Am J Hum Genet 31:574- 580, 1979

Arylsulfatase A Modulation with pH in Multiple Sulfatase Deficiency Disorder Fibroblasts ARVAN L. FLUHARTY,12 RICHARD L. STEVENS,"12 SYLVIA D. DE LA FLOR, 12 LARRY J. SHAPIRO,"13 AND HAYATO KIHARA'2

SUMMARY

It has been observed that multiple sulfatase deficiency disorder (MSDD) fibroblasts contained from profoundly deficient to near normal amounts of arylsulfatase (ARS) A depending on the medium in which they were cultured. Our present findings show that the major factor determining the enzyme level is the pH of the medium during growth. In media which became acidic or was maintained at low pH (< 7), the cells expressed the enzymopathy, while in high pH media (7.4), the cells produced enzyme. The high and low enzyme states were reversible. The ARS A deficiency in MSDD must, therefore, be a secondary manifestation of a mutation in another system.

INTRODUCTION

Multiple sulfatase deficiency disorder is a recessively transmitted disorder in which there is a simultaneous deficiency of a number of sulfatases in tissues of affected patients [1]. This is reflected in the clinical manifestations, which include a spectrum of features associated with disorders where each of the sulfatases are deficient individually. Deficiencies of all the sulfatases tested have also been observed in cultured fibroblasts derived from MSDD patients [2]. The molecular basis of the multiple enzyme deficiency is ambiguous. At least two of the deficient sulfatases are X-linked, while others are known to be autosomal. Most of the deficient sulfatases are associated Received-January 1, 1979; revised April 25, 1979. This study was supported by grants NS- 11665 and HD-4612 from the National Institutes of Health and grant 5-65 from the National Foundation-March of Dimes. 1 School of Medicine, University of California at Los Angeles, Los Angeles, Calif. 2 Neuropsychiatric Institute-Pacific State Hospital Research Group, Pomona, CA 91766. 3Division of Medical Genetics, Department of Pediatrics, Harbor General Hospital Campus, Torrance, Calif. © 1979 by the American Society of Human Genetics. 0002-9297/79/3105-0011$00.91

574

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575

with the catabolic complement of lysosomes, but at least one is associated with the anabolic system of microsomes. There have been a number of speculations on the possible mechanism for the concurrent deficiency of this group of enzymes, but there is a notable lack of experimental support for any of the postulates. Recently, we made the paradoxical observation that MSDD fibroblasts were deficient in ARS A when cultured in MEM-C02 medium, but cells cultured in MEM-HEPES medium contained near normal amounts of ARS A [3]. Other sulfatases were present at levels ranging from mildly deficient to near normal and did not appear to show the media dependent modulation. The ARS A from MSDD fibroblasts was indistinguishable from enzyme of control fibroblasts on DEAE-cellulose chromatography, ratio of activity with several substrates, thermal inactivation, sensitivity to inhibitors, and precipitation by antiserum to human ARS A. Therefore, we concluded that the ARS A genome was intact in fibroblasts and, by extension, in MSDD patients. The MEM-C02 and MEM-HEPES media differed in three respects. The MEM-CO2 medium contained nonessential amino acids and pyruvate which MEM-HEPES did not, so the former was somewhat richer. The MEM-C02 medium relied on bicarbonate-CO2 as the buffer system, while in MEM-HEPES the organic ampholyte acted as the buffer. The pH profile during growth of fibroblasts in MEM-CO2 showed a significant downward shift, while the pH in MEM-HEPES was quite stable and remained near 7.4. Conditions which induced MSDD fibroblasts to produce ARS A, thus, appeared to be a restricted nutritional state, the organic buffer, and/or the stable pH during growth. Present studies show that neither the nutritional state nor the buffer system has any effect, and enzyme modulation is largely dependent on the pH of the medium during growth of the fibroblasts. MATERIALS AND METHODS

