Inhibitory properties of low molecular mass cysteine proteinase inhibitors from human sarcoma

Biochimica et Biophysica Acta. 993 (1989) 63-73

63

Elsevier BBAGEN 23191

Inhibitory properties of low molecular mass cysteine proteinase inhibitors from human sarcoma T a m a r a T. L a h 1.., J o h n L. C l i f f o r d z, K e n t M . H e l m e r 1, N a n c y A. D a y 1, K a m i a r M o i n 1, K e n n e t h V. H o n n 3.4, J o h n D . C r i s s m a n 5 a n d B o n n i e F. S l o a n e 1.3,4 Departments of I Pharmacolog),, 2 Biological Sciences and 3 Radiation Oncology, Wc~vneState Unit,ersity and 4 the Gershenson Radiation Oncology Center, Harper/Grace Hospitals. Detroit. Michigan 48201 and ~ the Department of Pathology, Henry Ford Hospital, Detroit, MI (U.S.A.)

(Received 22 May 1989)

Key words: Cystatin; Stefin; Cysteineproteinase; Cancer; (Human liver);(Human sarcoma) Elevated activities of cysteine proteinases such as cathepsins B and L and cancer procoagulant have been linked to tumor malignancy. In the present study we examined the hypothesis that these elevated activities could be due to impaired regulation by the endogenous low molecular mass cysteine proteinase inhibitors (cystatins). lnhibitors from human sarcoma were compared to those from human liver, a normal tissue in which ,the inhibltors had been characterized previously. An extract of cystatins from sarcoma was less effective against papain ~md cathepsin B (liver or tumor) than was an extract from liver. This reduced inhibitory capacity in sarcoma was not due to a reduction in either the concentrations or specific activities of the cystatins or an absence of any family or ..'sofo~ of cystatins. We pu~f[ed, ,",vo mere-bets of the cystatin superfamiiy (stefin A and stefin B) to homogeneity and detc.~ined their individual ..'nhibitory properties. Siefins B from iiver and sarcoma exhibited comparable inhibition of papain and cathepsin B. In contrast, stefin A from sarcoma exhibited a reduced ability to inhibit papain, human liver cathepslus B, H and L and human and murine tumor cathepsin B. The Kt for inhibition of liver cathepsin B by sarcoma stefin A was 10-fold higher than that for inhibition of liver cathepsin B by liver stefin A, reflecting a reduction in the rate constant for association and an increase in the rate constant for dissociation. Can:er is now the third pathologic condition reported to be associated with alterations in cystatins, the other two being amyloidosis and muscular dystrophy.

llntroduct~on Among the cysteine proteinases whose activities have been reported to be elevated in tumors are cancer procoagulant [1,2], a cysteine proteinase that processes proapolipoprotein II extracellularly [3], cathepsin L (or MEP) and cathepsin B (for review see Refs. 4-7).

* On leave from the Jozef Stefan Institute. Ljubljana,Yugoslavia. Abbreviations: AMC, aminomethylcoumarin; CNBr. cyanogen bromide; CPI. cysteineproteinaseinhibitor; DTT. dithiothreitol; E-64, L.trans-epoxysuccinylleucylamino(4-guanidino)butane; FPI.C, fast protein liquid chromatography; IEF, isoelectric focusing; MeO/3NA, methoxy-/~-naphthylamide;MEP, majorexcreted protein; SDS-PAGE, sodium dodecyl sulfate-polyacrylamidegel electrophoresis;Z, N-benzyloxycarbonyl-. Correspondence: B.F. Sloane, Department of Pharmacology. Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, U.S.A.

However, accurate assessment of cysteine proteinase activities in biological fluids, homogenates or culture media is difficult for several reasons. (1) The exogenous substrates employed for assay of cysteine proteinases will compete with endogenous substrates. Therefore, in our recent studies of cathepsin B in tumors [8,9], we have measured activity at several concentrations of exogenous substrate. (2) In order for a cysteine proteinase to be fully active, the essential cysteine residue at the active site must be reduced. In vivo glutathione is hypothesized to be the activator whereas ex vivo cysteine or dithiothreitol must be added in excess to insure accurate quantitation of cysteine proteinase activity [i0,il]. (3) The ultimate control mechanism for cysteine proteinase activity in vivo is endogenous CPIs. Quantitation of cysteine proteinase activity in samples containing CPIs is clearly inaccurate. Use of an assay employing several substrate concentrations may not improve the quantitation, since CPIs are tight-binding inhibitors often classified as pseudo-irreversible (for

0304-4165/89/$03.50 © 1989 ElsevierScience Publishers B.V. (Biomedical Division)

