Purification of Plasmodium lophurae cathepsin D and its effects on erythrocyte membrane proteins

Molecular and Biochemical Parasitology, 8 (1983) 207-226

207

Elsevier

PURIFICATION OF PLASMODIUMLOPHURAE CATHEPSIN D AND ITS EFFECTS ON ERYTHROCYTE MEMBRANE PROTEINS

IRWIN W. S H E R M A N and L I N D A T A N I G O S H I

Department of Biology, University of California, Riverside, CA 92521, U.S.A. (Received 6 December 1983; accepted 17 January 1983)

The cathepsin D of Plasmodium lophurae was purified using a combination of CM-Sephadex, pepstatinagarose and Sephadex G-100 chromatography. The plasmodial enzyme was distinct from that of the host red cell and bovine spleen in its low isoelectric point (pl 4.3). The cathepsin D of P. lophurae, as well as p[asmodial extracts demonstrating such proteinase activity, were able to digest the membrane proteins of duckling and h u m a n red cells at pH 7.4; proteolysis was not inhibited in phosphate-buffered saline by 100 p_M pepstatin. Membrane proteins most susceptible to proteolysis were those of the cytoskeleton, notably bands 1 and 2 (spectrin), bands 2.1-2.6 (spectrin-binding proteins) and band 3. Membrane protein degradation by crude plasmodial extracts was partially inhibited by a combination of 10 mM FeCI3, and 10 mM phenylmethylsulfonyl fluoride in phosphate-buffered saline. The changes induced in erythrocyte membrane proteins by exposure to plasmodial cathepsin D parallel the alterations observed in red cell membranes obtained from malaria infected cells. Since the action of the plasmodial protease was confined to the inner surface of the red cell membrane, it is possible that protease-induced modifications in the red cell cytoskeleton could lead to merozoite release. Key words: Plasmodium lophurae; Malaria; Erythrocyte membranes; Cathepsin D; Pepstatin; Acid protease

INTRODUCTION

Intraerythrocytic malarial parasites ingest hemoglobin through a specialized organelle, the cytostome, and then digest it in food vacuoles pinched offfrom the base of the cytostome [ 1]. By this feeding process the intracellular plasmodium obtains amino acids for protein synthesis and leaves as a residue the pigment characteristic of malaria, hemozoin [2]. To degrade host cell proteins, notably hemoglobin, malarial parasites must possess proteolytic enzymes, and evidence for such proteinases has been provided by a number of investigators [3].

Abbreviations: DAN, diazo-acetyl-DL-norleucine methyl ester; DFP, diisopropylfluorophosphate; DTT, dithiothreitol; E D T A , ethylenediaminetetraacetic acid; IAA, iodoacetamide; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline, pCMB, p-chloromercuribenzoate; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; T L C K , N-c~-tosyl-L-lysine chloromethyl ketone; TPCK, L-tosylamide-2-phenylethyl chloromethyl ketone. 0166-6851/83/$03.00 © 1983 Elsevier Science Publishers B.V.

208 In studies on the isolation and characterization of an acid protease from the avian malarial parasite, Plasmodium lophurae, it was found that this enzyme not only hydrolyzed denatured hemoglobin, but was also capable of degrading membrane proteins of the erythrocyte [3]. In view of the fact that alterations of the host cell membrane in malarial infected red cells have been reported, it seemed of interest to compare the action of the P. Iophurae protease on human and duckling erythrocyte membranes. The present report describes the purification and properties of the P. lophurae cathepsin D and its effects on erythrocyte membrane proteins. MATERIALS AND METHODS

Parasitological. Erythrocyte-free Plasmodium lophurae were prepared by the hemolytic method of Trager [4]. Electron microscopic evaluation of these preparations showed the parasites to be surrounded by a parasitophorous vacuolar membrane, but to be essentially free of host cell membranes. Polyacrylamide gel electrophoresis (PAGE) of parasite extracts gave no evidence of the presence of red cell membranes [5]. Assays. Cathepsin D activity was determined by a radiometric method. 50 I,tl of enzyme was added to 40 l,tl of ~4C-labeled hemoglobin (0.2 laCi [methyl-14C]methemo globin, New England Nuclear, mixed with 2 mg human hemoglobin in 1 ml distilled water), and 10 ~1 of 1.0 M citrate buffer, pH 3.5. Reactants Were placed in a 1.5 ml microfuge tube, with the reaction initiated by the final addition of enzyme. Tubes were incubated at 38°C for 30 rain, and the reaction terminated by addition of 100 ~tl of ice-cold 10% (w/v) trichloroacetlc acid (TCA). Tubes were centrifuged for 4 rain in a Beckman microfuge, 100 ~tl aliquots of the supernatant were added to 10 ml Aquasol II (New England Nuclear), and radioactivity determined by liquid scintillation spectrometry. Protein was determined by the method of Lowry et al. [6] with bovine serum albumin as a standard. Isolation of normal and infected red blood cell membrane vesicles. Membrane vesicles were isolated by Nz cavitation [5]. Polyacrylamide gel electrophoresis (PAGE). Plasma membranes were isolated by N2 cavitation, as well as by Waring blender homogenization (normal ducklings only). Sealed and unsealed human red cell ghosts were prepared by the method of Steck and Kant [7]. Aliquots of the red blood cell membranes, duckling or human, in PBS ( 150 mM NaCI, 10 mM phosphate buffer, pH 7.4) or citrate buffer, pH 3.5, were incubated with protease-containing extracts or purified enzyme in microfuge tubes (see below). The membranes were collected by centrifugation in a Beckman microfuge, washed 3 times in PBS and solubilized [5]. Identical amounts of membrane protein were

