TIMP-3 Binds to Sulfated Glycosaminoglycans of the Extracellular Matrix
2000; Elsevier BV; Volume: 275; Issue: 40 Linguagem: Inglês
10.1074/jbc.m000907200
ISSN1083-351X
AutoresWei‐Hsuan Yu, Shuan-su C. Yu, Qi Meng, Keith Brew, J. Frederick Woessner,
Tópico(s)Connective tissue disorders research
ResumoOf the four known tissue inhibitors of metalloproteinases (TIMPs), TIMP-3 is distinguished by its tighter binding to the extracellular matrix. The present results show that glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfates A, B, and C, and sulfated compounds such as suramin and pentosan efficiently extract TIMP-3 from the postpartum rat uterus. Enzymatic treatment by heparinase III or chondroitinase ABC also releases TIMP-3, but neither one alone gives complete release. Confocal microscopy shows colocalization of heparan sulfate and TIMP-3 in the endometrium subjacent to the lumen of the uterus. Immunostaining of TIMP-3 is lost upon digestion of tissue sections with heparinase III and chondroitinase ABC. The N-terminal domain of human TIMP-3 was expressed and found to bind to heparin with affinity similar to that of full-length mouse TIMP-3. The A and B β-strands of the N-terminal domain of TIMP-3 contain two potential heparin-binding sequences rich in lysine and arginine; these strands should form a double track on the outer surface of TIMP-3. Synthetic peptides corresponding to segments of these two strands compete for heparin in the DNase II binding assay. TIMP-3 binding may be important for the cellular regulation of activity of the matrix metalloproteinases. Of the four known tissue inhibitors of metalloproteinases (TIMPs), TIMP-3 is distinguished by its tighter binding to the extracellular matrix. The present results show that glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfates A, B, and C, and sulfated compounds such as suramin and pentosan efficiently extract TIMP-3 from the postpartum rat uterus. Enzymatic treatment by heparinase III or chondroitinase ABC also releases TIMP-3, but neither one alone gives complete release. Confocal microscopy shows colocalization of heparan sulfate and TIMP-3 in the endometrium subjacent to the lumen of the uterus. Immunostaining of TIMP-3 is lost upon digestion of tissue sections with heparinase III and chondroitinase ABC. The N-terminal domain of human TIMP-3 was expressed and found to bind to heparin with affinity similar to that of full-length mouse TIMP-3. The A and B β-strands of the N-terminal domain of TIMP-3 contain two potential heparin-binding sequences rich in lysine and arginine; these strands should form a double track on the outer surface of TIMP-3. Synthetic peptides corresponding to segments of these two strands compete for heparin in the DNase II binding assay. TIMP-3 binding may be important for the cellular regulation of activity of the matrix metalloproteinases. extracellular matrix glycosaminoglycan matrix metalloproteinase phosphate-buffered saline tissue inhibitor of metalloproteinases N-terminal domain of TIMP-3 baby hamster kidney The extracellular matrix (ECM)1 provides mechanical support to cells and regulates signals reaching the cell that govern cell localization, differentiation, proliferation, and apoptosis. Components of the ECM, particularly the glycosaminoglycans (GAGs), are able to sequester bioactive molecules such as growth factors (1Iozzo R.V. Ann. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1344) Google Scholar), proteases (2Yu W.-H. Woessner Jr., J.F. J. Biol. Chem. 2000; 275: 4183-4191Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), and inhibitors. Turnover of the ECM is a highly regulated process necessary for movement of cells and for release of growth factors. Matrix metalloproteases (MMPs) are believed to be key participants in this remodeling; there are at least 20 MMPs, all able to digest various ECM components (3Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3890) Google Scholar, 4Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (701) Google Scholar). The MMPs, in turn, are regulated by tissue inhibitors of metalloproteinases or TIMPs. The major function of the TIMPs is to inhibit MMPs; any imbalance in which the activities of MMPs outweigh the TIMP levels will favor tissue destruction and pathological processes (5Gomez D.E. Alonso D.F. Yoshiji H. Thorgeirsson U.P. Eur. J. Cell Biol. 1997; 74: 111-122PubMed Google Scholar, 6Burger D. Rezzonico R. Li J.M. Modoux C. Pierce R.A. Welgus H.G. Dayer J.M. Arthritis Rheum. 