Brevican is degraded by matrix metalloproteinases and aggrecanase-1 (ADAMTS4) at different sites.

Brevican is a member of the lectican family of chondroitin sulfate proteoglycans that is predominantly expressed in the central nervous system. The susceptibility of brevican to digestion by matrix metalloproteinases (MMP-1, -2, -3, -7, -8, -9, -10, and -13 and membrane type 1 and 3 MMPs) and aggrecanase-1 (ADAMTS4) was examined. MMP-1, -2, -3, -7, -8, -10, and -13 degraded brevican into a few fragments with similar molecular masses, whereas the degradation products of aggrecanase-1 had apparently different sizes. NH(2)-terminal sequence analyses of the digestion fragments revealed that cleavages of the brevican core protein by these metalloproteinases occurred commonly within the central non-homologous domain. MMP-1, -2, -3, -7, -8, -10, and -13 preferentially attacked the Ala(360)-Phe(361) bond, whereas aggrecanase-1 cleaved the Glu(395)-Ser(396) bond, which are similar to the cleavage sites observed with cartilage proteoglycan (aggrecan) for the MMPs and aggrecanase-1, respectively. These data demonstrate that MMP-1, -2, -3, -7, -8, -10, and -13 and aggrecanase-1 digest brevican in a similar pattern to aggrecan and suggest that they may be responsible for the physiological turnover and pathological degradation of brevican.

Proteoglycans form a large gene family that can be classified into several groups according to the structural properties of the core proteins. The lecticans are a family of chondroitin sulfate proteoglycans that include aggrecan (cartilage proteoglycan), versican, neurocan, and brevican (1,2). Like other proteoglycans, lecticans are complex macromolecules that consist of a core protein and one or more types of glycosaminoglycan chains attached to the core protein (3). The core proteins of lecticans consist of two globular domains at the NH 2 and COOH termini and a central region connecting the two globular domains. The NH 2 -terminal globular domain represents a hyaluronic acidbinding domain comprising an immunoglobulin-like loop and two proteoglycan tandem repeats. The COOH-terminal globular domain forms a selectin-like C-type lectin domain complex consisting of epidermal growth factor-like repeats, a C-type lectin-like domain, and a complement regulatory protein-like domain (4). Unlike these globular domains, the central region is not conserved among the lectican family members.
Brevican is the most abundant lectican in the adult brain and is also subject to proteolytic processing (1,5). In the adult brain, brevican exists as full-length and proteolytically cleaved forms (5)(6)(7). The major cleavage site is at the Glu 395 -Ser 396 bond within the central domain of the core protein (6,7). This cleavage site bears sequence similarities to the aggrecanase cleavage site on the aggrecan core protein (7). It has been shown that brevican expression is up-regulated in invasive glioma cell lines (8) and that forced expression of brevican renders noninvasive glioma cells invasive (9). Interestingly, this invasion-stimulating activity requires truncation of brevican near the reported proteolytic cleavage site: expression of the NH 2 -terminal fragment of brevican renders noninvasive glioma cells invasive in vivo, whereas expression of full-length brevican does not have this effect (9). These results suggest that proteinases attacking brevican play an important role in glioma invasion. However, no information is available on the proteinases involved in this process.
Matrix metalloproteinases (MMPs) 1 are a gene family of structurally and functionally related zinc endopeptidases, which consists of at least 20 different members in humans (10). MMPs are involved in the physiological remodeling and pathological degradation of extracellular matrices, including proteoglycans (10). For the degradation of aggrecan, most MMPs digest the core protein at the Asn 341 -Phe 342 bond (11). On the other hand, Tortorella et al. (12) recently cloned aggrecanase-1 (ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs)), which preferably cleaves the aggrecan core protein at the Glu 373 -Ala 374 bond. Since aggrecan fragments with NH 2 termini of A 374 RGSVILTAKPDF and F 342 FGVG are detected in synovial fluids from arthritic patients (13)(14)(15), both MMPs and aggrecanase-1 are thought to be responsible for the aggrecan degradation in human arthritides (10). Interestingly, the sequence of aggrecanase-1 was originally deposited in the GenBank TM /EBI Data Bank (accession number KIAA0688) as an unidentified human gene from a set of size-fractionated human brain cDNA libraries (12). Thus, it seems likely that aggrecanase-1 is expressed in normal and pathological brains. Moreover, MMPs are expressed in the human astrocytic tu-mors, and some are also in normal brain tissues (16,17). Thus, these metalloproteinases are likely to be involved in brevican degradation in gliomas.

