Involvement of thiol proteases in galactosialidosis

CCA 03604

fnvolvc;ment of thiol protenses in galactosialidosis Fiji Takeda ‘, Yasuhiro Kuroda ‘, Kenji Toshima a, Etsuo Nai to ‘, ~~~h~n~~i Xto it5~a~uh~de h@au at Eiki Kominami ’ and Nobuhiko Katunuma b a ~epurtmen~ of Pediaxrics and b ~e~artme~lt of Enzyme Chemistry, Insritutefor Enzyme Research, School of Medicine, Unit~ersityof Tokwhima Kuramoto-cho 2, Tokushima City, Tokushima {Japnn) (Received July 24th. 1985; revision September 3&h, 1985)

The activities of Z-Phe-Arg-N~~c(~PA~ hydroiase, ~at~epsi~ I3 and cathepsin H tie concentration of endogenous thiol protease inhibitor in fibroblasts from padents with ~a~a~tosial~dos~swere found not to be sig~f~cant~y different from those in control fibroh~asts. Gulture for 5 days with thiot protease ~~hibjtors such as leupeptin, E-64 or Z-Phe-Phe-CHNz partially restored the &galactosidase activity of fibroblasts from patients, but did not affect the /3-galactosidase activity of fibroblasts from control subjects. However, culture with leupeptin, but not other protease inhibitors, increased the ZPA hydrolase and cathepsin B activities of f’ibroblasts from both patients and controls 2- to 4-fold. ~ephadex G-75 chromatography showed that the activity of high molecular weight ZPA hydrolase, which was initially predominant in fibmblasts, decreased markedly during their culture wit.h leupeptin, while the activities of lower molecular weight ZPA hydrolase and cathepsin B increased about Sfokd. These results suggest that high mol~~~ar weight ZFA hydrolase, which is presumably cathepsin 3; degrades ~-~al~~tos~dase~ and that the defect in gala~tosiaiidos~s is impaired protection of &gaiactosidase from degradation, and

Ga~a~~~s~a~idos~sis an autosomaf recessive inherited lysosomal storage disease associated with deficiencies of ,O-galactosidase and neuraminidase [l]. In fibroblasts Ab&&&&ti~:664, r.-trans-epoxysuccinyl-Ieucylamide CHN,, diazomethane; NMec, 4-methyl-%coumarylamide; a~no~etbylcoum~n; 4&W, ~methyium~]Iiferon~.

(rtguanidinoj butane; Z, be~zyloxyc~~~y~; EDTA, ethyfenediaminetetraacesic acid; AMC,

