Biochem. J. (1976) 155, 273-278 Printed in Great Britain
Human Cathepsin G CATALYTIC AND IMMUNOLOGICAL PROPERTIES By PHYLLIS M. STARKEY and ALAN J. BARRETT Tissuie Physiology Department, Strangeways Research Laboratory, Worts' Causeway, Cambridge CB1 4RN, U.K. (Received 28 November 1975)
1. The specificity of cathepsin G, a neutral proteinase from human spleen, was examined by use of low-molecular-weight substrates. The enzyme was found to hydrolyse several synthetic substrates also hydrolysed by chymotrypsin, but with different kinetic constants. 2. Maximal activity against benzoyl-DL-phenylalanine 2-naphthol ester and azo-casein was in the range pH7.5-8.0. 3. The sensitivity of cathepsin G to the action of potential inhibitors was determined, and compared with those of bovine chymotrypsin and subtilisin. Cathepsin G showed the characteristics of a serine proteinase, but was less affected by the chloromethyl ketone of tosylphenylalanine than was chymotrypsin. 4. A rabbit anti-(human cathepsin G) serum was raised, and precipitin lines formed in agarose gel were stained for activity of the enzyme. 5. Cathepsin G was shown to be immunologically identical with the chymotrypsin-like enzyme of the azurophil granules of the neutrophil granulocytes. The purification of two cationic serine proteinases from human spleen was described by Starkey & Barrett (1976a). One of these, now named cathepsin G, was active against Bz-DL-Phe-2-ONap,* a synthetic substrate of chymotrypsin, as well as against proteins. Chymotrypsin-like enzymes detected * Abbreviations: Ac-(Ala)3-AlaCH2CI, N-acetyl-triL-alanyl-L-alanine chloromethyl ketone; Ac-(Ala)2-ProAlaCH2CI, N-acetyl-L-alanyl-L-alanyl-L-propyl-L-alanine chloromethyl ketone; Ac-Phe-2-ONap, N-acetyl-Lphenylalanine 2-naphthyl ester; Ac-Tyr-OEt, N-acetylL-tyrosine ethyl ester; Boc-Ala-ONp, t-butyloxycarbonyl-L-alanine 4-nitrophenyl ester; Boc-Ala-2-ONap, t-butyloxycarbonyl-L-alanine 2-naphthyl ester; BZ-DLArg-Nan, N-benzoyl-DL-arginine 4-nitroanilide; BZ-DLArg-2-NNap, N-benzoyl-DL-arginine 2-naphthylamide hydrochloride; Bz-DL-Phe-2-ONap, N-benzoyl-DLphenylalanine 2-naphthyl ester; Bz-DL-Phe-2-NNap, N-benzoyl-DL-phenylalanine 2-naphthylamide hydrochloride; Bz-Tyr, N-benzoyl-L-tyrosine; Bz-Tyr-Nan, N-benzoyl-L-tyrosine p-nitroanilide; Bz-Tyr-OEt, N-benzoyl-L-tyrosine ethyl ester; ClAc-ONapAS-D, 2-(chloroacetyl)-3-naphthoic acid o-toluidide; Dip-F, di-isopropyl phosphorofluoridate; Glt-Phe-Nan, Nglutaryl-L-phenylalanine p-nitroanilide; NapAS, 3naphthoic acid anilide; NapAS-D, 3-naphthoic acid o-toluidide; PhPr-ONapAS, 2-(phenylpropionyl)-3naphthoic acid anilide; Pins-F, phenylmethanesulphonyl fluoride; Tos-LysCH2CI, 7-amino-1-chloro-3-tosylamidoL-heptan-2-one; Tos-PheCH2CI, 1-chloro-4phenyl-3tosylamido-L-butan-2-one; Z-Ala-2-ONap, N-benzyloxycarbonyl-L-alanine 2-naphthyl ester; Z-PheCH2Br,
an-2-one. Vol. 155