64 review see Refs. 12 and 13). Therefore an accurate assessment of cysteine proteinase activities in tumors will require information about the endogenous CPIs. Three families of endogenous CPIs have been identified: stefins, cystatins and kininogens (for review see Refs. 12 and 13). Stefins and cystatins comprise the low molecular weight CPIs of the cystatin superfamily. Stefin A isoforms with acidic isoelectric points and stefin B isoforms with neutral isoelectric points have been isolated from human spleen [14], liver [15] and urine [16]. Stefins A and B differ structurally and functionally [12,13], but both are more effective at inhibiting the lysosomal cysteine proteinases cathepsins H and L than cathepsin B; of the two, stefin A is the more petent inhibitor of cathepsin B [15,16]. One of the possible explanations for the increased cathepsin B activities our laboratory has observed in tumors [4,6] is a decrease in the concentrations or activities of the endogenous inhibitors of this enzyme. In the present study, we determined the inhibitory properties of tumor stefins A and B, using the cysteine proteinases papain, cathepsin B, cathepsin H and cathepsin L as target enzymes. Materials and Methods Materials Human liver tissue was obtained from cadavers less than 6 h postmortem and transported on ice to c,ux labo~'atory where it was either used immediately or frozen at - 2 0 ° C . Human sarcomas obtained from surgical resections were transported immediately to pathology where sections were frozen at - 7 0 o C. Tumor sections were transferred to our laboratory where they were stored at - 2 0 ° C prior to use. The sarcomas used were all spindle-cell sarcomas of the malignant histiocytoma or leiomyosarcoma type. If necessary, different specimens of the same type were pooled to provide an adequate amount of starting material. Chemicals and chromatographic media. CNBr activated Sepharose-4B, Sepharese-4B, Sephadex G-75 and G-50, phenyl-Sepharose and the PD-10 columns were obtained from Pharmacia, Piscataway, NJ. Chromatofocusing media (Polybuffer exchanger 94, Polybuffers 74 and 96) as well as FPLC columns (Superose 12, Mono P and Mono Q) were purchased from Pharmacia, Piscataway. NJ. Papain, CNBr, L-cysteine, E-64, DTT, sodium iodoacetate and Tris were purchased from Sigma, St. Louis, MO. Z-Atg-Arg-MeOflNA, Z-ArgArg-AMC, Z-Phe-Arg-MeOflNA, Z-Phe-Arg-AMC AMC and MeOflNA were obtained from Enzyme System Products, Livermore, CA. Human liver cathepsin L and H were gifts from Dr. V. Turk (Jozef Stefan Institute, Ljubljana, Yugoslavia). Murine melanoma (B16 amelanotic) subceUular fractions were prepared by Dr.

J. Rozhin using our published procedures [8,9]. All other chemicals were of analytical grade. Purification of low molecular mass CPI CPIs were r,trified using six steps: (1) homogenization and preparation of a cytosolic fraction; (2) alkali treatment; (3) acetone fractionation; (4) affinity chromatography on carboxymethylated papain Sepharose; (5) gel filtration; and (6) either chromatofocusing or anion-exchange chromatography. The methodology described in detail by Green et al. [15] was usec; for the first four steps. Steps 5 and 6 are described below. Inhibitory fractions from the affinity chromatography step were neutralized with 0.1 M HCI and then concentrated in an ArrJcon ultrafiltration apparatus using a YM 2 membrane. The proteins were resuspended in 1.5 ml of 0.05 M Tris-buffer (pH 7.5), containing 0.15 M NaC1 and applied on a gel-filtration column (Sephadex G-75 superfine column, i.5 × 90 cm) at 4 ° C. After sample application, the column was eluted with 0.05 M Tris-buffer (pH 7.5) containing 0.15 M NaCI at a flow rate of 12 ml/h. Alternatively, gel filtration was performed on an FPLC Superose 12 column ( H R / 3 0 ) at 25 ° C at a flow rate of 0.3 ml/min. Inhibitory fractions of low molecular mass (10-15 kDa) were pooled, concentrated as above, resuspended in 0.025 M Bistris-HCl buffer (pH 6.3) and subjected to chromatofocusing on an FPLC Mono P column (HR 5/20). Inhibitors were eiuted with Po!ybuffer 74 (diluted 10-fold and adjusted to pH 4.0 with 1.0 M HCI), active inhibitor being recovered in the void volume at the starting pH and in two peaks at pH 5.2 and 4.8. The unbound inhibitory protein was dialysed in Diaflo membranes (3500 Da cut off pore size) against 0.075 M Tris-acetate (pH 9.3), and subjected to a second chromatofocusing step on the same column. Inhibitors were eluted with Polybuffer 96 (diluted 12-fold and adjusted to pH 6 with 1.0 M acetic acid). Alternatively, anion-exchange chromatography was utilized. In this case the sample was applied on an FPLC Mono Q column that had been equilibrated with 0.025 M Tris buffer (pH 7.9). Elution was performed with a linear gradient of buffer and salt to 0.050 M "Iris and 0.25 M NaCI, respectively. Unbound inhibitory protein was concentrated as above, resuspended in 0.01 M Tris-buffer (pH 9.8), and then applied to the Mono Q column. The column was developed with a linear gradient of buffer and salt to 0.025 M Tris and 0.4 M NaCI, respectively. Enzymes and enzyme assays Commcrcial papain (2 × crystallized, Type III, Sigma) was purified prior to use by gel filtration on Sephadex G-50. Cathepsin B from normal human liver or human colon or breast carcinoma was purified to homogeneity by a modification of the Willenbrock and

65 Brocklehurst procedure [17] as has been described [18]. The specific activity of papain was determined fluorimetrically using Z-Phe-Arg-MeOflNA or Z-Phe-ArgA M C as substrate as descrit)ed by Turk and Kregar [19] and Barrett [20], respectively. The specific activity of cathepsin B was measured fluorimetrically using either Z-Arg-Arg-MeOflNA or Z-Arg-Arg-AMC as substrate [9]. The molar concentration of active papain or cathepsin B per unit of enzyme protein was determined by titration with E-64, an active site titrant for cysteine proteinases [21].

