The Mode of Action of Heparan and Dermatan Sulfates in the Regulation of Hepatocyte Growth Factor/Scatter Factor
2002; Elsevier BV; Volume: 277; Issue: 2 Linguagem: Inglês
10.1074/jbc.m107506200
ISSN1083-351X
AutoresMalcolm Lyon, Jon A. Deakin, John T. Gallagher,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoHepatocyte growth factor/scatter factor, in addition to binding to its specific signal-transducing receptor, Met, also interacts with both heparan and dermatan sulfates with high affinity. We have investigated the comparative role of these two glycosaminoglycans in the activation of Met by hepatocyte growth factor/scatter factor. Using glycosaminoglycan-deficient CHOpgsA-745 cells we have shown that growth factor activity is critically dependent upon glycosaminoglycans, and that heparan sulfate and dermatan sulfate are equally potent as co-receptors. Cross-linked 1:1 conjugates of growth factor and either heparan or dermatan sulfate do not dimerize under physiological conditions and are biologically active. This implies that a ternary signaling complex with Met formsin vivo. Native Met isolated from CHO pgsA-745 cells shows only very weak intrinsic affinity for heparin in vitro. Also, a heparin-derived hexasaccharide, which is the minimal size for high affinity binding to the growth factor alone, is sufficient to induce biological activity. Together these observations imply that the role of these glycosaminoglycan may be primarily to effect a conformational change in hepatocyte growth factor/scatter factor, rather than to induce a necessary growth factor dimerization, or to stabilize a ternary complex by additionally interacting with Met. Hepatocyte growth factor/scatter factor, in addition to binding to its specific signal-transducing receptor, Met, also interacts with both heparan and dermatan sulfates with high affinity. We have investigated the comparative role of these two glycosaminoglycans in the activation of Met by hepatocyte growth factor/scatter factor. Using glycosaminoglycan-deficient CHOpgsA-745 cells we have shown that growth factor activity is critically dependent upon glycosaminoglycans, and that heparan sulfate and dermatan sulfate are equally potent as co-receptors. Cross-linked 1:1 conjugates of growth factor and either heparan or dermatan sulfate do not dimerize under physiological conditions and are biologically active. This implies that a ternary signaling complex with Met formsin vivo. Native Met isolated from CHO pgsA-745 cells shows only very weak intrinsic affinity for heparin in vitro. Also, a heparin-derived hexasaccharide, which is the minimal size for high affinity binding to the growth factor alone, is sufficient to induce biological activity. Together these observations imply that the role of these glycosaminoglycan may be primarily to effect a conformational change in hepatocyte growth factor/scatter factor, rather than to induce a necessary growth factor dimerization, or to stabilize a ternary complex by additionally interacting with Met. hepatocyte growth factor/scatter factor glycosaminoglycan heparan sulfate dermatan sulfate Madin-Darby canine kidney Chinese hamster ovary fibroblast growth factor-1 fibroblast growth factor-2 phosphate-buffered saline degree of polymerization (i.e. number of monosaccharide units in the oligosaccharide) extracellular signal-regulated kinase 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride N-hydroxysulfosuccinimide high performance liquid chromatography 4-morpholineethanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Hepatocyte growth factor (HGF/SF)1 is a plasminogen-related growth factor secreted by stromal cells that acts primarily by a paracrine mechanism upon epithelial, endothelial, and hemopoietic progenitor cells (for review, see Ref. 1Bock G.R. Goode J.A. Plasminogen-related Growth Factors: Ciba Foundation Symposium 212. John Wiley, Chichester, UK1997Google Scholar). Some stromal cells are also now known to be responsive to HGF/SF (2Delehedde M. Sergeant N. Lyon M. Rudland P.S. Fernig D.G. Eur. J. Biochem. 