Calcium-induced activation and truncation of promatrix metalloproteinase-9 linked to the core protein of chondroitin sulfate proteoglycans Jan-Olof Winberg 1 , Eli Berg 1 , Svein O. Kolset 2 and Lars Uhlin-Hansen 1 1 Department of Biochemistry, Institute of Medical Biology, University of Tromsø, Norway; 2 Institute of Nutrition Research, University of Oslo, Norway In the leukemic macrophage cell-line THP-1, a fraction of the secreted matrix metalloproteinase 9 (MMP-9) is linked to the core protein of chondroitin sulfate proteoglycans (CSPG). Unlike the monomeric and homodimeric forms of MMP-9, the addition of exogenous CaCl 2 to the proMMP-9/CSPG complex resulted in an active gelatinase due to the induction of an autocatalytic removal of the N-terminal prodomain. In addition, the MMP-9 was released from the CSPG through a process that appeared to be a stepwise truncation of both the CSPG core protein and a part of the C-terminal domain of the gelatinase. The calcium-induced activation and truncation of the MMP-9/ CSPG complex was independent of the concentration of the complex, inhibited by the MMP inhibitors EDTA, 1,10-phenanthroline and TIMP-1, but not by general inhibitors of serine, thiol and acid proteinases. This indi- cated that the activation and truncation process was not due to a bimolecular reaction, but more likely an intra- molecular reaction. The negatively charged chondroitin sulfate chains in the proteoglycan were not involved in this process. Other metal-containing compounds like amino- phenylmercuric acetate (APMA), NaCl, ZnCl 2 and MgCl 2 were not able to induce activation and truncation of the proMMP-9 in this heterodimer. On the contrary, APMA inhibited the calcium-induced process, whereas high con- centrations of either MgCl 2 or NaCl had no effect. Our results indicate that the interaction between the MMP-9 and the core protein of the CSPG was the causal factor in the calcium-induced activation and truncation of the gel- atinase, and that this process was not due to a general electrostatic effect. Keywords: gelatinase B; MMP-9; proteoglycan; activation; calcium. The superfamily of matrixins or matrix metalloproteinases consists of at least 18 different mammalian zinc- and calcium- dependent metalloproteinases (MMPs) [1–4], of which monocytes/macrophages can express several types [5–9]. Together, the MMPs are able to degrade most extracellular matrix proteins [3,4,10], as well as regulating the activity of serine proteinases by digesting various serpins (serine proteinase inhibitors) [11], and the growth factor activity of insulin-like growth factor (IGF) by the ability to degrade IGF binding protein (IGHBP) [12]. Thus MMPs have broad substrate specificity, and have been shown to be involved in various regulatory processes in normal and pathological conditions in different tissues and organs. The activity of MMPs is regulated at the transcriptional, translational and post-translational levels. Most of the MMPs are synthesized in their latent pro-form, and must be converted to their active forms in the extracellular space. The cysteine in the conserved PRCG(V/N)PD sequence in the pro-domain binds to the active site zinc as a fourth ligand, and hence is involved in the mainten- ance of the latency of the enzymes [3,13]. During the activation, either parts of or the entire N-terminal pro- domain are removed. This process can be performed by various agents in vitro, including p-aminophenylmercuric acetate (APMA), SDS, urea, chaotropic agents, heat treatment and by proteinases [3,10,13–17]. A model for the latency and activation of MMPs has been proposed, called the Ôcysteine-switchÕ or ÔvelcroÕ model, which suggests that there is an equilibrium between a Ôswitch-openÕ and a Ôswitch-closedÕ form of the pro-enzyme [16]. The reaction of the free thiol group in the switch-open form with for example organomercurials has been suggested to drive the equilibrium toward the open form, which then undergoes an autolytic conversion to an active form. Once activated, the activity of MMPs can be regulated by endogenous inhibitors such as a2-macroglobulin and tissue inhibitors of MMPs (TIMPS) [3,4,10,13,18]. MMP-9 has been found as a monomer as well as in various dimeric forms [19–23]. In the homo- and hetero- dimeric forms, the proteins are either covalently linked to each other through disulfide bonds [20,22,23] or through a strong noncovalent and predominantly hydrophobic Correspondence to J O. Winberg, Department of Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway. Fax: + 47 77 646222, Tel.: + 47 77 645488, E-mail: janow@fagmed.uit.no Abbreviations: APMA, amino-phenylmercuric acetate; cABC, chon- droitin ABC lyase; CS, chondroitin sulfate; GAG, glycosaminoglycan; MMP, matrix metalloproteinase; PG, proteoglycan; TIMP, tissue inhibitors of MMP; SG, serglycin; SBTI, soybean trypsin inhibitor. (Received 1 July 2003, revised 4 August 2003, accepted 8 August 2003) Eur. J. Biochem. 270, 3996–4007 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03788.x dimerization contact [24]. Recently, we discovered that the leukemic monocyte cell line THP-1 produced a new type of reduction-sensitive heterodimer, in which MMP-9 is strongly linked to the core protein of a chondroitin sulfate proteoglycan (CSPG) [23]. Proteoglycans (PGs) constitute a distinct class of glycoconjugates, character- ized by a core protein substituted with highly negatively charged glycosaminoglycan (GAG) chains of which chondroitin sulfate (CS) is a major type [25–27]. The expression of various types of PG in THP-1 cells are not extensively studied, but it has previously been reported that serglycin is the major CSPG released from these cells [27]. As most cells produce several types of PG, one can expect that THP-1 cells also express various PG core proteins such as versican and syndecans. At present, it is not known which of these PG core proteins are bound covalently to MMP-9. The discovery of complexes of MMP-9 covalently linked to the core protein of CSPG expanded our view of how MMP-9 can interact with PGs, as it was known previously that both MMP-9 and MMP-2 binds to the negatively charged GAG chains in PGs through positively charged clusters in the C-terminal hemopexin-like domains of the MMPs [28,29]. Because calcium is known to maintain the structural integrity of MMPs including MMP-9 [30,31] and to be of import- ance for the activity in APMA activated MMP-9 [32,33], we investigated if either APMA, APMA in combination with calcium, or calcium alone could induce an in vitro activation of the proMMP-9/CSPG heterodimer and eventually influence on the enzymatic activity of the complex. The results presented show that proMMP-9 bound covalently to the CSPG has different character- istics compared to other MMP-9 forms. Experimental procedures Materials Safranin O (number S-2255), cetylpyridinuim chloride, phorbol 12-myristate 13-acetate, Triton X-100, chondroitin sulfate C, trypsin, soybean trypsin