Inhibitor assays Stopped assays were performed as described previously [8]. Inhibition of papain was determined using Z-Phe-Arg-MeOflNA as the substrate, and inhibition of cathepsin B using Z-Arg-Arg-MeOflNA as the substrate. The enzyme (8-30 nM) was preincubated with an equal volume (100 ~1) of the appropriate dilution of the inhibitor in the presence of 10 mM DTT ([lnal concentration) for 10 min at 2 5 ° C in 0.2 M citrate buffer (pH 6.2) containing 1 m M EDTA. After additian of 100/tl of substrate (0.5 mM final concentration), the reaction was allowed to proceed for 5-20 min at 25 ° C and then stopped by addition of 400 #i of 1 M HCI. The relative increase in fluorescence was measured at an excitation wavelength of 292 nm and emission wavelength of 410 nm. Substrate blanks were prepared by adding substrate after stopping the reaction whereas buffer blanks contained buffer instead of inhibitor solution. During the course of purification of CPIs, the activity of CPIs, i.e., the inhibi::ory activity, was determined in stopped assays using papain (itself titrated with E-64) as the standard target enzyme for the CPis and Z-PheArg-AMC as the substrate for papain (as described for assay of cathepsin L in Ref. 21). One inhibitory unit was defined as the amount of inhibitor preparat, on which totally inhibited one activity unit of papain, one activity unit representing the release of 500 /tmol of A M C per min. Continuous assays were performed to determine rate constants (k a and ka) as well as inhibition consta'ats ( K i) using a slight modification of the method employed by Nicklin and Barrett [22]. Briefly, the enzyme (200 ttl of 2-15 n M cathepsin B or 30 #I of 4 n M papain) was preincubated for 10 rain at 2 5 ° C in the fluorimeter cuvette in 0.2 M citrate buffer (pH 6.2) or 0.1 M acetate buffer (I,H 5.5), respectively, containing 1.5 mM DTT (final concentration), 1 mM EDTA and 0.05% Brij. 20 /~1 of substrate (Z-Arg-Arg-AMC or Z-Phe-Arg-AMC) was added to cathepsin B and papain, respectively, to a final concentration of 20 p.M in a total volume of 1 ml. After a linear increase in fluorescence was observed (Vo), inhibitor was added in a negligible volume (5-20 /tl). The reaction was followed for 20-30 min for papain

and 40-60 min for cathepsin B, i.e., until a new steadystate rate was reached; the slope (vi) was recorded. Prior to the assay the molar concentration of active inhibitor had been determined by microscale titration with papain assuming a 1 : 1 molar ratio of binding between papain and inhibitor as described by Anastasi et al. [23]. The molar concentration of the papain used for microscale titration had been determined using E-64 (see above). Rate and inhibition constants were calculated according to the method of Baici and GygerMarazzi [11] under the specific conditions delineated by Nicklin and Barrett [22] for low molecular mass CPIs.

Protein determination The concentration of protein was determined by the Bradford method [24] using bovine serum albumin as standard.

Electrophoresis (SDS-PA GE) Slab-gel electrophoresis was carried out in a 15% polyacrylamide gel in the presence of SDS as described by Laemmli [25]; 20-70 /tg of protein was loaded per lane. Electrophoresis was run at a constant current of 25 mA and staining was performed with Coomassie brilliant blue. The standard proteins applied were txlactalbumin (14.2 kDa), soybean, trypsin inhibitor (20 kDa), trypsinogen (24 kDa), carbonic anhydrase (29 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), egg albumin (45 kDa) and bovine albumin (66 kDa). For SDS-PAGE of inhibitors isolated by chromato focusing, Polybuffers were removed as follows: 1 r..fl o~ phenyl-Sepharose was washed with 5 ml of 80% ammonium sulphate solution to favor hydrophobic interaction. The inhibitor solution was applied and washed with 2 - 3 bed volumcs of saturated ammonium sulphate. Inhibitors were eluted with 20% glacial acetic acid and concentrated by lyophilization.

Isoelectric focusing (IEF) IEF was performed either on a DESAGA apparatus (F.R.G.) o : a Phast System (Pharmacia, Sweden). In the former case. 1 mm thick gels (10 × 10 cm), consisting of 5% polyacrylamide and 3% methylene bisacrylamide containing ampholytes in the pH range 3-16 (Pharmalyte, Pharmacia), were cast. In the latter case, precast gels were used as directed by the manufacturer. The former gels were stained with Coomassie blue and the latter silver-stained using the t~has '. System automated silver-staining procedure.

Fluorescence spectra Fluorescence spectra of inhibitor solutions (10-20 nM) were measured in a Perkin Elmer 650-10S fluorescence spectrophotometer in 1 cm quartz cells at 25~'C. Excitation and emission slits were set at 5 nm

a n d the emission s p e c t r u m was recorded at excitation wavelengths of 280 n m a n d 300 n m . D e p e n d i n g on the s a m p l e buffer the baseline was recorded with 50 m M Tris buffer (pH 7.5), Polybuffer 74 or Polybuffer 96. U n c o r r e c t e d emission spectra ar~. preset~ted.

Results O n e of the p r i m a r y interests of o u r laboratories h a s been the role(s) that a t u m o r m e m b r a n e - a s s o c i a t e d form of cathepsin B m i g h t play in focal dissolution of the extraccllular matrix d u r i n g t u m o r cell me)asta~is (see Ref. 6 for review). T h e physical c o n s t r a i n t s i m p o s e d by the m e m b r a n e would m a k e this e n z y m e a m o r e likely target for low molecular m a s s C P I s such as stefins a n d cystatins t h a n for high molecular m a s s CPIs. T h e extracell--'_.ar low molecular m a s s CPIs, cystatins, are m o r e effective inhibitors of c a t h e p s i n B )han are stefins a n d have been proposed to control c a t h e p s i n B activity in biological fluids [16]. T h u s cystatins m a y be of interest in matrix degradation. However, the e n h a n c e d cathepsin B activities we a n d others have m e a s u r e d in t u m o r ce~ls (for review see Refs. 4 a n d 6) m a y reflect alterations in the intracellular low molecular m a s s CPIs, i.e., stefins A a n d B. In the present s t u d y we have purified stefins A a n d B from two h u m a n tissues, n o r m a l liver a n d sarcoma. I n h i b i t o r y properties o f low molecular mass C P l s f r o m h u m a n liver a n d s a r c o m a