EXPERIMENTAL PROCEDURES
Preparation of Rat Recombinant Brevican-For preparation of recombinant brevican, Chinese hamster ovary cells were stably transfected with the expression vector pcDNA-rBV, which contains a fulllength rat brevican cDNA. Recombinant brevican was purified from culture supernatants by affinity column chromatography on the fibronectin type III 3-5 repeats of tenascin-R as described previously (18).
Construction of Aggrecanase-1 Expression Vectors-Aggrecanase-1 cDNA in the pBluescript II SK ϩ vector (GenBank TM /EBI Data Bank accession number KIAA0688) was provided by Dr. Takahiro Nagase (Kazusa DNA Research Institute, Kisarazu, Japan). The aggrecanase-1 cDNA fragments encoding Met 1 -Lys 837 were polymerase chain reaction-amplified using 5Ј-primers with an extra EcoRI site and a 3Јprimer with an extra BglII site. These fragments were inserted between the EcoRI and BglII sites of the mammalian expression vector pSG5 (Stratagene), giving rise to pSG0688. To add the FLAG epitope tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) to the COOH terminus of the aggrecanase-1 protein, the polymerase chain reaction products corresponding to both the FLAG epitope tag and stop codon were inserted into the BglII site of the pSG0688 vector, generating the pSG0688F vector. Nucleotide sequences of mutants were confirmed by direct sequencing. DNA sequencing analysis was performed by the polymerase chain reaction using fluorescent dideoxynucleotides and a MegaBase Model 1000 automated sequencer (Molecular Dynamics Japan, Tokyo).
Purification of Recombinant Aggrecanase-1-COS-7 cells were cultured in serum-free Dulbecco's modified Eagle's medium and cotransfected with the aggrecanase-1 expression vector (5.5 g/15-cm dish) and the furin expression vector (2.0 g/dish) in N- [1-(2,3-dioleoyloxy)propyl]-N,N,N,-trimethylammonium salts (45 g/dish; Roche Molecular Biochemicals, Mannheim, Germany). The culture media were harvested 3 days after transfection and used for purification. They were concentrated by an Amicon Diaflo apparatus fitted with a YM-10 membrane and applied to an anti-FLAG M2 affinity chromatography column (Eastman Kodak Co.) equilibrated with 50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10 mM CaCl 2 , 0.05% Brij 35, and 0.02% NaN 3 (TNCB buffer). Recombinant aggrecanase-1 was eluted with 6 M urea in TNCB buffer containing 3 M NaCl after washing the column with the buffer containing 3 M NaCl. The combined fractions of aggrecanase-1 were dialyzed against TNCB buffer to remove urea. Protein concentration was determined using BCA protein assay reagents (Pierce) with bovine serum albumin as a standard. The activity was demonstrated by detection of the specific cleavage of bovine aggrecan upon immunoblotting after digestion with the purified enzyme as shown previously (12,19).
Preparation of Human MMPs-The zymogens of MMP-1, -2, -3, -7, -8, -9, -10, and -13 were purified and activated by incubation with paminophenylmercuric acetate (Aldrich) according to previous methods (20 -26). Active forms of MT1-MMP and MT3-MMP were also purified as described previously (27,28). As MMP-13 was not stable after purification, 0.1% bovine serum albumin was added to the enzyme solution as a carrier protein. Concentrations of MMP-1, -3, -7, -8, -10, and -13 were determined in assays using fluorogenic synthetic substrates by titration of their activities against recombinant TIMP-1 (tissue inhibitor of metalloproteinases; concentration determined by amino acid analysis), and those of MMP-2, MMP-9, MT1-MMP, and MT3-MMP by titration against recombinant TIMP-2 (concentration determined by amino acid analysis) (27,28). Residual enzymatic activities were measured and plotted versus the TIMP concentrations. A linear plot of activity against the inhibitor molarity was extrapolated to be zero activity at the molarity of the enzyme solution.