inhibitor (SBTI), EDTA, 1,10-phenanthroline, gelatin, calf skin collagen, pepstatin, leupeptin, N-ethylmaleimide and alkaline phos- phatase-conjugated antibody were purchased from Sigma. Pefabloc was from Pentapharm Ltd. Human recombinant TIMP-1, Q-Sepharose, Sephadex 200, Sephadex G-50 (fine), Gelatin-Sepharose, Heparin-Sepharose, Amplify, 14 C-labeled Rainbow TM protein molecular mass standards, [ 35 S]sulfate and mouse monoclonal antibodies against human MMP-9 (#IM10L) were obtained from Amersham Pharmacia Biotech. According to the manufacturer, the MMP-9 antibody (#IM10L) detects only the latent 92 kDa form under both reducing and nonreducing conditions. Polyclonal antibodies against TIMP-1, MMP-7 and the C-terminal region and the hinge region of MMP-9 were obtained from Chemicon International, Inc. Chondroitin ABC lyase (proteinase free) was pur- chased from Seikagaku Kogyo Co. CDP-Star TM chemi- luminescent substrate was obtained from New England Biolabs. Unlabeled molecular mass standards were from Bio-Rad. Cronex 4 medical X-ray film was obtained from Sterling Diagnostic Imaging. Cells The human leukemic macrophage cell-line THP-1 was a kind gift from K. Nilsson, Department of Pathology, Uppsala University, Sweden. The cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 100 lgÆmL )1 of streptomycin, and 100 UÆmL )1 of penicillin. To isolate cell-synthesized MMP-9/CSPG complex, the cells were washed three times in serum-free medium and then cultured for 72 h in serum-free RPMI 1640 medium with 0.1 l M phorbol 12-myristate 13-acetate, and the conditioned medium was thereafter harvested and treated as described earlier [23]. This medium was then used directly for analyses or purification of CSPG (see below). Purification of proMMP-9/CSPG complexes The proMMP-9/CSPG complex was purified by Q-Seph- arose anion-exchange chromatography as described previ- ously [23]. Briefly, the column (bed volume 2 mL) was washed with 50 mL of 0.05 M sodium acetate, pH 6.0, containing 6 M urea and 0.35 M NaCl. During these conditions, both the 92 and 225 kDa forms of the MMP-9 passed through the column. Bound material was eluted with 1.5 M NaCl in 0.05 M sodium acetate, 6 M urea, pH 6.0. The CSPG-containing fractions, detected by the Safranin O method (see below), were pooled and diluted with 0.05 M sodium acetate/6 M urea to give a final NaCl concentration of 0.35 M . The material was then re-subjected to another column of Q-Sepharose. After extensive wash with the buffer containing 0.05 M sodium acetate, 6 M urea and 0.35 M NaCl, bound material was eluted with a gradient of 0.35–1.5 M NaCl in 0.05 M sodium acetate, 6 M urea, pH 6.0. The column was run at a flow rate of 0.5 mLÆmin )1 and fractions of 1 mL were collected. The fractions containing most CSPGs, as determined by Safr- anin O, were pooled and desalted on Sephadex G-50 (fine) columns run in H 2 O. The volume was reduced in a Speed Vac (Savant). In other experiments, the Q-Sepharose purified proMMP-9/CSPG complex was further purified by first incubating the complex in the presence of 10 m M of EDTA for 30 min at 4 °C. The EDTA and EDTA extracted material was then separated from the intact proMMP-9/ CSPG complex on gel permeation chromatography. Two hundred microlitres of this material was added to an 800 · 0.4 cm Sephacryl 200 column, and fractions of 50 lL were collected. The intact proMMP-9/CSPG complex was eluted in the void-volume. Purification of MMP-9 from the THP-1 cells The proMMP-9 in conditioned medium from the THP-1 cells was partly purified by subjecting the culture medium to a Gelatin-Sepharose column. Both the MMP-9 monomer and dimer forms bound to the column, while the MMP-9/ CSPG complex was detected in the pass-through fractions. Prior to elution of the bound proMMP-9 from the column with 10 m M of dimethylsulfoxide, the column was thor- oughly washed with 0.1 M Hepes buffer, pH 7.5. The eluted and pooled MMP-9 fractions were passed over a Sephadex G-50 (fine) column, run in 0.1 M Hepes, pH 7.5. Ó FEBS 2003 Activation of the proMMP-9/CSPG complex (Eur. J. Biochem. 270) 3997 Degradation of PG-bound CS-chains by chondroitin ABC lyase (cABC) treatment The PG-bound CS-chains were degraded by digestion for 2hat37°C with 0.2–1.0 units of cABCÆmL )1 of 0.05 M Tris/HCl, pH 8.0, containing 0.05 M sodium acetate. In some experiments, the degraded CS-chains were removed from the proMMP-9/PG core protein complex by gel chromatography on a Sephadex G-50 column. In other experiments, the resulting proMMP-9/core protein complex was separated from remaining intact complex or other impurities on either a new Q-Sepharose column pre- equilibrated with 0.35 M NaCl, or on a Gelatin-Sepharose column, or alternatively a combination of these two columns. During the conditions used, the proMMP-9/PG- core protein complex did not bind to the Q-Sepharose column. The proMMP-9/core protein complex was bound to the Gelatin-Sepharose column, and was eluted from the column using 10% dimethylsulfoxide, while the intact proMMP-9/CSPG complex passed through this column. Detection of PG-bound CS-chains PG-bound CS-chains were quantitated spectrophotometri- cally by the Safranin O method [34] as described previously [23]. Briefly, 30 lL was mixed with 300 lLof50m M sodium acetate, pH 4.75, containing 0.02% Safranin O. The mixture was subjected to a microsample filtration manifold using the slot-blotting process. After filtration through nitrocellulose filter (Millipore HA 0.45 lm), each sample was washed twice with 100 lLH 2 O by filling the wells and reapplying the vacuum. The nitrocellulose filter was removed and the individual dots were cut out and transferred to tubes containing 200 lL of 10% cetylpyridi- nium chloride in H 2 O. The precipitates were solubilized by incubation at 37 °C for 30 min. Vortexing was performed every 10 min during the incubation. The absorbance of the solubilized color was measured in a Pharmacia Ultrospec III spectrophotometer at 536 nm. The amount of GAGs in each sample was estimated from a standard curve of 4–40 lgÆmL )1 of chondroitin sulfate C. Gelatin zymography SDS/substrate PAGE was carried out as described previ- ously [23] with gels (7.5 · 8.5 cm · 0.75 mm) containing 0.1% (w/v) gelatin in both the stacking and the separating gel, 4 and 7.5% (w/v) of polyacrylamide, respectively. The gelatin zymograms were calibrated with both human gelatinase standards from capillary whole blood as des- cribed previously [35], protein standards and the condi- tioned serum-free THP-1 medium. Ten microlitres of conditioned medium or purified CSPG was mixed with 3 lL of loading buffer (333 m M Tris/HCl, pH 6.8, 11% SDS, 0.03% bromophenol blue and 50% glycerol). Eight microlitres of this nonheated mixture was applied to each gel, which was then run at 20 mA at 4 °C. Thereafter, the gel was washed twice in 100 mL of washing buffer (50 m M Tris/ HCl, pH 7.5, 5 m M CaCl 2 ,1l M ZnCl 2 and 2.5% (v/v) Triton X-100) and then incubated in 