209 analyzed by sodium dodecyl sulfate (SDS)-PAGE using a 4.5% stacking gel over an exponential 7.5 to 16% separating gel using the system of Laemmli and Favre [8]. Slab gels were stained with Coomassie blue.

Inhibition of enzyme activity in crude extracts with pepstatin. To determine the inhibitory effects of pepstatin, 10 Ixl of pepstatin (dissolved initially in 0.02 N NaOH) with final concentrations of ( 1 0 TM - 1 0 -17 M) were preincubated with crude enzyme for 30 min at 38°C before the addition of acid-denatured radioactive methemoglobin. The residual activity was then determined by incubating the mixture for 60 min in the standard protease assay (see above). Inactivation of cathepsin D and activity on membranes. Crude lysates (200 I.tl) of P. lophurae were incubated for 1 h at 38°C with 25 tal 1 M citrate buffer (pH 3.5) or 25 lal PBS with or without 25 lal 1 mM pepstatin. Aliquots of 50 p.1 were removed and assayed for protease activity in the standard manner, and an aliquot of 200 lal was added to 200 ~tl of normal duck red cell membranes and incubated for 2 h at 38°C. Purified cathepsin D ofP. lophurae (200 lal) was preincubated at 38°C for 30 min with 50 lal 1 mM pepstatin in citrate buffer, pH 3.5, the pH was raised to 7.4 by the addition of 0.1 N NaOH and PBS, and then 200 lal of duckling membranes added. Incubation of protease with membranes was carried out at 38°C for 2 h. To determine whether the cathepsin D activity present in crude lysates could be inhibited by compounds other than pepstatin, lysates were preincubated for 30 min with 10 mM FeC12 + 10 mM PMSF in PBS. Red cell membranes were added and incubation was continued for an additional 2 h at 38°C. Membranes were sedimented, washed twice in PBS, and analyzed by SDS-PAGE (see above). Chromatofocusing. Chromatofocusing was carried out using approx. 25 g (wet weight) polybuffer exchanger (PBE) 94, a starting buffer of 0.025 M imidazole-HC1, pH 7.4, and an eluent buffer PB 74, as described by Pharmacia. About 3 ml of crude plasmodial extract were placedon a 1 × 27 cm column. Flow rate of the eluent buffer was 40 ml h -~, and fractions of 3 ml were collected. The absorbance was read at 280 nm, protease activity was assayed (see above), and the pH determined for each fraction. Enzyme purification. Step 1: Preparation of a hemoglobin-free parasite lysate. Packed erythrocyte-free P. lophurae (8.25 ml) was added to an equal volume of distilled water, frozen and thawed twice, then centrifuged (27 000 × g, 15 rain). The supernatant was diluted 1:10 with distilled water and then sufficient 0.5 M phosphate buffer, pH 6.8, was added to bring the final concentration of phosphate to 5 mM. To the lysate was added 30 g (wet weight) of CM-Sephadex C-50 (non-bead form) which had been pre-equilibrated with 5 mM phosphate buffer, pH 6.8. The mixture was stirred for 10 min at 4°C, centrifuged (10 min, 1500 × g) and the supernatant treated with another 30