Degradation of Brevican by MMPs and Aggrecanase-1-Digestion of brevican was initially examined by incubation of the substrate (600 ng) at 37°C for 24 h with each MMP (20 ng) or aggrecanase-1 (20 ng) at an enzyme/substrate ratio of 1:30 in TNCB buffer. Since brevican was efficiently digested with MMP-2, -3, -7, -8, -10, and -13, time course digestion was performed by incubation with these MMPs for various amounts of time ranging from 0 to 24 h at the same enzyme/substrate ratio. The additive effects of aggrecanase-1 and MMP-2 on brevican digestion were examined by incubation of the substrate (600 ng) with aggrecanase-1 (20 ng) for 24 h and then with the addition of MMP-2 (20 ng) for another 24 h at 37°C or with both aggrecanase-1 (20 ng) and MMP-2 (20 ng) for 24 h at 37°C. These reactions were terminated with 20 mM EDTA, and the degradation products were analyzed by SDSpolyacrylamide gel electrophoresis (PAGE) under reducing conditions. NH 2 -terminal Sequencing of Brevican Degradation Products-Brevican (10 g) was incubated with MMP-1, -2, -3, -7, -8, -10, or -13 or aggrecanase-1 at an enzyme/substrate ratio of 1:30 at 37°C for 24 h and with MMP-7 and MMP-13 for 1 h in a total reaction volume of 100 l in TNCB buffer. Brevican (10 g) was also incubated with aggrecanase-1 (0.34 g) for 24 h and then with MMP-2 (0.34 g) for another 24 h at 37°C in a total volume of 100 l in TNCB buffer. These samples were subjected to SDS-PAGE under reducing conditions after treatment with 20 mM EDTA. Proteins in the gels were transferred to polyvinylidene difluoride membranes and located by staining with 0.1% Coomassie Brilliant Blue R-250. The bands of interest were excised and sequenced by Edman degradation using a Procise 491 protein sequencer (PerkinElmer Life Sciences, Tokyo). To analyze small fragments, the filtrate of a brevican sample digested with MMP-7 for 24 h was prepared by centrifugation using Ultrafree-MC centrifugal filter units with cutoffs of 10 kDa, Millipore Corp., Bedford, MA). Trifluoroacetic acid (5 l) was added to the filtrate (200 l) to make an acidic condition, and the sample was analyzed by reversed-phase high performance liquid chromatography using a TSKgel Super-ODS column (4.6 ϫ 50 mm; Tosoh Co., Tokyo). Chromatography was carried out in the presence of 0.5% trifluoroacetic acid with a linear gradient of acetonitrile to 40% (20 min) at a flow rate of 1 ml/min and at 50°C. By monitoring the absorbance at 210 nm, the unique fragment was recovered in the nearby range of 20% acetonitrile (data not shown). The amino acid sequence of the small separated fragment on a glass filter was analyzed by automated Edman degradation as described above.

RESULTS
Purification of Recombinant Aggrecanase-1-Purified aggrecanase-1 was homogeneous on SDS-PAGE, showing a single band of 69 kDa (data not shown). The activity of the purified enzyme was examined by an assay using bovine aggrecan. The enzyme generated a 60-kDa fragment, which was recognized by the polyclonal antibodies (I19C) specific to the COOH-terminal neoepitope (NITEGE 373 ) of aggrecan generated by the activity of aggrecanase-1 (19), verifying that the recombinant enzyme has the same specificity as aggrecanase-1 (data not shown).
