Eur. J. Biochem. 83, 87-97 (1978)

Characterisation of Cathepsin B and Collagenolytic Cathepsin from Human Placenta

Patricia EVANS and David J. ETHERINGTON

Comparative Pathology Laboratory, School of Veterinary Science, University of Bristol, and Meat Research Institute, Langford, Bristol

(Received September 12, 1977)

1. Human placental cathepsin B and collagenolytic cathepsin were separated by chromatography on columns of Amberlite CG-50. Collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50) and Sephadex G-100. Cathepsin B was purified by chromatography on CM-cellulose and Sephadex G-100.

2. Both enzymes required activation by thiol compounds and were bound to organomercurial- Sepharose-4B. Sulphydryl-blocking reagents were inhibitory, which confirmed an essential thiol group to be present.

3. The enzymes degraded soluble calf skin collagen and insoluble bovine tendon collagen in the telopeptide region at pH 3.5 and 28 "C to yield mainly a-chain components.

4. In contrast to cathepsin B, collagenolytic cathepsin was found not to hydrolyse any of the low-molecular-weight synthetic substrates that were tested.

5. Leupeptin, a structural analogue of arginine-containing synthetic substrates, and antipain, an inhibitor of papain, were strongly inhibitory to both enzymes.

6. The isoelectric points of the enzymes were similar, being 5.4 for cathepsin B and 5.1 for collagenolytic cathepsin.

7. From chromatography on Sephadex G-100 the molecular weight of cathepsin B was calculated to be 24500 and that of collagenolytic cathepsin to be 34600.

Physiological and pathological breakdown of col- lagen is probably a complex, multistep process [ l - 31. Attempts to elucidate the agents involved have im- plicated collagenase [l, 41, neutral proteinases [5,6] and the lysosomal cathepsins [7-91. All of these enzymes have been demonstrated to degrade native collagen, although the collagenases remain the only enzymes which will attack the triple helix of the native molecule. These latter enzymes, in conjunction with the neutral proteinases, are believed to reduce the collagen fibres to small fragments, which can then be ingested by phagocytic cells. Fragmented and partially degrad-

Abbreviations. Bz-Arg-2-Nap, a-N-benzoyl-DL-arginine 2-naph- thylamide. Bz-Arg 4-Nan, a-N-benzoyl-DL-arginine 4-nitroanilide. Z-Lys-4-ONp, N-carbobenzoxy-L-lysine 4-nitrophenyl ester. Z- Gly-4-ONp, N-carbobenzoxy-glycine 4-nitrophenyl ester. Z-Ala- 4-ONp, N-carbobenzoxy-L-alanine 4-nitrophenyl ester. Arg-2-Nap, L-arginine 2-naphthylamide. Arg-OMe, L-arginine methyl ester. Lys-OMe, L-lysine methyl ester. Ac-Lys-OMe, cc-N-acetyl-L-lysine methyl ester. Bz-Arg-OMe, cc-N-benzoyl-L-arginine methyl ester.

Enzymes. Cathepsin B (EC 3.4.22.1); collagenolytic cathepsin (EC 3.4.22.-); papain (EC 3.4.22.2).

ed collagen fibrils have been observed in phagolyso- somes [lo- 131 where the intracellular phase of digestion is completed by the lysosomal system.

Lysosomal cathepsin B (previously called cathepsin B1 [14]) and collagenolytic cathepsin have been shown to degrade soluble monomeric collagen and insoluble polymeric collagen in vitvo [15]. Cathepsin B has been isolated and characterised from a number of tissues including bovine spleen [8,16], human liver [17], human placenta [18] and human foetal membranes [ 191. The nature and occurrence of collagenolytic cathepsin, which appears to degrade collagen in a similar manner to cathepsin B, has been little studied and may be important in view of the reported synergistic effect between these two enzymes [15]. As part of a programme to elucidate the mode of action of collagenolytic cathepsin, this enzyme has been isolated from human placenta and its properties examined in comparison to cathepsin B from the same source. A preliminary report of these data has been presented [20].

88 Human Placental Cathepsin B and Collnpenolytic C'ithepsin

EXPERIMEKTAL PROCEDURE

Muter ials

Human full-term placentae were collected on ice,.. rinsed in ice-cold 0.9 Y O saline and stored at - 25 "C for up to 3 months until required. Calf skin acid- soluble collagen was prepared as described by Jackson and Cleary [21] and stored as a 0.5 "/, (w/v) solution in 0.01 M acetic acid. Insoluble bovine tendon collagen was obtained from Worthington Biochemical Corp. (Freehold, N. J.. U.S.A.).

