Missense mutations as a cause of metachromatic leukodystrophy Degradation of arylsulfatase A in the endoplasmic reticulum Peter Poeppel 1 , Matthias Habetha 2 , Ana Marca ˜ o 3 , Heinrich Bu ¨ ssow 4 , Linda Berna 5 and Volkmar Gieselmann 1 1 Institut fu ¨ r Physiologische Chemie, Rheinische-Friedrich-Wilhelms Universita ¨ t Bonn, Germany 2 Zoologisches Institut, Christian-Albrechts-Universita ¨ t zu Kiel, Germany 3 Instituto de Biologia Molecular e Celular, University of Porto, Portugal 4 Institut fu ¨ r Anatomie, Rheinische-Friedrich-Wilhelms Universita ¨ t Bonn, Germany 5 Institute of Inherited Metabolic Disorders, Charles University, Prague, Czech Republic Lysosomal storage diseases comprise a group of about 40 disorders, in most cases caused by the deficiency of a lysosomal enzyme involved in the degradation of, for example, lipids, glycosaminoglycans and oligo- saccharides. Much effort has been devoted to the identification of disease causing mutations in these dis- orders. Thus, a multitude of mutations has been identi- fied in recent years (for example [1]). Only a fraction of missense mutations, however, has been analysed at the biochemical level in order to understand the Keywords ERAD; proteasomal degradation; arylsulfatase A; metachromatic leukodystrophy Correspondence V. Gieselmann, Institut fu ¨ r Physiologische Chemie, Rheinische-Friedrich-Wilhelms Universita ¨ t Bonn, Nussallee 11, 53115 Bonn, Germany Fax: +49 22 873 2416 Tel: +49 22 873 2411 E-mail: gieselmann@institut.physiochem. uni-bonn.de Note P. Poeppel and M. Habetha contributed equally to this work. (Received 29 October 2004, revised 14 December 2004, accepted 4 January 2005) doi:10.1111/j.1742-4658.2005.04553.x Metachromatic leukodystrophy is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). Biosynthesis studies of ASA with vari- ous structure-sensitive monoclonal antibodies reveal that some epitopes of the enzyme form within the first minutes of biosynthesis whereas other epi- topes form later, between 10 and 25 min. When we investigated 12 various ASAs, with amino acid substitutions according to the missense mutations found in metachromatic leukodystrophy patients, immunoprecipitation with monoclonal antibodies revealed folding deficits in all 12 mutant ASA enzymes. Eleven of the 12 mutants show partial expression of the early epi- topes, but only six of these show, in addition, incomplete expression of late epitopes. In none of the mutant enzymes were the late forming epitopes found in the absence of early epitopes. Thus, data from the wild-type and mutant enzymes indicate that the enzyme folds in a sequential manner and that the folding of early forming epitopes is a prerequisite for maturation of the late epitopes. All mutant enzymes in which the amino acid substitu- tion prevents the expression of the late forming epitopes are retained in the endoplasmic reticulum (ER). In contrast, all mutants in which a single late epitope is at least partially expressed can leave the ER. Thus, irrespective of the missense mutation, the expression of epitopes forming late in biosyn- thesis correlates with the ability of the enzyme to leave the ER. The degra- dation of ER-retained enzymes can be reduced by inhibitors of the proteasome and ER a1,2-mannosidase I, indicating that all enzymes are degraded via the proteasome. Inhibition of degradation did not lead to an enhanced delivery from the ER for any of the mutant enzymes. Abbreviations ASA, arylsulfatase A; ER, endoplasmic reticulum; Lc, lactacystin; Kif, kifunensine; SOV, sodium orthovanadate; PAO, phenylarsine oxide; OA, okadaic acid; Lp, leupeptin; DNM, deoxynojirimycin; a1-AT, a1-antitrypsin; MLD, metachromatic leukodystrophy; BHK, baby hamster kidney; DMEM, Dulbecco’s modified essential medium; FBS, foetal bovine serum; LDL-receptor, low density lipoprotein receptor. FEBS Journal 272 (2005) 1179–1188 ª 2005 FEBS 1179 molecular basis of the enzyme deficiencies in greater detail. In many cases, missense mutations lead to an arrest and more rapid degradation of the encoded enzyme in the endoplasmic reticulum (ER) (for exam- ple [2]). In this respect lysosomal storage diseases are not special as this mechanism is responsible for protein deficiencies in many diseases. In fact, it has been esti- mated that