100 mL of assay buffer (50 m M Tris/HCl, pH 7.5, 5 m M CaCl 2 ,1l M ZnCl 2 and 1m M APMA) for approximately 20 h at 37 °C. Gels were stained with 0.2% Coomassie Brilliant Blue R-250 (30% methanol) and destained in a solution containing 30% methanol and 10% acetic acid. Gelatinase activity was evident as cleared (unstained) regions. The area of the cleared zones was analyzed with the GelBase/GelBlot TM Pro computer program from Ultra Violet Products. Western immunoblotting analysis Purified CSPG was electrophoresed on SDS/PAGE 4% (w/v) in stacking gel and 7.5% (w/v) in separating (gel) and electroblotted to a poly(vinylidene difluoride) membrane. After blockage of nonspecific binding sites with non fat milk (5% in Tris-buffered saline), blots were incubated for 1 h at room temperature with the appropriate antibody against human MMP-9. After washing, the blots were incubated for 1 h at room temperature with an alkaline phosphatase- conjugated antibody, diluted 1 : 20 000 in blockage solu- tion and developed with CDP-Star TM chemiluminescent substrate. All procedures were performed according to the manufacturer. The area and intensity of the stained bands was also analyzed with the GELBASE / GELBLOT TM PRO computer program from Ultra Violet Products. Activation of latent gelatinases The gelatin-degrading enzymes are secreted from THP-1 cells into the culture medium in a latent form and require proteolytic activation. The trypsin-titration of the latent enzymes in the THP-1 conditioned medium was mainly achieved as described previously [36], except that the activation time in the present work was between 15 and 30 min at 37 °C. The gelatinases associated with CSPG were activated by incubating the proMMP-9/CSPG complex at 37 °Cwith 0.1–100 m M of CaCl 2 for 2 h. In some experiments, the complex was incubated with either 1 m M APMA, 10 m M MgCl 2 , 0.001–10 m M ZnCl 2 or 10–200 m M NaCl for 2 h at 37 °C. To test for a possible prevention of the activation process, either 10 m M EDTA, 1 m M 1,10-phenanthroline, 1–20 n M human recombinant TIMP-1, 0.1–4.5 M urea, 1m M pefabloc, 2 lgÆmL )1 leupeptin, 1 lgÆmL )1 pepstatin or 1 m M N-ethylmaleimide was added to the proMMP-9/ CSPG complex prior to incubation with 10 m M of CaCl 2 for 2 h at 37 °C. 3 H-labeling of calf skin collagen Acid-soluble calf skin collagen was labeled with tritium by reductive methylation of the amino groups as described previously [37]. Collagen denatured for 5–10 min at 90 °C resulted in gelatin. Gelatinolytic proteinase activity Briefly, 50 lL of activated or non activated cell-conditioned medium or, activated or non activated purified MMP-9/ CSPG (5 lg), was mixed with 50 lLof0.1 M Hepes buffer, pH 7.5 and 50 lLofthe 3 H-labeled gelatin solution (2.3 mgÆmL )1 or 10 7 c.p.m.Æmg )1 ). In inhibition experi- ments, either 10 m M EDTA, 1 m M 1,10-phenanthroline or 3–20 n M of human recombinant TIMP-1 was added to 3998 J O. Winberg et al. (Eur. J. Biochem. 270) Ó FEBS 2003 CaCl 2 activated MMP-9/CSPG complex. The gelatinase assays were carried out at 37 °C for approximately 20 h. Twenty microlitres of the supernatants were subjected to SDS/PAGE. Thereafter, the gel was soaked in Amplify and dried. Nondegraded and degraded [ 3 H]gelatin were detected with autoradiography. Results Calcium-induced activation of the MMP-9/CSPG complex Experiments were performed to determine whether purified MMP-9/CSPG complex was active and able to degrade gelatin. 3 H-labeled gelatin was incubated with the MMP-9/ CSPG complex at 37 °C in the absence or presence of exogenously added CaCl 2 .After24hthesamplewas appliedtoSDS/PAGEandthegelwasanalyzedby autoradiography. No fragmentation of gelatin could be detected in the absence of CaCl 2 , while in the presence of 10 m M CaCl 2 , the gelatin was degraded to smaller frag- ments (Fig. 1A, lane 3 and Fig. 1B, lane 4). When APMA- treated MMP-9/CSPG complex was incubated in the absence or presence of 10 m M CaCl 2 , no degradation of gelatin was obtained (Fig. 1B, lanes 3 and 5). The CS chains were not involved in the activation of the complex, as the same results were obtained when the CS-chains were enzymatically degraded by cABC lyase and removed from the complex by Sephadex G-50 gel chroma- tography prior to incubation with gelatin. ThegelatinaseactivityoftheCaCl 2 activated MMP-9/ CSPG complex was totally inhibited in the presence of either 10 m M of EDTA (Fig. 2A) or 1 m M of 1,10- phenanthroline (data not shown). Also human recombinant TIMP-1 (3–20 n M ) inhibited the gelatinase activity in a concentration-dependent manner (Fig. 2B). The addition of calcium released low molecular size forms of MMP-9 from the MMP-9/CSPG complex As shown earlier [23], gelatin zymography of the MMP-9/ CSPG complex reveal bands in the stacking gel and a band around 300 kDa (Fig. 3A, lane 1). When the MMP-9/ CSPG complex was incubated with 10 m M CaCl 2 for 2 h at 37 °C prior to electrophoresis, both these bands either disappeared or were strongly reduced, and new bands with lower M r appeared (Fig. 3A, lane 2). Two weak bands at 80 and 85 kDa were seen along with a strong doublet at 74/76 kDa. The same pattern occurred when the MMP-9/ CSPG complex was incubated for 2 h at 37 °C with varying CaCl 2 concentrations from 0.1 to 100 m M (data not shown). However, the process was concentration dependent. The Fig. 1. Activation of proMMP-9/CSPG with CaCl 2 . Purified CSPG was incubated for 2 h at 37 °C with (+) or without (–) CaCl 2 (10 m M ), cABC or APMA (1 m M ) as indicated under each lane. Five micrograms of the CSPG was then mixed with [ 3 H]gelatin and incu- bated for 24 h at 37 °C as described in the method section. These samples (20 lL per lane) were then separated on a 7.5% SDS/PAGE gels, and the radioactivity of the labeled gelatin and its degradation products were detected by autoradiography. Lane 1 in (A) and (B) shows a [ 3 H]gelatin control, and both gels contained [ 3 H]gelatin incubated with either trypsin-activated THP-1 conditioned serum-free medium or trypsin as a positive control (not shown). At the left in each figure is shown the position of the rainbow standard markers and their M r in kDa. The arrowheads indicate the bottom of the application well. Fig. 2. Inhibition of the CaCl 2 -activated MMP-9/CSPG complex. (A) Five micrograms of purified MMP-9/CSPG was incubated for 2 h at 37 °C in the presence of 10 m M of CaCl 2 and then mixed with [ 3 H]gelatin, either with (+) or without (–) EDTA (10 m M ) or human recombinant TIMP-1 as indicated under each figure. These mixtures were thereafter incubated for 24 h at 37 °C as described in the Experimental procedures section. (B) The amount of TIMP-1 used was 3.3 n M (lane 5), 6.7 n M (lane 6) and 20 n M (lane 7). In (A) and (B), negative controls are shown in lane 1 ([ 3 H]gelatin) and in lane 3 ([ 3 H]gelatin incubated with 5 lg of unactivated MMP-9/CSPG). Lane 2 shows a positive control of [ 3 H]gelatin degraded by trypsin-activated THP-1 conditioned serum-free medium. At the left in each figure is shown the position of the rainbow standard markers and their M r in kDa. The arrowheads indicate the bottom of the application well. Ó FEBS 2003 Activation of the proMMP-9/CSPG complex (Eur. J. Biochem. 