THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 261, No. 4, Issue of February 5, pp. 1760-1765, 1986 Printed in (I. S. A.

The Major Excreted Protein of Transformed Fibroblasts Is an Activable Acid-Protease*

(Received for publication, September 13, 1985)

Susannah Gal and Michael M. Gottesman From the Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Malignant transformation of mouse cells by a variety of agents or treatment with the tumor promoter 12-0- tetradecanoylphorbol 13-acetate or platelet-derived growth factor results in increased synthesis and secre- tion of a 39,000-dalton protein termed major excreted protein (MEP). We report here that secreted MEP is an acid-activable protease. The secreted precursor form of the protease is auto-activated at low pH and is able to digest a variety of proteins, including the ex- tracellular matrix proteins fibronectin, collagen, and laminin. MEP protease activity has pH optimum of 3.3-3.6 and is temperature- and concentration-de- pendent. The activity is inhibited by sulfhydryl pro- tease inhibitors such as leupeptin and iodoacetic acid and not by metallo-, seryl-, or carboxyprotease inhib- itors. The MEP-derived protease has characteristics distinct from the cathepsins previously reported and thus may be a new acid-protease of mouse cells.

Tumor cells interact with their hosts in a variety of dele- terious ways by local invasion, metastasis, stimulation of angiogenesis, inhibition of immunological responses, and re- lease of bioactive materials. These effects are presumed to be caused by proteins or other substances synthesized by the tumor cells and either secreted or maintained on the surface of the tumor cell. A number of changes in the proteins which are on the cell surface and/or secreted into the medium have been described in association with malignant transformation such as increased release of plasminogen activator (1) and decreased expression of fibronectin (2) and collagen (3).

This laboratory has been studying a protein secreted from Kirsten virus-transformed NIH 3T3 mouse tissue culture cells (KNIH’). The MEP of these cells is a 39,000-dalton glycopro- tein (4) which contains mannose 6-phosphate and binds to the mannose 6-phosphate receptor (5). This receptor is re- quired for targeting of lysosomal enzymes to lysosomes in cultured fibroblasts (6). Three MEP-immunorelated polypep- tides of 39,000, 29,000, and 20,000 daltons are found in lyso- somes and Golgi of both parent and transformed cells (7). We have recently shown (7) that these peptides are related as precursor and products. Similar protein processing has been demonstrated for other lysosomal enzymes (8, 9). Despite considerable effort, we had not been able to assign a particular hydrolytic enzyme activity to the secreted MEP, but we

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: KNIH, Kirsten virus-transformed NIH 3T3 cells; HPLC, high performance liquid chromatography; MEP, major excreted protein of KNIH cells.

suspected that such an activity could reside in one of the processed forms.

In an attempt to obtain large amounts of lower molecular weight MEP peptides, we treated the secreted form with various proteases, including pepsin in formate buffer at pH 3.0. We observed the conversion of highly purified MEP to a lower molecular weight species which also occurred in the absence of pepsin. The generation of lower molecular weight forms of MEP correlated with the appearance of an acid- endoprotease activity which was able to digest a variety of proteins. The activation of secreted MEP and its resultant proteolytic activity have been characterized with respect to its pH optimum and its inhibitor specificity. We conclude that secreted MEP is an acid-activable cathepsin apparently distinct from those previously reported.

EXPERIMENTAL PROCEDURES Materials-All the materials used were obtained from Sigma unless

otherwise noted. BSA (PentaxO bovine albumin Fraction V; Miles), collagen (vitrogen 100 from Collagen Corporation, approximately 95% Type I and 5% Type 111 collagen), laminin (Bethesda Research Laboratories), fibronectin (gift of Dr. K. M. Yamada, National Cancer Institute), dithiothreitol (Schwarz/Mann) and pepstatin (Peninsula Laboratories) were used. Phenylmethanesulfonyl fluoride, leupeptin, and aprotinin (14 trypsin-inhibiting units/mg of protein) were ob- tained from Sigma.