previously in mammalian tissues include chymase from mast cells of several species (Lagunoff & Benditt, 1963; Kawiak et al., 1971; Vensel et al., 1971), an enzyme in human seminal plasma (Syner & Moghissi, 1972) and an enzyme that appears as a group of three or four of the most cationic proteins in the azurophil granules of human neutrophil leucocytes (Dewald et al., 1975; RindlerLudwig & Braunsteiner, 1975). The purpose of the present paper is to report some properties of cathepsin G, and to compare them with those of bovine chymotrypsin, subtilisin and other chymotrypsin-like enzymes mentioned above. Materials Cathepsin G was purified from human spleen as described by Starkey & Barrett (1976a); its specific activity was 107nkat/mg with Bz-DL-Phe-2-ONap as substrate and 144units/mg with azo-casein. Materials were generally as described previously (Starkey & Barrett, 1976a,b). Other chemicals were obtained as follows: Bz-Tyr-OEt and subtilisin Carlsberg(type VIII), from Sigma(London) Chemical Co. Ltd., Kingston-upon-Thames, Surrey KT2 7BH, U.K.; Bz-Tyr-Nan from Bachem, Liestal, Switzerland; chymotrypsin A4 from Boehringer (London) Corp. Ltd., London W5 2TZ, U.K.; Ac-Tyr-OEt from Aldrich Chemical Co. Ltd., Wembley HAO 1PY, Middlesex, U.K. The chymotrypsin-like enzyme from human neutrophil leucocytes, purified by chromatography on Trasylol-Sepharose and CM-cellulose (Baugh & Travis, 1976), was a gift K
274A from Dr. J. Travis, Department of Biochemistry, University of Georgia, GA, U.S.A. Methods Unless stated below, methods were as described previously (Starkey & Barrett, 1976a,b).
Enzyme assays Activity against PhPr-ONapAS. The method was the same as that described for Bz-DL-Phe-2-ONap (Starkey & Barrett, 1976a) except in that the substrate was PhPr-ONapAS (5mg/ml of dimethyl sulphoxide), and the mitures were incubated for 10mm at 400C. Activity was expressed in nkat, on the basis that NapAS gave As520 of 3.2x10'M-1 cm-1 in the colour reaction. Activity aganst ethyl esters. Activity against Bz-Tyr-OEt was measured spectrophotometrically by the method of Kang & Fuchs (1973). The 8256 of and activity was expressed Bz-Tyr is 964M'cm in nkat. For determination of kinetic constants, incubation mixtures contained Bz-Tyr-OEt (0.250.83mM) and chymotrypsin (3pg) or cathepsin G [16.8,ug on the basis of E2^ (1cm path length) = 10.0]. The kinetic constants for each enzyme were calculated as described by Wilkinson (1961). Activity against Ac-Tyr-OEt was measured spectrophotometrically by the method of Schwert & Takenaka
(1955). Inhibitor studies Assays of the proteolytic activity of cathepsin G (0.3unit), bovine chymotrypsin (1,g) and subtilisin (0.5ag) were made by the standard method against azo-casein. In general, the enzyme was preincubated with the inhibitor for 5min at room temperature before the addition of substrate, and the concentration of inhibitor given is that in the final reaction mixture. Inhibition by gold thiomalate and mercaptosuccinic acid was measured in 0.1 M-Tris/HCI buffer, pH7.5, without added KCI. For certain inhibitors (Dip-F, Tos-LysCH2CI, Tos-PheCH2CI, ZPheCH2Br, Pms-F, Ac-(Ala)3-AlaCH2Cl and Ac(Ala)2-Pro-AlaCH2CI) the enzyme was preincubated with the inhibitor at 4°C for 18h, and the concentration of inhibitor stated is that during this preincubation, The preincubation mixtures (0.75ml) contained 0.4m1 of 2.5M-KCI in 1.25M-Tris/HCI buffer, pH7.5, 0.25ml of 0.1 % bovine serum albumin in 0.02M-CaCI2, the enzyme in 0.05ml of 0.05% Brij 35, and 0.05ml of inhibitor solution. TosLysCH2CI was in aqueous solution; Dip-F, TosPheCH2Cl, Z-PheCH2Br and Pms-F were dissolved in propan-2-ol; Z-PheCH2Br, Ac-(Ala)3-AlaCH2CI and Ac-(Ala)2Pro-AlaCH2Cl were dissolved in dimethyl sulphoxide immediately before use.
