Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2561-2565, April 1992 Medical Sciences Multiple deficiency: Catalytically inactive are expressed from retrovirally introduced sulfatase cDNAs WINFRIED ROMMERSKIRCH AND KURT VON FIGURA Georg-August-Universitlit, Abteilung Biochemie II, Gosslerstrasse 12d, D-3400 Gdttingen, Federal Republic of Germany Communicated by Elizabeth F. Neufeld, December 18, 1991 (receivedfor review November 18, 1991)

ABSTRACT Multiple sulfatase deficiency (MSD) is an We studied the expression of two lysosomal sulfatases, inherited lysosomal storage disease characterized by the defi- A (ASA) and (ASB), and the ciency of at least seven sulfatases. The basic defect in MSD is microsomal sulfatase (STS). In accordance with the thought to be in a post-translational modification common to all current hypothesis we observed normal amounts of RNA for sulfatases. In accordance with this concept, RNAs of normal ASA, ASB, and STS in group I MSD fibroblasts. More size and amount were detected in MSD fibroblasts for three important, expression ofthe cDNAs for ASA, ASB, and STS sulfatases tested. cDNAs encoding , arylsulfa- led to the synthesis of inactive polypeptides in MSD fibro- tase B, or steroid sulfatase were introduced into MSD fibro- blasts and to catalytically active sulfatase polypeptides in blasts and fibroblasts with a single sulfatase deficiency by fibroblasts from patients with a single sulfatase deficiency. retroviral gene transfer. Infected fibroblasts overexpressed the Our results clearly demonstrate that the basic defect in MSD respective sulfatase polypeptides. While in single-sulfatase- affects a co- or post-translational modification that makes deficiency fibroblasts a concomitant increase of sulfatase ac- sulfatase polypeptides active or prevents their inactivation. tivities was observed, MSD fibroblasts expred sulfatase polypeptides with a severely diminished catalytic activity. From these results we conclude that the mutation in MSD MATERIALS AND METHODS severely decreases the capacity of a co- or post-translational Cell Lines and Cell Culture. The MSD fibroblasts were process that renders sulfatases enzymatically active or prevents obtained from J. Couchot (Clinic de Pediatrie et Puericulture, their premature inactivation. Reims) and E. Christenson (Rigshospitalet, Copenhagen). The MLD fibroblasts deficient in ASA activity, the Maro- Multiple sulfatase deficiency (MSD) is a rare autosomal teaux-Lamy fibroblasts deficient in ASB, and the chromo- recessively transmitted lysosomal storage disorder charac- some X-linked fibroblasts deficient in STS were terized by the accumulation of sulfated lipids and carbohy- obtained from R. Gitzelmann (Kinderhospital, Zurich), J. drates. The disorder is caused by partial deficiencies of at Zimmer (Humangenetik, Freiburg), and the Human Genetic least six lysosomal and one microsomal sulfatase (1). MSD is Cell Repository (Lyon). The packaging cell line PA 317 (12) clinically and biochemically heterogenous. On the basis of was kindly provided by K. Pfitzenmeyer (Klinische For- the residual activities of sulfatases, MSD patients can be schergruppe der Max-Planck-Gesellshaft, Gottingen) and the classified in two groups. Patients of group I exhibit severe retroviral vector pXT1 (13) by E. Wagner (Research Institute sulfatase deficiencies and a neonatal onset ofthe disease, and of Molecular Pathology, Vienna). patients ofgroup II show moderate sulfatase deficiencies (2). Human fibroblasts were maintained at 370C, under 5% CO2 The primary defect in MSD is unknown. The mutations in in minimal essential medium containing 15-20% fetal calf MSD and single sulfatase deficiencies [e.g., metachromatic