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Heparan Sulfate Proteoglycans as Extracellular Docking Molecules for Matrilysin (Matrix Metalloproteinase 7)

2000; Elsevier BV; Volume: 275; Issue: 6 Linguagem: Inglês

10.1074/jbc.275.6.4183

1083-351X

Wei‐Hsuan Yu, J. Frederick Woessner,

Cell Adhesion Molecules Research

Many matrix metalloproteinases (MMPs) are tightly bound to tissues; matrilysin (MMP-7), although the smallest of the MMPs, is one of the most tightly bound. The most likely docking molecules for MMP-7 are heparan sulfate proteoglycans on or around epithelial cells and in the underlying basement membrane. This is established by extraction experiments and confocal microscopy. The enzyme is extracted from homogenates of postpartum rat uterus by heparin/heparan sulfate and by heparinase III treatment. The enzyme is colocalized with heparan sulfate in the apical region of uterine glandular epithelial cells and can be released by heparinase digestion. Heparan sulfate and MMP-7 are expressed at similar stages of the rat estrous cycle. The strength of heparin binding by recombinant rat proMMP-7 was examined by affinity chromatography, affinity coelectrophoresis, and homogeneous enzyme-based binding assay; theK D is 5–10 nm. Zymographic measurement of MMP-7 activity is greatly enhanced by heparin. Two putative heparin-binding peptides have been identified near the C- and N-terminal regions of proMMP-7; however, molecular modeling suggests a more extensive binding track or cradle crossing multiple peptide strands. Evidence is also found for the binding of MMP-2, -9, and -13. Binding of MMP-7 and other MMPs to heparan sulfate in the extracellular space could prevent loss of secreted enzyme, provide a reservoir of latent enzyme, and facilitate cellular sensing and regulation of enzyme levels. Binding to the cell surface could position the enzyme for directed proteolytic attack, for activation of or by other MMPs and for regulation of other cell surface proteins. Dislodging MMPs by treatment with compounds such as heparin might be beneficial in attenuating excessive tissue breakdown such as occurs in cancer metastasis, arthritis, and angiogenesis. Many matrix metalloproteinases (MMPs) are tightly bound to tissues; matrilysin (MMP-7), although the smallest of the MMPs, is one of the most tightly bound. The most likely docking molecules for MMP-7 are heparan sulfate proteoglycans on or around epithelial cells and in the underlying basement membrane. This is established by extraction experiments and confocal microscopy. The enzyme is extracted from homogenates of postpartum rat uterus by heparin/heparan sulfate and by heparinase III treatment. The enzyme is colocalized with heparan sulfate in the apical region of uterine glandular epithelial cells and can be released by heparinase digestion. Heparan sulfate and MMP-7 are expressed at similar stages of the rat estrous cycle. The strength of heparin binding by recombinant rat proMMP-7 was examined by affinity chromatography, affinity coelectrophoresis, and homogeneous enzyme-based binding assay; theK D is 5–10 nm. Zymographic measurement of MMP-7 activity is greatly enhanced by heparin. Two putative heparin-binding peptides have been identified near the C- and N-terminal regions of proMMP-7; however, molecular modeling suggests a more extensive binding track or cradle crossing multiple peptide strands. Evidence is also found for the binding of MMP-2, -9, and -13. Binding of MMP-7 and other MMPs to heparan sulfate in the extracellular space could prevent loss of secreted enzyme, provide a reservoir of latent enzyme, and facilitate cellular sensing and regulation of enzyme levels. Binding to the cell surface could position the enzyme for directed proteolytic attack, for activation of or by other MMPs and for regulation of other cell surface proteins. Dislodging MMPs by treatment with compounds such as heparin might be beneficial in attenuating excessive tissue breakdown such as occurs in cancer metastasis, arthritis, and angiogenesis. matrix metalloproteinase phosphate-buffer saline recombinant rat proMMP-7 polymerase chain reaction Z-Phe-chloromethylketone Tosyl-Lys-chloromethylketone Tosyl-Phe-chloromethylketone Remodeling of the extracellular matrix is critical in many physiological processes such as embryonic development, bone growth, nerve outgrowth, ovulation, uterine involution, and wound healing; the family of matrix metalloproteinases (MMPs)1 plays a major role in such remodeling (1.Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3088) Google Scholar, 2.Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2639) Google Scholar). These proteases also have a prominent role in pathological processes such as cancer invasion and metastasis (3.Chambers A.F. Matrisian L.M. J. Natl. Cancer Inst. 1997; 89: 1260-1270Crossref PubMed Scopus (1436) Google Scholar), arthritis (4.Cawston T. Mol. Med. Today. 1998; 4: 130-137Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), and atherosclerosis (5.Celentano D.C. Frishman W.H. J. Clin. Pharmacol. 1997; 37: 991-1000Crossref PubMed Scopus (67) Google Scholar). The matrixin family comprises 20 members that differ in domain structure and specificity (6.Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3890) Google Scholar). Much is known about the regulation of these enzymes at the level of transcription (7.Borden P. Heller R.A. Crit. Rev. Eukaryot. Gene Expr. 1997; 7: 159-178Crossref PubMed Scopus (291) Google Scholar), activation of proenzyme (8.Nagase H. Biol. Chem. 1997; 378: 151-160PubMed Google Scholar), and inhibition by natural inhibitors such as the tissue inhibitors of metalloproteinases (9.Gomez D.E. Alonso D.F. Yoshiji H. Thorgeirsson U.P. Eur. J. Cell Biol. 1997; 74: 111-122PubMed Google Scholar). However, an important aspect of these enzymes has generally been overlooked, namely, how they are anchored outside the cell. An instructive example is found in articular cartilage: compression drives out fluid into the synovial space, and release of pressure leads to fluid imbibition. This tissue contains MMP-1 (collagenase 1), MMP-2 (gelatinase A), MMP-3 (stromelysin), MMP-8 (collagenase 2), and MMP-13 (collagenase 3) (4.Cawston T. Mol. Med. Today. 1998; 4: 130-137Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), yet the continual slow movement of fluid does not dislodge or wash away the bulk of these enzyme activities. MMPs are commonly studied by tissue culture methods, a situation in which there is a vast overproduction of enzyme and a paucity of matrix; here the MMPs appear to be readily soluble. However, early workers tried to extract MMPs from various tissues such as skin, cartilage, and uterus but had success only with chaotropic agents such as 5 murea (10.Wirl G. Connect. Tissue Res. 1977; 5: 171-178Crossref PubMed Scopus (36) Google Scholar) or 2 m guanidine HCl (11.Dean D.D Martel-Pelletier J. Pelletier J.-P. Howell D.S. Woessner Jr., J.F. J. Clin. Invest. 1989; 84: 678-685Crossref PubMed Scopus (543) Google Scholar). It was thought that MMP-1 bound to its substrate (12.Vater C.A. Mainardi C.L. Harris Jr., E.D. Biochim. Biophys. Acta. 1978; 539: 238-247Crossref PubMed Scopus (34) Google Scholar), but subsequent studies showed that the proMMP-1 could not bind collagen (13.Welgus H.G. Jeffrey J.J. Stricklin G.P. Roswit W.T. Eisen A.Z. J. Biol. Chem. 1980; 255: 6806-6813Abstract Full Text PDF PubMed Google Scholar) but was still difficult to extract. Anchoring MMPs to the cell surface or extracellular matrix would not only prevent them from rapidly diffusing away but would also enable the cell to keep them under close regulatory control. If there were specific binding sites, the cell could monitor these sites by receptors or integrins and respond by altering the synthesis of MMP. If the MMPs bind to the matrix, this could provide a reservoir for subsequent rapid tissue degradation. If they bind to the cell surface, they could be positioned for activation as in the case of gelatinase A (8.Nagase H. Biol. Chem. 1997; 378: 151-160PubMed Google Scholar), for interaction with cell surface adhesion molecules or receptors, for regulating the turnover of these molecules by shedding activity, or for a directed attack on the matrix as illustrated by MMP-14 bound on invadopodia (14.Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y.Y. Chen W.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7959-7964Crossref PubMed Scopus (364) Google Scholar). To explore the binding of MMPs to cells or matrix, we have selected the smallest member of the family, matrilysin (MMP-7), which has only two domains: the propeptide (9 kDa) and the catalytic domain (19 kDa). This enzyme is greatly elevated in the postpartum rat uterus but is quite difficult to extract; it requires the use of 0.1 m calcium salt and 60 °C heat (15.Sellers A. Woessner Jr., J.F. Biochem. J. 1980; 189: 521-531Crossref PubMed Scopus (51) Google Scholar). This method was originally developed for rat uterine collagenase 3 (16.Weeks J.G. Halme J. Woessner Jr., J.F. Biochim. Biophys. Acta. 1976; 445: 205-214Crossref PubMed Scopus (74) Google Scholar), an enzyme that was shown to bind tightly to heparin-Sepharose (17.Roswit W.T. Halme J. Jeffrey J.J. Arch. Biochem. Biophys. 1983; 225: 285-295Crossref PubMed Scopus (59) Google Scholar). This suggested that sulfated glycosaminoglycans might provide anchoring sites for MMP-7. Our present findings indicate that heparan sulfates on the cell surface and/or in the matrix may be the major binding site of MMP-7 and may also bind other MMPs such as MMP-1, -2, -9 (gelatinase B), and -13. Pregnant Sprague-Dawley rats were purchased from Harlan. Uteri were collected 1 day postpartum when MMP-7 levels are highest, weighed (∼2 g), washed three times 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 twice, resuspended in the same volume of cold 50 mm Tris, pH 7.5, 0.03% sodium azide, 50 μm ZPCK, 50 μm4-(2-aminoethyl)-benzenesulfonyl fluoride. This was divided at 0.5 ml/tube. Extractants (Sigma) were added, held at 4 °C for 30 min, and then centrifuged at 14,000 rpm for 10 min. For heat extraction, see Ref. 16.Weeks J.G. Halme J. Woessner Jr., J.F. Biochim. Biophys. Acta. 1976; 445: 205-214Crossref PubMed Scopus (74) Google Scholar. Bovine carboxymethylated-transferrin substrate was prepared (18.Nagase H. Methods Enzymol. 1995; 248: 449-470Crossref PubMed Scopus (70) Google Scholar). Pig gelatin type A (Sigma, 0.5 mg/ml) was embedded in 7.5% SDS-polyacrylamide gel and CM-transferrin (0.3 mg/ml) in 12.5% gel. Samples were treated with sample buffer without dithiothreitol at room temperature and electrophoresed. The gel was washed twice with 2.5% Triton X-100, 50 mm Tris, pH 7.5, 4 °C, 20 min each, to remove SDS and then washed twice with buffer plus 5 mm CaCl2. The gel was washed three times with incubation buffer (50 mm Tris, pH 7.5, 5 mm CaCl2) and then incubated in this buffer with added protease inhibitors (50 μm each of ZPCK, TPCK, TLCK, and 4-(2-aminoethyl)-benzenesulfonyl fluoride (Calbiochem)) for 18 h, 37 °C, with gentle shaking. Gels were stained with 0.1% Coomassie Blue in 40% MeOH, 10% acetic acid, for 45 min, and destained with 7% acetic acid. Two peptides of the sequences, (Cys)LRKFYLHDSKTKK (22.Guo X. Han I.S. Yang V.C. Meyerhoff M.E. Anal. Biochem. 1996; 235: 153-160Crossref PubMed Scopus (10) Google Scholar, 23.Cha J. Auld D.S. Biochemistry. 1997; 36: 16019-16024Crossref PubMed Scopus (62) Google Scholar, 24.Yang B. Zhang L. Turley E.A. J. Biol. Chem. 1993; 268: 8617-8623Abstract Full Text PDF PubMed Google Scholar, 25.Deprez P.N. Signorelli J. Inestrosa N.C. Biochim. Biophys. Acta. 1995; 1252: 53-58Crossref PubMed Scopus (6) Google Scholar, 26.Wilson, C. L., Matrisian, and L. M. (1998) in Matrix Metalloproteinases (Parks, W. C., and Mecham, R. P., eds) pp. 149–184, Academic Press, San DiegoGoogle Scholar, 27.Busiek D.F. Baragi V. Nehring L.C. Parks W.C. Welgus H.G. J. Immunol. 1995; 154: 6484-6491PubMed Google Scholar, 28.Mertens G. Van der Scheuren B. van den Berghe H. David G. J. 