1998; 41: 1748-1759Crossref PubMed Scopus (146) Google Scholar). The TIMPs also possess growth stimulatory and regulatory activities (7Murate T. Hayakawa T. Platelets. 1999; 10: 5-16Crossref PubMed Scopus (28) Google Scholar, 8Andreu T. Beckers T. Thoenes E. Hilgard P. von Melchner H. J. Biol. Chem. 1998; 273: 11385-13848Abstract Full Text Full Text PDF Scopus (42) Google Scholar). The four members of the TIMP family all have similar secondary structures of six loops stabilized by six highly conserved disulfide bonds. The TIMPs all bind tightly, albeit with widely varying affinity, to the various MMPs. The x-ray structure (9Gomis-Rüth F.X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (513) Google Scholar) shows that the N-terminal cysteine chelates the active site zinc. TIMPs have N- and C-terminal domains, each with three loops. The N-terminal domain of TIMP-1 folds readily and displays full inhibitory activity (10Murphy G. Houbrechts A. Cockett M.I. Williamson R.A.O. O'Shea M. Docherty A. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (285) Google Scholar). TIMP-3 has several features that distinguish it from the other TIMPs. First, it is the only TIMP to bind tightly to the ECM: it was first observed as a transformation-sensitive protein bound to the ECM of chick embryo fibroblasts (11Blenis J. Hawkes S.P. J. Biol. Chem. 1984; 259: 11563-11570Abstract Full Text PDF PubMed Google Scholar) and extractable with SDS or guanidine. This protein was subsequently shown to be TIMP-3 (12Pavloff N. Staskus P.W. Kishnani N.S. Hawkes S.P. J. Biol. Chem. 1992; 267: 17321-17326Abstract Full Text PDF PubMed Google Scholar). Second, TIMP-3 is the only TIMP to inhibit members of the ADAM (adisintegrin and metalloprotease domain) family such as tumor necrosis factor-α-converting enzyme (13Amour A. Slocombe P.M. Webster A. Butler M. Knight C.G. Smith B.J. Stephens P.E. Shelley C. Hutton M. Knäuper V. Docherty A.J.P. Murphy G. FEBS Lett. 1998; 435: 39-44Crossref PubMed Scopus (547) Google Scholar); this may account for its ability to induce apoptosis (14Smith M.R. Kung H.F. Durum S.K. Colburn N.H. Sun Y. Cytokine. 1997; 9: 770-780Crossref PubMed Scopus (171) Google Scholar). It is the only TIMP to inhibit shedding of l-selectin (15Borland G. Murphy G. Ager A. J. Biol. Chem. 1999; 274: 2810-2815Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and interleukin-6 receptors (16Hargreaves P.G. Wang F.F. Antcliff J. Murphy G. Lawry J. Russell R.G.G. Croucher P.I. Br. J. Haematol. 1998; 101: 694-702Crossref PubMed Scopus (70) Google Scholar). Third, TIMP-3 is the only TIMP directly implicated in a disease process: Ser-Cys mutants of TIMP-3 accumulate in Bruch's membrane of the eye and cause Sorsby's fundus dystrophy (17Fariss R.N. Apte S.S. Luthert P.J. Bird A.C. Milam A.H. Br. J. Ophthalmol. 1998; 82: 1329-1334Crossref PubMed Scopus (94) Google Scholar). TIMP-3 also promotes the detachment of transformed cells from the ECM (18Yang T.T. Hawkes S.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10676-10680Crossref PubMed Scopus (139) Google Scholar) and is involved in the formation, branching, and expansion of epithelial tubes and in regulating trophoblast invasion of the uterus (19Apte S.S. Hayashi K. Seldin M.F. Mattei M.G. Hayashi M. Olsen B.R. Dev. Dyn. 1994; 200: 177-197Crossref PubMed Scopus (125) Google Scholar). The present study is concerned with the mechanism of binding of TIMP-3 to the extracellular matrix. We recently reported that matrilysin could bind to heparan sulfate in rat uterine tissues (2Yu W.