T h e procedure we employed for purification of low molecular m a s s C P I s was a modification of o t h e r proce-

dures for their purilication from n o r m a l tissues [14,i5,26,27]. As has been observed by others [15,23], the mos~ crucial step in o u r purification of low molecular m a s s C P I s was affinity c h r o m a t o g r a p h y o n carboxym e t h y l a t e d papak,, Sepharose, resulting in ~, 40-fold increase in purification (Table I). T h e inhibitots b o u n d reversibly a n d were eluted wader alkaline conditions; the resulting affinity extract contained a m i x t u r e of stefins A a n d B. This affinity extract was tested against p a p a i n a n d ca,~hepsin B from h u m a n liver a n d colon adenocarcinor.aa. Low molecular m a s s C P l s from liver a n d s a r c o m a differed in tl,eir inhibitory properties (Fig. 1). T h e mixture of stefins A a n d B from liver strongly inhibited p a p a i n as well as both cathepsin Bs. In contrast, the mixture of stefins A a n d B from s a r c o m a exhibited a reduced ability to inhibit the cysteine proteinases, especially b o t h c a t h e p s i n Bs. At a 20 n M c o n c e n t r a t i o n ( d e t e r m i n e d by titration against papain), the inhibitory proteins in s a r c o m a a n d liver extracts reduced the relative activity of liver c a t h e p s i n B to 76 a n d 4% of control, respectively. C o n c e n t r a t i o n s of the affinity extract from s a r c o m a up to 160 n M did n o t reduce t u m o r c a t h e p s i n B activity below 40% of control. W e have also observed that a n affinity extract of stefins A a n d B form h u m a n colon a d e n o c a r c i n o m a s exhibited a reduced ability to inhibit papain, h u m a n liver c a t h e p s i n B a n d h u m a , colon a d e n o c a r c i n o m a ( d a t a n o t shown). T h e s e results suggest that the e n h a n c e d activities of c a t h e p s i n B observed in some t u m o r s m i g h t be d u e to altered regulation by stefins A a n d B, b u t did n o t establish w h e t h e r regulation by only one stefin or b o t h was altered.

TABLE I Results of typical purifications of human lieer and sarcoma stefins A and B

Protein concentration was measured according to Bradford [24]. Inhibitory activity was determined against active si:e-titrated papain as described in Materials and Methods. Tissue

Protein (mg)

Total act. (IU)

Yield (% IU)

Spec. act. dU/mg)

t'urification (-fold)

(A) Human liver (430 g) Cytosolic fraction Alkaline treatment Acetone fractionation ~ffinit y chromatography Gel filtration Mono Q. stefin A Mono Q, stefin B

6575 1 273 163 3.20 0.68 0.01 0.05

30380 10638 3600 2957 1 582 69 250

(100) 35.0 12.0 9.7 5.2 0.2 0.8

4.6 8.4 22 924 2326 6900 5000

1 1.8 4.8 201 506 1 500 108'/

(B) Human sarcoma (123 g) Cytosolic fraction Alkaline treatment Acetone fractionation Affinity chromatography Gel filtration Mono Q, stefin A Mono Q, stefin B

3 322 797 124 2 0.80 0.05 0.035

5 980 a 144 3100 2100 i 6(35 290 239

(100) 69 52 35 27 _~ 4

1.8 5.2 25 1050 2006 5 800 6 826

l 2.9 14 583 1114 3222 3794

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modified the procedure described by G r e e n et al. [15], first separating stefin A from stefin B on an acid p H gradient a n d then separating stefin B isoforms on an alkaline p H gradient. Representative c h r o m a t o f o c u s i n g elution profiles for the s a r c o m a stefins are presented in Figs. 2A a n d 2B. T h e elution profiles observed for liver stefins were similar (data ~lot shown). Generally, stefin A eluted as a s h a r p peak at p H 5.2, although in s o m e p r e p a r a t i o n s there was an additional inhibitory peak at lower p H (4.6-4.8) (Fig. 2A). W h e n the initial u n b o u n d protein peak was r u n on an alkaline gradient, peaks at p H 9.0 a n d 8.8 were resolved (Fig. 2B). Stefin B eluted in o n e isoform at p H 8.8 (and in s o m e cases a second isoform at p H 8.0). TV,e p H 9.0 peak probably represented cystatin C a n d was not investigated further. All of thc protein peaks from both g r a d i e m s exhibited inhibitory activity against papain. Preparations of inhibitors from liver a n d s a r c o m a varied in the relative a m o u n t s of stefin A and stefin B in the two tissues, however, the p H values at which the inhibitor') peaks eluted were highly reproducible. T h u s the isoforms of b o t h stefin A a n d stefin B present in s a r c o m a were qualitatively the s a m e as in liver; individual prepara-

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(stefins A and B) zfter elution from carboxymethylated papain Sepharose (affinity extract) were added in increasing concemradon:~ to papain (0.1 riM) and cathepsin B purified from human liver (3 nM) and from human colon carcinoma (8 riM). The molarity of the inhibitor mixture was established by microscale titrztion with active site-titrated papain as described in Materials and Methods. Cysteine proteinase activities were measured in stopped fluorimetric assays using Z-Phe-Arg-AMC and Z-Arg-Arg-MeO#NA as substrates for papain and cathepsin B, respectively. B, sarcoma inhibitors; ~. liver inhibitors.

