[CANCER RESEARCH 50, 7758-7764, December 15. 1990]

Purification and Characterization of Extracellular Matrix-degradingMetalloproteinase, Matrin (Pump-1), Secreted from HumanRectal Carcinoma Cell Line1

Kaoru Miyazaki,2 Yasuhisa Hattori, Fuminori Umenishi, Hidetaro Yasumitsu, and Makoto Umeda

Division of Cell Biology, Kifiara Institute for Biological Research, Yokohama City University, 2-120-3 Nakamura-cho, Minami-ku, Yokohama, Kanagawa 232, Japan

ABSTRACT

A metalloproteinase with M, 29,000 was purified to homogeneity as alatent proenzyme from the conditioned medium of a human rectal carcinoma cell line CaR-1. This enzyme hydrolyzed casein more potently thangelatin embedded in polyacrylamide gels in zymography assay. Calciumion was essential for the activity. It exerted the maximum activity at pH7-9. Its activity was stimulated by organomercurials, such as p-amino-phenyl mercuric acetate and p-chloromercuric benzoic acid, and wasinhibited by 1,10-phenanthroline but was hardly affected by diisopropylfluorophosphate and pepstatin. When the purified proenzyme was activated by the organomercurials, it effectively hydrolyzed fibronectin,laminin, type IV basement membrane collagen, and several types ofgelatins but not interstitial type I and III collagens. The treatment of thepurified proenzyme with /7-aminophenyl mercuric acetate or trypsinformed an active peptide with M, 20,000. The structural analysis indicated that it was most likely identical to putative metalloproteinase-1,the complementary DNA of which had been cloned from human tumormRNAs capable of hybridizing to a rat transin complementary DNA.Based on the fact that this enzyme is secreted extracellularly and degradesthe matrix proteins, we propose the name "matrin" for this newly

identified enzyme.

INTRODUCTION

ECM,3 which consists of fibrous structural proteins, glyco-

proteins, proteoglycans, and glycosaminoglycans, plays a fundamental role in the formation and maintenance of tissuearchitecture. The major structural proteins, collagens, supportthe basic structure of ECM, while the major adhesive glycopro-teins, fibronectin and laminin, mediate the binding of cells toECM (1-3). These ECM proteins affect proliferation, differentiation, morphology, substrate attachment, and motility ofcells in vitro (2, 4). There is increasing evidence indicating thatproteolytic degradation of ECM is required for tumor cells toinvade basement membranes, stremai matrix, and cell-cell junctions (5-7). Liotta et al. (8, 9) and other groups (10, 11) havedemonstrated that the secretion of type IV collagen-degradingenzymes by tumor cells is well correlated with the invasivepotentials. Furthermore, in vitro invasion experiments havesuggested that a cascade of serine proteinases and metallopro-teinases is required for tumor invasion into the basement membranes (12, 13).

It has been reported that tumor cells secrete metalloprotei-

Received 2/19/90; accepted 8/30/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by a Grant-in-Aid from the Ministry of Education,

Science and Culture of Japan.2To whom requests for reprints should be addressed.3The abbreviations used are: ECM, extracellular matrix; APMA, p-amino-

phenyl mercuric acetate; CBB, Coomassie brilliant blue R-250; DFP, diisopropylfluorophosphate; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PCMB, /7-chloromercuric benzoic acid; SDS,sodium dodecyl sulfate; MMP-7, matrix metalloproteinase-7; pump-1, putativemetalloproteinase-1; cDNA, complementary DNA; DME, Dulbecco's modifiedEagle's medium; FI2, Ham's F-12 medium.

nases (14-18), serine proteinases (19-22), thiol proteinases(23-25), and aspartic proteinases (26). Among the four classesof proteinases, the metalloproteinases appear to play a majorrole in matrix degradation. Recent studies have revealed afamily of secreted zinc metalloproteinases capable of degradingECM proteins. These include interstitial collagenase (27-29),type IV collagenases (or gelatinases) with M, 72,000 (16, 17)and M, 92,000 (18), and stromelysin (transin) (30-34). Theinterstitial collagenase preferentially hydrolyzes the interstitialcollagens of types I, II, and III, whereas type IV collagenasesdo hydrolyze type IV basement membrane collagen but not typeI collagen (16-18, 27). Stromelysin has a more broad substratespecificity: it hydrolyzes fibronectin, laminin, type IV collagen,and proteoglycans (30). These metalloproteinases are structurally related to each other and are secreted as latent proenzymes(zymogens) from various types of cells including tumor cellsand normal connective tissue cells. In addition to these enzymes, Woessner and Talpin (35) recently isolated a metalloproteinase with M, 28,000 from postpartum rat uterus, designated MMP-7, which was capable of degrading casein, fibronectin, and various types of gelatins. On the other hand, Mulleret al. (36) cloned cDNAs of two kinds of putative metalloproteinases, stromelysin-2 and pump-1, from human tumors withthe use of transin cDNA as a probe. Pump-1 has an exceptionally small molecular size (267 amino acid residues) comparedto other mammalian metalloproteinases. Recently, Quantin etal. (37) expressed the pump-1 cDNA in COS cells and demonstrated that the recombinant pump-1 protein was a precursorform of a metalloproteinase with a substrate specificity similarto that of MMP-7. They also showed that pump-1-relatedmRNA was expressed in postpartum rat uteri, suggesting thatpump-1 might correspond to rat MMP-7. However, the nativeform of pump-1 has not been identified in human sources.