-H. Woessner Jr., J.F. J. Biol. Chem. 2000; 275: 4183-4191Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). In this work, it was noted that heparin not only extracted matrilysin from the tissue, but also solubilized TIMP-3. The present results indicate that heparan sulfate and other sulfated glycosaminoglycans may be responsible for the binding of TIMP-3 to the ECM. Uteri were collected 1 day postpartum from Harlan Sprague-Dawley rats (Harlan), weighed (∼2 g), washed 3× with cold 50 mm Tris, pH 7.5, 0.03% sodium azide, and homogenized in 20 ml of this buffer containing 0.1% Triton X-100 with a Polytron for 6 min at 4 °C. The mixture was centrifuged at 11,000 rpm for 20 min. The pellet was washed 2× and resuspended in the same volume of cold 50 mm Tris, pH 7.5, 0.03% sodium azide, 50 μm Z-Phe-chloromethyl ketone, 50 μm aminoethyl-benzenesulfonyl fluoride. The suspension was divided at 0.5 ml per tube. Extractants (50 μl, Sigma) were added to a final concentration of 0.2 mg/ml of the various GAGs and 2 mg/ml of pentosan and suramin. Extraction at 4 °C for 30 min was followed by centrifugation at 14,000 rpm for 10 min. For the heat extraction procedure, see Ref. 20Weeks J.G. Halme J. Woessner Jr., J.F. Biochim. Biophys. Acta. 1976; 445: 205-214Crossref PubMed Scopus (74) Google Scholar. To destroy TIMPs, control extracts were reduced with 5 mm dithiothreitol for 25 min, 24 °C, and thiol groups were blocked with 10 mm iodoacetic acid. For the heparinase III (Flavobacterium heparinum, heparitinase I, Sigma) and chondroitinase ABC (Sigma) digestion, pellets were resuspended in 50 mm Tris, pH 7.5, 0.03% sodium azide, 5 mm CaCl2, 10 μmZnCl2, 50 μm Z-Phe-chloromethyl ketone, and 50 μm aminoethyl-benzenesulfonyl fluoride and incubated with 0.2 unit of enzyme/ml at 37 °C for 18 h. Controls were incubated without added enzyme to check for endogenous activity. Components for 12.5% SDS-polyacrylamide gel were mixed with gelatin (final concentration 1 mg/ml) and a proprietary mixture of gelatinases (University Technology International, Calgary) and cast as gels. Extracts containing TIMPs were electrophoresed, and the gels were then washed 3× with 2.5% Triton X-100/50 mm Tris/5 mmCaCl2/0.03% azide and 3× with 50 mm Tris, pH 7.5, 5 mm CaCl2, 50 μm Z-Phe-chloromethyl ketone, and 10 mmphenylmethylsulfonyl fluoride. The gels were incubated in this latter mixture at 37 °C for 18 h and stained with Coomassie Blue. The blue gelatin staining was cleared by gelatinase action except where TIMP bands blocked this activity. Marker TIMPs (mouse) were also obtained from the University Technology International, Calgary. Frozen tissue sections (5 μm) from 26- to 32-h postpartum rat uterus were air-dried and soaked in 95% ethanol for 10 min. Sections were prefixed with 100% methanol for 20 min and rinsed 3× with PBS. For the pretreated group, some sections were washed with 100 μl of heparin (20 mg/ml) for 1 h at 24 °C or digested for 18 h in a moist chamber at 24 °C with heparinase III, or chondroitinase ABC, or a combination of the two. These enzymes (0.2 unit/ml) were used in 50 mm acetate buffer, pH 6.5, containing 0.1 m ZnCl2, 5 mm CaCl2, 5 mm MgCl2, 10 mm phenylmethylsulfonyl fluoride, 0.05% sodium azide, and the following inhibitors at 0.1 mm: Z-Phe-chloromethyl ketone, tosyl-Phe-chloromethyl ketone, tosyl-Leu-chloromethyl ketone, antipain, and BB-94 (British BioTech Pharmaceuticals Ltd.). Control sections were treated with the same mixture without enzyme. The sections were rinsed twice with PBS. Sections that were not pretreated were postfixed for 5–10 min with 3.7% paraformaldehyde, rinsed with PBS, and blocked with 10% heat-inactivated normal rabbit serum at 24 °C in PBS for 40 min. They were then exposed to goat polyclonal antibody against human TIMP-3 (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal antibody (IgM) against heparan sulfate (Sagaku) for 1 h. The TIMP-3 antibody was raised against the C-terminal 20 amino acids and recognized both rat and human TIMP-3. Sections were washed 3× in PBS. Primary antibodies were reacted with a mixture in blocking reagent of rabbit anti-goat IgG polyclonal antibody conjugated to fluorescein isothiocyanate and rabbit anti-mouse IgM polyclonal antibody conjugated to Texas Red (both from Jackson ImmunoResearch Laboratories). Sections were washed 3× in PBS for a total of 45 min. Controls included sections in which the first antibody was omitted, sections treated only with goat anti-human TIMP-3 followed by rabbit