Cell Lines Fibroblast cultures MSDD- 1 and MSDD-2 were provided by Dr. U. 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]. Culture Media The formulations described are for one liter of media. Cultures in MEM-CO2 and 199-CO2 were incubated at 37°C in an atmosphere of 5% CO2 in flasks with vented caps. Cultures in MEM-HEPES and MEM-HHB (see below) were incubated in ungassed incubators, and flasks were tightly capped. Media, media supplements, and sera were obtained from Gibco (Grand Island, N.Y.), except for aureomycin from Lederle (Pearl River, N.Y.) and organic buffers from Calbiochem (La Jolla, Calif.). MEM-CO2 was formulated from minimum essential medium with Earle's salts (Gibco no. 410-1 100) and 10 ml nonessential amino acids (100-fold concentrate), 10 ml sodium pyruvate (100 mM), 20 ml L-glutamine (200 mM), 1.6 g NaHCO3, 20 ml pen-strep solution (penicillin, 5,000 U/ml; streptomycin, 5 mg/ml), and 100 ml fetal calf serum. This medium was placed in a CO2 incubator for 24 hrs and the pH adjusted to 7.2 prior to use. 199-CO2 was formulated from medium 199 with Earle's salts (Gibco no. 400-1100) and 0.1 g L-glutamine, 2.2 g NaHCO3, 50 mg aureomycin, and 100 ml fetal calf serum. The pH was adjusted to 7.4. MEM-HEPES was formulated from Gibco no. 410-1100 and 0.1 g L-glutamine, 2.2 g NaHCO3, 5.96 g N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 50 mg au-

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reomycin, and 100 ml fetal calf serum. The pH was adjusted to 7.4. MEM-HHB was formulated as MEM-HEPES with the organic buffer component modified to contain 3.57 g HEPES, 3.78 g N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid (HEPPS), and 5.33 g N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES). The pH was adjusted to the desired value (see RESULTS). Enzyme Assay Fibroblast cultures were harvested by trypsinization and washed twice with normal saline. Cells were suspended in an approximately equal volume of 25 mM Tris-HCI buffer, pH 7.5, lysed by six cycles of freezing and thawing, and centrifuged for 2 min at 14,000 g. Protein was determined on an aliquot of the supernatant fluid [4]. The ARS A activity in extracts was determined by the procedure of Baum et al. [5] as modified in this laboratory [6]. The incubation mixture contained 10 mM nitrocatechol sulfate (2-hydroxy-5-nitrophenyl sulfate; Sigma Chemical Co., St. Louis, Mo.); 0.5 M sodium acetate buffer, pH 5.0; 0.5 mM Na3P207; 1.7 M NaCl; and bovine serum albumin, 0.2 mg/ml. Five microliters of extract were added to 0.2 ml of this mixture, the complete mixture incubated for 30 min at 370C, and the reaction terminated with 0.2 ml 1 N NaOH. The released nitrocatechol was measured against a zero-time control by absorbence at 515 nm. RESULTS

The influence of nutrient complexity of the growth medium on the ARS A level was assessed by examining MSDD fibroblasts grown in 199-CO2. This medium contains a large variety of supplementary nutrients and is richer than either MEM-HEPES or MEM-C02. Cells grown to confluency in 199-CO2 were subcultured at a 1:2 ratio and grown in 199-CO2 for 3 weeks with a weekly medium change. A parallel set of cultures was grown in MEM-CO2 with the same refeeding schedule for comparative purposes. The MSDD fibroblasts cultured in 199-CO2 contained easily measurable levels of ARS A with specific activities in the range previously observed in cells cultured in MEM-HEPES [3] (table 1). As previously reported [3], MSDD cells cultured in MEM-CO2 contained marginal amounts of enzyme, the activity approaching that seen in metachromatic leukodystrophy fibroblasts (0.05 to 0.12 gmolhr per mg). Enzyme levels of control fibroblasts cultured in either media were the same. The high and low enzyme states, therefore, appeared to be independent of the richness of the growth medium. Furthermore, the expression of the high enzyme state in both 199-CO2 and MEM-HEPES indicated that the buffer system was also not involved. The pH of the medium during growth was next examined. TABLE I COMPARISON OF ARS A LEVELS OF FIBROBLASTS CULTURED IN MEM-CO2 AND 199-CO2 Medium*

Culture

ARS A (jimollhr/mg)

EM-CO2 MSDD-l ........................................M... 1199-CO2

0.18 0.79

MEM-CO2

0.06 0.40

........................MEM-C02 1199-CO2

2.43 2.30

MSDD-2 Control *

......................................

Cultures were refed once a week.

