Eur. J. Biochem. 123, 327-322 (1982) @) FEBS 1982

Enhanced Breakdown of Arylsulfatase A in Multiple Sulfatase Deficiency

Abdul WAHEED, Andre] HASILIK, and Kurt VON FIGURA

Physiologisch-Chemisches Institut der Westfalischen Wilhelms-Universitat Minster

(Received October 26/December 15, 1981)

Multiple sulfatase deficiency (mucosulfatidosis) is a lysosomal storage disorder characterized by the decrease in activities of all known sulfatases. To measure the apparent rate of synthesis and the half-life of arylsulfatase A in multiple sulfatase deficiency, fibroblasts from patients with the disease and from controls were subjected to pulse-chase labelling with radioactive amino acids. Arylsulfatase A and cathepsin D, a lysosomal enzyme that is not affected in multiple sulfatase deficiency, were isolated from cells and media by immunoprecipitation. The labelled polypeptides were separated by polyacrylamide gel electrophoresis, visualized by fluorography and quantified by liquid scintillation counting. Using single and double isotope techniques it was found that, as compared to cathepsin D, the apparent rate of synthesis of arylsulfatase A was 2- 5-times lower and the half- life 4 - 9-times shorter in multiple sulfatase deficiency than in control fibroblasts. In multiple sulfatase deficiency fibroblasts the rates of endocytosis and the stabilities of endocytosed arylsulfatases A isolated from human urine and bovine testes were equal to those in metachromatic leucodystrophy fibroblasts.

We postulate that in normal cells a gene product exists that affects the stability of sulfatases and that multiple sulfatase deficiency is due to a mutation in this gene.

Multiple sulfatase deficiency is a lysosomal storage dis- order biochemically characterized by the accumulation of sulfatides and glycosaminoglycans in tissues and cells [ 11. The storage is caused by the decrease in activities of at least six lysosomal sulfatases and one microsomal (arylsulfatase C) sulfatase [l - 31. The mutations in multiple sulfatase deficiency and in single sulfatase deficiencies (e.g. metachromatic leuco- dystrophy, Hunter syndrome and Sanfilippo syndrome type A) are non-allelic as shown by complementation studies [3,4]. The primary defect in the disorder is unknown. In multiple sulfatase deficiency fibroblasts the decrease in activities of arylsulfatase A and B is paralleled by that in the amount of cross-reacting materials [5] . The presence of reduced levels of catalytically normal sulfatases suggested a defect in a process that controls the levels of the sulfatases either by decreasing the rate of enzyme synthesis or by increasing the rate of enzyme breakdown (or a combination of both).

In the present communication we report on the apparent rate of synthesis and the half-life of arylsulfatase A in controls and multiple sulfatase deficiency fibroblasts. For comparison the synthesis and breakdown of cathepsin D, a lysosomal enzyme that is not affected by the mutation in multiple sulfatase deficiency was studied in the same cell lines. Pre- liminary reports of these data have been presented in abstract form [6,7] .

MATERIALS AND METHODS

Materials

~-[4,5-~H]Leucine (specific activity 50 Ci/mmol) and [I4C]- methylated standards were from New England Nuclear; [35S]niethionine (specific activity 11 50 Ci/minol) and [methyl- 3H]methionine (specific activity 12.9 Ci/mmol) from Amer- ~. ~

Enzymes. ArykdfdtaSe A (EC 3.1.6.1); cathepsin D (EC 3.4.23.5); arylsulfatase B (EC 3.1.6.-); N-acetylgalactosamine-6-sulfate sulfatase (EC 3.1.6.-); iduronide-2-sulfate sulfatase (EC 3.1.6.-); sulfamate sulfa- tase (EC 3.1.6.-); N-acetylglucosamine-6-sulfate sulfatase (EC 3.1.6--).

sham. The antiserum raised in goats against purified human placental cathepsin D has been described previously [ S ] .