Cell Culture-NIH 3T3 and KNIH cells obtained from C. Scher (University of Pennsylvania) were maintained at 37 “C in 5% COZ in Dulbecco’s modified Eagle’s medium (HEM Laboratories) supple- mented with penicillin (50 units/ml), streptomycin (50 pglml), and 10% calf serum (Colorado Serum). KNIH cells were passaged with 0.25% trypsin (Microbiological Associates) in Tris-dextrose buffer (National Institutes of Health media unit). NIH 3T3 cells were passaged with 0.25% trypsin and 0.2 mM EDTA in Tris-dextrose buffer.

Fluorescamine Protease Assay-The assay was performed in 13 X 100-mm glass tubes in a small volume (15 p l ) with 1-10 &I of MEP and 9 pg of BSA in 0.1 M sodium formate, pH 3.0. The reaction was stopped by adding 3 ml of 0.2 M sodium borate, pH 7.5. Quantitation of primary amino groups was obtained with fluorescamine as de- scribed by Udenfriend et al. (10) by adding l ml of a fluorescamine solution (0.1 mg/ml in acetone) and mixing immediately. The fluo- rescence was measured on a Perkin-Elmer LS-5 Fluorescence Spec- trophotometer with excitation and emission wavelengths set at 390 and 475 nm, respectively. Both slit settings were at 10 nm. The units of fluorescence were used directly with rates of reaction determined by subtracting zero time values. A blank contained buffer in place of the MEP and water in place of the BSA.

For inhibition assays, the inhibitor and BSA were added after preincubation of the MEP to activate the enzyme. Pepstatin was dissolved in methanol, leupeptin in ethanol, and phenylmethanesul- fonyl fluoride in 50% ethanol. The solvents had no effect on the reaction in the dilutions used. For the iodoacetic acid experiments, the solution was made fresh each day in potassium phosphate buffer, pH 7.0. After activating the MEP peptides, the iodoacetic acid was added at 0.1 mM final concentration in buffer to raise the pH to 7.0. The sample was incubated at 24 “C in the dark for the specified length of time and then this reaction was stopped by adding a 50-fold excess of P-mercaptoethanol. In the samples used to test inhibition of

1760

MEP Is an Activable Acid-Protease 1761

activity, BSA and sodium formate buffer were then added to bring the pH back to 3.0, and the protease activity was measured.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis-Sam- ples dissolved in sodium dodecyl sulfate dissociation buffer (0.25% sodium dodecyl sulfate, 10% glycerol, 5% P-mercaptoethanol, and 0.625 M Tris-HC1, pH 6.8) were boiled 3 min and clarified by centrif- ugation at 12,000 X g for 2 min. Electrophoresis was performed as described by Laemmli (11). Gels were stained with either Coomassie Blue or silver (12) and the method of Bonner and Laskey (13) was used for fluorography. The gels were exposed to preflashed X-Omat AR film (Kodak) a t -70 "C, radioactivity was quantitated by densi- tometry on a Joyce-Loebel 3CS microdensitometer, and the peak areas were measured by weighing excised peaks. Labeling and im- munoprecipitation of labeled proteins were performed as previously described (7). Western blot analysis using affinity-purified anti-MEP antibody and lZ5I-protein A (Amersham) was performed as reported previously (7). The radioactivity associated with the nitrocellulose paper was quantitated in the same manner as for the gels. Direct immunoprecipitation of MEP samples was performed with purified MEP samples (0.5 pg) incubated with anti-MEP anti-serum (5 pl) or with preimmune serum overnight a t 4 "C in 37 pl of 0.01 M Tris, pH 8.0. The samples were centrifuged at 15,000 X g for 30 min and the supernatants removed. The pellets were redissolved in 37 pl of 0.01 M Tris, pH 8.0, and assayed in the same manner as the supernatants. The assay was done by adding BSA (110 pg) and sodium formate (0.1 M, pH 3.0) to a total volume of 176 pl. The amount of primary amines was determined with fluorescamine at four time points.