In contrast, two major fragments of 83 and 56 kDa generated by aggrecanase-1 treatment had the NH 2 termini S 396 RGAIYS and D 23 DLKEDS, respectively (Table I). Since the sum of their molecular masses (83 and 56 kDa) corresponds to the molecular mass of intact brevican (140 kDa), aggrecanase-1 appears to hydrolyze at the single site of the Glu 395 -Ser 396 bond (Fig. 3). The 83-kDa fragment with the NH 2 terminus S 396 RGAIYS, which corresponds exactly to the NH 2 terminus of the fragment present in adult brains (6,7), was specific for aggrecanase-1 digestion: no MMPs tested in this study cleaved brevican at this site.
Time Course Digestion of Brevican by MMP-2, -3, -7, -8, -10, and -13-Since brevican was significantly digested by MMP-2, -3, -7, -8, -10, and -13, time course digestion of brevican with these MMPs was carried out. As shown in Fig. 2 (B and E), digestion of brevican with MMP-3 and MMP-10 resulted in the appearance of the 86-and 77-kDa fragments of F 360 SEASSP and the 51-kDa fragments of D 23 DLKEDS within 1 h, and the density of the latter two bands appeared to increase in a timedependent manner. This suggests that brevican is initially digested at the Ala 360 -Phe 361 bond into the 51-kDa NH 2 -terminal fragment and the 86-kDa COOH-terminal fragment (Fig.  3), the latter of which appears to be almost simultaneously processed to the 77-kDa fragment through the COOH-terminal cleavage. Similar changes in the time course digestion were observed with MMP-13 (Fig. 2F), but the 86-kDa fragment of F 361 SEASSP was further degraded into smaller fragments, indicating that this COOH-terminal fragment is susceptible to subsequent multiple cleavages. Upon digestion with MMP-2 and MMP-8 (Fig. 2, A and D), the 57-and 51-kDa NH 2 -terminal fragments of D 23 DLKEDS emerged simultaneously with the 86-and 77-kDa fragments of F 361 SEASSP, suggesting that in addition to the clip at the Ala 360 -Phe 361 bond, at least another cleavage site downstream of the bond, which was not identified in this study, is present with these MMPs.
In contrast, MMP-7 digested brevican into the 84-kDa fragment of L 385 QLPQE and into the 56-kDa fragment of D 23 DLKEDS within 30 min after incubation (Fig. 2C), indicating that the initial cleavage is at the Glu 384 -Leu 385 bond (Fig.  3). After longer incubations, the 63-and 60-kDa fragments of L 503 GASPSP and the 51-kDa fragment of D 23 DLKEDS emerged. Since a decrease in the density of the 84-kDa fragment of L 385 QLPQEA was associated with the appearance of the 63-and 60-kDa fragments of L 503 GASPSP, it is likely that  MMP-1, -2, -3, -7, -8, -9, -10, and -13  subsequent cleavages of the 84-kDa fragment at least at the Gln 502 -Leu 503 bond occurred (Fig. 3). The 51-kDa fragment of D 23 DLKEDS appeared to result from the COOH-terminal cleavage of the 56-kDa fragment of D 23 DLKEDS because it was produced after the appearance of the 56-kDa fragment. Additive Effects of Aggrecanase-1 and MMP-2 on Brevican Digestion-Among MMP-1, -2, -3, -7, -8, -10, and -13, capable of digesting brevican, only MMP-2 is so far known to be expressed and also activated in human gliomas (17,29). In addition, our preliminary study showed that aggrecanase-1 is expressed in gliomas as well as in normal human brain tissues. 2 Thus, to assess the additive effects of aggrecanase-1 and MMP-2, we carried out the sequential digestion of brevican by incubation first with aggrecanase-1 for 24 h and then with MMP-2 for an additional 24 h. As shown in Fig. 4, the 83-and 56-kDa fragments generated by the action of aggrecanase-1 were further hydrolyzed into major 56-and 51-kDa fragments with MMP-2. The identical digestion pattern was obtained when brevican was incubated simultaneously with aggrecanase-1 and MMP-2 for 24 h (Fig. 4). NH 2 -terminal sequence analysis demonstrated that the 56-and 51-kDa final products of these proteinases have the same NH 2 terminus D 23 DLKEDS. These data indicate that the 56-kDa NH 2 -terminal fragment generated by aggrecanase-1 is processed to the 51-kDa fragment through the COOH-terminal cleavage by MMP-2, whereas the 83-kDa COOH-terminal