CELL BIOCHEMISTRY AND FUNCTION VOL.
9: 55-62 (1991)
Study of Thiol Proteases of Normal Human Skin Fibroblasts HUSSAIN A. KHALFAN Discipline of Biochemistry, College of Medicine and Medical Science, Arabian Gurf University, P.O.Box 22979, State of Bahrain
The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein,the thiol inhibitors and the affinity for concanavalin A-Sepharose. and N-aThe majority of the activity to N-benzyloxycarbonyl-~-phenylalanyl-~-arginyl-7-amido-4-methy~-coumarin benzyloxycarbonyl-~-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-t-phenylalanyl-~-arginyl-7amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with t-arginine-7-amido-4methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractionseluted by a-methyl+-mannoside were significantlyhydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose. KEY WORDS-Cathepsin
L; cathepsin B; cathepsin H; cysteine proteinase; substrate specificity; tissue culture fibroblasts.
A, concanavalin A; Z-Phe-Arg-AMC, N-benzyloxycarbonyl-L-phenylalanyl-r-arginyl-7-amido-4-methylcournarin; Z-Arg-Arg-AMC, N-benzyloxycarbonyl-~-arginyl-arginyl-7-arnido-4-methylcoumarin; Z-Arg-AMC, N-a-benzyloxycarbonyI-~-arginine-7-amido-4-rnethylcoumarin; Arg-AMC, L-arginine-7-amido-4-methylcoumarin; DTT, dithiothreitol; 4 MU-casein, 4-methylumbelliferyl-casein.
ABBREVIATIONS-COn
INTRODUCTION The importance of lysosomal enzymes is underlined by the existence of several genetic disorders, in which the deficiency of a hydrolase leads to lysosoma1 storage of its undergraded substrate, t o cellular pathology, and to clinical disease. Lysosomal proteases play an important role in the turnover of cell constituents in fibroblasts.'. The various cathepsins are assayed differentially by the use of an appropriate range of synthetic substrates combined with the selective use of inhibitors and activator^.^ The recognition of a proteolytic enzyme as a cysteine proteinase is based on its inhibition by leupeptin and antipain, by thiolblocking reagents, and by activation by thiol compounds.' In some cases, native or modified proteins are necessary as substrates, making differential assays difficult. For instance, cathepsin L, acting upon sort-lived cytosol proteins has been 0263-6484/91 /O10055-08$05.00 0 1991 by John Wiley & Sons, Ltd.
suggested to have the highest intracellular activity of all the cysteine proteases, but is not as effective with synthetic substrate^.'^^ In addition to their lysosomal location, proteases also occur in the cytosol and on the external surface of the lysosomal membrane where they may react with cytosolic components without their entering the lysosomal ~ o m p a r t m e n t .Other ~ as yet poorly defined proteases are essential for the maturation of newlyformed lysosomal enzyme precursors by limited proteolysis and this function is probably also carried out in the lysosome.6 Characteristically, most fibroblast lysosomal hydrolases are glycoproteins having a specific mannose phosphate signal by which they are translocated to the lysosomal apparatus via a receptormediated process (for a review see reference 7). This rule may not, however, apply to some of the
56
H. A. KHALFAN
pH 6.0, containing 4 m EDTA ~ and, where required, 8 mM dithiothreitol. The method of assay involves incubation for 1 h at the optimum pH for protease activity, precipitation of unhydrolysed casein and adjustment of the filtrate to pH 10.0 for fluorimetry. The fluorescence was measured with excitation 360 nm and emission 460 nm against a standard 0.5 p~ solution of 4-methylumbelliferone3-acetic acid.9 Assays with Z-Phe-Arg-AMC (for cathepsins B and L) and Z-Arg-Arg-AMC (unspecified thiol proteases) were carried out in the same buffer. Enzyme suitably diluted in 0.1 per cent Brij 35 solution (500 pL) and buffer (250 pL) was equilibrated at 37 "C and the reaction started by the addition of 250pL of 2 0 p ~substrate solution. MATERIALS AND METHODS After incubation for lOmin, the reaction was 7-Amino-4-methylcoumarin was prepared accord- stopped by the addition of 1 mL of 100 mM sodium ing to the method of Zimmerman et a/." N-Cbz-L chloroacetate in 100 mM acetate buffer, pH 4.3, and -arginine hydrochloride and N-Cbz-L-phenyla- the fluorescence was measured with excitation lanyl-~-arginyl-7-amido-4-methylcoumarin were 360 nm and emission at 450 nm against a standard purchased from Cambridge Research Biochemicals 0-5,UM solution of 7-amino-4-methylcoumarin in (U.K.). ~-Arginine-7-amido-4-methylcoumarin was the same buffer. prepared by catalytic hydrogenation in the presAssays with L-Arg-AMC (for cathepsin H) were ence of palladium/charcoal from N-a-Cbz-L-argin- carried out in essentially the same way, except that ine-7-amido-4-methylcoumarin (Z-Arg-AMC) the pH for incubation was 6.8. Assays with Z-Argpreviously synthesized according to Zimmerman et AMC, to check for trypsin contamination after a/.' N-Cbz-~-arginyl-arginyl-7-amido-4-methyiharvesting the cells, were carried out in essentially coumarin (Z-Arg-Arg-AMC) was synthesized by the same fashion, in 50 mM Tris/HCl buffer, pH 8.2 containing 20 mM CaCI, and with incubation peri~-arginyl-7-amido-4-methylcoumarin acylating with Cbz-arginine HCl by the mixed anhydride ods extended for as long as 160 min. method using isobutylchloroformate. 