110

from patients with galactosialidosis, the half life of P-galactosidase 15 markedly reduced, while the rate of enzyme synthesis is normal [2,3]. and thus the reduction of ,&galactosidase activity is probably due to enhanced enzyme degradation. There are also reports [4,5] that the /3-galactosidase activity in fibroblasts from patients with this disease is restored by treatment of the fibroblasts with leupeptin, E-64 or other protease inhibitors, which inhibit thiol cathepsins such as cathepsin B, H and L. which are the most important proteases involved in intra-lysosomal proteolysis [6,7]. These findings suggest that these cathepsins degrade P-galactosidase. Our study was designed to elucidate which thiol protease participates in the degradation of /I?-galactosidase and whether the thiol cathepsin system is activated in galactosialidosis. Materials and methods Skin fibroblasts from two patients with galactosialidosis and seven controls were cultured at 37°C under 5% CO, in air in minimum essential medium (MEM; Eagle’s Medium, Nissui), supplemented with 10% heat-inactivated fetal calf serum (M.A. Bioproducts, Walkersville, MD, USA) and antibiotics. Cells were harvested with trypsin, washed three times with phosphate buffered saline, suspended in water. sonicated and centrifuged at 105000 X g for 1 h, and the supernatant was used for enzyme assays. Leupeptin [8], E-64 [9] (Peptide Research Foundation, Osaka, Japan) and Z-PhePhe-CHN, [lo] (Maruzen Oil Biochemical, Tokyo, Japan) were dissolved in culture medium before addition to cultures. Neuraminidase and /3-galactosidase were assayed by the methods of Potier et al [l l] and Sakuraba et al [12]. Hydrolases of Z-Phe-Arg-NMec(ZPA), Z-Arg-Arg-NMec and Arg-NMec were assayed by the method of Barrett and Kirschke [13]. Hydrolytic activities of Z-Arg-Arg-NMec and Arg-NMec represent the activities of cathepsin B and cathepsin H, respectively, while that on ZPA was measured because no specific substrate is available for cathepsin L [13-151. The incubation mixture for assay of ZPA hydrolysis consisted of 100 mmol/l sodium acetate buffer (pH 5.5), 1 mmol/l EDTA, 2 mmol/l dithiothreitol, 0.025% Brij, 5 pmol/l ZPA and 5-10 pg protein of supernatant. The mixtures for assays of cathepsin B and cathepsin H were the same except that 100 mmol/l sodium potassium phosphate buffer (pH 6.0), 100 mmol/l sodium potassium phosphate buffer (pH 6.8) and 5-10 pg of protein of supernatant were used with 5 pmol/l Z-Arg-Arg-NMec and Arg-NMec, respectively, as substrates. The incubation temperature was 37°C for assay of ZPA hydrolase and cathepsin B, and 30°C for assay of cathepsin H. Reactions were stopped by adding 1 ml of 100 mmol/l sodium acetate buffer (pH 4.3) containing 100 mmol/l monoiodoacetate. The fluorescence of free aminomethylcoumarin was determined with excitation and emission wave lengths of 370 nm and 460 nm, respectively. Endogenous thiol protease inhibitor was assayed as reported previously [16]. Protein was determined by the method of Lowry et al [17] with bovine serum albumin as a standard. Samples of 0.45 mg of protein of the fibroblast supernatant were applied to a

I

a Mean +

SD

inhibitors

+120 f 82 +295

n.d. n.d. n.d. n.d. n.d. n.d.

25 18 33

activity,

2.7 1.0 6.2

f 54 f 40 +117

f f f

4.5+ 2.2* 15.8k

352 246 346

102 64 100

n.d.; no detectable

30.2_+ 9.1 13.5* 1.1 52.3+ 19.0

684 357 755

f 70 = * 48 + 99

Cathepsin B (nmol/h per mg protein)

ZPA hydrolase (nmol/h per mg protein)

260 161 284

of ZPA hydrolase,

on the activities

determinations;

pmol/l)

for triplicate

Leupeptin (10 pg/ml) Patient 1 Patient 2 Controls E-64(10 pg/ml) Patient 1 Patient 2 Controls Z-Phe-Phe-CHN,(1.6 Patient 1 Patient 2 Controls

Patient 1 Patient 2 Controls (n = 7)

Effect of thiol protease

TABLE

not determined.

_ _

10.0 k 0.6 5.7 f 0.6 12.9+ 1.5

13.7+1.5 7.0 f 0.6 13.1 rto.7

11.6+4.6 6.5 k 4.5 12.1 * 4.5

H, P-galactosidase

34.9 58.3 78

50.4* 165.35 436 +

11.2 13.4 10.7

52.9* 18.2 47.2+ 10.9 371 &I18

95.2+ 152.2 k 464 *

11.4* 2.6 18.1 + 4.1 357 +186

/3-Galactosidase (nmol/h per mg protein)

B, cathepsin

Cathepsin H (nmol/h per mg protein)

cathepsin

0.2 0.3 8.8

1.3* 0.2 1.2* 0.3 45.9 f 12.4

1.0+ 0.3 1.2+ 0.2 52.6 f 10.1

1.1* 1.4* 56.3k

1.4* 0.2 1.3+ 0.2 51.8* 14.3

Neuraminidase (nmol/h per mg protein)