anti-mouse IgM antibody, and sections treated with mouse anti-heparan sulfate followed by rabbit anti-rabbit IgG antibody (to show absence of cross-species reaction). Washed sections were covered with SlowFade mounting solution (Molecular Probes, Eugene, OR), and a coverslip was applied. Sections were analyzed with a Zeiss confocal laser scanning microscope (LSM 510) equipped with a 25-milliwatt krypton-argon laser and a 10-milliwatt helium-neon laser. Excitation was at 488 nm and emission at 530 and 590 nm for fluorescein isothiocyanate and Texas Red, respectively. Images were captured at 0.5-μm increments along the Z axis and converted to composite images by LSM 510 software. BHK cells transfected with mouse TIMP-3 (mT3PNUT.BHK, University Technology International, Calgary) were grown at 37 °C in Dubecco's modified Eagle's medium/nutrient mixture F12 (Life Technologies, Inc.) containing 5% fetal calf serum and 1% penicillin and streptomycin. Conditioned medium (50 ml) was collected from 75-cm2 cell cultures confluent for 3–4 days. Medium (1 ml) from BHK-TIMP-3 cell cultures was mixed with 4 ml of buffer suspension of 0.5 mg of heparin-agarose beads (Sigma, H-0402) and stirred at room temperature for 4 h. The mixture was poured in a small column then washed with 50 mm Tris, pH 7.5, plus 0.15 m NaCl. Stepwise elution was then carried out using increasing amounts of NaCl or heparin in Tris buffer Human TIMP-3 cDNA from a placental cDNA library was kindly provided by Dr. H. Nagase, University of Kansas Medical Center. A set of primers, 5′-AGTCATATGTGCACATGCTCG3-′ (forward) and 5′-GCGGCCGCGTTACAACCCAGGTG-3′ (reverse), was used in a one-step polymerase chain reaction to amplify the cDNA insert encoding N-terminal TIMP-3 (residues Cys1-Asn121). The amplified insert was digested by NdeI and NotI and ligated into pET21b vectors (Invitrogen Inc.). The ligation reaction mixture was used to transform the Escherichia coli DH5α competent cells. Plasmid DNA from positive clones was purified using the Qiagen kit and digested byNotI and NdeI at 37 °C for 3 h. The correct clone was confirmed by DNA sequencing and used to transform into the expression host E. coli BL21(DE3). Cells containing pET3a-N-TIMP-3 were grown in 3 ml of LB/ampicillin medium at 37 °C overnight then inoculated into 1- to 6-liter batches. WhenA 600 reached 0.6–0.8, the culture was induced with isopropyl-β-d-thiogalactopyranoside (0.4 mm) for protein expression. Cells were grown for another 3 h and harvested by centrifugation. N-TIMP-3 was expressed as a fusion protein with a C-terminal His tail. Inclusion bodies were dissolved in 50 ml of loading buffer (5 mm imidazole, 0.5 mNaCl, 20 mm Tris-HCl, pH 7.9, 6 m guanidine) and centrifuged at 10,000 rpm for 40 min. The supernatant was loaded onto a Ni-NTA column (7 × 80 mm, Qiagen) equilibrated with loading buffer. The column was washed at room temperature with 60 mm imidazole, 0.5 m NaCl, 8 m urea, 20 mm Tris-HCl, pH 7.9, then eluted with 500 mmimidazole, 0.25 m NaCl, 8 m urea, 10 mm Tris-HCl, pH 7.9. Fractions from the Ni-NTA column were analyzed by SDS-polyacrylamide gel electrophoresis. Recombinant N-TIMP-3 in 8 murea, 0.15 m NaCl, 50 mm Tris, pH 7.5, 0.05% Triton X-100, and 5 mm dithiothreitol was diluted 1:10 in folding buffer A (20 mm acetate buffer, pH 5.6, 0.15m NaCl, 5 mm dithiothreitol, and 0.05% Triton X-100). Heparin-agarose beads (Sigma) were suspended in 50 mm Tris, 0.15 m NaCl, pH 7.8. A mixture of 1 ml of TIMP-3 plus 4-ml beads (0.5 mg) was transferred into 6-kDa cut-off dialysis tubing and dialyzed against folding buffer B (50 mm Tris, 0.15 m NaCl, pH 7.8, 0.05% Triton, 0.03% sodium azide, and 10 mm cystamine), 50 ml, with three changes. In the final dialysis, folding buffer C (50 mm Tris, 0.15 m NaCl, pH 7.8, 0.05% Triton, 0.03% sodium azide) was used. The mixture was poured into a small column then washed with 50 mm Tris, pH 7.5, plus 0.2m NaCl. Elution of folded N-TIMP-3 was then carried out using increasing amounts of NaCl or heparin in Tris buffer. In this assay, binding of heparin to DNase II was assessed (21Guo X. Han I.S. Yang V.C. Meyerhoff M.E. Anal. Biochem. 