ER degradation is the most frequent cause of protein deficiencies such that the term ‘conforma- tional diseases’ has been suggested [3]. The mechanisms of ER quality control, retention and degradation have been investigated in recent years (reviewed in [4]). Newly synthesized secretory, mem- brane or lysosomal glycoproteins interact sequentially with a number of membrane-bound or soluble glyco- sidases and chaperones of the ER. Modifications of N-linked oligosaccharide side chains play a major role in this process. The precursor of N-linked oligosaccharides is a Glc 3 - Man 9 -GlcNAc 2 dolichol pyrophosphate, from which the sugars are transferred en bloc to Asn ⁄ X ⁄ Ser(Thr) in newly synthesized polypeptide chains within the ER. Trimming of the Glc 3 -Man 9 -GlcNAc 2 side chains begins shortly after synthesis by the ER membrane- located glucosidase I to Glc 2 -Man 9 -GlcNAc 2 , followed by trimming of an ER-localized soluble glucosidase II to Glc 1 -Man 9 -GlcNac 2 . Glycoproteins bind to the ER-resident lectins calnexin and calreticulin, via the Glc 1 -Man 9 -GlcNAc 2 oligosaccharide. Glucosidase II then removes the remaining terminal glucose with the consequence that newly synthesized proteins no longer bind to the lectins and leave the ER. In case a protein is not folded correctly, it is recognized by the UDP-glu- cose:glycoprotein glucosyltransferase, which reglucosy- lates the Man 9 -GlcNAc 2 of misfolded proteins to Glc 1 -Man 9 -GlcNAc 2 [5]. Consequently the protein can bind to calnexin ⁄ calreticulin again and remains in the ER. This loop can be repeated several times and may enhance the chances of a protein folding correctly. Finally, a1,2-mannosidase I removes one mannose [6,7]. This removal of mannose by a1,2-mannosidase I has been suggested to be a signal for proteasomal de- gradation [7,8]. The proteasome seems to be the major pathway by which misfolded proteins are degraded, although the existence of an as yet poorly characterized nonproteasomal pathway has been demonstrated [6,7]. Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder which is caused by the deficiency of arylsulfatase A (ASA). This enzyme catalyses the first step in the degradation pathway of the glycosphingo- lipid 3-O-sulfogalactosylceramide. Deficiency of the ASA causes lipid accumulation leading progressive demyelination and various, ultimately lethal neurologi- cal symptoms (reviewed in [1]). The gene of human ASA has been cloned and more than 80 mostly missense mutations were identified. Some of these mutations were investigated more closely to reveal the effects of the amino acid substitutions on the mutant enzyme. According to these results two main mechanisms cause ASA deficiency. In about half of the examined cases the mutant enzymes are retained in the ER [2,9,10], in the other half, enzymes can leave the ER and be degraded after arrival in the lysosome [10–12]. Whereas the latter mechanism has been investigated thoroughly in view of potential therapeutic intralysosomal stabilization, nothing is known about the ER-associated degradation as a cause of MLD. Because it has been shown recently for Fabry disease [13] ) another lysosomal storage disorder ) the interference with the ER quality control mechanism can also be a therapeutic option, we decided to examine more closely these mechanisms of enzyme deficiency in MLD. Results Biosynthesis of wild-type ASA To examine the early events in ASA biosynthesis in more detail, baby hamster kidney (BHK) cells were transiently transfected with a plasmid encoding human wild-type ASA cDNA. Cells were pulse labelled with Fig. 1. Early stages of ASA biosynthesis. BHK cells transiently expressing the human wild-type ASA cDNA were pulse labelled with 18.5 MBq [ 35 S]methionine for 2.5 or 5 min, respectively, and chased for the times indicated (0, 2.5, 5, 10 and 25 min). Cell homo- genates were split into eight aliquots and precipitated with preim- mune serum (I), a polyclonal ASA antiserum (II), or six different mAbs (A2, A5, B1, C, E and F), which are directed against five dif- ferent epitopes. Arylsulfatase A degradation P. Poeppel et al. 1180 FEBS Journal 272 (2005) 1179–1188 ª 2005 FEBS [ 35 S]methionine for 2.5 or 5 min and chased for up to 25 min (Fig. 1). After harvesting, cell homogenates were