210 g of pre-equilibrated CM-Sephadex. The CM-Sephadex was centrifuged, washed with 200 ml of the 5 mM buffer, and the hemoglobin-free supernatants were combined and concentrated to 10 ml by ultrafiltration using an Amicon PM10 membrane. Step 2: Pepstatin-agarose affinity chromatography. The concentrated hemoglobinfree solution from Step 1 was dialyzed overnight against 3 changes of 50 m M sodium acetate buffer/1 M NaCI, pH 5.0, centrifuged (15 min, 20 000 Xg), and the supernatant was applied to a 1 × 13 cm column of pepstatin-agarose (Pierce Chemical)which had been pre-equilibrated with the same buffer-saline. Fractions of 2.8 ml were collected at a flow rate of 24 ml h -1. The column was washed with 50 m M sodium acetate/1 M NaC1, p H 5.0, until the absorption at 280 nm was minimal, and the enzyme was eluted with 50 mM Tris-HCl/1 M NaCI, pH 8.6. The fractions containing cathepsin D activity were pooled, and concentrated by ultrafiltration using the PM 10 membrane. Step 3: Sephadex chromatography. Approximately 4 ml of the concentrated eluate from Step 2 were applied to a Sephadex G-100 column (1.5 X 54 cm) previously equilibrated with 50 mM phosphate buffer, pH 7.0, containing 0.2 M NaC1; enzyme activity was eluted with this same buffer at a flow rate of 14 ml h -1, and 2 ml fractions were collected. Fractions containing peak enzyme activity were pooled and concentrated to 2 ml using the PM 10 membrane. The enzyme solution was stored either at -70°C or 4°C, with or without 10% (w/v) sucrose. All solutions contained 0.1% (w/v) sodium azide to prevent microbial growth. RESULTS

Purification. Preliminary attempts at purification of the P. lophurae cathepsin D using affinity chromatography with hemoglobin-Sepharose [9], as well as pepstatin-agarose, were unsuccessful in our hands. Commercially available bovine spleen cathepsin D (Sigma) however, behaved on the pepstastin-agarose column as described by Kreger et al. [10]. The enzyme dissolved in 50 m M sodium acetate buffer, p H 3.5, containing 0.2 M NaCI, was applied to the column, and eluted in a single peak by raising the p H to 8.6 with 50 mM Tris-HC1/0.2 M NaC1. The P. lophurae enzyme, in distinct contrast, bound so tightly to the column at p H 3.5 that it could not be eluted with 50 mM Tris-HC1, pH 8.6, containing either 0.2 M or 1.0 M NaCI. However, if P. Iophurae lysate was applied to the pepstatin-agarose in 50 mM sodium acetate buffer at p H 5.0, containing either 0.2 M or 1.0 M NaC1, it was then possible to elute the enzyme with 50 m M Tris-HC1, p H 8.6, containing 1.0 M NaC1. This tight binding of the parasite enzyme to the pepstatin-agarose suggested that it might be similar to the cathepsin D-I of primate lung tissue in having a very high affinity for pepstatin [11]. The proteolytic activity of both the P. lophurae extract and the spleen cathepsin D toward acid denatured hemoglobin was strongly inhibited by pepstatin (Fig. 1), and the concentration for 50% inhibition of the enzyme activity, under our assay conditions, was similar (approx. 10-7 M). The pattern of pepstatin inhibition of both enzymes with varying

211

100

5C

16 15 14 15 12 II I0 9 -log

8

7

6

5

4

[PEPSTATIN (M)]

Fig. 1. Effect of varying concentrations of pepstatin on the activity of cathepsin D from bovine spleen and the extract ofP. lophurae with cathepsin D activity. (The specific activity of the parasite extract was 3 × 104 dpm released h -~ mg-L) For experimental details see Materials and Methods.

concentrations of inhibitor strongly resembled that of cathepsin D-II, not the highly acidic cathepsin D-I [11]. The reason for the unusual elution pattern of the malarial parasite cathepsin D on pepstatin-agarose using conditions which were effective for the spleen enzyme remains unexplained. Purification of the P. lophurae cathepsin D was achieved by combining CM-Sephadex treatment with affinity chromatography (pepstatin-agarose) and Sephadex G- 100 chromatography. Using these techniques sequentially it was possible to achieve a 57-fold overall purification with 15% recovery (Table I). The enzyme was extremely sensitive to low pH after contaminating hemoglobin had been removed, and it was this lability that precluded further purification with high yield. The purified enzyme had a molecular weight of 32 000 by Sephadex chromatography, appeared to be nearly homogeneous by native PAGE and on SDS-PAGE had a molecular weight of 37 000 (Fig. 2). The enzyme showed greater stability at 4°C than at -70°C; stability at -70°C could be increased by addition of 10% (w/v) sucrose (Table II). The activity of the purified enzyme was linear at protein concentrations below 40 lag ml -~ (Fig. 3). Enzyme activity at pH 3.5 was linear for approximately 90 min of incubation (Fig. 4), however for convenience, the standard assay was conducted for 30 min. The purified enzyme had a pH optimum of 3.5 with human hemoglobin as a substrate when assays were carried out over the pH range 2-11 (Fig. 5). [Sodium citrate buffer was used for determinations in the pH range 2-6.5, sodium phosphate

38.0 4.9

1.7

3. Sephadex chromatography a. After ultrafiltration

15.2 184.0 370.0 11.0 8.6

Volume (ml)

