Am J Hum Genet 30:249-255, 1978

Presence of Arylsulfatase A (ARS A) in Multiple Sulfatase Deficiency Disorder Fibroblasts ARVAN L. FLUHARTY,1"2 RICHARD L. STEVENS,"12 LAUREL L. DAVIS,"12 LARRY J. SHAPIRO, 1"3 AND HAYATO KIHARA 1,2 INTRODUCTION

Multiple sulfatase deficiency disorder (MSDD) is an autosomal recessive trait in which affected individuals present clinical features of both metachromatic leukodystrophy and the mucopolysaccharidoses [1]. Biochemically, as implied by the name, there is a generalized deficiency of sulfatases in tissues and organs. Eto et al. [2] reported that fibroblasts from two patients with MSDD were also deficient in various sulfatases including arylsulfatase A (ARS A). We reexamined both cell lines and found that under certain culture conditions, these fibroblasts contained appreciable ARS A activity. This enzyme was indistinguishable from ARS A of normal fibroblasts by a variety of parameters. It is, therefore, suggested that the genome for ARS A is intact in MSDD fibroblasts. MATERIALS AND METHODS

Materials 4-Nitrocatechol sulfate and p-nitrophenyl sulfate were obtained from Sigma Chemical Co. (St. Louis, Mo.); [7 -3H ] dehydroepiandrosterone sulfate (20 Ci/mmol) from New England Nuclear (Boston, Mass.); and DEAE-cellulose (Whatman DE-32) from Reeve Angel (Clifton, N.J.). The preparation and isolation of 4-methylumbelliferyl sulfate [3], cerebroside [35S] sulfate [4], and 0-(a-L-idopyranosyluronic acid 2-sulfate)-(1-4)-2,5-anhydro-D- [1 - 3H]mannitol 6-sulfate [5] have been described.

Cell Lines

Fibroblast cultures MSDD- 1 and MSDD-2 were provided by Dr. Ulrich N. Wiesmann. They were designated as metachromatic leukodystrophy variant case 1 and case 2, respectively, in the report on biochemical findings by Eto et al. [2]. Control cultures were from normal subjects and from patients with known metabolic disorders. Received June 20, 1977; revised October 20, 1977. This study was supported by grants NS-1 1665 and HD-4612 from the National Institutes of Health and grant 5-65 from the National Foundation-March of Dimes. All authors: University of California, Los Angeles, School of Medicine, Los Angeles, California. 2 Neuropsychiatric Institute-Pacific State Hospital Research Group, Pomona, California 91766. 3 Division of Medical Genetics, Department of Pediatrics, Harbor General Hospital Campus, Torrance, California 90509. © 1978 by the American Society of Human Genetics. All rights reserved.

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FLUHARTY ET AL.

Culture Media The formulations described are for 10 liters of media. Cultures in MEM-CO2 were incubated at 370C in an atmosphere of 5% CO2 in flasks with vented caps. Cultures in MEM-HEPES were incubated in ungassed incubators, and flasks were tightly capped. Media, media supplements, and serum were obtained from Gibco (Grand Island, N.Y.), except for aureomycin from Lederle (Pearl River. N.Y.), and the organic buffer from Calbiochem (La Jolla, Calif.). MEM-CO2. Minimum essential medium with Earle's salts (Gibco No. F- I 1) was formulated with 100 ml nonessential amino acids (lOOx), 100 ml sodium pyruvate (100 mM), 200 ml L-glutamine (200 mM), 16 g NaHCO3, 200 ml pen-strep solution (penicillin 5,000 U/ml; streptomycin, 5.0 mg/ml), and 1,000 ml fetal calf serum. This medium was placed in a CO2 incubator for 24 hr prior to use (pH 7.2). MEM-HEPES. Minimum essential medium with Earle's salts (Gibco No. F- I 1) was formulated with 1 g L-glutamine, 22 g NaHCO3, 59.6 g HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 0.5 g aureomycin, and 1,000 ml fetal calf serum. The pH was adjusted to 7.4

Cell Extracts Fibroblast cultures were harvested by trypsinization and washed twice with normal saline. Cells were suspended in an equal volume of 25 mM Tris-HCl buffer, pH 7.5, lysed by six cycles of freezing and thawing, and centrifuged for 2 min at 14,000 g. The supernatant fluid was used for assessing activities of soluble enzymes. For assays of cerebroside sulfatase and iduronate sulfatase, chromatography on DEAE-cellulose and antibody precipitation, extracts were dialyzed overnight against two changes of 4 liters of the extract buffer. The pellet material was used for assessing activities of insoluble enzymes (ARS C and steroid sulfatase). Protein estimations of the extract and pellet material were carried out by the Lowry method [6]. Enzyme Assays Routine assays for ARS A were carried out by a modification [7] of the Baum et al. [8] assay for the A enzyme. Activity was also assessed by the hydrolysis of 4-methylumbelliferyl sulfate [3] and cerebroside sulfate [9]. ARS B activity was determined by a modification [7] of the Baum et al. [8] assay for the -B enzyme. The procedures for estimating ARS C and dehydroepiandrosterone sulfate sulfatase by suspended pellet material have been described [10]. The activity of iduronate sulfatase was estimated by the hydrolysis of sulfoiduronosyl sulfoanhydromannitol by a procedure similar to that of Liebaers et al. [11]. RESULTS

Fibroblasts Cultured in MEM-CO 2 and MEM-HEPES MSDD fibroblasts cultured in MEM-CO2 showed less than 10% of the normal ARS A which is comparable to the deficiency in metachromatic leukodystrophy fibroblasts. This is in agreement with the results of Eto et al. [2] who also used this medium. A typical set of results is shown in table 1. To our surprise, parallel MSDD fibroblasts which had been cultured in MEMHEPES did not show this deficiency. Analyses of cells from different subcultures indicated that ARS A was indeed present, and the level varied from 30% to 100% of normal (table 1). It was conceivable that this unexpected enzyme was a product of contaminant organisms, a possibility which was explored by several means. The cultures were maintained for several months in MEM-HEPES supplemented with the anti-PPLO