serum. The culture medium was changed every 3-4 days. leukodystrophy (MLD), mucopolysaccharidosis II, IIIA, and The packaging cell line PA 317 was cultured in Dulbecco's IV, and X-linked ichthyosis] are nonallelic as modified Eagle's medium with 10% fetal calf serum. shown by complementation studies (3-6). The primary defect RNA Isolation, Northern Blot Hybridization, and Probes. in MSD affects the stability and the catalytic properties of Total RNA of 1-3 x 107 cultured human fibroblasts was sulfatases to a variable extent depending on the type of prepared as described (14). Total RNA in 20 gl of 20 mM sulfatase and the MSD cell line (2, 7-9). This led to the morpholinopropanesulfonic acid (Mops), pH 6.8/6% form- hypothesis that the mutations in MSD affect a gene product aldehyde/33% formamide was heated for 10 min to 70'C and that interacts with sulfatases co- or post-translationally. This electrophoresed through a 1.3% denaturing agarose gel con- process-shared by all sulfatases-is thought to activate taining 0.7% formaldehyde in 20 mM Mops, pH 6.8, and and/or stabilize the sulfatases (2, 8). transferred to Hybond-N membranes (Amersham). To test this hypothesis we analyzed the expression of The radioactive probes for Northern blot analysis were sulfatases in MSD fibroblasts on the RNA level and intro- prepared by the multiprime DNA labeling system (Amer- duced the cDNAs of sulfatases into MSD fibroblasts via sham) with the ASA cDNA insert of pBEH/HT14-CP 18 (15) retroviral gene transfer. According to the working hypothe- and the STS cDNA insert pBEH-STS (16). Filters were sis, sulfatase genes and their transcription should be normal hybridized 16 hr at 420C in 10 mM Tris HCl, pH 7.4, con- in MSD, while expression ofthe endogenous genes and ofthe taining 48% (vol/vol) formamide, 10% dextran sulfate (Phar- introduced cDNAs should yield sulfatase polypeptides that macia LKB) 4.8x SSC, lx Denhardt's solution, salmon are inactive and/or unstable. On the other hand, expression sperm DNA at 100 pg/ml, 1% SDS, and 1-2 x 106 cpm of of the same cDNAs in cells with single sulfatase deficiencies [32P]DNA per ml (lx SSC = 0.15 M NaCl/0.015 M sodium caused by mutations in the sulfatase genes should yield sulfatase polypeptides with normal catalytic properties and Abbreviations: ASA, arylsulfatase A (cerebroside-3-sulfate 3-sulfo- stability, as was shown recently (10, 11). , EC 3.1.6.8); pdASA, pseudodeficiency allele of ASA; ASB, arylsulfatase B (N-acetylgalactosamine4-sulfate sulfohydro- lase, EC 3.1.6.12); STS, steroid sulfatase (steryl-sulfate sulfohydro- The publication costs of this article were defrayed in part by page charge lase, EC 3.1.6.2); MLD, metachromatic leukodystrophy; MSD, payment. This article must therefore be hereby marked "advertisement" multiple sulfatase deficiency; cfu, colony-forming units; U, in accordance with 18 U.S.C. §1734 solely to indicate this fact. unit(s). 2561 Downloaded by guest on October 1, 2021 2562 Medical Sciences: Rommerskirch and von Figura Proc. NatL Acad Sci. USA 89 (1992)

citrate, pH 7.0; lx Denhardt's solution = 0.02% bovine ASA RTS serum albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone). Filters were washed at 650C once with 2x SSC for 5 min and '1M MS three times for 20 min each with 0.1x SSC. Production of Recombinant Retroviruses. The construction of the retroviral vectors containing the cDNAs of ASA and ASB were described previously (10, 11). For the construct containing the coding part ofthe ASA pseudodeficiency allele (pdASA) a 1828-base-pair (bp) Xho I-Sal I DNA fragment was isolated from the eukaryotic expression vector pBEH- pdASA (17). It contains the entire cDNA for pdASA, 62 bp of 5' untranslated