We previously found that transformation of a rat liver epithelial cell line, BRL, with Rous sarcoma virus induced markedsecretion of a fibronectin-degrading metalloproteinase (38).Recently, we surveyed culture media conditioned by more than30 kinds of nonmalignant and malignant cell lines for secretedproteinases by the zymography technique. As a result, it hasbeen found that a human rectal adenocarcinoma cell line secretes a low molecular weight metalloproteinase capable ofdegrading ECM proteins. In the present paper, we report thepurification of this metalloproteinase, tentatively named "matrin," and its characterization. The structural analysis of the

purified enzyme demonstrated that it might be identical to theputative metalloproteinase pump-1.

MATERIALS AND METHODS

Cells and Culture. A human rectal carcinoma cell line CaR-1(JCRB0207), which had been established from a metastatic lymph nodeof a 70-year-old male patient with primary rectal adenocarcinoma byKaneko et al. (39), was used as the source of the low molecular weightmetalloproteinase matrin. Other human cancer cell lines tested for the

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secretion of the metalloproteinase were HLE (hepatoma), A549 (lungadenocarcinoma), T24 (bladder carcinoma), EJ-1 (bladder carcinoma),A431 (vulva epidermoid carcinoma), HeLa.S3 (cervix epitheloid carcinoma), HSC-4 (tongue squamous carcinoma), T98G (glioblastoma),YKG-1 (glioma), YST-2 (schwannoma), YST-3 (schwannoma),HT1080 (fibrosarcoma), NB-1 (neuroblastoma), VMRC-MELG (melanoma), and RPMI8226 (myeloma). These human cell lines, exceptA549, YKG-1, YST-2, YST-3, and NB-1, and an epithelial cell linederived from an African green monkey kidney BSC-1 were provided bythe Japanese Cancer Research Resource Bank.

These cells were cultured at 37 °Cin a humidified atmosphere of 5%

CO2 and 95% air. The basal medium (DME/F12) consisted of a 1:1mixture of Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) and Ham's F-12 medium (GIBCO), which was supplementedwith 15 mM AL2-hydroxyethylpiperazine-A''-ethanesulfonic acid, 1.2

mg/ml of NaHCOj, 100 units/ml of penicillin G, and 0.1 mg/ml ofstreptomycin sulfate. Cells were maintained in DME/F12 supplemented with 10% PCS (GIBCO) (10% PCS + DME/F12). Plasticculture wares were obtained from Becton, Dickinson Labware (Oxnard,CA).

Preparation of Conditioned Medium. CaR-1 cells were grown toconfluence in roller bottles (175 cm2) containing 200 ml of 10% PCS+ DME/F12. The cultures were then rinsed twice with Hanks' balanced

salt solution and incubated in serum-free DME/F12 overnight. Themedium was discarded and replaced with fresh serum-free DME/F12,and the cultures were continued. The serum-free conditioned mediumwas harvested twice/week and clarified by sequential centrifugation at800 x g for 15 min and at 15,000 x g for 30 min. The protein presentin the clarified conditioned medium was precipitated by 80% saturationof ammonium sulfate and collected by centrifugation at 15,000 x g for30 min. The protein precipitate was dissolved in a small volume of 10mM Tris-HCl (pH 7.5) containing 0.5 M NaCl and 0.01% Brij 35,dialyzed against the same buffer, and used as concentrated conditionedmedium.

Column Chromatographies. Molecular sieve chromatography wascarried out on a Cellulofine GCL2000 column (2.6 x 98 cm) (ChissoCo., Ltd., Tokyo, Japan), previously equilibrated with 10 mM Tris-HCl(pH 7.5) containing 0.5 M NaCl and 0.01% Brij 35. The concentratedconditioned medium of CaR-1 cells was applied to the column andeluted with the same buffer at a flow rate of about 25 ml/h.

Anion-exchange high performance liquid chromatography (HPLC)was carried out on a Shodex QA-824 column (8 x 75 mm; ShowaDenko Co., Ltd., Tokyo, Japan), preequilibrated with 10 mM Tris-HClbuffer (pH 7.5) containing 0.01% Brij 35. The proteinase pool fromthe molecular sieve chromatography was dialyzed against the samebuffer and applied to the column at a flow rate of 0.5 ml/min. Thecharged column was washed with the buffer and eluted with a lineargradient of NaCl from 0 to 0.5 M in 40 ml of the Tris-HCl buffer at aflow rate of 1 ml/min.

Molecular sieve HPLC was carried out on a TSK gel G3000SWcolumn (7.5 x 300 mm) (Tosoh Co., Ltd., Tokyo, Japan), preequilibrated with 0.1 M sodium phosphate buffer (pH 7.0) containing 0.2 MNaCl and 0.01% Brij 35. The proteinase-containing fractions from theanion-exchange HPLC were mixed, concentrated by ultrafiltrationthrough a Diaflo YM-10 membrane (Amicon; W. R. Grace & Co.,Danvers, MA), and then applied to the column at a flow rate of 0.5ml/min.

SDS-PAGE. Unless otherwise noted, SDS-PAGE was carried outon 12.5% polyacrylamide slab gels (90 mm long, 90 mm wide, 0.75mm thick) under nonreducing conditions according to the method ofLaemmli (40) with a Bio-Rad electrophoresis apparatus (Richmond,CA). The molecular weight markers used are rabbit muscle phospho-rylase b (M, 97,400), bovine serum albumin (M, 66,200), hen eggalbumin (M, 42,700), bovine carbonic anhydrase (M, 29,000), soybeantrypsin inhibitor (M, 20,100), and hen egg lysozyme (M, 14,300). Afterelectrophoresis, the gels were stained in 0.25% (w/v) CBB containing45% (v/v) ethanol and 10% (v/v) acetic acid for 30 min. The stainedgels were destained briefly with a mixture of 25% ethanol and 8% aceticacid and then with a mixture of 5% ethanol and 7.5% acetic acid.