Modified Purification of MEP-The preparation of pure secreted MEP was essentially that of Gottesman and Cabral (4) with the initial concentration of the media done on a DE52 batch column. The conditioned media from 10 roller bottles of cultured KNIH cells (200 ml) was diluted 10-fold with water, and 20 ml of a DE52 slurry was added. The pH was adjusted to 8.0 with 1 M Tris, pH 8.0, and the suspension was stirred gently for 1 h, then poured into a 10 X 2.5-cm column and rinsed with 0.1 M Tris, pH 8.0. The proteins were eluted with 0.3 M NaCl (100 ml) in Tris buffer; once eluted, the proteins were concentrated by pervaporation against 0.1 M Tris, pH 8.0, 1 M NaCl to approximately 1 ml and were then loaded onto a Sephadex G-75 column (0.9 X 50 cm) and eluted at a flow rate of 0.2 ml/min. The MEP fractions were pooled, concentrated, and then dialyzed against 0.01 M Tris, pH 8.0,O.Ol M NaCl. The final step of purification was done on a Bio-Gel TSK DEAE-5-PW HPLC column (Bio-Rad) eluting with a NaCl gradient. A Beckman HPLC system was used at a flow rate of 0.5 ml/min (13 p. s. i.). The column was washed for 2.1 min with 0.01 M NaCl to load the sample and then for 4 min with 0.05 M NaC1. This was followed by a linear salt gradient from 0.05 to 0.25 M lasting 8 min, followed by a wash with 0.5 M NaCl. The fractions were collected at 1-min intervals. The presence of protein eluting from the columns was determined by measuring the optical density at 280 nm while the concentration of protein in specific samples was quantitated by the method of Lowry et al. (14).

RESULTS

MEP Has General Protease Activity at Acid pH-In trans- formed cells the major form of MEP is a 39,000-dalton se- creted mannose 6-phosphate containing glycoprotein (5). A small amount of this protein is processed intracellularly, as an alternative to secretion, into 29,000- and 20,000-dalton forms which are localized to lysosomes (7). Incubations of the secreted protein in acid buffer (sodium formate, 0.1 M, pH 3.0 at 42 "C) resulted in the time-dependent loss of the MEP protein which could be inhibited by boiling the sample prior to the reaction. A similar degradation was observed using highly purified MEP (HPLC fraction, see below) suggesting that this was autodigestion and that MEP was itself a pro- tease.

When purified MEP was added to various other proteins and incubated in formate buffer, pH 3.0 at 37 "C, all of those tested were degraded to some extent by the MEP (Fig. 1). The degradation of some of the proteins resulted in discrete bands such as those from BSA (Fig. lA, lane 2) , ovalbumin (Fig. lA, lane 4 ) , glyceraldehyde 3-phosphate dehydrogenase (Fig. lA, lane 6), and fibronectin (Fig. lB, lane 2). Fig. 1B

A

1 2 3 4 5 6

1 2 3 4 5 6 7 8 9 1 0

FIG. 1. Digestion of proteins by MEP protease. Proteins (-3 pg) were incubated at 37°C for 1 h with MEP (0.22 pg) (euen- numbered lanes) and without MEP (odd-numbered lanes) in 0.1 M sodium formate buffer, pH 3.0. After the incubation, the proteins were separated on a 15% acrylamide (Panel A ) or a 7% acrylamide gel (Panel B ) and stained with Coomassie Brilliant Blue. The proteins digested in Panel A were BSA (lanes 1 and 2), ovalbumin (lanes 3 and 4 ) , glyceraldehyde-3-phosphate dehydrogenase (lanes 5 and 6 ) , cytochrome c (lanes 7 and 8), and histones (lanes 9 and IO). The proteins digested in Panel B were fibronectin (lanes I and 2) , laminin (lanes 3 and 4 ) , and collagen (lanes 5 and 6 ) .

shows that the extracellular matrix proteins fibronectin, lam- inin, and collagen were substrates for MEP protease.