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mo 0o4 Purification of stefin A and stefin B from human liuer and sarcoma Stefins A a n d B b o t h bind to papain-Sepharose. However, as they are structurally different proteins with distinctive isoelectric points, they can be efficiently separated from one a n o t h e r either by a n i o n - e x c h a n g e chrom a t o g r a p h y or c h r o m a t o f o c u s i n g [1,:1,15,26,27]. Initially, we separated stefins A and B by c h r o m a t o f o c u s i n g , as this m e t h o d allowed us to determine which isoforms of stefins A a n d B were present in liver a n d s a r c o m a as well as their relative quantities in the two tissues. W e

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anion-exchange chromatography on a Mono Q column. (A) Low molecular mass inhibitory fractions from the gel filtration were applied to tbe Mono Q column. Stefins A were eluted with a buffer gradient (0.025-0.050 M Tris) and salt gradient (0-0.25 M NaCI) as described in Materials and Methods. - - , protein (A2so); . . . . . . , inhibition of papain . . . . . . , [NaCI1. (B) The unbound inhibitory peak from A above was reapplied to the Mono Q column and stefins B ehited with a buffi:r gradient (0.01-0.025 Tris) and salt gradient (0-0.4 M NaCI) as described in Materials and Methods. - protein (A~s0); . . . . . . . inhibition of papain; . . . . . . , [NaC1].

tions differed slightly in relative amounts of each isoform. Having established via chromatofoeusing that the isoforms of stefins A and B did not differ in liver and sarcoma, we employed an a/tentative m e t h o d to separate the stefins, one that did not employ Polybuffers. This method using anion-exchange chromatogr:tFhy wa~ based on the procedure of others for the separador: of stefins A and B [27]. Stefin A was resolved from stefin B by chromatography on an F P L C M o n o Q column at p H 7.9, using a linear gradient of buffer and l'!aCl, stefin A ehiting at 0.08-0.10 M NaCI (Fig. 3A). The u n b o u n d inhibitory protein was reapplied to the M o n o Q column at p H 9.4-9.8 and stefin B isoforms were resolved using a linear gradient of buffer and NaCI (Fig. 3B). Stefin B ehited at 0.12-0.15 M NaCI. A n u n b o u n d inhibitory peak, possibly representing cystatin C, was obtained but not used for further studies. As

with chromatofocusing the M o n o Q ehition positions (i.e., NaCI molarity) for each stefin were reproducible and similar for b o t h liver and sarcoma, suggesting that stefins of roughly the same molecular properties were present in b o t h tissues. Protein yields varied among preparations, perhaps due to the presence of dimers of stefin B (Fig. 4A) a n d / o r of larger aggregates. Such dimers or aggregates may be present in vivo or may be formed during purification as previously reported [27,28]. Table I includes representative purification tables for liver and sarcoma stefins. From such tables compiled during each purification we were able to compare the relative amounts of low molecular mass CPIs in liver and sarcoma. After affinity chromatography we obtained an average of 12.3 #g inhibitor protein per g tissue (5-33 /~g/g) in six liver purifications and an average of 13.5 # g inhibitor protein per g tissue (7-23 / t g / g ) in four sarcoma purifications. Therefore we conclude that the total content of low molecular mass CPIs in liver and sarcoma was similar. The specific activities of the low molecular mass CPIs in the affinity extract and of the purified stefins A and B were also similar (Table I, compare A and B). The degree of purification achieved for sarcoma inhibitors appeared to be greater than that for liver inhibitors (compare Tables I and II). However, this may be misleading, as these values are based on quantitation of inhibitory activity in the cytosolic fraction, i,e., a fraction c o n t a m i n a t e d by cysteine proteinases released during tissue homogenization. The criteria we used to establish purity were S D S - P A G E , IEF and analytical chromatofocusing; these indicated that each isoform of stefin A and stefin B from liver and sarcoma had been purified to homogeneity. Stefin A or stefin B ran as a single b a n d on S D S - P A G E under reducing conditions, even when the gels were overloaded (Fig. 4B). This was ~he case for stefins A and B isolated by chromatoiocusing (Fig. 4B) or by anion-exchange chromatog. raphy (data not shown). Inhibitors purified by the latter technique were analyzed by chromatofoeusing to insure that each isoform eluted as a single peak at the appropriate pH. In b o t h liver and sarcoma, stefin B was present as two isoforms of p l 6.0 and 6.5 (Fig. 4C) as has been observed in other tissues [12,13,27]. Two isoforms of stefin A of p l 4.8 and 4.6 were f o u n d , , ,e 4.6 form being prevalent in b o t h tissues (Fig. 4C). Similar isoforms are found in h u m a n leukocytes [26]. Inhibitory properties of liver and saro ,ma stalin A a ~d stefin B Since the affinity extract from sarcoma containing both stefins A and B exhibited a redueea ability to inhibit cathepsin 13 (Fig. 1), we tested the ability of purified stefin A and stefin B to inhibit eathepsin B (Fig. 5). Molar concentrations of stefins A and B were

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4

3

Fig. 4. SOS-PAGE and lEE of human liver and sarcoma low molecular mass CPls. Electrophoresis (A and B) was performed according to Laemmli on 15% gels under reducing conditions [25]. 20-70 #g of protein was applied and stained with Coomassie brilliant blue. IEF (C) was performed as described in Materials and Methods. (A) Lanes represent: sarcoma acetone fraction before affinity chromatography (1), the same sample after affinity chromatography (2) and a liver sample after affinity chromatography (3). (B) Lanes represent: standard protein mixture (1). I;ver stefin A (2), sarcoma stefin B (3) and sarcoma stefin A (4). Stefins A and B were isolated by chromatofocusing and applied after the Polybu.fers were removed by phenyl-Sepharose chromatography as described in Materials and Methods. (C) Lanes represent: st mdard protein mixture (1 and 4). sarcoma stefin B (2) and sarcoma stefin A (3). Lanes 1 and 2 were stained with Coomassie blue; 3 and 4 were silver-stained.

