Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen

A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices.     

Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. Biosynthesis, isolation, characterization, and substrates

Rabbit synovial fibroblasts induced to undergo a specific switch in gene expression by agents that alter cell morphology secreted the neutral proteinase precursor procollagenase (apparent Mr of 53,000 and 57,000). A major Mr = 51,000 polypeptide that was always induced coordinately with procollagenase has now been identified as the proenzyme form of a metal-dependent proteinase active at neutral pH. We have named this proteinase stromelysin. Prostromelysin and procollagenase were the most prominent [35S]methionine-labeled secreted proteins of the induced fibroblasts. By the use of casein degradation as an assay for enzyme activity, stromelysin was isolated with high yield from the conditioned culture medium of 12-O-tetradecanoylphorbol 13-acetate-treated fibroblasts and migrated as an active form of Mr = 21,000 that was immunologically identical to the proteoglycan-degrading proteinase purified from rabbit bone. Immunoglobulin G from antiserum raised to purified rabbit bone proteoglycanase immunoprecipitated the Mr = 51,000 proenzyme form from conditioned medium of induced rabbit cells and also immunoprecipitated an Mr = 55,000 polypeptide from induced human fibroblasts. When rabbit prostromelysin was activated by trypsin or 4-aminophenylmercuric acetate, the proenzyme was converted to an active form of Mr = 41,000. During the course of the purification, prostromelysin was converted to an additional activatable form of Mr = 35,000 and additional active forms of Mr = 21,000-25,000, which had related peptide maps distinct from collagenase. All of these forms were immunologically cross-reactive. Purified stromelysin degraded casein, cartilage proteoglycans, fibronectin, alpha 1-proteinase inhibitor, and immunoglobulin G2a and had limited activity on laminin, elastin, type IV collagen, and gelatin, but did not degrade type I collagen. Stromelysin was inhibited by EDTA, 1,10-phenanthroline, and the specific glycoprotein tissue inhibitor of metalloproteinases isolated from human amniotic fluid and was therefore classified as a metalloproteinase.     

‘Gelatinase-like’ activity from articular chondrocytes in monolayer culture

In addition to releasing collagenase and proteoglycanase activity, rabbit articular chondrocytes in monolayer culture released into the culture medium, latent, neutral enzyme activity which when activated by p-aminophenylmercuric acetate degraded fluorescein-labeled polymeric rat tail tendon Type I collagen and the tropocollagen TC_A and TC_B fragments of human Type II collagen into smaller peptides at 37°C. Enzyme activity was abolished if p-aminophenylmercuric acetate-activated culture medium was preincubated with 1,10-phenanthroline, a metal chelator. Thus, articular chondrocytes in monolayer culture are capable of producing neutral proteinases which acting together can result in complete degradation of tendon and cartilage collagen to small peptides.     

Specific cleavage of human type III collagen by human polymorphonuclear leukocyte elastase

Purified polymorphonuclear leukocyte elastase degraded native human liver type III collagen at 27 degrees C by making a cleavage through the triple helix. The enzyme had no effect on human type I collagen. The reaction was inhibited by phenylmethanesulfonyl fluoride (PhCH2SO2F) but not by EDTA. The collagen reaction products were identical with those generated by human rheumatoid synovial collagenase when analyzed by polyacrylamide gel electrophoresis and gel filtration. NH2-trminal sequence analysis indicated that the enzyme cleaved at an isoleucyl-threonyl bond located 4 residues on the carboxyl side of the established cleavage site for animal collagenases. Therefore, it is likely that in pathologic states, type III collagen can be selectively depleted from the matrix by this enzyme.     

Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence

Polymorphonuclear leukocytes have been shown to contain proteolytic enzymes which are capable of degrading connective tissue proteins such as native collagen. In this study, proteolytic enzymes were extracted from human polymorphonuclear leukocytes and a neutral proteinase was extensively purified and characterized. The activity of this enzyme was monitored by degradation of denatured [ 3H ]proline-labeled type I collagen or by cleavage of a synthetic dinitrophenylated peptide with a Gly-Ile sequence. The enzyme was readily separated from leukocyte collagenase by concanavalin-A--Sepharose affinity chromatography and further purified by QAE-Sephadex ion-exchange chromatography and gel filtration on Sephacryl S-200. The purified enzyme had a molecular weight of approximately 105000, its pH optimum was about 7.8, and it was inhibited by Na2EDTA and dithiothreitol, but not by fetal calf serum. The enzyme degraded genetically distinct type I, II, III, IV and V collagens, when in a non-helical form, but not when in native triple-helical conformation. Dansyl-monitored end-group analyses, combined with digestion by carboxypeptidase A, indicated that the enzyme cleaved denaturated type I collagen at Gly-Xaa sequences, in which Xaa can be leucine, isoleucine, valine, phenylalanine, lysine, or methionine. Thus, the purified enzyme referred to here as Gly-Xaa proteinase, is a neutral proteinase, which may be of importance in inflammatory disease processes by degrading further collagen peptides which have been rendered non-helical as a result of collagenase cleavage.     

Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase.

The metalloproteinase 'gelatinase' stored in the granules of pig polymorphonuclear leucocytes has been purified in the latent form. The enzyme is secreted as an Mr 97,000 proenzyme that can be activated in the presence of 4-aminophenylmercuric acetate (APMA) by self-cleavage to generate lower-Mr species, of which an Mr 88,000 form was the most active. Trypsin-initiated activation generated different Mr gelatinases of much lower specific activity. Activation was slowed but not prevented by the presence of the tissue inhibitor of metalloproteinases, TIMP. The activated gelatinase formed a stable complex (Mr 144,000) with TIMP, in a Zn2+- and Ca2+-dependent manner, and complex formation was inhibited by the presence of the substrate gelatin. Similar to the human granulocyte gelatinase, the organomercurial-activated pig enzyme degraded gelatin and TCA and TCB fragments of type I collagen, as well as elastin and types IV and V collagen. The degradation of type IV collagen was shown, both by polyacrylamide-gel electrophoresis and by electron microscopic analysis, to generate 3/4 and 1/4 fragments as described for mouse tumour type IV collagenase. Furthermore, an antiserum raised to mouse type IV collagenase recognized the pig granulocyte gelatinase. An antiserum to the pig polymorphonuclear leucocyte gelatinase recognized other high-Mr gelatinases, including those from human granulocytes, pig monocytes and rabbit connective tissue cells, but not the Mr 72,000 enzyme from connective tissue cells. These data suggest that there are two distinct major forms of gelatinolytic activity that also cause specific cleavage of type IV collagen. These enzymes are associated with a wide variety of normal connective tissue and haemopoietic cells, as well as many tumour cells.     

Metalloproteinases from rabbit bone culture medium degrade types IV and V collagens, laminin and fibronectin.

Gel-filtration chromatography of culture medium from rabbit bone explants separates three latent metalloproteinases with activities against collagen, proteoglycan and gelatin respectively. The fractions degrading proteoglycan also degrade laminin, fibronectin and the polymeric products of pepsin-solubilized type IV collagen and can also solubilize insoluble type IV collagen. The fractions degrading gelatin are capable of degrading solubilized type V and 1 alpha,2 alpha,3 alpha (cartilage) collagens, as well as the lower-molecular-weight products of pepsin-solubilized type IV collagen. All activities can be inhibited by 1,10-phenanthroline and occur in either partially or totally latent forms that can be activated by 4-aminophenylmercuric acetate.     

Studies on the ability of 65-kDa and 92-kDa tumor cell gelatinases to degrade type IV collagen

Two major gelatinolytic metalloproteinases (gelatinases) of 65 kDa and 92 kDa were purified from a tumor cell line. Analysis of collagen degradation showed that native full-length Engelbreth-Holm-Swarm (EHS) type IV collagen was not cleaved by the purified gelatinases under conditions where native pepsin-extracted human placental type IV and V collagen and heat-denatured collagens were markedly degraded. However, EHS type IV collagen degradation was noted at 37 degrees C, i.e., under conditions that would favor denaturation of the collagen molecule in solution. The pattern of degradation of human placental type IV and V collagen appeared similar for both gelatinases. Zymogram analysis of gelatinase activity in the absence of sodium dodecyl sulfate (SDS) (to eliminate possible SDS-mediated denaturation of type IV collagen) confirmed the inability of 65 and 92-kDa gelatinases to degrade native full-length EHS type IV collagen. Under the same conditions and in SDS-polyacrylamide gel electrophoresis zymograms the gelatinases degraded pepsin-predigested EHS type IV collagen and pepsin-extracted human placental type IV collagen. These data suggest that the 65- and 92-kDa tumor cell gelatinases are not true type IV collagenases. Their ability to degrade pepsin-solubilized, or denatured, type IV collagen suggests a specificity for telopeptide precleaved or conformationally altered forms of this molecule.     