Cell Culture

Human diploid skin fibroblasts were maintained at 37 ’C in 5 % C02 in Eagle’s minimal essential medium supple- mented with antibiotics, non-essential amino acids and 10 % fetal calf serum (Boehringer, Mannheim) as described [9]. Fibroblasts from patients affected with multiple sulfatase deficiency were kindly provided by Drs J . Couchet (Reims), P. Durand (Genova), U. Wiesmann (Bern) and A. Greene (Human Genetic Mutant Cell Repository, Camden, NJ).

Pulse-Chase Labelling of’ Arylsulfatase A

The conditions for pulse-chase labelling of cells with ~-[4,5-~H]leucine, preparation of extracts from cells and media, immunoprecipitation, polyacrylaniide gel electropho- resis in presence of sodium dodecylsulfate and fluorography were those described by Hasilik and Neufeld [ S ] with the following modifications. Cells were labelled with 0.25 mCi [3H]leucine/25-cm2 flask. For immunoprecipitation, 20 p1 of antiserum raised in goats against arylsulfatase A purified from human placenta was used; 0.4 pg of partially purified arylsulfatase A from human placenta was used as carrier. The purification of arylsulfatase A, preparation of the anti- serum and identification of the arylsulfatase A polypeptides in the fluorogram will be described elsewhere. The antiserum precipitated specifically arylsulfatase A. Other sulfatases were not precipitated from extracts of human fibroblasts: aryl- sulfatase B, N-acetylgalactosamine-6-sulfate sulfatase, iduro- nide-2-sulfate sulfatase, sulfamate sulfatase, N-acetylglucos- amine-6-sulfate sulfatase. The antiserum precipitated also the residual arylsulfatase A in multiple sulfatase deficiency fibro- blasts. In the material precipitated by the antiserum from metabolically labelled fibroblasts, radioactive polypeptides resembling those in the purified enzyme were found. In

318

extracts from metachromatic leukodystrophy fibroblasts these polypeptides were absent. Fibroblasts were labelled for 5 and 16 h and subjected to a chase for 1 , 3 and 7 days. After separation of labelled polypeptides by gel electrophoresis and visualization by fluorography the radioactivity incorporated into arylsulfatase A was determined by liquid scintillation counting. The bands were cut out from the gels and incubated for 6 h with 0.1 ml water. Then 0.5 ml of NCS tissue solu- bilizer (Amersham) was added. After soaking for 6 h, the samples were mixed with 5 ml of Instagel (Packard) and counted.

Apparent Rate of Synthesis of Arylsulfatase A and Cathepsin D

Cells were labelled for 3 - 12 h in the presence of 10 mM NH4CI and of 0.4 mCi [3H]leucine/2S-cmz flask. Arylsulfa- tase A was immunoprecipitated from 9 parts (for details see above) and cathepsin D from 1 part of the medium as de- scribed [6] and the radioactivity incorporated into arylsulfa- tase A and cathepsin D, respectively, was determined as described above.

Half-Lives of Arylsulfatase A and Cathepsin D

Cells were incubated for 1 h in methionine-free medium and then labelled with either 1 .O mCi/flask of [met!~yl-~H]- methionine (specific activity 12.9 Ci/mmol) or 0.6 mCi/flask of [35S]methionine (specific activity 12.9 Ci/mmol). After incubation for 15 h 0.05 ml of sterile methionine solution (5 mg/ml) was added. After an 8-h chase, media of parallel cultures incubated with either [3H]methionine or [35S]- methionine were combined and processed for isolation of arylsulfatase A from 9 parts and cathepsin D from 1 part by immunoprecipitation, gel electrophoresis and fluorography as described above. The cells labelled with [35S]methionine were harvested and kept frozen. The cells labelled with [3H]- methionine were subjected to a chase with fresh medium for 10 days before harvesting. Cells from parallel cultures incu- bated with either [35S]methionine or [3H]methionine were combined and arylsulfatase A and cathepsin D were isolated as described above. The 3H/35S isotope ratio was determined in the gel section containing the labelled enzymes. To evaluate the rates of the decay of the enzymes, half-lives were cal- culated according to the equation t l p = In 2/k. The rate constant k was obtained from equation In N/No = - k . t , where N is the isotope ratio in the enzyme from cell extract, No that in the enzyme from medium and t the period of chase. The calculated half-lives are treated as an approximate measure of the rate of degradation. The decay of the enzymes studied is not necessarily logarithmic. The period of 8-h chase prior to initial analysis served to allow the newly synthesized enzyme to reach the medium or lysosomes and to exclude a possible short-term rapid phase from the studied long-time decay.