P. M. STARKEY AND A. J. BARRETIT Raising of antiserum A sample of purified human cathepsin G (4.5mg) in 3.0ml of phosphate-buffered saline (0.8% NaCl, 0.02% KCI, 0.02% KH2PO4, 0.12% Na2HPO4) was mixed with an equal volume of Freund's Complete Adjuvant and divided into three equal portions. A rabbit was immunizd by intramuscular injections of the enzyme into each thigh three times at fortnightly intervals.
Immunodiffusion Plates were made and stained for protein as described previously (Barrett, 1974); staining for enzymic activity was essentially as described for elastase (Starkey & Barrett, 1976a), but ClAcONapAS-D was used as substrate in some experiments. Results Specificity with low-molecular-weight substrates Cathepsin G showed no detectable activity against substrates of trypsin (Bz-DL-Arg-NNap and Bz-DLArg-Nan), elastase (Boc-Ala-ONp, Boc-Ala-2-ONap and Z-Ala-2-ONap), or some substrates of chymotrypsin (Ac-Tyr-OEt, Glt-Phe-Nan and BZ-DL-Phe2-NNap). Othersubstrates ofchymotrypsin, however, were hydrolysed by cathepsin G (Table 1), though 20-35-fold more slowly than by chymotrypsin. Two histochemical ester substrates, PhPr-ONapAS and CIAc-ONapAS-D were also susceptible. Values for Km and kcat. of cathepsin G and chymotrypsin for Bz-Tyr-OEt were determined. For cathepsin G, Km was 2.6±0.7mM (mean±s.E.; d.f. = 8), whereas the value for chymotrypsin was 0.5±0.1mM (mean±s.E.; d.f. = 10). On the basis of a mol.wt. of 28000 and El0- 10.0, kc,t. was 3.1 ± Table 1. Activity of cathepsin G and chymotrypsin against
Assay conditions were as described in the Methods section. n.d., Not determined. Enzymic activity (nkat/mg of enzyme)
Bz-Tyr-OEt Glt-Phe-Nan Bz-Tyr-Nan Bz-DL-Phe-2-ONap Ac-Phe-2-ONap Z-Phe-2-ONap PhPr-ONapAS ClAc-ONapAS-D
0.14 2.51 0.31 0.12 0.15 0.12 0.13 0.14
pH 7.8 7.5 7.5 7.5
7.5 7.5 7.5 6.0
G 59 0 0.21 90 98 28 0.66 13
trypsin 2100 3.8 23 2200 1900 n.d. 25 19
HUMAN CATHEPSIN 02
o 0~~~~~~~2 20~~~~~~~2.
Table 2. Effect ofsalts on the activity against azo-casein of purified cathepsin G Enzymic activity is expressed as a percentage of that in the abence of added salt. Ionic strength Proteolyticactivity Addition (mol/l) (°/) 100 None 0.09 1.0M-NaC1 110 1.09 1.0M-KCl 1.09 108 1.59 45 0.5M-CaCI2 1.59 0.5M-MgCl2 140 1.09 1 1.OM-NaSCN 1.59 71 0.5M-Na2SO4
pH Fig. 1. Effect ofpH on the enzymic activity of cathepsin G The activity against azo-casein (0) (0.43 unit of enzyme) and against Bz-DL-Phe-2-ONap (0) (0.05nkat of enzyme) is expressed as a percentage of the maximal ativity with that substrate. All buffers [sodium acetate (a); KH2PO4/
K2HPO4 (b); Tris/HCl (c); glycine/NaOQ (d)] were 0.1M, and those used with azo-casein contained 1.0MNaCl.