sequence of the ASA cDNA, and 65 bp of simian virus 40 5' untranslated sequence derived from the expression vector. This fragment was ligated into the Xho I site of the retroviral vector pXT1 (13). For construction of the plasmid pXT1STS a Xho I-Sal I DNA fragment from the eukaryotic expression vector pBEH- STS (16) was used. It contains the entire cDNA coding for of simian virus 40 5' untranslated sequence FIG. 1. Northern blot analysis oftotal RNA. (Left) RNA (10 ,g) STS and 65 bp isolated from the group I MSD fibroblasts (Co.) and control fibro- that is part of the expression vector. This fragment was blasts (Br.) was hybridized with an ASA cDNA probe. (Right) RNA ligated into the Xho I site of the retroviral vector pXT1. (5 Zg) isolated from group I MSD fibroblasts (Co.), chromosome For the production of replication-defective infectious ret- X-linked-ichthyosis fibroblasts (800), and control fibroblasts (Br.) roviruses the vectors pXT1ASA, pXT1ASB, and pXT1STS was hybridized with an STS cDNA. The size of the RNA species is were transfected into the helper-virus-free amphotropic indicated in kilobases (kb). Ethidium bromide staining and rehybrid- packaging cell line PA 317 (12) by the calcium phosphate ization with an actin probe revealed that comparable amounts of precipitation technique. Cells were selected with neomycin at RNA had been loaded (not shown). 0.4 mg/ml. Resistant cells were recultured to about 50%o confluency. Selection was withdrawn for 2 days, and the detects RNA species of 6.3, 4.6, and 2.5 kb. The latter tissue culture medium containing the retrovirus was har- represents a minor species detectable only after prolonged vested, centrifuged, filtered through a 0.45-,um-pore filter to exposure. In total RNA from fibroblasts carrying a deletion exclude remaining packaging cells, tested for colony-forming ofthe STS gene (X-linked ichthyosis), none ofthe STS RNA units (cfu) on NIH 3T3 tk- cells, and stored at -80°C until species is detectable. In total RNA from group I MSD used for infection of fibroblasts. Throughout this article the fibroblasts, ASA and STS transcripts of the same size as in retroviral vectors are designated by a p (e.g., pXT1). The p control RNA were detectable. Furthermore, the amounts of will be omitted to designate the corresponding viruses (e.g., ASA and STS transcripts in MSD and control fibroblasts XT1). were comparable (shown for cell line Co. in Fig. 1). Recently Retroviral Gene Transfer. Human fibroblasts were seeded we have reported similar findings for ASB (22). These results at 30%o confluency into a 75-cm2 culture flask 16 hr prior to show that the primary defect in MSD does not affect the infection. Cells were incubated for 16 hr with 5 ml of the transcription of ASA, ASB, and STS or the stability of their filtered culture supernatant of PA 317 cells producing XT1 transcripts. retrovirus (5 x 104 to 1 x 105 cfu/ml) in the presence of Retroviral Gene Transfer Does Not Restore Sufatae Activ- Polybrene at 10 ,ug/ml. After 48 hr infected cells were ities in MSD Fibroblasts. We introduced the sulfatase cDNAs selected by the addition of Geneticin (G418, GIBCO) at 0.2 into human diploid fibroblasts by infection with recombinant mg/ml. Selection efficiency was checked by comparison to a retroviruses. cDNA fragments containing the coding se- mock-infected control. Between 40% and 60% ofthe infected quences of human ASA, ASB, and STS were cloned in the fibroblasts survived the selection process. retroviral expression vector pXT1 (10, 11), yielding the Enzyme Assays, Metabolic Labeling, and Immunoprecipi- plasmids pXT1ASA, pXT1ASB, and pXT1STS (Fig. 2). The tation. ASA (18), ASB (19), and STS (20) activities