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Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar) and (Cys)QKLYGKRNKL (238–247), corresponding to two putative heparin-binding sequences of rat MMP-7 in the propeptide and C-terminal region were synthesized and high pressure liquid chromatography-purified (Genemed). They were disulfide-linked to maleimide-activated keyhole limpet hemocyanin (Pierce). The conjugates (1 mg) or recombinant proMMP-7 (200 μg) emulsified with Freund's complete adjuvant were used to immunize New Zealand female rabbits. Antibody titers were monitored by proMMP-7 dot blot assay. Antibody specificities were characterized by immunoblotting (see next section). Antibodies were designated RM7-P (to propeptide 22–34), RM7-C (to peptide 238–247 near the C terminus), and RM7-W (to whole recombinant rat matrilysin). RM7-W did not cross-react with human MMP-1, -2, -3, -7, -8, -9, or -13 zymogen or active forms. RM7-P reacts only with the proform of at MMP-7 and not with the active form. RM7-C reacts with both proform and active rat MMP-7 but not any other species tested from the MMP family. MMP-1 and -3 were kindly provided by Dr. H. Nagase (Kansas City, MO); MMP-8 was provided by Dr. H. Tschesche (Bielefeld, Germany); MMP-7 was provided by Dr. S. Shapiro (St. Louis, MO); and rat MMP-13 was provided by Dr. J. Jeffrey (Albany, NY). The protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad). Then 0.1% Tween-20 (TTBS) containing 5% nonfat milk was used to block the membrane for 3 h at 24 °C. The first antibody (e.g. RM7-P) was applied at 4 °C overnight. After washing three times with TTBS/milk, the second antibody (e.g. goat anti-rabbit IgG-alkaline phosphatase) was applied for 2 h. Then the transblot was washed three times with TTBS/milk and stained with NBT/BCIP (Pierce). If horseradish peroxidase-conjugated secondary antibody was used, the transblot was visualized by chemiluminescence (DuPont Renaissance kit) Pieces of mature rat uterus at various points in the estrous cycles (staged by cervical smears) were fixed in paraformaldhyde and embedded. Sections 0.5 μm thick were cut, dewaxed in xylene for 5 min, and fixed in 100% methanol for 20 min. Sections were washed with PBS three times and then submitted to directly immunofluorescence staining or pretreatment of sections for glycosaminoglycan digestion. Digestion conditions were 0.2 units/ml of heparinase III (bacterial, Sigma) and/or chondroitin ABC lyase (Sigma) in buffer containing 0.05% sodium azide 100 μm zinc chloride, 5 mmCaCl2/MgCl2, 10 mmphenylmethylsulfonyl fluoride, 0.1 mm ZPCK/TPCK/TLCK, 50 μm BB94 (British Biotech), 0.1 mm antipain 0.15 m NaCl acetate buffer (pH 6.5) at 37 °C for 18 h. Controls were incubated with buffer only. Samples were gently washed twice in PBS, then post-fixed with 2% formaldehyde in PBS with 0.1 mm CaCl2 and 1 mm MgCl2for 10 min, and washed twice with PBS for 10 min. The tissue sections were blocked with 3% bovine serum albumin and 10% goat serum at room temperature in PBS for 40 min and subsequently exposed to first antibodies: rabbit polyclonal antibodies (IgG) to rat MMP-7 (RM7-C) and mouse monoclonal antibodies (IgM) to heparan sulfate (Sagaku) for 1 h at room temperature. Sections were washed three times in PBS for 45 min. Primary antibodies were detected by using goat polyclonal antibodies to rabbit IgG and mouse IgM conjugated to either fluorescein isothiocyanate or Texas Red, respectively (Jackson ImmunoResearch Labs., Inc.). Sections were washed three times in PBS for 45 min. The samples were mounted on slides and analyzed by confocal microscopy (inverted Nikon with Multiprobe 2001, Molecular Dynamics). Excitation was at 488 nm, and emission was at 530 and 590 nm, for fluorescein isothiocyanate and Texas Red, respectively. Images were captured at 0.6 nm increments along the z axis and converted to composite images by ImageSpace 3.10 software (Molecular Dynamics). The full-length rat proMMP-7 coding region (19.Abramson S.R. Conner G.E. Nagase H. Neuhaus I. Woessner Jr., J.F. J. Biol. Chem. 1995; 270: 16016-16022Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) was cloned into the NdeI andBamHI sites of PET3a vector (Novagen). The cDNA insert of the proMMP-7 expression construct was completely sequenced by using the Sequenase version 2.0 kit (U. S. Biochemical Corp.). The expression plasmid was transfected into E. coli