_ _ _

_

_

_ _

106k13 185 + 25 127 + 32

Endogenous thiol protease inhibitor (nmol/h per mg protein)

and neuraminidase

112

column (1.4 X 60 cm) of Sephadex G-75 (Pharmacia). The column was developed with 50 mmol/l sodium acetate buffer (pH 5.0). containing 0.15 mol/l NaCl and 1 mmol/l EDTA. and fractions of 1.0 ml were collected. Results As shown in Table I, the activities of ZPA hydrolase, cathepsin B and cathepsin H and the level of endogenous thiol protease inhibitor in fibroblasts of patients with galactosialidosis were not significantly different from those in control fibroblasts. The fi-galactosidase activity of fibroblasts from patient 1 and 2 increased significantly on culture of the cells with leupeptin (10 pg/ml), E-64 (10 pg/ml) or Z-Phe-Phe-CHN, (1.6 pmol/l) for 5 days. reaching 26.7 f 9.8% and 42.6 + 16.3%, 14.8 _+ 5.1% and 13.2 + 3.1%, and 14.1 f 3.1% and 46.3 _+ 3.X%, respectively, of the mean levels of controls not treated with inhibitors, whereas on similar treatment. the /3-galactosidase activity of control fibroblasts did not change significantly. The activities of cathepsin H, neuraminidase, and several other acid hydrolases (data not shown) did not change during culture with any of these protease inhibitors. The activities of ZPA hydrolase and cathepsin B in fibroblasts from patients and controls were markedly reduced by culture with E-64 or Z-Phe-Phe-CHN,, but were increased 2- to 4-fold by culture with leupeptin. On gel filtration on a Sephadex G-75 column the supernatant fractions from fibroblasts of both patients and controls gave two distinct peaks of ZPA hydrolase activity (high and low molecular weight forms) (Fig. 1). In crude extracts, the high molecular weight ZPA hydrolase was unstable and its activity decreased 80% in 12 h at - 70°C, but after chromatographic separation, it became stable. The ratio of the activities of the high and low molecular weight forms was about 2.2 in fibroblasts of

,

Fig.

1. Separation of ZPA 0) from fibwblasts

(Ochromatography.

Conditions

cathepsin B (Ahydrolases (0 -0). of controls (A) and patients with galactosialidosis were as described

in ‘Materials

and Methods’.

A) and

P-galactosidase

(B) by Sephadex

G-75

(A)

&I ?

1 \\

. J!

40

30

20

t,u,,on

5”

Y”l”l”e

6”

,l”l,

ZLl

.

^\.i

uo

I” El”,i”ll

““l”lne

50

60

(ml,

and /3-galactosidase cathepsin B (A -A) 2. Separation of ZPA hydrolases (0 -o), 0) from fibroblasts of controls (A) and patients with galactosialidosis (B) after incubation for (O5 days with 10 pg/ml of leupeptin. The experimental procedure was as described in ‘Materials and

Fig,

Methods’.

both patients and controls. Cathepsin B gave one peak of activity, corresponding in position to that of low molecular weight ZPA hydrolase. After culture with leupeptin for 5 days, the high molecular weight hydrolase activity of fibroblasts of both patients and controls had decreased to 10.5 + 2.5% of the initial level, while their activities of low molecular weight ZPA hydrolase and cathepsin B had increased to 510 k 53% and 488 f 90%, respectively of the initial levels (Fig. 2). E-64 and Z-Phe-Phe-CHN, markedly and continuously inhibited the activities of high and low molecular weight hydrolases and cathepsin B (data not shown). The leupeptin concentration for 50% inhibition of the high molecular weight ZPA hydrolase was about 10% of that for 50% inhibition of the low molecular weight ZPA hydrolase. These inhibitory concentrations of leupeptin were the same for the enzymes in fibroblasts of patients and controls (Table II).