d , " " - r n i n e d b y t i t r a t i o n a g a i n s t p a p a i n . T h e ID50 v a l u e s t, ,,ahibition of p u r i f i e d h u m a n l i v e r c a t h e p s i n B b y human liver stefin B and human sarcoma stefin B were c o m p a r a b l e (Fig. hA: 25 a n d 30 n M , r e s p e c t i v e l y ) . I n c o n t r a s t t h e 1I:)50 v a l u e s for i n h i b i t i o n o f p u r i f i e d h u m a n liver c a t h e p s i n B b y h u m a n l i v e r s t e f i n A a n d h u m a n s a r c o m a s t e f i n A w e r e a n o r d e r of m a g n i t u d e d i f f e r e n t (Fig. 5B: 2 a n d 20 n M , r e s p e c t i v e l y ) . T h e s e

1.0

r e s u l t s s u g g e s t e d t h a t t h e a b i l i t y o f s a r c o m a s t e f i n A to regulate cathepsin B activity was reduced. T h e r e f o r e w e f u r t h e r a n a l y z e d the i n h i b i t o r y p r o p e r ties o f p u r i f i e d s t e f i n B a n d s t e f i n A f r o m l i v e r a n d s a r c o m a b y d e t e r m i n i n g the k i n e t i c c o n s t a n t s for i n h i b i tion of two cysteine proteinases, papain and cathepsin B ( T a b l e II). T h e k i n e t i c c o n s t a n t s d e t e r m i n e d w e r e a p parent kinetic constants, since they were not corrected

1.0.

l,.O

0.8 ~

- 30 '

O8

0.6

~o2o

00.6

>- 0./~

10

0.2

0

0

50

100 150 [Inh,bitor] [riM]

200

2.0

t.5

7

50

tOO

t50

~1.o

>Of, 0.2 ~k

0.5 0

[~or.~b~to,-]pM]

0

I0 20 [Inhibitor] [riM]

I0

20

[,,,h~b;to,']CnM]

30

Fig. 5. Inhibitory activity of human li~er and sarcoma stefins B and stefins A against human liver cathepsin B. Purified stefins B and A were added in increasing concentrations to cathepsin B. The rnolarity of tile purified inVdbitois was established by microscale titration with active site-titrated papain as described in Materials and Methods. CatheF=in B activity was measureg in a continuous ,quorimetric assay using Z-Arg-Arg-AMC as substrate. ~,. liver inhibitors; t,, sarcoma inhibitors. Vo, enzyme activity in the absence of inhibitor; Yi; enzyme activity in tile presence of inhibitor; Ill, inhibitor concentration. The slope of the right-hand graph is equal to the reciprocal of the apparent K i (A) b'h,~_apparent K, values for inhibition of cathepsin B by liver and sarcoma stefins B were 25 and 32 riM. respectively. (B) The apparent K values i'or inhibition of cathepsin B by liver and sarcoma stefins A were 2.4 and 21 riM. respectively.

for possible competition with the substrate for b i n d i n g to papain or cathepsin B. M o l a r c o n c e n t r a t i o n s of the inhibitors were d e t e r m i n e d against active site-titrated papain. Prior to kinetic analysis, we r e c h r o m a t o g r a p h e d ,~ingle isoforms of stefins A or B to insure their purity. O u r kinetic analyses of liver stefins A a a d B confirmed the findings of others [15,16] that liver stefins A a n d B differ in their inhibitory activities towards p a p a i n a n d cathepsin B (Table II) a n d that the a p p a r e n t inhibition c o n s t a n t s for inhibition of papa_in a n d c a t h e p s i n B by liver stcfin B were greater t h a n for liver stefin A [15]. ]'he a p p a r e n t K~ values for inhibition of p a p a i n or o f c a t h e p s i n B by liver stefin B a n d s a r c o m a stefin B were similar (Table II). However, the K~ values foi inhibition of p a p a i n a n d h u m a n c a t h e p s i n B by s a r c o m a stefin A were greater (an order of m a g n i t u d e greater in the case of liver c a t h e p s i n B) t h a n the K i values for inhibition by liver stefin A (Table II). T h e values of the a p p a r e n t rate c o n s t a n t s for the association a n d dissociation of stefins A a n d B a n d either p a p a i n or c a t h e p s i n B were similar to those reported for the association a n d dissociation of leupeptin a n d cathepsin B, suggesting that the stefins like leupeptin are slow, tight-binding inhibitors [1 ! ]. T h e interaction betwecn ~arcoma stefin A a n d liver c a t h e p s i n B was characterized by a decreased rate of association (3-fold) a n d a n increased rate of dissociatitan (3-fold) w h e n c o m p a r e d to the interaction between liver stefin A a n d liver cathepsin B. T h u s the 19-fold

FABLE 111 Inhibition constants for the interactions of stefins A purified from human ~rit,er and sarcoma with human lioer cathepsin L and cathepsin H or routine tumor fractions containing cathepsin B

PM, plasma membrane fraction; LYS, lysosomal fraction. For details see legend to Table II. Enzyme Cathepsin L Catbepsin H PM cathepsin B LYS cathepsin B