Generation of MEP Protease Activity Correlates with Auto- processing of MEP-To quantitate the protease activity of MEP, a fluorescamine assay for primary amines generated in the protease reaction was used. BSA was chosen as a substrate since it appears to be degraded into several small peptides (Fig. lA, lane 2 ) resulting in a large signal. With the BSA assay, using our most highly purified MEP preparations (see below), a lag in the appearance of fluorescent products was observed (Fig. 2 A ) . This lag correlated directly with the lag in appearance of lower molecular weight MEP peptides during this reaction (Fig. 2B). Prior incubation of MEP in the pH 3.0 buffer without BSA eliminated the lag in activity of MEP against BSA. Fig. 3 shows the time-dependent change in the shape of the BSA activity curve as a function of MEP prein- cubation at pH 3.0. Short periods of incubation of MEP at pH 3.0 (10-30 s) eliminated the lag period and generated a simple hyperbolic activity curve. Continued incubation of MEP (5-20 min) destroyed the activity (Fig. 3). This conver- sion was also followed with a Western blot to detect MEP immunoreactive peptides, which showed the appearance of lower molecular weight proteins as early as 30 s after incu- bation (data not shown). Only a very small amount of the 29,000-dalton protein could be detected in this assay, suggest- ing it may be very unstable under these conditions. The same kinetics of activation and the same appearance of lower molecular weight MEP forms were seen with boiled BSA (data not shown), suggesting that a protease activity in the BSA is not responsible for the conversion of the MEP.

Characterization of MEP Protease Activity-We have char- acterized both the activation of MEP protease and the result- ant protease activity. The appearance of fluorescent products is time- and MEP-dependent (Fig. 4A). The initial rate of the reaction is dependent on the amount of MEP with a linear relationship up to 0.65 pg protein (Fig. 4B). The activity is temperature-dependent with an optimum at 37 "C (Fig. 4). The pH profile for the activity (Fig. 4 0 ) is maximal in the

1762 MEP I.7 an Activable Acid-Protease

A

160 r 140 -

120 -

E l o o - :: m

L

20 ‘ 1 I I I I I

0 10 20 30 40 50 60

time (min)

B

39K -

29K -

20K -

a b c d e f g h i

FIG. 2. Lag in the protease activity of MEP. A sample of HPLC-purified MEP (0.20 pg) was mixed with 9 pg of BSA in 0.1 M sodium formate bulter, pH 3.0, and the activity (Panel A ) and the change in MEI’ immunoreactive forms (Panel H ) were observed. The reaction was allowed to take place a t 37 “C for various lengths of time and the fluorescent products were measured (Panel A ), or the sample was analyzed on a 12% acrylamide gel and a Western blot performed (Panel H ) . In Panel H , the time of reaction was 0 min (lane a ) , 30 s (lone b ) , 1 min (lone c) , 2 min (lane d ) , 5 min (lane e ) , 10 min (lane f ) , 20 min ( lanep) , 30 min (lone h ) , and 60 min (lane i). The positions of the three cellular MEP forms are indicated with their molecular mass in daltons noted ( K = 1000).

range of pH 3.5 with all three buffers tested. Several protease inhibitors and thiol protease activators

were added to observe their effect on BSA proteolysis. The per cent of control activity for the various compounds is shown in Table I. Very little effect was seen with pepstatin, aprotinin, phenylmethanesulfonyl fluoride, or EDTA. How- ever, leupeptin was a powerful inhibitor with a K, about 0.025 pg/ml or 0.5 X 10” M using 0.125 pg of MEP. The irreversible sulfhydryl protease inhibitor, iodoacetic acid, was also a very effective inhibitor of the activity. Sulfhydryl protease activa- tors such as dithiothreitol, cysteine, and @-mercaptoethanol had little or no effect.

Co-purification of MEP and Protease Actiuitv-To prove that the protease activity associated with MEP is intrinsic to MEP and not the result of a contaminating protease, we repurified MEP from the conditioned medium of KNIH cells. We modified the procedure as described under “Experimental Procedures” by concentrating the medium with DE52 and

TIME FIG. 3. Activation of the protease activity of MEP. Samples

of MEP (0.22 pg) were preincuhated without BSA in 0.1 M sodium formate, pH 3.0, for 0 min (u), 30 s (M), 1 min t u ) , or 5 min (W), then BSA was added, and the amount of primary amines generated was measured over the next hour.

eluting the proteins with 0.3 M NaCI. The eluted proteins were concentrated further to about 1 ml and run over a Sephadex G-75 column. All of the BSA hydrolyzing activity in this sample was leupeptin-sensitive (data not shown). MEP was eluted as a broad peak between fractions 54 and 62 and the protease activity coeluted with the MEP (Fig. 5). The MEP peak, which showed one major protein band of 39,000 daltons with some minor protein contaminants, was concen- trated and dialyzed against 0.01 M Tris, pH 8.0, 0.01 M NaCl. This sample was fractionated on Bio-Gel TSK DEAE-5-PW as described under “Experimental Procedures.” Pure MEP eluted as a single sharp peak and the activity co-chromato- graphed (Fig. 6). Overstaining of this fraction showed some minor protein components but the 39,000-dalton protein is a t least 99% pure. The homogeneous MEP from this HPLC separation was used in the characterization of protease activ- ity reported above.