Zymography of Proteinases. Caseinolytic activities of secreted pro-teinases were analyzed by zymography according to the methods ofChin et al. (30) with some modifications. SDS-polyacrylamide gelscontaining 1 mg/ml casein were prepared with 1.3 mg/ml ammoniumpersulfate (2.7-fold the usual amount). Samples to be tested were mixedwith an equal volume of the concentrated SDS sample buffer [4% (w/v) SDS, 125 HIM Tris-HCl (pH 6.8), 10% (v/v) glycerol] and thenelectrophoresed on the casein-containing gels without heating the mixture in boiling water. After electrophoresis, the proteinases separatedon the gels were renatured by gently shaking the gels in 2.5% TritonX-100 containing 50 mM Tris-HCl (pH 7.5) and 0.1 M NaCl at roomtemperature for l h to remove SDS, followed by incubation in 250 mlof 50 mM Tris-HCl (pH 7.5) containing 10 mM CaCl2 and 0.02% NaN3at 37°Cfor about 18 h. The resultant gels were stained with CBB and

destained as described above.Preparative SDS-PAGE. For structural analysis, the metalloprotei

nase obtained from the Cellulofine GCL2000 column was furtherpurified by preparative SDS-PAGE on 10% polyacrylamide slab gels(160 mm long, 150 mm wide, 1 mm thick). After the electrophoresis,the gels were briefly stained with CBB, destained, and soaked in waterfor 30 min. The CBB-stained band of the metalloproteinase was excisedfrom the gel and cut into about 5 x 5-mm pieces. The gel pieces wereplaced in a dialysis bag with a small volume of 10 HIMTris-HCl (pH8) containing 0.1% SDS and dialyzed overnight against the same bufferat room temperature. The protein eluted from the gel pieces wasprecipitated by adding 4 volumes of cold acetone and leaving themixture at -20°Covernight, collected by centrifugation, dissolved in a

small volume of 0.1% SDS, and subjected to reverse-phase HPLC ona phenyl-5PW RP column (Tosoh Co.). The eluted protein was usedfor analysis of NH2-terminal amino acid sequence and amino acidcomposition.

Structural Analysis. The NH2-terminal amino acid sequence of thepurified metalloproteinase (about 20 ¿ig)was analyzed with an AppliedBiosystems gas-phase protein sequencer provided courtesy of Drs. R.Muramatsu and S. Abe (Bioscience Research Laboratories, NipponMining Co., Ltd., Saitama, Japan). The amino acid composition of thepurified proteinase (25 ¿tg)was analyzed with a Hitachi amino acidanalyzer model 385 after hydrolysis in 6 N HC1 at 110°Cfor 22 h in

Toray Research Center, Inc. (Kamakura, Japan).Determination of Protein Concentrations. Protein concentrations were

determined by the dye method with a Bio-Rad protein assay kit, usingbovine serum albumin as the standard.

Reagents. Native type IV collagen purified from an EHS tumor wasa kind gift from Dr. Y. Ohba (Bioscience Research Laboratories,Nippon Mining Co., Ltd.). Bovine plasma fibronectin, mouse laminin,and bovine lens type IV collagen were purchased from Nitta GelatinCo., Ltd. (Tokyo, Japan), bovine skin type I collagen and bovineplacenta type III collagen from Koken Co., Ltd. (Tokyo, Japan), DFPand PCM B from Wako Chemicals, Ltd. (Osaka, Japan), APMA fromTokyo Kasei Co., Ltd. (Tokyo, Japan), and pepstatin from PeptideInstitute, Inc. (Osaka, Japan), respectively.

RESULTS

Purification of Matrin. When secreted proteinases were surveyed with various cultured cell lines by zymography on casein-containing SDS-polyacrylamide gels, the conditioned mediumof a human rectal adenocarcinoma cell line CaR-1 showed acaseinolytic band with M, 29,000 (Fig. 1). When a gelatin-containing gel was used, the conditioned medium showed afaint gelatinolytic band with M, 29,000, while showing strongbands with M, 66,000-90,000. This indicated that casein was abetter substrate than gelatin for the M, 29,000 proteinase. Thestrong gelatinolytic bands seemed due to the gelatinases characterized by other groups (16-18). The caseinolytic activitywith A/r 29,000 was hardly detected with the other 15 kinds ofhuman cancer cell lines tested. However, a nonmalignant epithelial cell line from an African green monkey kidney, BSC-1,

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97k-

66k-

43k-

29k-

1 2 3Fig. 1. Analysis of proteinases secreted from CaR-1 and BSC-1 cells by

zymography on casein- and gelatin-containing gels. The conditioned media fromthe serum-free cultures of CaR-1 (lanes I and 2) and BSC-1 (lane 3) wereconcentrated 30-fold by ammonium sulfate precipitation and dialyzed against 10mM Tris-HCl (pH 7.5) containing 0.01% Brij 35. A 10-^1aliquot of each dialyzedmaterial was applied to a polyacrylamide gel containing 1 mg/ml casein (lanes 1and 'I or gelatin (lane 2) after SDS treatment. Other experimental conditionsare described in the text. Ordinate, molecular weight in thousands.

secreted a high activity with a similar molecular weight. TheM, 29,000 proteinase in the CaR-1-conditioned medium alsoshowed a proteolytic band on a fibronectin-containing gel (datanot shown), suggesting that it may be a matrix-degrading enzyme. We tentatively named this enzyme matrin, and its purification was attempted from the serum-free conditioned medium of CaR-1 cells.