2001; 269: 4423-4429Google Scholar). Activation of its specific tyrosine kinase receptor, Met, elicits a diverse range of cellular activities, including proliferation, motility, morphogenesis, and protection from apoptosis (for review, see Ref. 1Bock G.R. Goode J.A. Plasminogen-related Growth Factors: Ciba Foundation Symposium 212. John Wiley, Chichester, UK1997Google Scholar). HGF/SF is considered to be an important mediator of mesenchymal-epithelial interactions during organogenesis, as well as in any subsequent organ repair. Developmental studies have identified the essential role of the HGF/SF-Met system in the formation of the liver (3Schmidt C. Bladt F. Goedecke S. Brinkmann V. Zschiesche W. Sharpe M. Gherardi E. Birchmeier C. Nature. 1995; 373: 699-702Google Scholar) and placenta (4Uehara Y. Minowa O. Mori C. Shiota K. Kuno J. Noda T. Kitamura N. Nature. 1995; 373: 702-705Google Scholar), as well as in the migration of both motor neurons (5Ebens A. Brose K. Leonardo E.D. Hanson Jr., M.G. Bladt F. Birchmeier C. Barres B.A. Tessier-Lavigne M. Neuron. 1996; 17: 1157-1172Google Scholar) and myogenic precursors (6Bladt F. Riethmacher D. Isenmann S. Aguzzi A. Birchmeier C. Nature. 1995; 376: 768-771Google Scholar). There is also increasing evidence of an involvement of HGF/SF-Met in the growth, invasiveness, and metastasis of both carcinomas and sarcomas (for review, see Ref. 7Jeffers M. Rong S. Vande Woude G.F. J. Mol. Med. 1996; 74: 505-513Google Scholar). Overexpression of wild-type Met and/or HGF/SF, sometimes involving the induction of an autocrine stimulatory loop, is common in cancer tissues. In a minority of cases there is evidence for mutations of Met leading to a dysregulated activity, although these often still remain ligand-dependent (8Michieli P. Basilico C. Pennacchietti S. Maffe A. Tamagnone L. Giordano S. Bardelli A. Comoglio P.M. Oncogene. 1999; 18: 5221-5231Google Scholar). Inhibition of Met activity may thus be beneficial in cancer treatment (9Trusolino L. Pugliese L. Comoglio P.M. FASEB J. 1998; 12: 1267-1280Google Scholar). Moreover, HGF/SF is itself considered to have considerable therapeutic potential in wound healing and specific organ regeneration after disease, damage, or surgery (10Matsumoto K. Nakamura T. Kidney Int. 2001; 59: 2023-2038Google Scholar). There is increasing evidence of a role for glycosaminoglycans (GAGs)/proteoglycans in HGF/SF activity, as suggested to varying degrees for a number of growth factors/cytokines (for review, see Ref.11Gallagher J.T. Lyon M. Iozzo R.V. Proteoglycans: Structure, Biology and Molecular Interactions. Marcel Dekker, New York2000: 27-60Google Scholar). In vitro, HGF/SF interacts with heparan sulfate (HS) (12Lyon M. Deakin J.A. Mizuno K. Nakamura T. Gallagher J.T. J. Biol. Chem. 1994; 269: 11216-11223Google Scholar, 13Ashikari S. Habuchi H. Kimata K. J. Biol. Chem. 1995; 49: 29586-29593Google Scholar, 14Rahmoune H. Rudland P.S. Gallagher J.T. Fernig D.G. Biochemistry. 1998; 37: 6003-6008Google Scholar) and dermatan sulfate (DS) (15Lyon M. Deakin J.A. Rahmoune H. Fernig D.G. Gallagher J.T. J. Biol. Chem. 1998; 273: 271-278Google Scholar) with sufficiently high affinities (KD values of 0.2–20 nm (14Rahmoune H. Rudland P.S. Gallagher J.T. Fernig D.G. Biochemistry. 