1996; 235: 153-160Crossref PubMed Scopus (10) Google Scholar). Competitive test compounds were added at 4 °C for 20 min in pH 4.8 acetate buffer + 5 mm dithiothreitol; then substrate was added for digestion at 37 °C. Heparin concentration was adjusted to inhibit DNase II activity by 90%; compounds binding heparin reversed this inhibition. The percentage inhibition observed in the presence of heparin and added test species was plotted versus log concentration to yield the dose-response curve for the given species. Several peptides from the A and B strands of TIMP-3 and RHAMM401–411 (a heparin-binding peptide from the Receptor for Hyaluronic Acid-Mediated Mobility) were synthesized (Genemed). Various sulfated compounds were tested as extractants; Fig. 1 illustrates that heparin/heparan sulfates and chondroitin sulfates were effective extractants at 0.2 mg/ml, but keratan sulfate showed only a weak effect. Sulfated compounds such as pentosan polysulfate and suramin also liberated TIMP-3, but higher concentrations (2 mg/ml) were required (Fig. 1). Heat extraction, effective for matrilysin (2Yu W.-H. Woessner Jr., J.F. J. Biol. Chem. 2000; 275: 4183-4191Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), was much less so for TIMP-3. Two bands of TIMP-3 (27 and 22 kDa, corresponding to glycosylated and nonglycosylated forms (22Langton K.P. Barker M.D. McKie N. J. Biol. Chem. 1998; 273: 16778-16781Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), appeared in the reverse zymogram. Most of the TIMP-1 and TIMP-2 appeared in the initial Triton extract (not shown). A band corresponding in position to TIMP-2 was also extracted by GAGs, suramin, and pentosan (Fig. 1); this may be TIMP-2 that was bound to gelatinase A, but positive identification of the inhibitor has not been made. Heparin and suramin also extracted a small inhibitory protein of 16 kDa that was not sensitive to reduction/alkylation and has not been identified. Optimal extraction of TIMP-3 was achieved at 2–4 mg of heparin/ml or 1–2 mg of suramin/ml (not shown). A parallel gel was run on SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue (right portion of Fig. 1) to show that the dark bands interpreted as TIMPs in the reverse zymograms were not due to nonspecific protein bands. The intensity of the glycosylated TIMP-3 (T3*) band appears to be less in the heparan sulfate/heparin lanes than in theCS lanes. This is attributed to interaction with a comigrating band of matrilysin extracted by heparin but not by chondroitin sulfate; this protease band can be directly visualized in the heat extract lane. TIMP-3 was identified by its position on the reverse zymogram and by its sensitivity to destruction by reduction and alkylation (Fig.2). A polyclonal antibody to human TIMP-3 (Santa Cruz) was used to show that heparin removed the factor from the uterus (see Fig. 4 below), but the antibody proved unsuitable for Western blotting of rat TIMP-3. Therefore, the identification of TIMP-3 depended on size, sensitivity to reduction and alkylation, and binding to matrix as shown by loss from tissue sections following treatment that increases TIMP-3 in reverse zymograms. TIMP-3 is the only TIMP that binds to the extracellular matrix (5Gomez D.E. Alonso D.F. Yoshiji H. Thorgeirsson U.P. Eur. J. Cell Biol. 1997; 74: 111-122PubMed Google Scholar).Figure 1Extraction of TIMP-3 from postpartum rat uterus by sulfated compounds. Extract aliquots (10 μl) were analyzed by reverse zymography as described under “Experimental Procedures.” The lanes contain: Mw, Marker 12 protein standards (Novex); T-3, authentic TIMP-3 marker from University Technology International, Calgary. T3*, glycosylated TIMP-3; R/A, reduced and alkylated to destroy TIMP; CS-A, CS-B, CS-C, chondroitin sulfates A, B, and C; All GAGS, extraction with heparin and CS-A, -B, and -C combined. Left axis,M r values × 10−3. The gel to the right serves as a control: samples were electrophoresed in a gel without added gelatinase, held for 18 h at 4 °C and stained together with the reverse zymogram. Coomassie Blue shows intensity of the protein bands at each position.