BL21(DE3) cells (Novagen). Cells were cultured for 6 h (A 600 = 0.2∼0.4) followed by 2 h with isopropyl-1-thio-β-d-galactopyranoside. Cells were collected, passed through a French press, and centrifuged at 13,000 rpm for 20 min. The pellets were washed three times with 10 ml of inclusion body wash solution (50 mm Tris, pH 7.5, 10 mmdithiothreitol, 1% Triton X-100). The pellets suspended in 8m urea, 50 mm Tris, pH 7.5, 0.02% sodium azide. After 48 h at 4 °C, the sample was centrifuged. The soluble fraction was passed through a PD10 column (Amersham Pharmacia Biotech) to remove dithiothreitol and then applied to a zinc chelate-Sepharose 6LB column (Amersham Pharmacia Biotech). The bound proMMP-7 was eluted by stepwise decreasing pH. The product had a purity greater than 95%. The proMMP-7, still in 8 m urea, was diluted dropwise 10-fold in ice-cold refolding buffer (20 mm acetate, pH 5.6, 10 mm CaCl2, 1 μm ZnCl2, and 0.05% Triton X-100). After 30 min it was centrifuged at 14,000 rpm for 10 min to remove the insoluble portion. Storage was at −70 °C in 18% glycerol. Proper folding was shown by aminophenyl mercuric acetate activation of latent form, specific activity, and specificity of bond cleavage in gelatin and transferrin. The E198Q mutant of proMMP-7 was prepared by the Quick Change site-directed mutagenesis method (Stratagene). The PET3a.proMMP-7 expression vector (above) was used with the inserts of rat proMMP-7 as templates and a pair of synthetic oligonucleotide primers containing the desired mutation site (GAA to CAA), the 3′-oligonucleotide primer (−)-strand) 5′-ACCCAGAGAGTGGCCAAGTTGATGAGTGGC-3′ and 5′-oligonucleotide primer (+)-strand) 5′-GCCACTCATCAACTTGGCCACTCTCTGGGT-3′ for PCR amplification. The PCR product was digested with DpnI endonuclease. This PCR extended nicked vector was then transformed intoE. coli, Epicurian Coli XL1-Blue supercompetent cells (Stratagene). The ampicillin resistant colonies were screened by PCR and NdeI/BamHI digestion, and the positive plasmids were purified and transformed into the expression host BL21(DE3) strain. The total sequence including the desired mutation site was confirmed by DNA sequencing. Purification and folding was as described above. rproMMP-7 in 8 murea was diluted 1:10 in refolding buffer (20 mm acetate buffer, pH 5.6, 10 mm CaCl2, 1 μmZnCl2, and 0.05% Triton X-100). Heparin-agarose beads (Sigma, H0402) were suspended in 50 mm Tris, 5 mm CaCl2, pH 7.8. A mixture of 1 ml of enzyme plus 4-ml beads (0.5 mg) was stirred at room temperature for 4 h. The mixture at pH 7.5 was poured into a small column and then washed with 50 mm Tris, pH 7.5, plus 0.15 m NaCl. Elution was then carried out in steps of 0.5 ml with increasing concentrations of NaCl or heparin in Tris buffer. The enzyme did not bind to agarose beads lacking heparin. Using the method of Smith and Knauer (20.Smith J.W. Knauer D.J. Anal. Biochem. 1987; 160: 105-114Crossref PubMed Scopus (18) Google Scholar), heparin (Grade I, porcine intestinal mucosa,M r 5,000∼ 16,000, Sigma) was modified with 0.07 mol fluoresceinamine/uronic acid residue. This corresponds to about 3.8 fluoresceinamines:1 heparin of M r12,000. Fluoresceinamine-heparin (F-heparin) was stored at −70 °C. This F-heparin was radioiodinated using the IODO-BEADSTMIodination Reagent (Pierce). The specific activity was approximately 100,000 cpm/ng uronic acid. Affinity coelectrophoresis was carried out as previously documented (21.Lee M.K. Lander A.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2768-2772Crossref PubMed Scopus (155) Google Scholar) with F-heparin migrating through lanes containing varying concentrations of rproMMP-7(E198Q). This assay system is based on the method of Guo et al. (22.Guo X. Han I.S. Yang V.C. Meyerhoff M.E. Anal. Biochem. 