divided into eight aliquots, which were immuno- precipitated with an ASA polyclonal antiserum or six various mAbs [14]. These mAbs recognize only native ASA and are directed against different structure-sensi- tive surface epitopes termed A, B, C, E and F [14]. After a pulse of 2.5 or 5 min, ASA can be readily detected with the polyclonal antiserum. As this serum also recognizes denatured ASA, it precipitates ASA irrespective of the enzyme’s three-dimensional struc- ture. After 2.5 and 5 min pulse only mAbs A2, A5 and B1 recognize ASA, whereas no or minute amounts of ASA are precipitated by the mAbs C, E and F. Epi- topes recognized by mAbs C, E and F start to develop slowly within 10 min of chase and have matured after another 15 min of chase. Thus, in the early stages of ASA biosynthesis, epitopes recognized by mAbs A2, A5 and B1 appear before those recognized by C, E and F, demonstrating that ASA folds in a sequential manner. Recognition of amino acid-substituted ASAs by mAbs We have previously identified various missense muta- tions in the ASA gene and we have examined the biochemical effects of the corresponding amino acid substitutions on ASA. In a number of mutants, the amino acid substitution causes an arrest of ASA in the ER [2,9,10], whereas others can leave the ER [10–12]. We have expressed these mutant ASAs transiently in BHK cells. Cells were labelled for 3 h with [ 35 S]methi- onine and after harvesting, cell homogenates were again divided into eight aliquots, which were immunoprecipi- tated with the mAbs or polyclonal antiserum (Fig. 2). The analysis of 12 amino acid-substituted ASAs reveals that, according to their reactivity with the mAbs, these mutants can be divided into three groups. One group includes mutant ASAs which react weakly with mAbs A2 and A5 and more strongly with B1 (Gly86Asp, Tyr201Cys, Pro377Leu, Asp335Val, Pro136Leu, Asp255His). None of these mutants, however, is recog- nized by any of the antibodies C, E or F. Substituted ASAs of the second group (Gly309Ser, Glu312Asp, Arg84Gln, Arg370Gln, Arg370Trp) react slightly better with A2, A5 and B1 and react ) although weakly ) with at least one of the mAbs C, E or F. Finally, the third group has only one member (Thr274Met) which ASA is not recognized by any of the mAbs. In a previous publication we located the epitopes recognized by the various mAbs (Table 3 in [14]). According to these data amino acid residues 85 and 86 may be part of the epitope recognized by mAbs A2 and A5, and amino acid residues 202–206 by mAb C, respectively. For this reason the reduced reactivity of mAbs A2 and A5 with Gly86Asp and Arg84Gln sub- stituted ASA and mAb C with the Tyr201Cys substi- tuted ASA, respectively, may reflect changes in the epitopes rather than conformational alterations. We could show in the meantime, however, that amino acids 202–206 are not part of the epitope recognized by mAb C (P. Poeppel, unpublished data), so that this cautionary notion does not apply to the immunopre- cipitation of Tyr201Cys substituted ASA with mAb C. Degradation of amino acid-substituted ASAs via the proteasome In order to investigate the degradation pathway of amino acid-substituted ASAs in the ER, we used Ltk – Fig. 2. Immunoprecipitation of amino acid-substituted ASAs with structure-sensitive mAbs. Wild-type ASA and 12 amino acid-substi- tuted ASAs were transiently expressed in BHK cells. Cells were labelled for 3 h with 1.85 MBq [ 35 S]methionine, harvested and aliquots of cell homogenates were immunoprecipitated as des- cribed in Fig. 1. + ⁄ – indicates whether or not the mutant enzymes according to previous publications (references in brackets; [2,9,10,25–28]) are retained in the ER. Polypeptides of lower appar- ent molecular mass, which can be seen in some of the experi- ments are unrelated to ASA. P. Poeppel et al. Arylsulfatase A degradation FEBS Journal 272 (2005) 1179–1188 ª 2005 FEBS 1181 cells which stably express the ER-retained ASA mutant enzymes (Gly86Asp, Tyr201Cys, Pro377Leu, Asp335Val, Pro136Leu, Asp255His, Thr274Met). We selected those clones with a medium level of over- expression and examined them by electron microscopy for normal ER morphology, in order to exclude the possibility that enzymes were being unphysiologically overexpressed. The examined cells