The purification and characterization of a glomerular-basement-membrane-degrading neutral proteinase from rat mesangial cells.

A neutral proteinase, capable of degrading gelatin, has been found in both an active and a latent form in the medium from the culture of rat mesangial cells. The latent form had an Mr of 80,000-100,000 and could be activated with either 4-aminophenylmercuric acetate or prolonged incubation at neutral pH. The active form of the enzyme was extensively purified. The estimated Mr of the purified enzyme on gel filtration was approximately 200,000, indicating that the active enzyme formed aggregates. However, analysis by SDS/polyacrylamide-gel electrophoresis under reducing conditions showed two protein bands, with Mr 68,000 and 66,000. Both proteins were found to contain proteolytic activity when run on SDS/substrate gels. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by inhibitors for cysteine, serine or aspartic proteinases. The enzyme did not digest fibronectin, bovine serum albumin, proteoglycan or interstitial collagen. The enzyme degraded pepsin-solubilized placental type V collagen at 31 degrees C, whereas similarly solubilized type IV collagen was only degraded at higher temperatures. In addition, the neutral proteinase degraded native soluble type IV collagen. It also had activity on insoluble type IV collagen of glomerular basement membrane. The above properties suggest that the mesangial neutral proteinase belongs to the gelatinase group of metalloproteinases and that it may play a role in the normal turnover of extracellular glomerular matrix.     

Degradation of native type IV procollagen by human neutrophil elastase. Implications for leukocyte-mediated degradation of basement membranes

Leukocyte-derived proteases may contribute to the destruction of basement membranes during inflammation. We have, therefore, examined the degradation of human type IV procollagen (PC) by purified human neutrophil elastase (HLE). Native [14C]proline-labeled type IV PC was isolated from cultures of human HT-1080 cells and incubated with HLE for various times at 25 or 37 degrees C. Cleavage products were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by CNBr peptide mapping. Incubation of type IV PC with HLE (less than 1:10 HLE:type IV weight ratio) resulted in cleavage of the pro alpha 1 (IV) and pro alpha 2 chains (Mr 180,000 and 175,000) to discrete components of Mr greater than 140,000. Peptide mapping indicated that the carboxy-terminal collagenase-resistant domains of both chains were rapidly and preferentially degraded. Longer incubations or incubations at higher enzyme:substrate ratios resulted in extensive and asymmetric internal cleavage with the generation of fragments similar in size distribution to the major pepsin-resistant fragments of type IV collagen. Our findings indicate that soluble, native human type IV PC is a substrate for HLE and is preferentially cleaved within the globular carboxy-terminal domains of the pro alpha 1 and pro alpha 2 chains. We suggest that even limited cleavage of type IV PC by HLE may disrupt intermolecular carboxy-terminal interactions believed to be important for basement membrane assembly and for maintaining basement membrane structure in vivo.     

Cathepsin B1. A lysosomal enzyme that degrades native collagen

1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released (14)C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human alpha(2)-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5-5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an alpha-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.     

Cleavage of type VII collagen by interstitial collagenase and type IV collagenase (gelatinase) derived from human skin

Type VII collagen is the major structural protein of anchoring fibrils, which are believed to be critical for epidermal-dermal adhesion in the basement membrane zone of the skin. To elucidate possible mechanisms for the turnover of this protein, we examined the capacities of two proteases, human skin collagenase, which degrades interstitial collagens, and a protease with gelatinolytic and type IV collagenase activities, to cleave type VII collagen. At temperatures below the denaturation temperature, pepsin cleaves type VII collagen into products of approximately 95 and approximately 75 kDa. Human skin collagenase cleaved type VII collagen into two stable fragments of approximately 83 and approximately 80 kDa, and the type IV collagenase (gelatinase) produced a broad band of approximately 80 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleavage of type VII collagen was linear with time and enzyme concentration for both enzymes. Although the Km values were similar for both enzymes, the catalytic rate of cleavage by type IV collagenase is much faster than by interstitial collagenase, and shows a greater rate of increase with increasing temperature. Sequence analysis of the cleavage products from both enzymes showed typical collagenous sequences, indicating a relaxation in the helical part of the type VII collagen molecule at physiological temperature which makes it susceptible to gelatinolytic degradation. Interstitial collagenase from both normal skin cells and cells from patients with recessive dystrophic epidermolysis bullosa, a severe hereditary blistering disease in which both an anchoring fibril defect and excessive production of collagenase can be observed, produced identical cleavage products from type VII collagen. These data suggest a pathophysiological link between increased enzyme levels and the observed decrease or absence of anchoring fibrils.     