Liver stefin A K i (nM) 5.0 0.4 2.0 1.0

Sarcoma stefin A K, (nM) 11.0 1.2 20.0 23.0

c h a n g e in the Ki o f s a r c o m a stefin A for liver c a t h e p s i n B s e e m s ~_o reflect bolh a decreased association of inhibitor a n d e n z y m e a n d a n increased dissociation of inhibitor f r o m i n h i b i t o r - e n z y m e complexes. T h e red u c e d ability of s a r c o m a stefin A to inhibit p a p a i n a n d h u m a n c a t h e p s i n B was n o t restricted to these cysteine proteinases. A p p a r e n t Ki values for inhibition of h u m a n liver c a t h e p s i n L a n d c a t h e p s i n H or c a t h e p s i n B in subcellular fractions of m u r i n e t u m o r s were 2 - 2 3 - f o l d greater for s a r c o m a stefin A t h a n for liver stefin A (TaMe III). T h e s e differences in kinetic c o n s t a n t s for the inhibition of cysteine proteinases by s a r c o m a stefin A suggest that stefin A m a y be responsible for the reduced inhibitory activity we observed with an affinity extract of s a r c o m a C P I s (see Fig. 1). F l u o r e s c e n c e s p e c t r a o f stefins A

TABLE I1 Inhibition and rate constants for the interactions of stefins A and B purtfied from human liver and sarcoma with papain (A). human hcer cathepsin B (B) and humcn breast tumor cathepsin B (C)

~pparent K i and k d values ,~',-'redetermined from continuous rate assays at 25 °C as described in Materials and Methods. The k a values were calculated from the relationship ka= k d / K i. n.d., not determined. Inhibitor concentrations for the calculations were based on their active concentration as determined by titration against papain (itself titrated against E-6¢). Inhibitor (A) Papain: Liver stefin A Liver stefin B Sarcoma stefin A Sarcoma stefin B

K~ (nM) 0.027 0.128 0.110 0.163

k~ (M-t.s -1 )

k d (s -l)

n.d. 4.4-106 2.6-106 3.0.106

n.d. 5.6-10- a 2.9.10-4 3.9-10 -4

1.3-105 1.1.105 0.4.105 0.9.105

3.1-10 -a 21.0-10 -4 10.0.10- a 9.0.10 -4

(C) Human carcinoma cathepsin B: Liver stefin A 10.9 0.3.105 Liver stcfin B 31.2 0.2.105 Sarcoma stefin A 19.2 n.d. Sarcoma stefin B 15.3 n.d.

3.7.10-4 7.3.10- 4 n.d. n.d.

(B) Human liver cathepsin B: Liver stefin A 2.4 Liver stefin B 19.1 Sarcoma stefin A 25.0 Sarcoma stefin B 10.0

T h e increased K~ a n d k j a n d the reduced k a for s a r c o m a stefin A could indicate that this inhibitor m a y differ from liver stefin A in structure. C h a n g e s in the s t r u c t u r e of m e : a b e r s of the c y s t a t m s u p e r f a m i l y have been p o s t u l a t e d in m u s c u l a r d y s t r o p h y a n d c o n f i r m e d :n amyioidosis t301. T h e fiuoresccnce spectra of hver stefin A a n d s a r c o m a stefin A were m e a s u r e d in order to assess possible c o n f o r m a t i o n a l differences. Spectra of liver a n d s a r c o m a stefins A that were isolated by chrom a t o f o c u s i n g are s h o w n in Fig. 6. T h e s e spectra were identical to those of stefins A isolated by anion-exc h a n g e c h r o m a t o g r a p h y , c o n f i r m i n g that the two m e t h o d s of isolation were c o m p a r a b l e (data not shown). M a x i m u m fluorescence at wavelength 302-305 n m was observed w h e n the protein was excited at 280 nm, whereas n o distinct fluorescence s p e c t r u m was observed w h e n only T r p was excited (300 nm). T h i s c a n be explained by the lack of T r p in this family of inhibitors a n d similar to the observed fluorescence properties of low molecular m a s s C P I s from n e w b o r n rat epidermis [31] w h i c h are closely related to h u m a n stefin A [26]. T h e s p e c t r u m of s a r c o m a stefin A differed f r o m liver stefin A in that the m ~ x i m u m fluorescence peak was b r o a d e r a n d shifted to 310 n m . A red-shift a n d a decrease in fluorescence intensity of a protein s p e c t r u m

2o

.~o

i

300

i

i

i

i

320

340

360

380

WAVELENGTH (nm)

Fig. 6. Fluorescence spectra of stefins A from human liver and sarcoma. Excitation wavelengthof 280 rim: , liver stefin A; . . . . . . , sarcoma stefin A. Excitationwavelengthof 300 nm: . . . . . . , liver and sarcoma stefins A; no spectra were observed at the latter wavelength.

might indicate a difference in conformation, possibly as a result of differences in primary structure. Discussion Many investigators have hypothesized that cysteine proteinases play a role(s) in tumor malignancy (see Refs. 4-7 for review). However, few have focused on the role(s) of the endogenous inhibitors of cysteine proteinases. Partial purification of low molecular mass CPIs has been achieved from human melanomas [32], human melanoma cell culture media [33], exudates of human pleural and ovarian cancers [34] and human lung cancers [35]. The properties of these partially purified low molecular mass CPIs are similar to those reported for low molecular mass CPIs in other tissues, e.g., inhibition of cysteine proteinases, stability at alkaline pH [12,13]. However, the kinetic properties of tumor low molecular mass CPIs have not been evaluated systematically. Our choice of tissues in this study was dictated by the following considerations. The distributions, but not the properties, of stefin A and stefin B differ from tissue to tissue, with stefin A being more prevalent in epithelial tissues [12]. Thus the nfixed composition of liver (approx. 65% epithelial (hepatocytes) and approx. 30% mesenchymal (Kupffer and endothelial cells)) would enable us to purify sufficieut quantities of stefii~ A and B for comparative studies with tumors of either epithelial or mesenchymal origins. Normal liver also was readily available and, most importantly, a detailed purification and kinetic analysis of stefins A and B from normal human liver had been published [15]. In terms of tumors, human sarcomas were the only tumor