fragment is digested by MMP-2 into multiple small fragments. DISCUSSION This study is the first to demonstrate the digestion of brevican by MMP-1, -2, -3, -7, -8, -9, -10, and -13, MT1-MMP, MT3-MMP, and aggrecanase-1. Of the MMPs and aggrecanase-1 examined, brevican was degraded by MMP-1, -2, -3, -7, -8, -10, and -13 and aggrecanase-1. MMP-1, -2, -8, and -13 contribute primarily to the degradation of fibrillar collagens such as collagen types I-III through the specific cleavage of the collagen molecules into the characteristic 3 ⁄4-and 1 ⁄4-length fragments by MMP-1, -8, and -13 and the subsequent degrada-tion of the denatured fragments by MMP-2 (10, 30). However, this study demonstrates that these MMPs are also active on brevican, as demonstrated for aggrecan (10,11). Since MMP-3 and MMP-10 are structurally related and share activity for many substrates including aggrecan (10), one could expect their degradative activity for brevican. Actually, both MMP-3 and MMP-10 cleaved brevican into almost identical fragments. On the other hand, the proteolytic activity of MMP-7 for brevican was different from that of other MMPs. MMP-7 quickly digested brevican into smaller fragments. Although kinetic analysis was not carried out in this study, MMP-7 appears to have the highest activity among these MMPs capable of degrading brevican. This character of MMP-7 has been demonstrated with many substrates such as elastin (22), aggrecan (11), decorin (31), and tenascin (32). This can be explained by the structural characteristics of MMP-7 since MMP-7 may readily have access to substrates for lack of the COOH-terminal hemopexin-like domain. MMP-13 is also known to be strongly active on aggrecan (33). As expected, this was the case with brevican digestion. MMP-9, MT1-MMP, and MT3-MMP have proteolytic activity for aggrecan (11,24,27,28). Contrary to our expectation, however, all these MMPs had no or negligible activity for brevican. NH 2 -terminal sequence analyses of the brevican digestion fragments provided evidence that the central non-homologous domain of brevican is commonly attacked by these MMPs and aggrecanase-1. The 51-kDa fragment observed in all the samples digested with the MMPs showed the identical NH 2 -terminal sequence of D 23 DLKEDS, the same NH 2 -terminal sequence of intact brevican. This fragment appears to be a stable end product after digestion with the MMPs since there was no further progressive degradation by the MMPs. Because of the glycosylation of brevican, it was difficult to calculate the molecular masses of the fragments from the amino acid sequence data. However, the molecular mass of the NH 2 -terminal fragment encoding the hyaluronic acid-binding domain of rat brevican (Asp 23 -Gly 371 ) is known to be ϳ50 kDa when it is expressed in 9L gliosarcoma cells (9). Thus, it seems likely that the COOH terminus of the 51-kDa fragment may end around Gly 371 , although information about the COOH terminus is not available. One of the most interesting findings in the NH 2terminal sequence data on the MMP digestion products is the appearance of the fragments with the NH 2 terminus F 361 SEASSP. The fragments emerged as bands of 86 and 77 kDa in the reaction products with MMP-1, -2, -3, -8, -10, and -13. In the analyses of the filtrate of the digestion products of MMP-7, a very low molecular mass (probably Ͻ10 kDa) fragment with the same NH 2 terminus was also demonstrated. Thus, the Ala 360 -Phe 361 bond of the central non-homologous domain is considered to be the common cleavage site susceptible to most MMPs (Fig. 3). Based on the data of the time course digestion of brevican and NH 2 -terminal sequences of the diges-tion fragments, MMP-3, -10, and -13 appear to cleave faster at the Ala 360 -Phe 361 bond than at other sites, although cleavages of brevican at least at two sites, including the Ala 360 -Phe 361 bond, may occur almost simultaneously with MMP-2 and MMP-8. On the other hand, the data on the time course digestion with MMP-7 indicate that cleavage at the Ala 360 -Phe 361 bond is preceded by those at the Glu 384 -Leu 385 and Gln 502 -Leu 503 bonds.