Direct Immunoprecipitation with Anti-MEP Antibody Pre- cipitates the Protease Actiuity-Purified MEP samples were directly immunoprecipitated with antibody to MEP and preimmune serum, and the protease activity in the pellet and supernatant fractions were assayed. Table I1 shows the results of this analysis. Antibodies to the MEP precipitate about 90% of the protease activity in these samples, whereas the preim- mune samples precipitate only 7%. Control incubations with no added serum showed a similar partition of the protease activity.

DISCUSSION

In an attempt to understand the ways in which tumor cells interact with their hosts, our laboratory has been studying the major excreted protein of Kirsten virus-transformed mouse cells. This protein is synthesized in about 20-100-fold greater amounts by these cells compared to their normal parent cells and makes up about 1% of the total protein synthesized by KNIH cells (15). More moderate increases (3- 10-fold) in synthesis of MEP have been seen with various other treatments, including transformation by chemicals or other tumor viruses (Harvey and Moloney sarcoma viruses and SV40) (15), phorbol ester tumor promoters (16, 17), and the platelet-derived (18) and fibroblast (19) growth factors. We have recently cloned a cDNA encoding this protein and found under these conditions that the increased synthesis is due to an increase in the mRNA for MEP (17, 20, 21). In the case of phorbol ester and growth factor induction, we have also shown that this effect is at the level of transcription using nuclear runoff experiments (22). In addition, a temper-

MEP Is an Activable Acid-Protease 1763

FIG. 4. Characteristics of the MEP protease. Panel A, the reaction is MEP- and time-dependent. The gener- ation of primary amines in the presence of MEP (0.6 pg) (C”-O) is much greater than without MEP (W). The appearance increases with time of incubation. Panel B , the amount of ac- tivity is dependent on the amount of MEP added. MEP concentration was 0.22 mg/ml. Panel C, the protease activ- ity is temperature-dependent. Panel D, the pH profile was measured with three different buffers and several pH values. Sodium salts were used and the concen- tration of the buffer was 0.1 M in all cases.

s 1001 ._

TABLE I Effect of inhibitors and potential activators on MEP activity

MEP (0.12 pg) was first activated by preincubating a t pH 3 for 30 s, and then BSA, inhibitor, and buffer were added and the activity was measured.

Inhibitor Concentration Activity”

% Cysteine P-mercaptoethanol Dithiothreitol Phenylmethanesulfonyl fluoride Aprotinin Pepstatin EDTA MgCL MnC12 CaC1, Leupeptin

Iodoacetic acid’

2 mM 0.5% 1 mM

3.6 mTIU” 1 mM

4 PM 1 mM 2 mM 2 mM 2 mM 0.05 pg/ml 0.025 pg/ml 0.01 pg/ml 0.003 pg/ml 2.7 mM 0.5 mM 0.1 mM

110 82 110 81 92 81

107 85 86 92 19 53 81 97 6

21 41

a % activity is the per cent of activity in the presence of the inhibitor

” Trypsin-inhibiting units. compared to controls.

Preincubation of iodoacetic acid with MEP was done at pH 7.0 for 9 min and then the pH was shifted down to measure protease activity.

ature-sensitive, Kirsten virus-transformed 3T3 cell line (ob- tained from B. Peterkovsky, National Cancer Institute) syn- thesizes greater amounts of MEP protein’ and MEP message (20) at the permissive temperature. Thus, there is a good correlation between MEP synthesis and enhanced growth and/or malignancy in mouse cultured cells. We have also observed a similar phenomena in mouse skin cells treated in vivo with the phorbol ester 12-0-tetradecanoylphorbol 13-

S. Gal, B. R. Troen, and M. M. Gottesman, unpublished obser- vations.

VOLUME OF MEP It111

120

1M)

80

60

4a

20

2.5 3.0 3.5 4.0 4.5 5.0

acetate (23). In the mouse tissue culture system used by this laboratory, about 5% of the MEP synthesized is retained by the cells and processed to two lower molecular size proteins of 29,000 and 20,000 daltons (7). These proteins are localized in Golgi and lysosomes by biochemical and cytochemical localization techniques (7).