available to us in both quantities and sizes sufficient to enable us to develop optimal procedures for purification of tumor stefins A and B. Since large quantities of stefins A and B were required for the kinetic analyses, we had to limit the present study to stefins A and B from one human tumor, i.e., to the study of stefins A and B in sarcoma, a tumor of mesenchymal origin. We have established that low molecular mass CPIs from human sarcoma and colon adenocarcinoma (affinity extract) exhibited a reduced ability to inhibit papain and cathepsin B (liver and tumor). Further analysis indicated that sarcoma stefin A had a reduced ability to inhibit cysteine proteinases (papain, cathepsin B, cathepsin H and cathepsin L). Since stefin A is the most potent intracellular inhibitor of cathepsin B [15,16], a reduced effectiveness of stefin A in tumors might account for the elevated activities of cathepsin B in tumors measured by several investigators including ourselves (see Refs. 4 - 7 for review). However, elevated activities of cathepsin B in metastatic murine tumors also appear to reflect increases in mRNA levels [36]. Although the reduced ability of sarcoma stefin A to regulate cathepsin B activity in vitro would be consistent with an elevation of cathepsin B activity in tumors in vivo, such a prediction does not necessarily follow. At present we do not know the concentrations of stefin A or stefin B in tumors in vivo. The half-life of free cathepsin B in vivo is dependent on both the cor~centration of the inhibitor in vivo and the rates for the association between cathepsin B and the inhibitor (tl/2 = In 2/k,,.[l]) [37]. The k a fcr the association between liver stefin A and cathepsin B was 3-fold greaser than that for sarcoma stefin A and cathepsin B. Therefore if the concentration of stefin A in liver and sarcoma were the same in vivo, one might hypothesize that the half-life of free cathepsin B in sarcoma would be 3-times longer. However, the interaction between cathepsin B and stefin A in vivo may be influenced by many factors. Stefin B, although a less potent inhibitor of cathep~in B than is stefin A [15,16], could be the physiological regulator 6f cathepsin B activity in many tissues due to its presence in higher amounts [38]. In sarcoma where there may be an impaired effectiveness of stefin A, stefin B may be present in sufficient concentration to regulate cathepsin B's activity. In vivo, in addition to the competition between stefin A and stefin B for bir~ding to cathepsin B, there will also be competition between cathepsin B and odaer cysteine proteinases for binding to stefins A and B; two of these cysteine proteinases (cathepsins H and L) bind more tightly to the stefins than does cathepsin B [15]. An additional influence on inhibition in vivo will be the competition between physiological substrates and the stefins for binding to cathepsin B, an interaction that will in turn be influenced by the subcellular localizations of cathepsin B and stefins A and B.

'Defective' forms of cystatins have been observed in two other disease states, amyloidosis [30] and muscular dystrophy [29]. G o p a l a n et al. [29] compared low molecular mass CPIs from skeletal muscle of normal and dystrophic mice and d e m o n s h a t e d that those from dystrophic muscle inhibit papain, cathepsin L and cathepsin H, but not cathepsin B. This is similar to the observation in the present study that sarcoma low molecular mass CPIs were still relatively effective inhibitors of papain, but not of cathepsir. B (see Fig. 1). G o p a l a n et al. [29] speculated that the functional differences in CPIs from normal and dystrophic mu~cic may reflect a change in their tertiary structure due to an a m i n o acid substitution a n d / o r a posttranslationai modification of an amino acid(s). In amyloidosis a single amino acid substitution in cystatin C has been s h o w n to be related to the deposition of cystatin C in vessel walls [30]. O u r finding of an alteration in the fluorescence spectrum of sarcoma stefin A would be consistent with a change in tertiary structure. To date due to the limited availability of the sarcoma stefins we have utilized only non-destructive methods for structural analysis. in our laboratory we have demonstrated that a significant p r o p o r t i o n of cathepsin B activity is associated with a plasma m e m b r a n e fraction of metastatic t u m o r cells [6,8,9]. Localization of cathepsin B at the surface of metastatic t u m o r cells may increase the access of cathepsin B to potential substrates in the extracellular matrix. We have established that t u m o r cathepsii~ B can degrade such extracellular matrix proteins as laminin [!8], fibronect~_n and type IV collagen (Buck, M,R, and Sloane, B.F., unpublished data) in vitro under physiologica~ conditions. Liver stefins can inhibit this degradation in vitro [18], Their ability to do so in vivo may be reduceo if there ts close physical contact between membrane-~ssociated cathepsin B and its substrate(s) el if there is a 'defective' form of stefin A in tumors. Ac "knowledgements This work was supported in part by G r a n t s CA 36481 (B.F.S.) and CA 482":0 (B.F.S.) from the Department of Health, Education and Welfare and a grant from H a r p e r / G r a c e Hospitals (K.V.H. and B.F.S.). B.F.S. is the recipient of Research Career Development Award CA 00921 from the National Institutes of Health. We thank Mrs. Sharon Hearn for her expert typing, and V. Turk and J. Rozhin for providing the purified h u m a n liver cathcpsin. L and the isolated fractions of plasma membrane-associated and lysosomal cathepsin B from murine B16 mnelanotic melanoma, respectively.

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