CM-cellulose (CM-52), Amberlite CG-50 and bovine serum albumin were supplied by BDH Chemi- cals Ltd (Poole, Dorset, U. K., BH12 4"). DEAE- Sephadex (A-50), Sephadex G-100, Sepharose-4B and blue dextran 2000 were purchased from Pharmacia (G.B.) Ltd (London, I?. K., W5 5SS). Ampholine solutions for electrofocusing were obtained from L.K.B. Instruments Ltd (South Croydon, U. K., CR2 8YD). The following chemicals were obtained from Sigma (London) Chemical Company, Kingston- on-Thames ([: .K. . KT2 7BH), 2,2'-dithiodipyridine, sperm whale niyoglobin, Bz-Arg 2-Nap, Bz-Arg- 4-Nan, Z-Lys-.l-ONp, Z-Gly-4-ONp, Z-Ala-4-ONp, Arg-2-Nap, Arg-OMe, Lys-OMe, Ac-Lys-OMe, Bz- Arg-OMe, bovine erythrocyte carbonic anhydrase, mersalyl acid and Brij 35.

Chicken ovotransferrin and ovalbumin were given by Dr R. Evans. Dept. of Biochemistry, University of Bristol.

NADH and cytochrome c were supplied by Boehringer Corp. (London) Ltd (Ealing, London W5 2TZ) and 4-amino-2',3-dimethylazobenzene was a product of Merck-Schuchardt supplied by Cambrian Chemicals Ltd (Suffolk House, George St., Croydon, U. K., CR9 3QL).

Leupeptin was obtained from a wild strain of Streptom?~cc~.v ;is described by Etherington [I 51. Anti- pain was kindly given by Prof. H. Umezawa, Institute of Microbial Chemistry, Shinagawa-Ku. Tokyo, Ja- pan.

Extraction of C'uthepsins

The extraction procedure of Etherington [15] was used for both enzymes with several modifications. Placentae were partially thawed, stripped of mein- branes and large blood vessels and then minced. Approximately 1 .S kg minced tissue (corresponding to three placentae) were added to 2 volumes of ice- cold 0.1 M sodium acetate buffer at pH 5.0 containing 1 mM EDTA and 1 mM 2-mercaptoethanol and homogenized in a Silverson homogenizer. Triton X- 100 (0.2",, v ' ~ ) was then added and the homogenate stirred for 16 h. All operations were carried out at 4 -C and all solutions supplemented with 1 mM EDTA and 1 mM 2-nicrcaptoethanol except where otherwise stated.

Tissue debris was removed by centrifugation at 2800 x g for 45 min in an MSE Mistral 4L centrifuge. The supernatant was acidified to pH 3.8 with 2 M HC1 and stirred for 30 min. The small amount of precipitated protein was then removed by centrifuga- tion (1 5 000 x g for 20 min in an MSL High Speed I8 centrifuge). Ammonium sulphate was added to 30 "<)

saturation when the bulk of the contaminating pro- tein was precipitated and removed by centrifugation at 15 000 x g for 30 min. Cathepsin B and collage- nolytic cathepsin were precipitated when the ammo- nium sulphate concentration was raised to 70" , saturation. The crude enzymes were recovered by centrifugation (1 5000 x g for 30 min) and the pellet was suspended in a small volume of 0.09 M sodium citrate at pH 5.3 and dialysed against the same solu- tion. Any insoluble material was removed by centrifu- gation.

Coluniii Chromatography

The enzyme solution was applied to a column of Amberlite CG-50 ( 5 x 20 cm) previously equilibrated to the citrate buffer. Eluate fractions containing un- adsorbed cathepsin B were pooled and precipitated by addition of ammonium sulphate to 90 ";) saturation. Collagenolytic cathepsin was adsorbed to the column under these conditions and was decanted from the column by discharging the resin containing the ad- sorbed enzyme into an equal volume of 0.1 5 M sodium phosphate, pH 7.4. The pH was adjusted to 6.5 with 10 M NaOH and the supernatant containing the desorbed enzyme filtered off. The column residue was then washed with 0.5 volume of the phosphate buffer and the two supernatants combined. Col- lagenolytic cathepsin was then precipitated by the addition of solid ammonium sulphate to 90";) satura- tion. The 90 "/, saturated ammonium sulphate pre- cipitate was centrifuged at 15000 x g for 20 min and the pellet was then taken up in and dialysed against 10 mM sodium phosphate, pH 6.5. The clear solution was applied to a column of DEAE-Sephadex, A 4 0 ( 5 x 15 cm) equilibrated to the phosphate buffer, and eluted with a salt gradient of 0-0.4 M NaC1.

After separation on Amberlite CG-50, catliepsin B was applied to a column of CM-cellulose (2.6 x 22 cm) equilibrated to 20 mM sodium acetate buffer, pH 5.0, and eluted with a salt gradient of 0 - 0.2 M NaCI.

