pro-HGF and human plasminogen was calculated to be 38%. The a subunit of rat HGF has four "kringle" structures, like the A chain of plasminogen, which has five kringle domains. Kringle structures are also found in tissue plasminogen activator, urokinase, coagulation factor XII, and prothrombin (30). Although the kringle domain is thought to play a role in the interaction between macromolecules, its real functions are unknown at present. The / subunit of rat HGF shows a high degree of homology with various serine proteases, including the B chain of plasmin. However, the histidine and serine residues of the active sites of the proteases are replaced in the ,8 chain rat:
human: rat:
pro
MMWGTKLLPVLLLQHVLLHLLLLPVTIPYE GQKKRRNTLHEFKKSAKTT --V-----IA-IA--- -H------I---------a LTKE PLVKIKTKKVNSADECANRCIRNKGFPFTCKAFVFDKSRKRCYWY
human: -I-I +AL--------T--Q-----T--N-L-----------A--Q-L-F
50 49 100 99
PFNSMSSGVKKGFGHEFDLYENKDYIRNCIIGKGGSYKGTVSITKSGIKC 150 rat: human: -----------E----------------------R--------------- 149 rat:
human:
QPWNSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYE 200 199
rat: VCDIPQCSEVECMTCNGESYRGPMDHTESGKTCQRWDQQTPHRHKFLPER 250 human: -----------L--------I-----H------------ 249
rat: YPDKGFDDNYCRNPDGKPRPWCYTLDPDTPWEYCAIKMCAHSAVNETDVP 300 human: ----------------Q----------H-R-------T--DNT--D---- 299 rat: METTECIKGQGEGYRGTTNTIWNGIPCQRWDSQYPHKHDITPENFKCKDL 350 human: -------Q---------A---------------------M---------- 349
rat: RENYCRNPDGAESPWCFTTDPNIRVGYCSQIPKCDVSSGQDCYRGNGKNY 400 human: ----------S---------------------N--M-N------------ 399 rat: MGNLSKTRSGLTCSMWDKNMEDLHRHIFWEPDASKLTKNYCRNPDDDAHG 450 human: -----Q----------N-------------------NE------------ 449
28S-
m
W -HGF rat:
PWCYTGNPLVPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLI VVNGI 500
human: ---------I----------------------------------
18S-
----
499
rat: PTQTTVGWMVSLKYRNKHICGGSLIKESWVLTARQCFPARNKDLKDYEAW 550 human: --R-N----I--R-------------------------S- -------- 547 rat: LGIHDVHERGEEKRKQILNISQLVYGPEGSDLVLLKLARPAILDNFVSTI 600 human: -------G--------V--V--------------M------V--D--N-- 597 rat: DLPSYGCTIPEKTTCSIYGWGYTGLINADGLLRVAHLYIMGNEKCSQHHQ 650 human: ---N---------S--V----------Y---------------------R 647
FIG. 5. Northern hybridization analysis of poly(A)+ RNA (2 ,ug per lane) from various rat tissues. Samples from various rat tissues were electrophoresed in a 1% agarose/formaldehyde gel. RNAs were transferred to a Zeta-Probe filter and probed with the 32P-labeled 1.4-kb EcoRI insert of clone RBC1. Positions of 28S and 18S rRNA are shown at left. Lanes: 1, lung; 2, kidney; 3, pancreas; 4, brain; 5, submandibular gland; 6, thymus; 7, heart; 8, liver; 9, liver 15 hr after administration of CCl4.
rat:
GKVTLNESELCAGAEKIGSGPCEGDYGGPLICEQHKMRMVLGVIVPGRGC 700
human:
--------- I--------------------V-------------------
rat: AIPNRPGIFVRVAYYAKWIHKVILTYKL human: ---------------------I-----VPQS
697 728 728
FIG. 6. Alignment of amino acid sequences of rat and human HGF. Amino acid numbers are given at right. Identical amino acids are indicated by dashes in the sequence of human HGF.