were cDNA is under the control of the promoter from the herpes determined as described; a unit (U) is defined as the amount simplex virus thymidine kinase gene. The neomycin resis- of enzyme catalyzing the cleavage of 1 mmol ofp-nitrocate- tance gene, which in eukaryotes confers resistance to the chol sulfate per min. Metabolic labeling of fibroblasts and antibiotic Geneticin, is expressed from the Moloney murine immunoprecipitation ofASA and ASB from cell extracts (21) leukemia virus 5' long terminal repeat of the vector (13). and STS from the membrane fraction (16) were performed as Two group I MSD cell lines (Co. and Mo.) were infected previously described. with the virus XT1ASA. Infected fibroblasts exhibited ASA Indirect ELISA of ASA Protein. ASA protein contents in activities that were not significantly different from controls the different cell extracts were compared by an indirect that were mock infected or infected with the parent retrovirus ELISA (H. J. Sommerlade, personal communication) using XT1 (Table 1). To control for the ability ofthe XT1ASA virus affinity-purified rabbit antibodies to human ASA to coat the stock to introduce the ASA gene into fibroblasts we infected wells and a mouse antiserum against human ASA and goat in parallel fibroblasts from two patients with MLD (2301 and anti-mouse IgG/IgM conjugated to horseradish peroxidase to detect ASA. Under the conditions used the assay was linear T for up to 1 ,ug of ASA. J _ FIG. 2. Organization of the recombinant XT1 retroviruses en- RESULTS coding the sulfatase cDNAs. The cDNA fragments encoding ASA, ASB, and STS were inserted in the retroviral expression vector Sulfatase RNAs in MSD Fibroblasts. Total RNA from group pXTl. The sulfatase cDNAs are expressed from the internal herpes I MSD and control fibroblasts was subjected to Northern blot simplex virus thymidine kinase (TK) promoter and the neomycin analysis and hybridized with cDNA probes specific for ASA resistance gene (neoR) from the Moloney murine leukemia virus 5' and STS (Fig. 1). In total RNA of controls the ASA probe long terminal repeat (LTR). The location ofthe viral packaging signal detects transcripts of 4.8, 3.7, and 2.1 kb and the STS probe (O) is indicated. Downloaded by guest on October 1, 2021 Medical Sciences: Rommerskirch and von Figura Proc. NatL Acad. Sci. USA 89 (1992) 2563 Table 1. ASA activity in retrovirally infected fibroblasts Table 3. STS activity in retrovirally infected fibroblasts ASA, Cell line Retrovirus STS, nmol per hr per mg Cell line Retrovirus mU/mg* Control Control Ki. 1.4 Ki. 14.8 X-L ichth. XT1 12.8 800 - <0.05 MLD XT1STS 7.2 (2.6-18)* 2301 1.1 MSD XT1 1.4 Co. - <0.05 XTlASA 95 (45-127) XT1STS 0.06 XTlpdASA 52 (18-86) Mo. <0.05 La. 0.8 XT1STS 0.17, 0.10 XTlASA 84 (56-126) X-L chromosome X-linked 129 ichth., ichthyosis. XTlpdASA (49-250) *The mean and of 6 infected cultures are MSD range individually given. Co. 1.2 while in fibroblasts with a single sulfatase deficiency sulfatase XT1 0.4 activities more than 10-fold higher than in control fibroblasts XTlASA 1.7 (0.7-2.4) are obtainable. XTlpdASA 1.6 (0.4-2.7) Expression of Inactive Sulfatase Polypeptides in MSD Fi- Mo. 0.5 broblasts After Retroviral Gene Transfer. The sulfatase XT1 poly- 1.3 peptides expressed from the endogenous sulfatase genes are XTlASA 1.5 (1.0-2.3) catalytically inactive or/and unstable (2, 7, 9). One of the XTlpdASA 2.3 (0.9-3.0) more likely possibilities to explain the failure of retroviral *The mean (n = 2-5) and range of activities in individually infected gene transfer to restore sulfatase activities in MSD fibroblasts cultures are given. was therefore the synthesis of inactive or/and unstable sulfatase polypeptides. La.). Both