4-MU-casein was prepared according to the method of Khalfan Cell Culture et a/.' Palladium/charcoal(5 per cent) and polyethNormal human skin biopsies (1 cm') were obylene lauryl ether (Brij 35) were from BDH Poole, Dorset. Dithiothreitol, concanavalin A-sepharose tained at surgery. The skin was sliced into small 4B, Activated thiol-sepharose 4B, leupeptin, and pieces and the cells were dispersed following digesantipain were supplied by Sigma Chemical Compa- tion with collagenase (Boehringer Mannheim Biony Ltd., U.K. Pure cathepsin B was generously chemica, West Germany) at a concentration of given by Dr A. J. Barrett, Strangeways Laboratory, 2 mg mL-' at 37 "C for 6 h. The free cells were Cambridge, U.K. All other buffer constituents and cultured in buffered alpha-modification of minimal common chemicals were analytical grade. Tissue essential medium (Flow Laboratories, U.K.) supculture reagents were obtained from Flow Labora- plemented with 100 pg streptomycin, 100 units penicillin mL-', and 10 per cent fetal calf serum in tories, U.K. the presence of 5 per cent CO,. The confluent cells from the primary culture 10 days after culturing Protease Assays were harvested with 0.05 per cent buffered trypsinProtease assays with all the substrates and inhi- EDTA and subcultured. Cells were harvested with bitors were performed in triplicate and the mean 0.05 per cent buffered trypsin-EDTA, centrifuged at 600 g for 10 min, and washed four times at 4 "C values were recorded. Assays with 4-methylumbelliferyl casein were with 5 mL of 10 mM phosphate buffered saline, pH carried out in 0 . 4 ~KH,PO,/Na,HPO, buffer, 7.0. proteases with specific control functions mentioned above, particularly if they are membrane-associated, and at least one cathepsin (B), has been said not to be a typical glycoprotein on the basis of its lack of binding to concanavalin A.' The aim of this study was to examine the protease profile of cultured normal human skin fibroblasts with respect to their cellular location, using inhibitors and low molecular weight synthetic peptide substrates that have been said to be preferentially hydrolysed by one cathepsin or another, along with the conjugated protein substrate, 4methylumbelliferyl casein, that we have described for fluorimetric assay^.^
'
THIOL PROTEASES OF HUMAN FlBROBLASTS
57
The immobilized concanavalin A was then collected by transferring the suspension into a 1 x In experiments that measured the activity of 5 cm column and washed with the cold 50 mM proteases with synthetic and protein substrates, the sodium acetate/l mM EDTA buffer at a flow rate of cell pellet harvested as above was resuspended in 24mL h-' until no protease activity could be 50 mM sodium acetate buffet, pH 5.0, containing detected in the washings using Z-Phe-Arg-AMC 1 mM EDTA and sonicated by two 15-s pulses and Z-Arg-Arg-AMC as substrates. The column (amplitude 10pm) in an MSE ultrasonic disinte- was then eluted with 0.2 M a-methyl-D-mannoside grator while being cooled in crushed ice. The in 50 mM sodium acetate/l mM EDTA buffer, with supernatant was collected by centrifugation at collection of fractions 2 mL. 600 g for 10 rnin and the pellet discarded. In experiments involving measuring the activity of proteases in subcellular fractionation, harvested cells were Activated Thiol-Sepharose 4B Binding resuspended in 1 mL of ice-cold 0.25 M sucrose Experiments containing 2 mM EDTA and homogenized with a Washed cells from six flasks (surface area glass pestle in a Wheaton homogenizer, capacity 125 cm') were sonicated as described above in 7 mL. The homogenate was centrifuged at 650 g for 3 mL of cold 50 mM sodium acetate/l mM EDTA 10 min at 4 "C and the supernatant decanted and buffer, pH 5.0 and centrifuged at 650 g for 10 min. retained. This process was repeated twice more The supernatant was collected and stirred slowly with the sedimented unbroken cells and debris, with activated thiol-sepharose 4B for 1 h at 4 "C. using 0.5 mL of the above sucrose solution, and the The suspension was transferred into a 1 x 5cm three supernatants were pooled. The sediment was column and washed with cold 50 mM sodium aceresuspended in the above sucrose solution (2 mL). tate buffer containing 1 mM EDTA, pH 5.0 at a The pooled supernatants were then centrifuged flow rate of 24mL h-' until all the detectable at 19 OOO g for 30 rnin and decanted from the pellet protein emerged from the column and absorbance which was resuspended in 2 mL of ice-cold 0.25 M at 280 nm was zero. The washing was also assayed sucrose/:! mM EDTA (lysosome fraction). with Z-Phe-Arg-AMC substrate until no protease The decanted supernatant from this last step was activities were detected. The column was then termed the microsomal/cytoplasmic fraction. eluted with 25 mM L-cysteine in 50 mM sodium One mL of each of the three fractions was used acetate/l mM EDTA buffer, pH 5.0 with collection directly for enzyme assay, and the remaining ali- of 2 mL fractions and assayed again with the same quots were sonicated with two 15-s pulses at 10 pm substrate. amplitude in the MSE ultrasonic disintegrator while cooled in crushed ice. The sonicates were centrifuged at 650 g for 10 rnin and the superna- RESULTS tants collected for enzyme assay. Assays of pure cathepsin B at a concentration of 1 pg and 2 pg per assay showed no activity towards Concanavalin A Binding Experiments 4-MU-casein at pH 6.0. Sonicates of whole cells showed no activity toCells harvested from 16 flasks (125 cm2 surface area), were washed five times with cold 10mM wards 2-Arg-AMC or Arg-AMC when assayed for phosphate-buffered saline, pH 7-0.The pellets were up to 160 rnin at pH 6.0 at an enzyme concentraresuspended in 4 mL of 50 mM sodium acetate/ tion equivalent 3 x lo4 cells/assay. This suggests 1 m M EDTA, pH 5.0 and sonicated by two 15-s that there is no significant contamination with pulses as described above. The sonicateswere centri- trypsin from the harvesting procedure and that fuged at 650 g for 10 rnin and the supernatants were cathepsin H, for which Arg-AMC is a preferred collected and dialysed overnight against 2 L of the substrate12 does not make a significant contribusame buffer containing in addition 1 mM MgCI,, tion to the protease activity. The latter substrate 1 m M CaCl,, 1 mM MnCI,, and 0.2 M NaCI. An was tested again at a 100-fold increased enzyme aliquot (0.25 mL) of the dialysed solution was concentration with the same result. Z-Phe-Arg-AMC was, on the contrary, readily retained for enzyme assay and the remaining 3.75 mL were stirred with 5 mL packed volume of hydrolysed with detectable activity at these concenconcanavalin A-sepharose 4B for 60 rnin at 4 "C. trations within 10 min (2.5 x background) and sigCell Fractionation
58
H. A. KHALFAN
100
,5.1000
-
80
. 7
E
800
M
_I
.A 5
c
c
I d
e
60
a
v
600 d
: 2
40
400
0
u
: 200
20
-
p
y.
