272

Biochimica et Biophysica Acta, 497 ( 1 9 7 7 ) 2 7 2 - - 2 7 9 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 2 8 1 8 7

CATHEPSIN D OF MOUSE LEUKEMIA L12010 CELLS U N U S U A L I N T R A C E L L U L A R LOCALIZATION AND BIOCHEMICAL PROPERTIES W I L L I A M E. BOWERS, C A R L F. B E Y E R a n d N A G A S U M I Y A G O *

The Rockefeller University, New York, N.Y. 10021 (U.S.A.) (Received S e p t e m b e r 7 t h , 1 9 7 6 )

Summary

Mouse leukemia L1210 cells contain lysosomes, but cathepsin D, a typical lysosomal enzyme, has an unusual localization. After fractionation of homogenates of L1210 cells by isopycnic density gradient centrifugation, most of the activity for all of the acid hydrolases studied, except cathepsin D, is sedimentable and shows a similar density distribution around a peak having a modal density of 1.16. In contrast, much more of the total activity for cathepsin D is not sedimentable, while the sedimentable activity has a distribution around a peak at a higher density of 1.18. After chromatography on Sephadex G-100 of cell extracts, two molecular weight forms of cathepsin D are found. One has an apparent molecular weight of approx. 45 000, similar to rat liver cathepsin D, while the apparent molecular weight of the second form is approx. 95 000. Both forms are 4--5 times more active than rat liver cathepsin D. The high molecular weight L1210 cathepsin D converts to the low molecular weight form with no loss in activity after treatment with ~-mercaptoethanol. In all respects the unusual intracellular localization and molecular weight forms of cathepsin D in mouse leukemia L1210 cells are similar to the situation found for rat thoracic duct lymphocytes. Introduction Fractionation of homogenates of rat thoracic duct lymphocytes by isopycnic centrifugation led to the unexpected finding that cathepsin D, a typical lysosomal enzyme, does n o t have the same distribution as that of the other acid hydrolases. Most of the cathepsin D activity was n o t sedimentable, in marked * Present address: Division of Physiology and Pathology, National Institute of Radiological S c i e n c e s , Chiba 280, Japan.

273 contrast to the other lysosomal enzymes which show mainly a sedimentable activity that is distributed around a peak having a modal density of 1.18 [1]. Further studies revealed that cathepsin D of rat lymphocytes and lymphoid tissues occurs in two forms, one (L) having about the same molecular weight as t h a t of rat liver cathepsin D (45 000) and the other (H) having a higher molecular weight (95 000). As demonstrated with the irreversibly bound inhibitor, sodium pepstatin, both forms are 4--5 times more active than rat liver cathepsin D, and neither of the two forms is inhibited by an anti-rat liver cathepsin D antiserum [2]. The presence of these enzymes in mouse lymphoid tissues [2] led us to examine mouse leukemia L1210 cells, a clonally derived mononuclear cell line. We report here that L1210 cells also contain an unusual cathepsin D that exists in the same two forms found for rat thoracic duct lymphocytes, and that the intracellular localization of these enzymes also differs from the other acid hydrolases studied. An established cell line bearing these unusual forms of cathepsin D thus provides a useful in vitro system for further studies on these enzymes and their relationship to lysosomal enzymes. Materials and Methods