Uptake of Arylsulfatase A

Arylsulfatase A was isolated from bovine testes. The tissue (2.2 kg) was homogenized in 3 vol. 25 mM sodium phosphate, pH 6.0. The homogenate was centrifuged for 30 min at 8900 x g. The residue was extracted in the same way a second time with 0.5 vol. phosphate buffer. The combined supernatant was subjected to precipitation with

Mu1 tiple S u I fa tase

Control Deficiency

92.5-

69 -

46 -

30 -

12.3-

Fig. 1. Pulse-chase labelling of arylsulfatase A . Control and multiple sulfatase deficiency fibroblasts were labelled for 5 and 16 h and sub- jected to a chase for 1, 3 and 7 days, respectively. The immuno- precipitates from cell extracts after a 16-h pulse and chase for 7 days are shown and the migration of [14C]methylated protein standards is indicated ( M , x given on left). The radioactivity incorporated into arylsulfatase A after 5-h and 16-h pulse in controls was 1010 and 1590 counts/min, respectively, and 470 and 750 counts/min in multiple sulfatase deficiency fibroblasts. The relative amount of labelled aryl- sulfatase A recovered after chase periods of 1, 3 and 7 days were 95 ?& 91 % and 103 ”/, in controls and 70 %, 41 % and 38 % in multiple sulfatase deficiency fibroblasts

(NHk)*SOd (400 g/l). The precipitate was collected by cen- trifugation as above, dissolved in the phosphate buffer, mixed with 250 ml concanavalin-A - Sepharose (Pharmacia) and poured into a column. After washing with 25 mM sodium phosphate, pH 6.0, containing 1 M NaCI, the enzyme was eluted with 10 methyl a-glucoside. The enzyme-con- taining fractions were concentrated by ultrafiltration, dialyzed against SO mM sodium phosphate, pH 7.6 and loaded on a DEAE-cellulose column in the same buffer. After washing the column with SO mM sodium phosphate, pH 7.5, the enzyme was eluted with 0.1 M NaCl in the same phosphate buffer. The enzyme-containing fractions were concentrated as above and repeatedly chromatographed on Sephacryl S-200 in 25 mM sodium phosphate, pH 7.5 and in 12 mM sodium citrate/2S mM sodium phosphate, pH 4.5. After discontinu- ous polyacrylamide gel electrophoresis in the anodic system number 1 of Maurer [lo], operating at pH 9 in the separation gel and at a total gel concentration of 8%, 17% of the starting enzyme activity was recovered with a specific activity of 21 U/mg protein. The enzyme was homogenous in poly- acrylamide gel electrophoresis in the presence of sodium dodecylsulfate [I I] and elicited formation of monospecific antibodies in rabbit.

319

Multiple Sulfatase

Aryl- Cathepsin D sulfatase A sulfatase A

92.5-

69 -

46 -

30 -

12.3-

Fig. 2. Secretion of' rrrylsulfatase A and cathepsin D in the presence of I0 m M NH4CI. Cells were labelled for 3 - 12 h in the presence of 10 mM NHLCI and of 0.4 mCi [3H]leucine/25-cm flask. Arylsulfatase A was immunoprecipitated from 9 parts and cathepsin D from 1 part of the medium. The fluoi-ogram shows the immunoprecipitates of a control cell line (81RD5) and multiple sulfatase deficiency cell line (Co.). Values of M , x are given on the left

Arylsulfatase A was partially purified from human urine by precipitation with (NH4)2S04 (chromatography on con- canavalin-A- Sepharose 4B and DEAE-cellulose at pH 7.5 as above. The final enzyme preparation had a specific activity of 0.5 U/mg of protein.