3.0
4.0
5.0
6.0
7.0
~
8.0
0.1
0
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
PH
.,
Figure 1. pH profiles of proteolytic activity in normal human skin fibroblasts towards synthetic peptides. Fibroblast sonicates were assayed in the presence of 8 mM DTT as described in the text. The substrates were 0 ,Z-Phe-Arg-AMC; A,Z-Arg-ArgAMC. 0, proteolytic activity in the absence of 8 mM DTT; Inhibition of protease activity with 1 ~ L Mleupeptin.
nificant hydrolysis in 40 min. The pH profile of this hydrolysis showed a maximum at 6.0 with less than 10 per cent of optimal activity at 3.0 (Figure 1). This activity was negligible in the absence of 8 mM DTT and was linear with time up to 60 min in its presence. The presence of leupeptin and antipain (Figure 2), at a concentration of 1 pm completely inhibited this thiol-dependent activity. Lower concentration of leupeptin produced 95 per cent inhibition at 0.25 p~ and 85 per cent inhibition at 0.1 p ~ Lower concentrations of antipain produced 90 per cent inhibition at 0.25 p~ and 80 per cent inhibition at 0.1 p ~ . Results with Z-Arg-Arg-AMC indicate a weakly positive activity in the presence of DTT and optimal at pH 6.0 (Figure l), and that extended periods of incubation (120 min) were necessary to achieve these results. When assayed with 4-methylumbelliferyl casein, the pH profile was biphasic, indicating activity at acid pH values as well as at 6.0. Significant hydrolysis was observed in the absence of DTT, but was however stimulated at both pHs in its presence (Figure 3). Cell fractionation studies using 4-methylumbelliferyl-casein as substrate are shown in Table 1. The results show that over 70 per cent the total activity towards this substrate is present in the lysosome
Inhibitor concentration (#If)
Figure 2. Inhibition characteristics of leupeptin and antipain on protease activity in normal human skin fibroblasts towards Z-Phe-Arg-AMC as described in text. 0 , leupeptin; A,antipain.
fraction and is only fully potentiated after sonication and in the presence of DTT. About half of this activity is expressed in the absence of DTT, and approximately 20 per cent of the total activity is represented by leupeptin-resistant activity in the lysosomal fraction.
.
u
60
:
00
2
20 0
3.0
.,
0.0
5.0
6.0
PH
7.0
8.0
.,
9.0
Figure 3. pH profiles of proteolytic activity in normal human skin fibroblasts towards 4-MU-casein. Experiments were performed as described in the text: A, no DTT; with 8 m ~ DTT; Inhibition of leupeptin (20 p M ) on protease activity towards 4-MU-casein.
59
THlOL PROTEASES OF HUMAN FIBROBLASTS
Table 1 .
Proteolytic activity of cell fractions from fibroblasts on 4 MU-casein
Fraction Debris + unbroken cells Lysosomes Microsomes + supernatant
+
( DTT) ( - DTT) ( DTT) ( - DTT) ( DTT) ( - DTT)
+ +
Sonicated
Unsonicated
12.8% ( 9.7- 15.6) 8.6 % ( 7.3 - 10.6) 72.9% (70.1-77.3) 38.9 % (33.1-45.2) 14.3% (13.0-16.7) 10.1 % ( 8.0-12.2)
5.2% (1.7-10.6) 4.0 % (1.2- 7.0) 11.0% (4.8-21.4) 7.8 % (2.7- 15.3) 8.8 % (8.0-10.0) 7.0% (5.8- 8.0)
Sonicated + leupeptin 6.3 % ( 5 4 7 . 9 ) -
19.7%(18.6-21.5) -
8.3% ( 5.5-11.1) -
Activity is expressed as a percentage of the total activity present in all fractions after sonication and in the presence of 8 mM DTT. Each result shown is the mean of three experiments (the range is in parentheses). Cell fractionation as was described in the text.
In order to assess what proportion of the total protease activity could be attributed to enzymes with glycoprotein characteristics, the degree of binding to immobilized concanavalin A was examined. Fibroblasts were grown to confluency in 16 flasks (1 25 cmz surface area), harvested, pooled, sonicated, and centrifuged as described, to yield 4 mL of supernatant. The equivalent of one flask (0.25 mL) was retained for total protease assay and the remainder adsorbed onto concanavalin Asepharose and eluted first with buffer and then with cr-methyl-D-mannoside solution as described. All fractions (2 mL) were assayed for protein (absorbance at 280 nm) and for protease activity towards Z-Phe-Arg-AMC (10-min incubation) and Z-ArgArg-AMC (30-min incubation). All the detectable protein emerged from the column in the first few fractions and absorbance was reduced to zero before application of the a-methyl-D-mannoside eluant. Figure 4 shows the elution profile for the two activities measured and indicates that the relative proportions of bound and unbound activity were the same and roughly equal, regardless of substrate, although as noted earlier the activity towards Z-Arg-Arg-AMC is approximately onetenth in all cases that for the more favourable substrate, Z-Phe-Arg-AMC. The fractions constituting the two peaks were pooled separately and assayed for their total activity compared with the retained sample of the original supernatant. These data are shown in Table 2, along with the corresponding results with 4-methylumbelliferyl casein. Both bound and unbound fractions were inhibited completely by antipain at 1 P M and more than 80 per cent inhibited at a final concentration of 0.1 ~ L M (Figure 4). The sonicate from six flasks (surface area 125 cm’) of confluent fibroblasts was loaded onto the activated thiol-sepharose 4B column, 2 mL-
fractions were collected for protein estimation (absorbance at 280 nm), and protease activity towards Z-Phe-Arg-AMC. The results showed that all detectable proteins emerged from the column in the first few fractions and absorbance was reduced to zero before application of the L-cysteine eluant. Figure 5 shows the elution profile for the two activities measured with Z-Phe-Arg-AMC substrate. It should be noted that the above experiments were successfully repeated. DISCUSSION The pH profile when assayed with 4-methylumbelliferyl casein indicated activities at both pH 3.3 and pH 6.0 (Figure 3), and significant hydrolysis was observed in the absence of DTT, but a further stimulation was observed in its presence. The thiolindpendent activity at pH 3.3 may indicate cathepsin D, while the stimulation at this pH in the presence of DTT is representative of cathepsin N. I assume the thiol-dependent component observed at pH 6.0 represents cathepsin L, since cathepsin B does not attack this substrate. The question arises whether the activity at pH 6.0 with this substrate, that is measurable in the absence of DTT, is the result of partially active thiol proteases interacting with endogenous activators, or some non-thiol-dependent species. The effect of leupeptin was therefore tested on this activity both in the absence and the presence of DTT. I t was found that a significant amount of hydrolytic activity was resistant to leupeptin inhibition even at concentrations as high as 20 ,UM. This result suggests that a further uncharacterized protease, functioning at pH 6.0 on the macromolecular substrate (4-methylumbellifery1casein) and not thiol-dependent, is present. This was further investigated in the cell fractionation studies using 4-methylumbelli-
60
H. A. KHALFAN
3000 h
m
Y
rl
c a
2000
01 U C
1000
01 0
m
k 01
0
a
3
k
0
10
20
30
40
SO
60
70
80
90
100
110
Fraction n u m b e r
Figure 4. Fractionation of proteolytic activity of normal human skin fibroblasts on concanavalin A-sepharose 4B. Fibroblast sonicate was adsorbed onto con A-sepharose as described under Methods, and eluted with 50 mM sodium acetate/l mM EDTA buffer, pH 5.0 to zero AZBObefore applying 0.2 M a-methyl-D-mannoside at the point indicated. Individual fractions were 2 mL: 0, assayed with Z-Phe-Arg-AMC (10-min incubation); A,assayed with Z-Arg-Arg-AMC (30-min incubation). 0 , Inhibition by antipain ( I PM) on protease activity in both bound and unbound fractions.
