Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6496-6499, December 1979 Genetics

Genetic complementation studies of multiple deficiency (cell fusion//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 (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 . 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 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 ( lippo A or 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 . A generalized Three milliliters of cold serum-free medium containing 10,000 deficiency of 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), (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 of one class (sulfatases) and is not re- taining serum. Fusion with polyethylene glycol 6000 was by stricted to lysosomal (ARSC activity, which includes a modification of the method of Davidson et al. (9). The cell 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 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 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, ; 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. The combined effluent, which (iduronate-sulfate sulfatase deficiency) failed to result in ac- contained desulfated reaction product, was assayed for radio- tivity above that expected from the sum of the individual ac- activity with 15 ml of Aquasol (New England Nuclear). All tivities. These findings indicate that MSD is unlikely'to be due enzyme assays were carried out under conditions in which ac- to a lack of a diffusable . tivity was linear with time and protein concentration. Heterokaryon formation by cell fusion was followed by staining of duplicate plates with hematoxylin. When no fusion RESULTS agent was used, less than 0.2% of the cells had two or more Characterization of MSD Fibroblasts. Cultured MSD fi- nuclei. After fusion with Sendai virus, more than 50% of the broblasts displayed normal levels of ,B-galactosidase (Table 1), cells were multinucleate. Fusion induced with polyethylene f3-glucuronidase, a-L-, f3-N-acetylglucosaminidase, glycol 6000 showed variable degrees of fusion with 35-50% of and a-N-acetylglucosaminidase. In both MSD cell lines, there the cells fused. Polyethylene glycol treatment resulted in cell was a marked decrease in activity of ARSA, ARSB, ARSc, sulf- loss as evidenced by a lesser cell density on stained plates and amidase, iduronate-sulfate sulfatase, and GIcNAc-6-SO4 sul- fatase. Extracts of C.S. cells showed GalNAc-6-SO4 sulfatase Table 2. Sulfatase activity in mixtures of extracts from MSD and activity in a normal range although, on repeated assays of normal fibroblasts various cultures, the levels were at the low end of the normal Activity, pmol/hr range. These deficiencies of ARSA, ARSC, sulfamidase, and Enzyme Normal MSD Normal + MSD iduronate-sulfate sulfatase are similar to those previously re- ported (3, 5, 6). Levels of ARSB have been shown to be variable ARSA 247 4 204 177 14 183 but have been in the range reported here (3, 6). When extracts ARSB Sulfamidase* 1980 65 1940 were to acetate electrophoresis of C.S. cells subjected cellulose Iduronate-sulfate sulfatase 24.2 0.6 22.6 et band was de- by the method of Rattazzi al. (15), the ARSg GalNAc-6-SO4 sulfatase 30.7 0.6 28.5 tectable by the hydrolysis of 4-methylumbilliferone sulfate, but GlcNAc-6-SO4 sulfatase 11.6 1.0 9.8 there was less activity than in extracts of normal fibroblasts. Mixed ARSA was not detectable by electrophoresis. Assay of ARSA utilized 200 ,g of fibroblast extract protein. normal + MSD assays were done with 200 of each. Incubations for extracts with extracts of normal ,gg Mixing of MSD fibroblast the other sulfatases were done with 100 Ag of protein; 100 ,ug of each fibroblasts did not result in significant inhibition of activities were in mixtures. MSD cells were C.S. except that for GlcNAc-6-SO4 of any of the sulfatases (Table 2). Mixtures with a 3-fold excess sulfatase the mixing was with GM2407. of MSD extract over normal extract still showed the activity * cpi/hr. Downloaded by guest on September 26, 2021 6498 Genetics: Horwitz Proc. Nati. Acad. Sci. USA 76 (1979)