CaR-1 cells were cultured in a serum-free DME/F12 medium. Eight liters of the conditioned medium was concentratedby ammonium sulfate precipitation and subjected to molecularsieve chromatography on a Cellulofine GCL2000 column (Fig.2A). The resultant fractions were analyzed by SDS-PAGE and

proteinase zymography (Fig. 2B). The caseinolytic activity withM, 29,000 was mostly eluted in fractions 57-65. The analysisby SDS-PAGE showed a CBB-stained protein band with M,29,000 in these fractions, which corresponded to the caseinolytic activity in the zymogram.

The active fractions (fractions 59-63) from the CellulofineGCL2000 column were combined, dialyzed against 10 mw Tris-HCl buffer (pH 7.5) containing 0.01% Brij 35 and then subjected to anion-exchange HPLC on a Shodex QA-824 column(Fig. 3). About 70% of the M, 29,000 proteinase was adsorbedto the column and eluted at approximately 0.1 M NaCl beforeother adsorbed proteins, while the remainder passed throughthe column without adsorption.

The proteinase fractions (fractions 7-10) eluted from theShodex QA-824 column were combined, concentrated by ultra-filtration, and finally subjected to molecular sieve HPLC on aTSK gel G3000SW column (Fig. 4). The proteinase activitywas eluted as a major peak with an apparent molecular weightof lower than 10,000. The abnormally retarded elution effectively separated this proteinase from contaminating proteins.Thus, the secreted proteinase matrin was purified to homogeneity by the simple three-step procedure (Fig. 5). About 0.7 mgof matrin was obtained from 8 liters of the CaR-1 cell-conditioned medium with a 110-fold enrichment. The electrophoreticmobility of the purified enzyme was not affected by the presenceof 2-mercaptoethanol, indicating that it is a single-chain pep-

tide.

Structural Analysis. In order to clarify the relationship ofmatrin with other known proteinases, its NH2-terminal aminoacid sequence was analyzed (Fig. 6). The sample for analysiswas obtained by using preparative SDS-PAGE after the firstmolecular sieve chromatography. This alternative purificationprocedure gave pure matrin with a high yield (0.3 mg/liter) butas a denatured form. The NH2-terminal amino acid sequenceto the 25th amino acid residue was almost perfectly consistentwith an NH2-terminal moiety (residues 18-42) of the putativemetalloproteinase pump-1 (35). This strongly suggested thatmatrin might be identical to pump-1. The amino acid composition of the purified matrin was also in good agreement withthat of a pump-1 peptide (residues 18-267) without the 17NH2-terminal amino acids, supporting the above possibility(Table 1).

Properties of Matrin. The optimum pH of matrin was examined by incubating the casein-containing gels at various pHsafter electrophoresis and subsequent renaturation of the purified matrin. It exerted the maximum activity in a pH range of7-9.

Effects of various proteinase inhibitors on the activity ofmatrin were examined using the zymography assay (Fig. 7).The activity of matrin was significantly increased by PCMB (athiol proteinase inhibitor) but hardly affected by DFP (a serineproteinase inhibitor) and pepstatin (an aspartic proteinase inhibitor). One mM 1,10-phenanthroline completely inhibited theproteinase activity even in the presence of 10 mM Ca2+, suggesting that this enzyme had a metal ion other than Ca2+ at the

active site. Matrin showed no activity in a reaction mixturewithout Ca2+. These results indicate that matrin belongs to the

metalloproteinase family.In general, mammalian metalloproteinases are secreted as

latent proenzymes (zymogens) and unable to hydrolyze substrates unless activated by organomercurial compounds, proteinases, or some other reagents (17,18, 28, 30). In zymographyassays, however, these proenzymes are able to hydrolyze substrates embedded in polyacrylamide gels without the above-mentioned treatments. This seems to be caused by the confor-mational changes of the proenzymes induced by SDS-PAGEand subsequent renaturation procedure. In the case of matrin,it hydrolyzed casein embedded in polyacrylamide gels in thezymography assay, but it could not hydrolyze casein in a freereaction mixture containing 50 mM Tris-HCl (pH 7.5) and 10mM CaCl2 unless an organomercurial compound, APMA orPCMB, was supplemented to the mixture. This indicated thatmatrin was also secreted as a latent proenzyme. When thepurified proenzyme was treated with 1 HIM APMA in thepresence of 10 mM CaCl2 at 30°Cfor l h and then analyzed by

SDS-PAGE, it was converted to a peptide with MT 20,000 andanother trace peptide with M, 23,000 (Fig. 8). Such lowermolecular weight forms of caseinolytic activities were alsoobserved in the zymograms of crude matrin preparations duringpurification. Therefore, the autolytic peptide with Mr 20,000seemed to be an active form of matrin, while the M, 23,000peptide seemed to be its intermediate form. Similar productswere also obtained when the proenzyme was treated with 1 mMPCMB or with a small amount of trypsin.