1199-CO2

ARYLSULFATASE A MODULATION

577

In one test, fibroblasts were cultured in MEM-HEPES, MEM-CO2, and 199-CO2. The medium of one set of cells in each of the three media was adjusted daily to pH 6.8, and the medium of another set to 7.4 for 2 weeks. MSDD fibroblasts maintained at the higher pH contained higher levels of ARS A than cells cultured at the lower pH (data not shown). In another test, MEM-HEPES was reformulated to include additional organic ampholytes with pKa' s between pH 6 and 8 to provide buffering over the pH range of interest (MEM-HHB). Fibroblasts grown to confluency in MEM-HEPES were subcultured at a 1:2 ratio and allowed to attach in MEM-HEPES. On the following day, the medium was replaced with MEM-HHB adjusted to different pH's between 6.6 and 7.4. The cultures were refed once a week. The pH's were monitored daily by the shade of the indicator and weekly by measurement of used media with a pH meter. There was a slight acidic shift of the pH, but it did not exceed 0.2 units over the 1 week period between feedings. The cells were harvested and analyzed after 3 weeks. Cultures at pH 6.6 remained at relatively low cell densities and never attained the confluent monolayers seen with the other cultures. Therefore, ARS A activities were compared by using different reference parameters: total units, ratio to a second lysosomal hydrolase (,f-N-acetylhexosaminidase [,JHA]) and ratio to extractable protein. By all parameters, the greatest depression of ARS A in MSDD fibroblasts occurred at the lowest pH. The total amount and relative activity of the enzyme increased as the pH was raised (fig. 1). The relative ARS A activity in control fibroblasts was not affected by pH, although the total enzyme per flask at pH 6.6 was reduced somewhat (results not shown). The pH dependence of ARS A in MSDD fibroblasts was clearly indicated. To establish whether the high and low enzyme states were reversible, MSDD fibroblasts were alternated between MEM-HEPES and MEM-CO2. Cells grown to confluency in MEM-HEPES were subject to the regimen indicated in table 2. Examination of specific enzyme activity showed that the enzyme level of the cells in MEM-HEPES was reduced by about one-half after a 2-week period in MEM-CO2, and the higher level was restored by a subsequent 2 weeks in MEM-HEPES. Examination by total enzyme content, extrapolated to include all cells derived from the original culture, provided a much better perspective of the effects of the two media. There was only a small (- 25%) increase of ARS A in fibroblasts changed from MEM-HEPES to MEM-CO2. However, there was more than a 300% increase of enzyme in cells changed back to MEM-HEPES from MEM-CO2. With both changes, cellular protein doubled in the expected manner. The high and low enzyme states were, therefore, reversible. DISCUSSION

The media dependent modulation of ARS A activity in MSDD fibroblasts was shown to be predicated largely by the pH of the medium during growth of the cells. Growth at low pH yielded cells with diminished enzyme content and growth at high pH produced cells with increased enzyme. Two other parameters considered, nutrient complexity and buffering agent, proved to have no effect on enzyme levels. Both the high and low enzyme states could be achieved in either of the media formulations by manipulation of pH. Similarly, both states were achieved whether the media were buffered by bicarbonate-CO2 or by organic ampholytes.

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Units ARS A/Flask X102 (eo) in

0

U'

00 N

N

ARS A to,8FiA Rati io O

(.--o)

-

*

c

?

I

e I' 001

N 1"I, N

6

I

0

Units ARS A/rmng Protein (..tb FIG. 1. -Effect of media pH on ARS A activity of MSDD fibroblasts. Closed symbols: initial pH; open symbols: final pH. Fibroblasts were cultured in MEM-HHB adjusted to indicated pH for 3 wks with weekly refeeding. Unit of ARS A activity is defined as 1 gmole of nitrocatechol sulfate hydrolyzed/hr under the assay conditions.

It was shown that a given population of cells could be shifted from high to low and back to the high enzyme state by alternating the growth medium. The period in each medium was restricted to 2 weeks and the split ratio at each subculture was limited to 1:2 in order to minimize possible selective effects. This paradigm resulted in only moderate changes in specific activity because of the long half-life of ARS A (> 3 weeks, unpublished results). However, the total enzyme content (column 4, table 2) shows an almost complete cessation of enzyme production in MEM-CO2, followed by a dramatic increase on return to MEM-HEPES. The pH dependence of metabolic activities of cells in culture, such as growth rate, contact inhibition, and cell motility, is well documented [7-9]. In each instance, the underlying mechanism for the pH dependence is unclear. Of particular relevance is the

579

ARYLSULFATASE A MODULATION TABLE 2 REVERSIBILITY OF HIGH AND Low ARS A LEVELS IN MSDD FIBROBLASTS SPECIFIC ACTIVITY OF ARS A WK

0. 1. 2.