MLD cell lines lack ASA mRNA and ASA activity When MSD due to fibroblasts infected with XT1ASB or XT1STS homozygosity for a splice donor site mutation of ASA were metabolically labeled, polypeptides corresponding to exon 2 (23). Infection of these two MLD cell lines with the- mature 47-kDa form of ASB and the mature 63-kDa form XT1ASA retrovirus and subsequent selection yielded cells of STS were detectable in the with ASA activities about cell extracts (Fig. 3). The 10-fold higher than normal fibro- amounts of ASB and STS polypeptides were 10 and 3 times blasts (Table 1). higher, respectively, than in noninfected MSD fibroblasts. Experiments similar to those with the ASA cDNA- These observations suggest that retroviral gene transfer transferring virus were done with viruses transferring the induced the synthesis of sulfatase in MSD cDNAs for ASB and STS. The capacity of the recombinant polypeptides viruses fibroblasts as in fibroblasts with a single sulfatase deficiency. XT1ASB and XT1STS to restore either ASB or STS Furthermore, the accumulation of mature forms indicates activity in deficient fibroblasts was examined by infection of that the sulfatase fibroblasts with a deficiency of ASB (Maroteaux-Lamy syn- polypeptides are correctly transported to drome/mucopolysaccharidosis type VI) or STS (X-linked lysosomes. ichthyosis). The ASB activity in infected Maroteaux-Lamy To demonstrate unequivocally that the sulfatase polypep- fibroblasts was up to 20-fold higher than in normal fibroblasts tides expressed in retrovirally infected MSD fibroblasts orig- (Table 2), and the STS activity in infected X-linked ichthyosis inate from the transferred and not from the endogenous fibroblasts was up to 12-fold higher (Table 3). Infection of sulfatase genes, we introduced a phenotypic marker into one group I MSD fibroblasts with XT1ASB slightly increased the of the transferred sulfatase cDNAs. This was done by delet- residual ASB activity, but not to the level found in normal ing one of the utilized N-glycosylation sites of ASA. From fibroblasts (Table 2). Infection with XT1STS did not increase studies of the ASA pseudodeficiency allele it is known that the residual STS activity in the MSD fibroblasts (Table 3). It loss of the third N-glycosylation site of ASA (caused by is apparent from these results that retrovirus-mediated gene change ofAsn-350 to Ser) affects neither catalytic activity nor transfer cannot restore sulfatase activities stability of arylsulfatase A. It reduces, however, the size of to normal levels, the ASA polypeptides by 2.5 kDa (17). Table 2. ASB activity in retrovirally infected fibroblasts When MLD fibroblasts were infected with XT1pdASA, the retrovirus the ASA Cell line carrying pseudodeficiency allele, as in- Retrovirus ASB, mU/mg sert, ASA activity increased to a level similar to that after Control Ho. 5.7 ASB STS MPS VI 5343 <0.1 Infection - ASB - STS XT1ASB 220 Ya. <0.1 ASB _- - _- STS XT1ASB 124 5123 XT1 <0.1 XT1ASB 108 FIG. 3. Synthesis of ASB and STS polypeptides in infected MSD MSD fibroblasts. Group I MSD fibroblasts (Mo.), uninfected or infected Co. <0.1 with XT1ASB or XT1STS, were labeled for 16 hr with [35S]methio- XTlASB 0.6, 2.5 nine (50 ,uCi/ml; 1 Ci = 37 GBq). ASB and STS were immunopre- Mo. 0.5 cipitated from cell extracts containing equal amounts of trichloro- XT1 0.9 acetic acid-insoluble radioactivity. The positions of the mature XTlASB 1.1, 3.5 47-kDa form of ASB and of the mature 63-kDa form of STS are indicated. The other labeled polypeptides are unrelated to ASB or MPS VI, mucopolysaccharidosis type VI. STS. Downloaded by guest on October 1, 2021 2564 Medical Sciences: Rommerskirch and von Figura Proc. Natl. Acad