Substrate specificity of matrin against five kinds of ECMproteins was examined in the reaction mixture supplementedwith 1 mM APMA (Fig. 9). Matrin effectively hydrolyzedfibronectin and laminin. A relatively short incubation withmatrin converted fibronectin and laminin to partially digestedfragments, but prolonged incubation caused their complete

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Fig. 2. Molecular sieve chromatography ofconcentrated conditioned medium of CaR-1cells on Cellulofine GCL2000 column. Eightliters of the serum-free conditioned mediumwas concentrated to 30 ml and divided intotwo equal portions. Each portion, which contained 280 mg of protein, was applied to thecolumn. A. elution pattern of proteins in thechromatography; O, A2M value of each fraction; arrows, elution positions of ferritin (M,450,000), albumin (M, 66,000), and cyto-chrome c (M, 12,500); bar, fractions 59-63from the two runs of the chromatography werepooled and used for further purification. Otherexperimental conditions are given in the text.B, SDS-PAGE (left) and proteinase zymogra-phy on casein-containing gel (right) of the fractions obtained from the molecular sieve column. Fr., fraction; ordinate, molecular weightin thousands; arrows, proteinase band with M,29,000 in fractions 57-65.

4 -

o00CM

2 -

20 40 60

Fraction number (6 ml each)

B (Proteins) (Proteinases)

97k-

66k-

43k-

29k-

20k-

Fr. 41 45 49 53 57 61 65 41 45 49 53 57 61 65

6 8 10 14

0.010 20

Fraction number (0.5 ml each)7761

Fig. 3. Anion-exchange HPLC on ShodexQA-824 column of matrin obtained by molecular sieve chromatography. The proteinase poolfrom the Cellulofine GCL2000 column (Fig. 2),which contained 20 mg of protein, was dialyzedagainst 10 ITIMTris-HCl (pH 7.5) containing0.01% Brij 35 and applied to the column asdescribed in the text. Inset, SDS-PAGE profilesof the column fractions; abscissa, fractions 6-15; ordinate, molecular weight in thousands.The proteinase-containing fractions 7-10 weremixed and used for further purification.

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digestion. Among two subunits of laminili, the A chain wasmore susceptible than the B chain. Matrin significantly degraded native and pepsin-treated type IV basement membranecollagens but hardly affected interstitial collagens of type I andtype III. Although matrin showed a very low proteolytic activityon gelatin-containing gels (Fig. 1), it digested types I, III, andIV gelatins dissolved in the reaction mixtures (data not shown).

0.5

0.4

0.2

0.1

0.0

290k 142k 67k 32k 12.4k

10

Elution volume (ml)15

Fig. 4. Molecular sieve HPLC on TSK gel G3000SW column of matrinobtained by anion-exchange HPLC. The proteinase pool obtained from theShodex QA-824 column (Fig. 3) was concentrated to 1 ml by ultrafiltration anddivided into two equal portions. Each portion, which contained approximately 2mg protein, was applied to the TSK gel G3000SW column as described in thetext. Top abscissa, elution positions of molecular weight markers (in thousands):glutamate dehydrogenase (290,000), láclate dehydrogenase (142,000), enolase(67,000), adenylate kinase (32,000), and cytochrome c (12,400); bar, protein peakwas collected and used as purified matrin.

1 2 3Fig. 5. SDS-PAGE of matrin preparation from each purification step. Lane

1, concentrated conditioned medium; lane 2, from first molecular sieve chroma-tography; lane 3, from anion-exchange HPLC; lane 4, from molecular sieveHPLC (pure matrin). Ordinate, molecular weight in thousands.

1 23456789 10Leu Pro Leu Pro Gin Glu Ala Gly Gly Met

11 12 13 14 15 16 17 18 19 20Ser Glu Leu Gin Trp (Glu) Gin Ala X (Asp)

21 22 23 24 25Tyr Leu Lys Arg Phe

Fig. 6. NH2-terminal amino acid sequence of electrophoretically purifiedmatrin. Parentheses, possible but not conclusive amino acids. The unidentified19th amino acid corresponds to the 36th amino acid Gin in pump-1. Other aminoacids including those in parentheses are consistent with the sequence of an NH2-terminal moiety (residues 18-42) in pump-1.

Table 1 Amino acid compositions of matrin and pump-1Pump-l°Matrin

Amino acid mol% No." mol% No.

Asp +AsnThrSerGlu

+GinGlyAlaValCysMetIleLeuTyrPheLysHisArgProTrp(Total)11.254.776.429.2510.286.774.200.273.024.339.304.504.586.273.114.225.791.67(100)28121623261711181123111116811IS4(252)10.84.87.28.410.06.44.40.43.24.48.84.44.46.83.24.46.02.0(100)27121821251611181122111117811155(250)

°The amino acid composition of the possible proenzyme form (residues 18-267) of pump-1 (Ref. 36).

* Estimated number of amino acids/matrin molecule.

29k-

123456Fig. 7. Effects of proteinase inhibitors on caseinolytic activity of purified

matrin. The purified proteinase was electrophoresed on a casein-containing gel,and the resultant gel was renatured and cut into strips, followed by incubation inthe Ca2*-containing (lanes 1-5) or Ca:*-free (lane 6) reaction mixture with or

without the indicated proteinase inhibitor. Lane I, no addition; lane 2, 5 mMDFP; lane 3, 1 mM PCMB; lane 4, 5 Mg/ml pepstatin; lane 5, 1 mM 1,10-phenanthroline; lane 6, no addition (—Ca2*). Ordinate, molecular weight in

thousands. Other conditions are the same as given in the text. The apparentdoublet of the caseinolytic activity is an artifact caused by the CBB staining ofthe enzyme protein at the center of the protein band.