270) 3999 low M r bands were much weaker at 0.1 m M calcium compared to the higher concentrations, and at 0.01 m M calcium, no low M r bands appeared. This calcium-induced conversion of the MMP-9/CSPG complex to lower M r forms was not affected by the presence of 0.05% Brij-35, a compound known to inhibit autoactivation and autolytic degradation of MMP-2 and MMP-9 [38]. Thus, treatment of the MMP-9/CSPG complex with calcium resulted in proteolytic cleavage and the release of the gelatinase from the complex. When the complex was incubated for 2 h at 37 °C in the presence of either 0.001–10 m M ZnCl 2 ,10m M MgCl 2 or 10–200 m M of NaCl, no conversion to low molecular size forms could be detected in gelatin zymogra- phy (data not shown). The calcium-induced activation and truncation of the MMP-9/CSPG complex was not affected by the presence of either 10 m M MgCl 2 or 200 m M NaCl (data not shown). Thus, the salt-induced processing of the complex is not due to a general electrostatic or ionic strength effect, but appears to be a unique effect of the chemical properties of calcium. As expected, the degradation and removal of CS-chains from the complex resulted in the disappearance or a large reduction of the bands in the stacking gel and the band at 300 kDa, and new bands around 120–150 kDa appeared (Fig. 3A, lane 4). Treatment of this MMP-9/PG-core protein complex with 10 m M of CaCl 2 for 2 h at 37 °C prior to electrophoresis resulted in the appearance of new bands with the same M r as the bands from the cABC untreated material (Fig. 3A, lane 5). The same result was obtained if the degraded CS-chains were removed or not removed from the sample, demonstrating that the CS-chains were not involved in the calcium-induced processing and release of the gelatinase from the complex. The CaCl 2 -induced conversion of the MMP-9/CSPG complex to lower molecular size forms was inhibited by the presence of either 10 m M of EDTA (Fig. 3A, lanes 3 and 6), 1–20 n M of human recombinant TIMP-1 (Fig. 3B) or 1 m M of 1,10-phenanthroline (data not shown). The addition of these inhibitors to the complex after calcium activation, but prior to electrophoresis gave the same pattern as without inhibitors (data not shown). The CaCl 2 induced conversion of the MMP-9/CSPG complex to lower molecular size forms was not inhibited by the presence of either pefabloc, leupeptin, N-ethylmaleimide or pepstatin (data not shown), i.e. general inhibitors of serine, thiol and acid proteinases. As EDTA inhibited the calcium-induced conversion of the MMP-9/CSPG complex to lower M r forms, we used this inhibitor to investigate the kinetics of the processing of the complex. The CaCl 2 -induced conversion to lower molecular size forms was stopped by 10 m M EDTA after 1, 5, 10, 15, 30, 60 and 120-min incubation, which showed that the bands at 80, 85 and 100 kDa were intermediates, with maximum intensity between 5 and 15 min (Fig. 4A). The intensity of the 74/76 kDa doublet increased during the entire incubation period (Fig. 4A). However, in a few other preparations of the MMP-9/CSPG complex, the induced conversion to lower M r forms by CaCl 2 was slower, and the conversion seemed to involve at least one additional step, i.e. the formation of a transient species at 180 kDa (Fig. 4B). As with the other preparations, the intensity of the 74/76 kDa bands increased during the entire incubation period. The released forms of MMP-9 from the proMMP-9/CSPG complex were N- and C-terminally truncated To determine whether the different bands obtained after CaCl 2 treatment lacked either the N- or the C-terminal regions, Western blots were performed using various antibodies against MMP-9. Under reducing conditions, only the 92 kDa band was obtained with all antibodies used (Fig. 5). When the complex had been incubated for 2 h in the presence of CaCl 2 , this 92 kDa band was weaker than in the untreated control (Fig. 5). As no bands with an M r lower than 92 kDa appeared in the blots treated with antibodies against the proform of MMP-9 (Fig. 5A), these results show that the calcium-induced truncated forms of MMP-9 must lack the N-terminal pro-domain. A strong band at approximately 70 kDa appeared in the CaCl 2 - treated material when the polyclonal antibody that recog- nizes the hinge region was used (Fig. 5B, lane 2). Intact MMP-9 was only weakly stained by this antibody (Fig. 5B, lane 1). As proMMP-9 in the serum-free conditioned medium from THP-1 cells also was weakly stained by this antibody (data not shown), it appears that the epitope in the hinge region is partly hidden in the intact enzyme. The fact Fig. 3. Activation of proMMP-9/CSPG with CaCl 2 results in the release of low M r forms of the gelatinase. Gelatin zymography of 4 lg per lane of MMP-9/CSPG, which has been incubated for 2 h at 37 °C with (+) or without (–) cABC, CaCl 2 (10 m M ), EDTA (10 m M )or human recombinant TIMP-1 as indicated under the figure. (A) Arrowheads show the 74/76 kDa doublet and 80 and 85 kDa forms of gelatinase in the CaCl 2 treated material. (B) The amount of TIMP-1 used was 1 (lane 3), 10 (lane 4) and 20 n M (lane 5). In (A) and (B), at the left side is shown the position of the 225 and 92 kDa forms of proMMP-9 in serum-free culture medium of THP-1 cells and the 72 kDa form of proMMP-2 in serum-free culture medium of human skin fibroblasts. Arrow shows the border between the stacking and the separating gel. Due to the high glycosylation of the proMMP-9/CSPG complex, the proteins migrate as if they initially are distributed to the edges of the stacking gel well, and two spots in the separating gel appears instead of a clear band. This is typical for highly glycosylated proteins as described by Carlsson [50]. 4000 J O. Winberg et al. (Eur. J. Biochem. 