feryl-casein as substrate as shown in Table 1. The activity toward this substrate in the lysosomal fraction amounted to over 70 per cent of the total activity and is fully potentiated after sonication and in the presence of DTT. About half of the activity was measured in the absence of DTT, and aproximately 20 per cent of the total activity is represented by leupeptin-resistant activity in the lysosomal fraction. The effects of sonication, thiol activation, and leupeptin sensitivity are less evident in the other fractions, but some potentiation in the pellet after
sonication can be attributed to unbroken cells and lysosomes present because of the gentle homogenization procedures. Since the 4-methylumbelliferylcasein substrate used is insensitive to cathepsin By the activity measured here (Table 1) is probably due to cathepsin L or a similar activity. The fact that this macromolecular substrate (4methylumbelliferyl-casein) cannot penetrate the lysosomal membrane by diffusion explains the high degree of latency observed. This suggests that the small amount of activity recorded in the lysosomal fraction before sonication may be significant and
Table 2. Proportional binding to concanavalin A of proteolytic activity of sonicated fibroblasts
Substrate Original sample Unbound fractions Bound fractions
nmol of free 7-amino-4-methylcoumarin min-' (nmol rnin-' liberated) Z-Arg-Arg-AMC ( 5 POM) Z-Phe-Arg-AMC ( 5 PM) 10.4 5.6
4.8
0.57 0.33 0.22
nmol min-' 4-MU liberated as TCA soluble peptides 4-MU-casein 0.2 x 10-3
0 0.2 x 10-3
61
THlOL PROTEASES OF HUMAN FIBROBLASTS
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30 32
34 36
38
F r a c t i o n number
Figure 5. Fractionation of proteolytic activity of normal human skin fibroblasts on activated thiol-sepharose 4B. Fibroblast sonicate was adsorbed onto activated thiol-sepharose 4B as described under Methods, and eluted with 50 mM sodium acetate/l mM EDTA buffer, pH 5.0 to zero A2*,, before applying 25 mM L-cysteine at the point indicated. Individual fractions (2 mL) were assayed with Z-Phe-Arg-AMC (10-min incubation) as described under the Methods.
represent proteases such as cathepsin M, which are said to be located in the cytosolic surface of the membrane. The fractions constituting the two peaks in concanavalin A binding experiment (Figure 4) were pooled separately and assayed for total activity toward Z-Phe-Arg-AMC and Z-Arg-Arg-AMC substrates. In this case, only the bound (glycoprotein) fractions showed significant proteolytic activity towards 4-MU-casein substrate compared with total activity from the retained original sample (Table 2). Presumably the first fractions may represent cathepsin B enzyme activity which is said to be a non-glycoprotein in nature, whereas the second fractions eluted by a-methyl-D-mannoside may represent cathepsin L activity. In order to ensure that lack of binding was a characteristic of the unretained peak and not an artefact of overloading the immobilized concanavalin A, these fractions were re-treated with a fresh sample of the immobilized lectin in the same
fashion. Over 95 per cent of the applied activity remained unbound. The fractions constituting the two peaks in the activated thiol-sepharose 4B binding experiment (Figure 5) were pooled separately and assayed with the Z-Phe-Arg-AMC substrate. The first fractions collected before the application of L-cysteine may be attributed to the presence of some endogenous thiol in the sonicate suspension or due to overloading the capacity of the immobilized activated thiolsepharose 4B. The second fraction resulting from elution with L-cysteine was obviously due to the presence of thiol proteases which act on the Z-PheArg-AMC substrate. The main conclusions of this study are that thiol proteases, optimally active on synthetic substrates at pH 6.0, constitute the bulk of the lysosomal activity. This activity is probably largely accounted for by cathepsin B and cathepsin L. The thioldependent component which degraded the protein substrate (4-MU-casein) observed at pH 6.0 repre-
62
H. A. KHALFAN
sents cathemin L. since cathemin B does not attack this substrate. Cathepsin H dould not be detected, was present in comparatively and cathepsin small amounts. There was evidence that an as yet unidentified non-thiol protease may contribute significantly to the activity at pH 6.0.
5.
6. 7.
REFERENCES Kirschke, H., Langner, J., Riemann, S., Wiederanders, B., Ansorge, S. and Bohley P. (1980) Lysosomal cysteine proteinases. In: Protein Degradation in Health and Disease (Ciba Foundation Symposium 7 9 , North-Holland: Amsterdam, pp. 15-35. Cockle, S. M. and Dean, R. T. (1982). The regulation of proteolysis in normal fibroblasts as they approach confluence. Evidence for the participation of the lysosomal system. Biochem. J., 208,795-800. Barrett, A. J. (1977). Cathepsin B and other thiol proteinases. In: Proteinases in Mammalian Cells and Tissues (Barret, A. J., ed.), North Holland: Amsterdam, pp. 18 1-208. Kirschke, H., Schmidt, I. and Wiederanders, B. (1986). Cathepsin S. The cysteine proteinase from bovine lymphoid tissue is distinct from cathepsin L. Biochem. J., 240, 455-459.
8. 9.
10. 11.
12
Pontremoli. S.. Melloni. E.. Damiani. G.. Michetti. M.. Salamino, F., Sparatore, B.' and Horecker, B. L. (1984): Binding of monoclonal antibody to cathepsin M located on the external surface of rabbit lysosomes. Arch. Biochem. Biophys., 233,267-271. Frisch, A. and Neufeld, E. F. (1981). Limited proteolysis of the beta-hexosaminidase precursor in a cell-free system. J . Biol. Chem., 256, 8242-8246. Strawser, L. D. and Touster, 0. (1980). The cellular processing of lysosomal enzymes and related proteins. Rev. Physiol. Biochem. Pharmacol., 87, 169-210. Barrett, A. J. and Kirschke, H. (1981). Cathepsin B, cathepsin H and cathepsin L. In: Methods in Enzymology,VoL 80, (Lorand, L., ed.) Academic Press: New York,pp. 535-561. Khalfen, H., Abuknesha, R. and Robinson, D. (1983). Fluorigenic method for the assay of proteinase activity with the use of 4-methyl-umbelliferyl-casein. Biochem. J., 209, 265-267. Zimrnerman, M., Yurewica, E. and Patel, G. (1976). A new fluorogenic substrate for chymotrypsin. Anal. Biochem., 70, 258-262. Zimmerman, M., Ashe, B., Yurewicz, E. C. and Patel, G. (1977). Sensitive assays for trypsin, elastase and chymotrypsin using new fluorogenic substrate. Anal. Biochem., 78, 47-51. Schwartz, W. N. and Barrett, A. J. (1980). Human cathepsin H. Biochem. J., 191 ,487-497.
