Identification of heparin-binding stretches of a naturally occurring deleted variant of hepatocyte growth factor (dHGF)

Biochimica et Biophysica Acta 1384 Ž1998. 93–102

Identification of heparin-binding stretches of a naturally occurring deleted variant of hepatocyte growth factor ždHGF / Masahiko Kinosaki ) , Kyoji Yamaguchi, Akihiko Murakami, Masatugu Ueda, Tomonori Morinaga, Kanji Higashio Research Institute of Life Science, Snow Brand Milk Products, Ishibashi-machi, Shimotsuga-gun, Tochigi, Japan Received 19 November 1997; accepted 19 December 1997

Abstract A deleted variant of hepatocyte growth factor ŽdHGF. is a naturally occurring major variant of HGF, which lacks five consecutive amino acid residues in the first kringle domain. While both HGF and dHGF bind to heparin, the residues involved in the binding to heparin have not been identified in either protein. To identify the residues involved in the binding, we made a series of dHGF mutants in which basic residues in the N-terminal and the first kringle domains were replaced with alanine residue. The analysis of heparin-binding ability revealed that three stretches, 42 RCTRNK in the hairpin loop structure, and 2 RKRR and 27 KIKTKK in the N-terminal basic region, are involved in the binding. Alanine substitution of each basic residue except 3 K and 27 K in the stretches reduced the heparin-binding ability of dHGF, and the decrease was additive. Conversely, lysine substitution of 37 D, 38 Q or 64 Q in the N-terminal domain increased heparin-binding ability. These results suggest that stretches distant from each other in the primary structure come into close proximity when the polypeptide folds into protein, and form a heparin-binding site with clusters of basic residues. q 1998 Elsevier Science B.V. Keywords: Hepatocyte growth factor; Heparin-binding; Site-directed mutagenesis

1. Introduction Hepatocyte growth factor ŽHGF. is a heparin-binding basic protein with an approximate molecular mass of 80 kDa w1,2x, and is also designated as scatter

Abbreviations: HGF, Hepatocyte growth factor; dHGF, Deleted variant of hepatocyte growth factor; IMDM, Iscove’s modified Dulbecco’s medium ) Corresponding author. Research Institute of Life Science, Snow Brand Milk Products, 519 Ishibashi-machi, Shimotsugagun, Tochigi 329-05, Japan. Fax: q81-285-53-1314.

factor ŽSF. w3x, or fibroblast-derived tumor cytotoxic factor ŽF-TCF. w4x. HGF is a disulfide-linked heterodimer composed of an a chain with a molecular mass of 52 to 56 kDa and a b chain of 30 to 34 kDa w5x. A structural analysis based on the amino acid sequence deduced from the cDNA w1,2x predicted that human HGF consists of 6 major domains: the Nterminal domain Žincluding the N-terminal basic region and the hairpin loop structure. and four kringle domains in the a chain, and a serine protease-like domain in the b chain. An alternatively spliced variant of HGF, which lacks five consecutive amino

