proteases of the nematode caenorhabditis elegans

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 261, No. 1, February 15, pp. 80-90, 1988

Proteases of the Nematode Caenorhabditis elegans

GARY J. SARKIS, MICHAEL R. KURPIEWSKI, JAMES D. ASHCOM, LINDA JEN-JACOBSON. AND LEWIS A. JACOBSON’

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania l5.%0

Received August 10, 1987, and in revised form October 21,1987

Crude homogenates of the soil nematode Caenorhabditis elegant exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Cel and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe- Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin- insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited >95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate. o 1988 Academic press. I”~.

Proteolytic processes play a number of important roles in eukaryotic cells, among which are the digestion of extracellular proteins for nutritional purposes, the mat- uration of primary translation products, the regulation of the steady-state levels of specific gene products by selective degradation (l), and the disposal of damaged proteins produced by mutation, chemical modification, or premature termination of translation (2, 3). In very few cases, however, has it been possible to assign specific in vivo functions in protein turnover to specific proteolytic enzymes. Indeed, even the unequivocal quantitative allocation of proteolytic processes to either the cytosol or the lysosomal compartment has proven difficult (4, 5).

In order to ascertain the involvement of specific proteases in specific functions in vivo, we have undertaken a genetic analysis of protease function. We believe that specific mutants which alter the in vivo activity of single proteolytic enzymes offer

1 To whom correspondence should be addressed.

promise for identifying the function(s) of each enzyme in various cellular processes,

For this analysis, we have chosen the small soil nematode Caenorhabditis elegans. The self-fertilizing hermaphroditic mode of reproduction of this organism permits the relatively simple isolation of mutant strains carrying recessive alleles in homozygous form (6), and its small size (1.5 X 0.07 mm at full size) and short life cycle (3-4 days at 16”C, Ref. (7)) permit rapid and convenient genetic experiments. Rare male animals (X0 sex chromosome composition), arising by nondisjunction of the X chromosome, will mate with XX hermaphrodites, allowing genetic map- ping through heterosexual crosses (6). These favorable properties have been ex- ploited in many laboratories to construct a genetic map consisting of more than 400 genes on six linkage groups (8).

As an essential preliminary step to a mutational analysis of protease function, we report here a description of the major proteolytic activities found in crude extracts of C. elegans. Using both macromolecular and synthetic substrates in con-

0003-9861/8X $3.00 80 Copyright @ 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Caewrhabditis elcgans PROTEASES 81

junction with a variety of inhibitors, we have identified at least four distinct enzymes. The most important of these are cathepsin2 D (a pepstatin-sensitive car- boxy1 protease), two leupeptin-sensitive thiol proteases which have properties somewhat analogous to the cathepsins B and L of vertebrates, and a thiol-independent, leupeptin-insensitive protease. We will report elsewhere (L. A. Jacobson et al., in preparation) the purification and characterization of the cathepsin D of C. ele- gan.s and the isolation of mutants deficient in cathepsin D activity (L. A. Jacobson et al, submitted for publication).

MATERIALS AND METHODS

Growth of nematodes. C. elegant strain N2 (wild type), also known as the Bristol strain (6), was ob- tained from the collection of R. L. Russell. Stock cultures and small numbers of experimental animals were grown in loo-mm petri dishes containing NG agar with Escherichia coli OP50 as food source (6).

For growth of large-scale cultures, the prototro- phic E. coli strain NA22 was grown overnight in a liquid medium suggested to us by P. D. Gardner and R. L. Russell (110 mM phosphate, pH ‘7; 25 mM NH&l; 8 mM MgS04; 3% (w/v) casamino acids; 2% (w/v) glycerol; 5 WM EDTA; 2.5 pM FeSO,; 1 pM MnClz, ZnS04, and CuSOJ and harvested by centrifugation, and the cell pellet was resuspended in about one-half the original volume of medium S (9). Medium S does not support the growth of E. coli. Cultures were then inoculated with C. elegans washed from several agar plates and incubated with forced aeration for 3-5 days at 20°C. A11 experiments used nematodes which were asynchronous with respect to age and develop- mental stage; the cultures contained mixed popula- tions ranging from adults to juveniles.

When the E. coli food source appeared by inspec- tion to have been nearly exhausted, the nematodes were settled out under gravity, part of the spent medium was decanted, and the nematodes were harvested by centrifugation for 5 min at 7000 rpm in a Servall GSA or GS-3 rotor at 5°C. The pellets containing both nematodes and some unconsumed bacteria were resuspended in buffer (70 mM potassium phosphate, 70 mM NaCl, pH 7) containing 35% (w/v) sucrose and centrifuged for 5 min at 8500 rpm in a Servall SS34 rotor at 5°C. Under these conditions, adult nematodes and many juveniles float to the top of the sucrose solution, whereas bacteria, dead ne-

z The term cathepsin is used generically to denote a cellular endoprotease (15).

matodes, and some small juveniles sediment to the bottom of the tube. The nematodes were collected with a pipet, washed three times with buffer, and resuspended in the buffer (see below) used for preparation of extracts. These suspensions were either used immediately or stored at -80°C for later use.