Tumor cell inoculation and culture. The t u m o r line was passaged by injecting 106 L1210 cells intraperitoneally into female DBA/2 mice (Microbiological Associates, Bethesda, Md.). Ascites fluid was removed under sterile conditions, the cells were sedimented at 375 X gay for 10 min, washed once in Hanks' balanced salt solution, and then either resuspended in the same solution for injection into mice or resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum (Associated Biomedic Systems, Inc., Buffalo, N.Y.) for initiation of cultures. L1210 cells were grown at 37°C in Spinner culture and used for experiments when they reached a density of approx. 1 • 106/ml. Cell counts were made with a Coulter Counter, model B (Coulter Electronics, Inc., Hialeah, Fla.). Homogenization and fractionation o f L1210 cells. L1210 cells removed from culture were washed three times in Hanks' balanced salt solution at room temperature and finally resuspended in 5 ml of ice-cold 0.15 M KC1. They were vigorously homogenized at 0°C in a Dounce homogenizer (Kontes Glass Co., Vineland, N.J.) with 40 up-and-down strokes. For fractionation studies, the resulting homogenate was centrifuged at 650 X gav for 10 min at 4 ° C, the supern a t a n t removed, and the pellet suspended in 5 ml of 0.15 M KC1 and rehomogenized according to the above procedure. After a second centrifugation at 650 X gav for 10 min, the supernatants were combined, and the pellet again homogenized according to the above procedure. After a third centrifugation at 650 X g~v for 10 min, the supernatants were combined to form the postnuclear extract, and the remaining nuclear pellet was taken up in ice-cold 0.15 M KC1. In some experiments, the postnuclear extract was fractionated further into high speed pellet and supernatant by centrifugation at 100 000 X g~v for 30 min. The pellet was resuspended in ice-cold 0.15 M KC1. Fractionation of the postnuclear extract by isopycnic density gradient centrifugation was performed with the automatic zonal rotor designed by Beaufay

274 [3,4]. 12 ml of postnuclear extract were layered over 24 ml of a sucrose gradient extending linearly with respect to volume between density limits of 1.10 and 1.20. The gradient rested on a 6 ml cushion formed by a sucrose solution with a density of 1.25. All sucrose solutions contained 0.15 M KC1. All operations and calculations were performed according to previously published detailed descriptions [ 1,4,5]. Enzyme assays. All enzyme assays were performed as described previously [ 6 ] . In the assay for cathepsin D, the trichloroacetic acid-soluble products of proteolysis were measured either by the fluorescamine method [7] as described by Yago and Bowers [2] or by the automated Lowry method described by Leighton et al. [4]. Enzyme activities are expressed in units, 1 unit being the amount of enzyme needed to hydrolyze 1 pmol of substrate per min under the assay conditions, except for cathepsin D activity, which is given either as fluorescence equal to that of 1 pmol of leucylleucine released per min or as the chromogenic equivalence of 1 mg of bovine serum albumin released per min. A unit obtained by the fluorescamine method is converted to the corresponding unit determined by the Lowry method by multiplying it by 2.1. Enzyme extraction and Sephadex column chromatography. L1210 cells taken up in 0.1% Triton X-100 were homogenized in a Potter-Elvehjem homogenizer (A.H. Thomas Co., Philadelphia, Pa.) and then centrifuged at 100 000 X gay for 30 min. The pellet was resuspended in 0.1% Triton X-100, rehomogenized, and centrifuged as above. The combined supernatants, containing 99% of the total cathepsin D activity, were applied directly to the Sephadex G-100 column. Chromatography was performed as described previously [2]. Inhibition of cathepsin D by sodium pepstatin. Tubes containing 10--30 munits of cathepsin D were preincubated in duplicate with varying amounts of sodium pepstatin (0.5--2 ng) for 5 min at 37°C in a volume of 0.4 ml containing 0.1 M lactate buffer, pH 3.6. To each tube was added 1.6 ml of substrate mixture. After incubation for 20 min, the reaction was stopped by the addition of 4.0 ml of ice-cold 4.5% trichloroacetic acid, and the proteolytic products in the trichloroacetic acid supernatant determined by the fluorescamine method [7,2]. The degree of inhibition by pepstatin was determined from the initial slope of the straight line and expressed as munits of enzyme inhibited by I ng of pepstatin. Materials. All substrates were purchased from Sigma (St. Louis, Mo.): ~-glycerophosphate (Grade I), hemoglobin (Bovine, Type I, twice crystallized), nitrocatechol sulfate (dipotassium salt), p-nitrophenyl-N-acetyl-~-D-glucosaminide (Grade III), p-nitrophenyl-a-mannoside, and phenolphthalein ~-glucuronide (free acid). Sodium pepstatin was generously provided by Dr. Arthur M. Dannenberg, Jr. (Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, Md.) and by Professor H. Umezawa (Institute of Microbial Chemistry, Tokyo, Japan). Results