It is notable that aggrecanase-1 digested brevican into two fragments of 83 and 56 kDa, which were distinct from any proteolytic fragments generated by MMPs. Moreover, these fragments remained even after complete digestion of the intact molecule, suggesting that aggrecanase-1 activity is restricted to some specific sites of the substrate. NH 2 -terminal sequence analyses demonstrated that the 83-kDa species represents the COOH-terminal fragment S 396 RGAIYS, whereas the 56-kDa species is the NH 2 -terminal fragment D 23 DLKEDS. These data suggest that cleavage of brevican by aggrecanase-1 occurs only at the Glu 395 -Ser 396 bond to form NH 2 -and COOH-terminal fragments of 56 and 83 kDa, respectively. During the revision of this paper, Matthews et al. (34) also reported that aggrecanase-1 (ADAMTS4) processes brevican into two fragments of similar sizes (90 and 50 kDa) by cleavage at the Glu 395 -Ser 396 bond, which was demonstrated by an antibody (B50) specific to the COOH-terminal neoepitope (QEAVESE 395 ) of brevican. A previous study indicated that normal adult rat brains contain intact brevican of ϳ145 kDa and an ϳ80-kDa COOH-terminal fragment with the NH 2 terminus S 396 RGAIYSIPITE (7). Although this study did not identify the NH 2 -terminal fragment (7), the transfection of rat brevican cDNA into 9L gliosarcoma cells demonstrated the NH 2 -terminal fragment of ϳ50 kDa together with the COOH-terminal fragment (9). These data indicate that brevican is readily processed by proteinases at the Glu 395 -Ser 396 bond into the ϳ50-kDa NH 2 -terminal and ϳ80-kDa COOH-terminal species in brain tissues and/or glioma cells (4,7). The present data, together with the expression of aggrecanase-1 in human brains (12), suggest that aggrecanase-1 is a candidate for the brevican-processing enzymes in the brain.
Sequential digestion of brevican with aggrecanase-1 and MMP-2 produced a stable NH 2 -terminal fragment of 51 kDa through the processing of the 56-kDa aggrecanase-1 digestion fragment by MMP-2. The same result was obtained by the simultaneous digestion with these proteinases, indicating that brevican is initially attacked by aggrecanase-1 and subsequently by MMP-2 in their coexistence. Previous studies have shown that overexpression of the ϳ50-kDa NH 2 -terminal fragment of brevican is associated with an invasive phenotype of glioma cells (9), although its molecular mechanism is unknown. Chondroitin sulfate proteoglycans exert an inhibitory influence on neurite outgrowth through their chondroitin sulfate moieties (36). Actually, chondroitin sulfate chains of brevican are known to be essential to inhibit neurite outgrowth from cerebellar granule neurons (5). Since the four potential glycosaminoglycan attachment sites of brevican are located in the central non-homologous domain (4), the NH 2 -terminal fragments generated by aggrecanase-1 and/or MMP-2 are considered to lack glycosaminoglycan. Thus, it is possible to speculate that these metalloproteinases may promote glioma cell invasion through formation of the brevican fragments without glycosaminoglycan chains. On the other hand, recent studies suggested that brevican plays a key role in maintaining the integrity of the brain extracellular matrix by forming molecular bridges linking hyaluronan and tenascin-R through the NH 2 -and COOHterminal globular domains of brevican, respectively (1). Thus, the disruption or weakening of the brevican-hyaluronan and/or brevican-tenascin-R interactions may be one of the factors that accelerate the invasiveness of glioma cells. Such disruption can be induced by cleavage of the brevican core proteins by aggrecanase-1 and/or MMP-2, both of which are present in human glioma tissues (17,29). 2 These hypotheses remain to be eluci-dated by further studies of how the brevican fragments generated by the action of the MMPs and/or aggrecanase-1 affect glioma cell invasion.