For chromatography on Sephadex (3-100, both enzymes were precipitated with ammonium sulphate and then taken up in and dialysed against 50 m M sodium acetate, pH 5.5. containing 0.2 M NaCl. Affinity chromatography on organomercurial-Sepha- rose-4B was carried out exactly as described by Bar- rett [17]. The final preparation of each enzyme was dialysed against a solution of 1 mM EDTA and 1 mM 2-mercaptoethanol before further examination.

P. Evans and D. J. Etherington 89

All chromatographic separations were carried out at 4 "C and column effluents were monitored at 280 nm with a Uvicord I1 spectrophotometer (LKB Instruments Ltd, Stockholm, Bromma-1, Sweden). Conductivity measurements on effluents were made using a Chandos Linear Conductivity meter [Chandos Products (Scientific) Ltd, New Mills, Derbyshire, U. K.].

Determination of Protein

Protein concentrations were measured by the method of Lowry et al. [22] with bovine serum albumin as standard.

Isoelectric Focusing

This was performed in a 110-ml LKB apparatus set up and operated as described by the manufacturers. The column was stabilized by a glycerol gradient; sucrose was unsatisfactory as it charred in the hydro- lysis step used in the assay for collagenolytic activity. Ampholines of pH range 3.5 - 10.0 were incorporated at a concentration of 1 yi and the focusing time was 48-60 h with a maximal voltage of 800 V for at least the latter 24 h. The column was maintained at 4 "C and the pH of the effluent fractions determined without delay. Analytical isoelectric focusing in poly- acrylamide gels was performed with an LKB Multi- phor electrophoresis unit. Samples (5- 10 pg) were applied to a half-width gel containing Ampholines from pH 3.5- 10.0. This was focused and stained with Coomassie blue as described by Barrett [17].

Assay of Enzymes

Collagenolytic activity was determined as described by Etherington [8]. Cathepsin B was routinely assayed using the method of Barrett [23] with Bz-Arg-2-Nap as substrate. Both enzymes were also tested for hydrolytic activity against the following substrates, Z-Lys-4-ONp, Z-Gly-4-ONp, Z-Ala-4-ONp, Arg- OMe, Bz-Arg-OMe, Lys-OMe, Ac-Lys-OMe [24] ; Arg-2-Nap [23] ; Bz-Arg-4-Nan [25]. For the synthetic substrates, 1 unit of activity is defined as the release of 1 pmol chromogen in 1 min at 37 "C.

The effect of pH on the collagenolytic activity of the separated enzymes was investigated by the method of Etherington [8]. Unless otherwise stated all units of cathepsin B refer to activity with the Bz-Arg-2-Nap substrate.

Sodium Dodecyl Sulphatel Polyucrylamide-Gel Electrophoresis

insoluble tendon collagen were performed at 28 "C. Enzymatic digests of calf skin soluble collagen and

Aliquots of soluble collagen (3.6 mg/ml) were dialysed into 0.1 M sodium formate containing 5 mM cysteine, which had been adjusted to the appropriate pH value with an equimolar mixture of sodium acetate and NazHP04. Samples were then incubated for 5 h with the enzymes and the digestion products analysed directly by sodium dodecyl sulphate/poly- acrylamide-gel electrophoresis according to Sykes and Bailey [26].

Insoluble collagen as a suspension in 0.1 M formate buffer, 5 mM cysteine at pH 3.5 was likewise incubated with the separate enzymes. The super- natants of such suspensions were sampled at intervals after centrifugation to separate the remaining insoluble collagen. The supernatant samples were analysed as described above, after dialysis against 0.01 M acetic acid and concentration by freeze-drying.

Testing of Potential Inhibitors

The cathepsins were dissolved in 1 mM EDTA, 5 mM 2-mercaptoethanol and then dialysed overnight against 1 mM EDTA to remove the 2-mercapto- ethanol. Samples were preincubated for 5 min with each inhibitor in three vol. 0.1 M sodium phosphate buffer, pH 6.0, and 37 "C [17]. Residual collagenolytic activity of each enzyme was determined at pH 3.5 and cathepsin B activity with Bz-Arg-2-Nap was tested at pH 6.0. Appropriate blanks were set up for each compound [17].

RESULTS Fractionation of Cutlzepsin B and Collagenolytic Cathepsin

The extraction procedure for the bovine spleen enzymes was followed [lS] but several differences were noted. The acidification step caused only a slight precipitation of protein, but was retained since denaturation caused gross precipitation at 30 "/, am- monium sulphate saturation. A greater yield of both enzymes was obtained by taking an ammonium sul- phate cut of 30 - 70 "/, saturation.

A flow diagram summarizing the extraction pro- cedure is presented in Fig. 1. The enzymes were com- pletely separated on Amberlite CG-50 with cathepsin B eluting in the breakthrough peak and collagenolytic cathepsin remaining firmly bound to the column. Since cathepsin B may be unstable near neutrality [27] and the collagenolytic cathepsin was tightly bound to the column, this latter enzyme was obtained by decanting the resin at pH 6.5 rather than by the pH elution described earlier [15].