Preparation of extracts. Fresh or frozen nematodes were resuspended in small volumes of 0.1 M sodium acetate buffer, pH 5.0. In some experiments, either phosphate buffer (pH 7.2) or distilled water was used instead, but this made no difference to the recovery of enzymatic activities.

For small-scale experiments, extracts were prepared by sonication using a Branson sonifier while the sample tubes were maintained on ice. For larger- scale experiments, the nematode suspension was passed once through a French press at an operating pressure of 7000 psi. In either case, the extracts received 0.1% Nonidet-P40 (NP-40), were held on ice for 10 min, and then centrifuged for lo-20 min at 27,OOOg to remove unbroken nematodes and cell debris.

Preparation of jluorescein isothiocyanate-protein conjugates. Substrate proteins were labeled with fluorescein isothiocyanate (FITC)3 essentially accord- ing to Rinderknecht (10). Protein solutions (l-5%, w/v) in 50 mM NaHC03 buffer, pH 8.6, were treated with 9 mg/ml of FITC adsorbed on Celite (Sigma Chemical Co.) for 1 to 5 h at 25°C. The solution was centrifuged briefly to remove the Celite then applied to a column of Sephadex G-25 equilibrated with 20 mM ammonium acetate and eluted under gravity with the same buffer. During chromatography, a faster-moving band of fluorescent protein and a slower-moving band of free fluorescein separated on the column. The protein peak was collected and con- centrated by lyophilization. Stock solutions at l-5% (w/v) were made in distilled water or in 0.1 M sodium formate, pH 4, and stored at -80°C.

For preparation of total C. elegant protein as substrate, crude extracts prepared as described above were treated with 10% trichloroacetic acid for 10 min at 100°C and the precipitated protein was collected by centrifugation. The protein was redissolved in 50 mM NaHC03, pH 8.6, and dialyzed overnight against 50-100 vol of the same buffer prior to FITC labeling as described above.

Enzyme assays. For assay of general proteolytic activity using FITC-labeled protein substrates, reactions (0.2 ml) contained 0.1 M buffer at the indicated pH, FITC-protein at a final concentration of 0.2-0.5

3 Abbreviations used: FITC, fluorescein isothiocyanate; MCA, 7-amino-4-methycoumarin amide; Z, benzyloxycarbonyl; Boc, tert-butyloxycarbonyl; EP-64, L-trans-epoxysuccinyl-leucylamido-(4guani- dino)butane.

82 SARKIS ET AL.

mg/ml, 8 mM dithiothreitol, and inhibitors (when present) at the indicated concentrations. Reactions were initiated by the addition of 5-20 ~1 of crude extract, then incubated for l-5 h at 25 or 37°C.

Reactions were stopped by the addition of an equal volume of 10% trichloroacetic acid and the precipitated proteins were removed by centrifugation at 10°C for 5 min in a Fisher microcentrifuge (approx 12,000g). A sample (0.1 ml) of the supernatant was withdrawn and diluted with 0.9 ml of 0.2 M NaOH, and the fluorescence was measured in a Farrand Mark I spectrophotofluorometer at an excitation wavelength of 490 nm and an emission wavelength of 515 nm. Control reactions without extract, or with extract added after the trichloroacetic acid, were included in each experiment and the average fluorescence of four such blanks was subtracted from each experimental value. All assays were conducted within the range in which measured activity was linear with time and with amount of extract added.

Assays with synthetic substrates used various peptide amide derivatives of 7-amino-4-methylcoumarin (peptide-MCA substrates). These were prepared as 1 mM stock solutions, usually in dimethyl sulfoxide, and diluted 50-fold with buffer immediately before use. A sample of enzyme (5-20 ~1) was added to 305-320 ~1 of 120 mM buffer at the indicated pH, followed by the addition of 50 ~1 of a freshly prepared solution of 80 mM dithiothreitol. These solutions were incubated for 10 min at 25°C to reacti- vate thiol proteases, then reactions were initiated by the addition of 125 ~1 of 0.02 mM substrate. Inhibi- tors, when present, were added prior to the substrate and the buffer volume was adjusted accordingly.

Standard assays for the thiol proteases cathepsins Cel and Ce2 used Z-Phe-Arg-MCA as substrate and were conducted in acetate buffer at pH 5.0.