2. Affinity chromatography a. After Pepstatin-agarose b. Alter ultrafiltration

1. Removal of hemoglobin a. Crude lysate diluted 1 : 1 b. Adjust pH to 6.8 c. After CM-Sephadex d. After ultrafiltration e. After dialysis at pH 5

Step

Purification of P. lophurae cathepsin D

TABLE I

0.5

5.6 1.8

163.0 118.0 60.0 47.1 34.3

Total protein (mg)

1.4

2.2 2.6

8.9 7.1 6.6 6.3 3.3

Total activity (dpm X 10TM)

3062

392 1487

54 61 110 133 96

Specific activity (dpm p_g-l)

57.0

7.3 27.5

1.0 1.1 2.0 2.5 1.8

Purification (-fold)

15

25 29

100 80 74 71 37

Recovery %

213 TABLE II Stability of the purified P. lophurae cathepsin D Temp ( ° C ) 4 4 -70 -70 -70 -70

Additions

No. days

% initial activity

lO%sucrose 10%sucrose

13 20 13 20 13 20

76 59 11 6 81 72

buffer for p H 7 to 8, Tris-HC1 for the range 7 to 9 a n d s o d i u m c a r b o n a t e buffer for the p H range 9.5 to 11.]. The purified P. lophurae p r o t e a s e was strongly inhibited b y low c o n c e n t r a t i o n s o f p e p s t a t i n , b y FeC13, a n d b y d i a z o a c e t y l n o r l e u c i n e methyl ester in the presence o f c o p p e r ions (Table III). P h e n y l m e t h y l s u l f o n y i fluoride ( P M S F , 10 m M ) i n h i b i t e d the activity o f the enzyme by 65% whereas 100 m M c h l o r o q u i n e inhibited enzyme activity

Fig. 2. SDS-PAGE of the purified cathepsin D of P. lophurae.

214

4000

3000

0 2000

1000

L 0

I 8

i

1 16 /zg

F i g . 3.

I

I 24

I

I 32

i

PROTE IN

Effect of enzyme concentration on the activity of the purified P. lophurae cathepsin D. Incubation

and assays were carried out as described under Materials and Methods.

by 50%. Cyanide, leupeptin, chymostatin, p-chloromercuribenzoate (pCMB), iodoacetamide (IAA), dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), mercaptoethanol, o-phenanthroline, sodium tetrathionate, and salts of lead, mercury, zinc and magnesium had no effect on enzyme activity. Protease effects on erythrocyte membrane proteins. Duckling erythrocyte membranes, prepared by Waring blender homogenization, were incubated in PBS for 120 min at 38°C in the presence of various dilutions of protease-containing extracts, washed in

3000

•

2000

•

I ooo

,3

5

i0

60

30

90

TqME (MqN)

Fig. 4. Time course of hemoglobin proteolysis by the P. Iophurae cathepsin D. Specific activity of the preparation = 70 × 105 dpm released h-~ mg-L

215

6000

5000

4000

e,_ 3000 ,.m

2000

1000

0

I

I

4

~-~,-.

. . . . . . . -. T . .-

6

8

TI0

?

pH

Fig. 5. Effect of pH on the activity of the purified P. Iophurae protease against hemoglobin. Method of assay is described in Materials and Methods.

PBS, and, when analyzed by SDS-PAGE, clearly showed proteolysis of membrane proteins (Fig. 6). The membrane proteins most susceptible to attack were spectrin (bands 1 and 2), the spectrin-binding proteins (bands 2.1-2.6) and band 3. Proteolysis of these high molecular weight membrane proteins produced an increase in the intensity of staining of approx. 12 bands of ~ 43 kDa. The degradation of membrane proteins was not inhibited by the addition of 100 laM pepstatin to the PBS-containing incubation mixture. This lack of effect was not unexpected since it was reported that above pH 7 the binding site of cathepsin D for pepstatin is abolished, presumably due to a conformational change in the enzyme molecule [12]. It should be noted that even with the extract showing the intermediate cathepsin D activity (lane 3) there was a striking reduction of bands in the region of 200 kDa (spectrin), a replacement of bands between 200 and 117 kDa (commonly referred to as bands 2.1-2.6, the spectrin-binding proteins), the appearance of a doublet moving in advance of band 3 (called 3'), and an intensification of bands migrating in advance of the 43 kDa band. With the extract of the highest activity (lane 2)there was a complete loss of spectrin, bands 2.1-2.6, as well as band 3, and the concomitant appearance of

216 TABLE III Effect of various compounds on the proteolytic enzymeactivity of a purified enzymefrom P.

lopkurae Compound

Final concn.