Purification of Cuthepsin B

After separation from collagenolytic cathepsin on Amberlite CG-50, cathepsin B was applied to CM-

90 P. Evans and D. J. Etherington: Human Placental Cathepsin B and Collagenolytic Cathepsin

,' ,' ,/' -

,I ,' 20 - ,'

,' - E 0

cellulose (Fig. 2 ) . Enzymatic activity was present as a single peak eluting at a conductivity of 10 mS (0.11 M NaCI) on gradient elution. The fractions indicated were pooled and concentrated by ammonium sulphate precipitation before chromatography on Sephadex G-100 (Fig. 3A). This step separated the enzyme from yellow-coloured material which eluted in the void volume.

Extract ion at pH 5.0 and 4OC

w i t h 0 2% i v M Tr i t on X 100

Cen t r i f uga t ion

- T 16 rn -

z . jl, 2 x 3 - c

Acid precipi tat ion at pH 3.8

Superna tan t

r --F A m m o n i u m sulphate f ract ionat ion

L 30'70% sat. precipi tate '- I r I

Cathepsin B

Not adsorbed

CM -C el I u lose Sephadex A-50

Organomercu r ia l -Sepharose

I Sephadex G-!IO -3 ~. Sephadex G-100

____ Cathepsin B Col lagenolyt ic cathepsin

Fig. 1 , Floic dirr,yrnn .sun~mur.ising the purification procedurt, ,fiii* cathepsin B and collagenolytic cathepsin fkom human placenta. Experimental details are given in the text and quantitative results are presented in Table 1

This purification procedure yielded cathepsin B with a specific activity 0.42 unitimg protein and with 0.012 unit collagenolytic activityimg (Table 1). On polyacrylamide gel isofocusing, the final cathepsin B preparations showed two major and two faint protein bands.

Purification of Collagenolytic Cuthepsin

The crude collagenolytic cathepsin was loaded on to a DEAE-Sephadex column equilibrated to phosphate buffer at pH 6.5. Bound enzyme was eluted in the salt gradient when the conductivity reached 24 mS (0.3 M NaC1) (Fig.4). This step was found to result in a 32-fold increase in specific activity (Table 1). Gel chromatography also greatly improved the specific activity. A typical elution profile of collagenolytic cathepsin on Sephadex G-100 is shown in Fig.3B. The final specific activity after chromatography on Sephadex G-100 was 0.12 unit/ mg protein, which represents a purification of 1000- fold over the crude extract (Table 1). Such preparations were still slightly impure when analysed by sodium dodecyl sulphateipolyacrylamide-gel electrophoresis. A single diffuse band was obtained when the enzyme was examined by isoelectric focusing in polyacryl- amide gel.

Organomercurial-Sepharose-4B affinity chroma- tography [I71 was often interposed before Sephadex G-100 fractionation. Both enzymes were bound by the adsorbent, but the step did not give large increases in specific activity, the maximum increase observed being fivefold. It was, however, useful as a concentra- tion step.

Some Properties of the Purijied Enzymrs. Isoelectric Focusing

Samples of purified cathepsin B (0.05 unit) and collagenolytic cathepsin (0.08 unit) were isofocused

Or A 91

0 8 * O I

' t Effluent volume (ml)

r B 0

' 1 40

E 3 . 21

> V

c .- .- c

Effluent volume (ml)

Fig. 3. Sephadex G-100 gel chromatography. (A) Cathepsin B (0.74 unit) in 0.5 ml was applied to a column of Sephadex G-100 (1 x 100 cm) equilibrated to 50 mM sodium acetate buffer, pH 5.5, containing 0.2 M NaC1, 1 mM EDTA and 1 mM 2-mercaptoethanol and chromatographed using a flow rate of 5 ml/h. (B) Distribution of collagenolytic cathepsin activity (0.02 unit) on a column (1.6 x 62 cm) of Sephadex G-100 equilibrated to the same buffer. The flow rate was 4.5 ml/h. (---) Protein; (0--0) enzyme activity

Table 1. Purification of cathepsin B and collagenolytic cathepsin ,from human placenta Values given are means based on four separate preparations. Yields of protein and enzymatic activity are expressed per kg tissue. OM, organo- mercurial

Stage Protein Collagenolytic activity Bz-Arg-2-Nap activity Ratio ~~__. ~ . . . ~ :activity -

activity lo3 x purifica- yield activity lo3 x purifica- yield cathepsin specific tion specific tion B/col- activity activity lagenolytic

activity

mg Initial extract 41 000

30 ~ 70 satd (NH4)2SO4 fraction 3 800

Protein not adsorbed on Amberlite CG-50 440 CM-cellulose fraction 80 OM-Sepharose fraction 35 Sephadex G-100 fraction 24