Fig. 1 presents the distribution obtained for cathepsin D activity after chromatography on Sephadex G-100 of a high speed extract of mouse leukemia L1210 cells. The distribution is bimodal, and after repeated Sephadex G-100

275

50 S

L

enzyme

J= 3 20

E

A c .9 ~6 4 0 o

50 E

c

o o c

10

~-

0

-E

25

50

55

40 Fr(:]c t ion

45

number

50

55

I0

0

0.5

1.0

1.5

20

Sodium pepstotin (ng/tube)

Fig. 1. C h r o m a t o g r a p h i c b e h a v i o r o f c a t h e p s i n D f r o m m o u s e l e u k e m i a L 1 2 1 0 cells. 9 8 . 6 % o f t h e t o t a l e a t h e p s i n D a c t i v i t y w a s r e c o v e r e d in the high s p e e d e x t r a c t , w h i c h w a s applied t o a S e p h a d e x G - 1 0 0 c o l u m n and e l u t e d w i t h 20 m M s o d i u m - p o t a s s i u m p h o s p h a t e b u f f e r (0.1 M, p H 6 . 9 ) , as d e s c r i b e d previo u s l y [ 2 ] . 2-ml f r a c t i o n s w e r e c o l l e c t e d e v e r y 2 0 rain. R e c o v e r y f r o m t h e c o l u m n w a s 9 4 . 5 % . T h e a r r o w i n d i c a t e s the p o s i t i o n o f t h e void v o l u m e . Fig. 2. E f f e c t o f p e p s t a t i n o n p r e p a r a t i o n s o f c a t h e p s i n D f r o m m o u s e l e u k e m i a L 1 2 1 0 cells a n d f r o m rat liver. I n h i b i t i o n b y p e p s t a t i n w a s p e r f o r m e d a c c o r d i n g to Materials and M e t h o d s . S y m b o l s in t h e figure are: e , rat liver c a t h e p s i n D ( 3 2 0 0 m u n i t s / m g p r o t e i n ) ; o, l o w m o l e c u l a r w e i g h t (L) c a t h e p s i n D ( 1 7 8 m u n i t s / m g p r o t e i n ) o f L 1 2 1 0 cells; A, high m o l e c u l a r w e i g h t (H) c a t h e p s i n D ( 4 9 9 m u n i t s / m g p r o t e i n ) o f L 1 2 1 0 cells; o, high s p e e d e x t r a c t o f L 1 2 1 0 cells ( 3 2 m u n i t s ] m g p r o t e i n ) . R a t liver c a t h e p s i n D w a s partially p u r i f i e d a c c o r d i n g to the d e s c r i p t i o n o f Y a g o and B o w e r s [ 2 ] . T h e l o w a n d high m o l e c u l a r w e i g h t e n z y m e s o f L 1 2 1 0 cells w e r e partially purified b y r e p e a t e d ( t h r e e t i m e s ) S e p h a d e x c h r o m a t o g r a p h y . C a t h e p s i n D activities w e r e d e t e r m i n e d b y t h e f l u o r e s c a m i n e m e t h o d [ 2 , 7 ] .

chromatography of the pooled fractions surrounding each of the two peaks, two distinct enzymes having apparent molecular weights of approx. 45 000 and 95 000 were obtained. Fig. 2 shows the sensitivity of these two partially purified enzymes to inhibition by sodium pepstatin, a potent inhibitor of pepsin and cathepsin D produced by Streptomyces argenteolus var. Toyokaensis, an Actinomycete [8,9]. Both mouse leukemia L1210 enzymes are considerably more sensitive to inhibition by pepstatin (approx. 5 munits of cathepsin D inhibited per ng of sodium pepstatin) than is rat liver cathepsin D (approx. 1 munit inhibited per ng pepstatin). In addition, the high molecular weight form found in mouse leukemia L1210 cells converts to the low molecular weight form without any loss in enzyme activity after treatment with 40 mM fl-mercaptoethanol for 30 min at 37°C. In all these respects, therefore, the two forms of cathepsin D present in mouse leukemia L1210 cells are identical to those detected in rat lymphocytes [2]. Homogenates of mouse leukemia L1210 cells were then fractionated by differential and isopycnic density gradient centrifugation, and the distribution of various enzymes was determined. In addition to cathepsin D a number of other acid hydrolases were also measured, and the total activity of these enzymes in homogenates of L1210 cells is given in Table I. Specific activities are nearly the