0.7s-1 for cathepsin G, whereas a value of 19.6±2.0s-' was obtained for chymotrypsin. The latter value is uncorrected for presumed partial activity (commonly 60-80%) of the commercial chymotrypsin. Dependence ofactivity on pH and inorganic ions The pH-dependence of the activity of purified cathepsin G was measured with both azo-casein and Bz-DL-Phe-2-ONap as substrates. The pH-dependence for both substrates (Fig. 1) show maxima at pH7.5-8.0, with little activity below pH5.0, or above pH9.0. It was not possible to make assays above. pH8.5 with Bz-DL-Phe-2-ONap, because of the alkali-lability of the substrate. The purified cathepsin G (0.4unit) was assayed against azo-casein in incubation mixtures containing 0.1 M-Trs buffer, pH7.5, together with various salts at 0.5 and 1.OM concentration. The results are given in Table 2. There was moderate stimulation by MgCl2, whereas CaCl2 and Na2SO4 were inhibitory at high concentration. Activity was abolished by 1.OM-NaSCN. In a separate experiment cathepsin G was stored for 24h at 4'C in 0.5 M-NaSCN, and assayed against azo-casein with 0.375M-NaSCN in the incubation mixture. Activity was identical with that of a control sample not exposed to NaSCN. Vol. 155
Inhibitors The results of inhibition experiments are presented in Table 3. All three enzymes were completely inhibited by Pms-F and Dip-F, although initial experiments with an aged sample of Dip-F failed to show inhibition of cathepsin G, whereas chymotrypsin was totally inhibited. The effect of halomethylketones on cathepsin G was less specific than that on trypsin and chymotrypsin (Shaw, 1970) in that partial inhibition was produced by b6th Tos-LysCH2Cl and Tos-PheCH2Cl. The increased inhibition by the bromomethyl ketone was consistent with its greater general reactivity (Shaw & Ruscica, 1968). Cathepsin G was unaffected by the peptide chloromethyl ketone inhibitors of elastase, whereas chymotrypsin and subtilisin were more susceptible. Cathepsin G was unaffected by Ca2+, Zn2+ and 4-chloromercuribenzoate at the concentrations used, but was inhibited by much lower concentrations of gold in the form of its thiomalate. The inactivity of mercaptosuccinate (thiomalate) shows that inhibition was due to the gold itself. The inhibitory activity of gold thiomalate was abolished by 1.OM-KC1. Of the natural inhibitors tested against cathepsin G, soya-bean trypsin inhibitor and lima-bean trypsin inhibitor were very effective, but the pancreatic inhibitor, turkey ovomucoid and chicken ovo-
inhibitor, which strongly inhibited chymotrypsin and subtilisin, were relatively ineffective. Antiserum to cathepsin G In Ouchterlony gel diffusion, in agarose gel equilibrated with 1.0M-NaCI, the antiserum gave a strong major precipitin line against purified cathepsin G and against crude spleen extract. A minor degree of polyvalence was detectable under certain conditions. Washed precipitin lines were stained for enzymlc activity against ClAc-ONapAS-D. The purple precipitate indicative of enzymic activity was clearly
P. M. STARKEY AND A. J. BARRETT
Table 3. Effect ofpotential inhibitors on cathepsin G, bovine chynmtrypsin and subtilisin Enzymic activity is expressed as a percentage of activity in the absence of the inhibitor. Assays were of proteolytic activity, except those marked (c) that were with Bz-DL-Phe-2-ONap. Inhibitors marked (a) were made up as stock solutions in dimethyl sulphoxide, and those marked (b) were dissolved in propan-2-ol, before being diluted into the incubation mixture; for each the final concentration of solvent was 4% (v/v). All other inhibitors were in aqueous solution. n.d., Not determined. Activity (%)
Compound Dip-F Tos-LysCH2Cl Tos-PheCH2Cl Z-Phe-CH2Br
(a) (b) (b) (a)
CaC12 ZnSO4 EDTA
Cysteine 4-Chloromercuribenzoate Heparin Gold thiomalate
Mercaptosuccinate Pepstatin Soya-bean trypsin inhibitor (Kunitz) Lima-bean trypsin inhibitor Bovine pancreatic trypsin inhibitor (Kunitz) Turkey ovomucoid Chicken ovoinhibitor
(mM) 1.0 1.0 1.0 1.0 0.5 1.0 0.33 0.10 0.01 0.001 1.0 0.1 0.01 0.001 5.0 1.0 5.0
5mg/ml 1.0 0.1 0.005 1.0 0.001 mg/mil 0.1 mg/ml 0.01 mg/ml 0.001 mg/ml 0.1 mg/ml 0.01 mg/ml 0.001 mg/ml
0.5mg/ml 0.1 mg/ml 0.1 mg/ml
visible only over the major precipitin line. No precipitin lines were formed between cathepsin G and the antiserum at the lower salt concentrations normally used on immunodiffusion plates, probably because of adsorption of the enzyme on to the gel. Because of the similarities between the properties of cathepsin G and the chymotrypsin-like enzyme of neutrophils, an extract of blood leucocytes was tested for the presence of an identical antigen (Plate 1). An antigen immunologically identical with spleen cathepsin G was found to be present and again the precipitin line
1 70 71 5 1 0 95 n.d. n.d. n.d. 97 n.d. n.d. n.d. 99 100 100 58 96 96 n.d. 42 (c) 56 (c) 93 100 1 15 74 0 2 33 51 101 78
Bovine chymotrypsin 1 90 4 6 0 0 32 65 91 99 2 33 79 83 127 62 101 56 97 103 103 n.d. n.d.