Cathepsin B

Collagenolytic cathepsin Protein adsorbed on Amberlite CG-50 1890 DEAE-Sephadex fraction 55.2 OM-Sepharose fraction 22 Sephadex G-100 fraction 5.1

units

4.7

1.46

0.7

0.69

0.4

0.3

0.81

0.76

0.72

0.6

units/mg

0.11

0.4

1.6

8.6

11.4

12.5

0.43

13.8

32.7

118

-fold

(1)

3.6

14.5

78

104

114

3.9

125

297

1070

%

31

15

15

8.5

6.4

17.2

16.2

15.3

12.8

units

15.4

16.3

15.1

11.7

11.1

10.0

2.3

0.2

0

0

units/mg

0.4

4.3

34

146

317

417

1.2

3.6

-fold % units/unit

(1) (100) 3.3

11 100 11.2

85 98 21.6

365 76 16.9

792 72 27.7

1040 65 33.3

2.8

0.26

0

0

92

4.0

3.6

-- 3.2

Tc 2.8

5 2.4

2.0

1.6- e 2 1 . 2 - e z

' _ E

- 0

- U

- P

- Q

0.8

0.4

0 2

Human Placental Cdlhepm B and ColLigenolq tic Cdthepsin

-

-

-

-

-

-

-

.o

Effluent volume (ml)

Fig. 4. C%i.on?rrrorrru/,h~. of' c~o/lrrgcno/yric cathepsin 017 DEAL-SepYI7udex. Collagenolytic cathepsin (0.6 unit) was applied to a DEAE- Sephadex column ( 5 x 15 cm) equilibrated to 10 mM sodium phosphate, pH 6.5, containing I m M 2-mercaptoethanol and 1 mM EDTA. Elution war by a linear gradient (800 ml) of 0-0.4 M NaCl with a flow rate of 17 ml/h. (--) Protein; (---- ) conductivity; (0 ~ e) collagenolytic activity

-9 -20 - 8 - 3 0 -7

-40 -6

-50 151" -60 - 4

- 3

-70 - 2 - -802

90 E

150 z 0

F- 8

m- 6

- 20 .g- -30

- 40 +- 5

-50 - 4 - 3

60

70

I 1:. 0 -._'-_ I ' 100 'L 100 150

Effluent volume (ml)

Fig. 5. I.roeic~ r u ( f i ic, i isi /rf i o/ the c t r ~ ~ ~ i i i e s in an Amnpholinr pH g d i o / ? r of'.?.?--- /O.O. Proteins were focused at up to 800 V over 48- 60 h in an L K B 110-ml column. which was then unloaded at a flow rate of 3.5 nil inin. (A) Cathcpsin B (0.05 unit). (B) Collageno- lytic cathepsin (0.08 unit). I 0) enzyme activity; ( ~ ) p f j

) Protein : (0-

separately in a 110-ml L K B column (Fig.5). Col- lagenolytic cathepsin was focused to a single peak of activity at pH 5.1 and cathepsin B to a peak at pH 5.4. Isoelectric focusing of crude samples of the latter enzyme (0.35 unit. derived from a 30-70% ammo- nium sulphate fraction) again showed a single peak of enzymatic activity near pH 5.5 and the presence of isoenzyme forms was not observed.

I 1 / \ 1 \\

I \ \

1 \\

2.4 2.8 3.2 3.6 4.0 4.4 PH

Fig. 6. T/7r elf& o f p H 017 co//ufic'no/~,ric m i i ~ i r r . Cathepsin B (0.002 collagenolytic unitlml) and collagenolytic cathcpsin (0.005 unit h i ) wcrc incubated 2.25 h and 3 h respectively at 37 C in 0.1 M sodium formate buffer containing 5 mM cysteine and 3.3 mg ml dispersed insoluble bovine tendon collagen. Release of solu ble hydrox! proline was determined as an index of collagenolytic activity. (0 0 ) Cathepsin B ; (A- A) collagenolytic cathepsin

p H Depmdmce and Action on Collrgrn Substrates

The pH activity profiles for cathepsin B and collagenolytic cathepsin with insoluble collagen as the substrate are shown in Fig.6. Both enzymes showed maximum activity near pH 3.3 with very little activity outside the pH range 2.5-4.0. To investigate the site of action of the enzyme in the collagen chains,

P. Evans and D. J. Etherington 93

S 3.0 3.5 4.0 4.5 5.0 6.0 7.0 S

S 3.0 3.5 4.0 4.5 5.0 6.0 7.0 S

Fig. 7. Sodium dodecyl suljiliuteipolyurr~~lurni~e-gel &ctrophor.rsis ofcollagen chains after enzymatic digestion. (A) Digestion of insoluble bovine tendon collagen at 28 'C and pH 3.5 by (a) cathepsin B (0.012 collagenolytic unitjml) and (b) collagenolytic cathepsin (0.011 unit/ml) for 7 h and 24 h. Untreated soluble collagen (S) was used as a standard. The positions of the sample origin (0) and the positions of the collagen chains (c(, [I, 7 ) are indicated. (B) Digestion of acid-soluble collagen by cathepsin B (0.014 collagenolytic unitjml) at 28 "C for 5 h at pH 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 and 7.0. (C) Digestion by collagenolytic cathepsin (0,011 unit/ml) under the same conditions

enzymatic digests of insoluble collagen were made at 28 "C pH 3.5, and analysed directly by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Predom- inantly a-chains were released into the supernatant in a

and show that intact (but slightly shortened) a-chains were released from the cross-linked fi and y-com- ponents over the pH range 3.0 - 6.0.