Reactions were incubated for 0.5-8 h at 3O”C, then terminated in one of two ways: Specific assays for thiol proteases were stopped by the addition of 500 pi of 100 mM chloroacetic acid, 100 mM acetic acid, pH 4.3. Other assays were stopped by the addition of 0.5 ml of 0.2 M Na2C03. In all cases, fluorescence was then measured at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. Measured fluorescence, corrected for control reactions as described above, was converted to moles of 7-amino-4- methylcoumarin liberated by comparison with a standard curve. One unit of enzyme activity is defined as that amount which liberates 1 picomol of product per hour under these conditions. In all experiments, activity was verified to be linear with time and with amount of added enzyme.

Cleavage studies with a model hexapeptide. For digestion with cathepsins Cel or Ce2, 0.1 mg of Leu- Trp-Met-Arg-Phe-Ala was incubated for various lengths of time in 120 mM acetate, pH 5.0, 1.5 mM EDTA, 8 mM dithiothreitol, and appropriate

amounts of enzyme in a total volume of 0.25 ml. Reac- tions were stopped at intervals as described above. Digestions with cathepsin Ce3 were under similar conditions, except that the pH was 5.5 and dithiothreitol was omitted. Digestions with trypsin or chy- motrypsin were conducted in 0.1 M borate buffer, pH 8.0. Samples of each reaction were spotted on a silica gel GHL plate (Analtech, Inc.) and chromatographed in n-butanol:acetic acid:water (4:1:1, by vol). Peptide products were visualized by spraying the dried plate with 1% fluorescamine (Roche Diagnostics) in anhy- drous acetone and viewed under long-wave ultravio- let illumination.

Partial purification of leupeptin-sensitive proteases. The starting material for these purifications was a byproduct of purification of cathepsin D (L. A. Jacobson et al, in preparation). Briefly, extracts were prepared in citrate buffer at pH 3.0 in the presence of various glycosidase inhibitors and traps, then passed through a column of pepstatinyl-diaminodipropyl- amine-agarose (Pierce Chemical Co.). The material passing through this column at pH 3, devoid of cathepsin D activity, was then used for chromatographic resolution of the leupeptin-sensitive proteases.

Lipids were removed by extracting the solution twice with one-half volume of n-hexane:isopropanol (3:2, v:v) at 5°C (11). This procedure resulted in sig- nificantly improved flow rates during subsequent column chromatography; recovery of enzyme activities ranged from 50 to 85% in various preparations. The recovered aqueous phase was then dialyzed overnight at 5°C against buffer (20 mM maleic acid, pH 6.8, 1.5 mM EDTA, 10% glycerol) and applied to a column of DEAE-Sephadex A-25 equilibrated with the same buffer. Elution was at 5°C with a linear gradient of 0 to 0.5 M NaCl in the same buffer. Frac- tions were collected into tubes containing sufficient glycerol to give a final concentration of 50% (v/v) in the fractions and stored at -20°C.

Slab gel isoelectric focusing. A horizontal slab gel (10 X 12.5 cm X 0.15 cm thick) was prepared consisting of 5% acrylamide, 0.14% methylene(bis)acrylamide, 0.2% agarose, and a l/20 dilution of pH 3-10 ampholyte mixture (Bio-Rad Laboratories). The catholyte was 1 M NaOH and the anolyte was 1 M HaPO,. The gel was prerun for about 30 min at a constant power of 6 W, during which time the voltage increased from about 400 V to about 1200 V. Samples were prepared by exchanging into l/20 diluted am- pholytes using a Centricon- microconcentrator (Amicon Corp.), then applied near the center of the gel. Isoelectric focusing proceeded for about 1.5 h at 1200-1600 V, with the gel cooled to 5°C. The pH gradient in the gel was determined by the use of marker proteins in separate lanes, as follows: amyloglucosi- dase (pl 3.55), soybean trypsin inhibitor (pZ 4.55), @-lactoglobulin A (~1 5.13), bovine carbonic anhy-

Caenorhabditis elegant PROTEASES 83

drase (pI5.85), human carbonic anhydrase (pI6.5’7), myoglobin (~16.76 and 7.16), and lactic dehydroge- nase (~18.55). The positions of these markers were established by staining as described below.