270) Ó FEBS 2003 that no band was detected at 80/84 kDa in the CaCl 2 - treated sample, but only a strong band at approximately 70 kDa indicates that also a certain degree of C-terminal processing is necessary to fully expose the epitope. The 70 kDa band was not detected by the polyclonal antibody against the C-terminal region of MMP-9 (Fig. 5C, lane 2), showing that the 74/76 kDa bands lack large parts of their C-terminal region. This antibody detected a weak band at around 80 kDa in the CaCl 2 treated material (Fig. 5C, lane 2). Thus, calcium induced both an N- and a C-terminal truncation of CSPG bound proMMP-9. As a control, the proMMP-9/CSPG complex was treated with either 10 m M EDTA, 1 m M 1,10-phenanthroline or various amounts of TIMP-1 (50, 100 and 200 n M )priorto incubation of these mixtures with 10 m M of CaCl 2 in 2 h at 37 °C. These samples were thereafter treated with 0.1 M dithiothreitol and subjected to SDS electrophoresis and analyzed by Western blotting, using the two polyclonal antibodies against the hinge and C-terminal region, respect- ively. As seen in Fig. 5B,C (lanes 3 and 4), only the 92 kDa species is seen in the samples treated with either EDTA or 1,10-phenanthroline, and the intensity corresponds to the untreated sample. Increasing concentrations of TIMP-1 resulted in a successively reduced amount of the 70 kDa form of the enzyme (using the antibody against the hinge region, data not shown). The released forms of MMP-9 from the heterodimer are active The proMMP-9/CSPG complex treated with 10 m M CaCl 2 for 2 h at 37 °C was applied to Q-Sepharose chromato- graphy. The released truncated forms of MMP-9 were collected in the flow-through fraction, whereas the MMP-9 bound to CSPG were eluted from the column by 1.5 M NaCl. The released forms of MMP-9 degraded [ 3 H]-labeled gelatin, both in the presence and absence of 10 m M CaCl 2 (Fig. 6A, lanes 5 and 6), whereas the MMP-9 complexed to the CSPG needed CaCl 2 for activity (Fig. 6A, lanes 7 and 8). Gelatin zymography revealed that this second addition of CaCl 2 resulted in a further release of low M r forms of the gelatinase from the complex (data not shown). The flow-through fraction from the Q-Sepharose column of the CaCl 2 -treated proMMP-9/CSPG complex contained the 74/76 kDa, 80/85 kDa and 100 kDa forms of MMP-9. To determine which of these forms were active in solution, proMMP-9/CSPG complex was incubated with CaCl 2 at various time intervals and then mixed with a2-macroglo- bulin, an inhibitor known to bind and trap active MMPs but not the proform of the enzymes [39]. Gelatin zymography (Fig. 6B) showed that all the released forms of MMP-9 (74/76, 80/85 and 100 kDa) reacted with a2-macroglobulin, which resulted in a partial or total disappearance of the gelatinolytic zones. This indicated that all four forms were active in solution. However, no change in intensity of the MMP-9 complexed to CSPG was observed in the presence of a2-macroglobulin. CaCl 2 -induced activation of the MMP-9/CSPG complex was not abolished by removal of potential contaminating proteins bound to the CS-chains Some MMP-9/CSPG preparations needed about 20–24 h incubation with 10 m M of CaCl 2 at 37 °C to release low M r forms from the complex, while for most preparations release of low M r forms was obtained after 2 h incubation. It is known that CS chains can bind various proteins [40]. The variation in time needed for activation of the proMMP-9 in different preparations could therefore be due to different amounts of contaminating proteins that inhibits the CaCl 2 -induced activation of the proMMP-9 in the CSPG Fig. 4. Time dependent release of low M r forms of the gelatinase during the activation of the proMMP-9/CSPG complex with CaCl 2 . Gelatin zymography of 4 lg per lane of CSPG (A and B represent two dif- ferent CSPG preparations), which has been incubated with 10 m M of CaCl 2 for various time points at 37 °C as indicated under the figure. The activation was stopped at the indicated time points by the addition of 10 m M EDTA to the reaction mixture. At the right is shown the position of the 225 and 92 kDa forms of proMMP-9 in serum-free culture medium of THP-1 cells and the 72 kDa form of proMMP-2 in serum-free culture medium of human skin fibroblasts. Arrow shows the border between the stacking and the separating gel. Ó FEBS 2003 Activation of the proMMP-9/CSPG complex (Eur. J. Biochem. 270) 4001 complex. Alternatively, the activation could be due to the presence of another metalloproteinase that cleaves and activates the proMMP-9 in the complex in the presence of CaCl 2 . As TIMP-1 inhibits the CaCl 2 -induced activation of the MMP-9/CSPG complex, we investigated whether the THP-1 cells produced TIMP-1. Western blots showed that the serum-free conditioned THP-1 medium contained TIMP-1 (Fig. 7, lane 2), and from ELISA the amount of TIMP-1 in the conditioned medium was estimated to be approximately 8.4 lgÆmL )1 . We also investigated whether some of the TIMP-1 was bound to the proMMP-9/CSPG complex in spite of the dissociating conditions used to avoid unspecific binding during the isolation procedure. In some of the purified MMP-9/CSPG preparations, a small amount of TIMP-1 was detected (Fig. 7, lane 1), while no TIMP-1 was detected in other preparations (data not shown). The various amounts of TIMP-1 in the different proMMP-9/ CSPG preparations may therefore explain the variations in the time needed to activate the complex with CaCl 2 . During purification, 6 M urea was present to prevent aggregation and to remove proteins reversibly bound to the proMMP-9/CSPG complex. Urea and salts were finally removed by gel filtration, followed by a concentration step of the purified complex. If urea and salts were not properly removed in all preparations, this might affect the CaCl 2 - induced conversion of the complex to lower M r forms of the gelatinase, as well as the cABC degradation of the CS-chains of the PG. The presence of 4.5 M urea in the proMMP-9/CSPG preparation had no effect on the posi- tion of the two main gelatinase bands (Fig. 8, lanes 1 and 2), suggesting that neither the band seen in the stacking gel nor the 300-kDa band at the top of the separating gel are due to aggregation. However, 4.5 M urea inhibited both the CaCl 2 -induced conversion of the complex to lower M r forms (Fig. 8, lanes 5 and 6) and also cABC-degradation of the CS-chains (Fig. 8, lanes 3 and 4). Up to 0.5 M urea had no effect on the CaCl 2 -induced conversion to lower M r forms, while a concentration-dependent effect was seen from 1 M andupto4.5 M (data not shown). In contrast to this, it was first at a concentration of 4.5 M that urea had an effect on the cABC degradation of the CS-chains. Thus, remnants of urea in some preparations may explain the delay in the kinetics of the CaCl 2 -induced conversion of the proMMP-9/CSPG complex to lower M r forms. Previously, we have shown that conditioned THP-1 medium did not contain MMP-1, MMP-2 and MMP-8 [23]. The two former enzymes are known activators of MMP-9 [13]. Another MMP that is known to activate MMP-9 is matrilysin (MMP-7) [13] that is also known to bind strongly to GAG-chains, especially to heparin and heparan sulphate [41]. However, neither the THP-1 medium nor the purified proMMP-9/CSPG complex revealed bands between 18 and 30 kDa in zymograms of gels containing either gelatin, casein or carboxymethylated-transferin (data not shown); nor was MMP-7 detected with Western blots (data not shown). The proMMP-9/CSPG complex was treated in ways that were expected to result in the release and separation of potential contaminating proteins bound to the CS-chains of the complex. In those experiments both intact and cABC- treated proMMP-9/CSPG complexes were subjected to the same chromatography columns, but in different experi- ments. Conditions were used so that only the intact or the cABC-treated complexes bound to the column. Intact proMMP-9/CSPG bound to the Q-Sepharose column, while the proMMP-9/PG-core protein passed through this column. The opposite was the case when these complexes Fig. 5. Western blots showing N- and C-terminal truncation of the low M r forms from the CaCl 2 -activated MMP-9/CSPG complex. Purified CSPG (30 lg) was treated for 2 h at 37 °Cwith(+)orwithout(–)10m M CaCl 2 ,10m M EDTA, 1 m M 1,10-phenanthroline (OP) as indicated under the figure. Western blots were run under reducing conditions, i.e. samples were treated with 0.1 M dithiothreitol prior to electrophoresis. In A, the monoclonal MMP-9 antibody (IM10L) from Amersham was used. This antibody detects only the pro-form of the enzyme. In B, a polyclonal antibody against the hinge region of MMP-9 was used. In C, a polyclonal antibody against the C-terminal region of MMP-9 was used. M r standard markers are shown at the left, and the arrowhead indicates the position of the 92 kDa band in the serum-free culture medium from THP-1 cells. 