Assay of the Enzymes time-dependent manner (Fig. 7A). There were no soluble products in a control sample obtained from the incubation of insoluble collagen in the absence of Synthetic Low-Molecular- Weight Substrates

- added enzymes. Highly purified preparations of collagenolytic

Calf skin acid-soluble collagen was similarly di- cathepsin possessed no detectable Bz-Arg-2-Nap hy- gested over a wider pH range at 28 "C. The final con- drolase activity on prolonged incubation, although centrations of cathepsin B and collagenolytic cathepsin the spleen enzyme has been reported to possess a in these digests were 0.014 unit/ml and 0.011 unit/ml very low intrinsic activity against this substrate [15]. respectively. The results are presented in Fig. 7B, C Several specific substrates of cathepsin B having

94 Human Placental Cathepsin B and Collagenolytic Cathepsin

Table 2. Behaviour of’ the enzymes t0ward.v synthetic substrates Cathepsin B (0.06 unitlml) and collagenolytic cathepsin (0.030 unit/ ml) were incubated for up to 4 h with the above substrates at the stated concentrations and pH values. The activity values are expressed as the percentages of the value calculated for the Bz-Arg- 2-Nap substrate

Substrate Concn pH Activity Activity with col- lagenolytic cathepsin

Bz-Arg-2-Nap Z-Lys-4-ONp Z-Ala-4-ONp Z-Gly-4-ONp Arg-2-Nap Bz-Arg-4-Nan Arg-OMe Bz-Arg-OMe Lys-OMe Ac-Lys-OMe

m M

2.33 6.0 0.078 5.1 0.078 5.1 0.078 5.1 2.33 6.0 0.75 6.5 5.0 5.1 5.0 5.1 5.0 5.1 5 0 5.1

% 100 0

2218 0 1130 0 29 1 0 47 0 12 0 0 0 0 0 0 0 0 0

I \

ICathepsin B

i o l lagenolytic 0.6 1

2 tathepsin

3.5 10.5 11.5 12.5 loge (Molecular weight)

Fig. 8. Drtc~rminci/ion of’ rnolec~ulrr weight hy exclusion chromatog- ruphy on ,St,phac/i,.r G-IOO. Collagenolytic cathepsin (0.03 unit in 0.5 ml) and cathepsin B (0.07 unit in 0.5 ml) were applied separately to a column (1 x 100cm) of Sephadex G-I00 and pumped through the column at a rate of 5 ml/h. The ratio of the elution volume to the total fluid volume (VJV,) was plotted against log, (niolccular weight) for the marker proteins: (1) cyto- chrome c; (2) m5oglobin: (3) carbonic anhydrase; (4) ovalbumin; ( 5 ) bovine scruin albumin; (6) ovotransferrin; (7) blue dextrdn 2000 ( VO). Elution position for the two enzymes are marked by arrows

relatively fast turnover rates [24] were therefore tried as substrates for collagenolytic cathepsin (Table 2). No activity bas detected with any of the substrates and the collagenolytic assay still remains the sole means for detecting this enzyme.

Table 3. Effect of thiol-blocking reagents andinhihitors on cathepsin B and collagenolytic cathepsin Stock solutions of cathepsin B and collagenolytic cathepsin were preincubated at pH 6.0 with the listed substances and residual activity was determined against Bz-Arg-2-Nap at pH 6.0 and collagen at pH 3.5

Substance Final Inhibition ~~~ concn

cathepsin B collageno lyt1c cathepsin

Bz-Arg- collagen collagen 2-Nap pH 3.5 pH 3.5 pH 6.0

0 ’ mM /o

Iodoacetic acid 1 .O 97 91 86 Mercuric chloride 0.1 96 87 73 2,2’-Dipyridyl

disulphide 2.0 97 32 16

Antipain 0.1 90 88 87 Leupeptin 0.1 82 88 80

Effect of Potential Inhibitors

Stock solutions of cathepsin B (0.07 units/ml) and collagenolytic cathepsin (0.03 unitlml) were used. After preincubation with each inhibitor, samples were removed and assayed for activity against Bz-Arg-2- Nap (cathepsin B) and against collagen (cathepsin B and collagenolytic cathepsin). Inhibitors of thiol enzymes were tested, since both enzymes are bound to organomercurial-Sepharose-4B and cathepsin B has been shown previously to possess an essential thiol group [17,27,28]. Table 3 shows that both enzymes were strongly inhibited by the thiol reagents iodo- acetate and mercuric chloride and that both activities of cathepsin B were inhibited. 2,2’-Dipyridyl di- sulphide was slightly inhibitory to the collagenolytic activity of these enzymes possibly on account of the more prolonged assay for this activity which allows time for secondary effects to occur. The microbial inhibitors leupeptin and antipain [29] were potent inhibitors for both enzymes.