1998; 37: 6003-6008Google Scholar,15Lyon M. Deakin J.A. Rahmoune H. Fernig D.G. Gallagher J.T. J. Biol. Chem. 1998; 273: 271-278Google Scholar)) to support a physiological interaction. The apparently similar affinities of HGF/SF for these two structurally distinctive GAGs is rather unusual, as most HS-binding proteins display relatively weak affinities for DS. It is partly explained by the lack of requirement for either N-sulfate or 2-O-sulfate groups as major binding determinants (12Lyon M. Deakin J.A. Mizuno K. Nakamura T. Gallagher J.T. J. Biol. Chem. 1994; 269: 11216-11223Google Scholar, 15Lyon M. Deakin J.A. Rahmoune H. Fernig D.G. Gallagher J.T. J. Biol. Chem. 1998; 273: 271-278Google Scholar, 16Merry C.L.R. Bullock S.L. Swan D.C. Backen A. Lyon M. Beddington R.S.P. Wilson V.A. Gallagher J.T. J. Biol. Chem. 2001; 276: 35429-35434Google Scholar). This raises the question as to whether the binding of HS or DS has the same functional consequences for HGF/SF activity? Indeed, the degree of involvement, and putative role, of GAGs in HGF/SF activity has been somewhat confused. We have previously reported that Madin-Darby canine kidney (MDCK) cells become completely unresponsive to HGF/SF when treated with chlorate, which metabolically inhibits the sulfation of endogenous GAGs (17Deakin J.A. Lyon M. J. Cell Sci. 1999; 112: 1999-2009Google Scholar). Responsiveness to HGF/SF can be restored by exogenous HS, although it has to be presented in the form of an immobilized heparan sulfate proteoglycan substratum, and, interestingly, HS does not work as a soluble ligand in MDCK cells (17Deakin J.A. Lyon M. J. Cell Sci. 1999; 112: 1999-2009Google Scholar). This suggests an obligate requirement for GAGs/proteoglycans for HGF/SF activity. An identical pattern of behavior was subsequently demonstrated with human mammary myoepithelial-like cells (18Sergeant N. Lyon M. Rudland P.S. Fernig D.G. Delehedde M. J. Biol. Chem. 2000; 275: 17094-17099Google Scholar). Namalwa Burkitt's lymphoma cells co-transfected with Met and a HS-bearing form of CD44 respond to HGF/SF, but not when a HS-lacking CD44 isoform is used (19Van der Voort R. Taher T.E.I. Wielenga V.J.M. Spaargaren M. Prevo R. Smit L. David G. Hartmann G. Gherardi E. Pals S.T. J. Biol. Chem. 1999; 274: 6499-6506Google Scholar). In contrast, Met-transfected BaF3 lymphoblastoid cells, which are reputedly completely deficient in HS, are reported to still respond to HGF/SF, although activity is significantly enhanced by the addition of exogenous heparin (20Schwall R.H. Chang L.Y. Godowski P.J. Kahn D.W. Hillan K.J. Bauer K.D. Zioncheck T.F. J. Cell Biol. 1996; 133: 709-718Google Scholar), a commonly used GAG analogue of the sulfated domains of HS. These same cells are completely refractory to the truncated NK1 and NK2 isoforms of HGF/SF (which are partial agonists of Met) unless exogenous heparin is present. The xylosyl transferase-deficient CHO pgsA-745 mutant cells are similarly unresponsive to NK1 in the absence of heparin (21Sakata H. Stahl S.J. Taylor W.G. Rosenberg J.M. Sakaguchi K. Wingfield P.T. Rubin J.S. J. Biol. Chem. 1997; 272: 9457-9463Google Scholar). It has also been reported that a double arginine reverse charge HGF/SF mutant with a consequent 50-fold reduction in heparin affinity had unimpaired biological activity (22Hartmann G. Prospero T. Brinkmann V. Ozcelik O. Winter G. Hepple J. Batley S. Bladt F. Sachs M. Birchmeier C. Birchmeier W. Gherardi E. Curr. Biology. 