Uptake of arylsulfatase A was determined as described [12] with the modification that the enzyme concentration in the medium was 100 mU/ml. After incubation for 24 h, the cells were washed twice with medium without arylsulfatase A and maintained in this medium up to 20 days.

Arysulfatase A activity was determined by incubating 50 - 60 pg of cell homogenate for 30 min in 240 pl of 0.5 M sodium acetate, pH 5.0, containing 8.3 mM p-nitrocatechol sulfate, 10 % NaCl and 0.05 M sodium pyrophosphate. After adding 200 pl of 1 M NaOH, the absorbance was determined at 515 nm. The unit of enzyme activity is the amount of enzyme hydrolyzing 1 pmol substratte/min.

RESULTS AND DISCUSSION

In an initial experiment, skin fibroblasts from a patient with multiple sulfatase deficiency and control fibroblasts were grown in the presence of [3H]leucune for 5 h and 16 h and then subjected to a chase for up to seven days. Arylsulfatase A was immunoprecipitated from cell extracts and media and subjected to polyacrylamide gel electrophoresis. The labelled polypeptides were visualized by fluorography and quantified by liquid scintillation counting of the excised arylsulfatase A polypeptides. Arylsulfatase A was synthesized in controls and diseased fibroblasts as a 62000-M, precursor (Fig. 1). During pulse and chase less than 7 of the newly synthesized

arylsulfatase A were secreted by both cell lines. Within the chase period intracellularly arylsulfatase A became shortened to a 60500-M, product (Fig. 1). The radioactivity incorpo- rated into arylsulfatase A in the diseased fibroblasts in 5-h and 16-h pulse was 3-4-times lower than in controls and within a chase period of seven days 60 % of the labelled aryl- sulfatase A was degraded in the diseased fibroblasts, whereas in the control less than 10 % of arylsulfatase A was degraded (see legend of Fig. 1). These results suggested that both the apparent rate of synthesis and the half-life of arylsulfatase A were reduced in multiple sulfatase deficiency fibroblasts.

To determine the rate of synthesis of arylsulfatase A under conditions that minimize concomitant degradation in lyso- somes, multiple sulfatase deficiency (n = 5) and control (n = 2) fibroblasts were grown for up to 12 h in the presence of [3H]leucine and 10 mM NH4CI. In the presence of NH4CI more than 90 % of the newly synthesized arylsulfatase A was secreted and remained undegraded in the medium. Aryl- sulfatase A and cathepsin D, a lysosomal enzyme not affected by the mutation in multiple sulfatase deficiency, were immuno- precipitated from the media and the radioactivity quantified after polyacrylamide gel electrophoresis (Fig. 2 and 3). After a lag phase of about 2.5 h and 1.5 h, labelled arylsulfatase A and cathepsin D, respectively, appeared in the medium and were secreted at a continuous rate for the following 10 h. The amount of secreted arylsulfatase A was related to that of cathepsin D. The relative amount of secreted arylsulfataseA was 2- 5-times lower in diseased fibroblasts than in controls (Table I), suggesting a correspondingly lower apparent rate of arylsulfatase A synthesis in multiple sulfatase deficiency fibroblasts.

320

50 - c .- E

40 ;;; .- C 2

u 30 0

C

w Q .-

20 s .- m V I -

10 2

0 m

0 0 3 6 9 1 2 0 3 6 9 12

Incubat ion (h)

Fig. 3. Synthesis vfaryI.sulfutuse A and cathepsin D. The radioactive bands seen in Fig. 2 containing arylsulfatase A and cathepsin D, respectively, were cut out, solubilized and quantified by liquid scintillation counting. (A) [3H]Arylsulfidtase A ; (B) [3H]Cathepsin D ; (0) control cell line (8lRD5); (0) multiple sulfatase deficiency cell line (Co.)