Gel slabs to be stained were fixed for 30 min in 20% methanol, 10% trichloroacetic acid, 5% sulfo- salicylic acid, then stained with silver nitrate ac- cording to Morrissey (12). For localization of enzyme activities, gels were sliced in 0.5-cm increments using a razor blade, and each slice was soaked in assay buffer without substrate for 30 min. Reactions were initiated by the addition of substrate and incubated overnight. Casein zymograms to detect proteolytic activity were prepared by placing an unfixed gel slab in contact with a 1% agarose slab containing 1% casein in 0.2 M acetate buffer, pH 5.3, as modified from Foltmann et al. (13). The isoelectric focusing gel was presoaked in acetate buffer, pH 5.3, for 10 min before being placed in contact with the casein-agarose gel.

Biochemical reagents. Model substrates were pur- chased from the following sources: Z-Phe-Arg-MCA, Boc-Gln-Arg-Arg-MCA, and Z-Arg-Arg-MCA from Peninsula Laboratories; succinyl-Ala-Ala-Ala- MCA, Arg-Arg-MCA, Arg-MCA, and glutaryl-phe- MCA from Vega Biochemicals; Z-Arg-MCA from Calbiochem; and Leu-Trp-Met-Arg-Phe-Ala from Bachem, Inc. Pepstatin A, leupeptin (synthetic, as the hemisulfate), antipain, elastatinal and L-trans- epoxysuccinyl-leucylamido-(4-guanidino)butane (EP-64) were from Sigma Chemical Co.

RESULTS

Proteolysis by Crude Extracts

Figure 1 shows the pH dependence of proteolysis by crude extracts of C. elegans, using three different kinds of FITC-labeled protein substrates. Proteolysis pro- ceeds quite rapidly at acid pH with all substrates, but there was little or no proteolysis above pH 7. The pH profile depends to some extent upon the choice of protein substrate: Proteolysis of FITC-hemoglobin showed a more acid pH optimum than did the proteolysis of FITC-casein or a crude mixture of acid-precipitated C. elegans proteins. Similar experiments with 35S-labeled E. coli proteins (data not shown) gave results substantially similar to those with FITC-C. elegans proteins.

To identify the responsible protease activities, we tested the effects of two protease inhibitors. Pepstatin inhibits car-

0.5 x

r \

FITC-C. elegam Protein

0.4

FITC- Hb

0.21 FITC-CASEIN

i

PH

-I

FIG. 1. Effect of pH and inhibitors on hydrolysis of FITC-labeled proteins by crude extracts of C elegant. Reaction conditions are given under Materials and Methods. The ordinate shows the measured fluorescence, corrected for blanks lacking enzyme extract, produced after 1 h of reaction at 25°C. Measured product fluorescence was linear with time and with amount of added extract throughout the range of these experiments. (X) Total activity; (0) pepstatin- sensitive activity, calculated as total activity minus the activity in the presence of 0.1 mM pepstatin; (0) leupeptin-sensitive activity, calculated as total activity minus the activity in the presence of 0.5 mM leupeptin.

boxy1 proteases such as pepsin (14) and cathepsin D (15), whereas leupeptin inhibits a number of thiol proteases, includ- ing cathepsin B (16), cathepsin L (1’7), and cathepsin H (17), as well as trypsin-like serine proteases (18).

Pepstatin was the most powerful inhibitor at all pH values and with all substrates (Fig. 1). At pH 4.5 to 5.0, which approxi- mates the probable intralysosomal pH (19), pepstatin inhibited proteolysis by >95%. This shows that a carboxyl protease is necessary for the overwhelming majority of proteolytic events in vitro

84 SARKIS

under these conditions. We will report elsewhere the purification and characterization of this enzyme, which closely resembles cathepsin D in its cleavage specificity, by affinity chromatography on im- mobilized pepstatin (L. A. Jacobson et al., in preparation).

The effects of leupeptin were more limited. A substantial amount of leupeptin- sensitive activity was evident with FITC- casein as substrate, but was less significant when FITC-hemoglobin was the substrate. For digestion of FITC-casein or FITC-C. elegant proteins, the leupeptin- sensitive activity showed a well-defined pH optimum around pH 5.5. This activity is attributable to at least two distinct enzymes (see below).

The sum of pepstatin-sensitive and leupeptin-sensitive proteolysis rates in the range pH 4-6 is greater than the total proteolysis rate. This indicates that cathepsin D and the leupeptin-sensitive proteases act synergistically in vitro to reduce protein substrates to acid-soluble fragments, as has been observed for crude mixtures of lysosomal proteases from vertebrate tissues (20). In this case, even with a hetero- geneous mixture of protein substrates (FITC-C. elegans proteins), the inhibition by pepstatin is nearly complete, suggest- ing that some peptide bond cleavage by cathepsin D is prerequisite for attack of intact proteins by the leupeptin-sensitive proteases.