Sci. USA 89 (1992) infection with XT1ASA, while infection of MSD fibroblasts Table 4. Specific activity of ASA in retrovirally with XT1pdASA did not restore ASA activity (Table 1). To infected fibroblasts follow the synthesis of ASA polypeptides, infected control, ASA Specific activity MLD, and MSD fibroblasts were labeled for 16 hr with [35S]methionine. Incorporation of radioactivity into trichlo- mU/mg of Pg*/mg of U/mg % of roacetic acid-insoluble material was comparable in all cell Cell line Retrovirus cell protein cell protein of ASA control lines. In MSD and MLD cells infected with the ASA- or Control pdASA-carrying retroviruses the amount of [35S]ASA poly- Ho. - 17.2 0.29 59.3 100 peptides was 5- to 7-fold higher than in control fibroblasts MLD infected with the parent virus XT1. In MSD and MLD cells La. XT1ASA 55.6 0.71 83.0 132 infected with XT1pdASA the ASA polypeptides were 2.5 XT1ASA 125 1.99 62.8 106 kDa smaller. MLD fibroblasts infected with XT1 lacked ASA XT1pdASA 250 3.55 70.4 118 polypeptides, and MSD fibroblasts infected with XT1 con- MSD tained reduced amounts (about 40% of control) of normal- Mo. 0.8 0.14 5.7 10 sized ASA polypeptides (Fig. 4). This clearly indicates that in XT1 1.3 0.15 8.7 15 cells infected with the ASA- or pdASA-carrying retroviruses XT1ASA 2.3 0.79 2.9 5 the bulk of ASA polypeptides are expressed from the retro- XT1ASA 1.5 0.82 1.8 3 virally transferred ASA cDNAs and that the efficiency of XT1pdASA 3.0 1.21 2.5 4 expression is similar in MSD and MLD fibroblasts. Co. 2.0 0.17 11.8 20 When MSD and MLD fibroblasts expressing pdASA poly- XT1ASA 2.4 0.73 3.3 6 peptides were cultured with unlabeled methionine after the *Determined by ELISA with purified ASA as a reference. metabolic labeling for up to 14 days, the [35S]ASA polypep- tides decreased at comparable rates in the two cell types (t1/2 was 59 U/mg ofASA. This agrees with the specific activities of approximately 12 days; data not shown). The apparent of 30 and 64 U/mg that have been reported for ASA purified stability of ASA polypeptides expressed from retrovirally from human placenta and human urine (24-27). The specific transferred ASA cDNAs is therefore comparable in MSD and activity of ASA and pdASA expressed in MLD cells was in MLD fibroblasts. the same range (63-78 U/mg), while it was less than 1/20th A comparison of the ASA activity and the amount of as high in group I MSD cells (2-3 U/mg). radioactivity incorporated into ASA in the cell lysates shown in Fig. 4 suggests that catalytically inactive ASA polypep- DISCUSSION tides are synthesized in MSD fibroblasts. The ratio of ASA activity and 3S radioactivity in ASA polypeptides in MSD Retroviral infection is an effective means to restore the fibroblasts is less than 1/20th of that in control and MLD sulfatase deficiencies in fibroblasts with a single deficiency of fibroblasts. To obtain a more precise estimate for the specific ASA, ASB, or STS (this study; refs. 10 and 11). In this study activity ofASA we quantified the ASA crossreacting material we show that infection of group I MSD fibroblasts with the by an ELISA and compared it to the ASA enzyme activity same viral stocks fails to restore the deficient sulfatase (Table 4). The specific activity of ASA in control fibroblasts activities. The transferred cDNAs led to an overexpression of sulfatase polypeptides but-as examined in detail for ASA- ro.Iall linc Co. MLD MSD the catalytic activity of these polypeptides was greatly re- duced. These observations strongly suggest that the basic K!. 2301 1I defect in MSD affects a co- or post-translational process that