In this report we have presented evidence that secreted MEP is an acid-activable cathepsin which is able to digest a variety of proteins including extracellular matrix proteins. Using BSA digestion as an assay for this protease, we found the activity to be optimal at pH 3.5 and to be inhibited by thiol reagents such as leupeptin and iodoacetate. The evidence to suggest that the source of proteolytic activity is indeed MEP and not some contaminating protein is derived from two separate methods. First, the MEP protein and the BSA proteolytic activity co-elute on two different columns (Seph- adex G-75 and DEAE HPLC). Second, antibody to secreted MEP immunoprecipitates this BSA proteolytic activity. These lines of evidence, taken together with the general protease resistance of MEP (15) and its similarity in size to other cathepsins (24), lead to the conclusion that MEP is a cathepsin.

Secreted MEP is not active as the 39,000-dalton protein but requires activation at pH 3.0 to show simple kinetics of proteolysis. This activation occurs rapidly in pure MEP prep- arations (Fig. 3) and slower in the presence of other proteins (Fig. 2). These data suggest an autoprocessing of MEP. The appearance of activity during activation is correlated with the appearance of lower molecular weight MEP peptides in a Western blot (Fig. 2). Our data suggest, therefore, that this species is the active enzyme. This form is similar in size to the 29,000-dalton cellular form of MEP which, along with the 20,000-dalton MEP protein, is found in lysosomes (7).

Many proteases are synthesized as precursor molecules and then processed to lower molecular weight active species. Spe- cific examples are trypsin (25), chymotrypsin (26), pepsin (27), and plasminogen activator (28). Processing of lysosomal enzymes into lower molecular weight species has been seen for a variety of enzymes, including cathepsins D (29, 30) and

1764

A

MEP Is an Activable Acid-Protease

A 6

0.40 r t

0.30

- 0.25 30

20 0.10

0.05

fraction

FRACTION ST F14 F15 F16 F17 F18

FIG. 6. Co-purification of BSA protease and MEP protein on a DEAE HPLC column. This column was operated as described under “Experimental Procedures.” Panel A shows the optical density (solid line) and BSA protease activity (a”.) profiles of the frac- tions, whereas Panel B shows the silver-stained gel of these fractions. The fraction numbers have been indicated. ST is the starting mate- rial.

TABLE I1 Direct immumprecipitatwn ofprotease activity with anti-MEP

antibodies MEP samples were incubated overnight with or without serum,

then centrifuged at 15,000 X g for 30 minutes. The pellet and super- natant fractions were analyzed as described in the text.

rescence/60-min reaction) Activity (change in fluo-

Supernatant Pellet

Control (no serum) 23 7 Preimmune serum control 26 2 Anti-MEP serum 3 25

St F38 41 44 47 50 52 54 56 58 60 62 64 66 68 71 FIG. 5. Co-purification of BSA protease and MEP protein

on a Sephadex G-75 column. A sample of concentrated medium was run over a Sephadex G-75 column as described under “Experi- mental Procedures.” Panel A shows the optical density (solid l i n e ) and BSA protease activity (.”-.) of the fractions. Panel B shows the silver-stained gel of the various fractions with fraction numbers indicated. St is thestarting material. The arrow indicates the position of MEP.