276 TABLE

I

ACID HYDROLASE

ACTIVITIES

IN MOUSE

LEUKEMIA

L1210

CELLS

V a l u e s given are m e a n s ± S . D . C a t h e p s i n D w a s assayed b y m e a n s o f t h e a u t o m a t e d L o w r y m e t h o d [ 4 ] . Enzyme

Nos. of determinations

munits/109 /

Acid phosphatase ~-Mannosidase A r y l sulfatase ~-Glucuronidase N-Acetyl-~-glucosaminidase Cathepsin D

5 6 9 5 9 6

81 33 193 74 569 7450

Protein

3

cells

+ 6 ± 16 ± 69 + 26 ± 191 ± 970

101 +

1 7 ( m g / l O 9 cells)

same as those found for rat thoracic duct lymphocytes [1]. Fractionation of L1210 cell homogenates by differential centrifugation into nuclear, high speed pellet and high speed supernatant fractions yielded the results presented in Table II. All of the enzymes contribute less than 10% of their total activity to the nuclear fraction. Cathepsin D, however, differs markedly from the other acid hydrolases in its relative contribution to the two remaining fractions. Nearly three-fifths of its total activity is recovered in the high speed supernatant fraction, whereas the other acid hydrolases are associated almost entirely with the high speed pellet fraction. Fig. 3 presents the distributions obtained after isopycnic density gradient centrifugation of a postnuclear extract of L1210 cells. They show, in confirmation of the results found after differential centrifugation, that most of the total activity for all of the acid hydrolases, except cathepsin D, is sedimentable and has a broad distribution with a peak around a modal density of 1.16. The sedimentable activity of cathepsin D also shows a broad distribution, but the peak occurs at a modal density of 1.18. In addition, much more of the cathepsin D activity is soluble and not sedimentable than is the case for the other acid hydrolases. TABLE

II

FRACTIONATION CENTRIFUGATION

OF HOMOGENATES

OF MOUSE

LEUKEMIA

L1210

CELLS

BY DIFFERENTIAL

F o r p r e p a r a t i o n o f t h e f r a c t i o n s , see Materials and M e t h o d s . V a l u e s are m e a n s ± S . D . o b t a i n e d f r o m t w o f r a c t i o n a t i o n s , e x c e p t for N - a c e t y l - ~ - g l u c o s a m i n i d a s e ( t h r e e f r a c t i o n a t i o n s ) ; t h e y w e r e c a l c u l a t e d as the p e r c e n t c o n t r i b u t i o n o f e a c h f r a c t i o n t o t h e Sum o f t h e t h r e e f r a c t i o n s . R e c o v e r y o f t h e t h r e e f r a c t i o n s relative t o the u n ~ a c t i o n a t e d h o m o g e n a t e ( d e t e r m i n e d for o n e ITactionation) was: acid p h o s p h a t a s e , 8 7 . 9 % ; aryl sulfatase, 9 1 . 1 % ; N - a c e t y l - ~ - g l u c o s a m i n i d a s e , 9 2 . 4 % ; and c a t h e p s i n D , 1 0 3 . 5 % . Fraction

Nuclear High s p e e d p e l l e t High s p e e d supernatant

Percent of total activity of Acid phosphatase

~.rylsulfatase

N-Acetyl-~glucosaminidase

Cathepsin D

5.4 + 0.8 85.6 + 7.3

5.4 ± 0.9 80.7 ± 10.2

4 . 6 -+ 3 . 8 84.7 ± 5.4

10.0 ± 3.5 32.4 ~ 3.5

13.9 ±

10.7 ± 1.9

57.6 ± 0.0

9.0 ± 8.0

9.3

277

lOICathepsinD (4)