showed an ER with normal morphology (results not shown). Stably trans- fected Ltk – cells were pulse labelled for 2 h and chased for various time periods to determine the half-life of the individual enzymes. According to these experiments, chase times were chosen for the following experiments so that in most cases about 80–90% of the enzyme was degraded within the chase periods. Various inhibitors were added during pulse and ⁄ or chase periods. Inhibits lactacystin (Lc) irreversibly the 20 S proteasome, leu- peptin (Lp) is an inhibitor of cysteine and serine prote- ases, and okadaic acid (OA), phenylarsine oxide (PAO) and sodium orthovanadate (SOV) are phosphatase inhibitors. The latter two were used as it has been repor- ted that misfolded a1-antitrypsin (a1-AT) mutants or immunoglobulin chains can be stabilized by these com- pounds [7,8]. Lp has been shown to stabilize some mutant ASAs, which are degraded in the lysosome [12]. Under the conditions of the experiment Lp should not inhibit the proteasome and was used as a nonproteasomal control inhibitor. Figure 3 shows an experiment performed with seven different amino acid-substituted ASAs. The results demonstrate that all of these mutant ASAs can be partially stabilized by proteasome inhibition and that the extent of stabilization varies between the substituted enzymes. Other inhibitors, in particular phosphatase inhibitors, showed no effect. Effects of glycosidase inhibitors on the stability of amino acid-substituted ASAs In order to elucidate the role of trimming reactions of the N-linked oligosaccharide side chains in ER associated degradation of mutant ASAs, stably trans- fected Ltk – cells were incubated with deoxynojirimy- cin (DNM), an inhibitor of ER glucosidases I and II and with kifunensine (Kif), an inhibitor of ER a1,2- mannosidase I. Cells were pulse labelled for 2 h and chased for various times, depending on the half-life of the mutants (Fig. 4). The mutant ASAs were sta- bilized by Kif, whereas inhibition of glucosidases I and II causes a more rapid degradation. Thus, all substituted enzymes showed a uniform pattern of stabilization or more rapid degradation upon addition of inhibitors. Influence of ER a1,2-mannosidase I inhibition on ER exit of amino acid-substituted ASAs In order to investigate whether stabilization of mutant ASAs through inhibition of ER a1,2-mannosidase I via Kif can lead to an enhanced exit of mutant enzyme from the ER, stably transfected Ltk – cells were pulse- labelled for 15 h in the presence of Kif and ⁄ or ammo- nium chloride. After leaving the ER, lysosomal enzymes including ASA are specifically recognized by a phosphotransferase in the Golgi apparatus [14]. This enzyme initiates the phosphorylation of mannose in the N-linked oligosaccharide side chains of lysosomal enzymes, yielding mannose-6-phosphate (M6P). In the trans-Golgi these M6P residues bind to M6P receptors, which mediate the further vesicular transport of lyso- somal enzymes from the Golgi to the lysosomes. Ammonium chloride interferes with this sorting and causes increased secretion of newly synthesized lyso- somal enzymes into the medium [15]. Thus, if newly synthesized lysosomal enzymes appear in secretions in the presence of ammonium chloride, they must have left the ER. The addition of ammonium chloride cau- ses secretion of wild-type ASA to the medium, whereas it has no effect on ER-retained mutant enzymes. Figure 5 shows Pro377Leu-substituted ASA as an example. Also the stabilization of amino acid-substi- tuted ASA with Kif and simultaneous addition of ammonium chloride does not cause increased secretion into the medium, indicating that stabilization of enzymes does not lead to an escape from quality con- trol mechanisms and increased exit of the ER. Only after prolonged exposure can minute amounts of mutant ASAs be detected in the medium, showing a marginal effect of Kif. We estimate that this accounts for less than 5% of the enzyme synthesized during the pulse period. Discussion Missense mutations are by far the most frequent type of mutations in the ASA gene [1]. The effects of these mutations have been shown to be rather uniform. Either the amino acid substitutions lead to an arrest of the mutant enzyme in the ER, or the enzyme is degra- ded in the lysosome after