0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 8 . 0 0 0 0 2 - 8

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acid residues Žthe FLPSS sequence. in the first kringle domain of HGF, has been isolated and termed deleted variant of HGF ŽdHGF. w6,7x. Both HGF and dHGF are biologically multifunctional w4,8–11x. dHGF is different from HGF in target cell specificity w7,12,13x, and physicochemical and immunological properties w13x. Several monoclonal antibodies raised against dHGF failed to recognize HGF and 2-mercaptoethanol-reduced dHGF, demonstrating that HGF and dHGF differ in their tertiary structure w13x. Recent studies have indicated that the binding of heparin-binding growth factors to the cell surface heparin-like molecules is of great significance to their biological activity w14–20x. It is known that the binding of HGF to its signaling receptor, c-Met, is modulated by cell surface heparin-like molecules w21x. In addition, a recent study showed that heparin induces dimerization of the HGF antagonists, NK1 and NK2, and confers proliferative activity on them w22x. Consensus for heparin-binding sequences in a variety of proteins has been proposed w23x. These include motifs XBBXBX and XBBBXXBX, where B and X represent basic and hydrophilic residues, respectively. In contrast, in some heparin-binding proteins, multiple stretches are combined to form a cluster of basic amino acid residues and interact with heparin w24,25x. Analysis of various domain-deleted mutants of HGF revealed that the N-terminal basic region contains a heparin-binding site w26x. Involvement of the second kringle domain of HGF in the binding is not clear since the elution profile of NK1, a mutant consisting of the N-terminal domain and the first kringle domain, from a heparin column does not differ from that of HGF w27x. These findings, however, were obtained through studies of HGF mutants with domain deletion, which may bear gross structural changes. Despite an accumulation of knowledge on the interaction between HGF and heparin, no systematic work has been carried out to identify amino acid residues involved in heparin-binding in either HGF or dHGF. In the present study, we identified amino acid residues involved in the binding to heparin in three stretches located in the N-terminal domain of dHGF by generating a series of dHGF mutants and analyzing their heparin-binding ability. We also demonstrate that the stretches work collectively in the binding.

2. Materials and methods 2.1. Materials Oligonucleotides used in in vitro mutagenesis and DNA sequence analysis were synthesized using the Applied Biosystem 384 DNA Synthesizer. DNA sequence analysis was carried out using Taq Dye Deoxy Terminator Cycle Sequencing kit Ž Perkin-Elmer, USA.. All restriction enzymes were purchased from Takara Shuzo ŽJapan. . 2.2. Expression of dHGF mutants SR a promoter-based vector termed pcDNASR296 w28x was a gift from Dr. H. Takebe. The vector was digested with restriction enzymes Pst I and BamHI, and the ends were filled-in with DNA polymerase I. A dHGF cDNA fragment, prepared by digesting the plasmid pcDNAI-TCF w6x with BamHI and SphI and treating the ends with DNA polymerase I, was inserted into the vector. The resultant dHGF expression vector was named pSR a-dHGF. Mutagenesis was carried out using PCR with mutagenetic oligonucleotides as primers, and pcDNAI-TCF as a template w29x. Each PCR product with site-directed mutation was digested with restriction enzymes Bst PI and EcoRV, and the digest was applied on a 1% agarose gel to separate fragments. Each fragment carrying a mutation was extracted from the gel and ligated with pSR a-dHGF which had been digested with the same enzymes. Chinese hamster ovary ŽCHO. cells Ž1 = 10 7 . were transfected with a mixture of 200 m g of a expression plasmid and 10 m g of blasticidin resistant gene, pSV2bsr Ž Funakoshi, Japan. , by the electroporation method Ž330 V, 960 m F. using Gene Pulser Cuvette with a 0.4 cm electrode Ž Bio-Rad. . The cells were cultivated in IMDM ŽGibco. with 10% fetal calf serum Ž FCS. at 378C in a CO 2 incubator. After three days of cultivation, the cells were suspended in IMDM containing 10% FCS and 5 m grml blasticidin Ž Funakoshi. and were seeded on 96-well plates at an initial density of 10 4 cellsr200 m lrwell. After 2 weeks of incubation, the amount of the dHGF mutant in each well was measured by an ELISA using rabbit anti-dHGF polyclonal antibody, and the cells expressing the mutant were selected.