DISCUSSION

In the present study, we isolated and characterized a newmember of the matrix-degrading metalloproteinase family fromthe conditioned medium of a human rectal adenocarcinoma cellline, CaR-1. This proteinase was secreted as a latent proenzymewith A/r 29,000 and converted to an active form with M, 20,000by treatment with organomercurials or trypsin. The activatedenzyme required Ca2+ for the activity and appeared to have

another metal ion at the active site. Like stromelysin, thepurified enzyme degraded various ECM proteins. It effectivelyhydrolyzed fibronectin, laminili, type IV basement membranecollagen, and several types of gelatins but hardly type I and IIIcollagens.

The NH2-terminal amino acid sequence and amino acidcomposition of the purified M, 29,000 proenzyme strongly

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-29k

-20k

Fig. 8. Effect of APMA treatment on molecular size of matrin. The purifiedproteinase was incubated with (+) and without (—)1 HIMAPMA at 30°Cfor 60min and then analyzed by SDS-PAGE. Ordinate, molecular weight in thousands.

suggest that it is identical to pump-1 reported by Muller et al.(36). Like other mammalian metalloproteinases such as colla-genases and stromelysin, the cDNA-derived primary structureof pump-1 contains a putative zinc-binding site homologous tothe zinc-binding sequence of thermolysin. Therefore, it is verylikely that the purified enzyme is a zinc metalloproteinase.Pump-1 was recently suggested to correspond to the rat uterusmetalloproteinase (MMP-7), which had been purified from theextract of postpartum rat uteri by Woessner and Talpin (35).These three enzymes share common properties such as molecular weight values of the precursor and active forms, optimumpH and Ca2"1"requirement for activity, but differences also exist

among them. MMP-7 and recombinant pump-1 were unable todigest type IV collagen (36, 37), whereas the CaR-1-derivedenzyme effectively digested the basement membrane collagen.It seems possible that this difference was derived from thedifference in the experimental conditions such as the amountsand specific activities of the used enzymes. In addition, the

molecular weight value of the rat uterus enzyme estimated bySDS-PAGE was 35,000 under nonreducing conditions and28,000 under reducing conditions, whereas the electrophoreticmobility of the CaR-1-derived enzyme (M, 29,000) was not

affected by the presence or absence of a reducing reagent.Further studies are required to explain these discrepancies andto establish their identity.

So far the following four members of the matrix-degradingmetalloproteinase family have been characterized by both primary structures and enzymological properties: interstitial col-lagenase, type IV collagenases (gelatinases) with M, 72,000 and92,000, and stromelysin. The CaR-1-derived enzyme is the fifthmember of the metalloproteinase family. With the exception ofinterstitial collagenase, these metalloproteinases can more orless hydrolyze type IV collagen and other ECM proteins (17,18, 30, 31). Therefore, the nomenclature of these proteinasesseems rather contradictory or confusing. More systematic nomenclature should be considered when their enzymologicalproperties and functions are well established. The name "pump-1" seems unsuitable for the CaR-1-derived enzyme, since it is

derived from a putative metalloproteinase. At present, we propose to refer to the CaR-1 cell-derived metalloproteinase asmatrin.

In general, the matrix-degrading metalloproteinases arehighly produced by connective tissue cells. For example, normalhuman skin fibroblasts are known to constitutively secreteinterstitial collagenase and stromelysin in vitro (31 ). Althoughit seems doubtless that these enzymes play an essential role inthe maintenance and remodeling of ECM, their roles in tumorgrowth and invasion remain to be clarified. Matrisian et al. (32,33) found that transcription of a gene encoding an unknownprotein, termed transin, was induced when fibroblasts weretransformed by polyomavirus, Rous sarcoma virus, or H-rasoncogene or treated with epidermal growth factor. Later, transin was shown to be a rat homologue of human stromelysin(34). We have reported that a rat liver epithelial cell line, BRL,secretes a high activity of a fibronectin-degrading metalloproteinase when transformed with Rous sarcoma virus (38). Recently, this proteinase was purified and identified as transin

12 34 5678 9 10 11 12Fig. 9. Substrate specificity of purified ma

trin. Substrate proteins were incubated with(+) or without (—)10 Mg/ml of the purifiedmatrin in SOjil of a reaction mixture of 20 HIMTris-HCl (pH 7.5), 10 mM CaCU, 0.01% Brij35, and 1 mM APMA at 37°Cfor 6 h (lanes1-4) or at 30°Cfor 18 h (lanes 5-12). After

the incubation, the reaction mixtures weremixed with the SDS sample buffer containing5% 2-mercaptoethanol, heated in boiling waterfor 3 min, and subjected to SDS-PAGE on7.5% polyacrylamide gels. Lanes 1 and 2, 0.2mg/ml bovine plasma fibronectin; lanes 3 and4, 0.2 mg/ml mouse laminin; lanes 5 and 6,0.5 mg/ml pepsin-treated bovine skin type Icollagen; lanes 7 and 8, 0.5 mg/ml pepsin-treated bovine placenta type III collagen; lanes9 and 10, 0.5 mg/ml pepsin-treated bovine lenstype IV collagen; lanes 11 and 12, 0.5 mg/mlEHS tumor-derived type IV collagen. Arrowheads, partially digested fragments of fibronectin (M, (ordinate) 220,000, 150,000, 88,000,68,000, 50,000, 38,000, 36,000, and 32,000]and laminin (M, 170,000, 150,000, 58,000,and 34,000).

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METALLOPROTEINASE SECRETED BY HUMAN RECTAL CARCINOMA

(41). These results suggest the involvement of this metallopro-teinase in the malignant transformation of some types of cells.