Table 3. Sulfatase activity in mixtures of MSD extracts and Table 5. Correction of iduronate-sulfate sulfatase deficiency in extracts of fibroblasts from various sulfatase deficiencies MSD by Hunter disease heterokaryons Activity Iduronate-SO4 sulfatase Iduronate- activity, ARSA, Sulfami- S04 pmol/hr/mg Enzyme nmol/hr/ dase, sulfatase, Fibroblasts Exp. 1 Exp. 2* source mg cpm/hr/mg pmol/hr/mg Unfused: Normal 1420 12,500 373 Normal 87.6 68.5 MSD 116 400 9.5 Hunter 4.8 3.5 Metachromatic MSD 22.5 13.5 leukodystrophy 37 MSD + Hunter 12.2 10.1 Sanfilippo A 20 Fused: Hunter 3.5 Normal 97.4 75.4 MSD + MLD 87 Hunter 6.8 5.6 MSD + Sanfilippo A 120 MSD 16.5 12.4 MSD + Hunter 6.0 MSD + Hunter 47.9 39.4 ARSA and sulfamidase were assayed with 70 ,ug of extract protein Cells were treated as in Table 4. Both experiments utilized MSD or 35 jsg of each extract in mixtures. Iduronate-sulfate sulfatase assays line C.S. were performed on 40 ,g of protein for individual extracts or 20 ,g * Harvested 3 days after fusion (day 5). each in mixtures. and fused normal cells. The activity of het- per were labeled with similarly plated less cellular protein plate. MSD cells 3- to 4-fold over that of the unfused mixed [3H]thymidine according to the method described by Rattazzi erokaryons increased fused cells. Polyethylene glycol-fused heterokaryons also showed such et al. (16), mixed with unlabeled Sanfilippo A cells, and complementation but activity was less than that observed for with inactivated Sendai virus. Autoradiography demonstrated Sendai virus-fused cells. that 62% of the multinucleated cells (31% of all cells) contained Similar fusions were attempted with mixtures of MSD fi- nuclei of both cell types. broblasts and those from a patient with metachromatic leuko- The results of two experiments involving fusion of MSD fi- dystrophy. ARSA activity in the heterokaryons was not signif- broblasts with Sanfilippo A cells are shown in Table 4. Mixed icantly higher than in unfused mixed cells when assayed 2 days unfused cells showed insignificant activity (<4% of normal). after fusion. When equal numbers of the two MSD cell lines After fusion, the heterokaryon-containing plates demonstrated were mixed and fused, the resulting heterokaryon mixture did 32% and 22% of normal sulfamidase activity, respectively, in not show increased activity of ARSA, iduronate sulfatase, or the two experiments listed. In unmixed fused cultures of MSD sulfamidase. cells, sulfamidase activities remained <5% of that of fused normal cells. Heterokaryons formed by polyethylene glycol showed a similar trend but a lesser degree of correction of ac- DISCUSSION 16% and 25% of normal). tivity (between to Table 5 shows the results of iduronate-sulfate sulfatase assays These studies extend the spectrum of deficiencies in MSD after similar fusion experiments using MSD and Hunter fi- two additional sulfatases. GlcNAc-6-S04 sulfatase, which may in extracts of MSD, Hunter, and mixed be involved in the degradation of both and broblasts. The activities lines. However, one but unfused MSD and Hunter cells all were <18% of normal. keratan sulfate, was defective in both cell of the MSD lines displayed a deficiency of GalNAc-6-SO4 sul- Homokaryons produced by fusion of MSD or Hunter cells alone of remained at that level. Extracts of the fused mixed cultures fatase whereas the other (C.S.) consistently showed activity heterokaryons displayed 40-50% of the activity of this enzyme within the normal range. Thus, there may be containing heterogeneity in the deficiencies. Variations have been reported in the levels of ARSB in tissues and fibroblasts in this syndrome Table 4. Correction of sulfamidase deficiency in MSD-Sanfilippo (3, 6). Although excess heparan sulfate and A heterokaryons in tissues and urine has been reported (1, 2), there have been Sulfamidase activity, no reports of abnormalities of keratan sulfate in cpm/hr/mg MSD as would be expected from deficiencies of the 6-sulfa- Fibroblasts Exp. 1 Exp. 2 tases. The appearance of active enzyme in the heterokaryons may Unfused: be due to Hunter or Sanfilippo A components' causing the ac- Normal 5890 14,900 tivation or production of enzymes of the MSD cell genome. The Sanfilippo A 60 420 possibility exists, however, that the increased enzymic activity MSD 55 260 in the heterokaryons results from some MSD-derived factor MSD + Sanfilippo A 240 580 affecting enzymes derived from the Sanfilippo A or Hunter Fused Cells: me- Normal 5780 13,700 cells. Although many lysosomal storage diseases, such as Sanfilippo A 59 720 tachromatic leukodystrophy, result from production of de- MSD 300 500 fective enzymes, as evidenced by the presence of antigenically MSD + Sanfilippo A 1894 3,025 crossreacting material (17), such studies have not been carried out for Hunter and Sanfilippo A syndromes. Cells (106 cells of individual types or 5 X 105 each of mixed cells) The results reported here indicate that complementation were plated in 60-mm dishes. On day 2, cells were fused with Sendai A as as virus and harvested for assay on day 4. Exp. 1 is the result of fusion occurs between MSD fibroblasts and Sanfilippo well of GM2407 cells with Sanfilippo A cells. Exp. 2 was carried out with Hunter syndrome fibroblasts. It is unlikely that a "corrective C.S. cells and the Sanfilippo A cells. factor" diffusable in culture medium similar to that described Downloaded by guest on September 26, 2021 Genetics: Horwitz Proc. Natl. Acad. Sci. USA 76 (1979) 6499