correct sorting [2,9–12]. Here we have investigated wild-type and mutant ASAs by immunoprecipitation with six structure-sensitive mAbs. These mAbs have recently been shown to recognize five different ASA epitopes, termed A to F. These epi- topes, which were recently delimited more closely [14], depend on the native structure of ASA. Examinations of the early biosynthetic events reveal that epitope B Arylsulfatase A degradation P. Poeppel et al. 1182 FEBS Journal 272 (2005) 1179–1188 ª 2005 FEBS forms rapidly after synthesis. Already after a 2.5-min pulse, the newly synthesized ASA is efficiently precipi- tated by mAb B1. At the same time point precipitation with mAbs A2 and A5 is possible but is less efficient, indicating that the epitope may be less matured than the B1 epitope. Figure 1 shows that the ratio of the signals obtained with mAb A2 ⁄ A5 and B1 is constant up to 10 min of chase (densitometric analysis, data not shown), indica- ting that no further maturation of epitopes A2 ⁄ A5 occurs within this time period. Epitopes C, E and F are only weakly expressed until 10 min and mature between 10 and 25 min after synthesis. The maturation of these late forming epitopes is accompanied by a further maturation of epitopes A2 and A5. After 25 min of chase, precipitation with mAbs A2 and A5 is almost as efficient as with mAb B1. The location of epitopes suggests that folding of ASA starts within a central part of the molecule [14]. This is accompanied by a partial expression of epitopes in the N-terminal part. The C-terminal part folds late in biosynthesis, but its folding is not an isolated event, because epi- topes A2 and A5 mature concomitantly. Studies on low density lipoprotein receptor (LDL-receptor) folding Fig. 3. Effects of protease or phosphatase inhibitors on the stability of mutant ASAs. Ltk – cells stably expressing the indicated amino acid-substituted ASAs were incuba- ted in the presence of various inhibitors (Lc, Lp, OA, PAO, SOV). Cells were pulse labelled for 2 h and chased for various times depending on the half-life of the respective mutant (Asp335Val, 4.5 h; Gly86Asp, 05.25 hours; Pro377Leu, 4 h; Tyr201Cys, 6 h; Thr274Met, 4.5 h; Asp255His, 4.5 h; Pro136Leu, 8 h). After the chase ASA was immunoprecipitated from the homogenates with the polyclonal ASA antiserum. Precipi- tated ASA was quantified after SDS ⁄ PAGE with a bio-imaging analyser (Fujifilm). Col- umns show mean and SD of arbitrary units of quadruple experiments. Under each dia- gram representative immunoprecipitates are shown. P. Poeppel et al. Arylsulfatase A degradation FEBS Journal 272 (2005) 1179–1188 ª 2005 FEBS 1183 have shown recently [16] that its folding does not proceed in a vectorial, domainwise process from the N terminus to the C terminus. Instead, folding occurs via intermediates with disulfide bridges involving distant parts of the protein. In addition, the N-terminal part of the LDL-receptor forms late in biosynthesis. Data on ASA are in agreement with this folding scheme. Early detectable epitopes are constituted by amino acid residues between positions 165 and 240 in the central part of the protein [14]. N-terminal epitopes are also detectable at an early stage, but do not mature before the C-terminal part of the protein folds correctly. As in case of the LDL-receptor, this suggests interactions of distant parts of ASA during folding. In addition to wild-type ASA, we also immunopre- cipitated 12 mutant ASAs, whose underlying missense mutations were previously found in MLD patients. Since the mAbs only recognize the native wild-type enzyme, we reasoned that the reactivity with the mAbs should provide a measure of the structural integrity of the substituted enzymes. Surprisingly, the mutant enzymes did not show an individual reaction pattern but according to their immunoprecipitation pattern they can be classified into three groups. One group has only one member, mutant Thr274Met, which does not react with any of the mAbs but is readily precipi- table with the polyclonal antiserum. This reveals a severe misfolding of this mutant. The second group of mutants (Gly86Asp, Tyr201Cys, Pro377Leu, Asp335Val, Pro136Leu, Asp255His) reacts partially with antibodies recognizing the early epitopes A and B and not with those recognizing the late