3204
Cell Biology: Tashiro et al.
of rat HGF by glutamine and tyrosine, respectively. Similar replacements ofthe amino acids in the active site of the serine protease domain are found in protein Z (31) and haptoglobin (32), which have lost their proteolytic activities. On the other hand, neither plasminogen nor plasmin has HGF activity. Recently, other laboratories have also purified similar factors, hHGF (33) and HPTA (34), from human plasma. The molecular weights and subunit compositions of hHGF and HPT.A, estimated by SDS/PAGE, appear to be similar to that of HGF; all three factors are heat-labile proteins, and they bind to heparin and are eluted from it by '1 M NaCl. Also they reported the whole amino acid sequence of hHGF (35), which was deduced from the nucleotide sequence of cDNA derived from a placental cDNA library, and the N-terminal amino acid sequence of rabbit HPTA (36). There are a few differences between the amino acids sequence ofour human HGF and that of hHGF, but these differences are due to the existence of a few variants of human HGF (unpublished data). From the similarities of the biological and chemical properties and from the similarity of the amino acid sequences, we conclude that hHGF and HPTA are identical to HGF. Hepatotrophic factors for mature hepatocytes are known to increase in the sera of rats after partial hepatectomy (12, 14, 15). Epidermal growth factor and insulin have been shown to have growth-promoting activity for mature hepatocytes, but these factors did not increase in rat sera after partial hepatectomy. On the other hand, HGF increased in the sera of partially hepatectomized rats and hepatotoxin-treated rats (18). Recently, we found that the HGF that existed in normal rat liver markedly increased in liver injured by the administration of CC14 into rats, and the HGF activity in the injured liver paralleled the degree of liver injury (unpublished data). Northern blot analysis using the isolated HGF cDNA as probe confirmed that HGF mRNA was synthesized in the liver and that the amount of HGF mRNA dramatically increased in the injured liver after administration of CCl4 (Fig. 5). Recent work showed that HGF mRNA could be detected only in nonparenchymal cells in liver and that its level increased markedly after CCI treatment (37). From these observations, HGF appears to be a putative growth factor for mature hepatocytes, and hepatocyte growth during liver regeneration may be regulated by HGF in a paracrine fashion. In this paper, we showed that HGF mRNA is present not only in megakaryocytes and liver but also in various other tissues of the rat. It is possible that HGF produced in these other tissues also acts in liver regeneration via the blood circulation or that HGF plays an important role in the repair of these tissues themselves. The molecular cloning and expression of HGF should enable researchers to investigate the roles of HGF in the growth control of adult hepatocytes, to analyze and clone the HGF receptor, and to elucidate the molecular mechanism of liver regeneration in vivo. Moreover, HGF may be useful clinically for enhancing liver regeneration and for the diagnosis and therapy of patients with hepatitis. This work was supported by a Research Grant for Science and Cancer from the Ministry of Education, Science and Culture of Japan and by research grants from the Princess Takamatsu Cancer Research Fund, the Cell Science Research Foundation, and the Terumo Life Science Foundation. 1. Leong, G. F., Grisham, J. W., Hole, B. V. & Albright, M. L. (1964) Cancer Res. 24, 1496-1501. 2. Moolten, F. L. & Bucher, N. L. R. (1967) Science 158, 272274. 3. Fisher, B., Szuch, P., Levine, M. & Fischer, E. R. (1971) Science 171, 575-577. 4. Sakai, A. (1970) Nature (London) 228, 1186-1187.
Proc. Natl. Acad. Sci. USA 87 (1990) 5. Ichihara, A., Nakamura, T. & Tanaka, K. (1982) Mol. Cell. Biochem. 43, 145-160. 6. Guillouzo, A. & Guguen-Guillouzo, A. (1986) in Isolated and Cultured Hepatocytes (Libbey, London), pp. 1-397. 7. Tomita, Y., Nakamura, T. & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371. 8. Nakamura, T., Tomita, Y. & Ichihara, A. (1983) J. Biochem.