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2.3. Nomenclature of the mutants Mutants were named after both the position in the amino acid sequence of mature dHGF at which the mutation was introduced, and the amino acid sequence before alteration. The glutamine residue at the N-terminus of a-chain is denoted residue No. 1. For example, if the sequence RKRR, from the 2nd to 5th amino acid residues in the a chain, was altered to AAAA, the resultant mutant is termed 2RKRR. The superior figures with the sequence, for instance 27 KIKTKK, indicate the position in the amino acid sequence of dHGF. 2.4. Protein quantification of wild-type dHGF and mutants A polyclonal sandwich ELISA was used to quantify dHGF and mutants. One hundred microliters of 2 m grml rabbit anti-recombinant human dHGF ŽrhdHGF. antibody in 0.1 M sodium carbonate was added to each well of 96-well microtiter plates ŽNunc. and incubated overnight at 48C to coat the surface. After blocking the wells at room temperature for 2 h with 50% Block Ace ŽSnow Brand. , serially-diluted samples in 0.2 M Tris–HCl Ž pH 7.4. containing 40% Block Ace and 0.1% Tween 20 were added to the wells Ž100 m lrwell.. rhdHGF Ž1.5–50 ngrml. was used as a standard. The plates were incubated overnight at 48C, and subsequently the wells were washed with PBS containing 0.05% Tween 20. Horseradish peroxidase-conjugated polyclonal antirhdHGF antibody diluted with 0.1 M Tris–HCl ŽpH 7.4. containing 25% Block Ace and 0.1% Tween 20 was added to the wells Ž 100 m lrwell., and the plates were further incubated at 378C for 2 h. After washing the wells, 100 m l of substrate solution Ž0.4 ngrml of o-phenylenediamine and 0.006% H 2 O 2 in 0.1 M citrate–phosphate buffer, pH 4.5. was added to the wells, and the plates were kept at room temperature for 30 min before reading absorbance at 492 nm using a microplate reader. Immunoblotting was performed to determine the size of the mutants. SDS-PAGE and blotting of the mutants onto PVDF membranes were performed using Phast System ŽPharmacia.. Blotted membranes were probed with 1:200 diluted horseradish peroxi-

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dase-conjugated polyclonal antibody, and specific binding was detected by a chemiluminescent detection method ŽAmersham. according to the protocol supplied by the manufacturer. 2.5. Analysis for heparin-binding ability The affinity of each dHGF mutant for heparin was analyzed by FPLC using a Hi Trap heparin-packed column Ž1 ml gel, Pharmacia. as follows. Thirty milliliters of culture medium containing each dHGF mutant Ž50–2500 ng dHGF antigenrml. was applied to the column equilibrated with 20 mM Tris–HCl ŽpH 7.5. containing 0.01% Tween 20. The column was developed with a 22.5-ml linear gradient of 0 to 1.5 M NaCl in equilibration buffer at a flow rate of 0.5 mlrmin and fractions Ž0.5 ml. were collected. The amount of dHGF mutant in each fraction was measured by ELISA, and the NaCl concentration at which the mutant was eluted from the column was determined. The relative heparin-binding ability was defined by the ratio Ž%. of NaCl concentration eluting respective dHGF mutant to that eluting wild-type dHGF. 2.6. Assay for

125

I-dHGF binding to heparin

dHGF was radioiodinated using IODO-GEN. Na125 I Ž0.5 mCi. in 5 m l of 0.5 M phosphate buffer ŽpH 7.0. was added to a tube which had been coated with 50 m g of IODO-GEN. Ten micrograms of human recombinant dHGF in 10 m l of 0.5 M phosphate buffer ŽpH 7.0. containing 0.01% Tween 20 was added to the tube, and the mixture was allowed to stand at room temperature for 30 s. The mixture was transferred to a fresh tube containing 5 m l of 5% BSA in 0.5 M phosphate buffer and 80 m l of 10 mgrml KI. Radioiodinated dHGF was purified using a Sephadex G-25 spin-column. Competition assay was carried out as previously reported with some modification w26x. Briefly, polystyrene tubes were coated with 50 m l of 1000 IUrml heparin. After rinsing the tubes twice with PBS, 0.2% BSA in PBS was added, and the tubes were allowed to stand at room temperature for 2 h. One nanogram of 125 IdHGF with or without various concentrations of competitor in 50 m l of PBS containing 0.2% BSA was

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added to each tube, and the tubes were allowed to stand at room temperature for 2 h. After rinsing the tubes three times with PBS, the radioactivity in the tubes was measured using a g-counter.