Cleavage of Model Peptide Substrates

Vertebrate tissues contain three distinct thiol proteases which are sensitive to leupeptin: Cathepsin B has a carboxydipeptidase activity (21, 22) and attacks intact proteins rather poorly (23). Cathepsin H (24) has aminopeptidase and some en- dopeptidase activity, but also little activity toward intact proteins. Cathepsin L, by contrast, has a powerful endoprotease activity and has been shown to digest many test enzymes and structural proteins (25, 26). We sought to determine whether the leupeptin-sensitive proteolytic activities of C. elegant resembled any of these vertebrate enzymes.

ET AL.

The thiol proteases can to some extent be distinguished by their specificities for fluorogenic peptide model substrates (27). Figure 2 shows that crude extracts of C. elegans can hydrolyze Z-Phe-Arg-MCA, which is a substrate for cathepsins B and L. Neither Z-Arg-Arg-MCA, an excellent cathepsin B substrate (28), nor Z-Arg- MCA, a substrate for cathepsins B and H, was hydrolyzed under these conditions. This indicates that these extracts contain little or no activity, which is precisely analogous to cathepsins B or H.

The pH-dependence of Z-Phe-Arg-MCA hydrolysis (Fig. 2) was very similar to that of the leupeptin-sensitive proteolysis of macromolecular substrates (Fig. 1). The activity toward Z-Phe-Arg-MCA was it- self leupeptin sensitive and completely dependent upon the addition of a sulfhy- dry1 reagent such as dithiothreitol (Fig. 2). In conjunction with the substrate specificity, this suggested the presence of one or more enzymes with properties resembling those of cathepsin L.

FIG. 2. Hydrolysis of Z-Phe-Arg-MCA by crude extracts. Reaction conditions are as given under Ma- terials and Methods and in Table I. (0) Total activity; (Cl) activity in the presence of 0.2 mM leupeptin; (X) activity in the absence of dithiothreitol.

Caenm-habditis elegant PROTEASES

0 30 60 90 120

Fraction Number

FIG. 3. Separation of acid proteases by chromatography on DEAE-Sephadex. The sample applied to the column was the unadsorbed fraction from a pepstatin affinity column, prepared as described under Materials and Methods. The dashed line shows the NaCl gradient used for elution. The ordinate indicates measured fluorescence, corrected for blanks containing no enzyme, for assay with (0) Z-Phe-Arg-MCA as substrate (3.75-h assay) or for assay with @) undenatured FITC-casein as substrate (18-h assay).

Chromatographic Resolution of Leupeptin- Sensitive Proteases

We found that material rich in leupeptin-sensitive protease activity could be ob- tained as a by-product of purification of cathepsin D (L. A. Jacobson et ah, in preparation). Crude extracts were made at pH 3 (where 95-98s of the nematode proteins are removed as insoluble materials) and passed through an affinity column of im- mobilized pepstatin. The unretained protein from this column was devoid of cathepsin D activity, but the activity toward Z-Phe-Arg-MCA was strong and stable on long-term storge at -20°C.

Figure 3 shows that chromatography on DEAE-Sephadex resolved two distinct peaks of activity toward Z-Phe-Arg-MCA and a small late-emerging third peak. Upon assay with FITC-casein as substrate at pH 5, the third peak showed strong endoprotease activity.

The first two peaks proved to represent two distinct forms of thiol-dependent activity (see below) and are designated cathepsin Cel and cathepsin Ce2, in order of emergence from the column. The fact that the relative activities of Cel and Ce2 are different with Z-Phe-Arg-MCA and FITC-casein as substrates (in that cathepsin Cel has relatively more proteolytic activity; cf. Fig. 4) suggests that they are distinct enzymes rather than different

85

9 E E IL

6;

P x s

3

5 10 15 20

Fraclion Number

FIG. 4. Isoelectric focusing of acid protease activities. Details of isoelectric focusing in nondenaturing acrylamide-agarose composite gels and subsequent assay of the fractions are given under Materials and Methods. The nominal gradient in the gel was from pH 3 to 10; the actual gradient, based upon the positions of stained marker proteins (X) is shown. (m) Unfractionated material, corresponding to the starting material for the column of Fig. 3; (0) chromatographically purified cathepsin Cel; (0) chromatographically purified cathepsin Ce2. The inset (photo- graph) shows a casein zymogram of a similar gel. The upper lane is the unfractionated material, whereas the lower lane is chromatographically purified cathepsin Ce3, corresponding to fraction 100 of Fig. 3. The diffuse band of activity in the upper lane was visibly resolved into two bands at earlier reaction times. The one-fraction discrepancy between Cel activity in the crude and partially purified materials is not significant.

86 SARKIS ET AL.

chromatographic forms of the same enzyme.