Muller et al. (36) have surveyed 50 kinds of human primarytumors for the expression of stromelysin-related genes anddetected the mRNA of stromelysin or stromelysin-2 in 6 tumors, all of which are squamous carcinomas. Recently, weexamined proteinase species secreted from various cultured celllines using proteinase zymography on casein- and gelatin-containing gels. Among 16 kinds of human cancer cell lines tested,type IV collagi-naso with M, 72,000 was significantly secretedfrom 13 cell lines, type IV collagi-nasi- with M, 92,000 from 6cell lines, stromelysin-like metalloproteinase from 4 cell lines,and matrin from only one cell line.4 These results suggest thattype IV collagi-nasi- with M, 72,000 is the most common

metalloproteinase in human tumors, whereas matrin is restricted to specific cell types. Matrin-like metalloproteinase isalso secreted from the African green monkey kidney cell line,BSC-1. Therefore, matrin may play a more specific role thanother metalloproteinases, although the possibility cannot beexcluded that in other cells it may turn over rapidly or localizeon the cell surface. For clarifying the physiological role ofmatrin, it seems important to further investigate the geneexpression and distribution of this enzyme in various normaland malignant tissues.

REFERENCES

1. McDonald, J. A. Extracellular matrix assembly. Anna. Rev. Cell Biol., 4:183-208, 1988.

2. Ruoslahti, E. Fibronectin and its receptors. Annu. Rev. Biochem., 57: 375-414, 1988.

3. Martin, G. R., and Timple, R. Laminin and other basement membranecomponents. Annu. Rev. Cell Biol., 3: 57-85, 1987.

4. Watt, F. M. The extracellular matrix and cell shape. Trends Biochem. Sci.,//: 482-485, 1986.

5. Moscatelli, D., and Rifkin, D. B. Membrane and matrix localization ofproteinases: a common theme in tumor cell invasion and angiogenesis.Biochim. Biophys. Acta, 948: 67-85, 1988.

6. van den Hooff, A. Stromal involvement in malignant growth. Adv. CancerRes., 50: 159-196, 1988.

7. Liotta, L. A., Rao, C. N., and Wewer, U. M. Biochemical interactions oftumor cells with the basement membrane. Annu. Rev. Biochem., 55: 1037-1057, 1986.

8. Liotta, L. A., Tryggvason, K., Garbisa, S., Hart, I. R., Foltz, C. M., andShafìie,S. Metastatic potential correlates with enzymatic degradation ofbasement membrane collagen. Nature (Lond.), 284: 67-69, 1980.

9. Garbisa, S., Pozzatti, R., Muschel, R. J., Saffiotti, U., Ballin, M., Goldfarb,R. H., Khoury, G., and Liotta, L. A. Secretion of type IV collagenolyticprotease and metastatic phenotype: induction by transfectin with c-Ha-rasbut not c-Ha-ros plus Ad2-Ela. Cancer Res., 47: 1523-1528, 1987.

10. Yamagata, S., Ito, Y., Tanaka, R., and Shimizu, S. Gelatinase of metastaticcell lines of murine colonie carcinoma as detected by substrate-gel electro-phoresis. Biochem. Biophys. Res. Commun., 151: 158-162, 1988.

11. Ballin, M., Gomez, D. E., Sinha, C. C., and Thorgeirsson, U. P. Ras oncogenemediated induction of a 92kDa metalloproteinase; strong correlation withthe malignant phenotype. Biochem. Biophys. Res. Commun., 154: 832-838,1988.

12. Mignatti, P., Robbins, E., and Rifkin, D. B. Tumor invasion through thehuman amniotic membrane: requirement for a proteinase cascade. Cell, 47:487-498, 1986.

13. Reich, R., Thompson, E. W., Iwamoto, Y., Martin, G. R., Deason, J. R.,Fuller, G. C., and Miskin, R. Effect of inhibitors of plasminogen activator,serine proteinases and collagenase IV on the invasion of basement membranesby metastatic cells. Cancer Res., 48: 3307-3312, 1988.

14. Yamanishi, Y., Maeyens, E., Dabbous, M. K., Ohyama, H., and Hashimoto,K. Collagenolytic activity in malignant melanoma: physicochemical studies.Cancer Res., 33: 2507-2512, 1973.

15. Tarin, D., Hoyt, B. J., and Evans, D. J. Correlation of collagenase secretionwith metastatic-colonization potential in naturally occurring murine mammary tumors. Br. J. Cancer, 46: 266-278, 1982.

16. Salo, T., Liotta, L. A., and Tryggvason. K. Purification and characterizationof a murine basement membrane collagen-degrading enzyme secreted bymetastatic tumor cells. J. Biol. Chem., 258: 3058-3063, 1983.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

4 K. Miyazaki, F. Umenishi, N. Koshikawa, H. Yasumitsu, and M. Umeda,

unpublished data.

Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A.,Seltzer, J. L., Kronberger, A., He, C., Bauer, E. A., and Goldberg, G. I. H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secretea single metalloprotease capable of degrading basement membrane collagen.J. Biol. Chem., 263: 6579-6587, 1988.Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A.,and Goldberg, G. I. SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal humanmacrophages. J. Biol. Chem., 264: 17213-17221, 1989.Unkeless, J., Daño,K., Kellerman, G. M., and Reich, E. Fibrinolysis associated with oncogenic transformation. Partial purification and characterization of the cell factor, a plasminogen activator. J. Biol. Chem., 249: 4295-4303, 1974.Wilson, E. L., Becker, M. L. B., Hoal, E. G., and Dowdle, E. B. Molecularspecies of plasminogen activators secreted by normal and neoplastic humancells. Cancer Res., 40: 933-938, 1980.Gaylis, F. D., Keer, H. N., Wilson, M. J., Kwaan, H. C., Sinha, A. A., andKozlowski, J. M. Plasminogen activators in human prostate cancer cell linesand tumors: correlation with the aggressive phenotype. J. Urol., 142: 193-198, 1989.Kao, R. T., Wong, M., and Stern, R. Elastin degradation by proteinases fromcultured human breast cancer cells, Biochem. Biophy. Res. Commun., 105:383-389, 1982.Poole, A. R., Tiltman, K. J., Recklies, A. D., and Stocker, T. A. M.Differences in secretion of the proteinase cathepsin B at the edge of humanbreast carcinomas and fibrosarcomas. Nature (Lond.) 273: 545-549, 1980.Sloane, B. F., Rozhin, J., Hatfield, J. S, Crissman, J. D., and Honn, K. V.Plasma membrane-associated cysteine proteinase in human and animal tumors. Exp. Cell Biol., 55: 209-224, 1987.Troen, B. R., Ascherman, D., Atlas, D., and Gottesman, M. M. Cloning andexpression of the gene for the major excreted protein of transformed mousefibroblasts. A secreted lysosomal protease regulated by transformation. J.Biol. Chem., 263: 254-261, 1988.Briozzo, P., Morisset, M., Capony, F., Rougeot, C., and Rochefort, H. Invitro degradation of extracellular matrix with M, 52,000 cathepsin D secretedby breast cancer cells. Cancer Res., 48: 3688-3692, 1988.Welgus, H. G., Jeffrey, J. J., and Eisen, A. Z. The collagen substratespecificity of human skin fibroblast collagenase. J. Biol. Chem., 256: 9511-9515, 1981.Cawston, T. E., and Murphy, G. Mammalian collagenases. Methods En-zymol., «Õ:711-722, 1981.Goldberg, G. L, Wilhelm, S. M., Kronberger, A., Bauer, E. A., Grant, G. A.,and Eisen, A. Z. Human fibroblast collagenase. Complete primary structureand homology to an oncogene transformation-induced rat protein. J. Biol.Chem., 261: 6600-6605, 1986.Chin, J. R., Murphy, G., and Werb, Z. Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. Biosynthesis, isolation, characterization,and substrates. J. Biol. Chem., 260: 12367-12376, 1985.Wilhelm, S. M., Collier, I. E., Kronberger, A., Eisen, A. Z., Marmer, B. L.,Grant, G. A., Bauer, E. A., and Goldberg, G. I. Human skin fibroblaststromelysin: structure, glycosylation, substrate specificity, and differentialexpression in normal and tumorigenic cells. Proc. Nati. Acad. Sci. USA, 84:6725-6729, 1987.Matrisian, L. M., Claichenhaus, N., Gesnel, M.-C., and Breathnach, R.,Epidermal growth factor and oncogenes induce transcription of the samecellular mRNA in rat fibroblasts. EMBO J., 4: 1435-1440, 1985.Matrisian, L. M., Leroy, P., Ruhlmann, C., Gesnel, M.-C., and Breathnach,R. Isolation of the oncogene and epidermal growth factor-induced transingene: complex control in rat fibroblasts. Mol. Cell. Biol., 6: 1679-1686,1986.Whitham, S. E., Murphy, G., Angel, P., Rahmsdorf, H.-J., Smith, B. J.,Lyons, A., Harris, T. J. R., Reynolds, J. J., Herrlich, P., and Docherty, J. P.Comparison of human stromelysin and collagenase by cloning and sequenceanalysis. Biochem. J., 240:913-916, 1986.Woessner, J. F., Jr., and Taplin, C. J. Purification and properties of a smalllatent matrix metallo-proteinase of the rat uterus. J. Biol. Chem., 263:16918-16925, 1988.Muller, D., Quantin, B., Gesnel, M.-C., Millon-Collard, R., Abecassis, J.,and Breathnach, R. The collagenase gene family in humans consists of atleast four members. Biochem. J., 253: 187-192, 1988.Quantin, B., Murphy, G., and Breathnach, R. Pump-1 cDNA codes a proteinwith characteristics similar to those of classical collagenase family members.Biochemistry, 28: 5327-5334, 1989.Miyazaki, K., Ashida, Y., Kihira, Y., Mashima, K., Yamashita, J., and Horio,T. Transformation of rat liver cell line by Rous sarcoma virus causes loss ofcell surface fibronectin, accompanied with secretion of metalloproteinase thatpreferentially digests the fibronectin. J. Biochem., 102: 569-582, 1987.Kaneko, Y., Koura, M., and Yoshii, H. Characterization of a newly established, human rectal adenocarcinoma cell line. Acta Med. Univ. Kagoshima,19: 71-81, 1977.Laemmli, U. K. Cleavage of structural proteins during assembly of the headof bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.Umenishi, F., Yasumitsu, H., Ashida, Y., Yamauti, J., Umeda, M., andMiyazaki, K. Purification and properties of extracellular matrix-degradingmetallo-proteinase overproduced by Rous sarcoma virus-transformed rat livercell line, and its identification as transin. J. Biochem. (Tokyo), 108: 537-543, 1990.

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1990;50:7758-7764. Cancer Res   Kaoru Miyazaki, Yasuhisa Hattori, Fuminori Umenishi, et al.   from Human Rectal Carcinoma Cell LineMatrix-degrading Metalloproteinase, Matrin (Pump-1), Secreted Purification and Characterization of Extracellular

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