2.7. Assay for biological actiÕity The biological activity of dHGF and the mutants was assayed by measuring their ability to stimulate

Fig. 1. Heparin-binding ability of Ala-scanning mutants with amino acid substitution in the N-terminal basic region. ŽA. Elution profile from Hi Trap heparin column of each mutant: An aliquot of cultured medium of the cells producing each mutant was applied to a Hi Trap heparin column as described under Section 2. The protein was eluted from the column with a linear gradient of 0 to 1.5 M NaCl in the equilibration buffer. The amount of the mutant was measured by ELISA. ŽB. Relative heparin-binding ability of the Ala-scanning mutants: The N-terminal amino acid sequence of dHGF is given in the top of the figure. Each line below this sequence represents the amino acid sequence of the respective mutant. Amino acid residues replaced with an alanine residue are indicated by As. Relative heparin-binding ability was defined as the ratio Ž%. of the NaCl concentration eluting the mutant to that eluting the wild-type dHGF.

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DNA synthesis in primary cultures of rat hepatocytes. Adult rat hepatocytes were prepared by the method of Seglen w30x. The cells suspended in William’s E medium containing 10% FCS and 10 mM dexamethason were inoculated at an initial cell density of 10 4 cellsr100 m lrwell on 96-well plates and cultivated for 24 h at 378C. dHGF mutants were partially purified by heparin-Sepharose CL-6B chromatography, and desalted and concentrated using Centricut ŽKurabou, Japan. . Each dHGF mutant was serially diluted with the medium and then added to the culture of hepatocytes in each well Ž 10 m lrwell, final 1–64 ng dHGF antigenrml.. The plates were incubated for 20 h at 378C. Subsequently, 1 mCi of w methyl- 3 Hx thymidine Ž10 m l, Amersham. was added to each well, and the plates were further incubated for 2 h. The cells were washed with PBS and then trypsinized. The radioactivity incorporated into the cells was measured using a Direct Beta Counter MATRIX 96 ŽPackard, USA..

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3. Results

column ŽFig. 1A. . The heparin-binding ability of the three mutants with substitution in the N-terminal domain was considerably lower than that of dHGF. The mutants, 2RKRR, 27KIKTKK and 42R, were eluted from the column at NaCl concentrations of 0.78 M Žrelative heparin-binding ability of 68%. , 0.82 M Ž72%. and 0.84 M Ž74%. , respectively. Furthermore, the heparin-binding ability of two mutants, 45R and 47K, was slightly lower than that of the wild-type dHGF. These results are summarized in Fig. 1B. The results indicate that the basic amino acid residues in the sequence of 2 RKRR, 27 KIKTKK and 42 R are important in the binding of dHGF to heparin. Substitution of an alanine residue for another basic residue in the N-terminal domain did not affect the affinity for heparin Žsee 62RK for example.. This fact indicates that net positive charge of dHGF is not a major factor influencing binding to heparin. All mutants with alanine substitution in the first kringle domain were eluted from the heparin column at the same NaCl concentration as dHGF Ž data not shown. , indicating that no basic residues in the first kringle domain are involved in the binding to heparin.

3.1. Analysis of the mutants with Ala-substitution in the N-terminal and the first kringle domains

3.2. Substitution of an alanine residue for a basic residue in multiple heparin-binding stretches

To locate Ža. heparin-binding siteŽs. in dHGF, we applied Ala-scanning mutagenesis w32x to the Nterminal and the first kringle domains in the a-chain. The heparin-binding ability of the mutants was analyzed by measuring the NaCl concentration at which each mutant was eluted from the Hi Trap heparin

To examine whether the basic residues in the three stretches, 2 RKRR Ž stretch 1., 27 KIKTKK Ž stretch 2. and 42 R Žstretch 3., interact with one another in the binding to heparin, a mutation was introduced into the stretches in every possible combination. As shown in Fig. 2, the heparin-binding ability of all four

Fig. 2. Heparin-binding ability of Ala-scanning mutants with substitution in multiple heparin-binding stretches. Relative heparin-binding ability of the Ala-scanning mutants: The N-terminal amino acid sequence of dHGF is given at the top of the figure. Each line below this sequence represents the amino acid sequence of the respective mutant. Amino acid residues replaced by an alanine residue are indicated by As. An aliquot of cultured medium of the cells producing each mutant was chromatographed on a Hi Trap heparin column as described in Fig. 1 legend. Relative heparin-binding ability was defined as described in Fig. 1 legend.