Matrix degrading properties of sperm serine proteinase, acrosin.

The serine proteinase acrosin plays an important role in sperm penetration of the zona pellucida. In the present study we investigated the effect of the enzyme on various matrix proteins. Acrosin degraded proteolytically fibronectin, type IV collagen and heat denatured type I collagen, whereas neither native type I collagen nor laminin were cleaved by the enzyme. The specific activity of acrosin with type IV collagen as substrate (66.6 g/h/g) was 125-fold higher than that of known type IV collagenase or stromelysin. These results suggest that acrosin may act as a matrix-degrading proteinase.     

High molecular mass type i.v. collagen-specific metalloprotease from human carcinoma tissue

A protease degrading type IV collagen was purified more than 8000-fold from human stomach carcinoma tissue. This protease degraded type IV collagen, while type I, II, III and V collagen, laminin, fibronectin, casein, albumin and hemoglobin were not affected. This enzyme had a pH optimum of pH 7.0-8.0 and was inhibited completely by EDTA and o-phenanthroline, but not by seryl, thiol and carboxyl protease inhibitors. Furthermore, the molecular mass of this enzyme was estimated to be 1 MDa by Sepharose 6B column and HPLC-gel filtration. The molecular mass and substrate specificity of this metalloprotease from human carcinoma tissue indicate it to be a new protease.     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

Differential susceptibility of type X collagen to cleavage by two mammalian interstitial collagenases and 72-kDa type IV collagenase

We have studied the degradation of type X collagen by human skin fibroblast and rat uterus interstitial collagenases and human 72-kDa type IV collagenase. The interstitial collagenases attacked the native type X helix at two loci, cleaving residues Gly92-Leu93 and Gly420-Ile421, both scissions involving Gly-X bonds of Gly-X-Y-Z-A sequences. However, the human and rat interstitial enzymes displayed an opposite and substantial selectivity for each of these potential sites, with the uterine enzyme catalyzing the Gly420-Ile421 cleavage almost 20-fold faster than the Gly92-Leu93 locus. Values for enzyme-substrate affinity were approximately 1 microM indistinguishable from the corresponding Km values against type I collagen. Interestingly, in attacking type X collagen, both enzymes manifested kinetic properties intermediate between those characterizing the degradation of native and denatured collagen substrates. Thus, energy dependence of reaction velocity revealed a value of EA of 45 kcal, typical of native interstitial collagen substrates. However, the substitution of D2O for H2O in solvent buffer failed to slow type X collagenolysis significantly (kH/kD = 1.1), in contrast to the 50-70% slowing (kH/kD = 2-3) observed with native interstitial collagens. Since this lack of deuterium isotope effect is characteristic of interstitial collagenase cleavage of denatured collagens, we investigated the capacity of another metalloproteinase with substantial gelatinolytic activity, 72-kDa type IV collagenase, to degrade type X collagen. The 72-kDa type IV collagenase cleaved type X collagen at both 25 and 37 degrees C, and at loci in close proximity to those attacked by the interstitial enzymes. No further cleavages were observed at either temperature with type IV collagenase, and although values for kcat were not determined (due to associated tissue inhibitor of metalloproteinases-2), catalytic rates appeared to be substantial in comparison to the interstitial enzymes. In contrast, type X collagen was completely resistant to proteolysis by stromelysin. Type X collagen thus appears to be highly unusual in its susceptibility to degradation by both interstitial collagenase and another member of the metalloproteinase gene family.     