Percent inhibition

Pepstatin

10 ]aM 100 p_M 10 mM 10 mM 10 mM 10 mM 10 mM 100 pM 10 mM 100 ~tM 100 pM 5 mM 100 laM 10 mM 5 mM 100 mM 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM each

88 96 90 0 5 3 0 0 0 3 10 0 0 65 27 50 0 0 0 4 0 5 0 87

FeCI3 Pb(NO3)2 ZnC12 MgCI2 HgCI2 Leupeptin Na tetrathionate TLCK TPCK pCMB "Chymostatin PMSF Chloroquine KCN Iodoacetamide 2-Mercaptoethanol Dithiothreitol EDTA 1,10-Phenanthr01ine Diazoacetylnorleucine methyl ester ÷ Cu2~

several new b a n d s in the region formerly occupied by b a n d s 2.1-2.6 a n d 3. New b a n d s 43 k D a were also evident. The changes i n d u c e d in duckling erythrocyte m e m b r a n e proteins by exposure to p l a s m o d i a l extracts d e m o n s t r a t i n g cathepsin D activity parallel the changes observed in red cell m e m b r a n e s prepared from P. lophurae-infected erythrocytes (Fig. 7). The lanes labelled N R B C M in Fig. 7 represent m e m b r a n e vesicle p r e p a r a t i o n s from 3 n o r m a l ducklings; the lanes bracketed with the label I R B C M represent 6 m e m b r a n e vesicle p r e p a r a t i o n s from d u c k l i n g red cells parasitized with P. lophurae a n d having parasitemias > 8 0 % . Some, but not all, of the m e m b r a n e s derived from infected red cells showed a d i m i n u t i o n in the staining intensity of spectrin, b a n d s 2.1-2.6 a n d b a n d 3, a n d in those instances where high molecular weight m e m b r a n e proteins showed evidence of d e g r a d a t i o n , there was an intensification as well as the a p p e a r a n c e of new b a n d s r a n g i n g from 94 to 14 kDa. The b a n d at 43 k D a was unaltered by parasitization, a n d this same m e m b r a n e protein was unaffected by protease treatment. W h e n p l a s m o d i a l extracts or purified enzyme were i n c u b a t e d with n o r m a l d u c k l i n g

217

i¸ //!i(ii(!!ii

Fig. 6. Effect of differing cathepsin D activities on proteolysis of normal duckling membranes. (1) Control, duckling membranes incubated in water; (2) duckling membranes incubated in PBS with crude lysate (specific activity = 3 )< 104 dpm released h -~ mg-1; (3) duckling membranes incubated in PBS with crude lysate (specific activity = 3 X 103 dpm released h -j mg-~; (4) duckling membranes incubated in PBS with crude lysate (specific activity = 3 )< 102 dpm released h -~ mg-~; (5) membrane and lysate as in lane 2 plus 100 ~tM pepstatin; (6) membrane and lysate as in lane 3 plus 100 laM pepstatin; (7) membrane and lysate as in lane 4 plus 100 p_M pepstatin.

membranes (prepared by Waring blender homogenization) either in citrate buffer (pH 3.5) or PBS (pH 7.4), it was found that degradation of membrane proteins occurred in both, but proteolysis was dramatically enhanced in citrate buffer (Fig. 8). This suggested that although the P. lophurae cathepsin D had maximal activity against red cell membrane proteins at pH 3.5, considerable activity was also present under more alkaline conditions. This differs from the activity of the crude and purified enzyme at various pH values with hemoglobin as the substrate [3]. The differential susceptibility of proteins from a variety of sources to attack by acid proteinases has been recently reported by Pontremoli et al. [13]. The correlation between cathepsin D activity in extracts of P. lophurae and membrane protein degradation was absolute; when an extract had no cathepsin D activity, no evidence of membrane protein proteolysis was seen (data not shown).

218

Fig. 7. SDS-PAGE patterns of red cell membrane proteins derived from normal duckling erythrocytes (NRBCM) and P. lophurae infected red cells (IRBCM). Identical amounts of membrane protein were tested in each lane. P r e i n c u b a t i o n o f the purified enzyme with 100 JaM p e p s t a t i n at p H 3.5, with s u b s e q u e n t a d d i t i o n o f m e m b r a n e s a n d further i n c u b a t i o n in PBS c o m p l e t e l y prevented m e m b r a n e d e g r a d a t i o n , whereas p e p s t a t i n in PBS h a d no effect on the purified enzyme (Fig. 8). Of c o n s i d e r a b l e interest is the fact that 10 m M FeC13 + 10 m M P M S F r e t a r d e d the d e g r a d a t i o n o f m e m b r a n e proteins, whereas these c o m p o u n d s on their o w n did n o t p r e v e n t p r o t e o l y s i s o f m e m b r a n e proteins. The W a r i n g b l e n d e r m e t h o d o f p r e p a r i n g e r y t h r o c y t e m e m b r a n e s results in m e m b r a n e sheets, b r o k e n ghosts a n d vesicles; therefore, e x p o s u r e o f such a p r e p a r a t i o n to the p r o t e a s e w o u l d allow access to b o t h the inner a n d o u t e r surfaces o f the red b l o o d cell m e m b r a n e . T o delimit the p r o t e o l y t i c a t t a c k to a p a r t i c u l a r m e m b r a n e surface, sealed a n d unsealed h u m a n red cell ghosts were i n c u b a t e d with crude extracts of P.