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Table 1 Biological activity of the mutants with lower heparin-binding ability Mutant

Relative efficacy Ž%.

dHGF 2RKRR 27KIKTKK 42R 2RKRRr27KIKTKK 2RKRRr42R 27KIKTKKr42R 2RKRRr27KIKTKKr42R

100 106.1"4.24 101.8"6.03 106.7"5.18 62.9"4.17 78.6"9.65 101.7"2.94 37.4"8.37

Biological activity was measured as described under Section 2. Efficacy of each mutant in stimulation of DNA synthesis in rat hepatocytes is compared with that of dHGF. Ratio Ž%. values are the mean"S.D. of at least three independent determinations.

mutants, 2RKRRr27KIKTKK, 27KIKTKKr42R, 2RKRRr42R and 2RKRRr27KIKTKKr42R, was lower than that of the mutants with alanine substitution in only one of the heparin-binding stretches Ž Fig. 1B .. A m ong th e fo u r m u ta n ts , 2RKRRr27KIKTKKr42R with a substitution in all three stretches had the lowest relative heparin-binding ability Ž35%. . Although substitution of an alanine for the basic residues in any of the three stretches greatly decreased heparin-binding ability, all such mutants still possessed the ability to stimulate DNA

synthesis in primary cultures of adult rat hepatocytes ŽTable 1.. A mutant, 27KIKTKKr42R, was 50% in relative heparin-binding ability, but the mutant fully maintained its biological activity. 3.3. Substitution of an alanine residue for a single residue in two heparin-binding stretches To examine which particular basic residue in stretches 1 and 2 has significance in the binding of dHGF to heparin, we constructed various mutants in which a single basic residue in these two stretches was replaced with an alanine residue. Four mutants, each with a single alanine substitution in different positions in stretch 1, were prepared, and their heparin-binding ability was examined ŽFig. 3.. Of these, three mutants, 2R, 4R and 5R, were eluted from the column at NaCl concentrations of 1.04 " 0.012 M Ž91%., 1.05 " 0.025 M Ž92%. and 1.04 " 0.009 M Ž91%., respectively Ž n s 3.. A series of mutants with a single alanine substitution in stretch 2 was also made, and their heparin-binding ability was examined. Of these, three mutants, 29K, 31K and 32K, were eluted from the column at NaCl concentrations of 1.04 " 0.012 M Ž91%., 0.98 " 0.002 M Ž85%. and 1.08 " 0.002 M Ž94%. , respectively Ž n s 3. . The six mutants were eluted from the column at NaCl concentrations significantly Ž p - 0.01. lower than that at

Fig. 3. Heparin-binding ability of the mutants with substitution of a single residue in the N-terminal heparin-binding stretches. Relative heparin-binding ability of the mutants with a single alanine substitution: The N-terminal amino acid sequence of dHGF is given at the top of the figure. Each line below this sequence represents the amino acid sequence of the respective mutant. Amino acid residues replaced by an alanine residue are indicated by As. An aliquot of cultured medium of the cells producing each mutant was chromatographed on a Hi Trap heparin column as described in Fig. 1 legend. Relative heparin-binding ability was defined as described in Fig. 1 legend.