CULTURE REGIMEN

Split 1:2, MEM-HEPES

TOTAL ARS A*

(imol/hr/mg)

umol/hr

...

...

% increase

TOTAL PROTEIN* mg

% increase

...

Fresh MEM-HEPES

Split 1:2, MEM-CO2,

harvest 1/2 3.Fresh MEM-CO 2 4. Split 1:2, MEM-HEPES, harvest 1/2 5. Fresh MEM-HEPES 6. Harvest

0.58

2.32

0.36

2.88

... 0.68

... 12.5

...

25 ...

334

4.0

8.0

100

... 18.4

130

...

* Extrapolated to include all cells derived from original culture.

effect of pH on mucopolysaccharide catabolism [10]. Normal fibroblasts cultured in media where the pH remained high had a diminished rate of mucopolysaccharide turnover, as do cells from patients with genetic mucopolysaccharidoses. In media where there was an acidic shift, there was rapid turnover by normal fibroblasts, so mucopolysaccharidoses cells were readily identified by the [35S]-mucopolysaccharide accumulation test. The MEM-C02 medium used in the present study is frequently used for this test. Eto et al. [2], who first reported an ARS A deficiency in MSDD fibroblasts, used a medium very similar to MEM-CO2. Certain aspects of the present study complicated experimentation. The ARS A activity of MSDD fibroblasts fluctuated over a wider range than usually observed for fibroblast enzymes, so meaningful comparisons by specific activity between experiments were difficult. Experiments were, therefore, designed to be internally controlled. Within this framework, cells cultured at lower pH's consistently contained less enzyme than parallel cultures grown at high pH's. This pattern has now been replicated in four additional MSDD cell strains (unpublished results). The present observations indicate that prenatal diagnosis of MSDD could be problematical. The major portion of the allowable period in culture of amniotic fluid cells is at low cell densities, so significant acidic shifts of the media pH may not occur. Conditions could thus favor ARS A production. Parallel determination of another enzyme, such as iduronate sulfatase which is reduced in MSDD fibroblasts and does not appear to be affected by the culture medium [3], might be advisable. The deficiency of ARS A and other sulfatases in patients is incontrovertible. Nevertheless, these studies support our premise that the genome for ARS A is intact in MSDD and ARS A deficiency is a secondary manifestation of a mutation in another system. The information available provides no indication of the nature of the primary defect nor of the suppression of its manifestation at high pH's. However, experiments can now be designed to explore these parameters. ACKNOWLEDGMENT We are indebted to Dr. Ulrich N. Wiesmann of the University of Bern for the MSDD cultures.

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REFERENCES 1. DULANEY JT, MOSER HW: Sulfatide lipidosis: metachromatic leukodystrophy, in The Metabolic Basis of Inherited Disease, edited by STANBURY JB, WYNGAARDEN JB, FREDRICKSON DS, New York, McGraw-Hill, 1978, pp 770-809 2. ETO Y, WIESMANN UN, CARSON JH, HERSCHKOWITZ NN: Multiple sulfatase deficiencies in cultured skin fibroblasts. Arch Neurol 30:153-156, 1974 3. FLUHARTY AL, STEVENS RL, DAVIS LL, SHAPIRO LJ, KIHARA H: Presence of arylsulfatase A (ARS A) in multiple sulfatase deficiency disorder fibroblasts. Am J Hum Genet 30:249-255, 1978 4. LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ: Protein measurement with Folin phenol reagent. J Biol Chem 193:265-275, 1951 5. BAUM H, DODGSON KS, SPENCER B: The assay of arylsulphatases A and B in human urine. Clin Chim Acta 4:453-455, 1959 6. PORTER MT, FLUHARTY AL, KIHARA H: Metachromatic leukodystrophy: arylsulfatase A deficiency in skin fibroblast cultures. Proc Natl Acad Sci USA 62:887-891, 1969 7. CECCARINI C, EAGLE H: pH as a determinant of cellular growth and contact inhibition. Proc NatlAcadSci USA 68:229-233, 1971 8. EAGLE H: Buffer combination for mammalian cells. Science 174:500-503, 1971 9. RUBIN H: pH and population density in the regulation of animal cell multiplication. J Cell Biol 51:686-702, 1971 10. LIE SO, McKuSIcK VA, NEUFELD EF: Simulation of genetic mucopolysaccharidoses in normal human fibroblasts by alteration of pH of the medium. Proc Natl Acad Sci USA 69:2361-2363, 1972