Multiple Sulfatase Deficiency: Catalytically Inactive Sulfatases Are Expressed from Retrovirally Introduced Sulfatase Cdnas
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Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2561-2565, April 1992 Medical Sciences Multiple sulfatase deficiency: Catalytically inactive sulfatases 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- arylsulfatase A (ASA) and arylsulfatase B (ASB), and the ciency of at least seven sulfatases. The basic defect in MSD is microsomal steroid 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 arylsulfatase A, 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 ichthyosis 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 chromosome 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). hydrolase, 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, enzyme 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).