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Fig. 4. Heparin-binding ability of Lys-scanning mutants with substitution of a single residue in the N-terminal basic region. Relative heparin-binding ability of Lys-scanning mutants: The N-terminal amino acid sequence of dHGF is given at the top of the figure. Each line below this sequence represents the amino acid sequence of the respective mutant. Amino acid residues replaced by an alanine residue are indicated by Ks. An aliquot of cultured medium of the cells producing each mutant was chromatographed on a Hi Trap heparin column as described in Fig. 1 legend. Relative heparin-binding ability was defined as described in Fig. 1 legend.

which wild-type dHGF was eluted Ž1.14 " 0.028 M., indicating a significant decrease in the heparin-binding ability of the mutants. 3K and 27K showed no significant change in heparin-binding ability, indicating that 3 K and 27 K are not involved in the binding to heparin. The relative heparin-binding ability of 31KK was 78% ŽFig. 3.. 3.4. Lys-scanning analysis in the N-terminal domain Mutants with lysine substitutions for the polar residues in the N-terminal domain other than arginine and serine residues were constructed, and their heparin-binding ability was analyzed. As shown in Fig. 4, relative heparin-binding ability of three mutants, 37D, 38Q and 64Q, were 109, 107 and 109%, respectively. Other mutants had essentially the same ability as dHGF.

3.6. Competition of the mutants with binding to heparin

125

I-dHGF for

To further confirm the involvement of some of the residues identified in the present analysis in the binding to heparin, the competition of the mutants bearing amino acid substitution with 125 I-dHGF for immobilized heparin was assayed. Four mutants were chosen for this assay: 27KIKTKK and 32K have lower Ž about 70% and 90%, respectively. , 37D and 64Q have higher Žabout 110%. heparin-binding ability than does

3.5. Integrity and stability of the mutants To examine the stability of the mutants during the chromatographic analysis, the mutants were incubated at 378C in PBS containing 0.01% Tween 20 and subjected to immunoblotting analysis. As shown in Fig. 5, every mutant with decreased or increased heparin-binding ability gave ; 80 kDa bands, indicating that they were expressed correctly Ž lanes a. . Even after an 18-h incubation, none of the mutants had degraded Žlanes b., indicating they were as stable as the wild-type dHGF.

Fig. 5. Immunoblotting analysis of the dHGF mutants. The mutants were incubated at 378C for 18 h in PBS containing 0.01% Tween 20. After incubation, they were separated by SDS-PAGE Ž10–15% gel.. The separated proteins were blotted onto a membrane and detected as described under Section 2. Lane a, before incubation; lane b, after incubation.

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Table 2 Competition by the mutants of binding of immobilized heparin Competitor dHGF 27KIKTKK 32K 37D 64Q

125

I-dHGF to the

Residual radioactivity Ž%. 100-fold excess

1000-fold excess

80.4"6.3 117.8"30.4 b 104.8"23.4 a 50.0"16.5 b 50.7"9.9 b

18.1"1.4 83.3"18.6 b 30.2"3.9 N.D. N.D.

Tubes coated with heparin were incubated with 1 ng of 125 I-dHGF with or without the indicated excess concentration Ž=100 or =1000. of competitor. After rinsing the tubes, the bound 125 I-dHGF was measured by counting the radioactivity using a g-counter. The values represent the mean"S.D. of six independent determinations. a : p- 0.05 vs. dHGF. b : p- 0.01 vs. dHGF. N.D.: not determined.

the wild-type dHGF. As shown in Table 2, binding of 125 I-dHGF to the immobilized heparin was reduced to 80% and to 18% when a 100-fold and 1000-fold excess concentration of dHGF was added to the assay, respectively. 32K and 27KIKTKK were significantly less effective in the competition than dHGF. In addition, 27KIKTKK was significantly less effective in the competition than 32K. On the other hand, 37D and 64K were more effective in the competition than dHGF. These results are consistent with the results of the heparin-binding experiment obtained by chromatographic analysis using a heparin column ŽFigs. 1, 3 and 4..