Acid phosphatase(1)

/~- Glucuronidase(3) tJ _

c

N-Acetyl-,~- Glucosaminidose(4)

oi

i

1.1

L2

Equilibrium

11

12

density

Fig. 3. D i s t r i b u t i o n of acid h y d r o l a s e s a f t e r i s o p y c n i c c e n t r i f u g a t i o n in s u c r o s e d e n s i t y g r a d i e n t s o f p o s t n u c l e a r e x t r a c t s o f m o u s e l e u k e m i a L 1 2 1 0 cells. T h e b l o c k w i t h d e n s i t y b e l o w 1 . 1 0 has a n a r b i t r a r y d e n s i t y i n t e r v a l o f 0.1 and r e p r e s e n t s t h e p o s i t i o n o f t h e s a m p l e l a y e r . T h e d o t t e d line i n d i c a t e s t h e h i s t o g r a m o b t a i n e d if e n z y m e a c t i v i t y w e r e u n i f o r m l y d i s t r i b u t e d . T h e h i s t o g r a m s h o w s t h e a v e r a g e o f results w i t h s t a n d a r d d e v i a t i o n , a n d t h e n u m b e r o f e x p e r i m e n t s is given b e t w e e n p a r e n t h e s e s . P e r c e n t r e c o v e r i e s b a s e d o n t h e u n f r a c t i o n a t e d p o s t n u c l e a r e x t r a c t w e r e : c a t h e p s i n D, 8 1 . 5 + 15.6; a r y l s u l f a t a s e , 85.4 + 6.8; N - a c e t y l - f l - g l u c o s a m i n i d a s e , 99.7 -+ 1 7 . 2 ; acid p h o s p h a t a s e , 7 6 . 1 ; a n d ~ - g l u c u r o n i d a s e , 8 5 . 4 +- 7.4.

Discussion Earlier fractionation studies on rat thoracic duct l y m p h o c y t e s showed that these cells contain lysosomes and have an unusudl localization for cathepsin D [1]. Our evidence suggested that b o t h occur together in the same cell [10], b u t the possibility could n o t be ruled o u t that cathepsin D belonged to a minor cell population. The results presented in this paper on mouse leukemia L1210 cells clearly indicate that, at least in this case, both lysosomal acid hydrolases and cathepsin D are components of the same cell. The sedimentable activity of four acid hydrolases (N-acetyl-~-glucosaminidase, aryl sulfatase, ~-glucuronidase, and acid phosphatase) has a similar distribution after isopycnic centrifugation of a postnuclear extract of L1210 cells (Fig. 3), which indicates that lysosomes are present in these cells, thus confirming the report of Rundell et al. [ 11]. Sedimentable cathepsin D activity has a distribution overlapping that of the other acid hydrolases, but its peak occurs at a higher density. The question remains open as to whether the sedimentable cathepsin D activity is associated to a greater extent with dense members of the