Cathepsins Cel and Ce2 cannot be distinguished on the basis of specificity for model substrates, cofactor requirements, or inhibitor sensitivities. Both cathepsins Cel and Ce2 are thiol dependent and leupeptin sensitive (Table I). Their pH optima (pH 5) with Z-Phe-Arg-MCA as substrate are indistinguishable. Both are in- activated by the epoxide inhibitor EP-64 (29), which also inactivates cathepsins L and B from vertebrate sources (30) by stoi- chiometric reaction with the active site. Both cathepsins Cel and Ce2 are insensitive to pepstatin and PMSF. Neither enzyme hydrolyzes Z-Arg-Arg-MCA, Z-Arg- MCA, or succinyl-(Ala)3-MCA (Table II) at a significant rate.

Cel and Ce2 also have identical specificities in cleaving a larger peptide fragment (Leu-Trp-Met-Arg-Phe-Ala). Both cathepsin Cel and Ce2 rapidly cleaved the Arg-Phe bond (Fig. 5) to form Phe-Ala and Leu-Trp-Met-Arg, and cleaved the Trp-Met bond more slowly to form Leu- Trp and Met-Arg. This carboxydipeptidase activity resembles that of mammalian cathepsin B (22) rather than that of

cathepsin L (which cleaves the Met-Arg bond). On the other hand, both Cel and Ce2 prefer Z-Phe-Arg-MCA among the fluorogenic dipeptide substrates (Table II) and in this respect differ from cathepsin B.

Nonetheless, cathepsins Cel and Ce2 are distinct enzymes. We found that Cel and Ce2 had quite distinct isoelectric points under nondenaturing conditions (Fig. 4). The difference in PI, nearly two pH units, appears too large to be the result of post- translational modifications. We also infer that these are distinct enzymes from the fact that Ce2 activity declines much more rapidly in older animals than does Cel activity (G. Sarkis, J. Ashcom, J. Hawdon, and L. Jacobson, submitted for publication).

Both enzymes were completely inhibited when EP-64 was included in the assay after separation by isoelectric focusing. Casein zymography of these gels (Fig. 4, inset) showed relatively little proteolytic activity in the position of Ce2. The crude material showed a diffuse band of proteolytic activity, which at earlier times of digestion appeared as two distinguishable bands. One of these corresponded to Cel,

TABLE I

EFFECT OF INHIBITORS ON CATHEPSINS Cel, Ce2, AND Ce3

Percent activity

Additions Cathepsin Cel Cathepsin Ce2 Cathepsin Ce3

Complete 1ooa 100” loo* - Dithiothreitol 4 3 -

+ Dithiothreitol (8 mM) - 27

t Pepstatin (IO pM) 100 100 93 + Leupeptin (10 pM) 3 1 103 + EP-64 (10 I"M) 0 0 85 + Antipain (5 pg/ml) 0 0 96

D Reaction mixtures contained, in a total volume of 0.5 ml, 30 pmol sodium acetate buffer, pH 5.0; 0.5 pmol EDTA; 4 pmol dithiothreitol; 2.5 nmol Z-Phe-Arg-AMC; and 20 ~1 chromatographically purified cathepsins Cel or Ce2. The enzyme was preincubated for 10 min at 25°C prior to addition of substrate. Reactions were run for l-4 h at 30°C then stopped by addition of 0.5 ml of 0.1 M ClCHaCOOH, 0.1 M CHaCOOH, pH 4.3.

*Reaction mixtures contained, in a total volume of 0.2 ml, 24 rmol sodium acetate buffer, pH 5.5; 0.3 pmol EDTA; 0.2 mg undenatured FITC-casein; and lo-20 gl of chromatographically purified cathepsin Ce3. Reac- tions were run for 12-24 h at 25”C, then stopped by the addition of Cl,CCOOH as described under Materials and Methods.

87 Caenorhabditis elegant PROTEASES

TABLE II

HYDROLYSIS OF PEPTIDE SUBSTRATES BY CATHEPSINS Cel AND Ce2”

Cathepsin Cel Cathepsin Ce2

Substrate pmol/min Percent* pmol/min Percentb

Z-Phe-Arg-MCA 27.7 100 40.9 loo Z-Arg-Arg-MCA 0.1 0.4 3.6 8.7 Arg-Arg-MCA 0.1 0.4 0.5 1.2 Arg-MCA 0 0 0.4 0.9 BOC-Gln-Arg-Arg-MCA 0.2 0.6 1.0 2.5 Succinyl-Ala-Ala-Ala-MCA 1.5 5.5 0.4 0.9 Glutaryl-Phe-MCA 0 0 0 0

a Reaction conditions were as in Table I. The concentration of all substrates was 5 WM.

b Activity relative to that with Z-Phe-Arg-MCA as substrate.

and the other band, at more acidic PI, corresponded to the position of cathepsin Ce3 activity (see below).