In oction XT 1 XT1 xTI XT1 X T'. X -1 controls the catalytic properties of sulfatases. v'th ASA Pd A defect in the editing of sulfatase RNAs is one of the ASA AS:A mechanisms that could explain the phenotype of MSD. The colinearity of the nucleotide sequence of the ASA RNA and of the ASA gene (ref. 28; V. Gieselmann, personal commu- nication) has made it unlikely that sulfatase RNAs are subject to a common editing step. If sulfatase RNAs would require editing to code for catalytically active sulfatases and if this = ~~_ _ would be defective in MSD, sulfatase cDNAs, which derive from edited RNAs, introduced into MSD fibroblasts should encode catalytically active sulfatases. The failure to express catalytically active sulfatases from three different sulfatase cDNAs as observed in this study excludes the possibility that the primary defect in MSD affects the editing of sulfatases. In earlier studies a decreased stability of endogenous ASA ASA and ASB polypeptides had been observed (2, 7). In the - cp[p 260 - 1306 2136 ii 2196'I present study, using in part the same cell lines (e.g., MSD cell line Co.), we made several observations that are incompatible n-iU 203 - 50 380 0.01 0.2i: .I with a significantly decreased stability of ASA. The amount of ASA polypeptides and the apparent rate of ASA synthesis FIG. 4. Synthesis of ASA polypeptides in infected fibroblasts. in noninfected MSD cell lines were about half normal. Contror(Ki.), MLD (2301), and MSD (Co.) fibroblasts infected with Furthermore, the stability ofpulse-labeled ASA polypeptides retrovirus XT1, XT1ASA, or XT1pdASA were metabolically labeled followed during a chase for up to 14 days was not decreased as described for Fig. 3. ASA-specific polypeptides are indicated by in fibroblasts. It has been noted earlier that the residual arrows. The apparent size of the ASA polypeptides encoded by the MSD pdASA lacking the third potential N-glycosylation site is 2.5 kDa activity of sulfatases fluctuates greatly in MSD fibroblasts smaller than normal ASA. Below the lanes the radioactivity incor- and can be affected by cell culture variables (29, 30). It is porated into ASA polypeptides and the ASA activity in cell extracts conceivable that such variables also affect the stability of are given. sulfatase polypeptides in MSD fibroblasts.

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The specific activity (catalytic activity per mass ofenzyme) 6. Ballabio, A., Parenti, G., Napolitano, E., Di Natale, P. & of ASA is significantly lower in MSD fibroblasts. This is true Andria, G. (1985) Hum. Genet. 70, 315-317. by the endogenous ASA 7. Waheed, A., Hasilik, A. & von Figura, K. (1982) Eur. J. for the ASA polypeptides encoded Biochem. 123, 317-321. gene, which have 1/5th to 1/10th the specific activity, and for 8. Horwitz, A. L., Warshawsky, L., King, A. & Bums, G. (1986) the ASA polypeptides encoded by the retrovirally introduced Biochem. Biophys. Res. Commun. 135, 389-3%. ASA cDNA. Interestingly, the specific activity of the latter 9. Conary, J. T., Hasilik, A. & von Figura, K. (1988) Biol. Chem. was even lower, reaching only 3-6% of control values. Hoppe-Seyler 369, 297-302. Inactivity of ASA encoded by normal ASA cDNA can result 10. Rommerskirch, W., Fluharty, A. L., Peters, C., von Figura, K. from the absence of a co- or post-translational modification & Gieselmann, V. (1991) Biochem. J. 280, 459-461. 11. Peters, C., Rommerskirch, W., Modaressi, S. & von Figura, K. that renders ASA polypeptides catalytically active or pre- (1991) Biochem. J. 276, 499-504. vents their premature inactivation. The observation that 12. Miller, A. & Buttimore, C. (1986) Mol. Cell. Biol. 6, 2895-2902. overexpression of ASA polypeptides in MSD fibroblasts is 13. Boulter, C. A. & Wagner, E. F. (1987) Nucleic Acids Res. 15, associated with even further decrease of their specific activ- 7194. ity indicates that this modification is saturable in MSD. 14. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, The inactivity of sulfatase polypeptides could also be W. J. (1979) Biochemistry 18, 5294-5299. caused by a mutation that generates a mechanism by which 15. -Stein, C., Gieselmann, V., Kreysing, J., Schmidt, B., Pohl- sulfatases are inactivated. Such a mutation, however, would mann, R., Waheed, A., Meyer, E. H., O'Brien, J. S. & von Figura, K. (1989) J. Biol. Chem. 264, 1252-1259. be expected to have a dominant effect. Moreover, overex- 16. Stein, C., Hille, A., Seidel, J., Rijmbout, S., Waheed, A., pression of sulfatases in cells with such a mutation should Schmidt, B., Geuze, H. & von Figura, K. (1989) J. Biol. Chem. either not affect the residual specific activity of sulfatases or 264, 13865-13872. increase it (due to saturation), but it should not decrease it, 17. Gieselmann, V., Polten, A., Kreysing, J. & von Figura, K. as was observed. (1989) Proc. Natd. Acad. Sci. USA 86, 9436-9440. The nature of the modification that renders ASA polypep- 18. Baum, H., Dodgsen, K. S. & Spencer, B. (1959) Clin. Chim. tides active or prevents their inactivation is not clear. It could Acta 4, 453-455. consist in a covalent (stable or transient) modification. Alter- 19. Steckel, F., Hasilik, A. & von Figura, K. (1983) J. Biol. Chem. natively, it may be represented by a transient interaction with 258, 14322-14326. 20. Conary, J. T., Nauerth, P., Burns, G., Hasilik, A. & von a gene product controlling the folding and assembly of Figura, K. (1986) Eur. J. Biochem. 158, 71-76. sulfatases. The availability of cells that overexpress sulfa- 21. von Figura, K., Steckel, F. & Hasilik, A. (1983) Proc. Natl. tases with the phenotypic defect characteristic of MSD Acad. Sci. USA 80, 6066-6070. should facilitate the identification of this modification. 22. Peters, C., Schmidt, B., Rommerskirch, W., Rupp, K., Zuhl- dorf, M., Vingron, M., Meyer, H. E., Pohlmann, R. & von We thank Dr. G. Hunsmann for giving us the opportunity to use Figura, K. (1990) J. Biol. Chem. 265, 3374-3381. the L3 facility of the Deutsche Primatenzentrum Gottingen at the 23. Polten, A., Fluharty, A. L., Fluharty, C. B., von Figura, K. & beginning of our experiments. This work was supported by the Gieselmann, V. (1991) N. Engl. J. Med. 324, 18-22. Deutsche Forschungsgemeinschaft and the Fonds der Chemischen 24. Stevens, R. L., Fluharty, A. L., Skokut, M. A. & Kihara, H. Industrie. (1975) J. Biol. Chem. 250, 2495-2501. 25. Waheed, A., Hasilik, A. & von Figura, K. (1982) f-Loppe- 1. Kolodny, E. H. (1989) in Metabolic Basis ofInherited Disease, Seyler's Z. Physiol. Chem. -363, 425-430. eds. Scriver, C. R., Beaudet, A. L., Sly, W. S. & Valle, D. 26. Lujten, J. A. F. M., van der Heijden, M. C. M., Rijksen, G. & (McGraw-Hill, New York), pp. 1721-1750. Staal, G. E. J. (1978) J. Mol. Med. 3, 213-225. 2. Steckel, F., Hasilik, A. & von Figura, K. (1985) Eur. J. 27. Laidler, P. M., Waheed, A. & van Etten, R. L. (1985) Biochim. Biochem. 151, 141-145. Biophys. Acta 327, 73-83. 3. Horwitz, A. L. (1979) Proc. Natl. Acad. Sci. USA 76, 6486- 28. Fluharty, A. L., Stevens, R. L., Laurel, L. D., Shapiro, L. J. 6499. & Kihara, H. (1978) Am. J. Hum. Genet. 30, 249-255. 4. Chang, P. L. & Davidson, R. G. (1980) Proc. Natl. Acad. Sci. 29. Fluharty, A. L., Richard, L. S., De La Flor, S., Shapiro, L. J. USA 77, 6166-6170. & Kihara, H. (1979) Am. J. Hum. Genet. 31, 574-580. 5. Fedde, K. & Horwitz, A. L. (1984) Am. J. Hum. Genet. 36, 30. Kreysing, J., von Figura, K. & Gieselmann, V. (1990) Eur. J. 623-633. Biochem. 191, 627-631. Downloaded by guest on October 1, 2021