epitopes C, E and F. Interestingly, mutant ASAs of these two groups are completely retained in the ER. Retention in the ER due to incorrect folding leads to repetitive regluco- sylation and binding to the calnexin and calreticulin chaperones. Finally, a mannose is removed, which is considered to be a signal for reverse transport out of the ER into the cytosol. After the transfer of the mis- folded protein into the cytosol, N-glycans are removed and the protein is degraded by the proteasome. The last group is comprised of mutant ASAs which form, at least partially, one or more of the late epitopes C, E and F. Thus, except for Thr274Met, all mutant enzymes express partially the early epitopes whereas only a fraction expresses the late ones. This suggests that in general the latter are more sensitive to amino acid substitutions, irrespective of their localization. Also none of the mutants expresses the late epitopes only, or to a larger extent, than the early epitopes. This Fig. 4. Effects of glycosidase inhibitors on stability of mutant ASAs. Ltk – cells stably expressing the indicated amino acid-substi- tuted ASAs were incubated in the presence of the two inhibitors DNM or Kif. Cells were pulse labelled for 2 h and chased for various times depending on the expression and the half-life of the respective mutants (Asp335V- al, 4.5 h; Gly86Asp, 5.25 hours; Tyr201Cys, 5 h; Thr274Met, 4.5 h; Asp255His, 4.5 h; Pro136Leu, 4.5 h). After the chase, ASA was immunoprecipitated from the homogen- ates with the polyclonal ASA antiserum. Precipitated ASA was quantified after SDS ⁄ PAGE with a bio-imaging analyser (Fujifilm). Columns show mean, minimal and maximal deviation of arbitrary units of two independent experiments. Under each dia- gram representative immunoprecipitates are shown. Arylsulfatase A degradation P. Poeppel et al. 1184 FEBS Journal 272 (2005) 1179–1188 ª 2005 FEBS indicates that the formation of the epitopes of ASA is sequential in two aspects: (a) two epitopes (A, B) form rapidly after translation, whereas others need several minutes to mature; and (b) formation of early epitopes is a prerequisite for the maturation of the late epitopes. Interestingly, none of the mutants that react with at least one of the antibodies C, E or F is retained in the ER (Gly309Ser, Glu312Asp, Arg370Gln, Arg84Gln, Arg370Trp). Our results suggest that enzymes have reached a folding state which suffices to pass the ER quality control, when they express at least epitope C partially (Arg370Gln and Arg370Trp). In a separate study we have identified two additional mutations (Phe219Val, Pro425Thr) that generally also fit into this pattern ([11] A Marca ˜ o, unpublished data). It should be mentioned that one of these mutations (Phe219Val) was found in a patient with an unusual phenotype and encodes an enzyme that, like Thr274Met, does not react with any of the mAbs. This mutant ASA, how- ever, can leave the ER to an extent of about 20% of the newly synthesized enzyme, the remainder is retained in the ER. Recently it was reported that a certain mutant of a1-AT is retained in the ER and degraded by nonpro- teasomal pathways [7]. This mutant could be stabilized by the addition of phosphatase inhibitors PAO and SOV. The existence of such a pathway is supported by the fact that phosphatase inhibitors can also inhibit immunoglobulin chain degradation in the ER [8]. Here we examined whether different ASA mutants, which are retained in the ER, show differences in the ER degradation. For that purpose we have investigated the influence of various protease and glycosidase inhib- itors on the stability of the substituted enzymes. All these mutants are partially stabilized by the protea- somal inhibitor Lc but not by the serine and cysteine protease inhibitor Lp, or any of the phosphatase inhib- itors. All mutant ASAs seem to be uniformly degraded via the proteasome; there is no indication that different mutants may use different degradation pathways. It should also be mentioned, however, that in none of the cases could we achieve a full stabilization upon proteasome inhibition. In fact the degree of stabiliza- tion in some mutants (e.g., Thr274Met, Pro136Leu) was rather weak. Although the lack of full-scale stabil- ization was unchanged when we increased the protea- some