The third peak of activity resolved by ion-exchange chromatography (Fig. 3) is another distinct enzyme, which we designate as cathepsin Ce3. Its activity toward FITC-casein (see also Fig. 4) is optimal at pH 5.5, leupeptin insensitive (Table I), and thiol independent. The latter properties, as well as its very weak activity toward Z-Phe-Arg-MCA, show it to be clearly distinct from cathepsins Cel and Ce2. Its isoelectric point (Fig. 4) is close to, but clearly distinct from, that of Cel. So far, we have not succeeded in finding a useful fluorogenic or chromogenic peptide substrate for this enzyme. It does not hydrolyze Z-Arg-MCA, Z-Arg-Arg-MCA, succinyl-(Ala)3-MCA, or glutaryl-Phe-MCA to any detectable extent, even at very long times of incubation at high enzyme concentration. Cathepsin Ce3 did not hydrolyze the hexapeptide Leu-Trp-Met-Arg- Phe-Ala.

Other Activities

We have also detected an activity for hydrolysis of succinyl-(Ala)3-MCA, an elastase substrate. This activity is optimal at pH 5.5, and only partially inhibited by leupeptin or the omission of dithiothreitol. Elastatinal, an aldehyde analog of the substrate (31), inhibits totally at 100 PM.

These properties, and the absence of activity toward this substrate in the three enzymes resolve in Fig. 3, suggest that the elastase-like activity is attributable to yet another distinct enzyme.

DISCUSSION

As a first step toward mutational analysis of in vivo protease function in C. elegans, we have identified the presence of at least four distinct endoproteases. We have emphasized endoproteases on the premise that peptidases are unlikely to be rate lim- iting in the functional inactivation of in- tracellular proteins, although they may play an important part in protein degradation and recycling of protein-derived amino acids.

Proteases with neutral to alkaline pH optima make little contribution to the in vitro activity of crude extracts. This may be deceptive because neutral proteases in C. elegans may require special conditions or cofactors which were not present in our assays, or because homogenization brings a protease in contact with an endogenous inhibitor from which it was segregated in vivo. It is also possible that neutral proteases which attack a limited subset of the substrate proteins-and thus make little quantitative contribution in our assays- have highly significant functional roles by virtue of their attack on specific target proteins in vivo. We specifically searched

88

ieu-trp *

phe-ala * Hexapeptide +

leu-trp-met-arg -w

met-arg +

Origin +

SARKIS

(0 30’3h18h110 60’4hl8h]

Cath Cel Cath Ce2

FIG. 5. Cleavage of a hexapeptide by cathepsins Cel and Ce2. The hexapeptide Leu-Trp-Met-Arg-Phe- Ala was digested for the indicated times with chromatographically purified cathepsin Cel or cathepsin Ce2 as detailed under Materials and Methods, and the products were separated by thin-layer chromatography. The three left lanes contain standards of hexapeptide, Leu-Trp, and Phe-Ala (left to right). The first products produced correspond precisely in migration to the products produced by trypsin (Leu- Trp-Met-Arg and Phe-Ala). At later times, a peptide comigrating with Leu-Trp and another peptide (presumably Met-Arg) begins to appear.

for neutral proteases which required ATP for activity in vitro (5), and found no stim- ulation of proteolysis rates at ATP concentrations up to 2 mM (data not shown).

Proteolysis in vitro by crude extracts was apparently due to the combined action of at least four distinct enzymes. The most significant contribution was made by the aspartyl protease cathepsin D, as judged by the strong (95%) inhibition of overall proteolysis by the specific inhibitor pepstatin. A further significant contribution was made by leupeptin-sensitive activities; this may represent the sum of contributions from the proteases we designate as Cel and Ce2. Although we cannot dis- tinguish the separate contributions of these two proteases at present, it is clear that each of them, as well as cathepsin

ET AL.

Ce3, has endoprotease activity (as assayed with FITC-casein) after separation from other proteases (Fig. 3 and Fig. 4). We caution that the quantitative roles of these proteases should not be inferred from their relative endoprotease activities in partially purified preparations. For ex- ample, cathepsin Ce2 appears to have a predominant carboxydipeptidase activity and relatively little ability to attack intact proteins, but might make a significant contribution to overall proteolysis in crude extracts (or in vivo) where initial endoproteolytic attack is made by other enzymes.