Receiued in revisedjorm 13 July 1990 Accepted 27 August 1990
9: 55-62 (1991)
Study of Thiol Proteases of Normal Human Skin Fibroblasts HUSSAIN A. KHALFAN Discipline of Biochemistry, College of Medicine and Medical Science, Arabian Gurf University, P.O.Box 22979, State of Bahrain
The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein,the thiol inhibitors and the affinity for concanavalin A-Sepharose. and N-aThe majority of the activity to N-benzyloxycarbonyl-~-phenylalanyl-~-arginyl-7-amido-4-methy~-coumarin benzyloxycarbonyl-~-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-t-phenylalanyl-~-arginyl-7amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with t-arginine-7-amido-4methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractionseluted by a-methyl+-mannoside were significantlyhydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose. KEY WORDS-Cathepsin
L; cathepsin B; cathepsin H; cysteine proteinase; substrate specificity; tissue culture fibroblasts.
A, concanavalin A; Z-Phe-Arg-AMC, N-benzyloxycarbonyl-L-phenylalanyl-r-arginyl-7-amido-4-methylcournarin; Z-Arg-Arg-AMC, N-benzyloxycarbonyl-~-arginyl-arginyl-7-arnido-4-methylcoumarin; Z-Arg-AMC, N-a-benzyloxycarbonyI-~-arginine-7-amido-4-rnethylcoumarin; Arg-AMC, L-arginine-7-amido-4-methylcoumarin; DTT, dithiothreitol; 4 MU-casein, 4-methylumbelliferyl-casein.
ABBREVIATIONS-COn
INTRODUCTION The importance of lysosomal enzymes is underlined by the existence of several genetic disorders, in which the deficiency of a hydrolase leads to lysosoma1 storage of its undergraded substrate, t o cellular pathology, and to clinical disease. Lysosomal proteases play an important role in the turnover of cell constituents in fibroblasts.'. The various cathepsins are assayed differentially by the use of an appropriate range of synthetic substrates combined with the selective use of inhibitors and activator^.^ The recognition of a proteolytic enzyme as a cysteine proteinase is based on its inhibition by leupeptin and antipain, by thiolblocking reagents, and by activation by thiol compounds.' In some cases, native or modified proteins are necessary as substrates, making differential assays difficult. For instance, cathepsin L, acting upon sort-lived cytosol proteins has been 0263-6484/91 /O10055-08$05.00 0 1991 by John Wiley & Sons, Ltd.
suggested to have the highest intracellular activity of all the cysteine proteases, but is not as effective with synthetic substrate^.'^^ In addition to their lysosomal location, proteases also occur in the cytosol and on the external surface of the lysosomal membrane where they may react with cytosolic components without their entering the lysosomal ~ o m p a r t m e n t .Other ~ as yet poorly defined proteases are essential for the maturation of newlyformed lysosomal enzyme precursors by limited proteolysis and this function is probably also carried out in the lysosome.6 Characteristically, most fibroblast lysosomal hydrolases are glycoproteins having a specific mannose phosphate signal by which they are translocated to the lysosomal apparatus via a receptormediated process (for a review see reference 7). This rule may not, however, apply to some of the
56
H. A. KHALFAN
pH 6.0, containing 4 m EDTA ~ and, where required, 8 mM dithiothreitol. The method of assay involves incubation for 1 h at the optimum pH for protease activity, precipitation of unhydrolysed casein and adjustment of the filtrate to pH 10.0 for fluorimetry. The fluorescence was measured with excitation 360 nm and emission 460 nm against a standard 0.5 p~ solution of 4-methylumbelliferone3-acetic acid.9 Assays with Z-Phe-Arg-AMC (for cathepsins B and L) and Z-Arg-Arg-AMC (unspecified thiol proteases) were carried out in the same buffer. Enzyme suitably diluted in 0.1 per cent Brij 35 solution (500 pL) and buffer (250 pL) was equilibrated at 37 "C and the reaction started by the addition of 250pL of 2 0 p ~substrate solution. MATERIALS AND METHODS After incubation for lOmin, the reaction was 7-Amino-4-methylcoumarin was prepared accord- stopped by the addition of 1 mL of 100 mM sodium ing to the method of Zimmerman et a/." N-Cbz-L chloroacetate in 100 mM acetate buffer, pH 4.3, and -arginine hydrochloride and N-Cbz-L-phenyla- the fluorescence was measured with excitation lanyl-~-arginyl-7-amido-4-methylcoumarin were 360 nm and emission at 450 nm against a standard purchased from Cambridge Research Biochemicals 0-5,UM solution of 7-amino-4-methylcoumarin in (U.K.). ~-Arginine-7-amido-4-methylcoumarin was the same buffer. prepared by catalytic hydrogenation in the presAssays with L-Arg-AMC (for cathepsin H) were ence of palladium/charcoal from N-a-Cbz-L-argin- carried out in essentially the same way, except that ine-7-amido-4-methylcoumarin (Z-Arg-AMC) the pH for incubation was 6.8. Assays with Z-Argpreviously synthesized according to Zimmerman et AMC, to check for trypsin contamination after a/.' N-Cbz-~-arginyl-arginyl-7-amido-4-methyiharvesting the cells, were carried out in essentially coumarin (Z-Arg-Arg-AMC) was synthesized by the same fashion, in 50 mM Tris/HCl buffer, pH 8.2 containing 20 mM CaCI, and with incubation peri~-arginyl-7-amido-4-methylcoumarin acylating with Cbz-arginine HCl by the mixed anhydride ods extended for as long as 160 min. method using isobutylchloroformate. 4-MU-casein was prepared according to the method of Khalfan Cell Culture et a/.' Palladium/charcoal(5 per cent) and polyethNormal human skin biopsies (1 cm') were obylene lauryl ether (Brij 35) were from BDH Poole, Dorset. Dithiothreitol, concanavalin A-sepharose tained at surgery. The skin was sliced into small 4B, Activated thiol-sepharose 4B, leupeptin, and pieces and the cells were dispersed following digesantipain were supplied by Sigma Chemical Compa- tion with collagenase (Boehringer Mannheim Biony Ltd., U.K. Pure cathepsin B was generously chemica, West Germany) at a concentration of given by Dr A. J. Barrett, Strangeways Laboratory, 2 mg mL-' at 37 "C for 6 h. The free cells were Cambridge, U.K. All other buffer constituents and cultured in buffered alpha-modification of minimal common chemicals were analytical grade. Tissue essential medium (Flow Laboratories, U.K.) supculture reagents were obtained from Flow Labora- plemented with 100 pg streptomycin, 100 units penicillin mL-', and 10 per cent fetal calf serum in tories, U.K. the presence of 5 per cent CO,. The confluent cells from the primary culture 10 days after culturing Protease Assays were harvested with 0.05 per cent buffered trypsinProtease assays with all the substrates and inhi- EDTA and subcultured. Cells were harvested with bitors were performed in triplicate and the mean 0.05 per cent buffered trypsin-EDTA, centrifuged at 600 g for 10 min, and washed four times at 4 "C values were recorded. Assays with 4-methylumbelliferyl casein were with 5 mL of 10 mM phosphate buffered saline, pH carried out in 0 . 