278 lysosomal population or whether it constitutes a separate, distinct population. The question of the intracellular localization of the soluble cathepsin D recovered after isopycnic centrifugation also remains unanswered, but if it exists entirely inside particles, as seems probable, then we are dealing with an interesting example of a cell that possesses acid hydrolases in two different kinds of lysosomal populations. Relatively few such cases have been described, b u t one other example, that of Tetrahymena pyriformis, is of interest because this organism utilizes the acid hydrolases o f one population o f lysosomes for intracellular digestion and secretes the acid hydrolases belonging to the second population [12], Although secretion of acid hydrolases by mouse leukemia L1210 cells was n o t studied, the release of lysosomal enzymes by this process or by some other mechanism may have important physiological consequences. In this regard it is of great interest that Greenbaum and his co-workers [13-15] have found that cathepsin D of mouse leukemia L1210 cells is capable of forming leukokinins, p o t e n t vasoactive peptides, from a kininogen present in plasma and that the injection of pepstatin, a specific inhibitor of cathepsin D, into mice carrying L1210 leukemia cells brings about a significant reduction in the volume of ascites fluid. These results are especially intriguing in view of the fact that L1210 cells contain highly pepstatin-sensitive high (H) and low (L) molecular weight forms of cathepsin D. It should be stressed that, since sodium pepstatin binds to cathepsin D in an equimolar ratio [9], the steeper titration curves found for the H and L forms of L1210 cathepsin D indicate that these enzymes, under the conditions of the assay, are 4--5 times more active that rat liver cathepsin D (Fig. 2). Since the H form can be converted to the L form without any loss in enzyme activity after treatment with ~-mercaptoethanol, it is important to establish whether the H form is a dimer of L form or whether a precursorp r o d u c t relationship exists between these two enzymes. The existence of these enzymes in an established cell line should facilitate such studies. Acknowledgements It is a pleasure to thank Dr. Mikl6s Mfiller for a critical reading of the manuscript, Ms. Kathy Kohn and Mr. Yi-Sheng John Lin for their excellent technical assistance, and Mrs. Anna Polowetzky for her superb typing. Supported by U.S.P.H.S. Grants HD-05065 and CA-16875, N.S.F. Grant P2B1759, and by Grant IM-67 from the American Cancer Society. References 1 Bowers, W.E. (1972) J. Exp. Med. 136, 1394--1403 2 Yago, N. and Bowers, W.E. (1975) J. Biol. Chem. 250, 4 7 4 9 - - 4 7 5 4 3 Beaufay, H. (1966) La centrifugation en gradient de densitY. Th~se d'Agr~gation de l ' E n s e i g n e m e n t Sup~rieu~, Universitd Catholique de Louvain, Louvain, Belgium, Ceuterick, S.A., Louvain, Belgium 4 Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Coffey, J.W., Fowler, S. and de Duve, C. (1968) J. Cell Biol. 3 7 , 4 8 2 - - 5 1 3 5 Beaufay, H., Bendal], D.S., Baudhuin, P., Wattiaux, R. and de Duve, C. (1959) Biochem. J. 7 3 , 6 2 8 - 637 6 Bowers, W.E., Fin kenstaedt, J.T. and de Duve, C. (1967) J. Cell Biol. 32, 325--337 7 Schwabe, C. ( ! 9 7 3 ) Anal. Biochem. 53, 484--490

279 8 U m e z a w a , H., A o y a g i , T., M o r i s h i m a , H . , M a t s u z a k i , M., H a m a d a , M. a n d T a k e u c h i , T. ( 1 9 7 0 ) J . Antibiot. Tokyo 23,259--262 9 Barrett, A.J. and Dingle, J.T. (1972) Biochem. J. 127,439--441 1 0 B o w e r s , W . E . ( 1 9 7 3 ) J. Cell Biol. 5 9 , 1 7 7 - - 1 8 4 11 R u n d e i l , J . O . , S a t o , T., U e d a , H . a n d B r a n d e s , D. ( 1 9 7 4 ) L a b . I n v e s t . 3 0 , 2 7 9 - - 2 8 5 12 M/iller, M. ( 1 9 7 2 ) J. Cell Biol. 5 2 , 4 7 8 - - 4 8 7 1 3 G r e e n b a u m , L.M. ( 1 9 7 2 ) A m . J. P a t h o l . 6 8 , 6 1 3 - - 6 2 3 1 4 G r e e n b a u m , L . M . , F r e e r , R . , C h a n g , J., S e m e n t e , G. a n d Y a m a f u g i , K . ( 1 9 6 9 ) Br. J . P h a r m a c o l . 3 6 , 623--634 1 5 G r e e n b a u m , L . M . , G r e b o w , P., J o h n s t o n , M., P r a k a s h , A. a n d S e m e n t e , G. ( 1 9 7 5 ) C a n c e r R e s . 3 5 , 706--710