In the crude extracts the participation of the leupeptin-sensitive proteases in di- gesting a mixture of C. elegans proteins is largely dependent upon the simultaneous presence of cathepsin D activity. These observations are consistent with the independent endoprotease activities of Cel and Ce2 if casein is unusually sensitive to the action of the leupeptin-sensitive proteases. Indeed, studies on mammalian systems both in vitro (32-34) and in vivo (35, 36) also imply that the quantitative contributions of various lysosomal proteases to the degradation of some individual proteins may not be representative of their relative roles in the degradation of complex protein mixtures.

There is some precedent for initial cleavage by cathepsin D as prerequisite for subsequent attack by thiol proteases. Apoprotein B of human low-density lipo- protein was not degraded by lysosomal extracts in the presence of pepstatin, although leupeptin still had a pronounced (50%) inhibitory effect on the hydrolysis to acid-soluble products (20). It has also been proposed (37) that thiol compounds in the reaction mixture can reduce disul- fide bonds in the target protein only after initial proteolysis, thus presumably in- creasing susceptibility to further proteolysis. There was no evidence for synergis- tic action of cathepsin D and the thiol proteases, however, in studies of the proteolytic activity of rat liver lysosomes (32, 34). In the latter case, the quantitative contribution of cathepsin D was consider- ably smaller (about 40-45s of the total

Caenorhabditis elegans PROTEASES 89

proteolysis rate) than we observed with C. elegans extracts.

Data on proteolysis in vitro, however, cannot be used to infer the qualitative or quantitative roles of the various proteases in vivo. The acidic pH optima of the proteases we have found in C. elegant suggest that these enzymes may normally reside within lysosomes; this has been confirmed for cathepsins D, Cel, and Ce2 (G. Sarkis et al., submitted for publication). Thus many of the C. elegans proteins used in our substrate mixture may come into contact with these proteases only infrequently. Furthermore, the ability of proteases to attack any given protein may be quite dependent upon the tertiary and/or quater- nary structure of the target protein (38), and it cannot be assumed that the struc- tures normally found in vivo are retained in the proteins in our substrate mixture. Our strategy for assessing the functional roles of these proteases in viva relies instead upon analysis of the effects of mu- tations which alter the levels or activities of individual proteases.

The principal value of this exploration, therefore, is in identifying proteases as potential targets of genetic manipulation and in providing insights into their properties which may prove useful in the design of screening or selection techniques for mutant isolation.

The activity we designate as cathepsin D is defined here only by its sensitivity to the aspartyl protease inhibitor pepstatin. In a subsequent paper (L. A. Jacobson et al, in preparation), we will report on the purification of this enzyme based upon its affinity for pepstatin, and show that its substrate specificity is virtually identical to that of mammalian cathepsin D. Be- cause of microheterogeneity in the protein so isolated, proof that cathepsin D repre- sents the product of a single gene depends upon both biochemical and genetic analysis of mutants deficient in cathepsin D (L. A. Jacobson et aZ., submitted for publication).

We have identified two thiol-dependent proteases, designated cathepsins Cel and Ce2, which bear some resemblances to enzymes in mammalian tissues. Cathepsin

Cel is quite similar to mammalian cathepsin L (24,39) in its thiol dependence, relatively high endoproteolytic activity, substrate specificity, inhibitor sensitivities, and pH optimum. On the other hand, Cel has a carboxydipeptidase activity, and in this respect differs from cathepsin L.

Cathepsin Ce2 is also not precisely analogous to any single mammalian protease. It resembles both cathepsin L and cathepsin B (27) in its thiol dependence, inhibitor sensitivity, and pH optimum. In its specificity for model substrates, protease Ce2 most nearly resembles cathepsin L (24,27, 39) in preferentially cleaving Z-Phe-Arg- MCA rather than substrates lacking a hy- drophobic residue (e.g., Z-Arg-Arg-MCA), but resembles cathepsin B in that it cleaves dipeptides from the carboxyl ter- minus of a model hexapeptide (22) and has relatively low endoprotease activity.

The strong similarities between proteases Cel and Ce2 illustrate both the dangers inherent in a pharmacological analysis of protease function in vivo and the value of even preliminary biochemical characterization to the design of a genetic strategy for assessing enzyme function. The use of an inhibitor such as leupeptin for in vivo studies of protein degradation might result in simultaneous inhibition of proteases Cel and Ce2, with consequent uncertainty about the relative roles of these quite distinct enzymes.

The large difference in pI and the relatively greater endoprotease activity of Cel indicate that proteases Cel and Ce2 are probably the products of different genes. Thus, we would require the isolation of two distinct classes of mutants to begin assessing their roles in protein degradation in vivo. Because the properties of the two enzymes are so similar, the successful design of mutant screening techniques depends upon a continued search for charac- teristics which will permit distinction to be made between the two activities in crude extracts of mutant candidates.

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proteases of the nematode caenorhabditis elegans

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