inhibitor concentration (data not shown), we cannot exclude that the proteasome was inhibited only partially. Nevertheless, the lack of stabilization by the phosphatase inhibitors indicates that recently detected nonproteasomal pathways [7,8] do not contribute to ASA degradation in the cell type used in this examina- tions. Proteins may be degraded in an ubiquitin-independ- ent way by the 20S proteasome. In various experiments (not shown) we failed to detect ubiquitinylation of the ASA mutants, suggesting that they may be degraded in a ubiquitin-independent way by the 20S proteasome [17]. Glucosidases I and II, as well as ER a1,2-mannosid- ases, play a role in the targeting of misfolded proteins in the ER [6–8,18–20]. For this reason we investigated the influence of glucosidase and mannosidase inhibi- tion on the mutant ASAs (Gly86Asp, Tyr201Cys, Asp335Val, Pro136Leu, Asp255His, Thr274Met). In these experiments all of the mutant ASAs behaved rather uniformly. They could all be stabilized by Kif, an ER a1,2-mannosidase I inhibitor. In all cases the degradation was enhanced when glucosidases I and II were inhibited by DNM. Increased degradation upon inhibition of glucosidases and stabilization by inhibi- tion of mannosidases is a common phenomenon and has been demonstrated for various misfolded proteins [6–8,21,22]. The behaviour of ASA mutants in the ER in the presence of various inhibitors is identical, Fig. 5. Effects of Kif on secretion of mutant ASAs. Ltk – cells stably expressing wild-type ASA and Pro377Leu substituted ASA were incubated in the presence of Kif, ammonium chloride (NH 4 Cl) or a combination of both compounds. Cells were pulse labelled for 16 h. After the labelling ASA was immunoprecipitated from the homogenates and secretions with the polyclonal ASA antiserum. The right panel shows an overexposed sample of the immunopre- cipitates from the secretion of Pro377Leu, which demonstrates that only low amounts of Kif stabilized ASA appear in the medium. The same experiment was performed with all ER retained ASAs, all showed identical results. P. Poeppel et al. Arylsulfatase A degradation FEBS Journal 272 (2005) 1179–1188 ª 2005 FEBS 1185 showing that all mutants interact uniformly with com- ponents of the ER degradation pathway independent of the underlying mutations. The interference with ER quality control may open new therapeutic strategies in the treatment of genetic diseases. Thus, it has been shown that secretion of an otherwise ER retained mutant protein, an a1-AT, is enhanced upon inhibition of ER a1,2-mannosidase I [23]. For that reason we have examined whether in principal any of the mutant ASAs can be delivered from the ER upon inhibition of the degradation path- way through inhibition of ER a1,2-mann osidase I. Cells were treated with Kif and ⁄ or ammonium chlor- ide. The latter interferes with lysosomal enzyme sorting in the Golgi, so that newly synthesized lysosomal enzymes appear in the medium. In the case of mutants, the appearance in the medium is thus an indicator that the enzyme has left the ER. In none of the analysed mutants, however, does treatment with Kif lead to a substantial increase of ASA in the medium. Thus, in case of ASA, inhibition of the degradation pathway does not lead to enhanced secretion, which suggests it will not be a therapeutic option for MLD. Experimental procedures Materials, enzymes, chemicals, antibodies Enzymes used for DNA modification or synthesis were from New England Biolabs (Frankfurt am Main, Germany) or Invitrogen (Karlsruhe, Germany). [ 35 S]Methionine (spe- cific activity > 39 TBqÆ mmol )1 ) was from Amersham Bio- sciences (Buckinghamshire, UK). Oligonucleotides were from MWG Biotech (Ebersberg, Germany) or Eurogentec (Seraing, Belgium). The preparation and characterization of the mAbs has been described previously [14]. Cell culture and transfection Mouse fibroblast Ltk – cells (Ltk – ) and BHK cells were maintained in Dulbecco’s modified essential medium (DMEM) supplemented with 5 or 10% fetal bovine serum (FBS), penicillin and streptomycin. For transient transfec- tions, BHK cells were transfected by Lipofectamine TM (Gibco, Karlsruhe, Germany). Cells (2 · 10 5 ) were plated onto a 3.5-cm cell-culture dish. Next day, medium was removed and cells were washed