4 ~KH,PO,/Na,HPO, buffer, 7.0. proteases with specific control functions mentioned above, particularly if they are membrane-associated, and at least one cathepsin (B), has been said not to be a typical glycoprotein on the basis of its lack of binding to concanavalin A.' The aim of this study was to examine the protease profile of cultured normal human skin fibroblasts with respect to their cellular location, using inhibitors and low molecular weight synthetic peptide substrates that have been said to be preferentially hydrolysed by one cathepsin or another, along with the conjugated protein substrate, 4methylumbelliferyl casein, that we have described for fluorimetric assay^.^
'
THIOL PROTEASES OF HUMAN FlBROBLASTS
57
The immobilized concanavalin A was then collected by transferring the suspension into a 1 x In experiments that measured the activity of 5 cm column and washed with the cold 50 mM proteases with synthetic and protein substrates, the sodium acetate/l mM EDTA buffer at a flow rate of cell pellet harvested as above was resuspended in 24mL h-' until no protease activity could be 50 mM sodium acetate buffet, pH 5.0, containing detected in the washings using Z-Phe-Arg-AMC 1 mM EDTA and sonicated by two 15-s pulses and Z-Arg-Arg-AMC as substrates. The column (amplitude 10pm) in an MSE ultrasonic disinte- was then eluted with 0.2 M a-methyl-D-mannoside grator while being cooled in crushed ice. The in 50 mM sodium acetate/l mM EDTA buffer, with supernatant was collected by centrifugation at collection of fractions 2 mL. 600 g for 10 rnin and the pellet discarded. In experiments involving measuring the activity of proteases in subcellular fractionation, harvested cells were Activated Thiol-Sepharose 4B Binding resuspended in 1 mL of ice-cold 0.25 M sucrose Experiments containing 2 mM EDTA and homogenized with a Washed cells from six flasks (surface area glass pestle in a Wheaton homogenizer, capacity 125 cm') were sonicated as described above in 7 mL. The homogenate was centrifuged at 650 g for 3 mL of cold 50 mM sodium acetate/l mM EDTA 10 min at 4 "C and the supernatant decanted and buffer, pH 5.0 and centrifuged at 650 g for 10 min. retained. This process was repeated twice more The supernatant was collected and stirred slowly with the sedimented unbroken cells and debris, with activated thiol-sepharose 4B for 1 h at 4 "C. using 0.5 mL of the above sucrose solution, and the The suspension was transferred into a 1 x 5cm three supernatants were pooled. The sediment was column and washed with cold 50 mM sodium aceresuspended in the above sucrose solution (2 mL). tate buffer containing 1 mM EDTA, pH 5.0 at a The pooled supernatants were then centrifuged flow rate of 24mL h-' until all the detectable at 19 OOO g for 30 rnin and decanted from the pellet protein emerged from the column and absorbance which was resuspended in 2 mL of ice-cold 0.25 M at 280 nm was zero. The washing was also assayed sucrose/:! mM EDTA (lysosome fraction). with Z-Phe-Arg-AMC substrate until no protease The decanted supernatant from this last step was activities were detected. The column was then termed the microsomal/cytoplasmic fraction. eluted with 25 mM L-cysteine in 50 mM sodium One mL of each of the three fractions was used acetate/l mM EDTA buffer, pH 5.0 with collection directly for enzyme assay, and the remaining ali- of 2 mL fractions and assayed again with the same quots were sonicated with two 15-s pulses at 10 pm substrate. amplitude in the MSE ultrasonic disintegrator while cooled in crushed ice. The sonicates were centrifuged at 650 g for 10 rnin and the superna- RESULTS tants collected for enzyme assay. Assays of pure cathepsin B at a concentration of 1 pg and 2 pg per assay showed no activity towards Concanavalin A Binding Experiments 4-MU-casein at pH 6.0. Sonicates of whole cells showed no activity toCells harvested from 16 flasks (125 cm2 surface area), were washed five times with cold 10mM wards 2-Arg-AMC or Arg-AMC when assayed for phosphate-buffered saline, pH 7-0.The pellets were up to 160 rnin at pH 6.0 at an enzyme concentraresuspended in 4 mL of 50 mM sodium acetate/ tion equivalent 3 x lo4 cells/assay. This suggests 1 m M EDTA, pH 5.0 and sonicated by two 15-s that there is no significant contamination with pulses as described above. The sonicateswere centri- trypsin from the harvesting procedure and that fuged at 650 g for 10 rnin and the supernatants were cathepsin H, for which Arg-AMC is a preferred collected and dialysed overnight against 2 L of the substrate12 does not make a significant contribusame buffer containing in addition 1 mM MgCI,, tion to the protease activity. The latter substrate 1 m M CaCl,, 1 mM MnCI,, and 0.2 M NaCI. An was tested again at a 100-fold increased enzyme aliquot (0.25 mL) of the dialysed solution was concentration with the same result. Z-Phe-Arg-AMC was, on the contrary, readily retained for enzyme assay and the remaining 3.75 mL were stirred with 5 mL packed volume of hydrolysed with detectable activity at these concenconcanavalin A-sepharose 4B for 60 rnin at 4 "C. trations within 10 min (2.5 x background) and sigCell Fractionation
58
H. A. KHALFAN
100
,5.1000
-
80
. 7
E
800
M
_I
.A 5
c
c
I d
e
60
a
v
600 d
: 2
40
400
0
u
: 200
20
-
p
y.
3.0
4.0
5.0
6.0
7.0
~
8.0
0.1
0
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
PH
.,
Figure 1. pH profiles of proteolytic activity in normal human skin fibroblasts towards synthetic peptides. Fibroblast sonicates were assayed in the presence of 8 mM DTT as described in the text. The substrates were 0 ,Z-Phe-Arg-AMC; A,Z-Arg-ArgAMC. 0, proteolytic activity in the absence of 8 mM DTT; Inhibition of protease activity with 1 ~ L Mleupeptin.