219

Fig. 8. Inhibition of purified cathepsin D activity (lanes 1, 2, 3, 4 and 5) and inhibition of cathepsin D activity in extracts of erythrocyte-free P. lophurae (lanes 6 and 7). (1) Control, duckling membranes incubated in PBS; (2) purified cathepsin D of P. lophurae (total activity 6 X 103dpm released h-') incubated in PBS with duckling membranes; (3) as in lane 2, but incubated in citrate buffer pH 3.5; (4) as in lane 2, but in the presence of 100 p_Mpepstatin; (5) as in lane 3, but in the presence of 100 laM pepstatin; (6) lysate of P. lophurae (total activity 12 X 104 dpm released h -~) incubated in PBS with duckling membrane; (7) as in lane 6, but in the presence of 10 mM FeCI3 + 10 mM PMSF.

lophurae, with o r w i t h o u t p e p s t a t i n , a n d then a n a l y z e d by S D S - P A G E (Fig. 9). Proteolysis o f m e m b r a n e p r o t e i n s was n o t o b s e r v e d in the sealed ghosts [i.e. c o m p a r e the b a n d i n g o f c o n t r o l s (lanes 8 a n d 9) with the effects seen after treating m e m b r a n e s with 3 p l a s m o d i a l extracts with varying c a t h e p s i n D activity (lanes 10-15)]. On the o t h e r h a n d , u n t r e a t e d , u n s e a l e d ghosts when c o m p a r e d to sealed ghosts showed a loss o f only b a n d s between 4.2 a n d 5 (actin) (lane 1), but u p o n exposure to the extract m a r k e d m e m b r a n e p r o t e i n a l t e r a t i o n s occurred. The d y n a m i c s o f m e m b r a n e p r o t e i n proteolysis m a y be visualized by a c o m p a r i s o n o f the a c t i o n o f crude extracts o f low activity (lanes 2 a n d 3) with those o f greater c a t h e p s i n D activity (lanes 6 a n d 7). In the earliest stages o f m e m b r a n e p r o t e i n d e g r a d a t i o n b a n d 2 splits, b a n d 2.3 is lost, 5 new b a n d s a p p e a r in the region between b a n d s 2.3 a n d 3, b a n d 3 stains less intensely, b a n d s 4.1 a n d 6 are u n c h a n g e d , but b a n d 4.2 splits, new b a n d s a p p e a r in the regions between b a n d s 5 a n d 6, as well as b e l o w

220

Fig. 9. Proteolysis in sealed and unsealed human red cell ghosts in the presence or absence of pepstatin (100 laM). Controls are in lanes 1and 8. Extracts of lowest cathepsin D activity are in lanes 2/3 and 9/10, whereas highest activity is represented in lanes 6/7 and 14/15. b a n d 6. The most dramatic alterations in unsealed ghosts can be seen after exposure to the extract having the highest cathepsin D activity (lanes 6 and 7): bands 1, 2 (spectrin), and 3 are lost, a new b a n d appears in the region ahead of band 3 (band 3'), bands 4.1, 4.2 are degraded, new bands appear in the regions between bands 5 and 6, as well as below it; only b a n d 5 remaines unchanged. The pattern of m e m b r a n e protein degradation evident in the unsealed h u m a n ghosts treated with plasmodial extracts is similar to that described for protease-treated duckling red cell membranes (Fig. 8) as well as that of P. lophurae-infected duckling erythrocyte membranes (Fig. 7). Additionally, the action o f the plasmodial protease appears to be confined to the inner surface of the red cell m e m b r a n e since the banding pattern o f m e m b r a n e proteins of sealed ghosts showed almost no change after exposure to plasmodial extracts (Fig. 9). The elution profiles of the acid proteases of the duckling erythrocyte membrane, bovine spleen and P. lophurae from the c h r o m a t o f o c u s i n g column were distinctly different from each other (Fig. 10). The p I of the spleen enzyme was 7.2, that of the duckling erythrocyte was 6.5, and the pI of the P. lophurae protease was 4.3. DISCUSSION Proteases can be conveniently classified as exopeptidases and endopeptidases [ 14,15]. Endopeptidases are further subdivided according to the chemical nature of the active center. Serine proteinases, active at neutral pH, are inhibited by chymosta-