(Tokyo) 94, 1029-1035. 9. Michalopoulos, G., Cianciulli, H. D., Novotony, A. R., Kligerman, A. D., Strom, S. C. & Jirtle, R. L. (1982) Cancer Res. 42, 4673-4682. 10. McGowan, J. A., Strain, A. J. & Bucher, N. L. R. (1981) J. Cell. Physiol. 108, 353-363. 11. Hasegawa, K., Watanabe, K. & Koga, K. (1982) Biochem. Biophys. Res. Commun. 104, 259-265. 12. Nakamura, T., Nawa, K. & Ichihara, A. (1984) Biochem. Biophys. Res. Commun. 122, 1450-1459. 13. Michalopoulos, G., Houck, K. A., Dolan, M. L. & Luetteke, N. C. (1984) Cancer Res. 44, 4414-4419. 14. Thaler, F. J. & Michalopoulos, G. K. (1985) Cancer Res. 45, 2545-2549. 15. Diaz-Gil, J. J., Escartin, P., Garcia-Canero, R., Trilla, C., Veloso, J. J., Sanchez, G., Moreno-Caparros, A., Enrique De Salamanca, C., Lozano, R., Gavilanes, J. G. & Garcia-Segura, J. M. (1986) Biochem. J. 235, 49-55. 16. Russell, W. E., McGowan, J. A. & Bucher, N. L. R. (1984) J. Cell. Physiol. 119, 183-192. 17. Paul, D. & Piasecki, A. (1984) Exp. Cell Res. 154, 95-100. 18. Ando, T., Tomita, Y., Takai, S., Komi, N., Nakamura, T. & Ichihara, A. (1990) Hepatology, in press. 19. Selden, C., Jhostone, R., Darby, H., Gupta, C. & Hodgson, J. J. F. (1986) Biochem. Biophys. Res. Commun. 139, 361-366. 20. Nakamura, T., Teramoto, H. & Ichihara, A. (1986) Proc. Natl. Acad. Sci. USA 83, 6489-6493. 21. Nakamura, T., Nawa, K., Ichihara, A., Kaise, N. & Nishino, T. (1987) FEBS Lett. 224, 311-316. 22. Ling, N., Ying, S. Y., Ueno, N., Esch, F., Denorog, L. & Guillemin, R. (1985) Proc. Natl. Acad. Sci. USA 82,7212-7221. 23. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold
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24. Cox, R. A. (1968) Methods Enzymol. 12, 120-129. 25. Benton, H. C. & Davis, R. W. (1977) Science 196, 180-182. 26. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 27. Pless, D. D. & Lernnarz, W. J. (1977) Proc. Natl. Acad. Sci. USA 74, 134-138. 28. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K. & Shimizu, S. (1989) Nature (London) 342, 440-443. 29. Sotrup-Jensen, L., Claeys, H., Zajdel, M., Peterson, T. E. & Magnussen, S. (1978) Progress in Chemical Fibrolysis and Thrombolysis, eds. Davidson, J. F., Roman, R. H., Samana, M. M. & Desroyers, P. C. (Raven, New York), Vol. 3, pp. 191-209. 30. Furie, B. & Furie, B. C. (1988) Cell 53, 505-518. 31. Hojurp, P., Jensen, M. S. & Peterson, T. E. (1985) FEBS Lett. 184, 333-338. 32. Kurosky, A., Barnett, D. R., Lee, T. H., Touchstone, B., Hay, R. E., Arnott, M. S., Bowman, B. F. & Fitch, W. M. (1980) Proc. Natl. Acad. Sci. USA 77, 3388-3392. 33. Gohda, E., Tsubouchi, H., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Miyazaki, H., Hashimoto, S. & Daikuhara, Y. (1988) J. Clin. Invest. 81, 414-419. 34. Zarnegar, R. & Michalopoulos, G. (1989) Cancer Res. 49, 3314-3320. 35. Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi, K., Okigaki, M., Arakaki, N., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Gohda, E., Daikuhara, Y. & Kitamura, N. (1989) Biochem. Biophys. Res. Commun. 163, 967973. 36. Zarnegar, R., Muga, S., Enghild, J. & Michalopoulos, G. (1989) Biochem. Biophys. Res. Commun. 163, 1370-1376. 37. Kinoshita, T., Tashiro, K. & Nakamura, T. (1989) Biochem. Biophys. Res. Commun. 165, 1229-1234.