4. Discussion dHGF and HGF are distinguishable in their target cell specificity and immunogenicity w6,12,13x. With regard to HGF, several research groups have located the regions involved in the binding to heparin in the N-terminal domain Žincluding the N-terminal basic region and the hairpin loop structure. , and the second kringle domain by the analysis of deletion mutants w26,27,31x. However, no investigation has been done either with HGF or dHGF to determine amino acid sequences involved in the binding. Site-specific mu-

tagenesis in the N-terminal and the first kringle domains of dHGF identified three heparin-binding stretches. One of them was located in the hairpin loop structure. The corresponding region in HGF has been suggested as the region involved in the binding to heparin by Mizuno et al. w26x. The other two were present in the N-terminal basic region. In the present study, we identified, for the first time, sequences involved in the binding of dHGF to heparin. Mizuno et al. showed that neither a deletion of the entire first kringle domain nor a deletion of five amino acid residues ŽFLPSS. in the first kringle domain affects the heparin-binding ability of HGF w26x. In dHGF, none of the mutations introduced in the first kringle domain decreased heparin-binding ability Ždata not shown.. These observations suggest that the corresponding residues in the N-terminal basic region of HGF are involved in the binding to heparin. Net charge cannot be a major factor influencing the binding of dHGF to heparin since not all Alascanning mutants had significant change in the ability, and not all Lys-scanning mutants had increased ability. Analysis of the mutants with single alanine residue substitution indicates that three of the four basic residues in stretch 1, and three of the four in stretch 2 participated in the binding to heparin. The chromatographic analysis suggests that each residue contributes to the binding additively Ž Figs. 2 and 3. . Lys-scanning analysis indicates that substituting a lysine residue for one of the specific polar residues in the N-terminal basic region causes an increase in relative heparin-binding ability by 9% ŽFig. 4.. The increase in relative heparin-binding ability was significant as confirmed by the competition assay. Analysis of the mutants with alanine substitution in plural heparin-binding stretches also revealed that contribution of the basic residues to the heparin-binding ability is additive Ž Fig. 2.. Considering the fact that alteration of a single basic residue to an alanine residue decreased heparin-binding ability by about 10%, 45R and 47K may also be included in stretch 3. Taken together, it is conceivable that the major heparin-binding site of dHGF consists of a cluster of at least nine basic residues. Cardin and Weintraub identified two sequence motifs that they proposed as consensus sequences recognized by heparin w27x. Those are XBBXBX and XBBBXXBX, where B denotes basic amino acid

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mutant was about 70% ŽTable 1.. Furthermore, the highly purified mutants Ž) 95% purified. had potency 2 to 10-fold higher than dHGF, though they were almost equal in efficacy to dHGF in the stimulation ŽKinosaki, M. et al., manuscript in preparation.. These results indicate that full heparin-binding ability is not a prerequisite for dHGF to exert its biological activity.

Acknowledgements Fig. 6. Summary of the results. Heavily shaded residues, alanine substitution caused the decrease in heparin-binding ability; lightly shaded residues, lysine substitution caused the increase in heparin-binding ability.

We thank Dr. H. Takebe for generously providing the expression vector, and Fumiko Kobayashi and Fumie Kobayashi for excellent assistance.

References residues and X any other residues. None of the three heparin-binding stretches in dHGF matched the motifs. If X permits basic amino acid residues, the sequence 2 RKRR matches one of the consensus sequences, XBBXBX. In some heparin-binding proteins such as mucus proteinase inhibitor, a heparin-binding site is formed by combining residues distant from each other in the primary sequence w24,25x. Folding of the protein brings these residues into close proximity to form a heparin-binding site. Such may be the case with the three stretches in dHGF. The results obtained in the present study are summarized in Fig. 6. Stretch 3, one of three heparin-binding stretches identified in this study, is located at the N-terminus of the hairpin loop structure. Three polar residues, 37 D, 38 Q and 64Q are also located near a disulfide bond at 39C– 65 C in the hairpin loop structure. Alteration of any one of these to a lysine residue increased the heparin-binding ability. The region that includes the disulfide bond and N-terminus of the hairpin loop structure may form a core of a heparin-binding site. As shown in Fig. 2 and Table 1, all the alaninescanning mutants with decreased heparin-binding ability maintained the biological activity. The mutants with alanine substitution in either stretch 1 or stretch 2 were equivalent to dHGF in efficacy evaluated by stimulation of DNA synthesis in rat hepatocytes, although relative heparin-binding ability of the

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