221

tin, PMSF and DFP. Cysteine proteinases are inhibited by N-ct-tosyl-L-lysine chloromethyl ketone (TLCK), L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), IAA, pCMB, and leupeptin, and usually have a pH optimum o f p H 6.0. Metalloproteinases are inhibited by E D T A and o-phenanthroline. Aspartic proteinases, such as cathepsin D, have molecular weights of 30 000-60 000, are active at acid pH, readily degrade hemoglobin, and are inhibited by pepstatin and diazoacetylnorleucine methyl ester + Cu 2÷, but not KCN [16]. The purified enzyme from P. lophurae is not a metalloproteinase since Mg 2÷, Zn 2+, E D T A or o-phenanthroline had little or no effect on the enzyme. The enzyme is neither a thiol nor a serine proteinase since DTT, mercaptoethanol, iodoacetamide, pCMB, leupeptin and chymostatin were without effect (Table III). The enzyme is most appropriately classified as an aspartic proteinase of the cathepsin D type since pepstatin and diazoacetylnorleucine methyl ester inhibited enzyme activity, but cyanide was without effect. Moulder and Evans [17], Cook et al. [18], Levy et al. [19,20] and Gyang et al. [21] reported that extracts of P. gallinaceum, P. knowlesi, P. berghei and P. falciparum readily hydrolyzed denatured hemoglobin at acid pH. Levy et al. and Gyang et al. also found enzyme activity to be inhibited by pepstatin, suggesting that the parasite proteinase was a cathepsin D. However, such activity was also inhibited to varying degrees by chymostatin and leupeptin, a situation unknown for any single proteinase. Therefore, their descriptions are more compatible with extracts that consist of a mixture of host and parasite enzymes (see below). Of interest is the finding o f G y a n g et al. [21] that the acid proteinase activity was significantly inhibited at pH 3.5 by 33.5 mM chloroquine, an observation similar to that reported herein. The acid proteinase of P. lophurae had a molecular weight of 30-40 000, typical of cathepsins D, whereas Gyang et al. [21] reported a molecular weight of 148 000, a value never recorded for any cathepsin D. The discrepancy remains unexplained. Chan and Lee [22] and Cook et al. [18].using hemoglobin as a substrate, identified alkaline protease activity in extracts of P. knowlesi and P. berghei. Enzyme activity was unaffected by CN, pCMB, cysteine, or chelators, but was inhibited by DFP. Based on this, as well as the fact that the partially purified P. knowlesi enzyme liberated phenylalanine from the 13-chain of insulin and attacked L-tyrosinamide, but not benzoyl-r-arginamide or carbobenzoxyglutamic acid diamide, Cook et al. [18] concluded that the plasmodial enzyme was chymotrypsin-like. It is entirely possible however, that the alkaline protease activity was due to the presence of both nucleases and host proteases. We found no alkaline protease activity with hemoglobin as the substrate nor could we find cathepsin B activity using 13-naphthylamine as a substrate (Sherman, unpublished). In 1953 Morrison and Neurath [23] described an acid proteinase from human erythrocytes. Two decades later Bernacki and Bosmann [24] and Reichelt et al. [25] purified and characterized a red cell enzyme which had the properties o f a cathepsin D. At about this same time T6kes and Chambers [26] reported that red cells contained