by Fratantoni et al. (18) is active because unfused mixed cells 1. Murphy, J. V., Wolfe, H. J., Balazs, E. A. & Moser, H. W. (1971) are not corrected. Previous studies by Eto et al. (3) have shown in Lipid Storage Diseases, eds. Bernsohn, J. & Grossman, H. J. no correction of sulfated macromolecule accumulation i (Academic, New York), pp. 67-110. mixtures of MSD with Hunter and Sanfilippo A fibroblasts. 2. Austin, J. H. (1973) Arch. Neurol. 28, 258-264. Although experiments involving mixtures of cell extracts rule 3. Eto, Y., Rampini, S., Wiesmann, U., Carson, J. H. & Hersch- out a activator kowitz, N. N. (1974) Arch. Neurol. 30, 153-156. diffusible or inhibitor, they do not eliminate the 4. Austin, J. (1973) in Lysosomes and Storage Diseases, eds. Hers, possibility of some factor that processes or activates the enzyme H. G. & Van Hoof, F. (Academic, New York), pp. 411-437. in vio. 5. Neufeld, E. F., Liebaers, I. & Lim, T. W. (1976) in Current Complementation of MSD with metachromatic leukodys- Trends in Sphingolipidoses and Allied Disorders, eds. Volk, B. trophy fibroblasts has not been successful. It is not clear if this W. & Schneck, L. (Plenum, New York), pp. 253-276. failure is due to a different basis for the defect in ARSA in MSD 6. Fluharty, A. L., Stevens, R. L., Davis, L. L., Shapiro, L. J. & Ki- or to details of cell fusion conditions. It has been reported that, hara, H. (1978) Am. J. Hum. Genet. 30,249-255. in some MSD cell cultures, ARSA activity was increased to near 7. Horwitz, A. L. & Caswell, N. (1979) Fed. Proc. Fed. Am. Soc. normal levels by a modification of culture medium that led to Exp. Biol. -38,496. 8. Hug, G., Soukup, S. W., Schubert, W. K., Bove, K. & Walling, improved pH control (6). However, iduronate-sulfate sulfatase L. (1972) Pediat. Res. 6, 422A. and sulfamidase were not affected by such changes in cul- 9. Davidson, R. L. & Gerald, P. S. (1976) Somat. Cell Genet. 2, ture. 165-176. The defect of MSD is not likely to be due to the absence of 10. Milsom, D. W., Rose, F. A. & Dodgson, K. S. (1972) Biochem. J. a common enzyme subunit because ARSA and ARSB have been 128,331-336. purified and found not to have subunits related either struc- 11. Baum, H., Dodgson, K. S. & Spencer, B. (1959) Clin. Chim. Acta turally or antigenically (18). If sulfatase synthesis is controlled 4, 453-455. by a common regulator gene, it cannot be acting in a polycis- 12. Hall, C. W., Liebaers, I., DiNatale, P. & Neufeld, E. F. (1978) tronic manner because ARSA, ARSB, and iduronate-sulfate in Methods in Enzymology, ed. Ginsburg, V. (Academic, New sulfatase are present on Evidence York), Vol. 50, pp. 439-456. separate (20). 13. Horwitz, A. L. & Dorfman, A. (1978) Biochem. Biophys. Res. exists that mucolipidosis (II) ("I-cell" disease), which is a mul- Commun. 80,,819-825. tiple lysosomal deficiency, results from a defect in 14. Telser, A., Robinson, H. C. & Dorfman, A. (1966) Arch. Biochem. post-translational processing of enzymes (21, 22). A separate 116,458-465. enzyme modification step may be necessary for sulfatase pro- 15. Rattazzi, M. C.., Marks, J. S. & Davidson, R. G. (1973) Am. J. duction or stability, but it is likely to be on a different basis than Hum. Genet. 25,310-316. the mucolipidosis because I have not found excess sulfatase 16. Rattazzi, M. C., Brown, J. A., Davidson, R. G. & Shows, T. B. activity in culture medium of these cells. A recent report has (1976) Am. J. Hum. Genet. 28, 143-154. indicated that cultured MSD cells contain small amounts of 17. Neuwelt, E., Stumpf, D., Austin, J. & Kohler, P. (1971) Biochim. ARSA and ARSB that are antigenically crossreactive with normal Biophys. Acta 236, 333-339. 18. Fratantoni, J. C., Hall, C. W. & Neufeld, E. F. (1968) Science 162, specific activity (23). These later findings taken together with 570-572. the studies reported in the present paper suggest that the genetic 19. Shapira, E., DeGregorio, R. R., Matalon, R. & Nadler, H. (1975) defect in MSD involves either a regulatory process for pro- Biochem. Biophys. Res. Commun. 62,448-455. duction of sulfatases or a post-translational modification com- 20. DeLuca, C., Brown, J. A. & Shows, T. B. (1979) Proc. Nati. Acad. mon to sulfatases. Sci. USA 76, 1957-1961. 21. Champion, M. J. & Shows, T. B. (1978) Science 270, 64-66. 22. Hasilik, A., Rome, L. H. & Neufeld, E F. (1979) Fed. Proc. Fed. The author gratefully acknowledges the assistance of Ms. Nancy Am. Soc. Exp. Btol. 38,467. Caswell. This work was supported by U.S. Public Health Service Grants 23. Fiddler, M. B., Vine, D., Shapira, E. & Nadler, H. L. (1979) AM-05596, HD-09402, and HD-04583. Pediat. Res. 13, 419A. Downloaded by guest on September 26, 2021