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Confocal microscopy of postpartum rat uterus. Microscopy and antibody staining are detailed under “Experimental Procedures.” A, section stained with antibody to heparan sulfate; the uterine lumen is to theleft. B, same section stained with antibody to TIMP-3. C, superimposed staining showing colocalization of heparan sulfate and TIMP-3. D, section stained with heparan sulfate antibody following a heparin wash. E, same section stained for TIMP-3. F, section stained for heparan sulfate following treatment with chondroitinase ABC. G, same section stained for TIMP-3. H, section stained for heparan sulfate following heparinase III treatment. I, same section stained for TIMP-3. J, section (similar to that in H) stained for heparan sulfate following combined treatment with heparinase III and chondroitinase ABC. K, same section stained for TIMP-3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig. 3 illustrates further extraction studies; reverse zymography is not quantitative and permits only rough qualitative comparisons. It was also affected by MMPs that migrated to the same region and reacted with the TIMPs. The initial tissue extract in Triton X-100 (normally discarded) contained some TIMP-3. This might reflect binding of TIMP-3 to proteoglycans of the cell membranes, which were disrupted by Triton, or TIMP-3 in complex with gelatinase A and B (23Butler G.S. Apte S.S. Willenbrock F. Murphy G. J. Biol. Chem. 1999; 274: 10846-10851Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). SDS should have released the bulk of the inhibitor (based on its ability to release the more tightly binding MMP-7 (2Yu W.-H. Woessner Jr., J.F. J. Biol. Chem. 2000; 275: 4183-4191Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar)), although some might remain bound to MMP-2 through its hemopexin domain (23Butler G.S. Apte S.S. Willenbrock F. Murphy G. J. Biol. Chem. 1999; 274: 10846-10851Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The highly positively charged heparin antagonist, poly-d-lysine, extracted TIMP-3; it is suggested that it competed with TIMP-3 for binding sites on the GAG chains. Digestion with chondroitinase ABC released less TIMP-3 than did heparinase III digestion. The postpartum rat uterus contained both heparan sulfate (Fig. 4 A) and TIMP-3 (Fig. 4 B), which were largely localized near the uterine lumen, in the epithelial cells, and in their underlying basement membrane. There was little of either molecule in the deep stroma (right side of Fig. 4 C). Superimposition of images indicates colocalization of the two proteins with some small patches of green remaining, perhaps on chondroitin sulfate. Washing with heparin (Fig. 4 E) completely eliminated the TIMP-3 staining. Digestion with chondroitinase ABC gave some reduction in TIMP staining (Fig. 4 G); but a better estimate of the chondroitinase-sensitive binding is probably provided by the residual staining in Fig. 4 I. Digestion with heparinase III gave extensive losses of both heparan sulfate and TIMP-3 (Fig. 4, H and I). Both components were completely removed by digestion with the two enzymes together (Fig. 4,J and K). Incubation of sections without added enzyme did not lead to significant losses of either component (not shown). In reverse zymography (Fig.5 A), mouse TIMP-3 activity could be detected in BHK-TIMP-3 cells but not in the mock-transfected cell line. Two forms of TIMP-3 were found in the medium: a 27-kDa form, corresponding to the glycosylated form (24Apte S.S. Olsen B.R. Murphy G. J. Biol. Chem. 1995; 270: 14313-14318Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) and a 22-kDa nonglycosylated form. To see TIMP-3 in the medium, it was necessary to culture for several days, presumably until binding sites in the ECM are first filled. The denatured truncated human N-TIMP-3 (∼14 kDa) was prepared from E. coli and folded (“Experimental Procedures”). Similar amounts of protein were found before and after folding (Fig. 5 B) but only the folded form was inhibitory (Fig. 5 C). Culture medium containing recombinant mouse TIMP-3 was mixed with heparin-agarose beads and packed in a small column (see “Experimental Procedures”). The initial concentration of NaCl was 0.15m and the wash was with 0.2 m. Stepwise increases in salt