1996; 235: 153-160Crossref PubMed Scopus (10) Google Scholar). The binding of compounds to heparin was assessed by using fresh standard solutions of these substances in pH 4.8 acetate buffer + 5 mmdithiothreitol. The concentrations of enzyme and substrate were monitored at 280 and 405 nm, respectively. Heparin concentration was adjusted to inhibit 1.1 μm DNase II by 90%; compounds binding heparin reversed this inhibition. The percentage of inhibition observed in the presence of both the test species and heparin was plotted versus log concentration to yield the dose-response curve for the given species. Various sulfated compounds were tested to see whether they could extract MMP-7 from the rat uterus at 1 day postpartum (a rich source of enzyme). Fig.1 A illustrates that heparin and heparan sulfates are effective extractants at 0.2 mg/ml, but chondroitin and dermatan sulfates are less effective. Sulfated compounds such as pentosan polysulfate and suramin also liberate MMP-7, but higher concentrations (2 mg/ml) are required (Fig. 1 A,lanes 7 and 8). The heparin antagonist, protamine, which is highly positively charged, can also extract MMP-7 from tissues (lane 9). Blotting with a polyclonal antibody specific for a segment of the propeptide (RM7-P, see “Experimental Procedures”) established that the zymogram bands correspond to proMMP-7 (data not shown). The major forms of MMP-7 that appear are the 28-kDa proform and a partially activated form of about 25 kDa. Active enzyme did not appear where expected (18 kDa), but there is evidence, obtained by use of antibody RM7-W to the whole enzyme, that it aggregates to produce some of the upper bands on the zymogram. Pro- and active MMP-2, proMMP-9, and proMMP-13 were released by the same extractants as for MMP-7 and could be detected by gelatin zymography (data not shown). Next, the effect of heparin concentration on MMP-7 extraction was studied. Because high levels of heparin (>2 mg/ml or 20 μg/gel lane) affect the resolution and enzyme activity on the subsequent CM-transferrin zymography, samples above 1 mg/ml were diluted to that final concentration. The optimum extraction of proMMP-7 from the uterus is with 2–4 mg heparin/ml based on the dilution factor multiplied by band intensity measured by UVP image analysis of Fig. 1 B(left panel). The polyclonal antibody RM7-P confirmed that the 28-kDa band in Fig. 1 B (right panel) was due to proMMP-7. The levels of heparin (0.2 mg/ml) used in Fig.1 A were suboptimal but were chosen to emphasize the differences among the various compounds tested. Heparin (5 mg/ml) and EDTA (10 mm) extracted maximum amounts of proMMP-7 and direct extraction with SDS also released most of the enzyme (Fig. 1 C). EDTA presumably leads to enzyme unfolding because of removal of stabilizing calcium and zinc. SDS will unfold the enzyme and also swamp out positive charges that might bind heparin. SDS treatment following heparin extraction did not release any further enzyme. Heparinase III (heparitinase I) digestion released 80–85% of the enzyme, but 15–20% was also released by chondroitin lyase ABC (Sigma), similar to results with chondroitin sulfates (Fig.1 A). During digestion, serine protease inhibitors, ZPCK, phenylmethylsulfonyl fluoride (0.1 mm), and 1 mm Zn2+ (to inhibit MMPs) were used to inhibit proteolytic release of enzymes, and also blanks were incubated without heparinase III or chondroitinase (not shown). The results of the five treatments are consistent with the hypothesis that native proMMP-7 binds to sulfated proteoglycans, particularly to heparan sulfate. The physiological relevance of the extraction data was investigated by studying the expression and localization of heparan sulfate and MMP-7 in the hormonally regulated tissue of the rat uterus. The expression of MMP-7 in the estrous cycle was most prominent in the early proliferative (estrous II) and late secretory (dioestrous) stages and was restricted to the endometrial glandular and lining epithelial cells (Fig. 2). Its secretion is bidirectional, apical and basolateral, but is predominant on the apical surface and some appears to be in the lumen. Interestingly, the epithelial surface heparan sulfate appears predominantly in the same estrous stages as MMP-7 does (Fig. 2). MMP-7 and heparan sulfate are colocalized at the apical surface of epithelial cells lining the lumen (Fig. 3 C). The hypothesis that heparan sulfate and MMP-7 are directly interacting was tested by washing