n.d. n.d. 1 n.d. n.d. 2 n.d. n.d. 1 0 0
Subtilisin 1 98 105 21
49 3 5 5 4 18 5 2 32 83 98 70 106 69 90 98 108 n.d. n.d. n.d. n.d. 95 n.d. n.d. 113 n.d. n.d. 7 0 0
stained for activity against ClAc-ONapAS-D. Purified chymotrypsin-like enzyme from human neutrophils gave a reaction of complete immunological identity with cathepsin G. Discussion Cathepsin G is active against several of the synthetic substrates of chymotrypsin. The naphthol ester used in our routine assays, Bz-DL-Phe-2-ONap, is one that has been used for chymotrypsin (Ravin
The Biochemical Journal, Vol. 155, No. 2
(b) EXPLANATION OF PLATE I
G in human blood
A double immunodiffusion
plate on which (1) purified cathepsin G (1.3pug) and (2) an extract of human blood leucocytes containing Brij 35 (58 jig of protein) were run against (3) antiserum to cathepsin G. Duplicate plates were stained for enzymic activity against ClAc-ONapAS-D (a), and for protein (b). in 1 m-NaCI
P. M. STARKEY AND A. J.
(facing p. 276)
HUMAN CATHEPSIN G et al., 1954). The convenient and sensitive colorimetric step with Fast Garnet has not previously been
applied, however. The high sensitivity of the naphthol ester substrates must be weighed against their susceptibility to hydrolysis by arylesterases, at least in liver (Ravin et al., 1954), but we found no evidence of such enzymes in human spleen. Probably, the specificity of Bz-DL-Phe-2-ONap is quite adequate for most work on the chymotrypsin-like proteinases. The chymotrypsin-like enzyme of human neutrophil leucocytes has been reported to hydrolyse Bz-Tyr-OEt, its Km of 2.0mM comparing with 0.71 mm determined for bovine chymotrypsin under the same conditions (Gerber etal., 1974). These values agree well with those reported here for the Km of cathepsin G, 2.6mi, and that of chymotrypsin, 0.5mM. The neutrophil enzyme is also reported to hydrolyse Ac-Tyr-OEt, though the activity, which was measured by titration, was extremely low (Rindler et al., 1973; Gerber et al., 1974; RindlerLudwig & Braunsteiner, 1975). Our inability to detect any activity of cathepsin G against this substrate may have been due to the relative insensitivity of the spectrophotometric assay; the activity even of chymotrypsin appeared low. The use of ClAc-ONapAS-D as a histochemical reagent was first described by Gomori (1953), who reported that in human tissues, mast cells stained very rapidly and intensely, neutrophil leucocytes somewhat less rapidly and other cell types such as monocytes, lymphocytes and eosinophil leucocytes gave only very weak staining after prolonged incubation. In normal human spleen, Li et al. (1972) have shown that neutrophil leucocytes are the predominant cell type staining with ClAc-ONapASD, there being very few mast cells in this tissue. ClAc-ONapAS-D has been shown to be hydrolysed by the chymotrypsin-like enzyme of neutrophils (Rindler-Ludwig & Braunsteiner, 1975). The action of potential inhibitors on cathepsin G showed the enzyme to be a serine proteinase more similar to chymotrypsin than to trypsin or elastase. The chymotrypsin-like enzyme of human neutrophils is inhibited by Dip-F (Gerber et al., 1974) and by Pms-F (Schmidt & Havemann, 1974). Cathepsin G was weakly inhibited by Tos-PheCH2CI, an efficient inhibitor of chymotrypsin. Similar results have been obtained by Feinstein & Janoff (1975) with the neutrophil enzyme, whereas others have reported strong inhibition (Gerber et al., 1974; RindlerLudwig & Braunsteiner, 1975). Cathepsin G and bovine chymotrypsin are both strongly inhibited by the inhibitors from soya-bean and lima-bean. The chymotrypsin-like enzyme of human neutrophils also is inhibited by these proteins (Rindler-Ludwig & Braunsteiner, 1975). The bovine pancreatic trypsin inhibitor (Kunitz), which is ineffective against human chymotrypsin (Coan & Vol. 155