nificant hydrolysis in 40 min. The pH profile of this hydrolysis showed a maximum at 6.0 with less than 10 per cent of optimal activity at 3.0 (Figure 1). This activity was negligible in the absence of 8 mM DTT and was linear with time up to 60 min in its presence. The presence of leupeptin and antipain (Figure 2), at a concentration of 1 pm completely inhibited this thiol-dependent activity. Lower concentration of leupeptin produced 95 per cent inhibition at 0.25 p~ and 85 per cent inhibition at 0.1 p ~ Lower concentrations of antipain produced 90 per cent inhibition at 0.25 p~ and 80 per cent inhibition at 0.1 p ~ . Results with Z-Arg-Arg-AMC indicate a weakly positive activity in the presence of DTT and optimal at pH 6.0 (Figure l), and that extended periods of incubation (120 min) were necessary to achieve these results. When assayed with 4-methylumbelliferyl casein, the pH profile was biphasic, indicating activity at acid pH values as well as at 6.0. Significant hydrolysis was observed in the absence of DTT, but was however stimulated at both pHs in its presence (Figure 3). Cell fractionation studies using 4-methylumbelliferyl-casein as substrate are shown in Table 1. The results show that over 70 per cent the total activity towards this substrate is present in the lysosome
Inhibitor concentration (#If)
Figure 2. Inhibition characteristics of leupeptin and antipain on protease activity in normal human skin fibroblasts towards Z-Phe-Arg-AMC as described in text. 0 , leupeptin; A,antipain.
fraction and is only fully potentiated after sonication and in the presence of DTT. About half of this activity is expressed in the absence of DTT, and approximately 20 per cent of the total activity is represented by leupeptin-resistant activity in the lysosomal fraction.
.
u
60
:
00
2
20 0
3.0
.,
0.0
5.0
6.0
PH
7.0
8.0
.,
9.0
Figure 3. pH profiles of proteolytic activity in normal human skin fibroblasts towards 4-MU-casein. Experiments were performed as described in the text: A, no DTT; with 8 m ~ DTT; Inhibition of leupeptin (20 p M ) on protease activity towards 4-MU-casein.
59
THlOL PROTEASES OF HUMAN FIBROBLASTS
Table 1 .
Proteolytic activity of cell fractions from fibroblasts on 4 MU-casein
Fraction Debris + unbroken cells Lysosomes Microsomes + supernatant
+
( DTT) ( - DTT) ( DTT) ( - DTT) ( DTT) ( - DTT)
+ +
Sonicated
Unsonicated
12.8% ( 9.7- 15.6) 8.6 % ( 7.3 - 10.6) 72.9% (70.1-77.3) 38.9 % (33.1-45.2) 14.3% (13.0-16.7) 10.1 % ( 8.0-12.2)
5.2% (1.7-10.6) 4.0 % (1.2- 7.0) 11.0% (4.8-21.4) 7.8 % (2.7- 15.3) 8.8 % (8.0-10.0) 7.0% (5.8- 8.0)
Sonicated + leupeptin 6.3 % ( 5 4 7 . 9 ) -
19.7%(18.6-21.5) -
8.3% ( 5.5-11.1) -
Activity is expressed as a percentage of the total activity present in all fractions after sonication and in the presence of 8 mM DTT. Each result shown is the mean of three experiments (the range is in parentheses). Cell fractionation as was described in the text.
In order to assess what proportion of the total protease activity could be attributed to enzymes with glycoprotein characteristics, the degree of binding to immobilized concanavalin A was examined. Fibroblasts were grown to confluency in 16 flasks (1 25 cmz surface area), harvested, pooled, sonicated, and centrifuged as described, to yield 4 mL of supernatant. The equivalent of one flask (0.25 mL) was retained for total protease assay and the remainder adsorbed onto concanavalin Asepharose and eluted first with buffer and then with cr-methyl-D-mannoside solution as described. All fractions (2 mL) were assayed for protein (absorbance at 280 nm) and for protease activity towards Z-Phe-Arg-AMC (10-min incubation) and Z-ArgArg-AMC (30-min incubation). All the detectable protein emerged from the column in the first few fractions and absorbance was reduced to zero before application of the a-methyl-D-mannoside eluant. Figure 4 shows the elution profile for the two activities measured and indicates that the relative proportions of bound and unbound activity were the same and roughly equal, regardless of substrate, although as noted earlier the activity towards Z-Arg-Arg-AMC is approximately onetenth in all cases that for the more favourable substrate, Z-Phe-Arg-AMC. The fractions constituting the two peaks were pooled separately and assayed for their total activity compared with the retained sample of the original supernatant. These data are shown in Table 2, along with the corresponding results with 4-methylumbelliferyl casein. Both bound and unbound fractions were inhibited completely by antipain at 1 P M and more than 80 per cent inhibited at a final concentration of 0.1 ~ L M (Figure 4). The sonicate from six flasks (surface area 125 cm’) of confluent fibroblasts was loaded onto the activated thiol-sepharose 4B column, 2 mL-
fractions were collected for protein estimation (absorbance at 280 nm), and protease activity towards Z-Phe-Arg-AMC. The results showed that all detectable proteins emerged from the column in the first few fractions and absorbance was reduced to zero before application of the L-cysteine eluant. Figure 5 shows the elution profile for the two activities measured with Z-Phe-Arg-AMC substrate. It should be noted that the above experiments were successfully repeated. DISCUSSION The pH profile when assayed with 4-methylumbelliferyl casein indicated activities at both pH 3.3 and pH 6.0 (Figure 3), and significant hydrolysis was observed in the absence of DTT, but a further stimulation was observed in its presence. The thiolindpendent activity at pH 3.3 may indicate cathepsin D, while the stimulation at this pH in the presence of DTT is representative of cathepsin N. I assume the thiol-dependent component observed at pH 6.0 represents cathepsin L, since cathepsin B does not attack this substrate. The question arises whether the activity at pH 6.0 with this substrate, that is measurable in the absence of DTT, is the result of partially active thiol proteases interacting with endogenous activators, or some non-thiol-dependent species. The effect of leupeptin was therefore tested on this activity both in the absence and the presence of DTT. I t was found that a significant amount of hydrolytic activity was resistant to leupeptin inhibition even at concentrations as high as 20 ,UM. This result suggests that a further uncharacterized protease, functioning at pH 6.0 on the macromolecular substrate (4-methylumbellifery1casein) and not thiol-dependent, is present. This was further investigated in the cell fractionation studies using 4-methylumbelli-
60
H. A. KHALFAN
3000 h
m
Y
rl
c a
2000
01 U C
1000
01 0
m
k 01
0
a
3
k
0
10
20
30
40
SO
60
70
80
90
100
110
Fraction n u m b e r
Figure 4. Fractionation of proteolytic activity of normal human skin fibroblasts on concanavalin A-sepharose 4B. Fibroblast sonicate was adsorbed onto con A-sepharose as described under Methods, and eluted with 50 mM sodium acetate/l mM EDTA buffer, pH 5.0 to zero AZBObefore applying 0.2 M a-methyl-D-mannoside at the point indicated. Individual fractions were 2 mL: 0, assayed with Z-Phe-Arg-AMC (10-min incubation); A,assayed with Z-Arg-Arg-AMC (30-min incubation). 0 , Inhibition by antipain ( I PM) on protease activity in both bound and unbound fractions.
