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Genetic Complementation Studies of Multiple Sulfatase Deficiency (Cell Fusion/Arylsulfatase/Mucopolysaccharidoses) ALLEN L

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Genetic Complementation Studies of Multiple Sulfatase Deficiency (Cell Fusion/Arylsulfatase/Mucopolysaccharidoses) ALLEN L Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6496-6499, December 1979 Genetics Genetic complementation studies of multiple sulfatase deficiency (cell fusion/arylsulfatase/mucopolysaccharidoses) ALLEN L. HORWITZ Department of Pediatrics and Committee on Genetics, Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637 Communicated by Albert Dorfman, August 16,1979 ABSTRACr Cultured fibroblasts from two individuals with MATERIALS AND METHODS multiple sulfatase deficiency (MSD) were found to have de- creased activities of arylsulfatases (aryl-sulfate sulfohydrolase, Fibroblast Cultures. Culture medium was a modification EC 3.1.6.1) A, B, and C as well as iduronate-sulfate sulfatase, of Eagle's medium (formula 78-5005, GIBCO), which has salts, sulfamidase, and N-acetylglucosamine-6-sulfate sulfatase. The vitamins, and amino acids as in Dulbecco's modified Eagle's activity of N-acetylgalactosamine--sulfate sulfatase was de- medium but with ascorbic acid (100 ,gg/ml), ferric nitrate (0.1 creased in one line but not in the other. Mixtures of MSD cell NaHCO3 (4.2 mg/ml), and 20% fetal calf serum. extracts with extracts from normal cells did not result in inhi- ,gg/ml), bition of normal sulfatase activities. Mixtures of MSD cell ex- Growth on plastic dishes was in 10% CO2 in air at 370C. Fi- tracts with extracts of fibroblasts from patients with Hunter or broblast culture C.S. was obtained from H. Kihara and is de- Sanfilippo A syndrome did not activate iduronate-sulfate sul- rived from the patient described by Hug et al. (8). A second fatase or sulfamidase activity. Heterokaryons formed by fusion MSD culture was cell line GM2407 from the Institute for of MSD cells with Sanfilippo A fibroblasts demonstrated a Medical Research (Camden, NJ). Fibroblast cultures were es- partial correction of the enzyme deficiency. In similar manner, MSD-Hunter heterokaryons showed a significant increase in tablished from patients with typical clinical and biochemical iduronate-sulfate-sulfatase activity. Genetic complementation abnormalities of Sanfilippo type A syndrome (mucopolysac- in heterokaryons of MSD fibroblasts and cells of either Sanfi- charidosis IIIA), and Hunter disease (mucopolysaccharidosis lippo A or Hunter syndrome implies a genetic defect in MSD II). The medium was changed every 3 days. The pH of the different from that causing specific sulfatase deficiencies. culture medium was always >7.2. Cell Fusion. Cultured fibroblasts that had undergone 6-12 Multiple sulfatase deficiency (MSD) is an inherited disorder in passages were used. Cells were plated at a density of 106 cells which affected individuals display clinical features resembling per 25-cm2 culture dish or 5 X 105 of each of the two cell types both metachromatic leukodystrophy and the mucopolysac- for a mixed culture. After culture for 18 hr, the medium was charidoses (1, 2). Tissues of patients show abnormal accumu- removed and the plates were rinsed with serum-free medium. lations of sulfatides and glycosaminoglycans. A generalized Three milliliters of cold serum-free medium containing 10,000 deficiency of sulfatases has been described in tissues and cul- agglutination units of /3-propionolactone-inactivated Sendai tured skin fibroblasts (1-4). Arylsulfatase (aryl-sulfate sul- virus (Connaught Laboratories, Ontario, Canada) was added, fohydrolase, EC 3.1.6.1) A (ARSA), arylsulfatase B (ARSB), ar- and the plates were kept at 4°C for 15 min and then at 37°C ylsulfatase C (ARSC), iduronate-sulfate sulfatase (5, 6), and for 30 min. Three milliliters of medium containing 20% fetal sulfamidase (6) activities have been among the sulfatases found calf serum was then added and the cells were incubated for 16 to be decreased. This disease is unique in that it results in a hr; after this the medium was replaced by fresh medium con- deficiency of hydrolases of one class (sulfatases) and is not re- taining serum. Fusion with polyethylene glycol 6000 was by stricted to lysosomal enzymes (ARSC activity, which includes a modification of the method of Davidson et al. (9). The cell steroid sulfatase, is microsomal). I have extended the range of layers were washed with serum-free medium followed by sulfatase deficiencies to two additional enzymes that are in- treatment with 50% (wt/vol) polyethylene glycol in serum-free volved in glycosaminoglycan degradation, N-acetylgalactos- medium for 1 min at room temperature. The plates were rinsed amine-6-sulfate sulfatase (GalNAc-6-SO4 sulfatase) and N- twice with serum-free medium and twice with serum-con- acetylglucosamine-6-sulfate sulfatase (GlcNAc-6-SO4 sulfa- taining medium followed by medium changes