concentration eluted TIMP-3 between 0.3 and 0.8m NaCl, with the peak at about 0.5 m (Fig.6 A). Both the glycosylated and unglycosylated forms emerged at the same position. Further washing with 2% SDS removed a small amount of residual TIMP (about 5%). The purified and folded human N-TIMP-3 bound in similar fashion (Fig.6 B); both monomeric and dimeric forms were eluted with a peak also around 0.5 m NaCl but with somewhat longer tailing. This tailing, with a distinct band at 0.9 m NaCl was attributed to the propensity of the truncated TIMP to aggregate. Such aggregates may be eluted at higher salt, but the aggregates did not appear on the gel because of dissociation by SDS in the gel. The elution of full-length and truncated TIMP-3 around 0.5 mNaCl indicated that the major heparin binding site was located in the N-terminal part of the protein. Three peptides were synthesized based on the A and B strand sequences of rat TIMP-3 (25Douglas D.A. Shi Y.E. Sang Q.X.A. J. Protein Chem. 1997; 16: 237-255Crossref PubMed Scopus (82) Google Scholar): Pep1 = residues 19–32 (IRAKVVGKKLVKEG); Pep2 = residues 41–52 (IKQMKMYRGFSKM); and the spanning peptide Pep3 = residues 19–52 (IRAKVVGKKLVKEGPFGTLVYTIKQMKMYRFHSKM). This last peptide contained 9 basic residues potentially involved in heparin binding. It can be seen from Fig.7 that the two shorter peptides have similar affinity for heparin and that this is about 10-fold less than the binding affinity of the long peptide (IC50 = 3.5 μm). All three bound more firmly than the RHAMM401–410 peptide (KQKIKHVVKLK). N-TIMP-3 was unstable under the reducing conditions of this assay and could not be measured. There has been no systematic study of the extraction of TIMP-3 from the ECM. It was earlier noted that guanidine and SDS are effective extractants (26Breedveld F.C. Arthritis Rheum. 1997; 40: 794-796Crossref PubMed Scopus (12) Google Scholar). Here we show that negatively charged molecules (heparin, various GAGs, and polysulfated compounds) gave extensive extraction comparable to SDS, but positively charged compounds such as polylysine were also extractants. However, enzymatic treatment with heparinase III and chondroitinase ABC gave extensive extraction, pointing to the negatively charged GAG molecules as binding sites, so positively charged compounds such as polylysine probably competed with TIMP-3 for binding to heparin. A preponderance of basic over acidic residues (26:13) in TIMP-3 also supports this interpretation. It is interesting to note that cultured mouse mesangial cells produce TIMP-3 in the medium only after pentosan polysulfate or heparin treatment (27Elliot S.J. Striker L.J. Stetler-Stevenson W.G. Jacot T.A. Striker G.E. J. Am. Soc. Nephrol. 1999; 10: 62-68PubMed Google Scholar); we suggest this might be due to the release of TIMP-3 bound to the ECM of the cultures. The N-terminal domain of TIMP-3 contains 17 positive and 8 negative charges (25Douglas D.A. Shi Y.E. Sang Q.X.A. J. Protein Chem. 1997; 16: 237-255Crossref PubMed Scopus (82) Google Scholar) and exhibits the OB (oligosaccharide/oligonucleotide binding) fold (9Gomis-Rüth F.X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (513) Google Scholar). Because GAGs are similar in charge and linearity to oligosaccharides/nucleotide polymers, the N-terminal domain of TIMP-3 could be a binding site for GAGs. The binding of TIMP-3 is strong, but not nearly as strong as the binding of matrilysin: TIMP-3 is eluted from a heparin affinity column with 0.5m NaCl, whereas matrilysin is not eluted at 2 mNaCl (2Yu W.-H. Woessner Jr., J.F. J. Biol. Chem. 2000; 275: 4183-4191Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Matrilysin also has a great many more positive residues that might participate in binding. The fact that chondroitinase ABC appeared to release TIMP-3 from tissue but not as efficiently as heparinase III (Figs. 3 and 4) suggests that the binding is not highly specific. Several types of sulfated GAGs may serve as binding sites, but heparan sulfate chains are probably the major sites. Full-length TIMP-3 eluted from a heparin affinity column at 0.5 m NaCl. This matches ex
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