1997; 8: 125-134Google Scholar), although it was without activity in the Met/CD44 co-transfected lymphoma cells (19Van der Voort R. Taher T.E.I. Wielenga V.J.M. Spaargaren M. Prevo R. Smit L. David G. Hartmann G. Gherardi E. Pals S.T. J. Biol. Chem. 1999; 274: 6499-6506Google Scholar). It is thus unclear as to the true role of GAGs, and whether some of these conflicting observations primarily reflect differences between the properties of full-length HGF/SF and its truncated variants. Indeed, it has been suggested that the various forms of HGF/SF may engage the Met receptor in different ways (23Chirgadze D.Y. Hepple J. Byrd R.A. Sowdhamini R. Blundell T.L. Gherardi E. FEBS Lett. 1998; 430: 126-129Google Scholar, 24Matsumoto K. Kataoka H. Date K. Nakamura T. J. Biol. Chem. 1998; 273: 22913-22920Google Scholar, 25Miller M. Leonard E.J. FEBS Lett. 1998; 429: 1-3Google Scholar). The putative mechanism(s) by which GAGs can modulate HGF/SF activity is also unclear. HGF/SF can apparently bind with high affinity to a purified Met-IgG fusion protein in vitro, in the absence of GAG (22Hartmann G. Prospero T. Brinkmann V. Ozcelik O. Winter G. Hepple J. Batley S. Bladt F. Sachs M. Birchmeier C. Birchmeier W. Gherardi E. Curr. Biology. 1997; 8: 125-134Google Scholar, 26Mark M.R. Lokker N.A. Zioncheck T.F. Luis E.A. Godowski P.J. J. Biol. Chem. 1992; 267: 26166-26171Google Scholar, 27Zioncheck T.F. Richardson L. Liu J. Chang L. King K.L. Bennett G.L. Fugedi P. Chamow S.M. Schwall R.H. Stack R.J. J. Biol. Chem. 1995; 270: 16871-16878Google Scholar). This suggests that GAGs are not required for at least the initial binding of HGF/SF to Met (although in the fusion protein constructs the Met is already dimerized). GAGs may, however, still be needed for a subsequent Met activation step. However, the predominant view, mostly based on experiments with the NK1 variant, is that the role of GAGs may be primarily to induce dimerization of the protein ligand (20Schwall R.H. Chang L.Y. Godowski P.J. Kahn D.W. Hillan K.J. Bauer K.D. Zioncheck T.F. J. Cell Biol. 1996; 133: 709-718Google Scholar, 21Sakata H. Stahl S.J. Taylor W.G. Rosenberg J.M. Sakaguchi K. Wingfield P.T. Rubin J.S. J. Biol. Chem. 1997; 272: 9457-9463Google Scholar, 27Zioncheck T.F. Richardson L. Liu J. Chang L. King K.L. Bennett G.L. Fugedi P. Chamow S.M. Schwall R.H. Stack R.J. J. Biol. Chem. 1995; 270: 16871-16878Google Scholar, 28Zhou H. Casas-Finet J.R. Coats R.H. Kaufman J.D. Stahl S.J. Wingfield P.T. Rubin J.S. Bottaro D.P. Byrd R.A. Biochemistry. 1999; 38: 14793-14802Google Scholar), thereby facilitating the subsequent dimerization and activation of Met. To try and further clarify these issues we have specifically investigated the GAG dependence of HGF/SF activity in the CHOpgsA-745 mutant cells. We wished to address experimentally the following: (i) is the activity of full-length HGF/SF essentially dependent upon, or enhanced by, the presence of appropriate GAGs; (ii) do HS and DS differ in their functional properties; (iii) is GAG-induced HGF/SF dimerization essential for activity; and (iv) is there evidence for an additional interaction between Met and HGF/SF-binding GAGs? CHO pgsA-745 cells were provided by Dr. J. Esko (University of California at San Diego, CA), and were routinely cultured in RPMI medium supplemented with 10% (v/v) fetal bovine serum. MDCK cells were provided by Dr. E. Gherardi (MRC Center, Cambridge, UK), and were routinely cultured in Eagle's modified minimal essential medium with Earle's salts containing 5% (v/v) heat-inactivated donor calf serum. All cell cultures were supplemented with 1% (w/v) glutamine, 100 IU/ml of penicillin, and 100 μg/ml streptomycin sulfate, and maintained in a humidified atmosphere of 5% CO2 in air at 37 °C. All cell culture reagents were obtained from Invitrogen (Paisley, UK). Recombinant human HGF/SF was obtained from R&D Systems (Abingdon, UK). Heparinase I (Flavobacterium heparinum; EC 4.2.2.7), chondroitinase ABC (Proteus vulgaris; EC 4.2.2.4), and chondroitinase ACI (F. heparinum; EC number 4.2.2.5) were from Seikagaku Kogyo Co. (Tokyo, Japan). Heparinase II (F. heparinum; no EC number assigned) and heparinase III (F. heparinum; EC4.2.2.8) were from Grampian Enzymes (Orkney, UK). Porcine mucosal heparin, heparin-agarose, wheat germ agglutinin-agarose, and azure A were from Sigma (Poole, UK). Sized heparin oligosaccharides were donated by Iduron (Manchester, UK). Porcine mucosal HS was a gift of NV Organon (Oss, Netherlands), and murine skin DS was a gift of Dr. David Lane (Imperial College, University of London). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) were from Pierce & Warriner (Chester, UK). Bio-Gel P10 was from Bio-Rad Laboratories (Hemel Hempstead, UK). Sephadex G-50 and PD-10 desalting columns were from Amersham Bioscience Inc. (St. Albans, UK). Porcine mucosal HS (10 mg) was dissolved in 1 ml of 50 mm sodium acetate, 0.5 mm calcium acetate, pH 7.0. Heparinase III was added to a final concentration of 50 mIU/ml and the mixture incubated at 37 °C for 2 h, before the addition of a second fresh batch of enzyme and digestion for a further 18 h. The resulting oligosaccharide mixture was resolved by gel filtration on a Bio-Gel P10 column (1.5 × 163 cm) eluted with NH4HCO3at a flow rate of 12 ml/h. Fractions (1.4 ml) were collected and monitored on a spectrophotometer at 232 nm. Individual size populations of oligosaccharides were pooled, desalted on a PD-10 column eluted with distilled water, and then dried on a centrifugal evaporator. Murine skin DS (10 mg) was dissolved in 1 ml of 50 mmsodium acetate, pH 6.5. Chondroitinase ACI was added to a final concentration of 50 mIU/ml and incubated at 37 °C for 2 h, before the addition of a second fresh batch of enzyme and digestion for a further 18 h. The resulting oligosaccharide mixture was resolved on a ProPac PA-1 strong anion-exchange HPLC column (0.4 × 25 cm; Dionex) using a 0–1.5 m NaCl, pH 3.5, gradient at a flow rate of 1 ml/min and collection of 1-ml fractions. Elution was monitored by on-line UV absorbance at 232 nm, and fractions were pooled and processed as described above. Freshly trypsinized CHOpgsA-745 cells were seeded at high density in 1 ml of RPMI, 10% (v/v) fetal bovine serum in a 24-well plate and incubated at 37 °C for 24 h. Medium was removed and replaced with serum-free RPMI for 2 h, before addition of fresh serum-free RPMI containing known concentrations of HGF/SF, cross-linked HGF/SF-GAG conjugates, or GAGs. After 20 min at 20 °C, the supernatants were removed and cells were solubilized in 70 μl/well of boiling, nonreducing Laemmli SDS sample buffer. Equivalent loadings of each sample were electrophoresed on a nonreducing 15% (w/v) SDS-polyacrylamide gel with a 5% (w/v) polyacrylamide stacking gel, and then electroblotted to nitrocellulose (Schleicher & Schuell GmbH, Dassel, Germany). Blots were blocked for 1 h in PBS, 10% (w/v) nonfat dried milk powder before probing for 1 h with a 1:1000 dilution (in PBS, 3% (w/v) nonfat dried milk powder, 0.1% (v/v) Tween 20) of a mouse monoclonal antibody to dually-phosphorylated (Thr183/202/Tyr185/204) ERK-1/2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA). After thorough washing with PBS, a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG was added for 30 min. Bands were visualized by enhanced chemiluminescence (ECL; Amersham Bioscience Inc.). Freshly trypsinized CHOpgsA-745 or MDCK cells in 1 ml of culture medium were seeded at high density into the top chamber of a 24-well Transwell plate with 12-μm pore polycarbonate membranes (Costar, High Wycombe, UK). Bottom chambers received 1 ml of medium containing known concentrations of HGF/SF, cross-linked HGF/SF-GAG conjugates, or GAG alone. After 4–5 h incubation at 37 °C, wells were emptied and cells were fixed with cold (−20 °C) methanol, air dried, and then stained with 1% (w/v) aqueous crystal violet. Cells on the upper surface of the membrane were removed using a cotton