with DMEM devoid of supplements. Plasmid DNA (2 lg) was mixed with 750 lL DMEM containing 5 lL Lipofectamine TM reagent. After a 30-min incubation, the DNA–Lipofectamine TM complexes were added to the cells in a total volume of 1.5 mL. After a 5-h incubation, the Lipofectamine TM -containing medium was removed and replaced by DMEM ⁄ FBS. Cells were harvested and analysed for enzyme activity and protein concentration 48 h after transfection. In case of stable transfections, 1.2 · 10 6 Ltk – cells were plated onto a 6-cm cell-culture dish. The next day, medium was removed and 1.5 mL DMEM containing 5% FCS, penicillin and strepto- mycin was added. Plasmid DNA (5 lg) was mixed with 300 lL 150 mm NaCl. After vortexing, 15.5 lL ExGen 500 reagent (Fermentas, St. Leon-Rot, Germany) was added and incubated for 10 min. This solution was added to the cells and left for 7 h, after which the ExGen 500-containing medium was removed and replaced by DMEM in the pres- ence of 5% FBS, penicillin and streptomycin. In the case of stable transfections one tenth of the transfected plasmids was pSV 2 neo carrying a neomycin-resistance gene. Cells were selected in 800 lgÆmL )1 G-418 (Invitrogen) and single colonies were screened for expression of ASA mRNA by northern blot and protein by western blot analysis. ASA activity was measured with the artificial substrate 10 mm p-nitrocatecholsulfate in 170 mm NaCl, 500 mm sodium acetate pH 5, 0.3% TritonÒ X-100 and 1 mgÆmL )1 BSA. 200 lL of substrate solution was incubated with 5–50 lg protein of cell homogenates. Reaction was performed at 37 °C for various time periods and stopped with 500 lLof 1 m NaOH. Absorption was read at 515 nm. To obtain measurements in the linear range, only samples with an extinction below 0.7 were included; otherwise the determin- ation was repeated with shorter incubation times. Metabolic labelling and immunoprecipitation Metabolic labelling and immunoprecipitation have been described in detail elsewhere [24]. In the experiments in which the degradation pathway of the mutant enzymes were investigated the following inhibitors and final concen- trations were used: lactacystin (Lc) 25 lm (Calbiochem, Bad Soden, Germany), kifunensine (Kif) 100 lm (Calbio- chem), sodium orthovanadate (SOV) 50 lm (Sigma), phenyl- arsine oxide (PAO) 800 nm (Sigma, Munich, Germany), okadaic acid (OA) 100 nm (Calbiochem), leupeptin (Lp) 200 lm (Calbiochem), deoxynojirimycin (DNM) 1 mm (kindly provided by E. Bause, Institut fu ¨ r Physiologische Chemie, Rheinische-Friedrich-Wilhelm Universita ¨ t Bonn, Germany). Lc was present during the pulse and chase peri- ods, the others only during the chase periods. In the experiments in which the secretion of newly synthesized enzymes was enhanced by the addition of NH 4 Cl, the drug was added to a final concentration of 10 mm and was pre- sent during labelling periods. When immunoprecipitation was performed under nondenaturing conditions with the mAbs, SDS was omitted from all solutions and the immu- noprecipitation procedure was modified accordingly. In this case, cells were harvested in 50 mm Tris ⁄ HCl pH 7.0, 0.2% TritonÒ X-100 containing 25 lgÆmL )1 leupeptin, 1 mm phenylmethanesulfonyl fluoride, 5 mm iodoacetamide and Arylsulfatase A degradation P. Poeppel et al. 1186 FEBS Journal 272 (2005) 1179–1188 ª 2005 FEBS 5mm EDTA. After removing debris by centrifugation at 10 000 g for 10 min the supernatants were adjusted to 5% BSA, 0.2% TritonÒ X-100, 0.1% sodium deoxycholate and 150 mm NaCl (buffer A). The adjusted supernatants were preabsorbed twice for 30 min with 100 lL of a 10% Staphylococcus aureus (Calbiochem) suspension, which was removed by centrifugation at 10 000 g for 10 min. mAbs and antisera were added to the cleared supernatants and incubation continued for 16 h at 4 °C. 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Biochem J 367, 499–504. Arylsulfatase A degradation P. Poeppel et al. 1188 FEBS Journal 272 (2005) 1179–1188 ª 2005 FEBS . Missense mutations as a cause of metachromatic leukodystrophy Degradation of arylsulfatase A in the endoplasmic reticulum Peter Poeppel 1 , Matthias Habetha 2 ,. ASAs In order to elucidate the role of trimming reactions of the N-linked oligosaccharide side chains in ER associated degradation of mutant ASAs, stably