4002 J O. Winberg et al. (Eur. J. Biochem. 270) Ó FEBS 2003 were applied to Gelatin-Sepharose and Heparin-Sepharose columns. In other experiments, intact proMMP-9/CSPG was subjected to gel chromatography (Sephacryl 200) in the presence and absence of EDTA. In all these experiments, the obtained proMMP-9/CSPG complex and proMMP-9/ PG-core protein complex could be activated by the addition of CaCl 2 . However, none of the complexes could be activated by APMA or APMA plus CaCl 2 as shown by zymography and degradation of 3 H-labeled gelatin (data not shown). This strongly indicates that the calcium-induced activation of the proMMP-9 complexed to CSPG was not due to any CS-chain bound contaminants. Fig. 8. Urea inhibits both the CaCl 2 -induced release of low M r forms of the gelatinase and cABC degradation of the CS-chains in the complex. Four micrograms of purified CSPG was incubated for 2 h at 37 °C with (+) or without (–) 4.5 M urea, cABC and 10 m M CaCl 2 as indi- cated under the figure. Arrow shows the border between the stacking and the separating gel. At the left side is shown the position of the 225 and 92 kDa forms of proMMP-9 in serum-free culture medium of THP-1 cells and the 72 kDa form of proMMP-2 in serum-free culture medium of human skin fibroblasts. Fig. 7. Western blots showing TIMP-1 in purified MMP-9/CSPG and in serum-free THP-1 conditioned medium. Both the purified CSPG (30 lg) and the conditioned medium was treated with 0.1 M dithio- threitol prior to electrophoresis. M r standard markers are shown at the left. The position of commercial human recombinant TIMP-1 was identical with the bands seen in the figure. Fig. 6. Released forms of MMP-9 from the CaCl 2 -treated heterodimer are active. (A) Purified CSPG was treated with CaCl 2 for 2 h at 37 °C, and thereafter applied to a Q-Sepharose column as described in Experimental procedures. MMP-9 forms that were released from the CSPG were collected in the flow through (F) fractions, whereas the MMP-9 that was still bound to CSPG (B) was attached to the column. The bound material was eluted with 1.5 M NaCl. The flow through material (F), the bound material (B) and the starting material of intact CSPG (U) was either treated for 2 h at 37 °Cwith(+)orwithout(–) 10 m M CaCl 2 as indicated under the figure, and then incubated with [ 3 H]gelatin for approximately 24 h at 37 °C as described in Experi- mental procedures. At the left is shown the M r of the rainbow M r standard markers in lane 1, and the arrowhead indicates the bottom of the application well. Lane 2 shows the negative control of nondegraded [ 3 H]gelatin, and lane 3 shows a positive control of trypsin digested [ 3 H]gelatin. In lane 5, the bands at 30 kDa and below appear as two spots instead of a band due to a crack in the dried gel. (B) Gelatin zymography of CSPG treated with CaCl 2 at different time intervals as indicated under the figure. At the indicated time points, each sample was either untreated (–) or treated (+) with 800 lgÆmL )1 of a2-macro- globulin (a2MG) for 10 min at 37 °C, after which 10 m M EDTA was added to the reaction mixture to stop the activation. At the left is shown the position of the 225 and 92 kDa forms of proMMP-9 in serum-free culture medium of THP-1 cells and the 72 kDa form of proMMP-2 in serum-free culture medium of human skin fibroblasts. The arrow shows the border between the stacking and the separating gel. Ó FEBS 2003 Activation of the proMMP-9/CSPG complex (Eur. J. Biochem. 270) 4003 The calcium-induced activation of proMMP-9 is restricted to the proMMP-9 covalently bound to CSPG If the calcium-induced activation of proMMP-9 com- plexed to CSPG was due to any contaminations, it should be expected that such contaminants also could activate the monomeric/homodimeric proMMP-9. Monomeric and homodimeric forms of proMMP-9 were therefore purified from THP-1 conditioned medium. As shown in Fig. 9, the same pattern occurred when proMMP-9 was incubated with either CaCl 2 treated or untreated proMMP-9/CSPG complex in the presence or absence of 1,10-phenanthro- line. This was also the case when these mixtures were treated with or without Brij-35 (0.05%) or SBTI (data not shown). These experiments show that the MMP-9/CSPG complex in the presence of calcium is not able to activate and process externally added monomeric/homodimeric proMMP-9, which indicates that the calcium-induced activation and processing of the proMMP-9 is restricted to proMMP-9 complexed to CSPG. Further, the results strongly suggest that the calcium-induced activation does not involve any contaminating calcium-dependent pro- teinase, but is due to an autoproteolysis of the proMMP- 9/CSPG complex. To investigate if the calcium-induced activation of the proMMP-9/CSPG complex is a bimolecular reaction or not, various concentrations of the complex were incubated with and without 10 m M of exogenous CaCl 2 for 0–4 h at 37 °C. As shown in Fig. 10, the calcium-induced activation was concentration independent, which strongly suggests that the autoactivation and the truncation process is not a bimolecular reaction, but rather an intramolecular reaction. Discussion Previously, we have shown that a significant amount of the MMP-9 produced by THP-1 cells is linked to the core protein of CSPG [23]. Western blots indicated that it was the 92 kDa proform that was bound to the CSPG. In the present work we have investigated the activity and condi- tions for inducing activation of the MMP-9 bound to the CSPG core protein. The enzyme in the complex was inactive in the soluble activity assay in the absence of exogenously added calcium, supporting that the synthesized complex contains only the 92 kDa pro-form of the gelatinase. The addition of exogenous calcium resulted in the generation of N- and C-terminally truncated forms of MMP-9 which were enzymatically active. Inhibition studies showed that the truncation and activation of the pro-MMP-9 in the complex was due to a metalloproteinase, and most likely a matrix metalloproteinase