TABLE

II

Leupeptin

concentrations

for 50% inhibition

of high and low molecular

ZPA hydrolases

ZPA hydrolase

Patient 1 Patient 2 Controls (n=4) a Leupeptin b Mean+s~.

concentration,

High molecular

Low molecular

0.64 a 0.60 0.66 k 0.06 ’

4.70 6.40 6.03 & 0.97

ng/ml.

in vitro

114

Discussion Our findings show that the activities of ZPA hydrolase, cathepsin B and cathepsin H in fibroblasts of patients with galactosialidosis and of controls did not differ. Sakuraba et al reported [12] that cathepsin B activity measured with ZPA is increased in galactosialidosis. As we used the same compound as a substrate for ZPA hydrolase, the difference between our results and theirs might be due to the instability of the high molecular weight form of ZPA hydrolase in crude extracts. In this study, restoration of ,&galactosidase activity was observed when fibroblasts from patients with galactosialidosis were cultured with leupeptin and E-64, which are inhibitors of cathepsin L and B [8,9,18] or with Z-Phe-Phe-CHN,. which is a specific inhibitor of rat cathepsin L [IO]. E-64 and Z-Phe-Phe-CHN, inhibited the activities of both ZPA hydrolase and cathepsin B, while leupeptin caused increases in the activities of cathepsin B and ZPA hydrolase. These findings are in agreement with reported results [19,20,21]. The high molecular form of ZPA hydrolase activity separated by Sephadex G-75 chromatography, may be important in degradation of /3-galactosidase, because culture of fibroblasts with leupeptin, which restored their P-galactosidase activity, also inhibited the high molecular weight ZPA hydrolase activity and this form was more sensitive than the low molecular weight form to leupeptin in vitro. Liao and Lenney [15] recently reported that a thiol protease, cathepsin J, present in the particulate fraction of rat liver resembles cathepsin L in hydrolyzing ZPA but not Z-Arg-Arg-NMec. They found that cathepsin J is inhibited more strongly than cathepsins B and L by endogenous inhibitors, and that it has a higher molecular weight than cathepsin L. Thus the high molecular weight ZPA hydrolase found in our study might be cathepsin J. It seems unlikely that during culture with leupeptin, the high molecular weight form of ZPA hydrolase was converted to the low molecular weight form, because the activities of the two did not always change reciprocally and high molecular weight ZPA hydrolase might have been inhibited by endogenous inhibitors. Previous results indicated that in galactosialidosis, mutation of P-galactosidase is unlikely [22]. Furthermore, the chromatographic patterns of /3-galactosidase of patients and controls are always identical. Thus our results are consistent with the idea that in galactosialidosis, the thiol protease system is not activated and the primary defect is in secondary factors such as those proposed by Galjaard et al [J&-24] and Strisciuglio et al [25] that influence the rate of degradation of P-galactosidase. References 1 Low&n JA, O’Brien JS. Sialidosis:

a review of

human neuraminidase deficiency. Am J Hum Genet

1979; 31: 1-18. 2 Van Diggelen oP, S&am AW, Sinnott ML, Smith PJ, Robinson D. Galjaard H. Turnover of P-galactosidase in fibroblasts from patients with genetically different types of P-galactosidase deficiency. B&hem J 1981; 200: 143-151. 3 Van Diggelen, Hoogeveen AT, Smith PJ, Reuser AJJ, Galjaard H. Enhanced proteolytic degradation of normal P-galactosidase in the lysosomal storage disease with combined B-galactosidase and neuraminidase deficiency. B&him Biophys Acta 1982; 703; 69-76.