feryl-casein as substrate as shown in Table 1. The activity toward this substrate in the lysosomal fraction amounted to over 70 per cent of the total activity and is fully potentiated after sonication and in the presence of DTT. About half of the activity was measured in the absence of DTT, and aproximately 20 per cent of the total activity is represented by leupeptin-resistant activity in the lysosomal fraction. The effects of sonication, thiol activation, and leupeptin sensitivity are less evident in the other fractions, but some potentiation in the pellet after
sonication can be attributed to unbroken cells and lysosomes present because of the gentle homogenization procedures. Since the 4-methylumbelliferylcasein substrate used is insensitive to cathepsin By the activity measured here (Table 1) is probably due to cathepsin L or a similar activity. The fact that this macromolecular substrate (4methylumbelliferyl-casein) cannot penetrate the lysosomal membrane by diffusion explains the high degree of latency observed. This suggests that the small amount of activity recorded in the lysosomal fraction before sonication may be significant and
Table 2. Proportional binding to concanavalin A of proteolytic activity of sonicated fibroblasts
Substrate Original sample Unbound fractions Bound fractions
nmol of free 7-amino-4-methylcoumarin min-' (nmol rnin-' liberated) Z-Arg-Arg-AMC ( 5 POM) Z-Phe-Arg-AMC ( 5 PM) 10.4 5.6
4.8
0.57 0.33 0.22
nmol min-' 4-MU liberated as TCA soluble peptides 4-MU-casein 0.2 x 10-3
0 0.2 x 10-3
61
THlOL PROTEASES OF HUMAN FIBROBLASTS
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30 32
34 36
38
F r a c t i o n number
Figure 5. Fractionation of proteolytic activity of normal human skin fibroblasts on activated thiol-sepharose 4B. Fibroblast sonicate was adsorbed onto activated thiol-sepharose 4B as described under Methods, and eluted with 50 mM sodium acetate/l mM EDTA buffer, pH 5.0 to zero A2*,, before applying 25 mM L-cysteine at the point indicated. Individual fractions (2 mL) were assayed with Z-Phe-Arg-AMC (10-min incubation) as described under the Methods.
represent proteases such as cathepsin M, which are said to be located in the cytosolic surface of the membrane. The fractions constituting the two peaks in concanavalin A binding experiment (Figure 4) were pooled separately and assayed for total activity toward Z-Phe-Arg-AMC and Z-Arg-Arg-AMC substrates. In this case, only the bound (glycoprotein) fractions showed significant proteolytic activity towards 4-MU-casein substrate compared with total activity from the retained original sample (Table 2). Presumably the first fractions may represent cathepsin B enzyme activity which is said to be a non-glycoprotein in nature, whereas the second fractions eluted by a-methyl-D-mannoside may represent cathepsin L activity. In order to ensure that lack of binding was a characteristic of the unretained peak and not an artefact of overloading the immobilized concanavalin A, these fractions were re-treated with a fresh sample of the immobilized lectin in the same
fashion. Over 95 per cent of the applied activity remained unbound. The fractions constituting the two peaks in the activated thiol-sepharose 4B binding experiment (Figure 5) were pooled separately and assayed with the Z-Phe-Arg-AMC substrate. The first fractions collected before the application of L-cysteine may be attributed to the presence of some endogenous thiol in the sonicate suspension or due to overloading the capacity of the immobilized activated thiolsepharose 4B. The second fraction resulting from elution with L-cysteine was obviously due to the presence of thiol proteases which act on the Z-PheArg-AMC substrate. The main conclusions of this study are that thiol proteases, optimally active on synthetic substrates at pH 6.0, constitute the bulk of the lysosomal activity. This activity is probably largely accounted for by cathepsin B and cathepsin L. The thioldependent component which degraded the protein substrate (4-MU-casein) observed at pH 6.0 repre-
62
H. A. KHALFAN
sents cathemin L. since cathemin B does not attack this substrate. Cathepsin H dould not be detected, was present in comparatively and cathepsin small amounts. There was evidence that an as yet unidentified non-thiol protease may contribute significantly to the activity at pH 6.0.
5.
6. 7.
REFERENCES Kirschke, H., Langner, J., Riemann, S., Wiederanders, B., Ansorge, S. and Bohley P. (1980) Lysosomal cysteine proteinases. In: Protein Degradation in Health and Disease (Ciba Foundation Symposium 7 9 , North-Holland: Amsterdam, pp. 15-35. Cockle, S. M. and Dean, R. T. (1982). The regulation of proteolysis in normal fibroblasts as they approach confluence. Evidence for the participation of the lysosomal system. Biochem. J., 208,795-800. Barrett, A. J. (1977). Cathepsin B and other thiol proteinases. In: Proteinases in Mammalian Cells and Tissues (Barret, A. J., ed.), North Holland: Amsterdam, pp. 18 1-208. Kirschke, H., Schmidt, I. and Wiederanders, B. (1986). Cathepsin S. The cysteine proteinase from bovine lymphoid tissue is distinct from cathepsin L. Biochem. J., 240, 455-459.
8. 9.
10. 11.
12
Pontremoli. S.. Melloni. E.. Damiani. G.. Michetti. M.. Salamino, F., Sparatore, B.' and Horecker, B. L. (1984): Binding of monoclonal antibody to cathepsin M located on the external surface of rabbit lysosomes. Arch. Biochem. Biophys., 233,267-271. Frisch, A. and Neufeld, E. F. (1981). Limited proteolysis of the beta-hexosaminidase precursor in a cell-free system. J . Biol. Chem., 256, 8242-8246. Strawser, L. D. and Touster, 0. (1980). The cellular processing of lysosomal enzymes and related proteins. Rev. Physiol. Biochem. Pharmacol., 87, 169-210. Barrett, A. J. and Kirschke, H. (1981). Cathepsin B, cathepsin H and cathepsin L. In: Methods in Enzymology,VoL 80, (Lorand, L., ed.) Academic Press: New York,pp. 535-561. Khalfen, H., Abuknesha, R. and Robinson, D. (1983). Fluorigenic method for the assay of proteinase activity with the use of 4-methyl-umbelliferyl-casein. Biochem. J., 209, 265-267. Zimrnerman, M., Yurewica, E. and Patel, G. (1976). A new fluorogenic substrate for chymotrypsin. Anal. Biochem., 70, 258-262. Zimmerman, M., Ashe, B., Yurewicz, E. C. and Patel, G. (1977). Sensitive assays for trypsin, elastase and chymotrypsin using new fluorogenic substrate. Anal. Biochem., 78, 47-51. Schwartz, W. N. and Barrett, A. J. (1980). Human cathepsin H. Biochem. J., 191 ,487-497.
Receiued in revisedjorm 13 July 1990 Accepted 27 August 1990