B (31). Some laboratories have shown that this processing correlates with the activation of these enzymes (32, 33). In the case of cathepsin D, a brief acid treatment is necessary for activation of the cathepsinogen D (32). Mort and co- workers (33) have been characterizing a latent thiol protease found in ascitic fluid which is activated by pepsin treatment. The resultant activity is similar to cathepsin B with respect to substrate specificity and pH optimum. The purification of cellular cathepsin B and D has in the past included an overnight incubation at pH 4.5 termed autolysis (24), which may have obscured the presence of precursors for these en- zymes up to now. Our data on MEP are consistent with its activation requiring an autoproteolytic step which removes about a 10,000-dalton piece. Tryptic peptide maps of cellular processed MEP peptides (7) show that the cellular forms lack

several methionine-rich peptides found in the precursor MEP. This suggests that at least some of the processing of MEP involves the peptide backbone. We have not characterized changes in the sugar structure of these different MEP forms, but the processed forms appear to be glycosylated (5).

The proteolytic activity which is associated with processed MEP has properties that are similar to, but appear distinct from, the known lysosomal cathepsins. The observation that the activity can be inhibited by leupeptin and iodoacetic acid, but not by pepstatin, puts the MEP protease in the thiol protease class. This class includes cathepsins B (34), H (35), L (36), T (37), M (38), S (39), a collagenolytic cathepsin (40), and a recently reported protease induced by leupeptin in rats (41). Each of the members in this class has properties which distinguish it from each other and from MEP. The MEP protease has no activity against synthetic substrates N-ben- zoyl-DL-Arg-P-naphthylamide or N-carbobenzoxy-L-Arg-L- Arg-4-methoxy-P-naphthylamide (at pH 6) which are sub- strates for cathepsins B and H, respectively. Cathepsin B requires added thiol in the form of cysteine or dithiothreitol for full activity (24), which is not the case with the MEP protease activity. The pH optima of the protease activity of cathepsins L, T, and M are all near 6, which distinguishes these cathepsins from MEP. The leupeptin-induced protease of Tanaka et al. (41) has a similar size, pH optimum, and substrate specificity as MEP but is not inhibited by iodoacetic acid. MEP is stable in the medium of the KNIH cells and at pH 8.0 during the purification process, which distinguishes it from cathepsins L and T, which are not stable. Cathepsin S

MEP Is an Activable Acid-Protease 1765

and the collagenolytic cathepsin have similar properties to M E P optimal pH for reaction (3.5), inhibited by leupeptin and iodoacetic acid, and low activity with the synthetic sub- strates N-benzoyl-DL-Arg-P-naphthylamide and N-carbo- benzoxy-~-Arg-~-Arg-4-methoxy-~-naphthylamide. However, the molecular properties of these proteins are different from those of MEP. Cathepsin S from bovine sources has a mass of 14,000 daltons and the bovine collagenolytic cathepsin also from bovine has a mass of 20,000 daltons. Thus, of the reported thiol cathepsins, none are exactly the same as the MEP protease we have characterized.

It is reasonable to question whether all ofthe thiol proteases are indeed different or whether different sources, different assay conditions, or ,different isolation procedures have ob- scured their similarities. We have recently partially sequenced a cDNA coding for MEP? This partial sequence when com- pared with the available sequences published for cathepsin B (rat (42,43), pig (44), and human (45)), cathepsin H (rat (40) and pig (42)), and the non-thiol protease cathepsin D (46) revealed no obvious identities. Although these comparisons are cross-species, Takahashi et al. (44) have reported a great deal of homology in the NH2-terminal sequences of cathepsins from rat and pig. We would expect to see near identity among mammalian cathepsins if they are the same protein. We conclude from these preliminary data that MEP is not ca- thepsin B, H, or D.

Why would malignant cells secrete the precursor to a ca- thepsin? It is possible that under some conditions this enzyme might be active in the acidic environment of a tumor (47) and that its function is to digest the extracellular matrix proteins fibronectin, collagen, and laminin so as to allow invasion of the tumor. An alternative possibility is that secreted.MEP, which carries the mannose 6-phosphate lysosomal recognition marker and binds to the mannose 6-phosphate receptor (5) , is taken up by normal cells of the host. The protein would then be delivered to the lysosomes and could cause a delete- rious effect on these cells which may account for some of the syndromes associated with the growth of malignant tumors. We have found that secreted MEP from transformed fibro- blasts can be taken up by both NIH 3T3 and KNIH cells and is processed to the same forms as is nascent intracellular MEP.4 The finding that MEP is an acid-activable protease enables us to focus our studies on these and other possible functions for MEP.

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