bud. Membranes were excised using a scalpel blade, and the number of migrated cells on the underside of the membrane were counted under light microscopy. At least three representative fields were counted for each replicate membrane. The motility of MDCK cells was also assayed by the colony scatter assay, performed as described in Ref. 17Deakin J.A. Lyon M. J. Cell Sci. 1999; 112: 1999-2009Google Scholar. Zero-length cross-linking of HGF/SF to intact GAGs or oligosaccharides was performed essentially as originally described for protein-protein cross-linking by Grabarek and Gergely (29Grabarek Z. Gergely J. Anal. Biochem. 1990; 185: 131-135Google Scholar). HS or DS chains (50–400 μg), or defined oligosaccharide size fractions (10 μg), were dissolved in 0.1 ml of 0.1 m MES, 0.1m NaCl, pH 6.0. Sufficient EDC and sulfo-NHS were added to give 6 and 15 mm concentrations, respectively, and the mixture was incubated at 25 °C for 15 min. Excess reagents were rapidly removed by passage through a 1.5-ml Sephadex G-50 column eluted with 0.1 m MES, 0.1 m NaCl, pH 6.0. Recovered activated GAGs/oligosaccharides were combined with 1 μg of HGF/SF and incubated at 25 °C for at least 2 h. Free HGF/SF was removed by adsorption to 50 μl of heparin-agarose beads for 1 h at room temperature. Cross-linked conjugates were stored at 4 °C until further use. Aliquots of cross-linked conjugates were made up to 0.1 ml with water and 25 μg of bovine serum albumin was added. Proteins were precipitated with 10% (w/v) trichloroacetic acid at 4 °C for 15 min. Precipitates were pelleted by centrifugation, washed with ice-cold acetone, and then re-centrifuged. The final pellet was re-dissolved in either: (i) water (for subsequent degradation with pH 1.5 nitrous acid, according to the method of Shively and Conrad (30Shively J.E. Conrad H.E. Biochemistry. 1976; 15: 3943-3950Google Scholar)); (ii) 50 mm sodium acetate, 0.5 mm calcium acetate, pH 7.0 (for digestion with heparinase III); (iii) 50 mmTris-HCl, pH 8.0 (for digestion with chondroitinase ABC). Samples of HGF/SF, or cross-linked HGF/SF-GAG conjugates, were electrophoresed on a nonreducing 7.5% (w/v) SDS-polyacrylamide gel with a 5% (w/v) polyacrylamide stacking gel. Western blots were probed for 1 h with a goat polyclonal antiserum (4 μg/ml) against human HGF (R & D Systems), followed by a horseradish peroxidase-conjugated rabbit anti-goat IgG (1:5000 dilution) and enhanced chemiluminescent detection. A TSK G4000PW XL (300 × 7.8 mm; Tosoh Corp., Tokyo, Japan) size exclusion chromatography HPLC column was equilibrated in 0.15 m NaCl, 20 mm phosphate, pH 7.0, at a flow rate of 0.3 ml/min. The void (Vo) and total (Vt) volumes were determined with dextran blue and sodium dichromate, respectively. The column was calibrated for molecular mass using ovalbumin (45 kDa), hemoglobin (64.5 kDa), transferrin (80 kDa), and collagenase type 3 (110 kDa), which were monitored by on-line UV absorption at 280 nm. A plot of Kavversus Mr (on a log scale) was constructed. The elution positions of HGF/SF (500 ng) and a purified cross-linked HGF/SF-heparin conjugate (400 ng), applied in a 0.05-ml volume were determined by collecting fractions of 0.3 ml, followed by dot-blotting to nitrocellulose and probing with an antiserum against HGF/SF as described earlier. The elution position of free heparin was determined by dot blotting of 0.3-ml fractions to a cellulose acetate membrane followed by staining with 0.08% (w/v) aqueous azure A and subsequent destaining in water. CHO pgsA-745 cell monolayers were washed with PBS and then scraped into 100 μl of extraction solution, comprising 0.15 m NaCl, 25 mm HEPES, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1% (v/v) Nonidet P-40, 0.05% (w/v) SDS, 0.2% (w/v) sodium