Molecular Weight Determinations

A Sephadex G-100 column (1.0 x 100 cm) equil- ibrated in 50 mM acetate, containing 0.2 M NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol at pH 5.5 was calibrated with blue dextran 2000 (200 OOO), ovotransferrin (77 000), bovine serum albumin (66 500), ovalbumin (43 500), carbonic anhydrase (30 OOO), myo- globin (17816), and cytochrome L‘ (13400). NADH was used for the location of the total fluid volume (V,) by measurement of absorbance at 340 nm. Averages of six separate determinations were taken for

P. Evans and D. J. Etherington 95

each enzyme with two or three determinations for the standards.

In Fig. 8 the ratio of elution volume ( Ve) and total fluid volume (V,) for the standard proteins was plotted against the log, (molecular weight) to provide a calibration line [30]. The molecular weight values for the standard proteins were taken from Edmund- son [31] and Smith [32]. Cathepsin B had a calculated molecular weight of 24 500 (range 24000 - 25 000) in close agreement with other reported values [15,17,27, 331. The molecular weight of collagenolytic cathepsin was found to be 34600 (range 33000-37000).

DISCUSSION

Collagenolytic cathepsin and cathepsin B have been extensively purified leading to preparations of high specific activity but which still contain a small amount of contaminant protein. The enzymes were readily separated on Amberlite CG-50 with cathepsin B being unadsorbed as found previously for the bovine spleen enzymes [15]. In this earlier investiga- tion, chromatography on Sephadex A-50 was employ- ed to complete the resolution of the two bovine cathepsins, however, the respective human enzymes were found not to be resolved by this method. In the isolation procedures for these enzymes, exclusion chromatography on Sephadex G-100 was used as the final purification step. These differences in behaviour of the enzyme on Sephadex A-50 probably arise from differences in isoelectric point. Cathepsin B has been shown here to be isoelectric at pH 5.4 which is similar to the reported values for this enzyme from other species [15,17]. Human collagenolytic cathepsin, however, had its isoelectric point at pH 5.1, in contrast to the value of pH 6.5 for the bovine spleen enzyme

Although isoenzymes of cathepsin B have been demonstrated in the foetal. membranes of human placenta [19], no isoenzymes were detected on iso- electric focusing of crude samples of the placental enzyme. Swanson et a/. [I81 described a single activity band for purified human placental cathepsin B on gel electrophoresis. There was no evidence to indicate that more than one species of the collagenolytic cathepsin was present.

The enzymes degraded both soluble and insoluble collagen to yield similar products at 28 "C. From sodium dodecyl sulphate/polyacrylamide gel electro- phoresis it was shown that slightly shortened M- chains were released from the cross-linked f l and y components, which established that cleavage had occurred only in the non-helical telopeptide regions. For soluble monomeric collagen this was shown to occur over a wider pH range of 3-6. With insoluble collagen as the substrate the pH optimum for solubili-

1151.

zation was around pH 3.3 when intact a-components were also shown to be released into the medium. It has been previously suggested [8] that the differ- ence in the pH activity range for degradation of insoluble and soluble collagen is because the sus- ceptible bonds in the telopeptide regions of insoluble collagen are inaccessible to catheptic cleavage until the fibres are swollen at low pH.

Activity towards collagen and towards the synthetic substrate for cathepsin B was abolished by sulphydryl inhibitors, suggesting that both enzymes are thiol proteinases. Values of percentage inhibition with 2,2'-dipyridyl disulphide were lower for collagen degradation than for Bz-Arg 2-Nap hydrolase activ- ity. The longer time scale required for the assay of collagenolytic activity, which included 2-mercapto- ethanol, may have permitted secondary effects to occur possibly from the establishment of equilibrating disulphides between 2-mercaptoethanol, the enzyme sulphydryl group and the added inhibitor. Except in the case of dithiothreitol, the equilibrium constants of such reactions are near unity [34].