115 4 Suzuki y, Sakuraba H, Hayashi K, Suzuki K, Imahori K. /3-Galactosidase-neuraminidase deficiency: restoration of /3-galactosidase activity by protease inhibitors. J Biochem 1981; 90: 271-273. 5 Galjaard H, Hoogeveen A, Verheijen F, Van Diggelen OP, Konings A, DAz.zo A, Reuser AJJ. Relationship between clinical, biochemical and genetic heterogeneity in sialidase deficiency. In: Tettamanti G, Durand P, DiDonato S, eds. Perspectives in inherited metabolic diseases: sialidoses and sialidases, Vol. 4. Milano: Edi Ermes, 1981: 317-333. 6 Katunuma N, Kominami E. Structures and functions of lysosomal thiol proteases and their endogenous inhibitor. In: Horecker BL, Stadtman ER, eds. Current topics in cellular regulation, VoI 22. New York/London: Academic Press Inc., 1983: 71-101. 7 Katunuma N, Watanabe N, Takio K, Titani K, Kominami E. Structure, function, and regulation of endogenous thiol proteinase inhibitor. In: Katunuma N, Umezawa H, Holzer H, eds. Proteinase inhibitors: medical and biological aspects. Tokyo: Japan Sci Sot Press, Berlin: Springer Verlag, 1983: 135-145. 8 Umezawa H, Aoyagi T. Activities of proteinase inhibitors of microbial origin. In: Barrett AJ, ed. Proteinases in mammalian cells and tissues. Amsterdam: Biochemical Press, 1977: 637-662. 9 Hanada K, Tamai M, Yamagishi M, Ohmura S, Sawada J, Tanaka I. Isolation and characterization of E-64, a new thiol protease inhibitor. Agric Biol Chem 1978; 42: 523-528. 10 Leary R, Shaw E. Inactivation of cathepsin B, by diazomethyl ketones. Biochem Biophys Res Commun 1977; 79: 926-931. 11 Potier M, Beauregard G, Belisle M, MameIi L, Nguyen Hong V, Melancon SB, Dallaire. Neuraminidase activity in the mucolipidosis (type I, II and III) and the cherry-red spot myoclonus syndrome. CIin Chim Acta 1979; 99: 97-105. 12 Sakuraba H, Aoyagi H, Suzuki Y. Galactosiahdosis (/I-galactosidase-neuraminidase deficiency); a possible role of serine-thiol proteases in the degradation of /3-galactosidase molecules. Clin Chim Acta 1982; 125: 275-282. 13 Barrett AJ, Kirschke H. Cathepsin B, cathepsin H, and cathepsin L. Methods Enzymol 1981; 80: 535-561. 14 Kirschke H, Shaw E. Rapid inactivation of cathepsin L by Z-Phe-PheCHN, and Z-Phe-AIaCHN,. Biochem Biophys Res Commun 1981; 101: 454-458. 15 Liao JCR, Lenney JF. Catbepsin J and K: high molecular weight cysteine proteinases from human tissues. Biochem Biophys Res Commun 1984; 124: 909-916. 16 Komonami E, Wakamatsu N, Katunuma N. Purification and characterization of thiol proteinase inhibitor from rat liver. J Biol Chem 1982; 257: 14648-14652. 17 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J BioI Chem 1951; 193: 265-275. 18 Kominami E, Hashida S, Katunuma N. Inhibitors of degradation of rat liver aldolase and lactic dehydrogenase by N-[ N-(L-3-trans-carboxyoxirane-2-carbonyl)-r-leucyl] agmatine of leupeptin in viva. Biochem Biophys Res Commun 1980; 93: 713-719. 19 Kato T, Gkada S, Yutaka T, Yabuuchi H. Induction of P-galactosidase-a-neuraminidase deficiency: effect of leupeptin and sucrose. Biochem Int 1983; 6: 267-273. 20 Libby P, Ingwall JS, Goldberg AL. Reduction of protein degradation and atrophy in cultured fetal mouse hearts by leupeptin. Am J Physiol 1979; 237: E35-E39. 21 Tanaka K, Ikegaki N, Ichihara A. Purification and Characterization of hemoglobin-hydrolyzing acidic thiol protease induced by leupeptin in rat liver. J Biol Chem 1984; 259: 5937-5944. 22 D’Azzo A, Hoogeveen A, Reuser AJJ, Robinson D, Galjaard H. Molecular defect in combined P-galactosidase and neuraminidase deficiency in man. Proc Nat1 Acad Sci USA 1982; 79: 453554539. 23 Hoogeveen A, Verheijen EW, Galjaard H. The relation between human lysosomal /I-galactosidase and its protective protein. J Biol Chem 1983; 258: 12143-12146. 24 Hoogeveen A, D’AZZO A, Brossmer R, Galjaard H. Correction of combined /3_galactosidase/neuraminidase deficiency in human fibroblasts. Biochem Biophys Res Commun 1981; 103: 292-300. 25 Strisciuglio P, Creek KE, SIy WS. Complementation, cross correlation, drug correlation studies of combined Bgalactosidase neuraminidase deficiency in human fibroblasts. Pediatr Res 1984; 18: 167-171.