deoxycholate, 5 mm EDTA, 2 mm EGTA, 1 μg/ml soybean trypsin inhibitor, 0.05% (w/v) sodium orthovanadate, 1 mm NaF, 0.1 mm ammonium molybdate, 1 mm MgCl2, 0.2 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin A, pH 8.0. Cell extracts were mixed end-over-end for 1 h at 4 °C and then centrifuged (12,000 rpm for 10 min) to remove insoluble residues. Met receptor was partially purified by adsorption of the soluble cell extracts onto a suspension (packed bed volume of 300 μl) of wheat germ agglutinin-agarose. After washing with extraction solution, the bound glycoproteins (including Met) were eluted with either nonreducing Laemmli SDS-sample buffer (for SDS-PAGE), or 0.15 m NaCl, pH 2.0, followed by neutralization to pH 7.0 (for heparin affinity chromatography). Partially purified Met was analyzed on a nonreducing 7.5% (w/v) SDS-polyacrylamide gel with a 5% (w/v) polyacrylamide stacking gel. Western blots were probed with either a 1:1000 dilution of the DQ-13 murine monoclonal antibody against the intracellular C terminus of the Met β-chain (Upstate Biotechnology Inc., Lake Placid, NY), or a 1 μg/ml dilution of a goat antiserum against the ectodomain of murine Met (R & D Systems). These were followed by 1:5000 dilutions of horseradish peroxidase-conjugated goat anti-mouse IgG or rabbit anti-goat IgG, respectively, and enhanced chemiluminescent detection. Partially purified Met was applied to a heparin-agarose affinity column (0.5 ml volume) equilibrated with PBS, and re-circulated three times. After extensive washing with 0.5 mm CHAPS in PBS, pH 7.4, any bound material was eluted using 1-ml stepwise additions of 0.15–1m NaCl in 20 mm phosphate, 0.5 mmCHAPS, pH 7.4. The agarose-based Sepharose CL-4B was used as a parallel control column. Met was recovered and concentrated from the collected fractions by re-adsorption to wheat germ agglutinin-agarose (30 μl of gel suspension), and then released by treatment with nonreducing SDS sample buffer at 100 °C. Samples were electrophoresed on a 7.5% (w/v) SDS-polyacrylamide gel with a 5% (w/v) polyacrylamide stacking gel. Western blots were probed with the anti-Met ectodomain antiserum as described above. The CHO pgsA-745 mutant cells are functionally mutated in the xylosyltransferase gene, resulting in a failure to transfer xylose to targeted serine resides and thus to initiate the synthesis of the sulfated GAGs. Consequently these cells are deficient in both HS and DS (31Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Google Scholar), although they do possess Met (21Sakata H. Stahl S.J. Taylor W.G. Rosenberg J.M. Sakaguchi K. Wingfield P.T. Rubin J.S. J. Biol. Chem. 1997; 272: 9457-9463Google Scholar). However, they fail to respond to HGF/SF by either ERK activation (dual phosphorylation) (Fig.1A) or by cell migration across a porous Transwell membrane (Fig. 1B). Even elevated HGF/SF concentrations of up to 50 ng/ml failed to have an effect (responsive cells usually respond optimally to 1–10 ng/ml). However, upon the simultaneous addition of soluble exogenous heparin together with HGF/SF, these mutant cells then signal through ERK (Fig.1A) and also acquire a migratory phenotype (Fig.1B), although they do not incorporate [3H]thymidine (data not shown). Heparin alone, in the absence of HGF/SF, had no effect (Fig. 1, A andB). Both HS and DS are known to bind HGF/SF with similar high affinities (14Rahmoune H. Rudland P.S. Gallagher J.T. Fernig D.G. Biochemistry. 