tissue sections with heparin sulfate or digesting with heparinase III or chondroitinase ABC. Heparin sulfate completely washed MMP-7 from tissue sections (Fig. 3 E), consistent with the tissue extraction experiments (Fig. 1 C), but did not displace heparan sulfate as shown by the remaining positive stain (Fig.3 D). Greater than 80% of MMP-7 in tissue sections was released by heparinase III treatment (Fig. 3 I); however, chondroitinase did not release appreciable amounts of MMP-7 from tissue sections (Fig. 3 G). The combined action of the two enzymes completely removed MMP-7 (Fig. 3 K). The results support the hypothesis of binding of MMP-7 to proteoglycans containing heparan sulfate chains, with a possible small contribution from chondroitin sulfate chains.Figure 3Immunocolocalization of heparan sulfate and MMP-7 on the endometrial epithelium of rat uterus in estrous stage II. Sections were stained as in Fig. 2. Enzyme digestion is as described under “Experimental Procedures.” A, heparan sulfate. B, MMP-7. C, colocalization of MMP-7 and heparan sulfate (yellow). D and E, effect of heparin wash (2 mg/ml) on heparan sulfate (D) and MMP-7 staining (E). F and G, effect of chondroitinase ABC digestion on heparan sulfate (F) and MMP-7 staining (G). H and I, effect of heparinase III digestion on heparan sulfate (H) and MMP-7 staining (I). J and K, combined treatment with heparinase III and chondroitinase ABC on heparan sulfate (J) and MMP-7 staining (K).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Further investigation of the binding of rat proMMP-7 to heparin was facilitated by using recombinant methods to obtain large amounts of enzyme from E. coli (see “Experimental Procedures”). Human active MMP-7 lacking the propeptide (clone from Dr. S. Shapiro, St. Louis) was also expressed inE. coli BL21(DE3) cells, purified, and refolded. Rat rproMMP-7 was bound (>95%) to heparin-agarose beads as described under “Experimental Procedures” and then washed with Tris buffer to remove unbound material. Stepwise increasing salt concentration from 0.2 to 2.0 m NaCl failed to elute the bound enzyme (Fig.4 A). Heparin (20 mg/ml) gave complete elution (Fig. 4 A, lane 14); 2 mg/ml (lane 11) did not release all enzyme. The active form of rat rMMP-7 can be eluted by 0.6–0.8 m NaCl and the human active form, by 0.4–0.5 m (data not shown). EDTA also elutes rproMMP-7 efficiently but in a state that forms dimers and aggregates on SDS-polyacrylamide gel electrophoresis (data not shown). Because rproMMP-7 undergoes spontaneous autoactivation at high concentrations, we prepared the E198Q mutant by the same methods described for wild type rproMMP-7. This mutation is known to reduce activity of the active form of the enzyme by 400-fold (23.Cha J. Auld D.S. Biochemistry. 1997; 36: 16019-16024Crossref PubMed Scopus (62) Google Scholar) and should retard self-activation. The purified enzyme showed no significant autolysis in 8 h at 24 °C as shown by Western blot with the polyclonal antibody RM7-W, which reacts with all fragments of the enzyme (Fig. 4 B,lanes 4 and 5). Zymography at the usual enzyme levels showed no activity (Fig. 4 B, lanes 1 and2). When this mutant enzyme was embedded at various concentrations in gel slabs and a radioiodinated heparin probe was electrophoresed through the gel, retardation of heparin by the enzyme was visualized by radiography (Fig. 4 C).K D can be estimated from the protein concentration at which the heparin is half-shifted from being fully mobile to being maximally retarded. The K D is approximately 5–10 nm; at 10 nm rproMMP-7, the heparin is 80% retarded, and at 5 nm, heparin is fully mobile. Because of heterogeneity of the heparin, the migration front in Fig. 4 Cis fractionated into two populations: one binds tightly to MMP-7 and is retarded at the top of the gel, and the other appears half way down the lane; the excess unbound heparin migrates to the bottom half of the gel. All of these affinity retardation effects were eliminated by adding 500 μg/ml of cold heparin as a competitor (data not shown). The heparin affinity of two potential heparin-binding peptid