Table 1. Incorporation of [3H]leucine into arj~lsulfatase A and cathepsin D The radioactivity incorporated into arylsulfatase A and cathepsin D during incubation for 3- 12 h in the presence of [3H]leucine and 10 inM NH4CI was determined in two control and five multiple sulfatase de- ficiency fibroblast lines (see Fig.2 and 3). The rates of incorporation were extrapolated from the phase of steady secretion (see Fig. 3)

Cell line Radioactivity incorporated Labelling per hour and flask ratio of aryl-

sulfatase A/ aryl- cathepsin D cathepsin D sulfatase A

counts/min

. . . ~~~

Control 81RD5 490 3890 0.126 254 560 4200 0.133

Multiple sulfatase deficiency

G M 2407 160 3130 0.051 c o . 150 4000 0.038 Vi. 330 4780 0.069 G M 3245 110 4200 0.026 G M 4681 410 5780 0.071

To determine the half-life of arylsulfatase A and cathep- sin D, fibroblasts were pulse-chase labelled with either [3H]- methionine or [3sS]methionine. The isotope ratio was deter- mined for pulse-labelled enzymes (isolated from the media) and for pulse-chase-labelled enzymes (isolated from the cells) and the half-lives were calculated as described in Materials and Methods. The half lives were determined in five multiple sulfatase deficiency and six control fibroblast lines in four different experiments (Table 2). Some variation was noted for the half-lives of arylsulfatase A and cathepsin D and their ratio in different experiments (the three experiments with cell line G M 2407 were performed over a period of 2 1 months). The half-life of cathepsin D in multiple sulfatase deficiency (mean 10.6 days) was 1.5-times shorter than in controls (mean 16.4 days) while that of arylsulfatase A was 9-times shorter (mean 7.1 days) than in controls (mean 65 days). When related to the half-life of cathepsin D in the same cell lines,

Table 2. Half-lives of arylsulfatase A and catliepsin D The numbers in brackets refer to the experiments in which the cell lines were studied

Cell line Half-life of Rat10 O f half- - - lives of aryl- aryl- cathepsin D sulfatase A, sulfatase A cathepsin D

days

Controls H. ( 1 ) 43 254 (1) 111 K. ( 2 ) 48 80RD5 (3) 84 XlRD174 (3) 40 w. (4) 64

Multiple sulfiatase deficiency

G M 2407 (1) (2) (3)

(4) Co. (2)

(4) Vi. (3)

Be. (2)

G M 3245(4) G M 4681 (4)

mean 65

6.3 4.3 6.2 9.6 2.9

10.4 5.4

11.9 4.1 9.7

15 9 2 7 32 5 3 4

8 6 5 6 10 3 8 2 17 0 2 4 14 0 4 6

16 4 4 5

21.0 6.7

11.2 14.2 4.1 9.0

12.8 12.5 6.2 7.9

0.30 0.64 0.55 0.68 0.70 0.81 0.42 0.95 0.66 1.22

- -~

mean 7 1 10 6 0 69

Table 3. Residual activities of arylsulfutase A in multiple sulfatase defi- ciency fibroblasts Controls ( n = 13) liberated 13.3 nmol p-nitrocatechol min-' (mg cell protein)-' (range 7.5-24.4)

~

Cell line Residual activities of drylSUlfdtdSe A -~ ~~

calculdted found in cell from Tables 1 homogenate and 2

~

% control ~~

G M 3245 3 19 co 4 2 G M 2407 4 5 V1 11 15 G M 4681 15 24

the half-life of arylsulfatase A was found to be 4-9-times (mean 6.5) shorter in diseased fibroblasts than in controls.

Table 3 shows that the decrease in the apparent rate of synthesis and the shortening of the half-life of arylsulfatase A correlate well with the residual activities of arylsulfatase A in 4 of 5 multiple sulfatase deficiency cell lines. The present results indicate that the primary defect in the disease reduces the apparent rate of synthesis of arylsulfatase A and more profoundly the stability of the enzyme protein in the cells. The rate of synthesis was determined by quantifying the immunoprecipitable enzyme after a labelling period for at

321

- 2 4 c a, 0

._ +.