1998; 37: 6003-6008Google Scholar, 15Lyon M. Deakin J.A. Rahmoune H. Fernig D.G. Gallagher J.T. J. Biol. Chem. 1998; 273: 271-278Google Scholar), even though there are significant structural differences between these two GAGs. Both HS and DS, as soluble exogenous ligands, promote HGF/SF-mediated motility of CHO pgsA-745 cells in a Transwell migration assay, and they appear to act with similar potencies over a 103-fold range of concentrations (Fig.2). The crucial role of GAGs in the activation of Met by HGF/SF, as demonstrated in the CHO pgsA-745 cells, could result from a number of possible mechanisms. We have utilized a zero-length cross-linking technique which, by covalently linking HGF/SF to a bound GAG partner, can allow us to probe some of these putative mechanisms. Application of the zero-length cross-linking procedure gives rise to the efficient formation of covalent complexes between HGF/SF and all three GAG species that bind to it, i.e. HS (Fig.3A), DS (Fig. 3B), and heparin (not shown). These complexes display a larger and more heterogeneous molecular size on SDS-PAGE than native HGF/SF, reflecting a stable conjugation of the protein with a heterogeneous GAG population. Treatment of HGF/SF-HS complexes with either low pH nitrous acid or a mixture of heparinase enzymes, to specifically degrade the HS component, leads to a reduction in molecular size of the complex toward that of the initial HGF/SF monomer (Fig. 3A). Heparinase enzymes are less effective than nitrous acid, probably because of the larger steric exclusion they encounter in accessing the protein-conjugated HS. Similarly, HGF/SF-DS conjugates can be reduced in size by digestion with chondroitinase ABC (Fig. 3B). Covalent HGF/SF-GAG conjugates are unable to bind heparin-agarose, unlike native HGF/SF (see Fig. 3C for HGF/SF-HS conjugates; HGF/SF-DS conjugates behave the same (not shown)). This proves that the GAG is correctly positioned and cross-linked into the putative GAG-binding site in HGF/SF, thereby blocking any additional GAG interactions. Importantly, this means that when the conjugates are introduced into cell cultures, there can be no subsequent interaction with any endogenous GAGs. HGF/SF can also be effectively cross-linked to high affinity oligosaccharides such as HS- and DS-derived dp12–14s. However, there is only a very slight molecular weight shift on SDS-PAGE (not shown), and with smaller oligosaccharides it becomes imperceptible, because of the contrasting large size of HGF/SF (90 kDa). The formation of conjugates in these cases can only be confirmed by the proportionate loss of heparin affinity. As oligosaccharide size decreases the efficiency of cross-linking similarly decreases, and with dp8s only a small proportion of conjugate is formed, as assessed by binding to heparin-agarose (not shown). Importantly, the small size shifts upon SDS-PAGE after cross-linking with oligosaccharides of various sizes are only sufficient to indicate the formation of a 1:1 complex of HGF/SF and oligosaccharide, and there is no evidence of higher molecular weight oligomers. Even with intact GAGs the size of the great majority of the conjugate product would appear to be consistent with the presence of only one HGF/SF monomer per GAG chain (Fig. 3, Aand B). Cross-linked conjugates of HGF/SF with HS or DS are biologically active, in that they stimulate the activation of ERK (Fig.4) and the consequent motility of CHOpgsA-745 cells in the Transwell assay (Fig. 4). They also stimulate the motility of MDCK cells in both the scatter and Transwell assays (data not shown). Again, HS and DS display comparable levels of activity when conjugated to HGF/SF, as they do when noncovalently mixed with HGF/SF (compare Figs. 1 and 4). However, in absolute terms, the HGF/SF-GAG conjugates, with either HS or DS, display about 40–55% of the activity of HGF/SF mixed with an identical concentration of free GAG in the CHO pgsA-745 cells(Fig. 4), and about 35–40% of the activity elicited by HGF/SF alone in t
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