at 2 and 18 tase). hr. It has been suggested that the genetic defect in MSD is a The cells were harvested 48 hr after fusion by scraping with faulty regulatory mechanism for sulfatase production (1), al- in 2 ml 50 mM sodium ace- though a lack of a common cofactor or enzyme subunit has not a rubber policeman, suspended of been completely excluded. In order to better understand the tate (pH 6.0), sonicated for 20 sec (Branson Sonifier, micro- lack of sulfatase activity in MSD, complementation analysis probe), and centrifuged at 10,000 X g for 10 min. The super- using cell fusion was carried out with MSD fibroblasts and fi- natant solution was dialyzed against 5 mM sodium acetate at broblasts from patients with various types of sulfatase defi- pH 5.5 and lyophilized; the residue was dissolved in 0.2 ml of ciencies. A preliminary report of these findings has been pre- water prior to enzyme assay. sented (7). Abbreviations: MSD, multiple sulfatase deficiency; ARSA, arylsulfatase A; ARSB, arylsulfatase B; ARSC, arylsulfatase C; GaINAc-6-SO4 sul- The publication costs of this article were defrayed in part by page fatase, N-acetylgalactosamine-6-sulfate sulfatase; GlcNAc-6-S04 charge payment. This article must therefore be hereby marked "ad- sulfatase, N-acetylglucosamine-6-sulfate sulfatase; sulfamidase, hep- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate arin-N-sulfate sulfatase; iduronate-sulfate sulfatase, iduronic acid- this fact. 2-sulfate sulfatase. 6496 Downloaded by guest on September 26, 2021 Genetics: Horwitz Proc. Natl. Acad. Sci. USA 76(1979) 6497 Enzyme Assay. For assays, fibroblast cultures were grown Table 1. Sulfatase activities of MSD fibroblasts for 2-3 weeks, scraped from the dishes with a rubber policeman, Activity, nmol/hr/mg* disrupted by sonication in 0.05 M sodium acetate/0.15 M dd; Control pH 5.5, and centrifuged at 600 X g for 10 min to remove un- mean broken cells and debris. The ARSc assay was performed on the Enzyme (range) C.S. GM2407 et al. resulting supernatant solution by the method of Milsom ARSA 988 70 18 (10). For the remaining enzyme assays, 600 X g supernatant (530-1990) solution was dialyzed against 5 mM sodium acetate at pH 5.5 ARSn 900 150 31 and centrifuged at 10,000 X g for 10 min. No detectable en- (430-1300) zyme activities for any of the lysosomal enzymes were found ARSC 117 13 4 in the precipitate. ARSA and ARSB were assayed by the method (81-168) of Baum (11). Sulfamidase and iduronate-sulfate sulfafase were Sulfamidaset 10.3 0.4 0.5 assayed as described by Hall et al. (12). The assay for Gal- (8.3-14.0) NAc-6-SO4 sulfatase was performed by a modification of the Iduronate-sulfate sulfatase 0.25 0.013 0.033 previously described assay (13) but utilized the substrate (0.20-0.37) N-acetyl-galactosamine 6-sulfate-f1,4-glucuronic acid- GalNAc-6-SO4 sulfatase 0.508 0.41 0.01 /1,3-N-acetyl[3H]galactosaminitol 6-sulfate which was made (0.300-0.657) by reduction of chondroitin 6-sulfate tetrasaccharide (14) with GlcNAc-6-SO4 sulfatasel 78 1.3 0.4 boro[3H]hydride followed by digestion with f3-glucuronidase. (45-148) Incubation mixtures contained 40-60 Mug of enzyme protein, f3-Galactosidase 505 303 593 7.5 nmol of sodium acetate, 4 ug of bovine serum albumin, and (305-730) 13 nmol of trisaccharide in total volume of 50 MI at pH 4.5. After * Mean of at least three separate determinations of each enzyme. incubation at 370C for 6 hr, the monosulfated product was Controls are from extracts of skin fibroblasts from age-matched separated from the disulfated substrate on ECTEOLA-cellulose normal children. t In cpm X 103/hr per mg. microcolumns by the same procedure used to separate substrate pmol/hr per mg. from reaction product in the iduronate-sulfate sulfatase assay (12). The assay for activity of GIcNAc-6-SO4 sulfatase utilized a expected by addition of the activities assayed separately. Thus, disaccharide prepared from keratan sulfate as described (13) MSD does not result from the presence of a diffusable sulfatase and reduced by sodium boro[3H]hydride, resulting in N-ace- inhibitor. tylglucosamine 6-sulfate-131,4-[3H]galactitol. Incubation mix- The results of assay of mixtures of equal amounts of MSD tures contained 13.9 nmol (2.4 X 105 cpm) of disaccharide extract and extracts of fibroblasts from patients with various substrate, 15 nmol of sodium acetate, 20 Mg of bovine serum specific sulfatase deficiencies are shown in Table 3. Mixtures albumin and 40-60 ,g of enzyme protein in a final volume of of MSD extracts with those of Sanfilippo A (sulfamidase defi- 100 ,l at pH 6.0. After incubation at 370C for 16-18 hr, 1 ml ciency) fibroblasts showed sulfamidase activities that remained of water was added, the mixture was applied to a 0.7 X 1 cm <7% of normal. Mixing MSD extracts with those of metachro- column of AG 5OW X 8 resin (Bio-Rad), and the column was matic leukodystrophy (ARSA deficiency) or with Hunter disease washed with 2 ml of water.
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