222 two kinds of enzymes, one inhibited by pepstatin and the other by DFP. It was assumed by these authors that such cathepsins were associated with the red cell membrane. Since it was not clear from these earlier studies whether leucocyte contamination might affect the findings, Pontremoli et al. [12,27] reinvestigated and characterized the acid proteinases from membranes as well as the cytosol of mature human erythrocytes. The release of proteolytic activity from membranes required solubilization using detergents or butanol, and activity was associated with proteins of molecular weight 80 000, 40 000 and 30 000. The lowest and highest molecular weight forms had a pH optimum of 3.5, whereas the 40 000 molecular weight species had a pH optimum of 2.5. The 40 000 molecular weight species was inhibited by 1 I.tg m1-1 pepstatin whereas the activity of the 80 000 molecular weight proteinase was unaffected by pepstatin. The lowest molecular weight proteinase was sensitive to pepstatin and leupeptin as well as reagents known to inactivate serine proteases. In a subsequent paper these workers purified from freeze-thawed red cells 3 acidic cytosolic proteinases which had molecular weights and pH optima identical to those derived from membranes, however all were inhibited by pepstatin (1 gg ml-l), leupeptin (10 gg ml-l), PMSF (5 mM) and pCMB (1 mM). Pontremoli and coworkers state that 'the properties of these cytosolic proteinases and especially their molecular sizes, substrate specificities and the susceptibility to inhibitors are clearly reminiscent of the picture previously obtained following gel chromatography through the same column of the solubilized membrane from human erythrocytes' yet they mention nothing of the contradictions. Further, the cytosolic acidic endopeptidases were not inhibited by Fe 3÷ or Hg 2÷. When one compares the acid proteinases of human red ceil'membranes with the properties of the P. lophurae cathepsin D [3]: pH optimum 3.5, molecular weight 30-40 000, inhibition by pepstatin and Fe 3÷, but not by leupeptin, the enzymes appear to be distinct from one another. Indeed, a direct comparison of the P. lophurae enzyme with that of the duckling red cell membrane enzyme shows dissimilarity in several respects: (1) the enzyme from erythrocyte membranes is easily obtained by Triton X- 100 lysis, but is more difficult to extract by freezing and thawing, a method used for its extraction from free parasites, and (2) the pH optima are distinct from one another. It might be argued that the P. lophurae cathepsin D is not a plasmodial proteinase, but instead is derived from the membrane of the host red blood cell. If this were true then all of the activity recovered in the free parasites would have to be derived from the invaginated red cell membrane that surrounds the merozoite during invasion, and which represents less than 10% of the total membrane surrounding the parasite at maturity. It seems unlikely that this amount of host-derived membrane could produce the substantial activities recovered in free parasites. Further, there is no evidence of host cell proteins, save for hemoglobin, in the plasmodial extracts. And finally, the pI values of the red cell and plasmodial enzymes are distinct from one another (Fig. 10). Thus, based on quantitative considerations as well as qualitative differences (extractability, isoelectric point, pH optimum) the cathepsin D activity of P. lophurae appears

223

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cell membrane hemoglobinase activity.

to be of parasite origin, and does not represent host cell membrane contaminants. Several workers have studied the endogenous proteolysis of red cell membrane proteins. Murakami et al. [28] extracted with butanol an acid protease from human erythrocyte membranes that had a pH optimum of 3, was sensitive to pepstatin, and in self-digestion experiments conducted at pH 4, degraded spectrin and band 4a. Allen and Cadman [29], Carraway et al. [30], Quirk et al. [31] and Siegel et al. [32] reported on Ca 2÷ induced proteolysis of membranes at physiological pH with the major alterations occurring in the band 2.1 to 2.3 region, decreased staining of bands 3 and 4.1, and the appearance of a new component (3') which was the degradation product of the band 2.1 family of proteins. PMSF was shown to prevent band 2.1 digestion and the formation of 3'; maximum endogenous proteolysis occurred at pH 8.5, and Ca2*-stimulated proteolysis of band 2.1 to produce band 2.3. These changes are mimicked to a large degree by the action of the P. lophurae protease, but the degradation of erythrocyte membrane proteins by endogenous proteases requires 3 to 5 h incubation, a time considerably in excess of that required by plasmodial extracts. Based on the time required for action it seems unlikely that the activity in plasmodial extracts simply represents the activation of an endogenous protease of the erythrocyte.

224

Characteristically, malarial infected erythrocyte m e m b r a n e s show d e g r a d a t i o n of b a n d s 1, 2, a n d 3, increased staining as well as a p p e a r a n c e of new b a n d s < 43 kDa, a n d with some of the rodent malarias, a new b a n d with a molecular weight of 165 000 was seen (reviewed by S h e r m a n [2]). Such changes could be the result of the activation of e n d o g e n o u s proteases of the red cell, or more likely are a direct consequence of attack by p l a s m o d i a l cathepsin D. It should be emphasized that the parasite enzyme preferentially degraded those erythrocyte m e m b r a n e proteins that constitute the cytoskeleton [33], a n d it may be that very late in schizogony protease-induced alterations in the rigidity a n d shape of the red cell m e m b r a n e could lead to the release of merozoites. The cathepsin D ofP. lophurae is the major, a n d possibly its only endopeptidase, a situation akin to that reported for Tritrichomonasfoetus [34] a n d Artemia salina [35]. This is in distinct contrast to the presence of several lysosomal acid endopeptidases in m a m m a l i a n cells [36]. The subcellular location of the plasmodial enzyme described in the present work is u n k n o w n , but it is t e m p t i n g to suggest that the P. lophurae cathepsin D exists in the food vacuoles, a n d has as its m a j o r f u n c t i o n the d e g r a d a t i o n of h e m o g l o b i n ingested by the intraerythrocytic parasite. I n h i b i t i o n of the proteinase by c h l o r o q u i n e , which is c o n c e n t r a t e d in lysosomes, may in part a c c o u n t for its a n t i m a l a r i a l activity. ACKNOWLEDGEMENT This work was s u p p o r t e d by a research grant from the U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for Research a n d T r a i n i n g in Tropical Diseases. REFERENCES 1 2 3

4

5 6 7 8 9

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