as TIMP-1 inhibited the activation. Our results exclude calcium-induced truncation and activation as a bimolecular process that involves either another metallo- proteinase or an N-terminally truncated form of MMP-9. The process is more likely to be due either to an intramolecular autoactivation process (i.e. within one het- erodimer) or to two MMP-9/PG molecules existing as dimers where the MMP-9 in each subunit might proteo- lytically cross-activate each other. Although the autoacti- vation in the latter scenario involves two distinct MMP-9 molecules, the process will not follow the kinetics of a bimolecular reaction as the two MMP-9 molecules that act on each other occurs within the same dimer. This conclusion Fig. 10. Calcium-induced truncation and release of MMP-9 from the proMMP-9/CSPG complex was not dependent on the concentration of the complex. Various concentrations of the proMMP-9/CSPG com- plex were incubated for 2 h at 37 °C in the absence (–) or presence (+) of CaCl 2 (10 m M ) as indicated in the figure, after which 2.1 lgof CSPG was withdrawn and loaded to the gel. Shown are the released forms from 74 to 100 kDa in the calcium-treated material, while a very faintbandisseenintheuntreatedmaterial.Totheleftisshownthe position of the 92 kDa form of proMMP-9 in serum-free culture medium of THP-1 cells and the 72 kDa form of proMMP-2 in serum- free culture medium of human skin fibroblasts. At the bottom of the figure is shown the relative amount (± SD) of the CaCl 2 -induced 74–100 kDa forms from four independent experiments. The amount released from the CSPG with the lowest concentration (0.5 mgÆmL )1 ) was set to 100%. Fig. 9. Neither proMMP-9/CSPG nor calcium-activated proMMP-9/ CSPG activates endogenously added proMMP-9 monomer and homodimer. The monomer (92 kDa) and dimer (225 kDa) forms of proMMP-9 was isolated from conditioned THP-1 medium as des- cribed in Experimental procedures. The partly purified proMMP-9 was incubated for 24 h at 37 °Cwith3lgofpurifiedCSPGinthe presence (+) or absence (–) of 10 m M CaCl 2 and 1 m M 1,10-phen- anthroline (OP) as indicated under the figure. Aliquots corresponding to 0.27 lg of CSPG were then added to the gel, in order to prevent the appearance of the released forms of MMP-9 from the complex. The arrowhead shows the border between the stacking and separating gels, and the arrow shows the position of the 72 kDa form of proMMP-2 in serum-free culture medium of human skin fibroblasts. Similar results appeared when the CSPG was treated with CaCl 2 for 2 h at 37 °C prior to mixing with the partly purified proMMP-9. 4004 J O. Winberg et al. (Eur. J. Biochem. 270) Ó FEBS 2003 is based on the following observations: (a) neither calcium- treated nor -untreated proMMP-9/CSPG activated exo- genously added monomeric or homodimeric proMMP-9; (b) the calcium-induced activation of the proMMP-9 in the complex was not dependent on the concentration of the complex; (c) calcium-induced activation took place even in preparations of the proMMP-9/CSPG and proMMP-9/ PG-core protein complexes that were treated in such ways that possible CS-chain bound activators were released and removed from the complex. Large amounts of endogen- ously produced TIMP-1 in the conditioned medium prob- ably explains why the proMMP-9 in the complex was not activated and processed to low M r forms already during the 72 h cell synthesis period, and hence before the isolation procedure started. In the present work, it is shown that the interaction between proMMP-9 and the CSPG core protein causes changes in the proMMP-9 with respect to its ability to autoactivate. The first example is the response to urea. During the purification of the complex, large amounts of urea was added to the preparation to dissolve and remove reversibly bound contaminants from the complex. Although urea is known to induce autoactivation and processing of 92 kDa proMMP-9 to lower M r forms [42], the treatment of the proMMP-9/CSPG complex with urea during the purification procedure did not induce activation and processing of the proenzyme in the complex. Only the 92 kDa pro-form of MMP-9 was detected in the purified material under reducing conditions. The second example is the response to APMA alone or in combination with CaCl 2 , which did not result in truncation and activation of the enzyme in the proMMP-9/CSPG complex. Previous studies have shown that treatment of calcium-depleted proMMP-9 with APMA resulted in an inactive enzyme that had lost approximately 8–9 kDa of its N-terminal prodomain, with Met75 as the N-terminal residue [32,33]. This form of the enzyme had retained the 78PRCGVPD sequence that blocks the active site [32,43,44]. However, in the presence of Ca 2+ the APMA- treated enzyme was active. This was due to a further processing of the enzyme, such that its C-terminal end was autocatalytically removed [32,43,44]. It has been suggested that calcium induced a conformational change in the N-terminally truncated enzyme and unblocked the active site. Site-directed mutagenetic studies indicate that calcium interacted with Asp432 and probably a residue in the remaining prodomain or in the catalytic domain [33]. Several reports show that the APMA-induced autoactiva- tion of various proMMPs is a complicated process. To achieve an understanding of the mechanism behind the APMA induced activation, Cys75 was chemically modified, the prodomain was successively deleted and the amino acids in the 73PRCGVPD were changed through site-directed mutagenesis of proMMP-3 [45–47]. These studies indicated that APMA was first bound to residues other than Cys75 in the prodomain and induced a conformational change prior to binding to the Cys75, followed by autoactivation. It was also shown that if 63 or more amino acids in the prodomain of MMP-3 were deleted, addition of APMA no longer accelerated, but rather inhibited the autoactivation process. The third example that shows the interaction between proMMP-9 and the PG-core protein alters the ability of the gelatinase to autoactivate is the effect of calcium, which is a stabilizer of proMMP-9 and other MMPs, but induces truncation and activation of the proMMP-9/CSPG com- plex. The various and complex effects metals exert on MMPs can be visualized by a recent study, where it was shown that calcium and zinc, but neither of the metals alone, could activate a truncated form of human proMMP- 3 that lacked the first 34 N-terminal amino acids and the entire C-terminal hemopexin domain [48]. The difference in response between proMMP-9 and the proMMP-9/CSPG complex to the treatment of urea, APMA, APMA in combination with calcium, and calcium alone is most likely due to the interaction between the enzyme and the core protein of the CSPG, and not through a general electrostatic effect involving the CS-chains as 200 m M of NaCl alone could not induce activation of the complex, nor did high salt concentrations affect the calcium-induced activation. Our observations