An important difference in properties between cathepsin B and collagenolytic cathepsin was in their reactivity towards synthetic low-molecular-weight sub- strates. A number of such substrates have been tested for activity with collagenolytic cathepsin and none was found to be hydrolysed over the time scale normally employed for such assays. Cathepsin B hydrolysed glycine and alanine ester bonds as well as those provided by basic residues. The most susceptible peptide substrate for cathepsin B in this study was Z-Lys-ONp, which has been proposed previously as a sensitive substrate for this enzyme [24]. Comparing activity towards Bz-Arg-2-Nap and Arg-2-Nap, and the high activity towards the 4-nitrophenyl esters, it would appear that the aromatic N-acyl group facilitates binding of the substrate. Cathepsin B also shows leaving-group specificity wherein amino acids, simple aliphatic alcohols and amines are relatively ineffective in stimulating hydrolysis of Z-Lys-ONp in comparison to dipeptides [35]. This could explain why substrates such as Arg-OMe and Bz-Arg-OMe are not hydrolysed to any significant extent by this enzyme.

Leupeptin (Ac-Leu-Leu-ArgCHO) and antipain (Phe-Arg-Leu-ArgCHO) [29] were bound to both enzymes, since they cause inhibition both of the Bz-Arg-Nap hydrolase activity and the collageno- lytic activity. From its chemical structure antipain would be expected to be a more effective inhibitor for cathepsin B than leupeptin, but it may be that the con- centrations used (0.1 mM) were too high for such differences to be apparent. These inhibitors may form hemithioacetals at the essential thiol group. The forma- tion of hemithioacetals between aldehydes and active centre thiol groups of enzymes may be expected on chemical grounds, but has not been demonstrated in

96 Huindn Placental Cathepsin B and Collagenolytic Cathepain

proton nuclear magnetic resonance spectral studies of N-acetylaminoacetaldehyde-papain [36].

Collagenolytic cathepsin and cathepsin B possess significantly different molecular weights. The value for human placental cathepsin B was found to be 24500, which is in good agreement with value of 27500 for the human liver enzyme [17], and 26000 for the bovine spleen enzyme [I 51. Human collageno- lytic cathepsin exhibited a molecular weight of 34 600, whereas the molecular weight of the bovine enzyme was substantially lower at 20000 [15]. An enzyme described as collagenolytic cathepsin has been re- ported in experimental rat granuloma [37] and this had an estimated molecular weight of 38000. The possibility, therefore, that collagenolytic cathepsin may be a degradation product of cathepsin B as was originally suggested for the bovine enzyme [15] is clearly precluded in the case of the human enzyme, which had a higher molecular weight than cathepsin B. It seems unlikely that the catalytic centre of col- lagenolytic cathepsin is blocked in some way, since the enzyme is able to degrade collagen and the catalytic centre is accessible to low-molecular-weight inhibitors.

The isolation of two enzymes which degrade collagen in a similar manner raises questions of specif- icity. Collagenolytic cathepsins have been reported in rat and human liver [38], rat leukocyte granules [39], post-partum rat uterus [40], bovine spleen [15], rat granuloma [37 ] and rabbit polymorphonuclear leukocytes [41]. The location of this enzyme is probably lysosomal [42- 441. Etherington [I51 has found that, on mixing collagenolytic cathepsin with cathepsin B from bovine spleen, the resultant combined activity against type I collagen was greater than the sum of the individual activities. This suggests that the en- zymes may act on the substrate in a concerted manner by cleaving different bonds in the telopeptide region or conversely exhibiting different rates for the cleavage of the respective a1 and a2 chains. An ex- amination of the properties of the purified enzymes in regard to the site of degradation of the collagen molecule should elucidate the nature of this effect. Additional studies now in progress with specific inhibitors and defined substrates may also assist in an interpretation of the specificity differences at the active site of these two enzymes.

Both enzymes are active on collagen at low pH. The feasibility of such low extracellular pH values in processes involving collagen degradation is question- able, although Vaes [45] has shown that osteoclasts may produce a microenvironment into which lyso- soma1 enzymes are secreted. However, these enzymes are more probably involved in completing the dis- solution of collagen in the phagolysosomes subsequent to the initial extracellular action of collagenase and the neutral proteinases. In the phagolysosome the col-

lagenous fragments are already partially degraded which may facilitate acid-swelling of these fibrous fragments at the intralysosomal pH. It has been postulated recently [46] that lysosomal glycosidases may assist in the solubilization of fibrous collagen by removing proteoglycans. These associated substances which may stabilize fibres extracellularly could also provide a blockade to the enzyme sensitive regions in the collagen subunits.

This investigation was supported by a grant from the Arthritis and Rheumatism Council. We also thank Prof. I . A. Silver of the Department of Pathology for the provision of laboratory facilities and Prof. G. Dixon of the Department of Obstetrics and Gynaecolo- gy for making human placental tissue available to u5.

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P. Evans *, Comparative Pathology Laboratory, School of Veterinary Science, University of Bristol, Langford, Bristol, Great Britain, BS18 7DU

D. J . Etherington, Meat Research Institute, Langford, Bristol, Great Britain, BS18 7DY

* To whom correspondence should be addressed.