ARYLSULFATASE A IN MSDD FIBROBLASTS

251

TABLE I ACTIVITIES OF ARYLSULFATASES OF MSDD FIBROBLASTS CULTURED IN MEM-CO2 AND IN MEM-HEPES ARYLSULFATASE (,umol/hr/mg)

CULTURE

In MEM-C02: MSDD-1 ............................... MSDD-2 ............................... Metachromatic leukodystrophy .............. Control ................................. In MEM-HEPES: MSDD- I ............................... MSDD-2 ............................... Control .................................

A

B

C

0.18 0.06 0.05 2.43

0.41 0.35 1.00 1.04

0.035 0.052

0.052

0.7 2.0 2.1

1.1 1.0 1.2

0.034 0.016 0.033

...

agent Tylocine and the antifungal agent Mycostatin. ARS A activities remained in the same range. Media from the MSDD cultures were added to metachromatic leukodystrophy fibroblasts in an attempt to promote enzyme production by infecting these cultures. Neither filtered nor unfiltered media from either MSDD cultures elicited ARS A production by metachromatic leukodystrophy fibroblasts. The bulk of the ARS A activity of MSDD fibroblasts was precipitated by antiserum to human ARS A at about the same extent as enzyme from control fibroblasts (table 2). (Failure to obtain complete precipitation is attributed to incomplete exclusion of ARS B in the assay with nitrocatechol sulfate [13].) This antibody preparation does not crossreact with human ARS B, bovine ARS A, nor limpet ARS (unpublished results).

Comparison of MSDD and Normal ARS A The elution profile of MSDD fibroblast ARS A on DEAE-cellulose chromatography was identical with that of enzyme from control fibroblasts (fig. 1). ARS A was eluted as a sharp peak at the same NaCl concentration and was clearly separated from ARS B. The major form of the latter enzyme appeared in the void volume, and the minor anionic forms [3] were eluted before ARS A in close coincidence with enzyme of control fibroblasts. TABLE 2 PRECIPITATION OF MSDD ARS A BY ANTISERUM TO HUMAN ARS A

Culture

MSDD- I .............. MSDD-2 .............. Control .4.4

Specific ARS A Activity

ARS A Used

Nonprecipitated ARS A

(U/mg protein)

(11U)

(,zU)

Precipitated (%)

2.2 0.5

76 54 56

6 16 8

92 70 86

ARS A

NOTE. -ARS A = arylsulfatase A; U =1 zmol substrate hydrolyzed/hr. Enough antiserum to precipitate 630 ,U of pure ARS A was incubated with dialyzed fibroblast extracts, then excess goat anti-rabbit y-globulin was added and further incubated. The ARS A remaining in the nonprecipitated fraction was estimated by the usual reaction with nitrocatechol sulfate. The percent of ARS A precipitated was calculated from the ARS A used and nonprecipitated ARS A.

FLUHARTY ET AL.

252

Arylsulfatase B E

_ 15

-

0

E

5

0

Arylsulfatase A 5

5

0

10

15

20

25

FRACTION NUMBER FIG.

1.-The elution

profile

of MSDD fibroblast

arylsulfatases

on

DEAE-cellulose

chromatography.

Dialyzed cell extracts were chromatographed on DEAE-cellulose and monitored by the hydrolysis of 4-methylumbelliferyl sulfate by procedures previously described [3]. MSDD fibroblasts (A); control fibroblasts (0).

The MSDD ARS A derived from the DEAE-cellulose chromatography had ratios of activities toward nitrocatechol sulfate, methylumbelliferyl sulfate, and cerebroside sulfate which were quite similar to those of enzyme from control fibroblasts (table 3). Thermal inactivation profiles of MSDD and control enzymes were essentially identical. Enzyme from both sources remained active with 10 mM cyanide ions and both were inhibited by 2.5 mM phosphate and 2.0 mM silver ions.

Other Sulfatases The ARS B activity in MSDD fibroblasts was not severely depressed (cf. table 1), a finding also noted by Eto et al. [2]. The activity ranged between half-normal and normal whether the cells were cultured in MEM-C02 or MEM-HEPES. This range is significantly higher than the level seen in Maroteaux-Lamy syndrome fibroblasts [14]. The enzyme was similar to the normal B enzyme by its chromatographic profile (cf. TABLE 3

ACTIVITY RATIOS OF MSDD ARS A WITH DIFFERENT SUBSTRATES ACriVITY RATIO Nitrocatechol S04 Cerebroside S04

Methylumbelliferyl S04:

Methylumbeiliferyl S04 15 18

69 56

4.5 3.2

Nitratechol S04. MSDD Control

...........

...........

Cerebroside S04

ARYLSULFATASE A IN MSDD FIBROBLASTS

253

TABLE 4 ACTIVITIES OF STEROID AND IDURONATE SULFATASES IN MSDD FIBROBLASTS

Culture

MSDD-

.

Medium

Dehydroepiandrosterone Sulfate Sulfatase (nmol/hr/mg)

Iduronate Sulfatase (nmol/hr/mg)

0.45 0.24 0.18 0.14 0.01

0.33 0.51

MEM-CO2 MEM-HEPES

MSDD-2 ................

MEM-CO2

MEM-HEPES Steroid sulfatase deficiency .. MEM-CO2 Hunter syndrome ......... MEM-HEPES Control ....... ... MEM-CO2 MEM-HEPES