that the ionic strength is not of importance for the activation was also reflected by the fact that the activation was equally as effective at 100 m M as at 10 m M of calcium. The metal-induced activation of the MMP-9/ CSPG complex was specific for calcium as the activation process could not be mimicked by either NaCl, MgCl 2 , ZnCl 2 or mercury (APMA). Thus, the interaction between proMMP-9 and the CSPG core protein probably generates a binding site for exogenous calcium that causes destabili- zation in the proenzyme and allows for autoactivation of the enzyme. This interaction must also hide the epitopes that are normally involved in the APMA-induced activation of MMP-9, and expose epitopes that results in an APMA- induced inhibition of the calcium-induced activation. Recently it has been shown that the various forms of proMMP-9 are not equally susceptible to activation. The monomeric and homodimeric forms of MMP-9 respond differently to MMP-3-induced activation of these enzymes [22], while the proMMP-9/NGAL heterodimer was more effectively activated by mercurial compounds in the pre- sence of human neutrophil lipocalin (HNL) than both the monomeric and homodimeric forms of proMMP-9 [49]. Thus, formation of various dimers of MMP-9 results in enzyme variants with altered biochemical properties, which expand the biological properties and function of the enzyme that might be optimal under various conditions. The calcium-induced activation of the proMMP-9/CSPG complex resulted in truncated variants of MMP-9 that had lost both the N-terminal domain and large parts of the C-terminal domain. The truncated variants that were released from the CSPG had lost their N-terminal part and were active in solution, as they reacted with a2-macro- globulin and degraded 3 H-labeled gelatin. In addition, the smallest truncated variants contained the hinge region that connects the catalytic site and the C-terminal hemopexin- like domain, but at least a part of the C-terminal domain was lacking. These results indicate that the interaction between MMP-9 and the CSPG core protein must involve the most C-terminal part of the MMP-9 hemopexin-like domain. Some of the truncated variants that had lost their N-terminal pro-domain, were active and reacted with a2-macroglobulin, and had an M r that was larger than the 92 kDa proform of MMP-9. In those variants, a part of the core protein from the CSPG must still be linked to the N-terminal truncated MMP-9 molecule. Thus, the Ó FEBS 2003 Activation of the proMMP-9/CSPG complex (Eur. J. Biochem. 270) 4005 [...]... Biochem 270) autoactivation process must also involve cleavage of the CSPG core protein in such a way that the part that contains the CS-chains no longer binds to the gelatinase, but only a small processed part of the core protein remains bound to the enzyme The calcium-induced activation and processing of the proMMP-9 bound to the CSPG core protein appeared to be a stepwise process First the N-terminal... of the C-terminal domain It appeared that the core protein of the CSPG was cleaved prior to the processing and shortening of the C-terminal hemopexin-like domain, as the 100 kDa form appeared to be an intermediate form that preceded the final 74/76 kDa forms This also implies that both the C-terminal end of the MMP-9, as well as the covalently bound CSPG core protein, must be able to interact with the. .. active site of the bound gelatinase in order to be cleaved through an intramolecular process The fact that MMP-9 can exist in various dimeric forms, in addition to the monomeric form, suggests that the regulation of activity and targeting of the gelatinase is complex When MMP-9 is linked to the core protein of CSPG one can anticipate that the CSPG can localize and concentrate the bound gelatinase to target... sites other than those usually targeted by the free monomer or its other dimeric forms One such target site may be the CD44 receptor located on the surface of various cells The function and activity of the MMP-9/CSPG complex at a target site is probably, at least partly, regulated by calcium and TIMP-1 The calcium-induced activation and truncation is of potential physiological relevance as the concentration... Ó FEBS 2003 Activation of the proMMP-9/CSPG complex (Eur J Biochem 270) 4007 23 Winberg, J.O., Kolset, S.O., Berg, E & Uhlin-Hansen, L (2000) Macrophages secrete matrix metalloproteinase 9 covalently linked to the core protein of chondroitin sulphate proteoglycans J Mol Biol 304, 669–680 24 Cha, H., Kopetzki, E., Huber, R., Lanzendorfer, M & Brandstetter, H (2002) Structural basis of the adaptive... role of calcium in promatrix metalloprotease-3 (pro-MMP-3, prostromelysin-1) activation and thermostability of the low mass catalytic domain of MMP-3 J Biol Chem 268, 4481–4487 31 Seltzer, J.L., Welgus, H.G., Jeffrey, J.J & Eisen, A.Z (1976) The function of Ca2+ in the action of mammalian collagenases Arch Biochem Biophys 173, 355–361 32 Bu, C.H & Pourmotabbed, T (1995) Mechanism of activation of human... mechanisms of matrix metalloproteinases Biol Chem 378, 151–160 14 Matrisian, L.M (1990) Metalloproteinases and their inhibitors in matrix remodeling Trends Genet 6, 121–125 15 Woessner, J.F Jr (1991) Matrix metalloproteinases and their inhibitors in connective tissue remodeling FASEB J 5, 2145– 2154 16 Van Wart, H.E & Birkedal-Hansen, H (1990) The cysteine switch: a principle of regulation of metalloproteinase... enzymes can degrade barriers at a distance from the cell The fact that the various forms of MMP-9 respond differently to known activators and stabilizing reagents makes MMP-9 capable of acting optimally during various conditions Acknowledgements This work was supported in part by grants from The Norwegian Research Council, The Norwegian Cancer Society and the Erna and Olav Aakre Foundation for Cancer Research... with potential applicability to the entire matrix metalloproteinase gene family Proc Natl Acad Sci USA 87, 5578–5582 17 Nagase, H., Suzuki, K., Morodomi, T., Enghild, J.J & Salvesen, G (1992) Activation mechanisms of the precursors of matrix metalloproteinases 1, 2 and 3 Matrix Suppl 1, 237–244 18 Liotta, L.A & Stetler-Stevenson, W.G (1991) Tumor invasion and metastasis: an imbalance of positive and. .. concentration needed is within the physiological level of calcium in the extracellular matrix Once activated, the MMP-9 will be released from the complex and can diffuse into the surrounding tissue where it may act distantly from its original attachment site Such behavior may be beneficial for migration of cells Enzymes attached to the cell surface can act on its immediate environment, while the released enzymes . Calcium-induced activation and truncation of promatrix metalloproteinase-9 linked to the core protein of chondroitin sulfate proteoglycans Jan-Olof. appeared to be a stepwise truncation of both the CSPG core protein and a part of the C-terminal domain of the gelatinase. The calcium-induced activation and truncation