Travis, 1971), was less inhibitory for cathepsin G than for bovine chymotrypsin or subtilisin. Ovoinhibitor and turkey ovomucoid had little or no effect on cathepsin G, although they are inhibitors ofhuman and bovine chymotrypsin (Feinstein et al., 1974). The histone-degrading enzyme of rabbit polymorphonuclear leucocytes shows certain similarities to chymotrypsin and cathepsin G, though it is said to be distinct from the rabbit neutrophil enzyme active against Ac-Phe-2-ONap (Davies et a!., 1970, 1971). Heparin, at a concentration of 5mg/ml, has been reported to give a 3-fold stimulation of activity of the rabbit neutrophil enzyme (Davies et al., 1971); no such activation was found with cathepsin G, but it should be noted that the heparin activation reported by Davies et al. (1971) was with a crude cell lysate, not purified enzyme. Dewald et al. (1975) showed that the neutral proteinase activity of rabbit polymorphonuclear leucocytes is located in the azurophil granules, but there was no evidence for the presence of a cathepsin G-like enzyme in addition to the elastase. It therefore seems probable that the enzyme described by Davies et al. (1970, 1971) was the rabbit elastase. The antiserum raised against cathepsin G gave a single precipitin line that stained for enzymic activity against ClAc-ONapAS-D clearly demonstrating that the antiserum reacted with cathepsin G itself and not a contaminating protein. The antiserum was used to show that cathepsin G is present in human blood leucocytes, and is identical with the chymotrypsinlike enzyme. It is not yet clear whether mast cell 'chymase' also is identical with cathepsin G. Chymase resembles cathepsin G in many respects; it is a basic protein, its enzymic activity is stimulated by high salt concentrations and it is 'sticky' at low ionic strength. The substrate specificity of chymase also is similar to that of cathepsin G in that it hydrolyses Ac-Tyr-OEt, Bz-Tyr-OEt (Km 0.6mM), ClAc-ONapAS-D and PhPr-ONapAS (Benditt & Arase, 1959; Lagunoff & Benditt, 1963; Vensel et al., 1971); the activity of cathepsin G against the latter substrate was particularly significant as this was previously considered to be a substrate totally specific for chymase (Lagunoff & Benditt, 1964). The pH optimum and sensitivity to inhibitors of chymase (Pastan & Almqvist, 1966; Kawiak et al., 1971; Vensel et al., 1971) are generally similar to those of cathepsin G. Both enzymes are stable to 0.5M-NaSCN (Lagunoff & Benditt, 1963), although cathepsin G was inactive
in the presence of I.OM-NaSCN.
We thank Dr. Elliott Shaw, Brookhaven National Laboratory, New York, NY, U.S.A., and Dr. J. C. Powers, Georgia Institute of Technology, Atlanta, GA, U.S.A., for gifts of halomethyl ketone inhibitors. We also thank Professor H. Umezawa, Institute of Microbial Chemistry,
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