2 2 0 - - a, "

16 . 3 E - 12 a m In 2 8 - - 3 v)

,.4 a

0 I

0 5 10 15 2 0 0 5 10 15 2 0 Period of chase (days)

Fig. 4. Half-life of arjhdfatase A activity iiifibroblasts. Multiple sulfatase deficiency (0) and metachromatic leucodystrophy fibroblasts (0) were incubated for 24 h in the presence of 100 mU/ml arylsulfatase A from human urine (A) or from bovine testes (B) and than subjected to a chase for up to 20 days. After the periods indicated, the cells were harvested and assayed for activity of arylsulfatase A and protein. The values were corrected for the residual activity of arylsulfatase A in multiple sulfatase deficiency fibroblasts (cell line Vi.) and metachromatic leucodystrophy

least 3 h. Any newly synthesized arylsulfatase A converted within the labelling period into a sedimentable or non- immunoprecipitable material would escape the detection. Therefore the present results d o not discriminate between a decrease in the rate of synthesis and a condition in which some of the newly synthesized arylisulfatase A molecules are subjected to a rapid modification or degradation (presumably extralysosomally). The reduced half-life indicates an insta- bility of arylsulfatase A in the lysosomes since the newly synthesized enzyme is transferred into lysosomes as indicated by its shortening to a 60500-M, product within less than two days in both diseased and control fibroblasts.

To assay the stability of homologous and heterologous arylsulfatase A in multiple sulfatase deficiency fibroblasts, advantage was taken from the ability of fibroblasts to internalize arylsulfatase A from the extracellular fluid via a mannose-6-phosphate-specific recognition system [12]. Aryl- sulfatase A partially purified from human urine and aryl- sulfatase A purified to homogeneity from bovine testes were used as enzyme sources ; multiple sulfatase deficiency fibro- blasts and fibroblasts from patients affected with meta- chromatic leucodystrophy, which exhibit a single deficiency of arylsulfatase A, were used as recipient cells. During a 24-h incubation period both enzymes were internalized with a similar efficiency by either cell type (Fig. 4). The uptake of both enzymes was mediated by a mannose-6-phosphate- specific recognition system as indicated by inhibition of uptake by more than 90 % in the presence of 0.5 mM mannose 6-phos- phate (not shown). In a following chase period of up to 20 days the homologous and heterologous arylsulfatase A were in-

activated in multiple sulfatase deficiency fibroblasts at a rate similar to that in metachromatic leucodystrophy fibroblasts. The loss of catalytic activity of arylsulfatase A occurred with half-lives of 6- 8 days for the homologous enzyme and 10- 15 day dor the heterologous enzyme (Fig. 4). These results indicate that the decrease in the stability of sulfatases in multiple sulfatidosis is not due to the presence of a factor enhancing breakdown of sulfatases.

The basic defect in multiple sulfatase deficiency remains unknown. The deficiency of several sulfatases in this disorder may hypothetically be explained by the existence of a gene product that affects the stability of all sulfatase affected in multiple sulfatase deficiency. The stability of arylsulfatase A would appear to be affected by such a product both before and after the delivery into the lysosomes. It should, therefore, be of interest to study the stability of other sulfatases in multiple sulfatase deficiency and to search for a gene product that is conferring the stability on sulfatases under normal conditions. Furthermore, the disclosure of decreased stability of arylsulfatase A in multiple sulfatase deficiency provides a rationale for therapeutic trials aiming to increase the stability of sulfatases in diseased cells and may promote understanding of the reported effects of pH [13] and thio- sulfate [14] on arylsulfatase A levels in this disorder.

We thank Drs J . Couchet (Reims), P. Durand (Genova). [I. Wies- mann (Bern) and A. Greene (Human Genetic Mutant Cell Rcposilvry, Camden, NJ) for providing us with multiple sulfatase deficiency cell lines. This work was supported by the Deutsche For.schunb.sgemrinschaJt. A. Waheed is the recipient of an Alexander von Humboldt fellowship.

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A. Waheed, A. Hasilik, and K. von Figur.a, Physiologisch-Chemisches Institut der Westfiilischen Wilhelms-Universitat Miinster, WaldeyerstraRe 15, D-4400 Munster, Federal Republic of Germany