Termination of RNA by nucleotides of 9-beta-D-xylofuranosyladenine

The protease susceptibilities of recently identified cartilage collagens HMW, 1α, 2α, and 3α were investigated. Mammlian skin collagenase cleaved the 3α chain under conditions where HMW, 1α and 2α were not degraded. A tumor cell derived type V collagenolytic metalloproteinase degraded HMW, 1α and 2α, but not 3α. Plasmin or leucocyte elastase failed to significantly degrade any of the cartilage collagens when digestion was performed at 25°C (15 hours, enzyme to substrate ratio 1:100). At 36°C but not 33°C α thrombin degraded HMW, 1α and 2α, with little or no degradation of 3α. This pattern of protease susceptibility for HMW, 1α and 2α is therefore similar to type V collagen. The cleavage of 3α by skin collagenase but not by elastase is similar to type II collagen. These results suggest that HMW, 1α and 2α are part of the type V collagen family.     

Purification and properties of a specific collagenase from rabbit synovial fibroblasts.

1. A specific collagenase from the culture medium of rabbit synovial fibroblasts was purified by gel filtration and ion-exchange chromatography. 2. The enzyme was homogenous on polyacrylamide-gel electrophoresis and showed only traces of contaminants when tested in gels with a non-specific antiserum. 3. The rabbit fibroblast collagenase could hydrolyse collagen both in solution and in fibrillar form. Viscometry showed that at 35 degrees C the purified enzyme could hydrolyse greater than 50 nmol of collagen/min per mg of enzyme. 4. The purified collagenase cleaved collagen in solution at either 24 degrees or 35 degrees C into the characteristic 1/4 and 3/4-length fragments. However, as compared with the impure enzyme, the purified enzyme at 35 degrees C had a much decreased capacity to further degrade the initial specific cleavage products. 5. The specific rabbit collagenase had a mol. wt. of approx. 32000 as estimated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, and 35000 by gel filtration.     

The purification of protease IV of E.coli and the demonstration that it is an endoproteolytic enzyme

A strong proteolytic activity is unmasked and solubilized when $\text{E. coli}$ outer membrane fragments are preincubated with 0.083% sodium dodecyl sulfate. This proteolytic activity cleaves αS1 casein into the same degradation products as protease IV, a recently described protease of $\text{E.coli}$ located in the outer membrane (Ph. Régnier, preceding paper), it is concluded that sodium dodecyl sulfate solubilizes the same protease. Protease IV has been purified 11,200 fold, probably to homogenetiy, by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by elution of the protein from gel slices. The purified enzyme is fully active, its molecular weight, determined from its migration in denaturating gels is 23,500. αS1 casein is cleaved by protease IV into two large polypeptides which are not further degraded and some small peptides of about 5,000 daltons. The production of discrete polypeptide species suggests that protease IV is an endoproteolytic enzyme.     

Cleavage of fibromodulin in cartilage explants involves removal of the N-terminal tyrosine sulfate-rich region by proteolysis at a site that is sensitive to matrix metalloproteinase-13

Integrity of cartilage fails in joint disease. The current work aimed to identify candidate active proteinases in joint diseases using an in vitro model for cartilage degradation induced by interleukin-1. A critical event in the process of cartilage destruction in joint disease is the failure of the collagen fiber network to maintain integrity. Proteins binding to the surface of the fibers are likely early points of failure. Fibromodulin, a member of the leucine-rich repeat protein family, is one predominant protein in cartilage and is known for its roles in the formation of collagen fibrils and sustained interaction with these formed fibers. Cleavage removes the tyrosine sulfate-rich region in the N terminus of fibromodulin. Whereas fibromodulin bound to collagen in tissue was digested, purified fibromodulin was not cleaved. In contrast an N-terminal 10-kDa fragment, Gln19-Lys98, of the protein generated by Lys-C digestion contains the cleavage site and was a substrate cleaved by the enzyme in medium from stimulated cultures. In solution, digestion of this substrate with matrix metalloproteinase (MMP)-2, -9, -8, and -13 demonstrated that only MMP-13 was capable to efficiently cleave it. The cleavage product obtained after MMP-13 digestion was identical to that observed in cleaved fibromodulin from cartilage explant cultures stimulated with interleukin-1. MMP-13 treatment of fresh articular cartilage also produced the fragment under study. The elucidation of the enzyme responsible for such cleavage may lead to treatment modalities involving its selective inhibition for patients suffering from arthritis. The known structure of the fragments permits the generation of neo-epitope antibodies to the cleavage site, which can be used to detect ongoing cartilage degradation in patients with arthritic disease, an important adjunct in monitoring disease progression, active disease, and efficacy of treatment.